DESC:DESCRIPTION
FIELD OF THE INVENTION
[0001] This invention relates to the fields of Biology, Life science, Computational Biology, Biocatalysis, Molecular biology and Biochemistry.

BACKGROUND OF THE INVENTION
[0002] This invention significantly enhances the functionality of the Leucine dehydrogenase (LeuDH) enzyme through strategic engineering to expand its substrate scope and improve cofactor interactions. Specific amino acid substitutions at LeuDH's active site enable the accommodation of bulky keto acids, while incorporating the formate dehydrogenase (FDH) enzyme facilitates efficient NAD+/NADH recycling, essential for accelerating reductive amination reactions. Additionally, surface mutations on LeuDH optimise the spatial interaction with FDH, ensuring effective cofactor transfer. The inclusion of NADP Oxidase (NOX) in oxidative deamination processes further boosts the recycling of NADH to NAD+, thereby increasing the reaction speed and overall amino acid yield. These enhancements broaden LeuDH's applicability and operational efficiency in biochemical transformations. Leucine dehydrogenase (EC 1.4.1.9) is an oxidoreductase enzyme that depends on co-factor NAD+. It also belongs to the amino acid dehydrogenase (AADH) superfamily and can reversibly catalyse the oxidative deamination of aliphatic amino acids and asymmetric reductive amination of keto acids, which is a biological pathway for the synthesis of keto acids and amino acids. LeuDH is one of the widely used key enzymes in the preparation of a-amino acids by asymmetric reductive amination of keto acids and oxidative deamination of amino acids as shown in Figure 1 and Figure 2, respectively.
[0003] The oxidative deamination of LeuDH is a kinetically controlled reaction. In this reaction, the enzyme substitutes an amine group to an oxo group in the substrate while utilizing NAD+ as the cofactor. The cofactor NAD+ is reduced to NADH by the transfer of hydride from the substrate. During the mechanism, the amine group of the substrate is converted into an imine intermediate by abstraction of proton by the enzyme, causing the attack of the water molecule on the imine carbon atom. This leads to the formation of keto acid and ammonia.
[0004] The reductive amination of LeuDH is also a kinetically controlled reaction. In this reaction, the enzyme facilitates the conversion of the keto acid substrate and ammonium, leading to the formation of an imine intermediate. The resulting imine intermediate is reduced into amine product through the hydride transfer of NADH cofactor the imine intermediates during the reaction. (Zhu et al., 2016, Guo et. al., 2022)
[0005] LeuDH enzymes possess an octameric structure exhibiting D4 symmetry, resulting in a molecular mass of approximately 400 kDa (Baker et al., 1995; Turnbull et al., 1994; Yamaguchi et al., 2019; Zhao et al., 2012). They are comprised of 14 a-helices and 12 ß-strands, with a deep cleft that divides the two domains. The core domain is formed by residues from the adjacent subunit, while the nucleotide-binding domain features a Rossman fold and encompasses a dinucleotide-binding motif (Kim et al., 2022). As reported in many literatures, the LeuDH enzymes are limited to narrow substrate scope due to their smaller active site. LeuDH enzymes do not favor aromatic amino acids or bulky substrates because they are unable to accommodate them effectively within the active site.
[0006] Many engineering studies have been conducted to improve the substrate specificity of LeuDH. In recent years, active genome sequencing efforts have helped in discovering LeuDH genes in various sources. These include Bacillus cereus, Bacillus licheniformis, Bacillus sphaericus, Citrobacter freundii, Sporosarcina psychrophile, Exiguobacterium sibiricum, Alcanivorax dieselolei, Bacillus stearothermophilus, Terasakiella magnetica, Labrenzia aggregate, Planifilum fimeticola, and T.intermedius. These studies aim to explore the distinctive properties of LeuDHs for potential industrial applications. Notably, LeuDHs from Sporosarcina psychrophile and Alcanivorax dieselolei exhibit excellent cold-adaptive properties. On the other hand, LeuDHs from Exiguobacterium sibiricum and the halophilic thermophile Laceyella sacchari demonstrate good thermostability (Sanwal et al., 1961; Zhao, Ying, et al., 2012; Li, Jing, et al., 2014; Jiang, Wei, et al., 2015).
[0007] Furthermore, several studies have reported that LeuDH can perform both reductive amination and oxidative deamination reactions. The reductive amination reaction from LeuDH serves as an alternative to the transaminase reaction, offering a potential cost reduction by using ammonia as an alternative amine source instead of PMP. Zhou et. al., 2019 demonstrated that the requirement of higher catalytic efficiency and better stability could be satisfied at the same time by engineering LeuDH from Bacillus cereus. The mutations T45M/E116V improved the rigidity of the ß5 fold of the LeuDH structure which improved the hydrophobicity of the substrate entrance tunnel. These mutations improved the substrate specificity as well as its activity for substrate phenylglyoxylic acid Kcat/Km 0.191 s -1mM-1, 9.85 times higher than that of wild type.
[0008] The high-throughput screening methods for the engineering of LeuDHs have been carried out for L-tert-leucine and L-2-aminobutyric acid production with semi-rational engineering and site saturation mutagenesis (Zhu, Lin, et al., 2016, Xu, Jian-Miao, et al., 2016). Additionally, in 2021, Xiaoqing Mu et al explored LeuDH from Bacillus cereus, on which different keto acids and amino acids were explored for reductive amination and oxidative deamination reactions, respectively. Substrates such as 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxobutanoic acid, 2-oxobutanoic acid, 3,3-dimethyl-2-oxobutanoic acid, 2-oxopropanoic acid, 2-oxo-3-phenylpropanoic acid were explored for reductive amination and amino acids such as L-Leucine, L-valine, L-2-aminobutyric acid, L-tert-leucine, L-alanine, and L-phenylalanine were tested for oxidative deamination. The above-mentioned substrates are also a common set of substrates reported in most of the studies as it has limited substrate scope. The LeuDH enzyme did not show activity for 2-oxopropanoic acid, 2-oxo-3-phenylpropanoic acid, and L-alanine, L-phenylalanine (Guo et. al.,2022).
[0009] In addition to employing directed evolution strategies for the engineering of LeuDH, other methods like probe-based screening methods, rational design, and in-silico methods have been explored to identify LeuDH with excellent properties. LeuDH from Terasakiella magnetica PR1, Labrenzia alba CECT 7551, Labrenzia aggregata IAM 12614 and Bacteroidetes bacterium OLB10 were tested against trimethylpyruvic acid for reductive amination reaction, and these enzymes showed conversion of 46%, 24%, 1.5% and 5%, respectively, in the lab scale. In 2021, Jia et al studied LeuDH from Planifilum fimeticola which showed better activity for trimethylpyruvate, 2-oxobutyric acid, 2-oxo-4-methylpentanoic acid. The activity of reductive amination is high compared to oxidative deamination. L-Leucine, L-tert-Leucine, and L-phenylglycine were tested for oxidative deamination. Relative activity of 100% conversion was found only for L-Leucine and others were not converted more than 50% when they tested with the whole cell system.
[0010] LeuDH was engineered to enhance its catalytic activity in the synthesis of chiral amines. This was achieved by introducing two mutants, L52S and T143C, with improved enzyme activities of 1.55 U/mg and 2.06 U/mg, respectively, towards 2-pentanone (Lu et al., 2023). The LeuDH from B. stearothermophilus has mutations such as A113G, A115G/V291L, L40K, L40K/V294S, L40D, and L40D/V294S. The positions of A113, V291, L40 and V294 act as key residues for substrate recognition. A113 in LeuDH controls the volume of the side-chain binding pocket and determines the bulkiness of the side chain. The mutation A113G was tested against phenylpyruvate and a-ketobutyric acid substrates which showed slightly improved activity with Kcat 22 s-1 and Km of 7.1 mM. The single mutation shows better activity for oxidative deamination than reductive amination. The activity for reductive amination is higher than oxidative deamination, aliphatic substrates such as a-Keto-iso-caproate, a-Ketobutyrate, a-Keto-ß-methylvalerate have shown 100% conversion, the aromatic substrates didn’t convert in wild, mutant showed only 17% conversion. In the oxidative deamination, only L-leucine was found to be 100% converted, the other L-isoleucine and Valine showed lesser activity (Kataoka K., et al. 2003).
[0011] Enzymatic reduction of keto acids by LeuDH is a frequently used method for amino acid production on an industrial scale. To improve the maximum yield of amino acids, the recycling of the coenzyme NADH using formate dehydrogenase (FDH) has been used. Rongsheng Tao et al. 2014 studied, LeuDH from Bacillus cereus in combination with FDH and GDH enzymes for cofactor recycling and to study the impact of both to increase LeuDH activity. LeuDH enzyme with FDH provided a better activity for a-ketobutyric acid yet they discovered FDH was a cost-intensive recycling system. Additional NADH cofactor must be provided into the system every 9 hours, which improved the activity by 30 % from 52.3 to 68.4 g.l-1 in 15 h.
[0012] The substrates of reductive amination reaction, as shown in Figure 1 is converted into (2S)- amino-3-(3-hydroxyphenyl) propanoic acid, (2S)-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid and L-2-aminobutyric acid. (2S)-amino-3-(3-hydroxyphenyl) propanoic acid is also known as m-tyrosine is a nonprotein amino acid found in various grasses, which demonstrates a wide range of biological activities such as antibacterial and antifungal, herbicidal (BR., et.al.,2016). Furthermore, the nonprotein amino acid’s involvement in synthesizing macrocyclic compounds which have potent antiviral properties indicates its potential role in the inhibition of secondary tumour formation and offering tumour resistance highlighting the potential importance of (2S)-amino-3-(3-hydroxyphenyl) propanoic acid in cancer research and therapy (Kevin X. Chen et.al 2005). (2S)-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid also known as Mimosine, a toxic non-protein amino acid chemically similar to tyrosine, has been found and isolated from certain species of mimosa and all members of Leucaena glauca. The non-protein amino acid acts as an antineoplastic agent by inhibiting the growth of tumours. Mimosine inhibits DNA synthesis, affecting the S-phase of the cell cycle and leads to apoptosis. Perry C et.al. (2004) demonstrated that Mimosine disrupts the elongation of the replication fork by impairing deoxyribonucleotide synthesis by inhibiting the activity of the iron-dependent enzyme ribonucleotide reductase and the transcription of the cytoplasmic Serine hydroxymethyltransferase gene (SHMT). Additionally, Mimosine also serves as a tyrosinase inhibitor, impacting pigmentation processes in plants and possibly in other organisms. However, despite the toxicity of Mimosine, its allelochemical properties which empowers the inhibition of growth of competing species, also highlight its significant ecological role (Patrick K. K. Yeung et.al 2002). L-2-aminobutyric acid is a non-natural amino acid which has found a wide range of uses for the pharmaceutical synthesis of many chiral drugs, such as anti-epileptic Levetiracetam & Brivaracetam, and anti-tuberculotic Ethambutol. (Xu, JM.,et.al 2019).
[0013] The oxidative amination of substrates presented in Figure 2 yields Indole-3-pyruvic acid, 4-methyl-2-oxopropanoic acid and 2-oxohexanoic acid as products. Indole-3-pyruvic acid is an a-Keto analogue of tryptophan, involved in the biosynthesis of the plant hormone auxin, extensively used in the synthesis of chromo-pyrrolic acid by a heme-containing enzyme (Asamizu,.et.al 2006). It is also a natural Aryl Hydrocarbon Receptor (AHR) agonist which are promising immunomodulators which maintain immune tolerance. Indole-pyruvic acid also prevents chronic inflammation in colon by activating AHR receptor. (Reiji Aoki, et.al, 2018). Lapin I.P., Politi V (1993) proved that Indole-3-pyruvic acid has potential anxiolytic activity in mice and the activity was comparable to the standard anxiolytic diazepam. Indole-3-pyruvic acid also acts by detoxicating the cells from free radicals, reducing the activity of excitatory amino acids. 4-methyl-2-oxopropanoic acid also known as a-keto-isocaproic acid is a metabolic intermediate for the L-Leucine pathway. It has also been used as a supplement to gain muscle without fatigue (Someren, Ken A, et.al 2005). This molecule is also used as a biomarker for Maple Syrup Urine Disease, also used for chronic renal failure and in nutritional therapy in acute renal dysfunction. (Martin PM, et.al 2006). 2-oxohexanoic acid is a potent insulin secretagogue. This molecule along with glucose is an appropriate model compound for studying the ß-cell metabolism and for the initiation of insulin release. Heissig H, et.al (2005) demonstrated that 2-oxohexanoic acid directly inhibits the ATP-sensitive K+ channel (K-ATP channel) in pancreatic ß-cells. However, it is unknown whether direct K-ATP channel inhibition contributes to insulin release.
[0014] In this invention, the substrate scope of the leucine dehydrogenase is expanded by engineering the leucine dehydrogenase polypeptide for bulky substrates which include 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid and 2-oxobutanoic acid for reductive amination and L-tryptophan, L-leucine, and L-Nor-leucine for oxidative deamination. The resulting enzyme will henceforth be referred as artificially engineered leucine dehydrogenase polypeptide.
[0015] This invention presents an artificially engineered leucine dehydrogenase polypeptide that can perform both asymmetric oxidative deamination and reductive amination on bulky substrates, which are precursors for important pharmaceuticals.
[0016] To improve the yield of the product i.e. amino acids from the reductive amination reaction, a FDH cofactor recycling system was introduced, which seamlessly facilitated the NADH recycling. NADH is an important cofactor which plays a major role in accelerating the reaction. Facilitating more NADH to artificially engineered leucine dehydrogenase polypeptide can improve the reaction rate. The oxidised NAD+ molecule must diffuse to active site of FDH. Where NAD+ cofactor is reduced to NADH and subsequently diffuses to the active site of LeuDH.
[0017] There are multiple conserved residues in the active site which facilitate strong binding affinity of NAD+ in LeuDH enzymes. For instance, in engineered LeuDH, ASN181, GLY180, ASN281, CYS239, ASP205, ILE206, VAL203, ALA240, THR152, and VAL188 are found to be important residues for stabilizing the NAD+/NADH in the pocket. The artificially engineered leucine dehydrogenase polypeptide tends to push oxidised NAD+ from the active site, so that another reduced NADH is accommodated in the active site.
[0018] The substrate in the active site is anchored by LYS70 and ASN263, which recognizes the carboxylic acid group of the substrate. The LYS82 interacts with the ketone of the substrate where reductive amination happens. The ASP117 anchors the NH3 entering into the active site. The reaction is initiated by ASP117, which abstracts a hydrogen atom from NH3, which results in charged NH2 to attack the carbonyl carbon. The ketone oxygen of the substrate abstracts hydrogen from LYS70 and becomes the OH group. The unstable LYS70 abstracts a proton from Asp117 to maintain the basic charge. OH group of the substrate abstracts proton from the same LYS70 and leaves the pocket as a water molecule. Meanwhile, NADH provides a hydride to stabilize NH2 which results in the formation of the amino acid product. By providing a hydride NAD+ leaves the active site and enters the pocket of FDH for recycling.
[0019] By establishing a strong interaction with the FDH enzyme, the artificially engineered leucine dehydrogenase polypeptide also promotes a faster exchange of NAD+/NADH molecules. This accelerates the mobility of NAD+/NADH molecules. In contrast to conventional methods, which required enzyme concentration to be 5% of substrate concentration, to achieve >95% conversion for substrates like 3-hydroxyphenyl pyruvic acid and oxo-butyric acid, the concentration of FDH enzyme required in the reaction medium is now only 0.5%. This reduction is attributed to improved binding affinity and faster exchange of cofactor molecules.
[0020] In this invention, artificially engineered leucine dehydrogenase polypeptide was engineered to broaden its substrate scope, making it more effective to accommodate the bulky keto acids as substrates. This was achieved by introducing specific amino acid substitutions at the active site of leucine dehydrogenase. To enhance the overall yield of amino acids through the reductive amination process and to accelerate the reaction, the FDH enzyme was incorporated into the reaction medium. This inclusion facilitates the seamless recycling of NAD+/NADH, an essential cofactor that significantly contributes to the acceleration of the reaction. Furthermore, this invention introduces mutations on the surface region of the artificially engineered leucine dehydrogenase polypeptide, which are designed to optimize the spatial proximity between the active sites of LeuDH and FDH. This strategic positioning enables an efficient transfer or diffusion of NAD+/NADH between the active sites of the two enzymes, supported by the presence of both charged and hydrophobic residues on the surface of the artificially engineered leucine dehydrogenase polypeptide, thus ensuring a seamless transition of the cofactor.
[0021] In this invention, the artificially engineered leucine dehydrogenase polypeptide is engineered to broaden its substrate specificity, making it more effective in the incorporation of bulky amino acids for oxidative deamination. This was achieved by the introduction of specific amino acid substitutions within the active site. For the oxidative deamination reaction from artificially engineered leucine dehydrogenase polypeptide, the NADP Oxidase (NOX) enzyme is used for the recycling of NADH to NAD+. Facilitating an increased supply of NAD+ to LeuDH accelerates the reaction, resulting in a higher yield of the product.

OBJECTIVES OF THE INVENTION
[0022] The primary objective of the invention is to engineer a polypeptide for LeuDH activity against non-natural bulky substrates. The engineered LeuDH enzyme aims to showcase the reductive amination process on bulky substrates such as 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid, and 2-oxobutanoic acid, thereby yielding specific products such as (2S)-2-amino-3-(3-hydroxyphenyl)propanoic acid, (2S)-2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid, and (L)-2-aminobutyric acid, respectively. Additionally, the enzyme was engineered for oxidative deamination of L-tryptophan, L-leucine, and L-nor-leucine, resulting in the production of indole-3-pyruvic acid, 4-methyl-2-oxopentanoic acid, and 2-oxohexanoic acid, respectively.
[0023] The secondary objective of the invention is to engineer the active site of the leucine dehydrogenase enzyme to enable the accommodation of bulky substrates inside the active site and converting them into their respective products.
[0024] Additionally, the tertiary objective of the invention is the expansion of the engineering scope to improve the leucine dehydrogenase binding affinity with FDH enzyme to improve the cofactor exchange kinetics. The binding region of the leucine dehydrogenase with FDH enzyme was engineered to improve the rate of cofactor shuttling facilitating faster exchange. Ultimately the engineered Leucine dehydrogenase enzyme shows much improved activity for non-native bulky substrates, improves the binding affinity with FDH, reducing the utilization of the FDH enzymes, and
[0025] Furthermore, the polynucleotide that encodes engineered LeuDH is operably linked to one or more promoter sequences that promotes the production of recombinant LeuDH in a recombinant host cell using an expression vector and expressed in a recombinant host cell.

SUMMARY
[0026] The artificially engineered leucine dehydrogenase polypeptide reported here in this invention has a distinct and broadened substrate range, which can accommodate large keto acids and amino acids in its active site. This allows for the effective production of a wide variety of industrially important L-amino acids and a-keto acids. The enzyme shows outstanding catalytic efficiency in both asymmetric reductive amination and oxidative deamination of aliphatic, cyclic, and bicyclic substrates compared to previously reported conventional and engineered leucine dehydrogenase enzymes.
[0027] In some embodiments, the artificially engineered leucine dehydrogenase polypeptide is engineered specifically to catalyze the reductive amination of 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid, and 2-oxobutanoic acid yielding products such as (2S)-2-amino-3-(3-hydroxyphenyl)propanoic acid, (2S)-2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid, and (L)-2-aminobutyric acid, respectively. It also catalyzes the oxidative deamination of L-tryptophan, L-leucine, and L-nor-leucine to produce their respective keto acid derivatives: indole-3-pyruvic acid, 4-methyl-2-oxopentanoic acid, and 2-oxohexanoic acid, respectively (Figure 1 and 2)
[0028] In order to improve the activity, the active site of the artificially engineered leucine dehydrogenase polypeptide has been rationally mutated by site-directed mutations at specific sites, singly or in combination, to enable bulky substrates like 1a and 3a to be hosted in the catalytic pocket. (Figures 6 and 7)
[0029] The current disclosure provides an artificially engineered leucine dehydrogenase polypeptide -FDH coupled system for efficient chiral amino acid compound biosynthesis through an artificially engineered leucine dehydrogenase combined with a formate dehydrogenase (FDH) cofactor recycling system for conversion of NAD+ to NADH. The artificially engineered leucine dehydrogenase polypeptide has increased affinity to bind with FDH via targeted residue substitution. Particularly, the changes at X243 (Glu), X226 (Val), X49 (Ser or Ile), X52 (Lys or Asn), and X55 (Ser) increase the binding affinity to FDH, maximizing enzyme coupling and catalytic activity (Figures 8 and 9, Table 9). Moreover, these structural improvements allow for a more effective cofactor NADH recycling, enhancing cofactor utilization and substantially increasing overall reaction efficiency of engineered LeuDH (Figure 9).
[0030] In addition, the current disclosure offers an artificially engineered leucine dehydrogenase polypeptide-NOX coupled system for effective biosynthesis of a-keto acid compounds through the utilization of an artificially engineered leucine dehydrogenase polypeptide in conjunction with NADH oxidase (NOX) as a recycling enzyme for cofactors, specifically converting NADH to NAD+. The system provides a constant and effective supply of cofactors to make the process sustainable and industrially applicable.
[0031] In some embodiments, the invention discloses a method for reducing a keto acid compound (Formula 1a)—a substrate of the artificially engineered leucine dehydrogenase polypeptide —to a chiral amino acid (Formula 2a). The reaction mixture includes a keto acid compound (Formula 1a), an artificially engineered leucine dehydrogenase polypeptide, an ammonium ion donor, and an NADH cofactor regeneration system comprising FDH, and formic acid. In optimised reaction conditions, Formula 1a compounds are effectively transferred into their corresponding chiral amino acids (Formula 2a) and the co-substrate ammonium formate is converted into carbon dioxide. It increases efficiency and sustainability of manufacturing chiral amino acids with a potential for being used as highly effective enzymatic methodologies in the production of chemicals and pharmaceuticals for industrial usage.

[0032] In the present disclosure, the current invention provides a process for converting structural formula 1a to structural formula 2a, where the “R” group represents (3-hydroxyphenyl)methyl group, (3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)methyl group, and ethyl group molecules. Wherein the product of formula 2a is formed in enantiomeric excess.

[0033] In the present invention, the preparation of chiral amino acid compounds utilizes formate dehydrogenase (FDH), which may be either a wild-type or an engineered variant optimized for enhanced performance. The process is conducted in a cell-free system or whole cell or in crude lysate form, ensuring precise control over reaction conditions and maximizing efficiency. Depending on the specific application, the artificially engineered leucine dehydrogenase polypeptide may be utilized either as a crude extract or in a highly purified form, allowing flexibility in enzyme preparation and scalability. The reaction conditions are carefully optimized to support efficient catalysis, with a pH range of approximately 7.0 to 10.0 and a temperature range of 25°C to 65°C. These conditions ensure robust enzyme activity, maintaining stability while maximizing conversion rates, making the process highly adaptable for industrial and biotechnological applications.
[0034] The invention includes a method for converting an L-amino acid compound (Formula 3a)—a substrate for engineered LeuDH—into a-keto acids (Formula 4a). This conversion is carried out by reacting Formula 3a with a reaction medium having an artificially engineered leucine dehydrogenase polypeptide and an NADH-dependent cofactor regeneration system consisting of NOX. With these conditions, Formula 3a is converted effectively to a-keto acids (Formula 4a), while molecular oxygen (O2) is reduced to H2O as a byproduct of NADH consumption through the NOX enzyme.
[0035] In addition, the current disclosure describes a method for the conversion of structural Formula 3a to structural Formula 4a, wherein the R1 group is (1H-indol-3-yl)methyl, 2-methylpropyl, or butyl functional groups. The Formula 4a products thus formed are in the form of a-keto acids, thereby broadening the application of this system to the biosynthesis of useful biochemical intermediates.

[0036] The current invention reveals a method or process of preparing chiral amino acid and a-keto acid compounds using wild-type or modified NADH oxidase (NOX) within a cell-free system. The artificially engineered leucine dehydrogenase polypeptide may be used as a crude extract or in a highly purified state, depending on the particular application requirements. The process is conducted at optimized conditions, having a pH level of around 7.0 to 10.0 and temperature of 25°C to 65°C, having increased enzyme activity and stability.
[0037] Quantum mechanical dynamics (QMD) investigations were performed to explore the oxidative deamination of L-tryptophan by the artificially engineered leucine dehydrogenase polypeptide. The investigations yielded information on the reaction mechanism and energetics, providing insight into the enzyme's catalytic efficiency and substrate specificity (Figures 10 and 11).
[0038] The artificially engineered leucine dehydrogenase polypeptides of this disclosure exhibit higher catalytic efficiency, greater than 95% conversion and greater than 99% enantioselectivity of a-keto acids like 3-hydroxyphenylpyruvic acid and 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid into their corresponding L-amino acid products such as (2S)-2-amino-3-(3-hydroxyphenyl)propanoic acid, (2S)-2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid, and (L)-2-aminobutyric acid. Such polypeptides have an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1 so as to possess high functional conservation while maximizing substrate specificity and catalytic efficiency.
[0039] In addition, the current disclosure shows that the engineered LeuDH polypeptides catalyse the oxidative deamination of L-amino acids, such as L-tryptophan, L-leucine, and L-Nor-leucine and producing Indole-3-pyruvic acid, 4-methyl-2-oxopropanoic acid and 2-oxohexanoic acid, respectively with more than 99% conversion. The enzymatic conversion is carried out with high accuracy and efficiency and represents a useful tool for biosynthetic purposes. The engineered enzyme retains an amino acid sequence identity of at least 89% to 99% to SEQ ID NO: 1, retaining its best catalytic features.
[0040] The engineering of Leucine Dehydrogenase (LeuDH) involved a comprehensive computational pipeline to enhance its catalytic efficiency and substrate specificity for both natural and non-natural substrates. Sequences were sourced from databases such as UniProt, PDB, and PubMed etc., classified based on oxidative deamination (OD) and reductive amination (RA) activity, and modelled into 3D structures for substrate interaction studies using L-valine and trimethylpyruvate (Figure 5). Near-attack conformations provided insight into key residue interactions, which were further analyzed using a CNN-based model to identify top ranked four wild-type leucine dehydrogenases from Bacillus cereus, Peribacillus kribbensis, Legionella fallonii, and Cupriavidus basilensis which were considered as templates for developing the artificial sequence. The artificial sequences were designed by incorporating high-ranking motifs derived from network path analysis (Figure 4). These were tested with bulky non-natural substrates to ensure compatibility and optimized binding. Advanced tools such as Non-Covalent Interaction (NCI) analysis, Interaction profiling grid method, and pLDDT scoring guided hotspot identification and engineering to improve substrate affinity and enzyme stability. The outcome is optimized artificially engineered leucine dehydrogenase variants with enhanced substrate binding and increased catalytic efficiency (Figure 3).
[0041] In some embodiments, artificially engineered leucine dehydrogenase (LeuDH) variants were cloned into the pET28a(+) plasmid between NcoI and XhoI sites under the control of a T7 promoter (Figure 12) and transformed into E. Artificially engineered leucine dehydrogenase (LeuDH) variants were cloned into the pET28a(+) plasmid between NcoI and XhoI sites under the control of a T7 promoter (Figure 12) and transformed into E. coli BL21 (DE3). SDS-PAGE analysis revealed high expression for SEQ ID NOs: 1 and 90; moderate expression for SEQ ID NOs: 3, 23, and 77; and low expression for SEQ ID NOs: 25, 35, 81, 187, and 198. (Figure 13).

BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1: Reaction showing conversion of keto acids (1a) to amino acids (2a) by artificially engineered leucine dehydrogenase polypeptide performing reductive amination reaction with a cofactor recycling system containing the coenzyme FDH; the reaction substrate and product carrying R group wherein the possible structures of “R” are given herewith which is indicative of the substrate scope of the leucine dehydrogenases. The “R” group represents (3-hydroxyphenyl)methyl group, (3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)methyl group, and ethyl group molecules.
[0043] FIG. 2: Reaction involving the conversion of amino acid (3a) to keto acids (4a) by engineered LeuDH which is performing oxidative deamination reaction with a cofactor recycling system containing NOX; the reaction substrate and product carrying R1 group wherein the possible structures of “R1” are given herewith which is indicative of the substrate scope of the engineered leucine dehydrogenases. R1 group represents (1H-indol-3-yl)methyl group, 2-methylpropyl group, and butyl group molecules.
[0044] FIG. 3: The schema of steps involved in the generation of artificially engineered leucine dehydrogenase polypeptide for the increased selectivity and improved catalytic efficiency towards the non-natural bulky substrates for oxidative deamination and reductive amination reactions. A diverse set of 100 leucine dehydrogenase sequences was collected (Step 1a) and filtered them for oxidative deamination (OD) and reductive amination (RA) (Step 2a). 3-D structures were modelled for all the 100 collected leucine dehydrogenase sequences for understanding their residue spatial arrangements and to predict their fold, and interactions (Step 3a). Natural substrates such as “Trimethylpyruvate” were used for obtaining the near attack conformation of the substrate complex for reductive amination reaction, and L-valine was used as natural substrate for obtaining near attack conformation for the oxidative deamination reaction (Step 4a). The near-attack conformation obtained for these natural substrates, along with the modelled enzymes structures (Step 5a), were then analysed using AI/ML-based Convoluted Neural Networks (CNN) to identify the features of the enzyme-substrate complexes, such as the binding pose, binding energy, total number of conformations, etc. Additionally, the CNN based analysis used to identify the hotspots present within the active site that can improve the binding affinity, stabilize the conformation of the substrate or hindering the conformation, clashing with the substrate etc., (Step 6a) based on the analysis from CNN 4 leucine dehydrogenase were selected as template sequences (Step 7a), These template sequences were used to obtain computationally generated artificial sequence (Step 8a) and the detailed explanation of the stepwise process is given in (Fig. 4) .The computationally generated artificial sequence required further engineering to fit the non-natural and bulky substrates (Step 9a). The non-natural substrates such as 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid, and 2-oxobutanoic acid for reductive amination (RA) and L-tryptophan, L-leucine, and L-Nor-leucine for oxidative deamination were used to obtain near-attack conformation (Step 10a). The near attack conformation was again subjected to CNN to obtain the hotspots which can be engineered (Step 11a). The selected hotspots from the CNN were then subjected to different engineering methods such as NCI Analysis, Interaction profiling grid method and pLDDT-based engineering (Step 12a). If the engineered variants are not better compared to the computationally generated artificial sequence, which will not be selected. The improved list of variants (Step 13a), which was obtained after extensive engineering, was then expressed and checked for the activity (Step 14a). If the variant is not improved, then the CNN-predicted different hotspots will be chosen for the engineering.
[0045] FIG. 4: The process of obtaining the artificial sequence from the template structures. The stepwise process explains the generation of artificial sequence from template sequences. The stepwise method given in the Fig. 3, Step 7a to 8a is explained briefly in this figure. (A) The top 4 template leucine dehydrogenases which are “Bacillus cereus” (WP_035429507.1/AAP11078.1), “Peribacillus kribbensis” (WP_026694696.1), “Legionella fallonii” (WP_045096578.1), and “Cupriavidus basilensis” (AJG22019.1) were derived from steps 1a to 7a. These 4 templates were used to develop the artificial sequences. (B) The structure of these templates was compartmentalized into individual domains to determine which domain contributed most for stabilizing the substrate inside the active site. The circles highlight the different domains observed in the 3D structure of templates obtained using network path analysis which predicts the correlation between the catalytic residues and other residues of the protein. (C) The E-S complex of the same templates were used for obtaining the residue interactions in the near-attack conformations and validated using Non-Covalent Interaction (NCI) analysis to examine interactions which are responsible for stabilizing the substrate conformation inside the active site. The NCI Analysis provided 3D electronic density map and 2D reduced density maps for the templates E-S complexes. (D and E) From the above analysis, different domain and hotspots were captured and a score given to them based on domain details and interacting residues in the pocket. (F) These features with scores for individual domain and hotspots was submitted to AI model. (G) The ranked domains and ranked hotspots were processed by AI model and listed top ranked domains and hotspots. (H) These top ranked domains and hotspots were used to develop artificial sequences, (I) the developed sequence was computationally assed for the substrate fitting and higher stability and finally to derive an artificial sequence.
[0046] FIG. 5: The illustration above shows the three-dimensional structure of the near attack conformation involved in the reductive amination (A) and oxidative deamination (B) of natural substrates Trimethyl pyruvic acid and L-Valine with the naturally occurring Leucine Dehydrogenase. The illustration highlights the distance interactions between the active site residues and the substrate. In the reductive amination reaction, the NADH cofactor transfers a hydride to the Trimethyl pyruvic acid, while H2O is released as a secondary product while in oxidative deamination reaction, a hydride is transferred from the L-Valine to the NAD+ co-factor, and ammonia is released as a byproduct. It is observed that both Trimethyl pyruvic acid and L-Valine adopt energetically favourable conformations leading to stability within the active site. The distance between the active site residues of the Leucine Dehydrogenase enzyme and the substrates are depicted using dotted lines. The binding conformations of the substrates are as follows: A) Trimethyl pyruvic acid interacts with NADH at 3.95 Å, and the acid also interacts with catalytic LYS82 at 3.92 Å. B) L-Valine interacts with the NAD+ co-factor at a distance of 3.46 Å and with the catalytically active residue ASP117 at a distance of 3.23 Å.
[0047] FIG. 6: The illustration above shows the three-dimensional structure of the near attack conformation involved in the reductive amination reaction with the artificially engineered leucine dehydrogenase. It highlights the distance interactions between the active site residues and the substrate. In the reductive amination reaction, the NADH cofactor transfers a hydride to the substrate, while H2O is released as a secondary product. It has been observed that, after the engineering of artificially derived Leucine Dehydrogenase, the substrates adopt energetically favourable conformations, achieving stability within the active site of the engineered enzyme. The distance between the active site residues of the artificially engineered Leucine Dehydrogenase polypeptide and the substrates are depicted using dotted lines. The binding conformations of the substrates are as follows: A) 3-hydroxyphenylpyruvic acid interacts with NADH at a distance of 3.94 Å, and the substrate also interacts with the catalytic active LYS82 at a distance of 3.73 Å. B) 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid interacts with NADH at a distance of 3.94 Å, and the substrate (3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid) also interacts with the catalytically active LYS82 at a distance of 3.77 Å. C) 2-oxobutanoic acid interacts with NADH at a distance of 3.95 Å, and the substrate (2-oxobutanoic acid) also interacts with the catalytically active LYS82 at a distance of 3.97 Å.
[0048] FIG. 7: The diagram above shows the near attack conformation for the oxidative deamination reaction involving the artificially engineered Leucine dehydrogenase. The active site residues and substrate interactions are highlighted. In this reaction, a hydride is transferred from the substrate to the NAD+ co-factor, and ammonia is released as a byproduct. After engineering the artificially derived Leucine Dehydrogenase, the substrates adopt energetically favourable conformations, which stabilizes the substrate in the active site of the engineered enzyme. These interactions promote the enzyme reaction and enhance catalytic efficiency. The distance between the active site residues and substrates are represented by dotted lines. The binding conformations of the substrates are as follows: A) L-tryptophan interacts with the NAD+ co-factor at a distance of 3.95Å and with the catalytically active residue ASP117 at a distance of 3.09Å, B) L-leucine interacts with the NAD+ at 3.45Å and with the catalytically active residue ASP117 at 3.32Å, and C) L-Nor-leucine interacts with the NAD+ co-factor at 3.65Å and with the catalytically active residue ASP117 at 3.37Å.
[0049] FIG. 8: A) The artificially engineered leucine dehydrogenase polypeptide demonstrates improved binding affinity with FDH, as depicted in the graphical representation in the two-dimensional format. The artificially engineered leucine dehydrogenase polypeptide‘s hotspots are highlighted with asterisks, while the cofactor binding regions in both artificially engineered leucine dehydrogenase and FDH are represented by rectangles. By mutating the enzyme's periphery, the artificially engineered leucine dehydrogenase polypeptide enhances its binding towards FDH, resulting in a specific interaction that energetically favours the binding of FDH with artificially engineered leucine dehydrogenase polypeptide. This, in turn, increases the efficiency of cofactor recycling with the FDH-artificially engineered Leucine Dehydrogenase system, leading to improved catalytic efficiency. The incorporation of mutations enhances the recycling of the limiting agent, NADH, in the reductive amination reaction. Moreover, these mutations improve the utilization of FDH concentrations in the reaction mixture, making it more suitable for industrial applications. B) artificially engineered leucine dehydrogenase polypeptide demonstrates improved binding affinity with FDH, as depicted in the graphical representation in the three-dimensional ribbon structures. The artificially derived leucine dehydrogenase polypeptide has been specifically engineered to enhance its ability to bind with the FDH enzyme. The interactive hotspots in artificially engineered leucine dehydrogenase polypeptide that were engineered to enhance its binding affinity with FDH are shown as salmon spheres, and the residues of the FDH enzyme are depicted as light blue spheres. This enhancement facilitates a faster exchange of nicotinamide adenine nucleotide from artificially engineered leucine dehydrogenase polypeptide to FDH. This binding conformation facilitates the movement of NADH from one active site to another along the path of least resistance. Consequently, there is an increased exchange of NADH between the active sites of the artificially engineered leucine dehydrogenase polypeptide and FDH, improving the efficiency of cofactor recycling and the catalytic efficiency of reductive amination.
[0050] FIG. 9: Illustrates an enhanced cofactor recycling mechanism facilitated by a diffusion pathway established between an artificially engineered leucine dehydrogenase polypeptide and formate dehydrogenase (FDH). The figure demonstrates the spatial and temporal progression of NAD?/NADH diffusion events and the residue-level interactions mediating these transitions, following rational mutagenesis of surface residues to improve cofactor trafficking efficiency. (Fig.9A) Depicts the overall facilitated diffusion pathway, wherein the oxidized cofactor NAD? diffuses from the artificially engineered leucine dehydrogenase polypeptide to the FDH enzyme, undergoes reduction within the FDH active site, and the resulting reduced cofactor NADH returns through a distinct path into the catalytic site of the artificially engineered leucine dehydrogenase polypeptide. Two conformational states, denoted as Conformation 1A and Conformation 2A, represent intermediate spatial orientations of NAD? during its movement from the artificially engineered leucine dehydrogenase to FDH. Similarly, Conformation 1 and Conformation 2 correspond to intermediate states of NADH diffusion from FDH to artificially engineered leucine dehydrogenase. (Fig. 9B) shows the atomic-level interactions associated with Conformation 1, wherein NADH engages with residues ASP203, ILE204, and ALA208 of the artificially engineered leucine dehydrogenase, and with residues ALA199, ASP222, ARG223, THR241, ARG242, CYS256, PRO257, and HIS259 of FDH. (Fig. 9C) illustrates Conformation 2, a transitional state immediately prior to catalytic re-entry of NADH into the active site of artificially engineered leucine dehydrogenase polypeptide. Interactions are observed with residues VAL116, ASP117, ILE204, and ALA24 of artificially engineered leucine dehydrogenase polypeptide, and with residues THR241, ARG242, and GLU243 of FDH. (Fig. 9D) details Conformation 1A, corresponding to the initial transition of NAD? exiting artificially engineered leucine dehydrogenase polypeptide. NAD? interacts with residues ASP49, ARG60, VAL116, GLY240, ALA260, and ASN261 of artificially engineered leucine dehydrogenase polypeptide, and simultaneously with residues HIS224, ARG225, PRO227, and ALA238 of FDH. (Fig. 9E) depicts Conformation 2A, wherein NAD? enters the FDH enzyme through its cofactor channel. During this transition, NAD? interacts with residue ASP49 of artificially engineered leucine dehydrogenase polypeptide and with residues GLY201, ARG225, PRO227, and ASP228 of FDH.
[0051] FIG. 10: The reaction coordinates delineating the conversion of L-Tryptophan into Indole-3-pyruvic acid, as derived from Quantum Molecular Dynamics (QMD) simulations, reveal a multi-step process (A) Ground State (GS): The substrate is bound to the active site of artificially engineered leucine dehydrogenase polypeptide in a near attack conformer, serving as the initial configuration for the QMD calculation. At this stage, the reaction was initiated with the abstraction of a proton from the amine group in the zwitter ion form of the substrate. Simultaneously, one of the protons from a water molecule was transferred to the deprotonated LYS, a key catalytic residue within the artificially engineered leucine dehydrogenase polypeptide active site. Notably, the distance between the substrate's amine proton and the oxygen atom of the water molecule is measured to be 1.09 Å, while the distance between the proton of the water molecule and the nitrogen atom of the catalytic deprotonated LYS was found to be 1.02 Å. (B) Transition state 1 (TS1): During this phase, the proton transfer process reaches a critical transition stage, marked by simultaneous transfer of the amine proton from the substrate and a proton from the water molecule. The distance was measured to be 1.30 Å between proton of amine of the substrate and the oxygen of the water molecule and 1.33 Å between proton of the water molecule and catalytic deprotonated amine of the LYS, at the first transition stage of the QMD simulation. (C) Intermediate state 1 (IS1): The proton from water molecule and amine of deprotonated LYS was transferred resulting to a formation of stable intermediate. (D) Transition state 2 (TS2): In this step the hydride is being abstracted from the substrate to NAD+. At the intermediate state 1(IS1), the distance was measured to be 3.07 Å between NAD+ carbon and the hydride of the substrate was being decreased to 1.73 Å at this stage. (E) Intermediate state 2 (IS2): The hydride from the substrate was transferred to NAD+ and the substrate attained a stable imine intermediate. Due to the imine formation, the water molecule will be activated to attack the imine carbon in the next step. (F) Transition state 3 (TS3): The proton from the water molecule was abstracted by deprotonated LYS, where the distance between the proton of water molecule and deprotonated amine was measured to be 3.54 Å which reduced to 1.18 Å and the distance between oxygen of water molecule and the carbon of imine intermediate was measured to be 3.84 Å. (G) Intermediate state (IS3): The hydroxyl-amine intermediate was formed at the Ca atom of the substrate with the distance of 1.49 Å between the hydroxyl and the Ca atom and the substrate attained a stable intermediate in this step. (H) Transition state 4 (TS4): The amine’s lone pair electrons abstract the proton from the hydroxyl group. The distance was measured to be 3.04 Å at the IS3 state and was reduced to 1.30 Å. (I) Product (P): The stable product (a-keto-acid) was formed after the bond between the amine group and the Ca atom is cleaved where ammonia (NH3) was considered as a leaving group.
[0052] FIG. 11: The QMD simulations conducted to elucidate the mechanism of the oxidative deamination of L-Tryptophan converting into Indole-3-pyruvic acid. Specific mutations were introduced into the active site of the artificially engineered leucine dehydrogenase polypeptide to accommodate the bulky amino acid substrate effectively. The activation energy provided in the graph gives insights into the energy barriers associated with each step involved in conversion process. The energy required for L-Tryptophan to attain is TS1, TS2, TS3, and TS4 were 12.8 kcal/mol, 19.6 kcal/mol, 8.9 kcal/mol and 7.2 kcal/mol, respectively.
[0053] FIG. 12: Schematic representation of the plasmid designed to host artificially engineered leucine dehydrogenase gene in expression vector E. coli BL21a+(DE3).
[0054] FIG. 13: Analysis of Engineered Leucine Dehydrogenase Expression Profile via SDS-PAGE for crude lysate. The expression profiles of various engineered leucine dehydrogenase variants, identified by SEQID 1, 3, 23, 25, 35, 77, 81, 187 and 198, were analysed using SDS-PAGE. The gel illustrates the relative expression levels and molecular weights of the variants, providing insight into the effectiveness of the engineering process. The SEQID NO: 1 and 90 shows high expression and other 3, 23 and 77 shows moderate expression and 25, 35, 81, 187 and 198 have low expression levels.

DETAILED DESCRIPTION OF THE INVENTION
[0055] Terminologies Explained / Abbreviations
[0056] Unless otherwise defined, all technical and scientific terms used herein are generally understood to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, the following terms are intended to have the following meanings.
[0057] “Encoding”, “encode” is used to refer to process of transforming the coding polynucleotide sequence into a polypeptide sequence or protein or enzyme for performing the reaction.
[0058] “polypeptide” and “protein” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification.
[0059] “polynucleotide”, “nucleic acid” refers to covalently linked two or more nucleosides. The polynucleotide is either wholly comprised of ribonucleotides (RNA) or deoxyribonucleotides (DNA). The nucleotides are primarily linked with each other through standard phosphodiester linkages. The polynucleotide may either be single stranded or double stranded or may also include both single stranded and double stranded regions. The polynucleotide will comprise of naturally occurring encoding nucleobases (i.e., adenine, guanine, cytosine, thymine and uracil).
[0060] “Recombinant” is used herein to refer to a cell, nucleic acid or polypeptide which has been modified in a manner not existing in nature or produced in a natural manner through the derivation from synthetic forms and/or by manipulation through recombinant techniques. Non-limiting examples include, among others such as recombinant cells which are expressing genes which are not found in the native form of the cell or expression of native genes at a significantly different levels when compared to its expression in the native form.
[0061] “Recombinant artificially Engineered Leucine Dehydrogenase”, “recombinant engineered artificial leucine dehydrogenase”, “engineered LeuDH”, “artificially derived leucine dehydrogenase” , “artificially engineered leucine dehydrogenase”, “Recombinant artificial leucine dehydrogenase”, “artificial LeuDH”, “engineered LeuDH” or “engineered LeuDH polypeptide” are used interchangeably herein to refer to an artificial polypeptide capable of carrying out the conversion of a keto acid, in the presence of an electron donor, ammonium ion donor to an amino acid, and oxidized electron donor. In some embodiments, leucine dehydrogenase is also capable of conversion of an amino acid, in the presence of an electron acceptor, to a keto acid, NH3, and reduced acceptor.
[0062] “Formate dehydrogenase” or “FDH” or “Engineered or wild FDH” are used interchangeably here to refer to a polypeptide capable of catalysing the oxidation of formate to carbon dioxide, by donating the electrons to a second substrate, such as NAD+ in formate. These enzymes maybe originate from but are not limited to Candida boidinii, Psuedomonas Sp.101, Starkeya nomas, Escherichia coli, Clostridium ljungdahlii, D. gigas, D. vulgaris, D. desulfuricans, D. alaskensis organisms.
[0063] “NADPH oxidases” or “NOX” are used interchangeably here to refer to a polypeptide capable of catalysing the reduction of oxygen to water by utilization of NADH as the electron donor. NAD+ is formed as a by-product of the donation of electrons from NADH to the substrate oxygen. These enzymes may originate form but are not limited to Fructilactobacillus sanfranciscensis, Bacillus licheniformis organisms.
[0064] “Derived from” identifies the originating polypeptide, and/or the gene encoding such a polypeptide, upon which the engineering or processes are based upon.
[0065] “Substrate” refers to a substance or compound that is converted or meant to be converted into another compound by the action of an enzyme.
[0066] “Non-natural substrate” refers to a substance or compound that is not naturally converted or meant to be converted into another compound by the action of the enzyme. In some embodiments the term non-natural substrate refers to L-Nor-Leucine for oxidative deamination, 2-oxo butanoic acid for reductive amination.
[0067] “Bulky substrate” refers to a substance or compound that is large or has complex, rigid ring chemical structures. In some embodiments the term bulky substrate refers to 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid for reductive amination and L-tryptophan for oxidative deamination.
[0068] “Non-natural bulky substrate” refers to a substrate or compound that is large or has a complex, rigid ring chemical structures that is not naturally converted or meant to be converted into another compound by the action of the enzyme. In some embodiments, the term non-natural bulky substrate refers to 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid for reductive amination and L-tryptophan for oxidative deamination.
[0069] “Natural substrate” refers to a substance or compound that is naturally converted or meant to be converted into another compound by the action of the enzyme. In some embodiments the term natural substrate refers to trimethylpyruvate in reductive amination and L-Leucine and L-Valine for oxidative deamination.
[0070] “Keto acid group” or “a-keto acids” refers to a compound which contains both a carboxylic group and a ketone group adjacent to each other.
[0071] “Amino acid group” refers to a compound which contains both an amine group and a carboxylic group adjacent to each other.
[0072] “Conversion” refers to the enzymatic transformation of the substrate to the corresponding product. “Percentage conversion” refers to the percent of the substrate that is converted to the product within a period under specified conditions. Thus, the “enzymatic activity” or “activity” of an enzyme(s) can be expressed as “percentage conversion” of the substrate to the product.
[0073] “Enantiomeric excess (e.e %)” or “e.e” refers to the measure of chiral purity in a mixture of enantiomer, calculated as the absolute difference between the mole fractions of each enantiomer which is expressed as a percentage. In some embodiments, it indicates the relative abundance of one enantiomer compared to the other in a sample. In some embodiments herein, the e.e of 100% indicates a pure enantiomer over the other enantiomer, while 0% indicates a racemic mixture.
[0074] “Enzyme load” herein refers to the amount or concentration of an enzyme catalyst which is present or introduced in a reaction mixture for the facilitation of a specific biochemical transformation of the substrate. In some embodiments, the enzyme load also refers to the amount of enzyme required to achieve a desired conversion efficiency, reaction rate or product yield under defined reaction conditions. In some embodiments, the enzyme load is expressed in various units including but not limited to mg/ml, U/ml, weight percent (w/w or w/v) or molar concentration (mol/L).
[0075] “Cofactor” refers to a substance that is necessary or beneficial for the activity of an enzyme. In the context of leucine dehydrogenase, the cofactor is generally a nicotinamide cofactor.
[0076] “Nicotinamide cofactor” refers to any type of the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD+ and NADH, respectively) and the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP+ and NADPH, respectively).
[0077] “Cofactor regenerating system” and “cofactor recycling system” are used interchangeably herein to refer to a set of reactants that participate in a reaction that reduces the oxidized form of the cofactor (e.g., NAD+ to NADH or NADP+ to NADPH). In the embodiment herein, cofactors oxidized by leucine dehydrogenase catalysed reaction are regenerated in reduced form by the cofactor regenerating system. Cofactor regenerating systems comprise a stoichiometric reductant that is a source of reducing hydrogen equivalents and is capable of reducing the oxidized form of the cofactor.
[0078] “Ammonium source” or “ammonium ion donor” refers to a substance or formulation that, when introduced into a reaction medium, generates ammonia or ammonium ions.
[0079] “Reaction medium” refers to a solution comprising a mixture of two or more components (e.g., enzyme, substrate, cofactor, cofactor recycling enzyme, appropriate buffer. in which reaction takes place.
[0080] “Thermostable protein” refers to a polypeptide that maintains similar activity (more than e.g., 50% to 70%) after exposure to high or low temperature (10 – 20°C or 30 - 60°C) for a period of time (e.g., 0.5 – 24 hrs) compared to the untreated polypeptide.
[0081] “pH stable protein” refers to a polypeptide that maintains similar activity (more than e.g., 50% to 70%) after exposure to high or low pH (4.5 – 6 or 8 -12) for a period of time (e.g., 0.5 – 24 hrs) compared to the untreated polypeptide.
[0082] “Asymmetric reductive amination”
[0083] The process described involves the synthesis of chiral amines through reductive amination. This reaction takes place between a prochiral carbonyl compound and an amine.
[0084] “Fold” herein refers to the description of multiplicative changes in a measurable parameter such as activity, concentration, yield, potency, or expression level. In some embodiments, “-fold” typically involves a positive change or increase in a measurable parameter such as activity, concentration, yield, potency, or expression level relative. The non-limiting examples include but are not limited to, “5-fold” increase in enzyme activity of artificially engineered leucine dehydrogenase for catalysis of 2-oxobutanoic acid when compared to reported conventional leucine dehydrogenases indicate that artificially engineered leucine dehydrogenase has five times more enzyme activity towards the catalysis of 2-oxobutanoic acid when compared with all the known reported leucine dehydrogenase enzymes.
[0085] “QMD” or “Quantum Molecular dynamics” or “CPMD” is a method for performing ab-intio molecular dynamics simulations, which incorporates quantum mechanical effects within the framework of Density Functional Theory (DFT). This study is primarily conducted at the atomic level of molecules to provide insight into their dynamic properties, reactions and structures.
[0086] “Features” of an invention, as referred herein, denote the technical elements that collectively define the scope and substance of the claimed subject matter. In some embodiments, such features may comprise the structural, functional, or procedural characteristics of biological materials, compositions, or processes. These include, but are not limited to, amino acid or polynucleotide sequences, specific substitutions, chemical modifications, or other molecular configurations that contribute to the technical advancement or provide a technical solution to a problem.
[0087] “Expressed”, refers to the transcription and/or translation of a nucleic acid/polynucelotide sequence, typically such as deoxyribonucleic acid or ribonucleic acid, leading to the production of a functional biological molecule, typically a polypeptide or protein or enzyme, within a host system. The expression may occur either in vivo, within a living organism, or in vitro, within a controlled environment such as a cultured cell line or cell-free expression system.
[0088] “Concentration”, refers to the quantitative measure of a biologically relevant component—such as a nucleic acid, protein, enzyme, substrate, cofactor, or any other molecular entity—present within a defined volume of a solution, medium, or reaction system. In some embodiments, the concentration of such components is typically expressed using scientifically recognized units, including but not limited to molarity (M), mass per volume (e.g., mg/mL), activity units per volume (e.g., U/mL), or percentage (%), depending on the physical or functional nature of the substance.
[0089] “Used in the form of”, refers herein to a specific physical, chemical, or functional form in which a biological molecule, compound, or composition is prepared, applied, or administered for the purposes of the invention. In some embodiments, this term encompasses the different states or preparations of a substance that are relevant to the practice of the invention, which may include, but are not limited to salts, esters, or derivatives of a molecule, complexes or conjugates, including but not limited to antibody-drug conjugates, formulations, which may be in liquid, solid, or lyophilized form, carriers, delivery systems, or vectors for biological entities, prodrugs, isomers, analogues, or variants of a compound.
[0090] “Exhibits”, refers herein to the demonstratable or observable properties, behaviours, or characteristics of a biological molecule, compound, or composition that are presented or shown in the context of the invention. These may include, but are not limited to, experimental results, data, figures, or graphs that illustrate the performance, function, efficacy, or characteristics of the invention.
[0091] “Cofactor recycling efficiency” refers to the increase in NAD+/NADH recycling by the cofactor recycling system. Mutating the periphery regions of the artificially engineered leucine dehydrogenase polypeptide results in a specific interaction that energetically favours the binding of FDH. This, in turn, increases the cofactor recycling efficiency with the FDH-artificially engineered Leucine Dehydrogenase system, leading to improved catalytic efficiency.
[0092] The “cofactor recycling efficiency” of the recombinant engineered leucine dehydrogenase polypeptide is measured relative to that of wild-type leucine dehydrogenase polypeptides.
[0093] In the context of the polypeptides shown here, "amino acid" or "residue" refers to the particular monomer at a sequence position (for example, X45 indicates that the "amino acid" or "residue" at position 45 is a Met).
[0094] When an amino acid is incorporated in a peptide or polypeptide, it is referred to as a "acidic amino acid or residue" if the hydrophilic amino acid or residue has a side chain that shows a pKa value of less than roughly 6. Because of the loss of a hydrogen ion, acidic amino acids usually have negatively charged side chains at physiological pH. L-Glu (E) and L-Asp are acidic amino acids that are genetically encoded (D).
[0095] When an amino acid is incorporated in a peptide or polypeptide, it is referred to as a "basic amino acid or residue" if it is a hydrophilic amino acid or residue with a side chain showing a pKa value larger than roughly 6. Because of their connection with hydronium ions, basic amino acids usually contain positively charged side chains at physiological pH. L-Arg (R) and L-Lys are two basic amino acids that are genetically encoded (K).
[0096] "Polar amino acid or residue" describes a hydrophilic amino acid or residue with a side chain that is uncharged at physiological pH and at least one bond in which one of the two atoms holds the pair of electrons that they share more tightly. Polar amino acids L-Asn (N), L-Gln (Q), L-Ser (S), and L-Thr are among those that are genetically encoded (T).
[0097] A hydrophobic amino acid or residue with a side chain that is uncharged at physiological pH and bonds where the pair of electrons shared by two atoms is typically held evenly by each of the two atoms is referred to as a "non-polar amino acid or residue" (i.e., the side chain is not polar). Non-polar amino acids that are genetically encoded are L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M), and L-Ala (A).
[0098] A "hydrophilic amino acid or residue" is defined as an amino acid or residue that, in accordance with the normalised consensus hydrophobicity scale, has a side chain that exhibits hydrophobicity of less than zero. L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K), and L-Arg are hydrophilic amino acids that are genetically encoded (R).
[0099] According to the normalised consensus hydrophobicity scale, an amino acid or residue is considered "hydrophobic" if its side chain has a hydrophobicity value higher than zero. L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A), and L-Tyr are hydrophobic amino acids that are genetically encoded (Y).
[0100] A hydrophilic or hydrophobic amino acid or residue with a side chain containing at least one aromatic or heteroaromatic ring is referred to as an "aromatic amino acid or residue". L-Phe (F), L-Tyr (Y), and L-Trp are examples of aromatic amino acids that are genetically encoded (W). It is occasionally categorised as either an aromatic residue because of its side chain's heteroaromatic ring or as a basic residue due to the pKa of its heteroaromatic nitrogen atom, L-His (H).
[0101] A hydrophobic amino acid or residue with an aliphatic hydrocarbon side chain is referred to as an "aliphatic amino acid or residue". The aliphatic amino acids L-Ala (A), L-Val (V), L-Leu (L), and L-Ile are all genetically encoded (I).
[0102] "Amino acid difference or residue difference" describes how a polypeptide sequence differs from a reference sequence in terms of the residue at a certain location. For instance, a change in the residue at position X43 to any other residue than valine is referred to as a residue difference at position X116, where the reference sequence contains valine. According to the information provided here, an enzyme may differ in one or more residues from a reference sequence. When more than one residue differs from the reference sequence, it is usually indicated by a list of the specific places where the modifications are made.
[0103] “Charged residues” describe the amino acid residues which have charge in the side chain of the amino acids are known as changed residues. For example, L-Asp (D) and L-Glu (E) are known as negatively charged residues, and L-Arg (R), and L-Lys (K) are known as positively charged amino acid residues.
[0104] In some embodiments “salt bridges” refer to the electrostatic interaction between charged residues that is between L-Arg (R) or L-Lys (K) with either L-Asp (D) or L-Glu(E). The charged residues interactions are a type of interactions which stabilize the enzyme structures.
Method of deriving artificial sequences
[0105] The Leucine dehydrogenases contain a compact active site, which is not suitable for the conversion of bulkier substrates. The method given below is an AI implemented method which developed an artificially generated sequence which can convert bulky substrates into their respective products. The diverse list of LeuDH, which are present in different databases (Uniprot, GenBank and PDB), were collected and used for understanding the native binding complex with natural substrates. These complexes were further analysed using CNN-based models to study binding pose, binding energy, and the hotspots that anchor the substrate conformation. With the detailed analysis, the top four wild sequences were extracted and used for deriving the artificial sequence. The developed artificial sequence was used to obtain near attack conformation with non-natural substrates and the complexes were analysed using CNN-based models and the hotspots were extracted for engineering. These hotspots were further engineered using various technologies such as non-covalent interactions (NCI), Interaction profiling grid method (Kumar P. et al., 2023), and pLDDT based engineering method used to engineer the artificial sequence further to improve the activity (Figure 3).
[0106] Step 1a: Collection of reported Leucine dehydrogenase from different databases
[0107] The diverse set of leucine dehydrogenases from different databases (Uniprot, GenBank and PDBs) was obtained. Leucine Dehydrogenase (LeuDH) is an enzyme catalyzing the oxidative deamination and reduction amination of different substrates. The initial step in this process is retrieving data on LeuDH from multiple databases. Some of these are protein sequence databases such as UniProt, structural databases such as the Protein Data Bank (PDB), and literature databases such as PubMed (Table 1).
[0108] Step 2a: Classification of LeuDH for Oxidative Deamination (OD) and Reductive Amination (RA) activity
[0109] The activity profiles for different substrates in LeuDH enzymes was characterized. The collected diverse sequences were further categorized based on their reported activity for oxidative deamination (OD) and reductive amination (RA). This categorised sequence helped to obtain specific mutations for specific activity.
[0110] Step 3a: 3D Modelling of the categorized LeuDH
[0111] After the relevant LeuDH sequences were categorised, their three-dimensional structures were modelled. If experimental structures exist, they are utilized directly. Reliable 3D models are critical for studying enzyme-substrate interactions and for engineering purposes.
[0112] Step 4a: Natural substrates for oxidative deamination and reductive amination
[0113] The interactions of natural substrates with the amino acid residues present inside the active site pocket is crucial information to understand the importance and function of individual residues. Natural substrates such as “Trimethylpyruvate” were used for obtaining the near attack conformation of the substrate for reductive amination reaction, and “L-valine” was used as a natural substrate for obtaining near attack conformation for the oxidative deamination reaction. The interactions of both Trimethylpyruvate and L-valine when they are complexed in the active site of LeuDH is crucial to understand the role of individual amino acids present inside the pocket. (Figure 5).
[0114] Step 5a: Near attack conformations of the substrate with modelled structures
[0115] The near attack conformation of L-Valine and Trimethylpyruvate in the categorized LeuDH structures was obtained to study the interactions with surrounding residues. The substrates were compartmentalized into atoms for studying the interactions. The atomic interactions are anchoring points which align the substrate conformation as required for the reaction.
[0116] Step 6a: AI Based CNN model to extract and interpret the interactions of the binding poses
[0117] A Convolutional Neural Network (CNN)-based model is utilized to analyse the most important features of enzyme-substrate interactions. CNN is superior at recognizing patterns, therefore, suitable for applications like molecular interaction analysis with multiple features. The CNN model is utilized to extract patterns of interaction between the substrate and enzyme. Studying the binding poses, which describe how the substrate is accommodated in the active site. Finding key interacting hotspots, emphasizing amino acid residues important for binding, which assists in determining the stability and strength of the enzyme-substrate complex.
[0118] Step 7a: Selected four wild sequences from the Database as template
[0119] CNN is a robust model which was used to identify more prominent sources where the substrate conformation, binding pose, and atomic interactions with the nearby atoms were accounted, and the top four enzymes were selected for developing the artificial sequences. The CNN based model was used to predict the most favourable and interactive source where the near attack conformations of the substrates was energetically lower compared to other sources. The selected top four LeuDH wild templates were from “Bacillus cereus” (WP_035429507.1/AAP11078.1), “Peribacillus kribbensis” (WP_026694696.1), “Legionella fallonii” (WP_045096578.1), and “Cupriavidus basilensis” (AJG22019.1).
[0120] Step 8a: Computationally generated sequences
[0121] The top ranked complexes from the previous step were further analysed using network path analysis to predict different domains which contribute to the overall catalytic site stability and NCI Analysis for residue interactions in the near attack conformations. These two components were used to derive the artificial sequences. Network path analysis was used to identify different domains present on selected top 4 templates. These domain movements and correlation for stabilizing the active site was studied and individual domains was given a score. The Non-Covalent Interactions were used to understand residue substrate interactions between the natural substrate and its surrounding residues. The NCI Analysis provided 3D density maps and 2D reduced density gradient maps for the E-S complex highlighting different interactions around the substrate molecule. Those residual interactions which stabilized the substrate conformations within the pocket were ranked as a prioritization of the hotspots.
[0122] The ranked domains from all 4 different templates with its respective score and maps from NCI analysis were used as features and subjected to CNN model to analyse and predict the top ranked domains and hotspots. These ranked hotspots and ranked domain details were used to generate an artificial sequence from four base template structures (Figure 4). Multiple artificially engineered LeuDH sequences were generated which were tested against the better fitting of the substrate. SEQ ID NOs: 1, 212, 213, 214, 215, 216, 217, 218, 219, 220, and 221 were generated with this approach. The SEQ ID NO 1 sequence was selected based on the substrate fitting and compactness of the active site which was developed from the information extracted from the different domains of the top four templates. The position with substitution such as X69 is ARG, X159 is CYS, X212 is LYS, X272 is CYS, X288 is CYS and X334 is CYS were obtained as feature mutation and observed for stabilizing the active site configuration. Additionally, these mutations were observed to be present on different domains, spatially arranged and improving the catalytic function. These mutations were used as a starting point to develop the engineered artificial sequence for non-natural and bulky substrates. These mutations were included in the SEQ ID NO: 1 for further engineering to fit non-natural and bulky substrates in the active site.
[0123] Step 9a: Non-natural-bulky substrates for oxidative deamination and reductive amination
[0124] The non-natural-bulky substrates are those substrates whose products yield products which are industrially crucial for formation as intermediates for APIs or as APIs themselves. Substrates such as 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid, and 2-oxobutanoic acid for reductive amination and L-tryptophan for oxidative deamination were used for obtaining near-attack conformation. These substrates yield products such as M-tyrosine, which has anticancer, antibacterial and antifungal properties; Mimosine which is an antineoplastic agent having anti-tumour properties and is used as a herbicide in plants; and 2-aminobutaryic acid, which is a key intermediate in the formation of pharmaceutically important products such as ethambutol, brivaracetam and levetiracetam. Similarly, L-tryptophan yield Indole-3-pyruvic acid, which exhibits anti-inflammatory, analgesic and sedative effects.
[0125] Step 10a: Near-attack conformations of non-natural substrates with computationally generated sequence
[0126] Near-attack conformations were obtained to predict the most favourable conformations of the substrate within the active site of SEQ ID NO 1. Multiple binding poses were used to evaluate their stability based on binding energy and the atomic interactions. The results help identify the most probable near attack conformations, which are essential for enzymatic catalysis. The substrates such as 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid, and 2-oxobutanoic acid for reductive amination and L-tryptophan, L-leucine, and L-Nor-leucine for oxidative deamination were used for obtaining near attack conformations. (Figure 6 and 7).
[0127] Step 11a: CNN to compare and identify hotspots for engineering
[0128] Hotspot identification is important in enzyme engineering because mutation of these residues can improve/affect substrate specificity, catalytic efficiency, or stability, and destabilize the conformation of the substrate. Residues in direct contact with the substrate tend to have a specific functional role, and their interactions contribute to near-attack conformations that are optimized. Alteration of these positions can result in either positive or negative effects on enzyme function. CNN was used to identify hotspots for engineering, the local network of the hotspot amino acids should be analysed as it is highly important to maintain the catalytic architecture.
[0129] Step 12a: Engineering studies using NCI Analysis, Interaction profiling grid method and pLDDT
[0130] Once the hotspots were identified, technologies such as non-covalent interactions, Interaction profiling grid method, and pLDDT based enzyme engineering methods were applied for further engineering of SEQ ID NO 1.
[0131] NCI
[0132] Non-Covalent Interaction (NCI) Analysis examines weak intermolecular forces such as hydrogen bonds, van der Waals interactions, and electrostatic interactions to optimise substrate binding. Which is used to identify hotspots that favour the substrate binding affinity, and which are required to substitute with different amino acids. The NCI Analysis provides 3D reduced density gradient maps and 3D electronic maps which explain the possible non-covalent interactions which are present around the substrate.
[0133] Interaction profiling grid method
[0134] The Interaction profiling grid method was used to analyse the SEQ ID NO 1-substrate complexes, with an emphasis on the active site. Quantum mechanical probes are used by the Interaction profiling grid method to collect data regarding the enzyme-substrate complex and the catalytic reaction. The kinetic characteristics of the enzyme are then predicted by analysing this data. In addition to unique probes that assess the direct quantum mechanical interaction energy between the amino acids in the active site, the technology integrates quantum mechanical probes inside a polarisable continuum model. The Fragment Molecular Orbital (FMO) approach is used to determine Pair Interaction Energy (PIE). Until a higher cumulative PIE is reached. Following the grouping of these grid points into patches, an alignment process compares the generated query probe patterns for each patch to pre-existing patterns in an internal database. Protein mutations are introduced using the highest PIE probe-amino acid pairings from the matched patterns. Patches can be localised to any part of the protein. In order to rank the designed variations, the resulting library was analysed for energetics. Cumulatively, the grid base analysis for the amino acids network around the catalytic site provides a score which was used for further analysis and to introduce mutations.
[0135] PLDDT based engineering
[0136] A predicted local distance difference test (pLDDT)-based method was used to engineer the artificially generated leucine dehydrogenase enzyme. The pLDDT score is a measure of the local confidence of every residue in a predicted structure, ranging from 0 to 100, with higher scores reflecting higher confidence and generally more accurate predictions. This measure tests local structural confidence by estimating the extent to which prediction is matched against an experimental structure, using the local distance difference test Ca (lDDT-Ca), a measure that tests local distance accuracy without resorting to structural superposition.
[0137] The current invention offers a systematic approach for engineering the artificially generated leucine dehydrogenase enzyme using pLDDT to drive structural changes with accuracy, improving enzyme activity, stability, and specificity. The process commences with predicting the three-dimensional structure of the artificially generated leucine dehydrogenase enzyme using computational approaches, where pLDDT scores are computed to determine per-residue confidence. Higher pLDDT values correspond to accurate prediction of the structure, whereas lower values reflect areas of uncertainty. Regions with low pLDDT scores are recognized as structurally unstable and are thus targeted for alteration, while high-confidence regions are retained to preserve structural integrity. Mutating low pLDDT residues by means of site-directed mutagenesis or computational protein design to enhance pLDDT scores. Mutational alterations are made to augment enzyme function through the optimization of activity, stability, and specificity. The produced artificially generated leucine dehydrogenase enzyme variants are subsequently validated and screened, whereby the mutations are introduced with mutation primers and tested to measure their enhanced attributes. Effective variants are chosen to be developed further. For the purpose of continuous improvement of artificially generated leucine dehydrogenase enzyme biocatalytic properties, an iterative refinement process is utilized, with repeated structure prediction and refinement of modification. The cycle provides continuous improvement of artificially generated leucine dehydrogenase enzyme performance.
[0138] Step 13a: Engineered variants list
[0139] The final outcome of this workflow was development of an optimized artificial engineered leucine dehydrogenase variants that exhibit improved binding and catalytic efficiency for both natural, non-natural, and bulky substrates. These engineered enzymes found with energetically favourable substrate conformation, meaning that the enzyme-substrate interaction is strong and conducive to efficient catalysis.
[0140] Step 14a: Expression of the Variants and wet-lab validation
[0141] The optimized variants experimentally validated with computational analyses to confirm their improved performance and are transformed and expressed through the vector plasmid pET28a (+) in the heterologous expression system of the host cell E.coli BL21, which contains the artificial LeuDH gene organized between the NcoI and XhoI restriction sites, under the control of the T7 promoter. The transformation is achieved by incubating the host and plasmid DNA on ice for 30 minutes, followed by a heat shock at 42°C for 1 minute, and a 5-minute incubation on ice. The cells are then cultured at 37°C for 1 hour, pelleted, resuspended in LB broth, and plated on LB agar with kanamycin (50 µg/mL). For expression, 5 mL of overnight culture is inoculated into 100 mL of LB medium with kanamycin, and once the culture reaches an OD600 of 0.6, protein expression is induced by 0.1 mM IPTG at 25°C for 16 hours. The cells are harvested by centrifugation, resuspended in PBS buffer containing lysozyme (1 mg/mL), and incubated on ice for 1 hour. Lysis is performed using sonication, and the expression levels of the polypeptide are analyzed by SDS-PAGE. Furthermore, biochemical assays and kinetic studies are conducted on the expressed artificially engineered leucine dehydrogenase variants for against each of the substrates for both reductive and oxidative deamination and top variants are selected.
[0142] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide comprise an amino acid sequence that has one or more residue differences which are X69 is ARG, X159 is CYS, X212 is LYS, X272 is CYS, X288 is CYS and X334 is CYS which are present in all the artificial sequences generated in this invention. The residue differences can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions.
[0143] In some embodiments, the position with substitution such as X69 is ARG, X159 is CYS, X212 is LYS, X272 is CYS, X288 is CYS and X334 is CYS were obtained as feature mutation and observed stabilizing the active site configuration. Additionally, these mutations were observed to be present on different domains, spatially arranged and improving the catalytic function. These mutations were used as a starting point to develop the engineered artificial sequence for non-natural and bulky substrates.
[0144] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide contains one or more of the following residue differences when compared to the polypeptide of SEQ ID No. 1 in the first round of the engineering: X7 is substituted to GLU, ASP, THR or SER; X11 is substituted to LYS, HIS, ARG, VAL, ILE or ALA; X42 is substituted to LEU, ILE or VAL; X43 is substituted to GLY, ALA or VAL; X44 is substituted to GLY, ALA or VAL; X45 is substituted to THR, ALA, MET, LEU or CYS; X49 is substituted to SER or ILE; X51 is substituted to ASP, ALA, ASN, PRO, GLY or THR; X52 is substituted to LYS or ASN; X64 is substituted to ALA, SER, GLY or THR; X80 is substituted to GLY, ALA or VAL; X81 is substituted to ALA, GLY or VAL; X90 is substituted to ARG; X116 is substituted to VAL; X119 is substituted to ALA, SER, THR, GLU or ASN; X122 is substituted to GLU, ILE, VAL, ALA, LYS or ASN; X123 is substituted to ASP; X137 is substituted to GLY, VAL or ALA; X138 is substituted to ARG, THR, ILE or SER; X140 is substituted to GLU, THR, ASP, ASN or GLN; X141 is substituted to SER; X148 is substituted to VAL, HIS, PRO, ASN or ILE; X172 is substituted to ASN, SER, ASP, ALA or THR; X174 is substituted to GLU, ALA, GLN, SER, LYS, THR or ASP; X177 is substituted to GLU; X190 is substituted to LEU, MET, VAL or ALA; X191 is substituted to MET, CYS, LEU or ILE; X192 is substituted to LYS; X194 is substituted to LEU, ALA or ILE; X195 is substituted to HIS, ASN, TRP, ARG, GLN or SER; X198 is substituted to GLY, ARG, SER or LYS; X199 is substituted to ALA, VAL, GLY or THR; X200 is substituted to LYS, GLN, ASN, SER, ARG or HIS; X221 is substituted to SER, GLU, LYS, ARG, THR or GLN; X224 is substituted to GLU, ASP, ASN, GLY or ALA; X226 is substituted to ASN, VAL, TYR, ASP, GLU, SER or GLY; X230 is substituted to ASP, GLY, SER or ALA; X241 is substituted to THR, LEU or MET; X242 is substituted to PRO, GLY or VAL; X248 is substituted to GLU, PRO, GLY or ALA; X244 is substituted to THR; X261 is substituted to SER; X267 is substituted to ASP, GLY, LYS, ARG, GLU or ASN; X269 is substituted to ASP; X274 is substituted to ILE, GLN, ASN, LEU or THR; X276 is substituted to CYS, MET, HIS or ASP; X290 is substituted to ALA, SER or THR; X293 is substituted to THR, ALA, ILE, LEU , GLY or SER; X294 is substituted to ILE, SER, MET or CYS; X295 is substituted to ASN, GLN, ASP or GLU; X296 is substituted to MET, CYS, PHE or LYS; X310 is substituted to GLN, LYS, ARG, ALA or ASN; X309 is substituted to LEU; X311 is substituted to ARG; X312 is substituted to ILE, VAL or LEU; X314 is substituted to SER, GLU, THR or GLN; X318 is substituted to THR; X320 is substituted to ARG, ALA, SER, THR or GLU; X325 is substituted to CYS, ILE, LEU or PRO; X326 is substituted to SER, CYS, ALA or GLY; X336 is substituted to CYS, ALA, SER or GLY; X340 is substituted to LEU, GLU, MET or VAL; X348 is substituted to LEU, MET, ILE, VAL or ASN; X350 is substituted to ASN, ARG, LYS, GLN, HIS or MET; X352 is substituted to ARG, LYS, THR or GLN; X363 is substituted to ILE, VAL, SER, CYS or ALA and X364 is substituted to SER, ASN, GLY, THR or HIS;
[0145] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide contains one or more of the following residue differences when compared to the polypeptide of SEQ ID No. 1 in the second round of the engineering: X24 is substituted to GLU, GLN, ASN, THR, SER, ARG or LEU; X41 is substituted to CYS, ALA, VAL, GLY or ILE; X46 is substituted to ALA, ASN, LYS, SER, PRO, GLN or PHE; X55 is substituted to SER, THR, ASP or GLU; X63 is substituted to LEU, ILE or VAL; X66 is substituted to GLN, HIS, PHE, THR, ASN, SER, VAL, ILE or ALA; X67 is substituted to ALA, GLY, VAL, HIS, SER, THR, ASN, CYS, LEU or ILE; X75 is substituted to LYS, VAL, ALA, LEU or ILE; X83 is substituted to THR, ALA or VAL; X90 is substituted to ARG; X115 is substituted to ASN, GLU, LYS, ASP, GLN, ILE or LEU; X118 is substituted to SER, ALA, PHE, ASP, ASN, CYS, HIS or LEU; X121 is substituted to ASN, ALA, SER, THR, GLU, ALA, GLY or VAL; X125 is substituted to MET, CYS, VAL or ALA; X142 is substituted to LYS, HIS, SER, THR or VAL; X147 is substituted to ARG, ASP, ASN or GLU; X166 is substituted to GLU, ASP, GLN, VAL, HIS, SER or ASN; X170 is substituted to THR, SER, ASP, ASN, HIS, VAL or ALA; X189 is substituted to HIS, GLU, ASN, SER, ALA or LYS; X193 is substituted to GLU, HIS, LEU, PHE, TYR or ASP; X197 is substituted to GLU, ASP or ALA; X243 is substituted to GLU, ASP, GLN or ASN; X301 is substituted to GLN, GLU, ASP or ASN; X317 is substituted to ASP; X322 is substituted to LYS, VAL, ILE or THR; X354 is substituted to THR, GLN, LYS, MET or ASN; X357 is substituted to ARG, GLY, SER, THR, LYS or ALA; X361 is substituted to ASP, HIS, SER or ASN;
[0146] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide contains one or more of the following residue differences when compared to the polypeptide of SEQ ID No. 1 in the third round of the engineering: X48 is ALA, ARG, ASN, LYS, MET, TYR, ILE, VAL or PHE; X71 is ALA, SER, ASN, THR or GLN; X94 is SER, ASN, THR, ALA or GLY; X101 is LEU, PHE, ILE or VAL; X130 is LYS, THR, GLN, SER, ASP or GLU; X143 is LYS, HIS, ARG, VAL or GLY; X146 is GLY, VAL or ALA; X150 is ARG, PRO, HIS or ILE; X154 is LYS, TYR, PHE or HIS; X157 is LYS, TYR, PHE, LEU or CYS; X173 is GLU, ASP, LEU, VAL or ALA; X185 is HIS, SER, LYS, HIS, VAL, LEU, ILE, ALA, GLY or ARG; X196 is ALA, VAL, GLY or GLU; X201 is LEU, ILE, VAL or ALA; X213 is ARG, LYS, MET, SER or LEU; X218 is PHE, TYR, PRO or ALA; X227 is GLU, GLU, ASP, ALA, GLN or ASN; X245 is VALX252 is LYS, GLN, LEU, LYS or ARG; X264 is GLU, ASN, GLN, ASP or GLU; X285 is ASP or GLU; X286 is TYR, PHE or TRP; X299 is ASP, GLU, ASN or GLN; X305 is GLU, ASP, GLN, VAL or ILE; X313 is ARG, GLU, ASP or ASN; X324 is GLU, ASP, ALA, SER or ASN; X332 is ALA, CYS, PRO, SER or VAL; X335 is VAL, LYS, LEU, ALA or ARG; X346 is ALA, GLY, GLU, VAL or ILE; X349 is LYS, ARG, ALA or SER; X353 is SER, THR, GLY, LYS, ASN or ARG; X360 is HIS, GLN, LYS, ARG or MET;
[0147] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide given in SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 ,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 ,201, 202, 203, 204, 205, 206, 207, 208, 209 and 210 have an amino acid difference by one or more substitutions in combination with one or multiple that are at least about 90%, 91%, 92%, 93%, 94%, 95% identical to SEQ ID NO:1
[0148] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 ,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209 and 210 are used individually or in combination with FDH enzyme for the conversion of substrates such as 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid, and 2-oxobutanoic acid for reductive amination and L-tryptophan, L-leucine, and L-Nor-leucine for oxidative deamination under suitable reaction condition.
[0149] The present invention provides the recombinant engineered artificial leucine dehydrogenase polypeptide that is capable of performing both asymmetric reductive amination and oxidative deamination on bulky keto and amino acid substrates with improved properties than the previously reported conventional leucine dehydrogenase.
[0150] The present disclosure provides a process for reductive amination where the conversion of keto acids to chiral amino acids in the presence of an ammonium source in a reaction mediated by the recombinant engineered artificial leucine dehydrogenase polypeptide and a cofactor recycling system comprising an FDH as generally depicted in Figure 1.
[0151] The present disclosure provides a process for oxidative deamination where the conversion of L-amino acids to a-keto acids is mediated by the recombinant engineered artificial leucine dehydrogenase polypeptide and a cofactor recycling system comprising a NOX, as generally depicted in Figure 2.
[0152] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide are known to recognise naturally occurring amino acids, the R group can be any side chain attached to the alpha carbon of an amino acid of a naturally occurring amino acid.
[0153] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide are kinetically controlled to recognise a-keto acids. R1 group can be any side chain attached to the alpha carbon of the substrate of interest.
[0154] In the process herein, the keto acid compound of formula 1a is a substrate for the the recombinant engineered artificial leucine dehydrogenase polypeptide. Accordingly, the R group in the compound of formula 1a can represent (3-hydroxyphenyl) methyl group, (3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) methyl group, and ethyl group molecules, respectively yielding the substrates 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid, and 2-oxobutanoic acid.
[0155] In the process herein, the L-amino acid compound of formula 3a is a substrate for the recombinant engineered artificial leucine dehydrogenase polypeptide. Accordingly, the R1 group in the compound of formula 3a can represent (1H-indol-3-yl) methyl group, 2-methylpropyl group, and butyl group molecules, which yield the substrates L-tryptophan, L-leucine, and L-Nor-leucine molecules, respectively.
[0156] As noted above, for reductive amination, the ammonium ion donor in the process mediated by the recombinant engineered artificial leucine dehydrogenase polypeptide can be any suitable ammonium ion donor, which provides the NH3 for the formation of the amino acid. Exemplary ammonium sources include, among others, various ammonium salts, such as ammonium halide (e.g., ammonium chloride), ammonium formate, ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium tartrate, and ammonium acetate. In particular, the process for forming amino acid with leucine dehydrogenase can use ammonium formate or ammonium chloride as the ammonium donor.
[0157] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide is engineered to shows higher activity for the non-natural bulky substrates, bulky substrates, non-natural substrate and natural substrates.
[0158] In some embodiments, Non-natural substrate refers to a substance or compound that is not naturally converted or meant to be converted into another compound by the action of the recombinant engineered artificial leucine dehydrogenase polypeptide. In some embodiments the term non-natural substrate refers to L-Nor-Leucine for oxidative deamination, 2-oxo butanoic acid for reductive amination.
[0159] In some embodiments, Bulky substrate refers to a substance or compound that is large or has complex, rigid ring chemical structures. In some embodiments the term bulky substrate refers to 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid for reductive amination and L-tryptophan for oxidative deamination. In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide is capable to accommodate and convert these substrate molecules into respective products.
[0160] In some embodiments, Non-natural bulky substrate refers to a substrate or compound that is large or has a complex, rigid ring chemical structures that is not naturally converted or meant to be converted into another compound by the action of the recombinant engineered artificial leucine dehydrogenase polypeptide. In some embodiments, the term non-natural bulky substrate refers to 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid for reductive amination and L-tryptophan for oxidative deamination.
[0161] In some embodiments, “Natural substrate” refers to a substance or compound that is naturally converted or meant to be converted into another compound by the action of the recombinant engineered artificial leucine dehydrogenase polypeptide. In some embodiments the term natural substrate refers to trimethylpyruvate in reductive amination and L-Leucine and L-Valine for oxidative deamination.
[0162] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of SEQ ID NO: 10, 90, 129, 142, 145, 151 or 154 which shows higher activity towards 3-hydroxyphenol pyruvic acid which was converted into (2S)-2-amino-3-(3-hydroxyphenyl) propanoic acid through reductive amination. For the reaction, 3-hydroxyphenyl pyruvic acid substrate concentration was 5g/L to 100g/L wherein the concentration of enzyme added to the reaction medium was 0.5g/L to 2g/L (Table 2).
[0163] In some embodiments, the active site of the recombinant engineered artificial leucine dehydrogenase polypeptide engineered at one or more position at once or in combination to accommodate substrate 3-hydroxyphenyl pyruvic acid in the pocket and the mutations are X46 is ALA or GLN or PHE, X48 is VAL or PHE, X66 is ILE or ALA, X115 is LYS or ASP, X118 is HIS or LEU, or X293 is GLY or ILE. In some embodiments, the resulting engineered artificial leucine dehydrogenase polypeptide exhibits >95% conversion and 99% enantioselectivity.
[0164] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of SEQ ID NO: 10, 90, 129, 142, 145, 151 or 154 which shows higher activity towards 3-hydroxyphenol pyruvic acid which was converted into (2S)-2-amino-3-(3-hydroxyphenyl) propanoic acid through reductive amination with the activity as indicated in the Table 2. In some embodiments as an example from the table SEQ ID NO: 10 specifically shows >50% to <70% conversion which was indicated as “++++” in the table 2.
[0165] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of SEQ ID NO: 4, 23, 108, 139, 177, 189 or 191 which shows higher activity towards 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid which was converted into (2S)-2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid through reductive amination. For the reaction mixture, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid substrate concentration was 5g/L to 100g/L wherein the concentration of enzyme added to the reaction medium was 0.5g/L to 2g/L (Table 3).
[0166] In some embodiments, the active site of the recombinant engineered artificial leucine dehydrogenase polypeptide engineered at one or more positions at once or in combination to accommodate 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid and the mutations are X46 is PRO or ALA or GLN, X48 is TYR or ILE, X66 is SER or VAL, X115 is ILE or LEU, X118 is ASN or CYS or X293 is LEU. In some embodiments, the resulting engineered artificial leucine dehydrogenase polypeptide exhibits >95% conversion and 99% enantioselectivity.
[0167] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of SEQ ID NO: 4, 23, 108, 139, 177, 189 or 191 which shows higher activity towards (3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid which was converted into (2S)-2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid through reductive amination with the activity as indicated in the Table 3. In some embodiments as an example from the table SEQ ID NO: 139 specifically shows >80 to <90% conversion which was indicated as “++++++” in the Table 3.
[0168] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of SEQ ID NO: 19, 21, 28, 59, 69, 109, 119, 137 or 153 which shows higher activity towards 2-oxobutanoic acid which was converted into L-2-aminobutaryic acid through reductive amination. For the reaction mixture of 4L, 500 g of 2-oxobutanoic acid was treated with 3.8 g of crude or purified enzyme, 10 g of Formate Dehydrogenase from Candida boidini
[0169] (FDH), and 700 mg of NAD with 2M ammonium formate buffer of pH 7.5 and temperature of 30ºC for 3 hours. The recombinant artificial leucine dehydrogenase polypeptide exhibited an increase of at least 60% to 120% of substrate to product conversion rate as compared to wild type leucine dehydrogenase enzymes such as SEQ ID NO: 222 “Bacillus cereus” (WP_035429507.1/AAP11078.1), SEQ ID NO: 223 “Peribacillus kribbensis” (WP_026694696.1), SEQ ID NO: 224 “Legionella fallonii” (WP_045096578.1), and SEQ ID NO: 225 “Cupriavidus basilensis” (AJG22019.1) (Table 10).
[0170] In some embodiments, the active site of the recombinant engineered artificial leucine dehydrogenase polypeptide engineered at one or more position at once or in combination to accommodate substrate 2-oxobutanoic acid in the pocket and the mutations are X46 is LYS or SER, X48 is LYS, X115 is ASN or HIS, X118 is ASP, X293 is LEU or THR or ILE, or X296 is ILE or PHE. In some embodiments, the resulting engineered artificial leucine dehydrogenase polypeptide exhibits >99% conversion and 99% enantioselectivity.
[0171] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of SEQ ID NO: 19, 21, 28, 59, 69, 109, 119, 137 or 153 which showed higher activity towards 2-oxobutanoic acid which was converted into L-2-aminobutaryic acid through reductive amination with the activity as indicated in the Table 4. In some embodiments as an example from the table SEQ ID NO: 137 specifically shows >90% activity which was indicated as “+++++++” in the Table 4 and Table 10.
[0172] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of the SEQ ID NO: 29, 49, 81, 131, 154, 178, 182, 183, 186 or 188 which showed higher activity towards L-Leucine which was converted into 4-methyl-2-oxo-pentanoic acid through oxidative deamination. For the reaction mixture, L-Leucine substrate concentration was of 5g/L to 100g/L wherein the concentration of enzyme added to the reaction medium was 0.5g/L to 2g/L (Table 5).
[0173] In some embodiments, the active site of the recombinant engineered artificial leucine dehydrogenase polypeptide engineered at one or more position at once or in combination to accommodate substrate L-leucine in the pocket and the mutations are X115 is GLN or ILE, X118 is PHE, X148 is VAL, X293 is ILE, or X296 is MET. In some embodiments, the resulting engineered artificial leucine dehydrogenase polypeptide exhibits >95% conversion and >99% substrate enantioselectivity.
[0174] In some embodiments, The recombinant artificial leucine dehydrogenase polypeptide exhibited an increase of at least 60% to 120% of substrate L-Leucine to product 4-methyl-2-oxo-pentanoic acid conversion rate as compared to wild type leucine dehydrogenase enzymes such as SEQ ID NO: 222 “Bacillus cereus” (WP_035429507.1/AAP11078.1), SEQ ID NO: 223 “Peribacillus kribbensis” (WP_026694696.1), SEQ ID NO: 224 “Legionella fallonii” (WP_045096578.1), and SEQ ID NO: 225 “Cupriavidus basilensis” (AJG22019.1).
[0175] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of SEQ ID NO: 29, 49, 81, 131, 154, 178, 182, 183, 186 or 188 which showed higher activity towards L-Leucine which was converted into 4-methyl-2-oxo-pentanoic acid through oxidative deamination with the activity as indicated in the Table 5. In some embodiments as an example from the table SEQ ID NO: 178 specifically shows >80% to <90% conversion which was indicated as “++++++” in the table.
[0176] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of SEQ ID NO: 105, 123, 134, 148, 181, 185 or 193 which showed higher activity towards L-nor-Leucine which was converted into 2-oxohexanoic acid through oxidative deamination. For the reaction mixture, L-nor-Leucine substrate concentration was of 5g/L to 100g/L wherein the concentration of enzyme added to the reaction medium was 0.5g/L to 2g/L (Table 6).
[0177] In some embodiments, the active site of the recombinant engineered artificial leucine dehydrogenase polypeptide engineered at one or more position at once or in combination to accommodate the substrate L-nor-leucine in the pocket. The mutations are X115 is PHE or LEU, X118 is HIS or ALA, X148 is ALA or GLY, X293 is SER or LEU, and X296 is ASN. In some embodiments, the resulting engineered artificial leucine dehydrogenase polypeptide exhibits >95% conversion and >99% substrate enantioselectivity.
[0178] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of SEQ ID NO: 105, 123, 134, 148, 181, 185 or 193 which showed higher activity towards L-nor-Leucine which was converted into 2-oxohexanoic acid through oxidative deamination with the activity as indicated in the Table 6. In some embodiments as an example from the table SEQ ID NO: 105 specifically shows >50% to <70% conversion which was indicated as “++++” in the table 6.
[0179] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide engineered with the SEQ ID NO: 9, 27, 165, 197 or 200 which showed higher activity towards L-Tryptophan which was converted into Indole-3-pyruvic acid through oxidative deamination. For the reaction mixture, L-Tryptophan substrate concentration was of 5g/L to 100g/L wherein the concentration of enzyme added to the reaction medium was 0.5g/L to 2g/L (Table 7).
[0180] In some embodiments, the active site of the recombinant engineered artificial leucine dehydrogenase polypeptide engineered at one or more position at once or in combination to accommodate substrate L-tryptophan in the pocket, and the mutations are X46 is ALA or ASN, X48 is ALA or ASN, X66 is HIS, X115 is LYS or ASP, or X293 is ALA. In some embodiments, the resulting engineered artificial leucine dehydrogenase polypeptide exhibits >95% conversion and >99% substrate enantioselectivity.
[0181] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of SEQ ID NO: 9, 27, 165, 197 or 200 which showed higher activity towards L-Tryptophan which was converted into Indole-3-pyruvic acid through oxidative deamination with the activity as indicated in the Table 7. In some embodiments as an example from the table SEQ ID NO: 165 specifically shows >70% to <80% which was indicated as “+++++” in the Table 7.
[0182] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide of the present disclosure converts a-keto acids such as 3-hydroxyphenylpyruvic acid, 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid with greater than 99% conversion to their respective amino acid products with greater than 99% enantioselectivity and comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.
[0183] In the present disclosure, the recombinant engineered artificial leucine dehydrogenase polypeptide of the present disclosure converts 2-oxobutanoic acid to the respective amino acid product with greater than 99% conversion and greater than 99% enantiomeric excess, comprising of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.
[0184] In the present disclosure, the recombinant engineered artificial leucine dehydrogenase polypeptide of the present disclosure converts L-amino acids such as L-tryptophan with greater than 99% conversion to their respective a-keto acids products and comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.
[0185] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide are capable of converts L-leucine, and L-Nor-leucine with greater than 99% conversion to their respective a-keto acids products and comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.
[0186] In the conversion of L-amino acid to the a-keto acid process, a sacrificial substrate is used in the process for cofactor regeneration, which is making NADH to NAD+. The herein used sacrificial substrate can be O2, which is converted into H2O.
[0187] In the conversion of the a-keto acids to chiral amino acids, the recombinant engineered artificial leucine dehydrogenase polypeptide typically uses a cofactor, generally nicotinamide adenine dinucleotide (NAD+/NADH). To enhance the recombinant engineered artificial leucine dehydrogenase polypeptide mediated process, a cofactor regenerating system of formate dehydrogenase (FDH) has been used to convert the oxidized NAD+ to the reduced form NADH. By continual replenishment of the reduced NADH, the equilibrium of the recombinant engineered artificial leucine dehydrogenase polypeptide mediated process can be shifted towards product formation, thereby increasing the conversion of the keto acid to the amino acid product.
[0188] In the conversion of the L-amino acids to keto acids, the recombinant engineered artificial leucine dehydrogenase polypeptide typically uses a cofactor, generally nicotinamide adenine dinucleotide (NAD+/NADH). To enhance the artificially engineered leucine dehydrogenase polypeptide mediated process, a cofactor utilising enzyme NOX has been used to convert the reduced NADH to the oxidised form NAD+. By continual replenishment of the oxidised NAD+, the equilibrium of the engineered LeuDH-mediated process can be shifted towards product formation, thereby increasing the conversion of the L-amino acid to a-keto acid product.
[0189] The introduction of a cofactor recycling system with the recombinant engineered artificial leucine dehydrogenase polypeptide in the reductive amination process is pivotal for enhancing the catalytic efficiency of the conversion process. This system employs FDH, strategically attached to artificially engineered leucine dehydrogenase polypeptide to optimize cofactor movement towards the FDH active site. The recycling of cofactors, particularly NADH, is identified as both crucial and rate-limiting in the substrate-to-product conversion facilitated by the recombinant engineered artificial leucine dehydrogenase polypeptide. In this system, NADH donates a hydride to the product, converting it into NAD+, which is then reduced back into NADH by FDH. Computational studies, as well as experimental investigations, have elucidated the necessary mutations for the recombinant engineered artificial leucine dehydrogenase polypeptide-FDH module effectively, without the use of peptide linkers.
[0190] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide has features which facilitates the interaction with FDH and improves binding affinity between them, which in-turn facilitates the movement of NAD+/NADH molecules. Notably, the X243 is GLU, X226 is VAL, X49 is SER or ILE, X52 is LYS or ASN, and X55 is SER mutation promotes the conformation of the FDH, and the recombinant engineered artificial leucine dehydrogenase polypeptide complex, ensuring optimal orientation of their active sites and expediting NAD cofactor movement with minimal hindrance. (Figure 8 and 9, Table 9).
[0191] In some embodiments, the recombinant engineered leucine dehydrogenase polypeptide exhibits at least 5-fold improvement in the recycling efficiency with the feature mentioned in the above embodiment. In some embodiments, the recombinant engineered leucine dehydrogenase shows five-fold improvement compared to the wild-type leucine dehydrogenases such as SEQ ID NO: 222 “Bacillus cereus” (WP_035429507.1/AAP11078.1), SEQ ID NO: 223 “Peribacillus kribbensis” (WP_026694696.1), SEQ ID NO: 224 “Legionella fallonii” (WP_045096578.1), and SEQ ID NO: 225 “Cupriavidus basilensis” (AJG22019.1).
[0192] The recombinant engineered leucine dehydrogenase polypeptide exhibits improved cofactor recycling efficiency. In some embodiment, the improved efficiency of the recombinant engineered leucine dehydrogenase polypeptide for cofactor recycling is greater than 5 folds improvement.
[0193] These mutations not only enhance cofactor recycling but also boost the catalytic conversion rate of FDH, contributing significantly to the recombinant engineered artificial leucine dehydrogenase polypeptide's efficiency. Additionally, they stabilize the enzyme complex through quaternary structural formations, ensuring its integrality. Considering the cost implications raised by Rongsheng Tao et al. (2014) regarding the industrial usage of FDH as an enzyme recycling system, our mutations allow for a significant reduction in FDH usage. Employing just 0.5% of FDH relative to the recombinant engineered artificial leucine dehydrogenase polypeptide concentration, we achieve efficient cofactor recycling, leading to a substantial increase in overall catalytic efficiency.
[0194] Herein, in this invention, the recombinant engineered artificial leucine dehydrogenase polypeptide demonstrates the effectiveness of strategically engineered mutations in enhancing cofactor recycling within the recombinant engineered artificial leucine dehydrogenase polypeptide-FDH system and catalytic efficiency for non-natural bulky substrates. These findings offer promising implications for industrial applications, addressing concerns regarding enzyme cost while advancing biocatalytic processes.
[0195] The recombinant engineered artificial leucine dehydrogenase polypeptide with substitution at one or more positions facilitates a faster exchange of cofactor nicotinamide adenine dinucleotide (NAD+/NADH). In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide with substitutions at one or more positions would benefit the integrality of both the recombinant engineered artificial leucine dehydrogenase polypeptide and FDH enzyme by facilitating stronger interactions with FDH enzymes. The stronger binding of the recombinant engineered artificial leucine dehydrogenase polypeptide and FDH was evaluated with computational studies.
[0196] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide is engineered in the active site at one or more positions in combination or individually to improve the activity and affinity towards non-natural bulky substrates.
[0197] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide with substitutions at one or more positions facilitates a faster exchange of cofactor nicotinamide adenine dinucleotide (NAD+/NADH), which in turn allows the usage of a lower concentration of FAD enzymes. In some embodiments, a concentration of 0.5% of FDH enzymes is used with respect to the concentration of the recombinant engineered artificial leucine dehydrogenase polypeptide in the reaction media.
[0198] In the conversion of the keto acids to chiral amino acids process, a sacrificial substrate is used in the process for cofactor regeneration, i.e., making NAD+ to NADH. Herein used sacrificial substrates can be Sodium formate or formic acid. These sacrificial substrates are converted to carbon dioxide by the FDH enzyme.
[0199] In the conversion of the a-keto acids to chiral amino acids, the recombinant engineered artificial leucine dehydrogenase polypeptide typically uses a cofactor, generally nicotinamide adenine dinucleotide (NAD+/NADH). To enhance the artificially engineered leucine dehydrogenase polypeptide mediated process, a cofactor regenerating system of formate dehydrogenase (FDH) has been used to convert the oxidized NAD+ to the reduced form NADH. By continual replenishment of the reduced NADH, the equilibrium of the artificially engineered leucine dehydrogenase polypeptide mediated process can be shifted towards product formation, thereby increasing the conversion of the keto acid to the amino acid product.
[0200] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide engineered to use in lower concentrations in the reaction conditions.
[0201] In some embodiments, a method is provided for the generation of an artificially engineered leucine dehydrogenase polypeptide capable of catalysing both oxidative deamination and reductive amination reactions with improved efficiency toward bulky, non-natural substrates (Figure 3). The method comprises collecting sequences from multiple databases to build a diverse dataset of 100 naturally occurring leucine dehydrogenase sequences (Table 1), classifying said sequences based on substrate activity profiles, and modelling the three-dimensional structures of the sequences to analyse natural substrate interactions.
[0202] In some embodiments, the method further comprises analysing the interactions of natural substrates with amino acid residues within the active site pocket of the categorized leucine dehydrogenase enzyme structures. Natural substrates, including, but not limited to, Trimethylpyruvate and L-valine, are employed to generate enzyme-substrate complexes. The method further comprises deriving near-attack conformations of the respective natural substrates within the modelled three-dimensional structures of the leucine dehydrogenase sequences. The near-attack conformation represents the energetically favourable spatial orientation of the substrate relative to catalytic residues that is conducive to enzymatic catalysis. Near-attack conformations of Trimethylpyruvate and L-valine are derived for the LeuDH sequences, wherein the substrate molecules are computationally decomposed into atomic components, and detailed atomic-level interaction profiling is conducted to identify stabilizing interactions between the substrate atoms and the catalytic amino acid residues (Figure 5). A convolutional neural network (CNN)-based analysis is employed to predict binding poses, interaction energies, and hotspot residues critical for substrate anchoring. The top four wild-type leucine dehydrogenase sequences exhibiting favourable interaction profiles are selected as templates for artificial enzyme generation.
[0203] In some embodiments, the top four wild-type leucine dehydrogenase sequence are selected from “Bacillus cereus” (WP_035429507.1/AAP11078.1), “Peribacillus kribbensis” (WP_026694696.1), “Legionella fallonii” (WP_045096578.1), and “Cupriavidus basilensis” (AJG22019.1) as template sequences.
[0204] In some embodiments, the method further comprises subjecting the top-ranked enzyme-substrate complexes obtained from the preceding analysis to a combination of network path analysis and non-covalent interaction (NCI) analysis to facilitate the design of artificial leucine dehydrogenase polypeptides. Network path analysis is employed to identify, characterize, and rank discrete structural domains within each of the selected leucine dehydrogenase template sequences based on their respective contributions to the conformational stability of the catalytic site and the dynamic integrity of the overall enzyme scaffold. The ranked domains are recombined to generate a chimeric polypeptide backbone representing the structural core of the artificial leucine dehydrogenase sequences. Concurrently, non-covalent interaction analysis is performed to identify specific amino acid residues within the enzyme active site that engage in stabilizing interactions with the bound substrate under near-attack conformations. Such interactions include, but are not limited to, hydrogen bonding, van der Waals forces, p-p stacking, hydrophobic packing, and electrostatic attractions. Residues demonstrating high interaction strength and positional conservation across complexes are designated as interaction hotspots. The above-mentioned interactions were highlighted in the 3D electronic density maps and 2D reduced density gradient maps. The hotspots are prioritized for inclusion in the artificial sequence architecture based on their predicted contributions to substrate affinity and catalytic efficiency. The integration of ranked structural domains identified via network path analysis and prioritized hotspot residues derived from NCI analysis yields an artificially designed leucine dehydrogenase polypeptide sequence (Figure 4).
[0205] In some embodiments, the method of generating artificial sequences produces plurality of artificial sequences. SEQ ID NO: 1, 212, 213, 214, 215, 216, 217, 218, 219, 220, and 221 were generated with this approach. These artificially engineered sequences were tested for the better fitting of the substrate inside the active site. The SEQ ID NO 1 sequence was selected based on the substrate fitting and compactness of the active site which was developed from the information extracted from the different domains of the top four templates. SEQ ID 1 was further engineered to fit non-natural bulky substrates in the active site.
[0206] The SEQ ID NO 1 was further engineered to accommodate non-natural, bulky substrates such as 3-hydroxyphenylpyruvic acid and L-tryptophan, yielding industrially relevant products such as M-tyrosine and indole-3-pyruvic acid. AI-driven analyses, including CNN models, Interaction profiling grid enzyme engineering technology, and pLDDT-based structural confidence scoring, are employed to identify, mutate, and optimize key residues, thereby generating variants with enhanced binding affinity, catalytic efficiency, and stability while preserving overall structural integrity. The final engineered artificially engineered leucine dehydrogenase polypeptide variants are validated through computational energetics scoring and iterative refinement to achieve optimal performance. These variants exhibit significantly improved catalytic properties in asymmetric reductive amination and oxidative deamination of bulky substrates compared to reported leucine dehydrogenase enzymes.
[0207] In some embodiments, to accommodate the non-natural bulky substrates such as 3-hydroxyphenyl pyruvic acid and (3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) pyruvic acid, the artificially engineered leucine dehydrogenase polypeptide is complexed with the substrates and mutational studies are conducted. Furthermore, to computationally validate the mutations, their near attack conformations are derived (Figure 6). These conformations are further evaluated with non-covalent interactions (NCI) and Interaction profiling grid method to derive the least energetic substitutions for the hotspots. Further, they are evaluated in terms of stability through pLDDT calculations.
[0208] In some embodiments, the non-natural substrate 2-oxobutanoic acid is also accommodated by the artificially engineered leucine dehydrogenase polypeptide through induced fit and the complex’s mutational studies are conducted. To computationally validate the mutations, their near attack conformations are derived (Figure 6). These conformations are further evaluated with non-covalent interactions (NCI) and Interaction profiling grid methods to derive the least energetic substitutions for the hotspots. Further, they are evaluated in terms of stability through pLDDT calculations.
[0209] In some embodiments, to accommodate the non-natural bulky substrate such as L-Tryptophan, the artificially engineered leucine dehydrogenase polypeptide is complexed with the substrates and mutational studies are conducted. Furthermore, to computationally validate the mutations, their near attack conformations are derived (Figure 7). These conformations are further evaluated with non-covalent interactions (NCI) and Interaction profiling grid methods to derive the least energetic substitutions for the hotspots. Further, they are evaluated in terms of stability through pLDDT calculations. To further evaluate the enzyme kinetics of L-tryptophan with artificially engineered Leucine Dehydrogenase polypeptide, Quantum Mechanical Dynamics simulations were performed. These studies yielded both the reaction mechanism of the oxidative deamination of L-Tryptophan to Indole-3-pyruvic acid as well as the energy transition states during the reactions. The energy required for L-Tryptophan to attain is TS1, TS2, TS3, and TS4 were 12.8 kcal/mol, 19.6 kcal/mol, 8.9 kcal/mol and 7.2 kcal/mol, respectively. (Figure 10 and Figure 11).
[0210] In some embodiments, the non-natural substrate L-nor-Leucine is also accommodated by the artificially engineered leucine dehydrogenase polypeptide through induced fit and the complex’s mutational studies are conducted. To computationally validate the mutations, their near attack conformations are derived (Figure 7). These conformations are further evaluated with non-covalent interactions (NCI) and Interaction profiling grid methods to derive the least energetic substitutions for the hotspots. Further, they are evaluated in terms of stability through pLDDT calculations.
[0211] In some embodiments, the natural substrate L-Leucine is also accommodated by the artificially engineered leucine dehydrogenase polypeptide through induced fit and the complex’s mutational studies are conducted. This is primarily conducted to enhance the catalytic conversion of the L-Leucine to 4-methy-2-oxo-pentanoic acid. To computationally validate the mutations, their near attack conformations are derived (Figure 6). These conformations are further evaluated with non-covalent interactions (NCI) and Interaction profiling grid methods to derive the least energetic substitutions for the hotspots. Further, they are evaluated in terms of stability through pLDDT calculations.
[0212] The recombinant engineered artificial leucine dehydrogenase polynucleotides encoding the recombinant engineered artificial leucine dehydrogenase polypeptides given herein. The polynucleotide can include promoters and other regulatory elements useful for the expression of the encoded engineered leucine dehydrogenase and can utilize codons optimized for specific desired expression system.
[0213] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide transformed through a E. coli BL21 (DE3) host system using the plasmid pET28a(+) (Figure 12). In this plasmid, the artificial engineered LeuDH gene is organised between the restriction sites of NcoI and XhoI, controlled by the T7 promoter. The host system and plasmid DNA incubated on ice for 30 mins. Next, they were subjected to heat shock treatment wherein they were placed in water bath for a minute at 42° C and then placed on ice for five minutes. Then they were incubated at 37° C in the shaker incubator for an hour, following which they were pelleted at 3500 RPM for 5 mins and then further resuspended in leftover LB broth and plated on LB agar plates containing kanamycin (50µg/ml).
[0214] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptides present in the plasmid of pET28a(+) are expressed in E. coli BL21(DE3) system where the variants were inoculated in 5ml of LB+kanamycin median and grown overnight at 37° C. The 5ml overnight culture was further inoculated into 100ml LB medium in the presence of 50 µg/ml of kanamycin and once the culture reached OD600 = 0.6, artificial leucine dehydrogenase variants were induced by 0.1mM isopropyl-ß-D-1-thiogalactopyranoside (IPTG) and cells were further grown at 25°C for 16 hours. These induced cells were then harvested by centrifugation at 4700 RPM for 10 minutes. Further to this step, the cells were then resuspended in 1mL PBS buffer pH 7.5 (containing lysozyme of 1mg/ml) and incubated on ice for 1 hour. Then, the resulting mixture volume was made up to 5ml with PBS buffer and lysis were performed using sonication. The expression profiles of crude lysates were analyzed using SDS-PAGE. It is observed that the SEQID NO: 1 and 90 shows high expression and other SEQ ID NO: 3, 23 and 77 shows moderate expression and SEQ ID NO: 25, 35, 81, 187 and 198 have low expression levels (Figure. 13) .
[0215] Here, the recombinant engineered artificial leucine dehydrogenase polypeptide produced using well-established protein synthesis techniques. To be more precise, the process of creating an the recombinant engineered artificial leucine dehydrogenase polypeptide gene involves first preparing the host organism E. coli BL21 (DE3) and then inserting it using the plasmid pET28a(+). In this plasmid, the artificial LeuDH gene is organized between the restriction sites of NcoI and XhoI, controlled by the T7 promoter. Next comes the process of cultivating the host, which involves causing the expression of the artificially engineered leucine dehydrogenase polynucleotide within the host after the logarithmic growth phase. This is achieved by culturing the host at a temperature that is below what is ideal for host cell growth and survival. There are no restrictions on the use of other conventional promoters as an inducible promoter in the process of creating the recombinant engineered artificial leucine dehydrogenase polypeptide. For example, when transcription is employed with the expression host E. coli, an inducible promoter that is triggered by isopropyl -D-1-thiogalactopyranoside (IPTG) can be used. Examples of this kind of promoter include Lac, Tac, Trp, and Trc.
[0216] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide were expressed in E.coli BL21 (DE3) host system using the plasmid pET28a(+) and in some embodiments, the expression details are provided in Table 8 for SEQ ID NO 1, 2, 3, 4, 5 ,6 ,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112,113,114,115,116,117, 118, 119 ,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170 ,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209 and 210.
[0217] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide were expressed in E.coli BL21(DE3) host system using the plasmid pET28a(+) and in these embodiments, the relative expression are provided in Table 8 wherein where “+” indicates low expression, “++” indicates moderate expression and “+++” indicates good expression for SEQ ID NO 1, 2, 3, 4, 5 ,6 ,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 ,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170 ,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209 and 210.
[0218] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide were expressed in E.coli BL21(DE3) host system using the plasmid pET28a(+) and in these embodiments, the conversion is provided in Table 8 for SEQ ID NO 1, 2, 3, 4, 5 ,6 ,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 ,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170 ,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209 and 210.
[0219] In some embodiments of the process herein, the recombinant engineered artificial leucine dehydrogenase polypeptide can be present in the forms of whole cells, including whole cells transformed with polynucleotide constructs. In some embodiments, artificially engineered leucine dehydrogenase polypeptide can be present in the form of cell extracts and/or lysate thereof and may be employed in a variety of different forms, including solid (e.g., lyophilized, spray-dried) or semisolid (e.g., a crude paste). In some embodiments, the artificially engineered leucine dehydrogenase polypeptide is isolated and can be in a substantially purified form. In some embodiment of the process, both the artificially engineered leucine dehydrogenase polypeptide and FDH of the regenerating system can be present in the form of whole cells, including whole cells transformed with polynucleotide constructs such that the whole cells express both the artificially engineered leucine dehydrogenase polypeptide and FDH.
[0220] In some embodiments, the improved enzymatic activity of the recombinant engineered artificial leucine dehydrogenase polypeptide is also associated with other improvements in enzyme properties. In some embodiments, the improvement in enzyme property concerns thermal stability, such as at 25°C, 30°C, 40°C, 45°C, 50°C, 55°C, 60°C, and 65°C or higher.
[0221] Generally, the process with the recombinant engineered artificial leucine dehydrogenase polypeptide experimented at a pH of about 10 or below, usually in the range of about 8.0 to 11. In some embodiments, the process is carried out at a pH of about 9.0 or below, usually in the range of about 8.5 to about 9.0. In some embodiments, the process of forming amino acids with the recombinant engineered artificial leucine dehydrogenase polypeptide can be carried out within a pH range of about 9 to about 11, particularly at about pH 9 to 10, more particularly at about pH 9.5. In some embodiments, the process may be carried out at a neutral pH, i.e., about 7.0. It is to be understood that the pH of the reaction medium for the formation of amino acids can be determined based on the activities of the recombinant engineered artificial leucine dehydrogenase polypeptide and FDH at different pHs.
[0222] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide expressed through a E. coli BL21(DE3) host system using the plasmid pET28a(+), and the SEQ ID NO: 10, 90, 129, 142, 145, 151 or 154 is used in the catalysis of 3-hydroxypyruvic acid’s reductive amination where in the reaction mixture of 160 µL contains 3-hydroxyphenylpyruvic acid of 10mM concentration , NH4OH/NH4Cl (900mM, pH 9.5), NADH (0.2 mM) and crude or purified enzyme solution (2.5 mg/ml). The reaction was carried out at 32°C for 2 minutes and the absorbance change at 340 nm was recorded every 30 seconds for determination of enzyme activity through the consumption of NADH (Table 2). The analysis of the product was conducted through HPLC method. The concentration of (2S)- 2-amino-3-(3-hydroxyphenyl) propanoic acid, were assayed using a Agilent 1100 HPLC system equipped with a diode-array detector and a Supelco RP-18 Discovery column (length, 250 mm; diameter, 10 mm), which was eluted with methanol–water mixtures (starting with a mixture of 3% methanol, 67% water, and 30% of 0.05% aqueous trifluoroacetic acid for an initial period of 3 min, followed by a linear gradient reaching 50% methanol, 20% water, and 30% of 0.05% aqueous trifluoroacetic acid at 30 min, at a constant flow of 3.4 ml/min. The absorption was monitored at 280 nm.
[0223] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide expressed through a E. coli BL21(DE3) host system using the plasmid pET28a(+), and the SEQ ID NO: 4, 23, 108, 139, 177, 189 or 191 is used in the catalysis of towards 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid through reductive amination where in the reaction mixture of 160 µL contains 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid of 10mM concentration, NH4OH/NH4Cl (900mM, pH 9.5), NADH (0.2 mM) and crude or purified enzyme solution of concentration of 2.5 mg/ml (Table 3). The reaction was carried out at 34°C for 2 minutes and the absorbance change at 340nm was recorded every 30 seconds for determination of enzyme activity through the consumption of NADH. The resulting product was analyzed through HPLC method. The concentration of (2S) -2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid were assayed using Shimadzu HPLC equipment comprising quaternary LC-10A VP pumps, a variable wavelength programmable UV–visible detector, SPD-10AVP column oven, and a SCL 10AVP system controller. Samples with the volume of 20 µL were injected by means of a Rheodyne injector fitted with a 20-µL loop. The instrumentation was controlled by use of Class-VP 5.032 software. Compounds were separated on a 250 × 4.6 mm, 5-µm particle, C18 reversed-phase column. The mobile phase was water–orthophosphoric acid (98.8:0.2, v/v) filtered through a 0.22-µm membrane filter and sonicated before use; pH 3.0 was adjusted with orthophosphoric acid, (2S) -2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic was eluted isocratically with a flow rate of 1.0 mL min-1. The eluate was monitored by UV detector at wavelength of 284 nm.
[0224] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptides expressed through a E.coli BL21(DE3) host system using the plasmid pET28a(+), and the SEQ ID NO: 19, 21, 28, 59, 69, 109, 119, 137 or 153 is used in the catalysis of towards 2-oxobutanoic acid through reactive amination where in the reaction mixture of 160 µL contains 2-oxobutanoic acid (10mM), NH4OH/NH4Cl (900mM, pH 9.5), NADH (0.2 mM) and crude or purified enzyme solution (2.5 mg/ml) (Table 4). The reaction was carried out at 30°C for 2 minutes and the absorbance change at 340nm was recorded every 30 seconds for determination of enzyme activity through the consumption of NADH. The resulting product was analyzed through HPLC-GC method. The concentration of 2-ketobutyric acid and (L)-2-aminobutyric acid were assayed using a 1260 Infinity II liquid chromatography platform equipped with a COSMOSIL PBr column (4.6 mm ID x 250 mm) at 254 nm with 5mM ammonium dihydrogen phosphate at a flow rate of 0.8 ml/min. The enantioselectivity of (L)-2-aminobutyric acid was assayed by GITC precolumn derivatization equipped with an ZORBAX SB-Aq column (4.6 mm x 250 mm) at 254 nm using water (0.1% phosphate)/methanol (40:60, v/v) as the eluent at a flow rate of 1mL/min.
[0225] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptides expressed through a E. coli BL21(DE3) host system using the plasmid pET28a(+), and the SEQ ID NO: 10, 90, 129, 142, 145, 151 or 154 is used in the catalysis of 3-hydroxypyruvic acid’s reductive amination where in the reaction mixture of 160 µL contains 3-hydroxyphenylpyruvic acid of 10mM concentration , NH4OH/NH4COOH (900mM, pH 9.5), NADH (0.3 mM) and crude or purified enzyme solution (2.5 mg/ml) and 2.7mg/ml of formate dehydrogenase from Candida boidinii in 0.1 M PBS buffer (pH 7.5) at 32°C for 15 minutes . The analysis of the product was conducted through HPLC method. The concentration of (2S)- 2-amino-3-(3-hydroxyphenyl) propanoic acid, were assayed using a Agilent 1100 HPLC system equipped with a diode-array detector and a Supelco RP-18 Discovery column (length, 250 mm; diameter, 10 mm), which was eluted with methanol–water mixtures (starting with a mixture of 3% methanol, 67% water, and 30% of 0.05% aqueous trifluoroacetic acid for an initial period of 3 min, followed by a linear gradient reaching 50% methanol, 20% water, and 30% of 0.05% aqueous trifluoroacetic acid at 30 min, at a constant flow of 3.4 ml/min. The absorption was monitored at 280 nm.
[0226] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide expressed through a E. coli BL21 DE3 host system using the plasmid pET28a(+), and the SEQ ID NO: 4, 23, 108, 139, 177, 189 or 191 is used in the catalysis of towards 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid through reductive amination where in the reaction mixture of 160 µL contains 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid of 10mM concentration, NH4OH/NH4COOH (900mM, pH 9.5), NADH (0.3 mM) and crude or purified enzyme solution (2.5 mg/ml) and 2.7mg/ml of formate dehydrogenase from Candida boidinii in 0.1 M PBS buffer (pH 7.5) at 34°C for 15 minutes. The reaction was carried out at 34°C for 2 minutes and the absorbance change at 340nm was recorded every 30 seconds for determination of enzyme activity through the consumption of NADH. The resulting product was analyzed through HPLC method. The concentration of (2S) -2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid were assayed using Shimadzu HPLC equipment comprising quaternary LC-10A VP pumps, a variable wavelength programmable UV–visible detector, SPD-10AVP column oven, and a SCL 10AVP system controller. Samples with the volume of 20 µL were injected by means of a Rheodyne injector fitted with a 20-µL loop. The instrumentation was controlled by use of Class-VP 5.032 software. Compounds were separated on a 250 × 4.6 mm, 5-µm particle, C18 reversed-phase column. The mobile phase was water–orthophosphoric acid (98.8:0.2, v/v) filtered through a 0.22-µm membrane filter and sonicated before use; pH 3.0 was adjusted with orthophosphoric acid, (2S) -2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic was eluted isocratically with a flow rate of 1.0 mL min-1. The eluate was monitored by UV detector at wavelength of 284 nm.
[0227] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide having the SEQ ID NO: 19, 21, 28, 59, 69, 109, 119, 137 or 153 is used in the catalysis of 2-oxobutanoic acid through the reductive amination wherein the reaction mixture comprises of 0.05 M 2-oxobutanoic acid, NH4OH/NH4COOH (0.05 M) , 0.3mM NAD+, 0.09mg/ml crude or purified enzyme and 2.7mg/ml of formate dehydrogenase from Candida boidinii in 0.1 M PBS buffer (pH 7.5) .The reaction was conducted at pH 7.5 at temperature of 30°C in a stir flask of 200rpm for 45 minutes. These samples were further boiled for detection of product. The resulting product was analyzed through HPLC-GC method. The concentration of 2-ketobutyric acid and (L)-2-aminobutyric acid were assayed using a 1260 Infinity II liquid chromatography platform equipped with a COSMOSIL PBr column (4.6 mm ID x 250 mm) at 254 nm with 5mM ammonium dihydrogen phosphate at a flow rate of 0.8 ml/min. The enantioselectivity of (L)-2-aminobutyric acid was assayed by GITC precolumn derivatization equipped with a ZORBAX SB-Aq column (4.6 mm x 250 mm) at 254 nm using water (0.1% phosphate)/methanol (40:60, v/v) as the eluent at a flow rate of 1mL/min.
[0228] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide having the SEQ ID NO: 19, 21, 28, 59, 69, 109, 119, 137 or 153 is used in the catalysis of 2-oxobutanoic acid through the reductive amination through the efficient cofactor recycling system wherein the reaction mixture comprises of NH4OH/NH4COOH (0.5 M), 3mM NAD+, 0.9 mg/ml crude or purified enzyme and 4.5 µg/ml of formate dehydrogenase from Candida boidinii in 0.1 M PBS buffer (pH 7.5) at 30ºC for 45 minutes. These samples were further boiled for detection of product. The resulting product was analyzed through HPLC-GC method. The concentration of 2-ketobutyric acid and (L)-2-aminobutyric acid were assayed using a 1260 Infinity II liquid chromatography platform equipped with a COSMOSIL PBr column (4.6 mm ID x 250 mm) at 254 nm with 5mM ammonium dihydrogen phosphate at a flow rate of 0.8 ml/min. The enantioselectivity of (L)-2-aminobutyric acid was assayed by GITC precolumn derivatization equipped with a ZORBAX SB-Aq column (4.6 mm x 250 mm) at 254 nm using water (0.1% phosphate)/methanol (40:60, v/v) as the eluent at a flow rate of 1mL/min.
[0229] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide expressed through a E.coli BL21 (DE3) host system using the plasmid28a(+), and having the SEQ ID NO: 19, 21, 28, 59, 69, 109, 119, 137 or 153 is used in the catalysis of 2-oxobutanoic acid through the reductive amination wherein the reaction mixture of 4L comprising of 500g of 2-oxobutanoic acid , 10g of formate dehydrogenase (FDH) , 700mg of NAD and 3.8g of crude or purified enzyme and 2 M of NH4OH/NH4COOH at pH 7.5 and at 30ºC for 3 hours (Table 10). This asymmetric reaction lead to the increase in conversion rate of the substrate 2-oxobutanoic acid to L-2-amino butyric acid by at least 60% to 120% in comparison to wild type sequences SEQ ID NO: 222, 223, 224 and 225 respectively. The resulting product was analyzed through HPLC-GC method. The concentration of 2-ketobutyric acid and (L)-2-aminobutyric acid were assayed using a 1260 Infinity II liquid chromatography platform equipped with a COSMOSIL PBr column (4.6 mm ID x 250 mm) at 254 nm with 5mM ammonium dihydrogen phosphate at a flow rate of 0.8 ml/min. The enantioselectivity of (L)-2-aminobutyric acid was assayed by GITC precolumn derivatization equipped with a ZORBAX SB-Aq column (4.6 mm x 250 mm) at 254 nm using water (0.1% phosphate)/methanol (40:60, v/v) as the eluent at a flow rate of 1mL/min.
[0230] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide expressed through a E. coli BL21(DE3) host system using the plasmid pET28a(+), and the SEQ ID NO: 29, 49, 81, 131, 154, 178, 182, 183, 186 or 188 is used in the catalysis of L-Leucine through oxidative deamination reaction wherein the substrate mixture of 1.5 mL for the enzymatic activity analysis consisted of 5 mM of substrate L-leucine and 0.2 mM NAD+ and crude or purified enzyme solution (2.5 mg/ml) in the presence of a 100 mM glycine–NaOH buffer at pH 9.5 (Table 5). One unit of enzymatic activity was defined as the quantity of enzyme necessary to catalyse the reduction of 1 µM NAD+ per minute at a temperature of 30°C. The enzyme activity assays were performed using a spectrophotometer to measure the change in NADH absorbance at 340 nm. The product was analyzed using the HPLC method. The product was analyzed through its derivatization with Diamino-4,5-methylenedioxybenzene (DMB) where in 40µL of DMB was added to 40 µL of the product in the sealed tube.The solution is then heated to 85°C for 45 minutes and then cooled on ice for 5 minutes. Further, the solution is diluted fivefold with 65 mM NaOH aqueous solution and 25 µL was injected into HPLC. The HPLC system from Jasco was composed of a PU-980 pump, an LG-1580-02 ternary gradient unit, a DG-980-50 3-line degasser, AS-2057 PLUS autosampler, CO-1560 column oven, and FP1520S fluorescence detector. The analysis of 4-methyl-2-oxopentanoic acid was conducted on Inertsil ODS-4V column (250 × 3.0 mm, 5.0 µm). Fluorescence detection was performed at excitation and emission wavelengths of 367 nm and 446 nm, respectively. The Mobile phases were MeOH/H2O (30/70, v/v) and MeOH. A flow rate was 0.3 mL/min and the column temperature was maintained at 40°C.
[0231] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide expressed through a E. coli BL21(DE3) host system using the plasmid pET28a(+), and the SEQ ID NO: 105, 123, 134, 148, 181, 185 or 193 were used in the catalysis of L-nor-Leucine through oxidative deamination reaction where in the substrate mixture of 1.5 mL for the enzymatic activity analysis consisted of 5 mM of substrate L-Nor-leucine and 0.2 mM NAD+ and crude or purified enzyme solution (2.5 mg/ml) in the presence of a 100 mM glycine–NaOH buffer at pH 9.3 (Table 6). One unit of enzymatic activity was defined as the quantity of enzyme necessary to catalyse the reduction of 1 µM NAD+ per minute at a temperature of 27°C. The enzyme activity assays were performed using a spectrophotometer to measure the change in NADH absorbance at 340 nm. The product was analyzed using HPLC method. The product 2-oxo hexanoic acid were analysed on Agilent HPLC system 1100 series equipped with a model G1311A LC pump, model G1315B diode array detector (DAD) and model 7725 Rheodyne injector. Reversed-phase LC analysis was performed isocratically at room temperature using a Zorbax 300 SB-C18 (4.6mm x 150 mm) column. The product sample of 1ml was added into an SDA solution of 0.5mL which contains 2% w/v in methanol, acetic acid-sodium acetate buffer of pH 3.2. The contents were heated at 95-100°C for 30 minutes and volume was adjusted with methanol to 10mL. A mobile phase consisting of a mixture of methanol-water-acetonitrile-tetrahydrofuran (38.4:60:1:0.6, v/v/v/v) was used. The injection volume was 10 mL and the detection wavelength was set at 255 nm. The flow rate was set to 1 mL/min.
[0232] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide expressed through a E. coli BL21(DE3) host system using the plasmid pET28a(+), and the SEQ ID NO: 9, 27, 165, 197 or 200 were used in the catalysis of L-Tryptophan through oxidative deamination reaction where in the substrate mixture of 1.5 ml for the enzymatic activity analysis consisted of 5 mM of substrate L-tryptophan and 0.2 mM NAD+ and crude or purified enzyme solution (2.5 mg/ml) in the presence of a 100 mM glycine–NaOH buffer at pH 9.5 (Table 7). One unit of enzymatic activity was defined as the quantity of enzyme necessary to catalyse the reduction of 1 µM NAD+ per minute at a temperature of 25°C. The enzyme activity assays were performed using a spectrophotometer to measure the change in NADH absorbance at 340 nm. The product was analyzed using HPLC method. The product Indole-3-pyruvic acid were analysed by injecting 50?µL into a reverse-phase Zorbax Eclipse XDB-C8 (column (4.6 × 150 mm, 5?µm). Columns temperature was controlled at 30°C. Mobile phase was composed of methanol and 1% acetic acid (60:?40 v /v) in isocratic mode at a flow rate of 1?mL min-1. The detection was monitored at 360?nm.
[0233] In some embodiments, the recombinant engineered artificial leucine dehydrogenase polypeptide expressed through a E. coli BL21(DE3) host system using the plasmid pET28a(+), and the SEQ ID NO: 29, 49, 81, 131, 154, 178, 182, 183, 186 or 188 is used in the catalysis of L-Leucine through oxidative deamination reaction wherein the substrate mixture of 1.5 mL comprising of 5 mM of L-leucine and 0.2 mM NAD+ and crude or purified enzyme solution of 2.5 mg/ml in the presence of a 100 mM glycine–NaOH buffer at pH 9.5 at 30°C. The reaction was incubated for two hours. For the regeneration of the cofactor from NADH to NAD+ crude or purified NOX enzyme (2.5mg/L) solution was used. The product was analyzed using the HPLC method. The product was analyzed through its derivatization with Diamino-4,5-methylenedioxybenzene (DMB) where in 40µL of DMB was added to 40 µL of the product in the sealed tube.The solution is then heated to 85°C for 45 minutes and then cooled on ice for 5 minutes. Further, the solution is diluted fivefold with 65 mM NaOH aqueous solution and 25 µL was injected into HPLC. The HPLC system from Jasco was composed of a PU-980 pump, an LG-1580-02 ternary gradient unit, a DG-980-50 3-line degasser, AS-2057 PLUS autosampler, CO-1560 column oven, and FP1520S fluorescence detector. The analysis of 4-methyl-2-oxopentanoic acid was conducted on Inertsil ODS-4V column (250 × 3.0 mm, 5.0 µm). Fluorescence detection was performed at excitation and emission wavelengths of 367 nm and 446 nm, respectively. The Mobile phases were MeOH/H2O (30/70, v/v) and MeOH. A flow rate was 0.3 mL/min and the column temperature was maintained at 40°C.
Sl. No Accession Scientific Name
1 WP_214711448.1 Exiguobacterium
2 WP_128122304.1 Exiguobacterium sp. AM39-5BH
3 WP_214706116.1 unclassified Heliobacterium
4 WP_233004366.1 Exiguobacterium aurantiacum
5 WP_090775413.1 Shouchella lonarensis
6 WP_054949537.1 Numidum massiliense
7 WP_078391673.1 Shouchella patagoniensis
8 WP_413074288.1 unclassified Shouchella
9 WP_099301234.1 Bacillaceae
10 WP_026694696.1 Peribacillus kribbensis
11 WP_149458603.1 Macrococcus equipercicus
12 WP_187116891.1 Rubeoparvulum massiliense
13 WP_034144736.1 Desulfosporosinus sp. BICA1-9
14 WP_187136961.1 Listeria grandensis
15 WP_184402883.1 Geomicrobium halophilum
16 WP_319632013.1 Marinococcus sp. PL1-022
17 WP_204698326.1 Geomicrobium sediminis
18 WP_094907926.1 Marinococcus halophilus
19 WP_185371862.1 Listeria aquatica
20 AAP11078.1 Bacillus
21 WP_160652363.1 Salinicoccus hispanicus
22 WP_060382524.1 Flavobacterium
23 WP_374899963.1 Shewanella xiamenensis
24 WP_340117357.1 Pelagibius sp. 7325
25 Q60030 Thermoactinomyces intermedius
26 A0A011PRX2 Candidatus Accumulibacter appositus
27 A0A011RIX7 Accumulibacter regalis
28 A0A024HCG7 Pseudomonas knackmussii
29 A0A069QG62 Pseudomonas aeruginosa
30 A0A081FXR4 Marinobacterium lacunae
31 A0A090D2Z7 Criblamydia sequanensis CRIB-18
32 A0A097ATF5 Thermoanaerobacter kivui (Acetogenium kivui)
33 WP_045096578.1 Legionella fallonii
34 A0A099CWQ9 Oleiagrimonas soli
35 A0A099KGX7 Colwellia psychrerythraea (Vibrio psychroerythus)
36 A0A099LI32 Thalassotalea sp. ND16A
37 A0A0A7PGV6 Sphingopyxis fribergensis
38 A0A0A8UN70 Legionella hackeliae
39 A0A0B7MN77 Syntrophaceticus schinkii
40 A0A0C1E5R1 Parachlamydia acanthamoebae
41 A0A0C1HIP7 Neochlamydia sp. TUME1
42 A0A0C1HQ85 Neochlamydia sp. EPS4
43 A0A0C3IXD4 Thauera sp. SWB20
44 AJG22019.1 Cupriavidus basilensis
45 A0A0F7Y3V7 Pseudomonas sp. CCOS 191
46 A0A0H2X1R6 Chlamydia trachomatis serovar A
47 A0A0H3CBC2 Caulobacter vibrioides (
48 A0A0H5E6T3 Estrella lausannensis
49 A0A0J1FEE3 Peptococcaceae bacterium CEB3
50 WP_147786711.1 Bacillus sp. AY18-3
51 WP_001162675.1 Bacillus
52 WP_173780796.1 Bacillus toyonensis
53 WP_060632218.1 Bacillus cereus group
54 WP_060488314.1 Bacillus wiedmannii
55 WP_166701854.1 Bacillus albus
56 WP_144547613.1 Bacillus sp. X1(2014)
57 WP_088010905.1 Gottfriedia acidiceleris
58 WP_090637358.1 Neobacillus massiliamazoniensis
59 WP_328154539.1 Cytobacillus praedii
60 WP_144541285.1 Cytobacillus oceanisediminis
61 WP_066257451.1 Neobacillus
62 WP_126406912.1 Robertmurraya yapensis
63 WP_088072938.1 Gottfriedia luciferensis
64 WP_307194536.1 Neobacillus niacini
65 WP_335420595.1 Bacillus sp. JJ1566
66 WP_061791231.1 Cytobacillus
67 WP_091481676.1
Alkalibacterium pelagium
68 WP_072905754.1
Anaerobranca californiensis
69 WP_012801066.1
Kangiella koreensis
70 WP_374073233.1
Bdellovibrio
71 WP_017637023.1
Staphylococcus
72 WP_092351088.1
Candidatus Chrysopegis kryptomonas
73 WP_345784844.1
Roseisolibacter agri
74 WP_262551912.1
Staphylococcus pasteuri
75 WP_133566670.1
Bacteriovorax stolpii
76 WP_232133751.1
Staphylococcus warneri
77 WP_277578061.1
Bdellovibrio svalbardensis
78 WP_346025603.1
Alkalibacterium indicireducens
79 WP_142696327.1
Bdellovibrio sp. NC01
80 WP_088565218.1
Bdellovibrio bacteriovorus
81 WP_320146862.1
uncultured Anaeromusa sp.
82 WP_161878691.1
Alkalibacterium sp. MB6
83 WP_373063710.1
Gemmatimonas sp.
84 WP_305791194.1
Thermaerobacter sp. FW80
85 WP_256461060.1
Bdellovibrio reynosensis
86 WP_091347725.1
Anaerobranca gottschalkii
87 WP_101539324.1
Anaerococcus
88 WP_227947168.1
Staphylococcus capitis
89 WP_313909349.1
Rheinheimera sp. MMS21-TC3
90 WP_191728171.1
Luteimonas colneyensis
91 WP_281012623.1
Rhodanobacter sp. AS-Z3
92 WP_395375347.1
Marinicella sp. W31
93 WP_022967753.1
Arenimonas oryziterrae
94 WP_179477157.1
Rhodanobacter sp. K2T2
95 WP_324894866.1
Dyella sp.
96 WP_166051966.1
Thioalkalivibrio sp. XN279
97 WP_242274366.1
Staphylococcus hominis
98 WP_132582873.1
Rheinheimera
99 WP_203072044.1
Staphylococcus auricularis
100 WP_007806066.1
Rhodanobacter spathiphylli

[0234] Table 1: Contains the list of diverse Leucine dehydrogenase enzymes obtained from the different databases. These sequences were subjected to a series of computational calculations and AI based models to obtain the recombinant engineered artificial leucine dehydrogenase polypeptide.

Sl. No SEQ ID NO Substitutions Residue difference from SEQ ID NO: 1 Expression Conversion
1 10 X340E, X352R, X170T, X66I, X157K, X252K 6 + ++++
2 90 X7E, X48V, X66F, X115Q, X148V, X150R, X170T, X221S, X230D, X252K, X301Q, X305E, X320R, X348L 14 +++ +++
3 129 X221E, X340L, X248E, X118F, X48V, X66A, X314E, X350N, X353S, X326S, X320A, X189H, X293T, X115K, X335V, X352R, X46F, X157K, X310Q, X193E, X312I, X313R, X173E, X276C, X150R 25 + +++++
4 142 X24E, X170T, X148V, X349K, X274I, X332A, X46F, X248E, X350N, X314S, X352R, X348L, X353S, X189E, X118L, X226Y, X66A, X296M, X305E, X147D, X193E, X142K, X154K, X299D, X191M, X313R, X312I 27 + +++
5 145 X24E, X170T, X148V, X349K, X274I, X332A, X46F, X248E, X350N, X314S, X352R, X348L, X353S, X189E, X118L, X226Y, X66A, X296M, X305E, X147D, X193E, X142K, X154K, X299D, X191M, X313R, X312I 28 + ++
6 151 X314S, X350N, X226N, X357R, X170T, X296M, X46Q, X248E, X274I, X148V, X115L, X332A, X346A, X320R, X326S, X293G, X364S, X353S, X221E, X118S, X154K, X252K, X75K, X157K, X173E, X267G, X264E, X310Q, X301Q 29 + ++++++
7 154 X189E, X115I, X353S, X66T, X332A, X354T, X148V, X24E, X48R, X293G, X274I, X118C, X46K, X296M, X352R, X350N, X248E, X357R, X7E, X276C, X310Q, X305E, X230D, X41C, X241T, X191M, X140E 27 + ++++++
[0235] Table 2: Artificially engineered Leucine dehydrogenase variants specific activity towards 3 3-hydroxyphenylpyruvic acid resulting in the formation of (2S)-2-amino-3-(3-hydroxyphenyl)propanoic acid though reductive amination. The expression of the variants observed during the study mentioned with “+” indications where “+” indicates low expression, “++” indicates moderate expression and “+++” indicates good expression. The symbol “+” given in the conversion column indicates the % conversion of the substrate, specifically, “+” indicate the conversion of <10%, “++” indicates the conversion of 10 to 30%, “+++” indicates the conversion of >30% and <50%, “++++” indicates the conversion of >50% <70%, “+++++” indicates the conversion of >70 and <80%, “++++++” indicates the conversion of >80 and 90% and “+++++++” indicates conversion of >91% and up to 100% of the substrate.
Sl No SEQ ID NO Substitutions Residue difference form SEQ ID NO: 1 Expression Conversion
1 4 X24E, X46S, X115L, X241T 4 + +
2 23 X48Y, X138R, X276C, X296M, X332A, X346A 6 + ++++
3 108 X348L, X353S, X320A, X346A, X360H, X24E, X48N, X314E, X349K, X7E, X11K, X115L, X226N, X332A, X46P, X118N, X170T, X148V, X322K, X140E, X242P, X75K, X147R, X310Q, X41C, X154K 26 + +++++
4 139 X348L, X353S, X320A, X346A, X360H, X24E, X48N, X314E, X349K, X7E, X11K, X115L, X226N, X332A, X46P, X118N, X170T, X148V, X322K, X140E, X242P, X75K, X147R, X310Q, X41C, X154K 26 + ++++++
8 177 X357R, X66N, X352R, X189H, X350N, X221S, X348L, X46P, X226N, X48Y, X335V, X364S, X11K, X326S, X340E, X170T, X274I, X118D, X148V, X353S, X332A, X7E, X360H, X241T, X173E, X305E, X147D, X193E, X325C, X312I, X310Q, X276C, X154K 33 ++ +++++
9 189 X357R, X66N, X352R, X189H, X350N, X221S, X348L, X46P, X226N, X48Y, X335V, X364S, X11K, X326S, X340E, X170T, X274I, X118D, X148V, X353S, X332A, X7E, X360H, X241T, X173E, X305E, X147D, X193E, X325C, X312I, X310Q, X276C, X154K 33 ++ ++++++
10 191 X226Y, X357R, X346A, X326S, X353S, X66I, X364S, X7E, X24E, X352R, X320A, X11K, X148V, X354T, X296M, X46P, X348L, X350N, X115D, X314E, X360H, X274I, X170T, X142K, X305E, X75K, X41C, X301Q, X140E, X267G, X150R, X230D, X322K 33 + ++++++

[0236] Table 3: Artificially engineered Leucine dehydrogenase variants specific activity towards 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid resulting in the formation of (2S)-2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid though reductive amination. The expression of the variants observed during the study mentioned with “+” indications where “+” indicates low expression, “++” indicates moderate expression and “+++” indicates good expression. The symbol “+” given in the conversion column indicates the % conversion of the substrate, specifically, “+” indicate the conversion of <10%, “++” indicates the conversion of 10 to 30%, “+++” indicates the conversion of >30% and <50%, “++++” indicates the conversion of >50% <70%, “+++++” indicates the conversion of >70 and <80%, “++++++” indicates the conversion of >80 and 90% and “+++++++” indicates conversion of >91% and up to 100% of the substrate.
Sl No SEQ
ID NO Substitutions Residue difference form SEQ ID NO: 1 Expression Conversion
1 19 X354T, X364S, X293I, X173E, X336C 6 + +++
2 21 X115N, X314E, X357R, X293I, X143K, X142K 6 ++ ++++
3 28 X296M, X357R, X349K, X348L, X41C, X267D 6 ++ +++
5 59 X350N, X296M, X357R, X348L, X170T, X276C, X336C, X267D 8 ++ ++++
3 69 X364S, X248E, X353S, X170T, X24E, X346A, X293L, X267D, X154K, X193E 10 + ++++
4 109 X320R, X66T, X357R, X7E, X332A, X11K, X24E, X348L, X115N, X274I, X350N, X364S, X335V, X140T, X312I, X336C, X81A, X154K, X242P 19 ++ +++++
5 119 X48K, X11K, X353S, X314S, X348L, X352R, X320R, X189H, X293L, X346A, X46K, X274I, X332A, X349K, X248E, X264E, X299D, X276C, X301Q, X147R, X173E, X154K 22 ++ ++++++
6 137 X346A, X314S, X46F, X357R, X332A, X115K, X354T, X221S, X293I, X226Y, X350N, X360H, X274I, X118D, X326S, X66H, X24E, X276C, X193E, X336C, X325C, X301Q, X157K, X191M 24 ++ +++++++
7 153 X46S, X354T, X226N, X24E, X296M, X221E, X189E, X48F, X118N, X348L, X340L, X248E, X7E, X11K, X293T, X350N, X326S, X346A, X274I, X322K, X241T, X242P, X305E, X193E, X264E, X252K, X313R 23 + ++++++
[0237] Table 4: Artificially engineered Leucine dehydrogenase variants specific activity towards 2-oxobutanoic acid resulting in the formation of L-2-amino butyric acid though reductive amination the expression of the variants observed during the study mentioned with “+” indications where “+” indicates low expression, “++” indicates moderate expression and “+++” indicates good expression. The symbol “+” given in the conversion column indicates the % conversion of the substrate, specifically, “+” indicate the conversion of <10%, “++” indicates the conversion of 10 to 30%, “+++” indicates the conversion of >30% and <50%, “++++” indicates the conversion of >50% <70%, “+++++” indicates the conversion of >70 and <80%, “++++++” indicates the conversion of >80 and 90% and “+++++++” indicates conversion of >91% and up to 100% of the substrate.
Sl No SEQ ID NO Substitutions Residue difference form SEQ ID NO: 1 Expression Conversion
1 29 X296M, X221S, X11K, X314E, X41C, X81A 6 +++ ++++
2 49 X348L, X293I, X364S, X320A, X130T, X75K 6 + +++
3 81 X248E, X346A, X352R, X7E, X46N, X364S, X348L, X296M, X264E, X154K, X242P, X75K 12 + +++
4 131 X354T, X326C, X66T, X118H, X348L, X350N, X226Y, X248E, X320R, X349K, X357R, X7E, X221S, X46K, X332A, X352R, X170T, X173E, X336C, X193E, X138T, X143K, X41C, X325C, X310Q 25 ++ ++
5 154 X189E, X115I, X353S, X66T, X332A, X354T, X148V, X24E, X48R, X293G, X274I, X118C, X46K, X296M, X352R, X350N, X248E, X357R, X7E, X276C, X310Q, X305E, X230D, X41C, X241T, X191M, X140E 28 + +++++++
6 178 X326S, X360H, X340E, X7E, X314S, X353S, X335V, X11K, X221E, X226Y, X364S, X24E, X274I, X248E, X189E, X115I, X170T, X320A, X293G, X332A, X354T, X147D, X138T, X142K, X75K, X130K, X150R, X325C, X191M, X252K 30 ++ ++++++
7 182 X115L, X7E, X118N, X274I, X66V, X364S, X326C, X332A, X170T, X357R, X354T, X248E, X360H, X46F, X226Y, X320R, X24E, X148V, X189E, X314E, X48F, X296M, X313R, X142K, X299D, X193E, X305E, X143K, X267G, X173E, X310Q, X130K 32 ++ +++
8 183 X352R, X248E, X170T, X24E, X320A, X349K, X360H, X357R, X226Y, X296M, X46Q, X7E, X274I, X314E, X348L, X346A, X115D, X353S, X221S, X11K, X326C, X138R, X140T, X230D, X252K, X305E, X264E, X312I, X276C, X241T, X150R 32 ++ ++++++
9 186 X226N, X118S, X357R, X360H, X66H, X354T, X148V, X248E, X170T, X48K, X353S, X24E, X364S, X346A, X46K, X293G, X115I, X11K, X340L, X7E, X332A, X296M, X350N, X138T, X276C, X242P, X191M, X147D, X140T, X312I, X41C, X267D, X322K 33 + ++++++
10 188 X221S, X364S, X350N, X296M, X66T, X24E, X335V, X357R, X115N, X118D, X170T, X226N, X314E, X274I, X340L, X189E, X346A, X349K, X352R, X46K, X148V, X354T, X11K, X140T, X299D, X301Q, X313R, X325C, X75K, X138T, X193E, X143K 32 +++ +++++
[0238] Table 5: Artificially engineered Leucine dehydrogenase’s variants specific activity towards L-Leucine resulting in the formation of 4-methyl-2-oxopentanoic acid through oxidative deamination. The expression of the variants observed during the study mentioned with “+” indications where “+” indicates low expression, “++” indicates moderate expression and “+++” indicates good expression. The symbol “+” given in the conversion column indicates the % conversion of the substrate, specifically, “+” indicate the conversion of <10%, “++” indicates the conversion of 10 to 30%, “+++” indicates the conversion of >30% and <50%, “++++” indicates the conversion of >50% <70%, “+++++” indicates the conversion of >70 and <80%, “++++++” indicates the conversion of >80 and 90% and “+++++++” indicates conversion of >91% and up to 100% of the substrate.
Sl No SEQ ID NO Substitutions Residue difference form SEQ ID NO: 1 Expression Conversion
1 105 X335V, X115L, X326S, X364S, X320R, X296M, X314S, X350N, X348L, X357R, X352R, X360H, X267D, X305E, X157K, X154K, X193E, X130T 18 ++ ++++
2 123 X118N, X7E, X353S, X170T, X148V, X66T, X360H, X346A, X48M, X326C, X350N, X357R, X293T, X248E, X115L, X322K, X230D, X325C, X140T, X252K, X173E, X193E, X264E 23 ++ ++++
7 134 X340E, X226Y, X364S, X7E, X326S, X357R, X66A, X346A, X24E, X115D, X189H, X348L, X248E, X293L, X148V, X48Y, X325C, X138R, X154K, X193E, X173E, X140E, X305E, X242P 25 ++ +++
8 148 X326C, X48A, X274I, X349K, X248E, X66I, X226N, X348L, X170T, X296M, X189H, X118H, X148V, X357R, X350N, X354T, X221E, X360H, X115E, X352R, X314S, X320A, X138R, X130T, X140E, X41C, X312I, X276C, X157K, X143K, X193E 31 ++ ++++
9 181 X326C, X48A, X274I, X349K, X248E, X66I, X226N, X348L, X170T, X296M, X189H, X118H, X148V, X357R, X350N, X354T, X221E, X360H, X115E, X352R, X314S, X320A, X138R, X130T, X140E, X41C, X312I, X276C, X157K, X143K, X193E 29 + +++++
13 185 X293L, X352R, X320R, X118N, X346A, X332A, X46A, X354T, X314E, X364S, X226N, X274I, X326S, X7E, X349K, X115E, X148V, X350N, X189E, X170T, X335V, X11K, X276C, X143K, X241T, X301Q, X230D, X157K, X325C, X147R, X305E, X336C 32 + +++++
14 193 X360H, X332A, X248E, X357R, X340E, X353S, X314S, X352R, X293T, X221S, X170T, X335V, X11K, X349K, X226N, X320R, X148V, X118S, X115L, X350N, X24E, X354T, X66H, X138T, X322K, X299D, X191M, X130K, X310Q, X157K, X193E, X143K 32 ++ ++++++++
[0239] Table 6: Artificially engineered Leucine dehydrogenase variants specific activity towards L-nor-Leucine resulting in the formation of 2-oxohexanoic acid though oxidative deamination. The expression of the variants observed during the study mentioned with “+” indications where “+” indicates low expression, “++” indicates moderate expression and “+++” indicates good expression. The symbol “+” given in the conversion column indicates the % conversion of the substrate, specifically, “+” indicate the conversion of <10%, “++” indicates the conversion of 10 to 30%, “+++” indicates the conversion of >30% and <50%, “++++” indicates the conversion of >50% <70%, “+++++” indicates the conversion of >70 and <80%, “++++++” indicates the conversion of >80 and 90% and “+++++++” indicates conversion of >91% and up to 100% of the substrate.
Sl No SEQ ID NO Substitutions Residue difference from SEQ ID NO: 1 Expression Conversion
1 9 X360H, X46A, X118S, X293A, X193E, X142K 6 + +++
2 27 X46F, X226N, X293A, X352R, X230D, X150R 6 + ++++
3 165 X346A, X293G, X352R, X66F, X24E, X11K, X274I, X340L, X314E, X46N, X48N, X7E, X354T, X115I, X326S, X350N, X348L, X364S, X221S, X248E, X353S, X299D, X138R, X154K, X252K, X193E, X313R, X191M, X325C, X140E, X157K 31 + +++++
4 197 X115D, X326S, X357R, X293I, X189H, X66H, X348L, X332A, X226N, X296M, X340E, X360H, X350N, X48A, X170T, X221E, X346A, X349K, X320A, X24E, X353S, X352R, X140T, X143K, X150R, X276C, X241T, X299D, X305E, X310Q, X173E, X41C, X267D 34 + ++++++
5 200 X364S, X320A, X226N, X350N, X354T, X346A, X353S, X340L, X349K, X66S, X46N, X274I, X348L, X360H, X24E, X248E, X115L, X296M, X7E, X314E, X48N, X221S, X154K, X322K, X150R, X157K, X299D, X252K, X276C, X130K, X267D, X143K 33 ++ +++++++
[0240] Table 7: Artificially engineered Leucine dehydrogenase variants' specific activity towards L-Tryptophan resulting in the formation of Indoole-3-pyruvic acid though oxidative deamination the expression of the variants observed during the study mentioned with “+” indications where “+” indicates low expression, “++” indicates moderate expression and “+++” indicates good expression. The symbol “+” given in the conversion column indicates the % conversion of the substrate, specifically, “+” indicate the conversion of <10%, “++” indicates the conversion of 10 to 30%, “+++” indicates the conversion of >30% and <50%, “++++” indicates the conversion of >50% <70%, “+++++” indicates the conversion of >70 and <80%, “++++++” indicates the conversion of >80 and 90% and “+++++++” indicates conversion of >91% and up to 100% of the substrate.
Sl.No Sequence ID NO Substitutions Residue difference from SEQ ID No: 1 Expression Conversion
1 1 Artificial Sequence 0 +++ +
2 2 X170T, X326C, X314E, X150R, X154K 5 + +
3 3 X348L, X364S, X349K, X248E, X310Q, X230D 6 ++ +
4 4 X24E, X46S, X115L, X241T 4 + +
5 5 X293I, X274I, X170T, X322K, X140E 5 ++ +
6 6 X320R, X226Y, X326S, X305E 4 + +
7 7 X349K, X48R, X357R, X138T, X267D 5 + +
8 8 X274I, X24E, X221E, X138R, X230D 5 ++ +
9 9 X360H, X46A, X118S, X293A, X193E, X142K 6 + +
10 10 X340E, X352R, X170T, X66I, X157K, X252K 6 + +
11 11 X357R, X364S, X24E, X173E, X142K 5 + +
12 12 X118L, X360H, X170T, X332A, X191M 5 + +
13 13 X148V, X46N, X118C, X226Y, X147R, X150R 6 + +
14 14 X326C, X11K, X170T, X314S, X276C, X264E 6 + +
15 15 X364S, X350N, X353S, X314E, X191M, X230D 6 ++ +
16 16 X118L, X293I, X332A, X354T, X138R, X154K 6 + +
17 17 X332A, X189H, X7E, X293A, X193E, X230D 6 + +
18 18 X148V, X189H, X349K, X296M, X301Q 5 + +
19 19 X354T, X364S, X293I, X173E, X336C 5 + +
20 20 X189E, X360H, X115I, X7E, X242P, X313R 6 ++ +
21 21 X115N, X314E, X357R, X293I, X143K, X142K 6 ++ +
22 22 X354T, X221E, X226Y, X296M, X230D, X138R 6 ++ +
23 23 X48Y, X332A, X346A, X296M, X138R, X276C 6 ++ +
24 24 X332A, X340E, X350N, X349K, X312I, X130K 6 + +
25 25 X221E, X118F, X226Y, X296M, X336C, X191M 6 + +
26 26 X66N, X350N, X226Y, X340E, X242P, X157K 6 + +
27 27 X46F, X226N, X293A, X352R, X230D, X150R 6 + +
28 28 X296M, X357R, X349K, X348L, X41C, X267D 6 ++ +
29 29 X296M, X221S, X11K, X314E, X41C, X81A 6 +++ ++
30 30 X353S, X7E, X360H, X364S, X310Q, X267D 6 +++ ++
31 31 X350N, X349K, X66N, X314S, X75K, X325C 6 ++ ++
32 32 X189H, X248E, X118L, X293L, X193E, X150R 6 + ++
33 33 X296M, X332A, X226Y, X24E, X301Q, X241T 6 + ++
34 34 X170T, X296M, X11K, X354T, X276C, X305E 6 + ++
35 35 X189H, X7E, X115N, X296M, X81A, X252K 6 + ++
36 36 X115K, X293I, X221E, X320R, X301Q, X322K, X130T 7 + ++
37 37 X320R, X221E, X314S, X364S, X313R, X252K, X241T 7 + ++
38 38 X221S, X348L, X115I, X320R, X241T, X41C, X191M 7 + ++
39 39 X274I, X350N, X296M, X11K, X336C, X75K, X230D 7 + ++
40 40 X296M, X360H, X346A, X66V, X264E, X191M, X157K 7 ++ ++
41 41 X360H, X340E, X7E, X349K, X147R, X305E, X299D 7 ++ ++
42 42 X248E, X115K, X348L, X296M, X157K, X41C 6 ++ ++
43 43 X118S, X248E, X364S, X320A, X276C, X252K, X336C 7 +++ ++
44 44 X226N, X320A, X360H, X352R, X41C, X157K, X322K 7 ++ ++
45 45 X352R, X274I, X364S, X335V, X157K, X267G, X191M 7 +++ ++
46 46 X349K, X296M, X148V, X354T, X230D, X241T, X313R 7 + ++
47 47 X189E, X326C, X350N, X7E, X252K, X336C, X81A 7 + ++
48 48 X170T, X48R, X357R, X360H, X75K, X241T, X173E 7 + ++
49 49 X348L, X293I, X364S, X320A, X130T, X75K 6 + ++
50 50 X24E, X226Y, X349K, X354T, X173E, X150R, X267D 7 + ++
51 51 X248E, X170T, X118F, X11K, X357R, X81A, X193E 7 + ++
52 52 X353S, X348L, X7E, X340L, X346A, X173E, X130K, X142K 8 + ++
53 53 X115E, X189E, X48A, X360H, X332A, X75K, X230D, X312I 8 +++ ++
54 54 X66V, X340L, X332A, X293L, X314E, X299D, X276C, X267G 8 + ++
55 55 X353S, X352R, X274I, X189H, X332A, X264E, X147R, X41C 8 + ++
56 56 X118A, X364S, X326S, X354T, X314S, X193E, X142K 7 + ++
57 57 X320A, X189E, X221E, X7E, X352R, X313R, X276C, X173E 8 + ++
58 58 X170T, X46Q, X314S, X364S, X148V, X242P, X140E, X313R 8 ++ ++
59 59 X350N, X296M, X357R, X348L, X170T, X276C, X336C, X267D 8 ++ ++
60 60 X350N, X332A, X353S, X170T, X349K, X191M, X312I, X264E 8 + ++
61 61 X7E, X226N, X66T, X332A, X189H, X252K, X301Q 7 ++
62 62 X293T, X274I, X148V, X170T, X320R, X305E, X325C, X313R 8 ++ ++
63 63 X148V, X352R, X353S, X221S, X48N, X157K, X310Q, X252K 8 + ++
64 64 X274I, X350N, X24E, X354T, X148V, X320A, X41C, X301Q, X305E 9 ++ ++
65 65 X340E, X350N, X48M, X349K, X170T, X352R, X310Q, X147D, X267G 9 ++ ++
66 66 X364S, X66F, X349K, X46A, X48I, X118H, X154K, X310Q, X242P 9 + ++
67 67 X274I, X350N, X364S, X24E, X353S, X11K, X41C, X193E, X301Q 9 + ++
68 68 X226N, X170T, X360H, X189E, X48V, X332A, X142K, X143K, X267D 9 + ++
69 69 X364S, X248E, X353S, X170T, X24E, X346A, X293L, X267D, X154K, X193E 10 + ++
70 70 X248E, X326C, X189H, X350N, X221E, X360H, X226Y, X41C, X142K, X276C 10 + ++
71 71 X11K, X348L, X364S, X314E, X226Y, X115K, X81A, X191M, X313R 9 + ++
72 72 X170T, X364S, X274I, X24E, X7E, X148V, X326C, X242P, X241T, X157K 10 ++ ++
73 73 X357R, X296M, X354T, X66V, X364S, X24E, X115I, X130T, X313R, X305E, X138T 11 + ++
74 74 X148V, X320A, X346A, X332A, X7E, X274I, X118L, X147D, X305E, X193E, X322K 11 ++ ++
75 75 X353S, X348L, X326S, X66F, X148V, X221E, X189H, X130T, X147D, X143K, X157K 11 + ++
76 76 X66F, X118A, X364S, X170T, X353S, X48M, X274I, X142K, X41C, X252K, X322K 11 ++ +++
77 77 X66T, X349K, X115N, X353S, X46A, X248E, X332A, X138T, X142K, X312I 10 ++ +++
78 78 X364S, X350N, X274I, X118H, X352R, X348L, X357R, X115Q, X310Q, X276C, X147R, X154K 12 ++ +++
79 79 X293L, X189E, X24E, X360H, X348L, X320R, X66T, X221E, X75K, X305E, X336C, X242P 12 ++ +++
80 80 X326C, X248E, X118S, X148V, X274I, X354T, X221S, X170T, X242P, X310Q, X322K, X142K 12 ++ +++
81 81 X248E, X346A, X352R, X7E, X46N, X364S, X348L, X296M, X264E, X154K, X242P, X75K 12 + +++
82 82 X274I, X352R, X148V, X346A, X314S, X360H, X296M, X75K, X312I, X276C, X264E 11 +++ +++
83 83 X349K, X340E, X226Y, X348L, X115E, X353S, X248E, X326C, X314E, X41C, X267G, X276C, X150R 13 +++ +++
84 84 X293T, X46Q, X360H, X332A, X66F, X314S, X340E, X296M, X81A, X264E, X276C, X325C 12 + +++
85 85 X115Q, X226Y, X314E, X66F, X360H, X353S, X148V, X346A, X364S, X313R, X322K, X154K, X75K 13 + +++
86 86 X350N, X118A, X296M, X221E, X66T, X353S, X340L, X357R, X349K, X191M, X242P, X267G 12 + +++
87 87 X335V, X349K, X293G, X346A, X115N, X189E, X148V, X352R, X7E, X301Q, X140T, X81A, X142K 13 ++ +++
88 88 X357R, X360H, X274I, X364S, X170T, X189H, X352R, X314E, X148V, X130T, X142K, X267D, X143K, X157K 14 + +++
89 89 X115E, X364S, X66H, X24E, X148V, X48I, X189H, X332A, X354T, X154K, X81A, X142K, X241T, X143K 14 + +++
90 90 X320R, X170T, X148V, X348L, X115Q, X7E, X66F, X221S, X48V, X150R, X305E, X252K, X301Q, X230D 14 +++ +++
91 91 X66N, X348L, X346A, X320A, X48F, X118F, X221S, X248E, X24E, X310Q, X312I, X299D, X241T, X264E 14 ++ +++
92 92 X296M, X274I, X314E, X350N, X348L, X7E, X357R, X11K, X252K, X230D, X142K, X154K, X325C 13 ++ +++
93 93 X354T, X226N, X48A, X148V, X189H, X248E, X314E, X24E, X332A, X325C, X157K, X242P, X130K, X150R 14 ++ +++
94 94 X348L, X340E, X7E, X314S, X115N, X354T, X24E, X248E, X296M, X350N, X140T, X301Q, X252K, X276C, X230D 15 ++ +++
95 95 X350N, X296M, X11K, X364S, X332A, X118H, X357R, X7E, X66N, X352R, X336C, X313R, X193E, X241T 14 ++ +++
96 96 X340E, X274I, X360H, X326C, X148V, X46Q, X349K, X353S, X66V, X357R, X138R, X276C, X193E, X241T, X252K 15 +++ +++
97 97 X148V, X360H, X293I, X326S, X24E, X314E, X364S, X346A, X340L, X335V, X264E, X142K, X191M, X140E 14 ++ +++
98 98 X357R, X352R, X11K, X364S, X354T, X7E, X46A, X226Y, X248E, X335V, X360H, X264E, X142K, X138R, X130K, X75K 16 +++ +++
99 99 X274I, X335V, X314S, X226N, X340L, X11K, X148V, X332A, X353S, X115Q, X364S, X276C, X313R, X193E, X154K, X252K, X138R 17 ++ +++
100 100 X354T, X348L, X360H, X350N, X364S, X326S, X226Y, X314S, X320R, X221E, X293T, X130K, X154K, X336C, X138T, X313R 16 + +++
101 101 X332A, X189H, X346A, X46Q, X326C, X7E, X48A, X24E, X274I, X357R, X226Y, X142K, X301Q, X75K, X252K, X336C 16 + +++
102 102 X340E, X320A, X360H, X354T, X115E, X296M, X7E, X148V, X332A, X346A, X357R, X276C, X325C, X41C, X193E, X157K, X312I 17 ++ +++
103 103 X296M, X320A, X274I, X189H, X326S, X118H, X352R, X24E, X11K, X46N, X221E, X346A, X242P, X41C, X150R, X336C, X264E, X241T 18 ++ +++
104 104 X274I, X148V, X350N, X360H, X326S, X66Q, X11K, X24E, X332A, X357R, X226Y, X115Q, X75K, X154K, X81A, X241T, X140T, X130K 18 ++ +++
105 105 X335V, X115L, X326S, X364S, X320R, X296M, X314S, X350N, X348L, X357R, X352R, X360H, X267D, X305E, X157K, X154K, X193E, X130T 18 ++ +++
106 106 X46S, X66Q, X296M, X340E, X221S, X326C, X293T, X248E, X335V, X314E, X48N, X7E, X81A, X193E, X299D, X310Q, X142K, X154K 18 + +++
107 107 X24E, X189E, X360H, X226Y, X364S, X350N, X7E, X11K, X296M, X346A, X314E, X248E, X221S, X147D, X336C, X130K, X157K, X305E, X312I 19 + ++++
108 108 X364S, X170T, X320R, X314E, X350N, X7E, X66F, X346A, X274I, X357R, X118N, X349K, X241T, X313R, X140T, X310Q, X267D, X252K 18 ++ ++++
109 109 X320R, X66T, X357R, X7E, X332A, X11K, X24E, X348L, X115N, X274I, X350N, X364S, X335V, X140T, X312I, X336C, X81A, X154K, X242P 19 ++ ++++
110 110 X346A, X340E, X364S, X353S, X352R, X248E, X189E, X349K, X115Q, X148V, X170T, X320A, X335V, X138T, X299D, X301Q, X147D, X41C, X325C 19 ++ ++++
111 111 X248E, X115E, X346A, X221S, X118H, X274I, X360H, X332A, X353S, X357R, X11K, X66H, X252K, X264E, X322K, X41C, X157K, X81A 18 ++ ++++
112 112 X170T, X340E, X353S, X296M, X66A, X335V, X46F, X332A, X226N, X320R, X248E, X24E, X346A, X326S, X252K, X310Q, X305E, X264E, X299D, X173E 20 + ++++
113 113 X221E, X340L, X360H, X346A, X348L, X293G, X11K, X350N, X296M, X115D, X118C, X332A, X354T, X267D, X305E, X310Q, X322K, X75K, X312I 19 + ++++
114 114 X170T, X226N, X148V, X118S, X293I, X320A, X274I, X348L, X349K, X354T, X248E, X350N, X48M, X7E, X230D, X322K, X154K, X299D, X150R, X140T, X142K 21 +++ ++++
115 115 X148V, X332A, X170T, X349K, X352R, X66S, X226Y, X24E, X348L, X335V, X274I, X221S, X118F, X314S, X276C, X242P, X230D, X305E, X252K, X140T, X264E 21 ++ ++++
116 116 X66S, X326C, X350N, X46K, X296M, X7E, X346A, X293L, X320R, X115I, X314E, X170T, X332A, X130K, X173E, X313R, X41C, X191M, X305E, X75K 20 + ++++
117 117 X118S, X350N, X7E, X148V, X335V, X360H, X11K, X346A, X115E, X364S, X66F, X320R, X293I, X226N, X348L, X325C, X41C, X322K, X173E, X252K, X299D, X157K 22 ++ ++++
118 118 X226N, X364S, X349K, X352R, X335V, X348L, X293G, X353S, X46S, X66V, X11K, X340E, X357R, X115N, X314E, X75K, X81A, X130T, X305E, X154K, X230D, X173E 22 ++ ++++
119 119 X48K, X11K, X353S, X314S, X348L, X352R, X320R, X189H, X293L, X346A, X46K, X274I, X332A, X349K, X248E, X264E, X299D, X276C, X301Q, X147R, X173E, X154K 22 ++ ++++
120 120 X170T, X360H, X352R, X48I, X335V, X364S, X296M, X332A, X189H, X350N, X7E, X118D, X346A, X24E, X349K, X150R, X157K, X191M, X154K, X242P, X325C, X230D 22 ++ ++++
121 121 X293I, X46F, X354T, X226Y, X348L, X350N, X296M, X314S, X357R, X320A, X118S, X170T, X221S, X352R, X360H, X325C, X252K, X75K, X140T, X242P, X138R 21 + ++++
122 122 X346A, X7E, X326S, X221S, X352R, X348L, X349K, X340E, X66T, X350N, X226Y, X296M, X353S, X148V, X314S, X293T, X81A, X150R, X267D, X173E, X75K, X147D, X230D, X322K 24 + ++++
123 123 X118N, X7E, X353S, X170T, X148V, X66T, X360H, X346A, X48M, X326C, X350N, X357R, X293T, X248E, X115L, X322K, X230D, X325C, X140T, X252K, X173E, X193E, X264E 23 ++ ++++
124 124 X118A, X364S, X314S, X221S, X66S, X189H, X340L, X332A, X296M, X7E, X226Y, X293I, X346A, X11K, X353S, X115I, X81A, X242P, X336C, X267D, X325C, X313R, X191M, X140T 24 ++ ++++
125 125 X364S, X189E, X346A, X48F, X170T, X248E, X326C, X24E, X349K, X226Y, X353S, X348L, X350N, X293G, X360H, X340L, X252K, X191M, X138R, X142K, X193E, X154K, X313R 23 ++ ++++
126 126 X346A, X24E, X364S, X320R, X118N, X326C, X354T, X350N, X353S, X115I, X248E, X11K, X46N, X360H, X296M, X170T, X41C, X336C, X130T, X267G, X150R, X301Q, X322K 23 ++ ++++
127 127 X148V, X293T, X7E, X11K, X189H, X350N, X357R, X274I, X353S, X352R, X346A, X296M, X320A, X66Q, X360H, X314S, X336C, X252K, X313R, X322K, X241T, X264E, X193E, X81A 24 + ++++
128 128 X314S, X346A, X293I, X364S, X354T, X221E, X320R, X115E, X46F, X349K, X357R, X340E, X350N, X226Y, X348L, X66Q, X193E, X322K, X299D, X325C, X142K, X75K, X310Q, X143K 24 + ++++
129 129 X221E, X340L, X248E, X118F, X48V, X66A, X314E, X350N, X353S, X326S, X320A, X189H, X293T, X115K, X335V, X352R, X46F, X157K, X310Q, X193E, X312I, X313R, X173E, X276C, X150R 25 + ++++
130 130 X348L, X340L, X346A, X332A, X335V, X364S, X248E, X349K, X221S, X352R, X314E, X115Q, X11K, X148V, X48A, X226Y, X357R, X150R, X310Q, X241T, X75K, X312I, X301Q, X191M, X336C 25 +++ ++++
131 131 X354T, X326C, X66T, X118H, X348L, X350N, X226Y, X248E, X320R, X349K, X357R, X7E, X221S, X46K, X332A, X352R, X170T, X173E, X336C, X193E, X138T, X143K, X41C, X325C, X310Q 25 ++ ++++
132 132 X170T, X115I, X248E, X352R, X7E, X274I, X354T, X118A, X66N, X360H, X364S, X350N, X11K, X335V, X189E, X221S, X24E, X230D, X252K, X267D, X310Q, X81A, X312I, X150R, X157K 25 + ++++
133 133 X314E, X274I, X226N, X349K, X221E, X170T, X332A, X46N, X353S, X354T, X293A, X360H, X340L, X350N, X364S, X66T, X154K, X81A, X241T, X173E, X252K, X130K, X325C, X299D 24 + ++++
134 134 X340E, X226Y, X364S, X7E, X326S, X357R, X66A, X346A, X24E, X115D, X189H, X348L, X248E, X293L, X148V, X48Y, X325C, X138R, X154K, X193E, X173E, X140E, X305E, X242P 24 ++ ++++
135 135 X332A, X170T, X348L, X118A, X46A, X314E, X320R, X293I, X346A, X357R, X226N, X11K, X274I, X360H, X352R, X340E, X150R, X143K, X325C, X41C, X191M, X305E, X130T 23 ++ ++++
136 136 X326C, X248E, X353S, X11K, X189E, X170T, X340E, X7E, X66T, X335V, X332A, X357R, X24E, X354T, X364S, X46K, X226N, X360H, X325C, X312I, X252K, X154K, X138R, X305E, X322K 25 ++ ++++
137 137 X346A, X314S, X46F, X357R, X332A, X115K, X354T, X221S, X293I, X226Y, X350N, X360H, X274I, X118D, X326S, X66H, X24E, X276C, X193E, X336C, X325C, X301Q, X157K, X191M 24 ++ ++++
138 138 X352R, X353S, X346A, X170T, X357R, X364S, X226N, X348L, X293I, X350N, X349K, X274I, X24E, X248E, X118H, X320R, X221E, X325C, X305E, X276C, X140E, X157K, X130T, X252K, X241T 25 + ++++
139 139 X348L, X353S, X320A, X346A, X360H, X24E, X48N, X314E, X349K, X7E, X11K, X115L, X226N, X332A, X46P, X118N, X170T, X148V, X322K, X140E, X242P, X75K, X147R, X310Q, X41C, X154K 26 + ++++
140 140 X293T, X353S, X314E, X326S, X7E, X364S, X189H, X296M, X221E, X48M, X24E, X248E, X320R, X332A, X46Q, X349K, X348L, X75K, X313R, X147D, X252K, X154K, X142K, X143K, X305E 25 + ++++
141 141 X293L, X148V, X340E, X314E, X248E, X353S, X346A, X350N, X332A, X48F, X24E, X66F, X115L, X189H, X274I, X364S, X326C, X11K, X267G, X193E, X81A, X140T, X336C, X301Q, X142K, X305E, X264E 27 + ++++
142 142 X24E, X170T, X148V, X349K, X274I, X332A, X46F, X248E, X350N, X314S, X352R, X348L, X353S, X189E, X118L, X226Y, X66A, X296M, X305E, X147D, X193E, X142K, X154K, X299D, X191M, X313R, X312I 27 + ++++
143 143 X11K, X221E, X66N, X364S, X360H, X46K, X340E, X293A, X320A, X148V, X274I, X314S, X326C, X118L, X357R, X7E, X170T, X48I, X24E, X130T, X313R, X41C, X301Q, X322K, X264E, X299D, X147D, X252K 28 +++ +++++
144 144 X340E, X332A, X24E, X357R, X360H, X354T, X7E, X349K, X189H, X170T, X274I, X48I, X296M, X115E, X221E, X314E, X11K, X326C, X325C, X230D, X336C, X305E, X147D, X276C, X41C, X130K, X299D 27 ++ +++++
145 145 X66S, X357R, X48I, X296M, X320A, X221S, X314S, X332A, X350N, X353S, X7E, X170T, X115N, X352R, X148V, X226N, X346A, X354T, X293L, X313R, X312I, X81A, X130T, X157K, X143K, X75K, X41C, X193E 28 + +++++
146 146 X7E, X320R, X170T, X348L, X326C, X293I, X360H, X364S, X346A, X354T, X314S, X148V, X24E, X46S, X353S, X350N, X189E, X352R, X267G, X41C, X322K, X230D, X313R, X173E, X157K, X242P, X336C 27 + +++++
147 147 X350N, X248E, X226N, X115K, X118F, X346A, X170T, X314E, X7E, X320R, X340L, X352R, X360H, X353S, X348L, X349K, X66H, X46P, X296M, X301Q, X241T, X142K, X310Q, X138R, X230D, X322K, X147D, X154K 28 ++ +++++
148 148 X357R, X332A, X46N, X346A, X66A, X118A, X189E, X248E, X296M, X352R, X274I, X226Y, X335V, X170T, X326C, X24E, X48V, X7E, X353S, X241T, X299D, X242P, X150R, X193E, X75K, X157K, X191M, X267G 28 ++ +++++
149 149 X248E, X274I, X118A, X350N, X226N, X170T, X364S, X335V, X24E, X326C, X221E, X349K, X7E, X357R, X314S, X11K, X352R, X48M, X296M, X313R, X230D, X41C, X322K, X301Q, X252K, X142K, X312I, X299D 28 ++ +++++
150 150 X189H, X360H, X340E, X274I, X350N, X46A, X348L, X248E, X357R, X353S, X364S, X326S, X24E, X48I, X332A, X296M, X148V, X66N, X115I, X241T, X310Q, X301Q, X264E, X336C, X312I, X75K, X299D, X230D 28 ++ +++++
151 151 X314S, X350N, X226N, X357R, X170T, X296M, X46Q, X248E, X274I, X148V, X115L, X332A, X346A, X320R, X326S, X293G, X364S, X353S, X221E, X118S, X154K, X252K, X75K, X157K, X173E, X267G, X264E, X310Q, X301Q 29 + +++++
152 152 X353S, X24E, X335V, X326C, X170T, X296M, X189E, X364S, X118S, X348L, X340L, X46P, X248E, X349K, X320R, X360H, X293T, X7E, X354T, X66S, X150R, X301Q, X230D, X138R, X75K, X267D, X325C, X173E, X191M 29 + +++++
153 153 X46S, X354T, X226N, X24E, X296M, X221E, X189E, X48F, X118N, X348L, X340L, X248E, X7E, X11K, X293T, X350N, X326S, X346A, X274I, X322K, X241T, X242P, X305E, X193E, X264E, X252K, X313R 27 + +++++
154 154 X189E, X115I, X353S, X66T, X332A, X354T, X148V, X24E, X48R, X293G, X274I, X118C, X46K, X296M, X352R, X350N, X248E, X357R, X7E, X276C, X310Q, X305E, X230D, X41C, X241T, X191M, X140E 27 + +++++
155 155 X364S, X296M, X314E, X66H, X346A, X320R, X24E, X335V, X148V, X360H, X248E, X48K, X46Q, X226N, X340E, X170T, X348L, X293I, X353S, X349K, X191M, X81A, X138T, X313R, X322K, X299D, X310Q, X336C 28 +++ +++++
156 156 X296M, X314S, X248E, X170T, X326S, X11K, X360H, X350N, X353S, X349K, X352R, X24E, X354T, X221E, X293T, X46Q, X48F, X7E, X148V, X364S, X191M, X150R, X142K, X140T, X276C, X81A, X264E, X154K, X305E 29 ++ +++++
157 157 X293A, X296M, X352R, X357R, X11K, X274I, X148V, X360H, X226N, X170T, X326S, X320R, X340L, X348L, X48M, X24E, X314S, X354T, X248E, X147R, X41C, X157K, X336C, X276C, X75K, X154K, X230D, X325C 28 + +++++
158 158 X346A, X46N, X296M, X115K, X332A, X66H, X348L, X349K, X357R, X274I, X326C, X350N, X48R, X226N, X320R, X354T, X118N, X314S, X340L, X248E, X173E, X313R, X142K, X154K, X140E, X241T, X242P, X41C, X143K 29 + +++++
159 159 X248E, X352R, X221S, X320A, X326C, X354T, X350N, X346A, X170T, X274I, X348L, X7E, X340E, X293L, X11K, X148V, X353S, X296M, X189E, X349K, X130K, X313R, X142K, X322K, X230D, X264E, X150R, X140E, X157K 29 + +++++
160 160 X170T, X248E, X314E, X46Q, X115E, X118S, X148V, X360H, X296M, X346A, X11K, X357R, X354T, X364S, X326S, X332A, X293A, X226Y, X335V, X320A, X350N, X41C, X336C, X193E, X75K, X150R, X230D, X173E, X143K, X276C 30 + +++++
161 161 X314E, X357R, X189H, X349K, X332A, X360H, X7E, X24E, X226N, X346A, X340E, X293A, X348L, X326S, X248E, X118L, X335V, X66I, X11K, X46K, X115N, X336C, X142K, X154K, X242P, X191M, X252K, X310Q, X305E, X264E 30 + +++++
162 162 X360H, X189E, X349K, X293T, X115D, X7E, X248E, X46S, X314E, X350N, X320R, X352R, X48I, X364S, X170T, X24E, X221S, X66V, X296M, X326C, X138T, X325C, X310Q, X241T, X154K, X267D, X75K, X140T 28 + +++++
163 163 X350N, X24E, X296M, X148V, X326S, X320R, X66I, X11K, X352R, X48R, X357R, X7E, X189E, X354T, X170T, X346A, X115I, X348L, X46Q, X221E, X349K, X143K, X322K, X301Q, X150R, X264E, X41C, X130T, X336C, X191M 30 +++ +++++
164 164 X349K, X346A, X326C, X115L, X148V, X274I, X357R, X364S, X314E, X46N, X353S, X226N, X296M, X348L, X24E, X332A, X354T, X293A, X170T, X66H, X320A, X41C, X312I, X154K, X264E, X325C, X142K, X191M, X75K, X305E 30 ++ +++++
165 165 X346A, X293G, X352R, X66F, X24E, X11K, X274I, X340L, X314E, X46N, X48N, X7E, X354T, X115I, X326S, X350N, X348L, X364S, X221S, X248E, X353S, X299D, X138R, X154K, X252K, X193E, X313R, X191M, X325C, X140E, X157K 31 + +++++
166 166 X357R, X332A, X170T, X115Q, X320R, X118C, X46K, X226N, X48K, X360H, X350N, X314E, X346A, X335V, X340L, X364S, X274I, X66A, X189E, X24E, X221S, X241T, X173E, X75K, X242P, X41C, X267D, X230D, X138T, X143K 30 ++ +++++
167 167 X115D, X352R, X320R, X189E, X226N, X353S, X170T, X332A, X118S, X346A, X357R, X296M, X148V, X340L, X354T, X293I, X24E, X350N, X221S, X364S, X143K, X310Q, X173E, X313R, X305E, X147D, X130K, X142K, X322K, X301Q 30 ++ +++++
168 168 X346A, X314S, X46K, X332A, X320A, X189E, X170T, X24E, X296M, X11K, X66S, X118F, X353S, X274I, X340E, X350N, X349K, X7E, X248E, X148V, X312I, X267D, X154K, X230D, X140T, X150R, X252K, X276C, X147R, X193E 30 ++ +++++
169 169 X326C, X314E, X48R, X118S, X226N, X352R, X24E, X66I, X170T, X349K, X320A, X274I, X360H, X293T, X221S, X353S, X340L, X332A, X189H, X335V, X148V, X142K, X313R, X264E, X276C, X312I, X322K, X336C, X143K, X173E, X150R 31 ++ ++++++
170 170 X332A, X226N, X115N, X46S, X296M, X346A, X348L, X118S, X357R, X360H, X353S, X340L, X148V, X293L, X326C, X248E, X314E, X221S, X66Q, X352R, X241T, X267D, X276C, X81A, X150R, X140E, X130T, X157K, X242P, X301Q 30 + ++++++
171 171 X314E, X118S, X66A, X248E, X293L, X352R, X320A, X364S, X7E, X350N, X346A, X148V, X326S, X332A, X360H, X221S, X226Y, X170T, X354T, X115E, X296M, X305E, X242P, X191M, X241T, X142K, X147R, X322K, X143K, X75K, X267G 31 ++ ++++++
172 172 X349K, X314E, X7E, X296M, X148V, X353S, X354T, X24E, X332A, X170T, X46S, X350N, X326C, X11K, X293L, X348L, X248E, X340E, X48A, X66A, X301Q, X230D, X81A, X75K, X191M, X252K, X305E, X264E, X41C, X142K 30 ++ ++++++
173 173 X352R, X349K, X46S, X189H, X221S, X354T, X66N, X340E, X148V, X115Q, X274I, X7E, X248E, X357R, X320A, X170T, X348L, X353S, X118D, X296M, X332A, X193E, X310Q, X142K, X264E, X150R, X276C, X325C, X305E, X313R, X299D 31 ++ ++++++
174 174 X364S, X332A, X7E, X221S, X348L, X340E, X352R, X226Y, X48A, X24E, X170T, X349K, X46S, X326S, X293A, X296M, X350N, X354T, X248E, X148V, X252K, X140E, X312I, X130K, X143K, X193E, X301Q, X81A, X336C, X325C 30 ++ ++++++
175 175 X326C, X360H, X346A, X352R, X48Y, X349K, X24E, X314S, X332A, X296M, X348L, X340E, X320R, X221S, X354T, X11K, X115Q, X248E, X357R, X118F, X310Q, X193E, X242P, X75K, X81A, X143K, X130T, X299D, X267G 29 + ++++++
176 176 X115D, X357R, X349K, X148V, X340E, X350N, X360H, X46K, X314S, X293A, X24E, X226Y, X348L, X7E, X274I, X248E, X346A, X11K, X221E, X326C, X296M, X41C, X150R, X138R, X173E, X305E, X130T, X301Q, X322K, X81A, X276C 31 +++ ++++++
177 177 X314S, X170T, X248E, X335V, X357R, X115L, X293I, X326C, X148V, X353S, X320A, X332A, X11K, X118F, X352R, X66V, X226Y, X274I, X360H, X189E, X24E, X75K, X313R, X242P, X276C, X191M, X143K, X138R, X81A, X305E, X230D 31 ++ ++++++
178 178 X326S, X360H, X340E, X7E, X314S, X353S, X335V, X11K, X221E, X226Y, X364S, X24E, X274I, X248E, X189E, X115I, X170T, X320A, X293G, X332A, X354T, X147D, X138T, X142K, X75K, X130K, X150R, X325C, X191M, X252K 30 ++ ++++++
179 179 X189E, X354T, X348L, X364S, X360H, X296M, X48A, X118N, X314E, X221E, X274I, X226N, X24E, X326S, X352R, X46N, X349K, X115I, X332A, X353S, X293I, X193E, X325C, X138T, X252K, X264E, X305E, X230D, X301Q, X143K, X142K 31 + ++++++
180 180 X7E, X320A, X346A, X115I, X348L, X170T, X340E, X248E, X353S, X354T, X350N, X46K, X335V, X24E, X189H, X293A, X66Q, X360H, X118D, X296M, X349K, X148V, X41C, X130T, X173E, X276C, X241T, X301Q, X230D, X81A, X310Q, X252K 32 ++ ++++++
181 181 X326C, X48A, X274I, X349K, X248E, X66I, X226N, X348L, X170T, X296M, X189H, X118H, X148V, X357R, X350N, X354T, X221E, X360H, X115E, X352R, X314S, X320A, X138R, X130T, X140E, X41C, X312I, X276C, X157K, X143K, X193E 31 ++ ++++++
182 182 X115L, X7E, X118N, X274I, X66V, X364S, X326C, X332A, X170T, X357R, X354T, X248E, X360H, X46F, X226Y, X320R, X24E, X148V, X189E, X314E, X48F, X296M, X313R, X142K, X299D, X193E, X305E, X143K, X267G, X173E, X310Q, X130K 32 ++ ++++++
183 183 X352R, X248E, X170T, X24E, X320A, X349K, X360H, X357R, X226Y, X296M, X46Q, X7E, X274I, X314E, X348L, X346A, X115D, X353S, X221S, X11K, X326C, X138R, X140T, X230D, X252K, X305E, X264E, X312I, X276C, X241T, X150R 31 ++ ++++++
184 184 X293T, X48F, X221E, X296M, X320A, X189E, X340L, X170T, X24E, X350N, X248E, X364S, X11K, X7E, X354T, X314E, X360H, X274I, X226N, X332A, X353S, X230D, X267D, X305E, X157K, X143K, X312I, X310Q, X191M, X242P, X193E 31 + ++++++
185 185 X293L, X352R, X320R, X118N, X346A, X332A, X46A, X354T, X314E, X364S, X226N, X274I, X326S, X7E, X349K, X115E, X148V, X350N, X189E, X170T, X335V, X11K, X276C, X143K, X241T, X301Q, X230D, X157K, X325C, X147R, X305E, X336C 32 + ++++++
186 186 X226N, X118S, X357R, X360H, X66H, X354T, X148V, X248E, X170T, X48K, X353S, X24E, X364S, X346A, X46K, X293G, X115I, X11K, X340L, X7E, X332A, X296M, X350N, X138T, X276C, X242P, X191M, X147D, X140T, X312I, X41C, X267D, X322K 33 + ++++++
187 187 X314E, X115K, X346A, X364S, X353S, X357R, X320R, X340E, X221S, X170T, X348L, X354T, X226Y, X24E, X274I, X148V, X248E, X46A, X48R, X66T, X350N, X360H, X142K, X336C, X81A, X138T, X264E, X173E, X301Q, X322K, X41C, X325C 32 + ++++++
188 188 X221S, X364S, X350N, X296M, X66T, X24E, X335V, X357R, X115N, X118D, X170T, X226N, X314E, X274I, X340L, X189E, X346A, X349K, X352R, X46K, X148V, X354T, X11K, X140T, X299D, X301Q, X313R, X325C, X75K, X138T, X193E, X143K 32 +++ ++++++
189 189 X357R, X66N, X352R, X189H, X350N, X221S, X348L, X46P, X226N, X48Y, X335V, X364S, X11K, X326S, X340E, X170T, X274I, X118D, X148V, X353S, X332A, X7E, X360H, X241T, X173E, X305E, X147D, X193E, X325C, X312I, X310Q, X276C, X154K 33 ++ ++++++
190 190 X226N, X314S, X11K, X332A, X221E, X353S, X326C, X66V, X364S, X148V, X346A, X189H, X48A, X320R, X349K, X115I, X24E, X46K, X7E, X274I, X350N, X340L, X170T, X242P, X276C, X230D, X157K, X81A, X322K, X325C, X301Q, X154K 32 + +++++++
191 191 X226Y, X357R, X346A, X326S, X353S, X66I, X364S, X7E, X24E, X352R, X320A, X11K, X148V, X354T, X296M, X46P, X348L, X350N, X115D, X314E, X360H, X274I, X170T, X142K, X305E, X75K, X41C, X301Q, X140E, X267G, X150R, X230D, X322K 33 + +++++++
192 192 X66A, X340E, X296M, X354T, X274I, X326S, X48M, X248E, X226Y, X348L, X46S, X148V, X360H, X170T, X346A, X352R, X364S, X320A, X11K, X115K, X7E, X314S, X357R, X193E, X325C, X150R, X138R, X191M, X230D, X305E, X140E, X130T, X242P 33 ++ +++++++
193 193 X360H, X332A, X248E, X357R, X340E, X353S, X314S, X352R, X293T, X221S, X170T, X335V, X11K, X349K, X226N, X320R, X148V, X118S, X115L, X350N, X24E, X354T, X66H, X138T, X322K, X299D, X191M, X130K, X310Q, X157K, X193E, X143K 32 ++ +++++++
194 194 X248E, X353S, X46A, X148V, X360H, X352R, X221S, X170T, X320A, X115N, X314S, X118F, X189H, X350N, X349K, X340E, X7E, X66H, X348L, X293I, X332A, X364S, X354T, X130T, X75K, X138R, X267D, X325C, X154K, X242P, X305E, X322K, X191M 33 ++ +++++++
195 195 X352R, X226N, X221E, X326S, X354T, X115L, X353S, X118N, X340L, X349K, X189H, X364S, X148V, X348L, X170T, X346A, X296M, X274I, X357R, X11K, X48N, X293A, X66T, X81A, X230D, X325C, X299D, X142K, X173E, X41C, X140E, X252K, X264E 33 ++ +++++++
196 196 X348L, X354T, X7E, X357R, X349K, X293I, X364S, X346A, X226N, X11K, X350N, X148V, X115K, X340L, X46P, X360H, X170T, X353S, X118F, X326S, X48F, X332A, X24E, X336C, X267G, X150R, X138R, X140E, X142K, X305E, X157K, X242P, X276C, X130K 34 + +++++++
197 197 X115D, X326S, X357R, X293I, X189H, X66H, X348L, X332A, X226N, X296M, X340E, X360H, X350N, X48A, X170T, X221E, X346A, X349K, X320A, X24E, X353S, X352R, X140T, X143K, X150R, X276C, X241T, X299D, X305E, X310Q, X173E, X41C, X267D 33 + +++++++
198 198 X320A, X346A, X348L, X360H, X314E, X48F, X332A, X349K, X353S, X24E, X115L, X350N, X7E, X148V, X170T, X293T, X189H, X226Y, X296M, X248E, X364S, X46Q, X312I, X191M, X193E, X150R, X276C, X301Q, X299D, X336C, X241T, X140T, X157K 33 + +++++++
199 199 X226N, X274I, X335V, X357R, X66I, X350N, X314E, X349K, X346A, X352R, X7E, X221E, X115D, X360H, X24E, X326S, X11K, X46Q, X364S, X348L, X353S, X248E, X293G, X305E, X299D, X230D, X140T, X310Q, X147R, X41C, X336C, X191M, X313R, X81A 34 +++ +++++++
200 200 X364S, X320A, X226N, X350N, X354T, X346A, X353S, X340L, X349K, X66S, X46N, X274I, X348L, X360H, X24E, X248E, X115L, X296M, X7E, X314E, X48N, X221S, X154K, X322K, X150R, X157K, X299D, X252K, X276C, X130K, X267D, X143K 32 ++ +++++++
201 201 X226V, X243E, X49S, X52K, X55S 5 +++ ++++
202 202 X352R, X349K, X46S, X189H, X221S, X354T, X66N, X340E, X148V, X115Q, X212R, X274I, X7E, X248E, X357R, X320A, X170T, X348L, X353S, X118D, X296M, X332A, X193E, X310Q, X142K, X264E, X150R, X276C, X325C, X305E, X313R, X299D 32 ++ ++++++
203 203 X320A, X346A, X348L, X360H, X314E, X48F, X332A, X349K, X353S, X24E, X115L, X350N, X7E, X148V, X170T, X293T, X189H, X226Y, X296M, X248E, X364S, X46Q, X312I, X191M, X193E, X150R, X276C, X301Q, X299D, X336C, X241T, X140T, X157K, X212R 34 + +++++++
204 204 X66A, X340E, X296M, X354T, X274I, X326S, X48M, X248E, X226Y, X348L, X46S, X148V, X360H, X170T, X346A, X352R, X364S, X320A, X11K, X115K, X7E, X314S, X357R, X193E, X325C, X150R, X138R, X191M, X230D, X305E, X140E, X130T, X242P, X212R 34 ++ +++++++
205 205 X314E, X115K, X346A, X364S, X353S, X357R, X320R, X340E, X221S, X170T, X348L, X354T, X226Y, X24E, X274I, X148V, X248E, X46A, X48R, X66T, X350N, X360H, X142K, X336C, X81A, X138T, X264E, X173E, X301Q, X322K, X41C, X325C, X212R 33 + ++++++
206 206 X293L, X352R, X320R, X118N, X346A, X332A, X46A, X354T, X314E, X364S, X226N, X274I, X326S, X7E, X349K, X115E, X148V, X350N, X189E, X170T, X335V, X11K, X276C, X143K, X241T, X301Q, X230D, X157K, X325C, X147R, X305E, X336C, X212R 33 + ++++++
207 207 X226N, X118S, X357R, X360H, X66H, X354T, X148V, X248E, X170T, X48K, X353S, X24E, X364S, X346A, X46K, X293G, X115I, X11K, X340L, X7E, X332A, X296M, X350N, X138T, X276C, X242P, X191M, X147D, X140T, X312I, X41C, X267D, X322K, X212R 34 + ++++++
208 208 X332A, X226N, X115N, X46S, X296M, X346A, X348L, X118S, X357R, X360H, X353S, X340L, X148V, X293L, X326C, X248E, X314E, X221S, X66Q, X352R, X241T, X267D, X276C, X81A, X150R, X140E, X130T, X157K, X242P, X301Q, X212R 31 + ++++++
209 209 X314E, X118S, X66A, X248E, X293L, X352R, X320A, X364S, X7E, X350N, X346A, X148V, X326S, X332A, X360H, X221S, X226Y, X170T, X354T, X115E, X296M, X305E, X242P, X191M, X241T, X142K, X147R, X322K, X143K, X75K, X267G, X212R 32 ++ ++++++
210 210 X349K, X314E, X7E, X296M, X148V, X353S, X354T, X24E, X332A, X170T, X46S, X350N, X326C, X11K, X293L, X348L, X248E, X340E, X48A, X66A, X301Q, X230D, X81A, X75K, X191M, X252K, X305E, X264E, X41C, X142K, X212R 31 ++ ++++++

[0241] Table 8: Contains the list of mutations generated during the LeuDH engineering process with the respective sequence IDs. The substitutions are mentioned in single letter amino acid code with the positions respective to the sequences. The total number of differences compared to SEQ_ID_NO: 1 was mentioned, along with the expression and conversion details of the variants library. The expression of the variants observed during the study mentioned with “+” indications where “+” indicates low expression, “++” indicates moderate expression and “+++” indicates good expression. The symbol “+” given in the conversion column indicates the % conversion of the substrate, specifically, “+” indicate the conversion of <10%, “++” indicates the conversion of 10 to 30%, “+++” indicates the conversion of >30% and <50%, “++++” indicates the conversion of >50% <70%, “+++++” indicates the conversion of >70 and <80%, “++++++” indicates the conversion of >80 and 90% and “+++++++” indicates conversion of >91% and up to 100%.
Sl.No Variant FDH concentration (%)
1 A226V 5
2 T49I-S52N 4
3 A55S 3.8
4 A243E-S52K 3.2
5 S52K 2
6 A226V, A243E, T49S, S52K, A55S 0.5

[0242] Table 9: Contains the list of mutations tested to improve the binding affinity and reduce the concentration of FDH enzymes. The mutations are in the surface of the artificially engineered leucine dehydrogenase polypeptide which provides strong interactions with amino acids present on the FDH. The single variants improved the affinity, but the additive effect of all the mutations reduced the required concentration to 0.5%.
Sl No SEQ ID NO Conversion
1 222 +
2 223 +
3 224 +
4 225 +
5 19 +++
6 21 +++
7 28 ++
8 59 ++
9 69 ++
10 109 ++
11 119 +++
12 137 ++++

[0243] Table 10: Specific activity of leucine dehydrogenases towards high substrate load (125g/L) of 2-oxobutanoic acid resulting in the formation of L-2-amino butyric acid though reductive amination. The symbol “+” represents the enzymatic activity indicating the % conversion of the substrate, Specifically “+” indicate the conversion of <10%, “++” indicates the conversion of 10 and 40%, “+++” indicates the conversion of >40% and <60%, “++++” indicates the conversion of >61% and <80%, and “+++++” indicates the conversion of >81% to 100%.

EXAMPLES
Example 1
[0244] Gene Cloning and Expression of engineered Leucine dehydrogenase
[0245] Engineered LeuDH genes were synthesized and constructed in between NcoI and XhoI restriction sites of pET28a(+) plasmid after codon optimization and further expressed in E. coli BL21 (DE3). The recombinant E. coli cells were cultured for about 3 h at 37 °C with 180 rpm and induced with IPTG (1mM) for another 8 h at 28 °C in LB medium supplemented with 50 µg/mL kanamycin. These recombinant cells were then centrifuged at 6,000g for 10 minutes at 4 °C and resuspended in PBS buffer (50 mM, pH 7.5). After that, the recombinant cells were disrupted on ice by ultrasonication and the cell disruption was centrifuged at 10,000g for 20 minutes at 4 °C to obtain the intracellular proteins.

Example 2
[0246] Generations of Mutations
[0247] All mutations were introduced by using the QuikChange® site-directed mutagenesis kit (Stratagene, Agilent Technologies Company, La Jolla, CA, USA) according to the manufacturer’s instructions. A pET-28a(+)-arFDH vector generated was used as template. DNA in the first rounds of mutagenic PCR. In this vector, the engineered LeuDH gene is cloned between NcoI and XhoI restriction sites. Mutations were combined by consecutive rounds of site-directed mutagenesis. All mutations were confirmed by sequencing.

Example 3
[0248] Enzyme Activity of artificially engineered Leucine Dehydrogenase for reductive amination of 3-hydroxyphenylpyruvic acid
[0249] To determine the enzymatic activity of artificially engineered Leucine Dehydrogenase, the concentration variation of NADH during the 3-hydroxyphenylpyruvic acid reduction was measured by monitoring the absorbance change at 340 nm (e=6220 M-1 cm-1). The reaction mixture (160 µL) contained 3-hydroxyphenylpyruvic acid (10mM), NH4OH/NH4Cl (900mM, pH 9.5), NADH (0.2 mM) and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells (2.5 mg/ml). The reaction was carried out at 32°C for 2 minutes and the absorbance change at 340nm was recorded every 30 seconds (Table 2).

Example 4
[0250] Enzyme Activity of artificially engineered Leucine Dehydrogenase for reductive amination of 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid
[0251] To determine the enzymatic activity of artificially engineered Leucine Dehydrogenase, the concentration variation of NADH during the 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid reduction was measured by monitoring the absorbance change at 340 nm (e=6220 M-1 cm-1). The reaction mixture (160 µL) contained 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid (10mM), NH4OH-NH4Cl (900mM, pH 9.5), NADH (0.2 mM) and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells (2.5 mg/ml). The reaction was carried out at 34°C for 2 minutes and the absorbance change at 340nm was recorded every 30 seconds (Table 3).

Example 5
[0252] Enzyme Activity of artificially engineered Leucine Dehydrogenase for reductive amination of 2-oxobutanoic acid
[0253] To determine the enzymatic activity of artificially engineered Leucine Dehydrogenase, the concentration variation of NADH during the 2-oxobutanoic acid reduction was measured by monitoring the absorbance change at 340 nm (e=6220 M-1 cm-1). The reaction mixture (160 µL) contained 2-oxobutanoic acid (10mM), NH4OH/NH4Cl (900mM, pH 9.5), NADH (0.2 mM) and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells (2.5 mg/ml). The reaction was carried out at 30°C for 2 minutes and the absorbance change at 340nm was recorded every 30 seconds (Table 4).

Example 6
[0254] Asymmetric synthesis of L-amino butyric acid forms the substrate 2-oxobutanoic acid through reduction amination using artificially engineered leucine dehydrogenase polypeptide using cofactor recycling system
[0255] The catalysis of 2-oxobutanoic acid through the reductive amination was conducted using artificially engineered leucine dehydrogenase. The reaction comprises of 0.05 M 2-oxobutanoic acid, 0.05NH4OH/NH4COOH (0.05 M) , 0.3mM NAD+, 0.09mg/ml of and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells and 2.7mg/ml of formate dehydrogenase from Candida boidinii in 0.1 M PBS buffer (pH 7.5) .The reaction was conducted at pH 7.5 at temperature of 30°C in a stir flask of 200rpm for 45 minutes. These samples were further boiled and 100µl of samples were sent through high performance liquid chromatography (HPLC) analysis of the product.

Example 7
[0256] Asymmetric synthesis of L-amino butyric acid from the substrate 2-oxobutanoic acid through reduction amination using artificially engineered leucine dehydrogenase polypeptide at reduced FDH concentrations.
[0257] The catalysis of 2-oxobutanoic acid through the reductive amination was conducted using artificially engineered leucine dehydrogenase. The reaction comprises of 0.05 M 2-oxobutanoic acid, NH4OH/NH4COOH (0.5 M), 3mM NAD+, 0.9 mg/ml of and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells and 4.5 µg/ml of formate dehydrogenase from Candida boidinii in 0.1 M PBS buffer (pH 7.5). The reaction was conducted at pH 7.5 at temperature of 30°C in a stir flask of 200rpm for 45 minutes. These samples were further boiled and 100µl of samples were sent through high performance liquid chromatography (HPLC) analysis of the product.

Example 8
[0258] Enzyme Activity of artificially engineered Leucine Dehydrogenase for reductive amination of 2-oxobutanoic acid
[0259] To determine the enzymatic activity of artificially engineered Leucine Dehydrogenase, an assay reaction system of 1mL contained 10µg/ml of and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells, 0.89M of NH4OH-NH4Cl buffer, 10mM 2-oxobutanoic acid and 0.2mM NADH. The reaction was initiated by the addition of enzyme and carried out at 50ºC for 30 mins. One unit of artificially engineered Leucine Dehydrogenase activity (U) was defined as the amount of enzyme required to catalyze the consumption of 1µmol NADH per min under the above assay condition.

Example 9
[0260] Asymmetric synthesis of L-amino butyric acid from the substrate 2-oxobutanoic acid through reduction amination using artificially engineered leucine dehydrogenase polypeptide at reduced FDH concentrations.
[0261] The catalysis of 2-oxobutanoic acid through the reductive amination was conducted using artificially engineered leucine dehydrogenase. The reaction mixture of 4L comprises of 500 g of 2-oxobutanoic acid was treated with 3.8 g of whole cells, or a cell extract or crude lysate of such cells, 10 g of Formate Dehydrogenase from Candida boidini (FDH), and 700 mg of NAD with 2M ammonium formate buffer of pH 7.5 and temperature of 30ºC for 3 hours (Table 10).

Example 10
[0262] Thermal and pH inactivation study of artificially engineered Leucine Dehydrogenase for reductive amination
[0263] The effect of temperature on the enzymatic reaction of artificially engineered Leucine Dehydrogenase was analysed by assaying for the reductive amination activity at temperatures ranging from 20 to 65°C. The reaction mixture was pre-incubated at the desired temperature for 4 min and the reaction was started by adding keto acid followed by incubating for 4 min.
[0264] The effect of pH on the artificially engineered leucine dehydrogenase was evaluated by incubation of enzyme at 37 ºC for twelve hours in the following buffers: 50 mM CH3COONa/CH3COOH buffer (pH 5.0-5.5), 50 mM Phosphate buffer (PBS buffer, pH 6.0-8.0), 50 mM NH4Cl/NH4OH (pH 8.0-10.0), 50 mM Sodium carbonate/Sodium bicarbonate buffer (pH 10.5). Further to the incubation, the artificially engineered leucine dehydrogenase polypeptides activities were measured for reductive amination in NH4Cl/NH4OH (900mM, pH 9.5) at 30 ºC.

Example 11
[0265] Biosynthesis of a-amino acid from a-keto acid using whole cell
[0266] The biocatalytic reaction was carried out at 10ml scale in 50ml flask employing freeze-dried whole cells as biocatalyst. The reaction mixture contains 500mM NH4Cl/NH3.H2O buffer (pH 9.5), 100mM a-amino acid, 200mM sodium formate, 0.1g freeze-dried whole cells. The reaction mixture was incubated at 30°C and 200 rpm for 35h, and 100µl samples were periodically removed for high-performance liquid chromatography (HPLC) analysis.

Example 12
[0267] Site-directed mutagenesis of Leucine Dehydrogenase
[0268] Site-directed mutagenesis of leucine dehydrogenase gene was conducted using whole plasmid two-step PCR with the primer containing required codons for mutations (sense strand) and antisense strand based on the expression vector pET-28a(+). The primers used for site-directed mutagenesis were synthesized from Agilent and mutated using Quick Change mutagenesis kits. Luria-Bertani (LB) broth containing ampicillin (100 µg/mL), or kanamycin (50 µg/mL) was used for recombinant bacteria growth.

Example 13
[0269] Enzyme Activity of artificially engineered Leucine Dehydrogenase for oxidative deamination of L-tryptophan
[0270] Enzyme activity assays were performed using a spectrophotometer to measure the change in NADH absorbance at 340 nm. The substrate mixture (1.5 ml) for the enzymatic activity analysis consisted of 5 mM of substrate L-tryptophan and 0.2 mM NAD+ and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells (2.5 mg/ml) in the presence of a 100 mM glycine–NaOH buffer at pH 9.5. One unit of enzymatic activity was defined as the quantity of enzyme necessary to catalyse the reduction of 1 µM NAD+ per minute at a temperature of 30°C. For the regeneration of the cofactor from NADH to NAD+ crude or purified NOX enzyme (2.5mg/ml) solution was used (Table 7).

Example 14
[0271] Enzyme Activity of artificially engineered Leucine Dehydrogenase for oxidative deamination of L-leucine using glycine-NaOH buffer
[0272] Enzyme activity assays were performed using a spectrophotometer to measure the change in NADH absorbance at 340 nm. The substrate mixture (1.5 mL) for the enzymatic activity analysis consisted of 5 mM of substrate L-leucine and 0.2 mM NAD+ and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells (2.5 mg/ml) in the presence of a 100 mM glycine–NaOH buffer at pH 9.5. One unit of enzymatic activity was defined as the quantity of enzyme necessary to catalyse the reduction of 1 µM NAD+ per minute at a temperature of 30°C (Table 5).

Example 15
[0273] Enzyme Activity of artificially engineered Leucine Dehydrogenase for oxidative deamination of L-leucine using glycerol/KCl/KOH buffer
[0274] Enzyme activity assays were performed using a spectrophotometer to measure the change in NADH absorbance at 340 nm. The enzyme activity of artificial engineered LeuDH for the oxidative deamination of L-Leucine was performed by using an assay reaction system of 200µl which consisted of 10 mM L-Leucine, 1mM NAD+, 0.1 M glycerol/KCl/KOH buffer at pH 10 and 10 µl of artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells. This reaction mixture was incubated at 30°C for two hours. Following this the activity of the enzyme was determined by the amount of enzyme required to catalyze the oxidation of L-Leucine under the above assay conditions.

Example 16
[0275] Enzyme Activity of artificially engineered Leucine Dehydrogenase for oxidative deamination of L-leucine using carbonate buffer
[0276] Enzyme activity assays were performed using a spectrophotometer to measure the change in NADH absorbance at 340 nm. The reaction assay mixture of 1.95ml consisted of 10mM L-Leucine, carbonate buffer of 100mM (pH 10.5) and NAD+ at the concentration of 3mM. To this reaction mixture 50 µl of artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells was added and the reaction was carried out at 60°C for 2hr. One unit of enzyme activity was determined as the amount of enzyme required to catalyze the formation of 1µmol NADH per minute under assay conditions.

Example 17
[0277] Enzyme Activity of artificially engineered Leucine Dehydrogenase for oxidative deamination of L-leucine using cofactor recycling system.
[0278] The substrate mixture (1.5 mL) for the enzymatic activity analysis consisted of 5 mM of substrate L-leucine and 0.2 mM NAD+ and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells (2.5 mg/ml) in the presence of a 100 mM glycine–NaOH buffer at pH 9.5 at 30°C. The reaction was incubated for two hours. For the regeneration of the cofactor from NADH to NAD+ crude or purified NOX enzyme (2.5mg/L) solution was used.

Example 18
[0279] Enzyme Activity of artificially engineered Leucine Dehydrogenase for oxidative deamination of L-Nor-leucine
[0280] Enzyme activity assays were performed using a spectrophotometer to measure the change in NADH absorbance at 340 nm. The substrate mixture (1.5 mL) for the enzymatic activity analysis consisted of 5 mM of substrate L-Nor-leucine and 0.2 mM NAD+ and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells (2.5 mg/ml) in the presence of a 100 mM glycine–NaOH buffer at pH 9.5. One unit of enzymatic activity was defined as the quantity of enzyme necessary to catalyse the reduction of 1 µM NAD+ per minute at a temperature of 27°C (Table 6).

Example 19
[0281] Thermal and pH inactivation study of artificially engineered Leucine Dehydrogenase for oxidative deamination
[0282] The effect of temperature on the enzymatic reaction of artificially engineered Leucine Dehydrogenase was analysed by assaying for the oxidative deamination activity at temperatures ranging from 20 to 65°C. The reaction mixture was pre-incubated at the desired temperature for 4 min and the reaction was started by adding L-amino acid followed by incubating for 4 min. The effect of pH on the artificially engineered leucine dehydrogenase was evaluated by incubation of enzyme at 37 ºC for twelve hours in the following buffers: 50 mM CH3COONa/CH3COOH buffer (pH 5.0-5.5), 50 mM Phosphate buffer (PBS buffer, pH 6.0-8.0), 50 mM NH4Cl/NH4OH (pH 8.0-10.0), 50 mM Sodium carbonate/Sodium bicarbonate buffer (pH 10.5). Further to the incubation, the artificially engineered leucine dehydrogenase polypeptide’s residual activities were measured for oxidative deamination in glycine–NaOH buffer (100mM, pH 9.5) ,5 mM of L-Leucine, 0.2 mM NAD+ and artificially engineered Leucine Dehydrogenase in whole cells, or a cell extract or crude lysate of such cells (2.5 mg/ml), purified NOX enzyme(2.5mg/ml) solution at 30 ºC.

Example 20
[0283] Cell expression protocol for the artificially engineered Leucine Dehydrogenase
[0284] The transformed cell colonies form the master plate were chosen and inoculated in 5ml LB+ kanamycin (50µg/ml) media and were grown overnight at 37°C. The inoculated culture was then seeded into the 100ml LB media with 50µg/ml of kanamycin. Once the culture reached an OD600 = 0.6, the protein induction was conducted using 0.1 mM isopropyl ß-d-1-thiogalactopyranoside (IPTG) and cells were further grown at 25 °C for 16 hours. From this medium the cells were harvested by pelleting them at 4700RPM for 10 minutes. The harvested cells were resuspended in 1ml PBS (pH 7.5) containing 1mg/ml lysozyme and were incubated on ice for 1 hour. Then the overall volume was made up to 5ml using PBS and the cell lysis was performed using sonication and crude lysates were analysed through SDS-PAGE.

Example 21
[0285] Biocatalysis of a-keto acid from a-amino acid using whole cell
[0286] The biocatalytic reaction was carried out at 10ml scale in 50ml flask employing freeze-dried whole cells as biocatalyst. The reaction mixture contains 100 mM glycine–NaOH buffer at pH 9.5, 5 mM of substrate (L-amino acid) and 0.2 mM NAD+, 0.1g freeze-dried whole cells. The reaction mixture was incubated at 30°C and 200 rpm for 35h, and 100µl samples were periodically removed for high-performance liquid chromatography (HPLC) analysis.

Example 22
[0287] Fed-batch synthesis (L)-2-aminobutyric acid
[0288] Synthesis of (L)-2-aminobutyric acid in a 5L fermenter was carried out as an example for upscale production. A 2L biotransformation was performed at 30°C, 200 rpm using crude enzymes containing about 2 g of artificially engineered Leucine Dehydrogenase and about 10 mg of formate dehydrogenase. The substrate was added by fed batch. 0.6 mmol NAD+ was fed into the solution at first and then at 5h. The pH was controlled at pH 7.5 with 15% NH4OH or 20% (v/v) formic acid. Samples were taken during the biotransformation and then boiled for the further detection. The determination of substrates and products was performed on the Agilent HPLC system. All the assays in this study were carried out in triplicates.

Example 23
[0289] HPLC Analysis Method for 2-ketobutyric acid and (L)-2-aminobutyric acid
[0290] The concentration of 2-ketobutyric acid and (L)-2-aminobutyric acid were assayed using a 1260 Infinity II liquid chromatography platform (Agilent Technologies, California USA) equipped with a COSMOSIL PBr column (4.6 mm ID x 250 mm) at 210 nm with 5mM ammonium dihydrogen phosphate at a flow rate of 0.8 ml/min. The enantioselectivity of (L)-2-aminobutyric acid was assayed by GITC precolumn derivatization equipped with an Agilent ZORBAX SB-Aq column (4.6 mm x 250 mm) at 254 nm using water (0.1% phosphate)/methanol (40:60, v/v) as the eluent at a flow rate of 1mL/min.

Example 24
[0291] HPLC Analysis Method for 3-hydroxyphenylpyruvic acid and (2S)-2-amino-3-(3-hydroxyphenyl) propanoic acid,
[0292] The concentration of (2S)- 2-amino-3-(3-hydroxyphenyl) propanoic acid, were assayed using a Agilent 1100 HPLC system (Agilent, Palo Alto, CA) equipped with a diode-array detector and a Supelco (Bellefonte, PA) RP-18 Discovery column (length, 250 mm; diameter, 10 mm), which was eluted with methanol–water mixtures (starting with a mixture of 3% methanol, 67% water, and 30% of 0.05% aqueous trifluoroacetic acid for an initial period of 3 min, followed by a linear gradient reaching 50% methanol, 20% water, and 30% of 0.05% aqueous trifluoroacetic acid at 30 min, at a constant flow of 3.4 ml/min. The absorption was monitored at 280 nm.

Example 25
[0293] HPLC Analysis Method for 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid and (2S)-2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid
[0294] The concentration of (2S) -2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid were assayed using Shimadzu HPLC equipment comprising quaternary LC-10A VP pumps, a variable wavelength programmable UV–visible detector, SPD-10AVP column oven, and a SCL 10AVP system controller. Samples with the volume of 20 µL were injected by means of a Rheodyne injector fitted with a 20-µL loop. The instrumentation was controlled by use of Class-VP 5.032 software. Compounds were separated on a 250 × 4.6 mm, 5-µm particle, C18 reversed-phase column (Phenomenex). The mobile phase was water–orthophosphoric acid (98.8:0.2, v/v) filtered through a 0.22-µm membrane filter (Millipore) and sonicated before use; pH 3.0 was adjusted with orthophosphoric acid, (2S) -2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic was eluted isocratically with a flow rate of 1.0 mL min-1. The eluate was monitored by UV detector at wavelength of 284 nm.

Example 26
[0295] HPLC Analysis method for L-leucine and a-keto isocaproic acid
[0296] The product was analyzed using the HPLC method. The product was analyzed through its derivatization with Diamino-4,5-methylenedioxybenzene (DMB) where in 40µL of DMB was added to 40 µL of the product in the sealed tube. The solution is then heated to 85°C for 45 minutes and then cooled on ice for 5 minutes. Further, the solution is diluted fivefold with 65 mM NaOH aqueous solution and 25 µL was injected into HPLC. The HPLC system from Jasco was composed of a PU-980 pump, an LG-1580-02 ternary gradient unit, a DG-980-50 3-line degasser, AS-2057 PLUS autosampler, CO-1560 column oven, and FP1520S fluorescence detector. The analysis of 4-methyl-2-oxopentanoic acid was conducted on Inertsil ODS-4V column (250 × 3.0 mm, 5.0 µm) (GL Sciences, Tokyo, Japan). Fluorescence detection was performed at excitation and emission wavelengths of 367 nm and 446 nm, respectively. The Mobile phases were MeOH/H2O (30/70, v/v) and MeOH. A flow rate was 0.3 mL/min and the column temperature was maintained at 40°C.

Example 27
[0297] HPLC Analysis method for L-nor-leucine and 2-oxohexanoic acid
[0298] The product 2-oxo hexanoic acid were analysed on Agilent HPLC system 1100 series (Agilent Technology Inc, USA) equipped with a model G1311A LC pump, model G1315B diode array detector (DAD) and model 7725 Rheodyne injector. Reversed-phase LC analysis was performed isocratically at room temperature using a Zorbax 300 SB-C18 (4.6mm x 150 mm) column (Agilent Technology Inc., USA). The product sample of 1ml was added into an SDA solution of 0.5mL which contains 2% w/v in methanol, acetic acid-sodium acetate buffer of pH 3.2. The contents were heated at 95-100°C for 30 minutes and volume was adjusted with methanol to 10mL. A mobile phase consisting of a mixture of methanol-water-acetonitrile-tetrahydrofuran (38.4:60:1:0.6, v/v/v/v) was used. The injection volume was 10 mL, and the detection wavelength was set at 255 nm. The flow rate was set to 1 mL/min.

Example 28
[0299] HPLC Analysis method for L-Tryptophan and Indole-3-pyruvic acid
[0300] The product Indole-3-pyruvic acid were analysed by injecting 50?µL into a reverse-phase Zorbax Eclipse XDB-C8 (column (4.6 × 150 mm, 5?µm). Columns temperature was controlled at 30°C. Mobile phase was composed of methanol and 1% acetic acid (60:?40 v /v) in isocratic mode at a flow rate of 1?mL min-1. The detection was monitored at 360?nm.

ADVANTAGES
[0301] The artificially engineered Leucine Dehydrogenase exhibits improved affinity for bulkier substrate score, being capable of accessing bulkier keto acids and aliphatic amino acids, making access to a much wider variety of compounds possible for synthesis. Engineered active site mutations enhance its reductive amination and oxidative deamination efficacy. Coupling with FDH into the reaction mixture also enhances the recycling of NAD+ efficiently, hence ensuring the activity of artificially engineered Leucine Dehydrogenase. Strategic surface mutations of artificially engineered Leucine Dehydrogenase increase its affinity for FDH, increasing cofactor transfer and increasing reaction velocities by minimizing the time required to reach maximum conversion and yield. This increase makes the process more efficient by lowering required FDH concentration. Overall, this work is a milestone in enzyme engineering with prospects for effective biocatalysis by offering actionable methods to increase the efficiency and usability of artificially engineered Leucine Dehydrogenase. By incorporating a cost-efficient cofactor recycling system, this approach significantly lowers the expenses of NAD+/NADH regeneration, making artificially engineered Leucine Dehydrogenase economically more feasible for application on an industrial scale.

OTHER PUBLICATIONS
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[0325] Martin PM, et.al (2006) Martin PM, Gopal E, Ananth S, Zhuang L, Itagaki S, Prasad BM, Smith SB, Prasad PD, Ganapathy V: Identity of SMCT1 (SLC5A8) as a neuron-specific Na+-coupled transporter for active uptake of L-lactate and ketone bodies in the brain. J Neurochem. 2006 Jul;98(1):279-88. https://doi.org/10.1111/j.1471-4159.2006.03878.x
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[0327] Kumar, P., Sigamani, G. & Roopa. L., (2023). METHOD FOR ENGINEERING PROTEINS (Indian Patent Application No. 202341064088). Indian Patent Office ,CLAIMS:CLAIMS
We Claim:
1. A recombinant engineered leucine dehydrogenase polypeptide, comprising an amino acids sequence that is at least 90% identical to SEQ ID NO :1 and that includes the feature of residues corresponding to X69 as ARG, X159 as CYS, X212 as LYS or ARG, X272 as CYS, X288 as CYS, and X334 as CYS

2. The recombinant engineered leucine dehydrogenase polypeptide of claim 1, which comprises an amino acids sequence having one or more of the following features:
X7 is GLU, ASP, THR or SER;
X11 is LYS, HIS, ARG, VAL, ILE or ALA;
X42 is LEU, ILE or VAL;
X43 is GLY, ALA or VAL;
X44 is GLY, ALA or VAL;
X45 is THR, ALA, MET, LEU or CYS;
X49 is SER or ILE;
X51 is ASP, ALA, ASN, PRO, GLY or THR;
X52 is LYS or ASN;
X64 is ALA, SER, GLY or THR;
X80 is GLY, ALA or VAL;
X81 is ALA, CYS, GLY or VAL;
X90 is ARG;
X116 is VAL;
X119 is ALA, SER, THR, GLU or ASN;
X122 is GLU, ILE, VAL, ALA, LYS or ASN;
X123 is ASP;
X137 is GLY, VAL or ALA;
X138 is ARG, THR, ILE or SER;
X140 is GLU, THR, ASP, ASN or GLN;
X141 is SER;
X148 is VAL, HIS, PRO, ASN or ILE;
X172 is ASN, SER, ASP, ALA or THR;
X174 is GLU, ALA, GLN, SER, LYS, THR or ASP;
X177 is GLU;
X190 is LEU, MET, VAL or ALA;
X191 is MET, CYS, LEU or ILE;
X192 is LYS;
X194 is LEU, ALA or ILE;
X195 is HIS, ASN, TRP, ARG, GLN or SER;
X198 is GLY, ARG, SER or LYS;
X199 is ALA, VAL, GLY or THR;
X200 is LYS, GLN, ASN, SER, ARG or HIS;
X221 is SER, GLU, LYS, ARG, THR or GLN;
X224 is GLU, ASP, ASN, GLY or ALA;
X226 is ASN, VAL, TYR, ASP, GLU, SER or GLY;
X230 is ASP, GLY, SER or ALA;
X241 is THR, LEU or MET;
X242 is PRO, GLY or VAL;
X244 is THR;
X248 is GLU, PRO, GLY or ALA;
X261 is SER;
X267 is ASP, GLY, LYS, ARG, GLU or ASN;
X269 is ASP;
X274 is ILE, GLN, ASN, LEU or THR;
X276 is CYS, MET, HIS or ASP;
X290 is ALA, SER or THR;
X293 is THR, ALA, ILE, LEU , GLY or SER;
X294 is ILE, SER, MET or CYS;
X295 is ASN, GLN, ASP or GLU;
X296 is MET, CYS, PHE or LYS;
X309 is LEU;
X310 is GLN, LYS, ARG, ALA or ASN;
X311 is ARG;
X312 is ILE, VAL or LEU;
X314 is SER, GLU, THR or GLN;
X318 is THR;
X320 is ARG, ALA, SER, THR or GLU;
X325 is CYS, ILE, LEU or PRO;
X326 is SER, CYS, ALA or GLY;
X336 is CYS, ALA, SER or GLY;
X340 is LEU, GLU, MET or VAL;
X348 is LEU, MET, ILE, VAL or ASN;
X350 is ASN, ARG, LYS, GLN, HIS or MET;
X352 is ARG, LYS, THR or GLN;
X363 is ILE, VAL, SER, CYS or ALA
X364 is SER, ASN, GLY, THR or HIS;
Wherein, optionally the amino acids sequence has one or more residue differences at other amino acid residues as compared to the 1

3. The recombinant engineered leucine dehydrogenase polypeptide of claim 2, wherein the amino acids sequence includes at least one or more of the following features:
X24 is GLU, GLN, ASN, THR, SER, ARG or LEU;
X41 is CYS, ALA, VAL, GLY or ILE;
X46 is ALA, ASN, LYS, SER, PRO, GLN or PHE;
X48 is ALA, ARG, ASN, LYS, MET, TYR, ILE, VAL or PHE;
X55 is SER, THR, ASP or GLU;
X63 is LEU, ILE or VAL;
X66 is GLN, HIS, PHE, THR, ASN, SER, VAL, ILE or ALA;
X67 is ALA, GLY, VAL, HIS, SER, THR, ASN, CYS, LEU or ILE;
X71 is ALA, SER, ASN, THR or GLN;
X75 is LYS, VAL, ALA, LEU or ILE;
X83 is THR, ALA or VAL;
X94 is SER, ASN, THR, ALA or GLY;
X101 is LEU, PHE, ILE or VAL;
X115 is ASN, GLU, LYS, ASP, GLN, ILE or LEU;
X118 is SER, ALA, PHE, ASP, ASN, CYS, HIS or LEU;
X121 is ASN, ALA, SER, THR, GLU, ALA, GLY or VAL;
X125 is MET, CYS, VAL or ALA;
X130 is LYS, THR, GLN, SER, ASP or GLU;
X142 is LYS, HIS, SER, THR or VAL;
X143 is LYS, HIS, ARG, VAL or GLY;
X146 is GLY, VAL or ALA;
X147 is ARG, ASP, ASN or GLU;
X150 is ARG, PRO, HIS or ILE;
X154 is LYS, TYR, PHE or HIS;
X157 is LYS, TYR, PHE, LEU or CYS;
X166 is GLU, ASP, GLN, VAL, HIS, SER or ASN;
X170 is THR, SER, ASP, ASN, HIS, VAL or ALA;
X173 is GLU, ASP, LEU, VAL or ALA;
X185 is SER, LYS, HIS, VAL, LEU, ILE, ALA, GLY or ARG;
X189 is HIS, GLU, ASN, SER, ALA or LYS;
X193 is GLU, HIS, LEU, PHE, TYR or ASP;
X196 is ALA, VAL, GLY or GLU;
X197 is GLU, ASP or ALA;
X201 is LEU, ILE, VAL or ALA;
X213 is ARG, LYS, MET, SER or LEU;
X218 is PHE, TYR, PRO or ALA;
X227 is GLU, ASP, ALA, GLN or ASN;
X243 is GLU, ASP, GLN or ASN;
X245 is VAL, GLN, LEU, LYS or ARG;
X264 is GLU, ASN, GLN, ASP or GLU;
X285 is ASP or GLU;
X286 is TYR, PHE or TRP;
X299 is ASP, GLU, ASN or GLN;
X301 is GLN, GLU, ASP or ASN;
X305 is GLU, ASP, GLN, VAL or ILE;
X313 is ARG, GLU, ASP or ASN;
X317 is ASP;
X322 is LYS, VAL, ILE or THR;
X324 is GLU, ASP, ALA, SER or ASN;
X332 is ALA, CYS, PRO, SER or VAL;
X335 is VAL, LYS, LEU, ALA or ARG;
X346 is ALA, GLY, GLU, VAL or ILE;
X349 is LYS, ARG, ALA or SER;
X353 is SER, THR, GLY, LYS, ASN or ARG;
X354 is THR, GLN, LYS, MET or ASN;
X357 is ARG, GLY, SER, THR, LYS or ALA;
X360 is HIS, GLN, LYS, ARG or MET;
X361 is ASP, HIS, SER or ASN;
Wherein optionally the amino acids sequence has one or more residue differences at other amino acid residues as compared to the 1

4. The recombinant engineered leucine dehydrogenase polypeptide of claims 1 to 3, wherein the amino acid sequence corresponds to the SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 ,205, 206, 207, 208, 209 and 210

5. The recombinant engineered dehydrogenase polypeptide of claims 1 to 4, wherein the recombinant engineered leucine dehydrogenase polypeptide converts:
a) 3-hydroxyphenylpyruvic acid to (2S)-2-amino-3-(3-hydroxyphenyl) propanoic acid exhibiting >95% conversion and >99% enantiomeric excess, wherein the recombinant engineered artificial leucine dehydrogenase polypeptide has one or more of the following features: X46 is ALA or GLN or PHE, X48 is VAL or PHE, X66 is ILE or ALA, X115 is LYS or ASP, X118 is HIS or LEU, or X293 is GLY or ILE
b) 3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl)-2-oxopropanoic acid to (2S)-2-amino-3-(3-hydroxy-4-oxo-1,4-dihydropyridin-1-yl) propanoic acid, exhibiting >95% conversion and >99% enantiomeric excess, wherein the recombinant engineered leucine dehydrogenase polypeptide has one or more of the following features: X46 is PRO or ALA or GLN, X48 is TYR or ILE, X66 is SER or VAL, X115 is ILE or LEU, X118 is ASN or CYS, or X293 is LEU
c) 2-oxobutanoic acid to (L)-2-aminobutyric acid conversion exhibiting >99.0% conversion and >99% percent enantiomeric excess, within 3 hours of reaction time wherein the recombinant engineered artificial leucine dehydrogenase polypeptide comprises one or more of the following features: X46 is LYS or SER, X48 is LYS, X115 is ASN or HIS, X118 is ASP, X293 is LEU or THR or ILE, or X296 is ILE or PHE
d) L-tryptophan to Indole-3-pyruvic acid, exhibiting >95% conversion and >99% substrate enantioselectivity, wherein the recombinant engineered artificial leucine dehydrogenase polypeptide has one or more of the following features: X46 is ALA or ASN, X48 is ALA or ASN, X66 is HIS, X115 is LYS or ASP, or X293 is ALA
e) L-nor-leucine to 2-oxohexanoic acid, exhibiting >95% conversion and >99% substrate enantioselectivity, wherein the recombinant engineered artificial leucine dehydrogenase polypeptide has one or more of the following features: X115 is PHE or LEU, X118 is HIS or ALA, X148 is ALA or GLY, X293 is SER or LEU, or X296 is ASN
f) L-Leucine to 4-methyl-2-oxopropanoic acid, exhibiting >95% conversion and >99% substrate enantioselectivity, wherein the recombinant engineered artificial leucine dehydrogenase polypeptide has one or more of the following features: X115 is GLN or ILE, X118 is PHE, X148 is VAL, X293 is ILE, or X296 is MET

6. The recombinant engineered leucine dehydrogenase polypeptides of claims 1 to 5 exhibit at least 5-fold improvement compared to the 222, 223, 224, and 225 in cofactor recycling efficiency, wherein recombinant engineered leucine dehydrogenase polypeptide has one or more of the following features: X49 is SER or ILE, X52 is LYS or ASN, X55 is SER, X226 is VAL, or X243 is GLU

7. The recombinant engineered leucine dehydrogenase polypeptides of claims 1 to 6, wherein the polypeptides are used for reductive amination under reaction conditions comprising a pH range of 7.0 to 11.0, a temperature range of 25°C to 65°C, NADH concentration of 0.1 mM to 3 mM and a buffer selected from the group consisting of NH4OH/NH4COOH buffer (50 mM to 2 M) or NH4Cl/NH4OH buffer (500 mM to 2 M)
8. The recombinant engineered leucine dehydrogenase polypeptides of claims 1 to 5, wherein the polypeptides are used for oxidative deamination under reaction conditions comprising a pH range of 7.0 to 11.0, a temperature range of 25°C to 65°C, a buffer selected from the group consisting of glycine-NaOH buffer (100 mM to 200 mM), carbonate buffer (100 mM), or glycerol/KCl/KOH buffer (100 mM), and an NAD? concentration ranging from 0.1 mM to 3 mM

9. The recombinant engineered leucine dehydrogenase polypeptides of claims 1 to 6 used in the reaction condition of claims 7 and 8 wherein at least 60% to 120% increase in substrate to product conversion rate is observed compared to 222, 223, 224, and 225 wherein 500 grams of 2-ketobutyric acid is treated with 3.8 grams of recombinant engineered leucine dehydrogenase polypeptide, 10 grams of formate dehydrogenase and 700 milligrams of NAD in 4 L of 2M ammonium formate buffer for 3 hours

10. The recombinant engineered leucine dehydrogenase polypeptide of claim 1 is used in the method of any of the claims 7 to 9, wherein the recombinant engineered leucine dehydrogenase polynucleotide of 211 is expressed in an E. coli BL21 host using the pET28a(+) expression vector under the control of a T7 promoter, in 20 mM phosphate buffer at pH 7.5, with 0.1 mM IPTG for induction at a temperature of 25°C, and wherein the recombinant engineered leucine dehydrogenase polypeptide is employed in the form of whole cells, or a cell extract or crude lysate of such cells