DESC:FIELD OF THE INVENTION:
The present invention relates to modified nitrilases for biocatalysis, method of preparation of the said nitrilases and uses thereof; and more specifically to modified nitrilases in various forms for biocatalysis, a method of preparation of the said nitrilases having enhanced catalytic properties and functionality for biocatalytic applications. The present invention further relates to creating a platform technology for conversion of nitrile substrates to carboxylic acids.

BACKGROUND OF THE INVENTION:
Nitrile compounds, characterized by the presence of cyano groups, constitute a category of organic chemical raw materials extensively utilized in the chemical, pharmaceutical, pesticide, and material industries. Nitrilases catalyze the hydrolysis of nitriles (organic compounds containing a -C=N group) into the corresponding carboxylic acids (R-COOH) and ammonia (NH3). This hydrolysis reaction is significant because it often occurs under mild conditions, which makes nitrilases attractive for synthetic processes in industry.
Nitrilases selectively hydrolyse one enantiomer of a racemic nitrile, converting it into the corresponding carboxylic acid. This enantioselectivity makes nitrilases valuable in the production of optically pure carboxylic acids, which are used as intermediates in the synthesis of various bioactive compounds, including pharmaceuticals and food additives. Their classification into aromatic nitrilases, aliphatic nitrilases, and arylaceto nitrilases reflects the broad diversity of nitrile substrates they can hydrolyse, each with distinct industrial applications.
The high chemical specificity and enantioselectivity of nitrilase enzymes make them particularly valuable in industries requiring precise chemical transformations for the production of fine chemicals, pharmaceutical intermediates, and bioorganic compounds. The ability of some nitrilases to catalyse nitrile hydrolysis adds another layer of versatility, expanding their potential in producing both amides and carboxylic acids. To fully leverage their potential, it is necessary to enhance the hydrolysis activity and reaction specificity of nitrilases through enzyme engineering; optimize the reaction conditions and utilize the technique of immobilization for scaling up their industrial use. By carefully regulating their specificity, nitrilases can be tailored for a wide range of applications, helping to meet the growing demand for sustainable, efficient, and selective biocatalytic processes.
Numerous studies have investigated nitrilase enzymes exhibiting significant potential for industrial synthesis of various valuable products and intermediates. There are reports in the prior art indicating the isolation of nitrilase enzymes and cloning the responsible genes and heterologous expression in a variety of bacteria and fungi. The nitrilase enzymes have been isolated and in some cases the genes responsible have been cloned and heterologously expressed from variety of bacteria such as Acidovorax faecalis 72W (Gagavan J. E. et al., 1999, Appl. Microbiol. Biotechnol., 52, 654-659), Alcaligens faecalis JM3 (Nagasawa T. et al., 1990, Eur. J. Biochem., 194,765-772), Comamonas testosterone (Levy-Schill, S. et al., 1995, Gene, 16, 15-20), Klebsiella pneumoniae ssp. ozonae (Stalker D.M. and McBride K. E., J. Bacteriol., 169, 955-960), Rhodococcus rhodochrous J1 (Kobayashi M. et al., Eur. J. Biochem., 182, 349-356), Pseudomonas fluorescence EBC191 (Layh N. A. et al, 1992, Arch. Microbiol., 158, 405-11) and fungi such as Fusarium oxysporum f. sp. melonis (Goldlust A. and Bohak Z., 1989, Biotechnol. and Appl. Biochem., 11, 581-601).
Production of industrially important chiral carboxylic acids by the biocatalytic processes involving the nitrilases has been reported from a variety of sources such as Alcaligenes faecalis (M. Ress-Löshke et al., US6869783 B1) and Acidovorax sps. (Payne M. S. et al., US7358071). Moreover, there are reports in the prior art indicating isolation of DNA from soil and acquisition of a gene encoding an enzyme with nitrilase activity.
The US Patent Application No. US6869783 B1 to Ress-Loeschke Marion et al. describes a process for producing chiral carboxylic acids from nitriles using nitrilase or Alcaligenes faecalis that contain a gene for the nitrilase. The US Patent Application No. US7358071 to Payne Mark S and others describes a process for hydrolysing a 3-hydroxynitrile to a 3-hydroxycarboxylic acid using nitrilase mutants. Further, the US Patent Application No. US2004053378A1 to Burk Mark J. teaches a process for making 4-cyano-3-hydroxybutyric acid from 3-hydroxyglutaronitrile using a polypeptide having nitrilase activity.
The nitrilases employed in the prior art possess limitations such as thermal instability, low substrate tolerance, and low enantioselectivity that restrict their industrial application. Moreover, the biocatalysts derived from nitrilase enzymes have additional challenges, such as rapid loss of catalytic activity after the initial use and poor catalyst recovery, hindering their widespread adoption on industrial scale.
The utilization of nitrilase enzymes for the hydrolysis of nitrile groups offers remarkable advantages, particularly in terms of mild reaction conditions, enantioselectivity, and nitrile reracemization. However, commercially available nitrilases exhibit low enantioselectivity. Therefore, it is of great significance to prepare nitrilase products providing high enantioselectivity and meet the requirements of preparing high value-added products.
Thus, there is a need to provide novel nitrilase enzymes and immobilize them using appropriate methods to provide biocatalysts having high catalytic activity and high enantioselectivity; thus, furnishing optically pure carboxylic acids with high volumetric productivity.

SUMMARY OF THE INVENTION:
The present invention relates to modified nitrilase enzymes for biocatalysis derived from Variovorax paradoxus. One of the modified nitrilase, SEQ ID NO. 2, is the amino acid sequence of the modified nitrilase, Vp-nit-T135A containing a codon change resulting in an amino acid substitution at residue position 135 (threonine to alanine) of the Variovorax paradoxus nitrilase. Another modified nitrilase is SEQ ID NO. 3, an amino acid sequence of the modified nitrilase Vp-nit-F192T/T135A containing two codon changes resulting in amino acid substitutions at residue positions 135 (threonine to alanine) and 192 (phenyl alanine to threonine) of the Variovorax paradoxus nitrilase. Yet another modified nitrilase is SEQ ID NO. 4, an amino acid sequence of the modified nitrilase Vp-nit-A286I containing a codon change resulting in an amino acid substitution at residue position 286 (alanine to isoleucine) of the Variovorax paradoxus nitrilase.
The present invention further discloses a method 100 of preparation of the said modified nitrilases for biocatalysis including several steps. In a first step of downloading sequences 105, the nitrilase polypeptide sequences for the enzyme classified under EC 3.5.5.1 are downloaded from a protein database. In a second step of analyzing the sequences 110, the downloaded polypeptide sequences are subjected to bioinformatic analysis. The third step of cloning and expression 115 includes cloning the nitrilase gene sequences corresponding to the selected nitrilase polypeptide sequences in a cloning vector and checking the expression of these gene sequences in an expression vector.
In a fourth step of analysis of expression and activity 120, the expression of the nitrilase gene sequences is analyzed, the activity of all the nitrilases is assessed, and the resistance of nitrilases to proteolytic degradation is studied. In fifth step of selection of the clone 125, the clone of Variovorax paradoxus nitrilase sequence having the nitrilase gene possessing maximum expression and high enantioselectivity is identified based on the results of the analysis of expression of the nitrilase sequences in step 120. In a sixth step of site directed mutagenesis 130, mutations are carried out at specific sites of the gene sequence of the clone selected in step 125 for optimal performance of the enzyme.
In a seventh step of cloning 135, the resultant mutant genes are cloned in a cloning vector and their expression is checked in an expression vector. In an eighth step of biotransformation 140, the various mutant enzymes obtained in step of cloning 135 are employed for biotransformation of the racemic nitrile substrates. In a ninth step of immobilization 145, the mutant enzymes are immobilized onto solid supports using suitable immobilization techniques. In a tenth step of evaluation of immobilized enzymes 150, the immobilized mutant enzymes are evaluated for biotransformation of nitrile substrates and their recycling capabilities.
The present invention provides nitrilase enzymes as biocatalysts that are stable, resistant to proteolytic degradation, active over a wide range of solvents and different nitrile substrates and provide optically pure carboxylic acids on an industrial scale.

BRIEF DESCRIPTION OF DRAWINGS:
The objectives and advantages of the present invention will become apparent from the following description read in accordance with the accompanying drawings wherein,
Figure 1 shows the method of preparation of the modified nitrilases for biocatalysis 100 in accordance with the present invention, and
FIG. 2 shows comparison of solid representation of surfaces of the active site cavity of Variovorax paradoxus nitrilase and Alcaligenes faecalis nitrilase.

DESCRIPTION OF THE INVENTION:
Nitrilase enzymes in bio-catalytic processes must demonstrate several key qualities: thermal stability, the ability to handle high substrate concentrations, tolerance to a wide pH range, and the production of carboxylic acids with high enantiomeric excess. Crucially, these nitrilase biocatalysts must be robust, reusable over multiple use-cycles while maintaining activity, easily separable from reaction media, and effective in both aqueous and non-aqueous environments.
In the present invention, novel nitrilase encoding genes are identified in the gene databases and their enzyme activity is studied by cloning and expressing in suitable heterologous hosts. Further, nitrilase enzymes are characterized and modified in regard to biocatalytic properties, followed by immobilization using suitable methods to convert the racemic nitrile substrates into enantiomerically pure carboxylic acids. The said immobilized enzymes are stable, tolerant to high substrate concentrations, capable of several cycles and ultimately provide an economical and industrially applicable process for preparation of various carboxylic acids having high enantiomeric excess.
The inventors focused on developing a cost-effective, industrial process for producing optically pure carboxylic acids from nitrile substrates. Their studies aimed at creating modified and immobilized nitrilase enzymes that could:
1. maintain activity across a wide range of aqueous and non-aqueous solvent conditions,
2. be recycled multiple times without loss of activity,
3. handle high substrate loads, and
4. produce carboxylic acids with high enantiomeric purity through dynamic resolution.
The inventors investigated the enzyme activity of novel nitrilase enzyme encoded by genes identified from gene databases; by cloning and expressing them in suitable heterologous hosts. They unexpectedly found that further characterization of the expressed enzymes, along with sequence modifications and enzyme immobilization, led to the development of highly effective biocatalysts. These biocatalysts proved to be ideal for converting various nitrile substrates into the corresponding optically active carboxylic acids.
As used herein, the term "nitrilase" refers to an enzyme, or a catalytically active portion, derivative, or analogue, that catalyzes the conversion of nitriles into carboxylic acids and ammonia. A "derivative" or "analogue" refers to a modified version or a portion derived from the original molecule. The nitrilase used in the present invention can be any enzyme that catalyzes the conversion of nitriles into carboxylic acids. A preferred type of nitrilase is the enzyme classified under EC 3.5.5.1, also known as "nitrile aminohydrolase." The nitrilase may display solely nitrilase activity or could be a multifunctional enzyme that exhibits nitrilase activity along with other enzymatic activities.
As used herein, the term "nitrile" refers to any compound containing at least one nitrile group (-CN functional group), which may be present in quantities of one or more -CN groups. The nitrile group can be attached to an aromatic moiety, such as an aryl group, which typically contains between 6 and 14 carbon atoms, preferably between 6 and 10 carbon atoms. The aryl group may consist of one, two, or three fused aromatic rings, with the phenyl group being a more preferred example. Additionally, the aryl group may be substituted with one or more substituents, that could range from one to several different substituents.
As used herein, the term "expression" means the transcription and translation to gene product from a gene coding for the sequence of the gene product, usually a protein.
As used herein, the terms “coding sequence of” or a “sequence encoding a particular polypeptide or protein”, is a nucleic acid sequence which is transcribed and translated into a polypeptide or protein when placed under the control of appropriate regulatory sequences.
As used herein, the terms "protein", "polypeptide", and "peptide" are used interchangeably to refer to the gene product expressed.
As used herein, the term "amino acid sequence" refers to a list of abbreviations, letters, characters or words representing amino acid residues.
As used herein, the abbreviations are conventional one letter codes for the amino acids following the standards established by the International Union of Pure and Applied Chemistry (IUPAC) - A: alanine; B: asparagine or aspartic acid; C: cysteine; D: aspartic acid; E: glutamate, glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine; Z: glutamine or glutamic acid (L. Stryer, Biochemistry, 1988, W. H. Freeman and Company, New York).
As used herein, the term “chiral carboxylic acids” refers to carboxylic acids having at least one carbon atom with four different substituents, making them chiral centers (i.e., they lack symmetry and exist as non-superimposable mirror images, called enantiomers). In a carboxylic acid, the general structure is R-COOH, where R is the rest of the molecule (a hydrocarbon chain or another substituent). For a carboxylic acid to be chiral, the R group must be attached to a carbon that is also attached to three other different groups, making it a chiral center.
As used herein, the term “enantioselective” process or enzyme refers to a reaction or enzymatic catalysis that selectively produces one enantiomer over the other. In these processes, the enzyme or catalyst favors the formation of one of the two possible mirror-image forms (enantiomers) of a chiral compound, usually leading to an excess of that enantiomer (greater than 50% but typically less than 100%).
As used herein, the term “enantiomeric excess (ee)” refers to the difference in concentration between the two enantiomers of a chiral compound, expressed as a percentage. It quantifies the level of asymmetry in a sample with respect to the two enantiomers. Enantiomeric excess (ee) of a substance is a measure of its optical purity, which indicates the relative amount of one enantiomer compared to the other in a mixture. This optical purity can be determined by measuring the compound's optical rotation, which is a physical property that varies depending on the chiral composition of the substance. The equation for calculating enantiomeric excess (ee) is: ee = [(A-B)/(A+B)] ×100
where A is the percentage of the major enantiomer (the enantiomer produced in greater quantity), and B is the percentage of the minor enantiomer (the enantiomer produced in lesser quantity). The result gives the percentage by which the major enantiomer exceeds the minor enantiomer in the mixture. A higher ee (close to 100%) indicates a stronger enantioselectivity in the process, meaning the catalyst (enzyme or chemical) is highly selective for one enantiomer.

BRIEF DESCRIPTION OF THE SEQUENCES PROVIDED
The following sequences are consistent with World Intellectual Property Organization (WIPO) Standard ST.26 and the sequence listing requirements as per rule 9(3) of the Patent Rules, 2003.
SEQ ID NO. 1 is the deduced amino acid sequence of the wild type Variovorax paradoxus nitrilase, Vp-nit-28a;
SEQ ID NO. 2 is the deduced amino acid sequence of the mutant nitrilase containing a codon change resulting in a single amino acid substitution at residue position 135 (threonine to alanine) of the Variovorax paradoxus nitrilase; to give modified nitrilase, Vp-nit-T135A;
SEQ ID NO. 3 is the deduced amino acid sequence of the mutant nitrilase containing two codon changes resulting in two amino acid substitutions, one at residue positions 135 (threonine to alanine) and other at residue position 192 (phenyl alanine to threonine) of the Variovorax paradoxus nitrilase; to give modified nitrilase, Vp-nit-F192T/T135A; and
SEQ ID NO. 4 is the deduced amino acid sequence of the mutant nitrilase containing a codon change resulting in a single amino acid substitution at residue position 286 (alanine to isoleucine) of the Variovorax paradoxus nitrilase; to give modified nitrilase, Vp-nit-A286I.
References in the specification to "one embodiment" or "an embodiment" means that a particular feature, structure, characteristic, or function described in connection with the embodiment is included in at least one embodiment of the invention. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment.
References in the specification to “preferred embodiment” means that a particular feature, structure, characteristic, or function described in detail thereby omitting known constructions and functions for clear description of the present invention.
The foregoing description of specific embodiments of the present invention has been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed and obviously many modifications and variations are possible in light of the above teaching.
The present invention provides novel polypeptide sequence for nitrilase enzyme, and modifications in the nitrilase gene sequence for improvement in the enzymes. Specifically, putative nitrilase polypeptide sequences selected from the Uniprot databank are analyzed using bioinformatics tools; and further the corresponding gene sequence is cloned and expressed in E. coli. The enzymes are screened for the aryl acetonitrile-specific enzyme activity and are also assessed for stability. The enzymes are further modified for optimal performance and the improved enzymes show quantitative conversion of aryl cyanohydrin and aryl acetaldehyde cyanohydrin substrates to yield the corresponding optically pure carboxylic acids, at high substrate concentration. The present invention also provides a platform technology for conversion of nitrile substrates to carboxylic acids.
The present invention describes a method of preparation of modified nitrilases for biocatalysis 100, hereinafter referred to as “method 100”. The method 100 includes several steps consisting of a first step 105 of downloading the sequences, a second step 110 of analyzing the sequences, a third step 115 of cloning and expression, a fourth step 120 of analysis of expression and activity, a fifth step 125 of selection of clone with Variovorax paradoxus nitrilase sequence, a sixth step 130 of site directed mutagenesis, a seventh step 135 of cloning, an eighth step 140 of biotransformation, a ninth step 145 of immobilization, and a tenth step 150 of evaluation of immobilized enzymes.
In the first step of downloading the sequences 105, the polypeptide sequences for the enzyme classified under EC 3.5.5.1 are downloaded from a protein database. The protein databases are selected from UniProt (Universal Protein Resource), Protein Data Bank (PDB), NCBI Protein Database, InterPro, Ensembl Genome Browser, ExPASy (Expert Protein Analysis System), KEGG (Kyoto Encyclopedia of Genes and Genomes), Swiss-Prot, Pfam and BLAST (Basic Local Alignment Search Tool), BRENDA (The Enzyme Database).
The second step of analyzing the sequences 110 includes analyzing the downloaded nitrilase polypeptide sequences. Accordingly, in the second step 110, the nitrilase polypeptide sequences are subjected to bioinformatic analysis. This bioinformatic analysis includes analysis of the downloaded nitrilase polypeptide sequences by several tools for sequence alignment, sequence annotation, homology modeling, phylogenetic analysis, optimization and site-directed mutagenesis, enzyme activity prediction, stability prediction, gene expression and cloning strategy, and systems biology and pathway analysis.
Accordingly, sequence alignment studies are performed with tools such as BLAST (Basic Local Alignment Search Tool) to compare nitrilase polypeptide sequences against existing databases. Bioinformatic tools are used to annotate the sequences, predict the functional domains (e.g., active sites or binding sites) and understand the potential enzyme mechanisms. The functional motifs within the nitrilase protein sequence are identified using tools like InterPro. Bioinformatic tools such as Swiss-Model or Phyre2 are employed to predict the 3D structure of the nitrilase enzyme. Bioinformatic tools like Clustal Omega or MEGA are used to generate phylogenetic trees to understand the evolutionary lineage.
Tools like Rosetta and FoldX are utilized to predict the effects of mutations on protein stability and function to identify potential sites for site directed mutagenesis. Further, Bioinformatic tools like IDT Codon Optimization Tool are used to adjust the gene sequence for efficient translation in the host organism. Tools like CATH and Pfam are used to predict the enzyme’s substrate specificity based on its sequence and structural domains.
Bioinformatic tools including I-Mutant and SDM (Site Directed Mutagenesis) help predict the impact of sequence modifications on the enzyme’s stability. Tools including SnapGene or Benchling are employed for visualizing and planning cloning strategies. Tools like pET vector optimization help ensure efficient expression of the gene in the bacterial host. Bioinformatic tools including KEGG or Reactome can be used to visualize how the nitrilase fits into metabolic networks.
In the third step of cloning and expression 115, the gene sequences corresponding to the nitrilase polypeptide sequences as identified by the bioinformatic analysis are cloned in a cloning vector and the expression of these gene sequences is checked in an expression vector. Examples of vectors which may be used include viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, fowlpox virus, pseudorabies and derivatives of SV40), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for the hosts of interest (e.g., Bacillus, Aspergillus and yeast). Suitable cloning vectors known to those skilled in the art and commercially available can be used.
Examples of viral vectors that can be used includes Baculovirus, Adenovirus, Fowlpox virus, Pseudorabies virus, Vaccinia virus, SV40 (Simian Virus 40) and the like. Examples of bacterial vectors that can be used include pQE vectors, pBluescript plasmids, pNH vectors, Lambda-ZAP vectors, pTRC99a, pKK223-3, pDR540, pRIT2T, pTrc His2B, pET21a, pET28a, pGEX4T1, pGEX6P1 vectors and the like. The gene sequences are expressed in vectors selected from E. coli BL21 (DE3), E. coli BL21, E. coli TOP10, E. coli DH10B and the like.
In the fourth step of analysis of expression and activity 120, the expression of the nitrilase gene sequences is analyzed and the activity of all the nitrilases are assessed. Further, the resistance of nitrilases to proteolytic degradation is also assessed.
In the fifth step of selection of the clone with Variovorax paradoxus nitrilase sequence 125, the clone with the gene sequence expressing nitrilase having specificity towards racemic nitrile substrates, good activity and enantioselectivity is selected based on the results of the analysis of expression of the nitrilase sequences in step 120. In the sixth step of site directed mutagenesis 130 mutations are carried out at specific sites of the gene sequence of the clone selected in step 125, for optimal performance of the enzyme.
In the seventh step of cloning 135, the resultant mutant genes are cloned in a cloning vector and their expression are checked in an expression vector. Suitable cloning vectors known to those skilled in the art and commercially available can be used. The expression vector is selected from E. coli BL21 (DE3), E. coli BL21 (DE3) star, E. coli BL21 (DE3) Rosetta, and the like. In the eighth step of biotransformation 140, various mutant enzymes obtained are used for biotransformation of the racemic nitrile substrates.
Further, in the ninth step of immobilization 145, the mutant enzymes are immobilized onto solid supports using suitable immobilization techniques selected from electrostatic droplet generation, electrochemical means, adsorption, covalent binding, cross-linking, chemical reactions, encapsulation, entrapment, calcium alginate, and poly (2-hydroxyethyl methacrylate), as described in Methods in Enzymology, Immobilized Enzymes and Cells, Part C. 1987, Academic Press, Edited by S. P. Colowick and N. O. Kaplan. Volume 136; and Immobilization of Enzymes and Cells, 1997, Humana Press, Edited by G. F. Bickerstaff, Series: Methods in Biotechnology, Edited by J. M. Walker. In the last step of evaluation of immobilized enzymes 150, the immobilized mutant enzymes are evaluated for biotransformation of nitrile substrates and their recycling capabilities.
In accordance with a preferred embodiment, in the step 105, the polypeptide sequences for the nitrilase enzyme are downloaded from the protein database UniProt. As per step 110, 241 prokaryotic sequences were selected for further bioinformatic analysis. This process involved multiple sequence alignments and the construction of a phylogenetic tree. The resulting phylogenetic tree was divided into 16 clades based on phylogenetic distances. From these clades, 12 nitrilase sequences, each representing different clades, were chosen for further study. The bioinformatic analysis of the selected nitrilase sequences resulted in shortlisting of eight nucleotide sequences.
In accordance with this embodiment, in step 115, the nitrilase gene sequences were codon optimized to be expressed in E. coli and cloned in pTrc His2B with BamHI and XbaI restriction sites and expressed in vectors selected from E. coli BL21 (DE3), E. coli BL21, E. coli TOP10, E. coli DH10B. A few of the genes were also cloned in vectors selected from pET21a, pET28a, pGEX4T1, pGEX6P1 and expressed in E. coli BL21 (DE3). The enzymes obtained by expressing all these genes in various expression systems were characterized in terms of their activity, substrate specificity and enantioselectivity.
In accordance with steps 120 and 125, the analysis of expression of the nitrilase gene sequences resulted in selecting the clone of Variovorax paradoxus having the putative nitrilase gene possessing maximum expression. The enzyme encoded by the gene is analyzed for its activity, revealing that it is stable and highly specific towards aryl acetonitrile substrates. This specificity enables the enzyme to efficiently convert racemic mandelonitrile into optically active mandelic acid, demonstrating its optimal performance.
It is found by the inventors that the nitrilases encoded by the putative gene sequence from Variovorax paradoxus show good enzyme activity. The said enzymes are also found resistant to proteolytic attack and are stable when stored in the form of whole cells making them suitable for whole cell biocatalysis.
On detailed comparison of the enzymes with the known nitrilases in terms of active site topography analysis, along with limited enantioselectivity of the enzyme towards the aryl acetonitrile such as mandelonitrile; the enzyme sequences were modified in step 130 by carrying out mutations at the specific sites of the gene sequence of Variovorax paradoxus nitrilase.
In accordance with the preferred embodiment, in step 130, the enzyme is modified using site directed mutagenesis of the gene sequence. Homology modelling techniques are used to predict these sites for mutagenesis. Protein Nit6803 (PDBid-3WUY) is selected as the template for bioinformatic analysis, and the modelling of both the said novel gene sequence and the earlier reported nitrilase gene sequence from Alcaligenes faecalis is carried out using Modeller 9.11. It is found that the gene sequence shared only 51% sequence identity with the nitrilase from Alcaligenes faecalis indicating that the enzyme is different from those reported in the prior art.
Further, specifically, structure guided sequence alignment with the nitrilase gene sequence from Alcaligenes faecalis indicated that the catalytic triad Glu47-Lys129-Cys163 is conserved in the said novel nitrilase enzyme from Variovorax paradoxus.
Hotspot wizard is used to map active site pockets of both the nitrilases, i.e. nitrilase enzyme obtained from Variovorax paradoxus and nitrilase of the prior art obtained from Alcaligenes faecalis. It is found that the active site cavity tunnel is broader in Alcaligenes faecalis nitrilase than that in Variovorax paradoxus nitrilase. The active site topography indicated that the active site cavity is tilted in the case of Alcaligens faecalis nitrilase; while in the case of Variovorax paradoxus nitrilase, it is upright. The differences in the active site topography, possibly responsible for the limited enantioselectivity exhibited by the Variovorax paradoxus nitrilase prompted further modification of the enzyme sequences and further immobilization of the same for use as biocatalysts.
FIG. 2 demonstrates solid representation of surfaces of the active site cavity of Variovorax paradoxus nitrilase and Alcaligenes faecalis nitrilase. The figure shows that the topography is tilted in Alcaligenes faecalis nitrilase, while it is upright in Variovorax paradoxus nitrilase. The analysis showed that cavity tunnel of Alcaligenes faecalis nitrilase is wider than that of Variovorax paradoxus nitrilase as shown by the larger bottleneck radius and tunnel length in Alcaligenes faecalis nitrilase than Variovorax paradoxus nitrilase.
Table 1 below represents the dimensions obtained from the Tunnel analysis done using hotspot wizard showing the dimensions of Alcaligenes faecalis nitrilase is larger than Variovorax paradoxus nitrilase.
Dimensions Variovorax paradoxus nitrilase Alcaligenes faecalis nitrilase
Mouth Radius 2.07Å 2.43Å
Tunnel Length 2.83Å 3.33Å
Curvature 1.09 1.14
Table 1: Tunnel analysis done using hotspot wizard showing the dimensions of the tunnel of Alcaligenes faecalis nitrilase is larger than Variovorax paradoxus nitrilase
BLAST analysis showed that Variovorax paradoxus nitrilase showed a 40% sequence identity against 84% sequence coverage with a nitrilase protein Nit6803 (PDBid-3WUY). Similarly, Alcaligens faecalis nitrilase shared a 34% sequence identity against 97% sequence coverage.
BLAST search against 2A6 nitrilase (PDBid-1J31) which Xue et al., 2015 had used to model NitA protein showed relatively less sequence identity of about 23% against Alcaligens faecalis nitrilase (80% query coverage) and Variovorax paradoxus nitrilase (60% query coverage) respectively.
Therefore, protein Nit6803 (PDBid-3WUY) is selected as the template for modelling Alcaligens faecalis nitrilase and Variovorax paradoxus nitrilase to make the comparative analysis on the scenario of active site cavity.
In accordance with the preferred embodiment, in step 130, the point mutations are carried out at the specific sites in the nitrilase gene sequence from Variovorax paradoxus. The point mutations include
a) replacing threonine at position 135 by alanine, to give modified nitrilase, Vp-nit-T135A;
b) further replacing phenyl alanine at position 192 by threonine to give modified nitrilase Vp-nit-F192T/T135A; and
c) replacing alanine at position 286 by isoleucine to give modified nitrilase Vp-nit-A286I.
In accordance with the preferred embodiment, in step 135, the resultant mutant genes are cloned in pET28a vector, and their expression are checked in E. coli strains selected from E. coli BL21 (DE3), E. coli BL21 (DE3) star, E. coli BL21 (DE3) Rosetta, and the like.
Based on the results obtained in step 135, various mutant enzymes obtained are used for biotransformation of the racemic nitrile substrates in step 140. The racemic nitrile substrates are selected from aryl nitriles, aryl acetonitrile, aryl cyanohydrins and aryl acetaldehyde cyanohydrins consisting of benzonitriles, 4-methylbenzonitrile, 4-chlorobenzonitrile, 4-nitrobenzonitrile, 2-methoxybenzonitrile, 3,4-dichlorobenzonitrile, phenoxybenzonitrile, phenyl acetaldehyde cyanohydrin, 4-methylphenyl acetaldehyde cyanohydrin, 4-chlorophenyl acetaldehyde cyanohydrin, 4-methoxyphenyl acetaldehyde cyanohydrin, 2,4-dichlorophenyl acetaldehyde cyanohydrin, 2-naphthyl acetaldehyde cyanohydrin, p-anisyl acetonitrile, 2,4-dichlorobenzyl cyanide, phenoxybenzyl cyanide, o-tolyl acetonitrile, mandelonitrile, 4-bromo mandelonitrile, 2-chloro mandelonitrile, 4-nitro mandelonitrile, 4-methyl mandelonitrile, 3-phenyl propane cyanohydrin, cyclohexyl mandelonitrile, hydroxyglutarylnitrile, 2-bromo mandelonitrile, 2-methyl mandelonitrile, 3-bromo mandelonitrile, 4-fluoro mandelonitrile and the like.
In accordance with the preferred embodiment, in step 145, the selected mutant enzymes having SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4 are further immobilized using one of the suitable methods selected from adsorption or entrapment using suitable resins or clay minerals, enzyme crosslinking using suitable crosslinking agents, or making cross-linked protein coated microcrystals.
In a further embodiment, in step 135, 140 and 145, the resultant mutant genes are cloned in pET28a vector, followed by checking their expression in E. coli BL21 (DE3) strains. The expressed enzymes are then immobilized using ion exchange resins and the resulting bio catalysts are used for conversion of various racemic nitrile substrates.
In another embodiment, in step 145, the nitrilase enzymes are subjected to immobilization using suitable methods including adsorption, adhesion/entrapment, ion exchange. Accordingly, the nitrilase enzymes are immobilized using ion exchange resins, clay minerals, polyvinyl alcohol-calcium alginate (PVA-alginate), etc. The enzymes are also subjected to suitable crosslinking agents, resulting in cross-linked protein coated microcrystals (CL-PCMCs). For immobilization using ion exchange resins, resins having ionic amino functionalities including quaternary amines, hexamethylenediamines etc. exemplified by LSF975 and LKZ218 (Sunresin & Amicogen, China) are used for the purpose.
In a further embodiment, the immobilization conditions for nitrilase enzymes are optimized with respect to different parameters including enzyme loading on the immobilization support, time for immobilization, stirring conditions, pH, buffer strength, temperature, adsorption time, stability of the resulting immobilized enzymes, as well as the methods for effective extraction of the product.
For immobilization using ion exchange resins, it is observed that when the said process of immobilization is carried out in the temperature range of 15 to 25°C using a phosphate buffer such as sodium phosphate (pH 7.5), wherein the enzyme loading is in the range of 400 to 800 U/g of beads, the immobilization efficiency in terms of the enzyme activity is substantially high (>95%). Further, the immobilized nitrilase enzymes are found to be stable for 1-2 months in the temperature range of 4 to 8°C.
In accordance with the preferred embodiment, in step 150, the biotransformation reactions with immobilized enzymes are found to be advantageous as compared to those with free enzymes. Further, the immobilized enzymes are recycled multiple times retaining the enzyme efficiency as against the free enzymes which cannot be reused or recycled.
The immobilized enzymes are recycled multiple times using the same set of reaction conditions as used for the first reaction, including the substrate concentration. It is observed that all reactions with immobilized recycled enzymes proceeded with remarkably good conversion, in the range of 85 to 95%. Further, fresh enzyme loading of only 5% in the subsequent recycles is sufficient for obtaining 85% conversion of the substrate. In addition, the said biocatalysts are able to withstand loading close to one mole of the nitrile substrate.
In an embodiment, the evaluation of immobilized enzymes in step 150 includes subjecting racemic mandelonitrile to treatment with various nitrilases including the wild type (WT) and the modified nitrilase. Accordingly, the evaluation of immobilized enzymes 150 further includes several steps. In a first step, the racemic mandelonitrile is added to a phosphate buffer such as sodium phosphate at pH 7.5, in temperature range of 29°C to 37°C. In a second step, the nitrilase enzyme is added to the buffer, then it is allowed to equilibrate to the set temperature and followed by addition of the nitrile substrate using fed-batch method. In a third step, the reaction is carried out in the temperature range of 29°C to 37°C and completion of the reaction is monitored by TLC and HPLC. In a fourth step, after completion of the reaction, the reaction mixture is centrifuged, the biomass is separated, and the resultant mass is concentrated under vacuum.
In the evaluation of immobilized enzymes 150, gradual addition of concentrated sulfuric acid to the concentrated reaction mass at low temperature, followed by filtration and drying of the precipitated solid provides the desired product, (R)-(-)- Mandelic acid.
In an embodiment, the evaluation of immobilized enzymes 150 includes subjecting racemic mandelonitrile to treatment with wild type (WT) and modified nitrilase, namely Vp-nit-T135A. It is observed that as compared to wild type nitrilase, Vp-nit-T135A, the modified nitrilase showed improved conversion and enantioselectivity.
In another embodiment, the evaluation of immobilized enzymes 150 includes subjecting racemic mandelonitrile to treatment with modified nitrilase, namely Vp-nit-F192T/T135A. It is observed that the doubly modified nitrilase, Vp-nit-F192T/T135A showed improvements in substrate tolerance, and enantioselectivity as compared to Vp-nit-T135A.
In a further embodiment, the evaluation of immobilized enzymes 150 includes subjecting racemic mandelonitrile to treatment with the modified nitrilase, namely, Vp-nit-A286I. It is observed that amongst the modified nitrilases, in case of Vp-nit-A286I, the reaction is advantageously carried out at substrate concentrations as high as 800 mM/L, maintaining the high enantioselectivity, making it a suitable biocatalyst for industrial application. It is noteworthy that the biocatalyst developed by immobilization of Vp-nit-A286I on LSF975 not only exhibits recycling ability of the catalyst but shows improved conversion, retaining the high enantioselectivity, with easier isolation of the final compound, making the immobilized catalyst more suitable and economical for industrial application.
In yet another embodiment, similar reactions using modified nitrilases and biocatalysts developed from the same are carried out on nitrile substrates such as aryl cyanohydrins and aryl acetaldehyde cyanohydrins. The results are similar with respect to conversion, enantiomeric excess, yield, isolation and purity of final compound etc. Both free and immobilized enzymes provide the corresponding carboxylic acids possessing high enantiomeric excess, purity with good conversion rates and ease of operation and recycling.
EXAMPLES:
Only a few examples and implementations are disclosed. Variations, modifications, and enhancements to the described examples and other implementations can be made based on what is disclosed.
Examples are set forth herein below and are illustrative of different amounts and types of reactants and reaction conditions that can be utilized in practicing the disclosure. It will be apparent, however, that the disclosure can be practiced with other amounts and types of reactants and reaction conditions than those used in the examples, and the resulting devices various different properties and uses in accordance with the disclosure above and as pointed out hereinafter.
Example 1: Immobilization of modified nitrilase, Vp-nit-A286I, using the ion exchange resin, LSF975
Preparation of crude lysate: Whole cell suspension of Vp-nit-A286I was prepared in 100 mM sodium phosphate buffer, pH 7.5, containing phenyl methyl sulfonyl fluoride (PMSF) and Dithiothreitol (DTT), (1mM each). Whole cells from the bacterial suspension were lysed in a sonicator. The lysed cell suspension was further centrifuged under cold condition to separate the lysate and cell debris. The crude cell lysate containing nitrilase enzyme was further used for the immobilization on LSF975 resin.
Preparation of resin: Resin LSF975 was activated by respectively using 4% HCl and 4% NaOH solutions wherein the resin was mixed and stirred for 15 minutes in each solution. The activated resin was further washed with sodium phosphate buffer, pH 7.5 containing 2mM DTT at room temperature. The cell free crude lysate containing nitrilase enzyme was added to the activated resin LSF975 and kept for stirring at 20 °C, 180 rpm for 24 hours. 10 ml crude lysate containing 75-80U/ml nitrilase activity was immobilized on 1g resin with 95% immobilization efficiency with reference to nitrilase activity and protein binding. After 24 hours, immobilized beads were washed with 20 mM phosphate buffer, 2mM DTT pH 7.5 to remove the unbound proteins.
Example 2: Preparation of (R)-(-)-Mandelic Acid using Wild Type nitrilase (WT)
A solution of sodium phosphate buffer (100 mM, 500 ml), pH 7.5 was placed in a three necked round bottom flask, fitted with an overhead stirrer and equilibrated at 33+ 4°C. Wild Type nitrilase enzyme (1150U) was added to it and allowed to equilibrate to the set temperature. Mandelonitrile (33.28 g/L) was then added to the reaction mass using fed batch method. pH of the reaction was maintained above 7.3 using alkaline component/ buffer and the progress and the completion of the reaction was monitored by TLC and HPLC analysis of the intermittent samples.
After completion of the reaction, the reaction mixture was centrifuged, the biomass was separated, and the resultant mass was concentrated. Dropwise addition of concentrated sulfuric acid to the concentrated reaction mixture at low temperature, followed by filtration and drying of the precipitated off white solid provided mandelic acid wherein the % conversion was 60%.
Purity: 99.9 %
Yield: 82.5 %.
Enantiomeric Excess: 96%
Example 3: Preparation of (R)-(-)-Mandelic Acid using modified nitrilase, Vp-nit-T135A
A solution of sodium phosphate buffer (100 mM, 500 ml), pH 7.5 was placed in a three necked round bottom flask, fitted with an overhead stirrer and equilibrated at 33+ 4°C. The modified nitrilase enzyme, Vp-nit-T135A (1150U) was added to it and allowed to equilibrate to the set temperature. Mandelonitrile (33.28 g/L) was then added to the reaction mass using fed batch method. pH of the reaction was maintained above 7.3 using alkaline component/ buffer and the progress and the completion of the reaction was monitored by TLC and HPLC analysis of the intermittent samples.
After completion of the reaction, the reaction mixture was centrifuged, the biomass was separated, and resultant reaction mixture was concentrated.
Drop-wise addition of concentrated sulfuric acid to the concentrated reaction mass at low temperature, followed by filtration and drying of the precipitated solid provided white crystalline mandelic acid wherein the % conversion is 95%.
Purity: 99%
Yield: 89.5%
Enantiomeric Excess: 98%
Example 4: Preparation of (R)-(-)-Mandelic Acid using modified nitrilase, Vp-nit-F192T/T135A
A solution of sodium phosphate buffer (100 mM, 500 ml), pH 7.5 was placed in a three necked round bottom flask, fitted with an overhead stirrer and equilibrated at 33+ 4°C. The double modified nitrilase enzyme, Vp-nit-F192T/T135A (1150 U) was added to it and allowed to equilibrate to the set temperature. Mandelonitrile (66.56 g/L), was then added to the reaction mass using fed batch method. pH of the reaction was maintained above 7.3 using alkaline component/ buffer and the progress and the completion of the reaction was monitored by TLC and HPLC analysis of the intermittent samples.
After completion of the reaction, the reaction mixture was centrifuged, the biomass was separated, and resultant reaction mixture was concentrated.
Drop-wise addition of concentrated sulfuric acid to the concentrated reaction mixture at low temperature, followed by filtration and drying of the precipitated solid provided white crystalline mandelic acid wherein the % conversion is 96%.
Purity: 99.9%
Yield: 95.2 %
Enantiomeric Excess: 99.5%
Example 5: Preparation of (R)-(-)-Mandelic Acid using modified nitrilase, Vp-nit-A286I
A solution of sodium phosphate buffer (100 mM, 500 ml), pH 7.5 was placed in a three necked round bottom flask, fitted with an overhead stirrer and equilibrated at 33+ 4°C. The advance modified nitrilase enzyme, Vp-nit-A286I (1150 U) was added to it and allowed to equilibrate to the set temperature. Mandelonitrile (99.86 g/L) was then added to the reaction mass using fed batch method. pH of the reaction was maintained above 7.3 using alkaline component/ buffer and the progress and completion of the reaction was monitored by TLC and HPLC analysis of the intermittent samples.
After completion of the reaction, the reaction mixture was centrifuged, the biomass was separated, and resultant mass was concentrated.
Drop-wise addition of concentrated sulfuric acid to the concentrated reaction mass at low temperature, followed by filtration and drying of the precipitated solid provided mandelic acid wherein % conversion is 95.2%.
Purity: 99.9%
Yield: 96 %
Enantiomeric Excess: 99.7 %
Example 6: Preparation of (R)-(-)-Mandelic Acid using modified nitrilase, Vp-nit-A286I
A solution of sodium phosphate buffer (100 mM, 500 ml), pH 7.5 was placed in a three necked round bottom flask, fitted with an overhead stirrer and equilibrated at 33+ 4°C. The advance modified nitrilase enzyme, Vp-nit-A286I (1150U) was added to it and allowed to equilibrate to the set temperature. Mandelonitrile (133.16 g/L) was then added to the reaction mass using fed batch method. pH of the reaction was maintained above 7.3 using alkaline component/ buffer and the progress and completion of the reaction was monitored by TLC and HPLC analysis of the intermittent samples.
After completion of the reaction, the reaction mixture was centrifuged, the biomass was separated, and resultant mass was concentrated.
Drop-wise addition of concentrated sulfuric acid to the concentrated reaction mass at low temperature, followed by filtration and drying of the precipitated solid provided mandelic acid wherein % conversion is 85%.
Purity: 98.0%
Yield: 92.0 %
Enantiomeric Excess: 99.5%
Example 7: Preparation of (R)-(-)-Mandelic Acid using nitrilase, Vp-nit-A286I immobilised on LSF975 ion exchange resin:
A solution of sodium phosphate buffer (100 mM, 500 ml), pH 7.5 was placed in a three necked round bottom flask, fitted with an overhead stirrer and equilibrated at 33+ 4°C. The immobilized nitrilase enzyme, Vp-nit-A286I (1150U) was added to it and allowed to equilibrate to the set temperature. Mandelonitrile (133.16 g/L) was then added to the reaction mass using fed batch method. pH of the reaction was maintained above 7.3 using alkaline component/ buffer and the progress and completion of the reaction was monitored by TLC and HPLC analysis of the intermittent samples.
After completion of the reaction, the reaction mixture was filtered by simple filtration and immobilized enzyme is separated. The immobilised beads after first cycle were washed with 20 mM phosphate buffer, 2mM DTT pH 7.5 to remove the unbound material if any. The immobilized nitrilase enzyme was further used for second cycle of biotransformation reaction with additional 5% (58U) whole cell nitrilase enzyme to compensate for the loss in enzyme activity during first cycle. The second cycle of biotransformation was performed using same set of reaction conditions, including the substrate quantity, and immobilized enzyme was separated after the reaction.
The third cycle of recycling of immobilized enzyme was performed after appropriate washing and with additional 5% (58U) whole cell nitrilase enzyme to compensate for the loss during second cycle. The biotransformation was performed at same set of reaction conditions, including the substrate quantity for third recycling of immobilised enzyme preparation and immobilised enzyme was separated after reaction.
The separated reaction mixture after each cycle of biotransformation was concentrated separately. Drop-wise addition of concentrated sulfuric acid to the concentrated reaction mixture at low temperature, followed by filtration and drying of the precipitated solid provided white crystalline mandelic acid separately from each cycle. The details of conversion, purity, yield and enantiomeric excess in each cycle are as provided in Table 2.
Parameters 1st cycle 2nd cycle 3rd cycle
% Conversion 95.2 92.5 85
% Purity 99.9 99.2 98.2
% Yield 96 95 92
Enantiomeric excess 99.6 99.5 99.5
Table 2: Details of conversion, purity, yield and enantiomeric excess in each cycle
Advantageously, the present invention provides nitrilase enzymes as biocatalysts that are stable, resistant to proteolytic degradation, active over a wide range of solvents and different substrates and provide optically pure carboxylic acids on industrial scale. The improved enzyme optimally hydrolyzes the racemic nitrile substrates, with high enantioselectivity required for industrial application, resulting in the production of chirally pure carboxylic acids in high yield. The modified and immobilized enzymes hydrolyze high concentration of various nitrile substrates in a fed batch process leading to high volumetric productivity. The modified and immobilized enzymes are recyclable multiple times, thereby leading to overall cost reduction of the process.
The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others, skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated.
It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the scope of the present invention.

Application Project
-------------------
<120> Title : MODIFIED NITRILASES FOR BIOCATALYSIS, METHODS OF PREPARATION AND
USES THEREOF
<130> AppFileReference : 202421003143
<140> CurrentAppNumber : 202421003143
<141> CurrentFilingDate : 2024-01-16

Sequence
--------
<213> OrganismName : Variovorax paradoxus
<400> PreSequenceString :
MPATVHPKLR VAAVQAAPVF LDLDGTIDKT IDLMAQAAGQ GVKLIAFPET WVPGYPWWIW 60
LDSPAWGMQF VQRYHDNALV VGSAEFDRVR EAARKHNIWV SLGYSEKAAG SLYIAQALID 120
DQGRTVQTRR KLKPTHVERT VFGEGDGSDL AVVETPIGNI GSLSCWEHLQ PLSKYAMYAQ 180
NEQIHCGAWP SFSLYRGAAY ALGPELNNAA SQVYAAEGQC FVIAPCATVS QSMSELMCTD 240
AGKQQMLRVG GGFARIYAPD GSPLGTPLAE DQEGLVIADI DLGMIALSKA AADPSGHYSR 300
PDVTQLLLNK TRREPVVLQR APEVERGAFE AIVAAPEPAP AAARQQPLAA 350
<212> Type : PRT
<211> Length : 350
SequenceName : SEQ 1 - WILD TYPE Vp Nitrilase Vp_Nit_28a
SequenceDescription :

Sequence
--------
<213> OrganismName : Variovorax paradoxus
<400> PreSequenceString :
MPATVHPKLR VAAVQAAPVF LDLDGTIDKT IDLMAQAAGQ GVKLIAFPET WVPGYPWWIW 60
LDSPAWGMQF VQRYHDNALV VGSAEFDRVR EAARKHNIWV SLGYSEKAAG SLYIAQALID 120
DQGRTVQTRR KLKPAHVERT VFGEGDGSDL AVVETPIGNI GSLSCWEHLQ PLSKYAMYAQ 180
NEQIHCGAWP SFSLYRGAAY ALGPELNNAA SQVYAAEGQC FVIAPCATVS QSMSELMCTD 240
AGKQQMLRVG GGFARIYAPD GSPLGTPLAE DQEGLVIADI DLGMIALSKA AADPSGHYSR 300
PDVTQLLLNK TRREPVVLQR APEVERGAFE AIVAAPEPAP AAARQQPLAA 350
<212> Type : PRT
<211> Length : 350
SequenceName : SEQ 2 - Vp Nitrilase Mutant sequence Vp-Nit-T135A
SequenceDescription :

Sequence
--------
<213> OrganismName : Variovorax paradoxus
<400> PreSequenceString :
MPATVHPKLR VAAVQAAPVF LDLDGTIDKT IDLMAQAAGQ GVKLIAFPET WVPGYPWWIW 60
LDSPAWGMQF VQRYHDNALV VGSAEFDRVR EAARKHNIWV SLGYSEKAAG SLYIAQALID 120
DQGRTVQTRR KLKPAHVERT VFGEGDGSDL AVVETPIGNI GSLSCWEHLQ PLSKYAMYAQ 180
NEQIHCGAWP STSLYRGAAY ALGPELNNAA SQVYAAEGQC FVIAPCATVS QSMSELMCTD 240
AGKQQMLRVG GGFARIYAPD GSPLGTPLAE DQEGLVIADI DLGMIALSKA AADPSGHYSR 300
PDVTQLLLNK TRREPVVLQR APEVERGAFE AIVAAPEPAP AAARQQPLAA 350
<212> Type : PRT
<211> Length : 350
SequenceName : SEQ 3 - Vp Nitrilase Mutant sequence Vp-Nit-F192T/T135A
SequenceDescription :

Sequence
--------
<213> OrganismName : Variovorax paradoxus
<400> PreSequenceString :
MPATVHPKLR VAAVQAAPVF LDLDGTIDKT IDLMAQAAGQ GVKLIAFPET WVPGYPWWIW 60
LDSPAWGMQF VQRYHDNALV VGSAEFDRVR EAARKHNIWV SLGYSEKAAG SLYIAQALID 120
DQGRTVQTRR KLKPTHVERT VFGEGDGSDL AVVETPIGNI GSLSCWEHLQ PLSKYAMYAQ 180
NEQIHCGAWP SFSLYRGAAY ALGPELNNAA SQVYAAEGQC FVIAPCATVS QSMSELMCTD 240
AGKQQMLRVG GGFARIYAPD GSPLGTPLAE DQEGLVIADI DLGMIILSKA AADPSGHYSR 300
PDVTQLLLNK TRREPVVLQR APEVERGAFE AIVAAPEPAP AAARQQPLAA 350
<212> Type : PRT
<211> Length : 350
SequenceName : SEQ 4 - Vp Nitrilase Mutant sequence Vp-Nit-A286I
SequenceDescription :
,CLAIMS:CLAIMS:
We claim:
1. A modified nitrilase enzyme derived from Variovorax paradoxus, having sequence selected from SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, wherein
SEQ ID NO. 2 being the amino acid sequence of the modified nitrilase, Vp-nit-T135A containing a codon change resulting in a single amino acid substitution at residue position 135 (threonine to alanine) of the Variovorax paradoxus nitrilase;
SEQ ID NO. 3 being the amino acid sequence of the modified nitrilase Vp-nit-F192T/T135A containing two codon changes resulting in two amino acid substitutions at residue positions 135 (threonine to alanine) and 192 (phenyl alanine to threonine) of the Variovorax paradoxus nitrilase; and
SEQ ID NO. 4 being the amino acid sequence of the modified nitrilase Vp-nit-A286I containing a codon change resulting in a single amino acid substitution at residue position 286 (alanine to isoleucine) of the Variovorax paradoxus nitrilase.
2. A method 100 of preparation of modified nitrilases for biocatalysis as claimed in Claim 1 including
a) a first step of downloading the sequences 105 including downloading the nitrilase polypeptide sequences for the enzyme classified under EC 3.5.5.1 from a protein database;
b) a second step of analyzing the sequences 110 including subjecting the downloaded polypeptide sequences to bioinformatic analysis;
c) a third step of cloning and expression 115 including cloning the nitrilase gene sequences corresponding to the selected nitrilase polypeptide sequences in a cloning vector and checking the expression of these gene sequences in an expression vector;
d) a fourth step of analysis of expression and activity 120 including analyzing the expression of the nitrilase gene sequences, assessing the activity of all the nitrilases, and resistance of nitrilases to proteolytic degradation;
e) a fifth step of selection of the clone with Variovorax paradoxus nitrilase sequence 125 including identifying the clone having the nitrilase gene possessing maximum expression and high enantioselectivity based on the results of the analysis of expression of the nitrilase sequences in step 120;
f) a sixth step of site directed mutagenesis 130 including carrying out mutations at specific sites of the gene sequence of the clone selected in step 125 for optimal performance of the enzyme;
g) a seventh step of cloning 135 including cloning the resultant mutant genes in a cloning vector and checking their expression in an expression vector;
h) an eighth step of biotransformation 140 including employing the various mutant enzymes obtained in step of cloning 135 for biotransformation of the racemic nitrile substrates;
i) a ninth step of immobilization 145 including immobilizing the mutant enzymes onto solid supports using suitable immobilization techniques; and
j) a tenth step of evaluation of immobilized enzymes 150 including evaluating the immobilized mutant enzymes for biotransformation of nitrile substrates and their recycling capabilities.
3. The method 100 of preparation of modified nitrilases for biocatalysis as claimed in Claim 2, wherein in the first step of downloading the sequences 105, the polypeptide sequences for the nitrilase enzyme being downloaded from the protein database UniProt.
4. The method 100 of preparation of modified nitrilases for biocatalysis 100 as claimed in Claim 2, wherein in the third step of cloning and expression 115, the shortlisted sequences being subjected to cloning in vector selected from pTrc His2B, pET21a, pET28a, pGEX4T1, pGEX6P1 and expression of these sequences being carried out in E. coli BL21 (DE3).
5. The method 100 of preparation of modified nitrilases for biocatalysis as claimed in Claim 2, wherein in the sixth step of site directed mutagenesis 130,
the site directed mutagenesis being carried out based on homology modelling techniques for predicting the sites for mutagenesis, and
the point mutations being carried out at the specific sites in the nitrilase gene sequence from Variovorax paradoxus including
a) replacing threonine at position 135 by alanine, giving modified nitrilase, Vp-nit-T135A;
b) replacing threonine at position 135 by alanine and further replacing phenyl alanine at position 192 by threonine, giving modified nitrilase Vp-nit-F192T/T135A; and
c) replacing alanine at position 286 by isoleucine, giving modified nitrilase Vp-nit-A286I.
6. The method 100 of preparation of modified nitrilases for biocatalysis as claimed in Claim 2, wherein in the seventh step of cloning 135, the resultant mutant genes being cloned in pET28a vector, and their expression being checked in E. coli strains selected from E. coli BL21 (DE3), E. coli BL21 (DE3) star, E. coli BL21 (DE3) Rosetta.
7. The method 100 of preparation of modified nitrilases for biocatalysis as claimed in Claim 2, wherein in the eighth step 140 of biotransformation, the substrate being selected from racemic nitrile substrates consisting of aryl nitriles, aryl acetonitrile, aryl cyanohydrins and aryl acetaldehyde cyanohydrins.
8. The method 100 of preparation of modified nitrilases for biocatalysis as claimed in Claim 2, wherein in the ninth step of immobilization 145, the selected mutant enzymes having SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4 being immobilized using methods selected from adsorption or entrapment using suitable resins or clay minerals, enzyme crosslinking using suitable crosslinking agents, or making cross-linked protein coated microcrystals.
9. The method 100 of preparation of modified nitrilases for biocatalysis as claimed in Claim 2, wherein in the ninth step of immobilization 145, the selected mutant enzymes having SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4 being immobilized using ion exchange resins wherein the process of immobilization being carried out in the temperature range of 15 to 25°C using sodium phosphate buffer of pH 7.5, the enzyme loading being in the range of 400 to 800 U/g of beads, the immobilization efficiency in terms of the enzyme activity being >95%, and the immobilized nitrilase enzymes being stable for 1-2 months in the temperature range of 4 to 8°C.
10. The method 100 of preparation of modified nitrilases for biocatalysis as claimed in Claim 2, wherein in the tenth step of evaluation of immobilized enzymes 150, the immobilized enzymes being recycled multiple times using the same set of reaction conditions, the reactions with immobilized recycled enzymes demonstrating conversion in the range of 85 to 95%, the fresh enzyme loading of 5% in the subsequent recycles being sufficient for obtaining 85% conversion of the substrate, and the biocatalysts being able to withstand loading close to one mole of the nitrile substrate.
Dated this 16th day of January 2024.
For Hi Tech Biosciences India Private Ltd.

Mahurkar Anand Gopalkrishna
IN/PA-1862
(Agent for Applicant)