DESC:TECHNICAL FIELD:
The present invention is generally related to enzyme/protein engineering. Specifically, the present invention provides recombinant transaminase polypeptides, fusion protein comprising the same, and process of designing the same. Further, the present invention provides a process of preparing chiral amines catalyzed by the recombinant transaminase polypeptides. Also, the present invention provides uses of the recombinant transaminase polypeptides.
BACKGROUND ART:
Transaminases are enzymes that enable transfer of an amine group from amino group donor to a keto group of an acceptor substrate. The cofactor, pyridoxal phosphate (PLP) facilitates catalysis with transaminase polypeptides. Transaminases have often been used in the synthesis of chiral amines and amino acids with high enantiomeric purity. ?-Transaminases, a subclass of transaminase enzymes produce chiral amines by resolution of the racemic amine or by asymmetric synthesis starting from the prochiral ketone, which is an amine acceptor, and uses an amine donor compound. Amongst different classes of transaminases, ?-transaminases show higher selectivity and activity towards ketoester substrates.
Chiral amines are valuable substrates for the production of a large number of bioactive compounds with pharmacological properties. Various new chemical entities (NCEs) among FDA approved drugs contain chiral amine moieties. Sitagliptin ((3R)-3-amino-1-[3-(trifluoromethyl)-6,8-dihydro-5H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one) is one such oral anti-hyperglycemic drug with chiral amine moiety, and belongs to the gliptin class of anti-diabetics, characterized by their dipeptidyl- peptidase-4 (DPP-4) inhibiting activity. It was developed and marketed by Merck & Co. This enzyme-inhibiting drug is used either alone or in combination with other oral anti-hyperglycemic agents (such as metformin or a thiazolidinedione) for treatment of diabetes mellitus type 2.
The original synthesis of Sitagliptin required the asymmetric hydrogenation of an enamine at high pressure using a rhodium-based chiral catalyst. The major drawbacks in this process are inadequate stereoselectivity and the rhodium catalyst contamination in the downstream processing, necessitating additional purification steps, at the expense of both enantiomeric excess (“ee”) and chemical purity.
There are currently existing technologies leveraging a transaminase scaffold along with various protein engineering techniques, which have improved the efficiency of Sitagliptin manufacturing by enzymatic catalysis.
The prior approaches to synthesize chiral amines use expensive metal catalysts along with extreme reaction conditions such as high pressure and high temperature, leading to lower product yields with longer reaction times. Moreover, these processes entail cumbersome purification along with generation of higher waste components rendering these processes non-environment friendly.
Patent numbers, US219372P and US2016/0304843A1, disclose transaminases and reactions catalyzed by them. However, these documents fail to disclose S Transaminase which results in R form of chiral amines. US219372P discloses conversion using R Transaminase which results in R product. As regards Patent number US2016/0304843A1, it has been shown by the present inventors that when polypeptide disclosed, is employed on the substrate of the present invention then it resulted in an S product, which is an inactive form, and is completely different from the present invention i.e. chiral amines in R form.
Use of substrate specific, selective transaminases is useful in industrial scale production of several compounds including compounds of pharmaceutical, nutraceutical interest. The current invention is directed to recombinant transaminases and biocatalytic synthetic routes for enantiopure chiral amines which are key intermediates for the synthesis of Sitagliptin, employing a transaminase enzyme. The recombinant transaminase of the invention leads to reduced cost of synthesis of enantiopure chiral amines along with high yield, purity and enantiomeric excess. The recombinant transaminases enable higher conversion of substrate into product requiring less enzyme loading. The synthesized intermediate can also be leveraged for synthesis of two other gliptins namely, Evogliptin and Retagliptin, for thetreatment of type 2 diabetes.

SUMMARY:
The present invention recombinant transaminase polypeptide which comprises an amino acid substitution selected from the group consisting of:
i. Phe55 substituted with Cys or Met or any Aliphatic, Polar or non-polar or Aromatic amino acid;
ii. Glu93 substituted with Pro or any Aliphatic, Acidic, or Polar or non-polar amino acid;
iii. Gly151 substituted with Ala, or any Aliphatic, non-polar, or Polar amino acid;
iv. Thr159 substituted with Ala, or any aliphatic or non-polar or polar amino acid;
v. Glu166 substituted with Ala, or any Aliphatic, Acidic, Polar or non-polar, amino acid;
vi. Val262 substituted with Met, or any Aliphatic, non-polar amino acid;
vii. Ser298 substituted with Gly, or any Aliphatic or polar amino acid;
viii. Ile427 substituted with Thr, or any Polar, or Aliphatic amino acid;
ix. Val385 substituted with Leu, or Ile, or any non-polar or Aliphatic amino acid;
x. Leu428 substituted with Ile, or any aliphatic or non-polar residue;
xi. Leu58 substituted with Ser or Thr or any aliphatic polar amino acid;
xii. Gly154 substituted with Val or Ala or Ile or Thr or any aliphatic amino acid; and
xiii. Gly323 substituted with Val or Ala or Ile or any aliphatic non-polar amino acid.
Further, the present invention provides a fusion protein comprising the same. Also, a process of designing polypeptide of the present invention is disclosed which process is performed in silico using QZyme WorkbenchTM. Furthermore, the present invention provides a process of preparing chiral amines catalyzed by the recombinant transaminase polypeptides wherein conversion rate of the recombinant transaminase polypeptides is high compared to wild type. Also, the present invention provides uses of the recombinant transaminase polypeptides.
BRIEF DESCRIPTION OF FIGURES AND DRAWINGS:
The accompanying drawings illustrate some of the embodiments of the present invention and, together with the descriptions, serve to explain the invention. The drawing (s) has been provided by way of illustration and not by way of limitation.

Figure 1 Shows the scheme of synthesis of BOC Butanoic acid ((R)-3-(tert- Butoxycarbonylamino)-4-(2,4,5-trifluorophenyl)butanoic acid). The present invention represents the enzymatic step required to convert beta keto ester to chiral amine
DETAILED DESCRIPTION:
The present invention is now described with reference to the tables/figures and specific embodiments, including the best mode contemplated by the inventors for carrying out the invention. This description is not meant to be construed in a limiting sense, as various alternate embodiments of the invention will become apparent to persons skilled in the art, upon reference to the description of the invention. It is therefore contemplated that such alternative embodiments form part of the present invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Some of the terms are defined briefly here below; the definitions should not be construed in a limiting sense.
The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.
The term “plurality” as used herein is defined as “one, or more than one”. Accordingly, the terms “one”, “at least one” would all fall under the definition of “plurality”.
Whether or not a certain feature or element was limited to being used only once, either way it may still be referred to as “one or more features” or “one or more elements” or “at least one feature” or “at least one element”.
Definitions:
As used herein the term "amine donor" refers to amine compounds such as isopropyl amine, amino acids like alanine. The “amine donor” can be accepted by a transaminase polypeptide, and which can supply an amino group.
As used herein the term "Sitagliptin" refers to a compound with CAS no. 486460-32-6. It also includes salts thereof like Sitagliptin phosphate monohydrate (CAS no. 654671-77-9). Chemically it is (R)-3-amino-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one.
As used herein the term "Native" or "Naturally occurring" or "wild type" polypeptide or polynucleotide sequence refers to naturally occurring or wild-type polypeptide or polynucleotide sequence that exists in nature in an organism that can be isolated from a source in nature, and which has not been intentionally modified by human manipulation.
As used herein the term "Recombinant" refers to polypeptide sequences or polynucleotide sequences encoding them that have been modified in their sequence to the naturally occurring or wild type sequences. These also include polypeptide sequences or polynucleotide sequences encoding them that have the same sequences as naturally occurring sequences but are obtained by means of recombinant techniques. As used herein the term "enantiomeric excess" refers to a parameter for measuring purity of chiral compounds and indicates abundance of one enantiomer over the other.

As used herein, "enzyme activity" is defined in units. 1 unit of enzyme (U, used interchangeably herein with IU or international units) is the amount of enzyme that catalyzes the reaction of 1 µmol of substrate per minute.

The fusion protein of the present invention comprises of tags. Tags are attached to proteins for various purposes, e.g. in order to ease purification, to assist in the proper folding in proteins, to prevent precipitation of the protein, to alter chromatographic properties, to modify the protein or to mark or label the protein. The tag used in the present invention is 6X His-tag.
The substrates used for activity assay can be any substrates known for the given enzymes. Moreover, the enzymes used can be from any of the known sources.
As used herein, the terms “Percentage of sequence identity,” “percent identity,” and “percent identical” are used interchangeably and herein refer to identity between a pair of polynucleotide or polypeptide sequences which is reflected as a quantitative measure. It measures the number of identical residues (“matches”) in relation to the length of the alignment of the polynucleotide or polypeptide sequences compared. "Percent identity" as used herein can be measured by an indigenous technology QZyme WorkbenchTM . This technology is a fully automated proprietary in silico protein engineering platform, evolved by integrating open-source computational chemistry and biology/bioinformatics tools in combination with customized algorithms and scripts. Further, this software is capable of tackling several important aspects of protein modelling and engineering including, but not limited to, structural refinement, ligand docking, conformational sampling, estimating substrate binding affinity, modelling catalytic reaction, identifying mutable hotspots, further hotspot optimization.

As used herein, the terms "Transaminase" and "Aminotransferase" are used interchangeably herein and refer to enzymes that catalyze the transfer of an amino group from an amino donor to an amino acceptor. This class of enzymes belongs to pyridoxal-phosphate dependent aminotransferase family. It includes ?-transaminases, a class of enzymes that belong to Class III aminotransferases which catalyze the transfer of an amino group from a non-a position amino acid, or an amine compound with no carboxylic group, to an amino acceptor. ?-transaminases have been often used in synthesis of various building blocks in chemical and pharmaceutical industry.

The present invention encompasses recombinant transaminase polypeptides and a process of preparing chiral amines catalyzed by the recombinant transaminase polypeptides.

Transaminase polypeptides catalyze the conversion of Beta-ketoester compounds to enantiopure chiral amines. Among different classes of transaminases, ?-transaminases show higher selectivity and activity towards ketoester substrates.
The recombinant transaminase polypeptides disclosed in the current invention enable highly selective, improved catalytic synthesis of chiral amines with high enantiomeric purity, high product yield and at high conversion rate. Further, the conversion of the substrate of present invention to R-product is not well characterized in prior art using the recombinant transaminase polypeptides of the present invention. The recombinant transaminase polypeptides disclosed herein have high enzymatic activity compared to the naturally occurring or a base variant.

Transaminases found in nature lack the substrate specificity and selectivity to enable catalysis with high product yield and purity.

Chiral amines, in general, are compounds that are of immense interest and value across diverse fields including agriculture, pharmaceutical, nutraceutical domains.

Transaminase polypeptides of the invention enable efficient synthesis of chiral amines. One such chiral amine, BOC butanoic acid ((R)-3-(tert- Butoxycarbonylamino)-4-(2,4,5-trifluorophenyl)butanoic acid.), is an intermediate in the synthesis of gliptins like Sitagliptin, Evogliptin, and Retagliptin.

In a first aspect, the present invention provides recombinant transaminase polypeptide which comprises an amino acid substitution selected from the group consisting of:
i. Phe55 substituted with Cys or Met or any Aliphatic, Polar or non-polar or Aromatic amino acid;
ii. Glu93 substituted with Pro or any Aliphatic, Acidic, or Polar or non-polar amino acid;
iii. Gly151 substituted with Ala, or any Aliphatic, non-polar, or Polar amino acid;
iv. Thr159 substituted with Ala, or any aliphatic or non-polar or polar amino acid;
v. Glu166 substituted with Ala, or any Aliphatic, Acidic, Polar or non-polar, amino acid;
vi. Val262 substituted with Met, or any Aliphatic, non-polar amino acid;
vii. Ser298 substituted with Gly, or any Aliphatic or polar amino acid;
viii. Ile427 substituted with Thr, or any Polar, or Aliphatic amino acid;
ix. Val385 substituted with Leu, or Ile, or any non-polar or Aliphatic amino acid;
x. Leu428 substituted with Ile, or any aliphatic or non-polar residue;
xi. Leu58 substituted with Ser or Thr or any aliphatic polar amino acid;
xii. Gly154 substituted with Val or Ala or Ile or Thr or any aliphatic amino acid; and
xiii. Gly323 substituted with Val or Ala or Ile or any aliphatic non-polar amino acid.
In one embodiment, said polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 to 45.
In another embodiment, said polypeptide is represented amino acid sequences selected from SEQ ID No. 1 to 45.
In yet another embodiment, said polypeptide further comprises an amino acid substitution selected from the group consisting of: Phe55Cys, Phe55Met, Glu93Pro, Gly151Ala, Thr159Ala, Glu166Ala, Val262Met, Ser298Gly, Ile427Thr, Val385Leu, Val385Ile and Leu428Ile, Leu58Ser, Leu58Thr, Gly154Val, Gly154Ala, Gly154Ile, Gly323Val, GLy323Ala, GLy323Ile.
In a second aspect, the present invention provides a polynucleotide encoding the recombinant transaminase polypeptide of the present invention, wherein said polynucleotide is selected from SEQ ID Nos. 46-56.
In a third aspect, the present invention provides a fusion protein comprising the recombinant transaminase polypeptide of the present invention and 6X His-tag.
In a fourth aspect, the present invention provides an expression vector comprising the polynucleotide of the present invention.
In a fifth aspect, the present invention provides a host comprising the expression vector of the present invention, wherein said host cell in a bacterial cell.
In a sixth aspect, the present invention provides a process of preparing a compound of structural formula I,

(I)

having the indicated stereochemical configuration at the stereogenic center marked with an *; in an enantiomeric excess of at least 70 % over the opposite enantiomer, wherein

X is OR2;

R1 is alkyl, aryl, heteroaryl, benzyl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, carbocyclic, heterocyclic, optionally being unsubstituted or substituted with one to five fluorine, or any halogen; and
R2 is alkyl, aryl, heteroaryl, benzyl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, optionally being unsubstituted or substituted; wherein the process comprising the step of contacting a prochiral ketone compound of structural Formula (II):

(II)
X is OR2;

R1 is alkyl, aryl, heteroaryl, benzyl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, carbocylclic, heterocyclic, optionally being unsubstituted or substituted with one to five fluorine, or any halogen; and
R2 is alkyl, aryl, heteroaryl, benzyl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, optionally being unsubstituted or substituted;
with the transaminase polypeptide of the present invention; in the presence of an amino group donor; and wherein the conversion is such that the product obtained has enantiomeric form “R”.

In an embodiment, R1 in the compound of structural formula I is a benzyl group, and wherein the phenyl group of benzyl is unsubstituted or substituted with one to five fluorines.

In another embodiment, the compound of formula I is (R)-3-amino-4- (2,4,5-trifluorophenyl) butyric acid methyl ester.

In yet another embodiment, the compound of formula I is produced in the range of 60-100% enantiomeric excess.

In an embodiment, the amount of recombinant transaminases required for conversion of compound of structural formula II to compound of formula I is 1.5 mg/mL

In another embodiment, the rate of conversion of compound of formula II to compound of formula I ranges from 10% to 95%.

In yet another embodiment, the amino group donor is selected from o-Xylylenediamine and isopropyl amine.

In an embodiment, said process further comprises a step of protecting the amino group by an amino protecting group selected from formyl, acetyl, trifluoro acetyl, benzyl, benzyloxy carbonyl ("CBZ"), tert-butoxy carbonyl ("BOC"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl (“SES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), and nitroveratryloxycarbonyl (“NVOC”).

In another embodiment, the amino protecting group is a BOC protecting group.

In yet another embodiment, the BOC protected chiral amine is (R)-3-(tert-Butoxycarbonylamino)-4-(2,4,5-trifluorophenyl)butanoic acid.

In an embodiment, the organic solvent is selected from dimethylsulfoxide (DMSO), Dimethylformamide (DMF), methyl tert- butyl ether (MTBE), isopropyl acetate, methanol, ethanol or propanol.

In another embodiment, the solvent is a mixture of water and dimethylsulfoxide (DMSO),

In another embodiment, the prochiral ketone of structural formula II is 3-oxo-4-(2,4,5 trifluorophenyl)butyricacid methyl ester.

In an embodiment, the process of preparing the compound of formula I further comprises the step of reacting the compound of formula I with 3-(trifluoromethyl)-5,6,7,8-tetrahydro [1,2,4]triazolo[4,3-a]pyrazine, 3-(trifluoromethyl)-6,8-dihydro-5H-imidazo[1,5-a]pyrazine, and3-[(2-methylpropan-2-yl)oxymethyl]piperazin-2-one to make Sitagliptin, Retagliptin, and Evogliptin, respectively.

In a seventh aspect, the present invention provides a method of designing the recombinant transaminase polypeptide of the present invention, wherein said method is performed in silico using QZyme WorkbenchTM and comprising the steps of
- structural refinement and modelling;
- ligand docking;
- conformational sampling;
- estimating substrate binding affinity;
- modelling catalytic reaction;
- identifying mutable hotspots and further optimizing the hotspot, and wherein said method provides an efficient recombinant transaminase polypeptide of the present invention having increased catalytic activity.

In eighth aspect, the present invention provides use of the recombinant transaminase polypeptide in manufacturing of chiral amines, wherein said chiral amines are essential intermediates in production of pharmaceutical drugs selected from Sitagliptin, Evogliptin and Retagliptin, which treats diabetes mellitus type 2.

In an embodiment, recombinant transaminase polypeptide in the form of whole cell, crude extract, isolated polypeptide, or purified polypeptide alone or in combination with another recombinant transaminase polypeptide.

In ninth aspect, the present invention provides a method of treating diabetes mellitus type 2, wherein said method comprising administering a subject a drug manufactured using intermediates obtained from the process as defined in the present invention.

The present disclosure with reference to the following accompanying examples describes the present invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. It is understood that the examples are provided for the purpose of illustrating the invention only, and are not intended to limit the scope of the invention in any way.
EXAMPLES:
Example 1: Method of Incorporating Mutations
An in silico enzyme engineering framework was used (QZyme WorkbenchTM) to engineer the transaminase enzymes to achieve high catalytic efficiency. The framework carries out different aspects of in silico protein engineering including structural refinement and modelling, ligand docking, conformational sampling, estimating substrate binding affinity, modelling catalytic reaction, identifying mutable hotspots, further hotspot optimization.
Three-dimensional structure of the protein (?-transaminase) was modelled to an appropriate oligomeric state. Additional structural refinement was carried out in this second step to ensure that the modelled structure satisfied catalytically competent conformation (open vs closed state). The modelled structure thus obtained was used to model the near-attack conformation of the substrate in the enzyme active site (Michaelis complex) by implementing several docking algorithms. In this step, the bottle neck of the enzymatic conversion of the substrate was also determined. In particular, three areas were investigated in detail:
a) Dynamics of Michaelis complex through classical molecular dynamics simulation to assess the stability of Michaelis complex;
b) Non-equilibrium molecular dynamics simulation to study the substrate entry, product exit and to estimate associated free energy barriers, and
c) The rate-limiting step of the reaction using hybrid quantum mechanics/ molecular mechanics (QM/MM) approach. The energy barriers associated with each step are further compared to identify the step that has the highest activation barrier (rate-limiting).
To address the bottleneck of enzymatic conversion identified in the previous step, the next step included discovery of key functional residues and evaluation of their mutability in an effort to improve the enzyme function. The step further incorporated a rapid screening method to know the contribution of each amino acid and all possible amino acid substitutions to the enzyme’s function and stability. Sequence analysis as well as contact score analysis was given priority for selection of hotspots. Hotspots were selected based on partial conserved residues obtained through Delta- BLAST using NR database. The conserved residues obtained through contact score analysis was selected. All the conserved residues were neglected whereas the partially conserved residues were checked to create a focused library. Apart from sequence alignment, stability analysis was carried out for selecting hotspots. The common mutations obtained through sequence alignment, and stability analysis were shortlisted for the designing step. Thus, a huge library of variants is created, followed by creation of a focused library.
In the designing step, the rate-limiting step (previously referred to as the bottleneck) was modelled for each of the variants belonging to the focused library, and compared with the variants of the wild type enzyme. In addition to that, binding energy calculations were then performed for all the variants from the focused library. The purpose of the designing step is to reduce the false positives and to increase the quality of the focused library. As a final outcome, top variants were shortlisted for further validation through lab experiments.
TABLE 1: Lists of the Mutations in Recombinant Transaminase Polypeptides:
Sr. no Sequence ID Mutations#
1 SEQ ID NO:1 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala
2 SEQ ID NO:2 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Thr159Ala, Val385Leu
3 SEQ ID NO:3 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Val385Ile
4 SEQ ID NO:4 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Thr159Ala, Glu166Ala
5 SEQ ID NO:5 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Phe55Met
6 SEQ ID NO:6 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Thr159Ala, Glu166Ala, Val385Ile, Ile427Thr
7 SEQ ID NO:7 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Gly151A, Val262Met, Leu428Ile
8 SEQ ID NO:8 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro
9 SEQ ID NO:9 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Val262Met
10 SEQ ID NO:10 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Gly151Ala
11 SEQ ID NO:11 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Ile427Thr
12 SEQ ID NO:12 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Leu428Ile
13 SEQ ID NO:13 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Ser298Gly
14 SEQ ID NO:14 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Phe55Cys, Gly151Ala, Glu166Ala, Val262Met, Ser298Gly, Ile427Thr
15 SEQ ID NO:15 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Val385Ile, Gly154Ala, Leu58Ser
16 SEQ ID NO:16 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Val385Ile, Gly154Ala, Leu58Thr
17 SEQ ID NO:17 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Val385Ile, Gly154Ile, Leu58Ser
18 SEQ ID NO:18 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Val385Ile, Gly154Ile, Leu58Thr
19 SEQ ID NO:19 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Gly154Ala, Leu58Thr
20 SEQ ID NO:20 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Gly154Ile, Leu58Thr
21 SEQ ID NO:21 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Thr159Ala, Val385Leu, Leu58Ser
22 SEQ ID NO:22 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Val385Ile, Leu58Ser
23 SEQ ID NO:23 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Val385Ile, Leu58Thr
24 SEQ ID NO:24 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Val385Ile, Gly323Val
25 SEQ ID NO:25 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala,Val385Ile, Gly154Ala
26 SEQ ID NO:26 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Val385Ile, Gly154Thr
27 SEQ ID NO:27 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Ile427Thr, Leu58Ser
28 SEQ ID NO:28 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Gly151Ala, Leu58Ser
29 SEQ ID NO:29 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Gly151Ala, Leu58Thr
30 SEQ ID NO:30 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Gly151Ala, Gly323Val
31 SEQ ID NO:31 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Gly151Ala, Gly154Val
32 SEQ ID NO:32 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Leu58Ser
33 SEQ ID NO:33 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Leu58Thr
34 SEQ ID NO:34 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Gly323Val
35 SEQ ID NO:35 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Gly154Val
36 SEQ ID NO:36 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Gly154Ala
37 SEQ ID NO:37 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Gly154Ile
38 SEQ ID NO:38 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Glu93Pro, Phe152Trp
39 SEQ ID NO:39 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Phe55Met, Leu58Ser
40 SEQ ID NO:40 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Phe55Met, Leu58Thr
41 SEQ ID NO:41 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Phe55Met, Gly323Val
42 SEQ ID NO:42 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Phe55Met, Gly154Val
43 SEQ ID NO:43 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Thr159Ala, Val385Leu, Leu58Ser, Gly323Val
44 SEQ ID NO:44 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Thr159Ala, Val385Leu, Leu58Ser, Gly323Val, Gly154Val
45 SEQ ID NO:45 Tyr59Trp, Tyr87Phe, Tyr152Phe, Thr231Ala, Phe55Met, Thr159Ala, Val385Leu, Leu58Ser
# The mutations in the present invention are designed from the wild type sequence of UniProt ID: Q1GD43.
Table 2 below, lists the polynucleotides of the present invention and the recombinant transaminase polypeptides which they encode:
Table 2:
Sr. no. Polynucleotide Sequences Recombinant transaminase polypeptide sequences
1 SEQ ID NO. 46 SEQ ID NO. 1
2 SEQ ID NO. 47 SEQ ID NO. 2
3 SEQ ID NO. 48 SEQ ID NO. 3
4 SEQ ID NO. 49 SEQ ID NO. 4
5 SEQ ID NO. 50 SEQ ID NO. 5
6 SEQ ID NO. 51 SEQ ID NO. 7
7 SEQ ID NO. 52 SEQ ID NO. 8
8 SEQ ID NO. 53 SEQ ID NO. 9
9 SEQ ID NO. 54 SEQ ID NO. 10
10 SEQ ID NO. 55 SEQ ID NO. 11
11 SEQ ID NO. 56 SEQ ID NO. 12

Example 2: Protein overexpression:
The plasmids of wildtype and mutants were transformed into E. coli BL21(DE3) competent cell. Single colony was inoculated to LB broth containing ampicillin (100 µg/mL). 2% of the inoculum was inoculated to TB broth containing ampicillin (100 µg/mL). Cells were grown at 37°C till OD600 reaches 0.8-1.0. cultures were induced with 0.5mM IPTG and grown further overnight at 25°C. Expressions were analysed via SDS-PAGE and total protein was estimated by Bradford method with BSA (Bovine Serum Albumin) as standard.
Example 3: Protein purification:
Pellet was resuspended in 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl and 0.1 mM PLP and lysed by sonication. Lysate was centrifuged and soluble fraction recovered was incubated with Ni-NTA beads pre-equilibrated with Tris buffer (pH 7.5) at 4 °C. Ni-NTA beads were subsequently washed with 50 mM Tris buffer (pH 7.5) containing 300 mM NaCl, 5 mM imidazole and 0.1 mM PLP. Beads are again washed with 50 mM Tris buffer (pH 7.5) containing 300 mM NaCl, 10 mM imidazole and 0.1 mM PLP. Protein elution was carried out using 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl, 200 mM imidazole and 1.0 mM PLP. Elution fraction obtained was desalted via buffer exchange with 75 mM Tris buffer (pH 7.5) containing 150 mM NaCl using 30 kDa Molecular weight centrifugal concentrator.
Example 4: Biochemical Assay:
a. Assay Conditions
The assay is performed with a total reaction mixture of 1.0 ml in a 2.0 ml vial. The reaction is carried out with an optimized Substrate: Amine donor ratio of 1:5. The working concentration of the reaction components is given below:
Table 3:
Reagent Working Concentration
Water To make up the final volume
Beta keto ester (substrate) 10 mM
Isopropylamine (amine donor) 50 mM
Enzyme 1.5 mg/mL
DMSO 5%
HEPES buffer, pH 7.5 1 M containing 1 mM Pyridoxal phosphate (PLP)

Reaction mixture was incubated at 40 °C for 48 h with continuous mixing at 1000 rpm in a thermomixer
Example 4: Quantitative Analysis of assay
a. RP-HPLC
An aliquot (100 µl) was quenched with 6 N HCl (10 µl) and diluted with 220 µl methanol (containing 3.75 mM benzoic acid) and shaken. The mixture was filtered over a syringe filter (0.2 µm) and subjected to C18 HPLC measurement.

Table 4:
Column Phenomenex Gemini-NX – C18 (250 x 3.0 mm), 3 µm
Mobile phase A: Water (0.1% Formic acid), B: Acetonitrile
Gradient flow needs to be followed as below:
Gradient Programming Time (min) Mobile Phase (A) % Mobile Phase (B) %
0 80 20
5 80 20
8 5 95
12 5 95
14 80 20
15 80 20
Diluent Mobile phase
Detection UV and QDa detector; UV at 210 and 254 nm
Flow rate 0.5 mL/min
Column temperature 40 °C
Total Run time/ sample 15 min

b. CHIRAL-GC
Lyophilized samples were treated with 500 µL AcCl at 40 °C for 2 hours. The AcCl was subsequently left to evaporate at 50 °C for approx. 30 minutes. The mostly solidified (gummy viscous) residue was extracted with 50 µL MeCN which was done by repeated washing of the solid material. The usually orange suspension was transferred into micro-Eppendorf tubes and centrifuged. The supernatant was measured using the chiral GC.

Table 5: Temperature profile for Chiral GC
Temperature (°C) Ramp [ °C /min] Hold [min]
150 - 1
150 0.5 -
200 20 -
220 - 15

GC analysis was performed on a BGB175 column gamma cylcodextrixn column (50% 2,3-diacetyl-6-tert-butyldimethylsilyl-gamma-cyclodextrin dissolved in BGB-1701 [14% cyanopropylphenyl-,86% methylpolysiloxane]). The gas flow was set to 1ml/min, detection was done using an FID detector.
Results:
The table 6 below includes results for recombinant transaminase polypeptides which were tested in-silico to determine its efficiency in converting a product in its R and S forms. The test conducted and results obtained extends to all the 45 polypeptides described in Table 1.

Table: 6 shows in silico conversion efficiency of the transaminases of the present invention into R and S form of product:

Sl. No SEQ ID NO % R product Conversion % S product Conversion
1 SEQ ID NO:4 20 80
2 SEQ ID NO:6 68 32
3 SEQ ID NO:7 100 0
4 SEQ ID NO:9 0 100
5 SEQ ID NO:14 21 79
6 SEQ ID NO:15 68 32
7 SEQ ID NO:16 53 47
8 SEQ ID NO:17 33 67
9 SEQ ID NO:18 67 33
10 SEQ ID NO:19 35 65
11 SEQ ID NO:20 85 15
12 SEQ ID NO:21 83 17
13 SEQ ID NO:22 98 2
14 SEQ ID NO:23 64 36
15 SEQ ID NO:24 0 100
16 SEQ ID NO:25 0 100
17 SEQ ID NO:26 97 3
18 SEQ ID NO:27 3 97
19 SEQ ID NO:28 98 2
20 SEQ ID NO:29 0 100
21 SEQ ID NO:30 94 6
22 SEQ ID NO:31 19 81
23 SEQ ID NO:32 58 42
24 SEQ ID NO:33 11 89
25 SEQ ID NO:34 4 96
26 SEQ ID NO:35 0 100
27 SEQ ID NO:36 4 96
28 SEQ ID NO:37 67 33
29 SEQ ID NO:38 27 73
30 SEQ ID NO:39 13 87
31 SEQ ID NO:40 70 30
32 SEQ ID NO:41 96 4
33 SEQ ID NO:42 0 100
34 SEQ ID NO:43 0 100
35 SEQ ID NO:44 96 4
36 SEQ ID NO:45 1 99
37 SEQ ID NO:46 8 92

The table 7 below includes results for recombinant transaminase polypeptides which were tested in vitro to determine its efficiency in converting a product in its R and S forms and also represents results for product conversion rate. The test conducted and results obtained extends to all the 45 polypeptides described in Table 1.
Table 7 shows in vitro efficiency of the transaminases of the present invention.
Sl.No. Sequence ID Product Conversion Enantiomeric excess
%R %S
1 SEQ ID NO:1 65% 0 100
2 SEQ ID NO:2 10% 0 100
3 SEQ ID NO:3 55% 0 100
4 SEQ ID NO:4 8% 0 100
5 SEQ ID NO:5 92% 60 40
6 SEQ ID NO:7 7% 40 60
7 SEQ ID NO:8 90% 40 60
8 SEQ ID NO:9 18% ND ND
9 SEQ ID NO:10 65% 40 60
10 SEQ ID NO:11 10% 0 100
11 SEQ ID NO:12 15% ND ND ,CLAIMS:1. A recombinant transaminase polypeptide, wherein said polypeptide comprises amino acid substitutions selected from the group consisting of:
i) Phe55 substituted with Cys or Met or any Aliphatic, Polar or non-polar or Aromatic amino acid;
ii) Glu93 substituted with Pro or any Aliphatic, Acidic, or Polar or non-polar amino acid;
iii) Gly151 substituted with Ala, or any Aliphatic, non-polar, or Polar amino acid;
iv) Thr159 substituted with Ala, or any aliphatic or non-polar or polar amino acid;
v) Glu166 substituted with Ala, or any Aliphatic, Acidic, Polar or non-polar, amino acid;
vi) Val262 substituted with Met, or any Aliphatic, non-polar amino acid;
vii) Ser298 substituted with Gly, or any Aliphatic or polar amino acid;
viii) Ile427 substituted with Thr, or any Polar, or Aliphatic amino acid;
ix) Val385 substituted with Leu, or Ile, or any non-polar or Aliphatic amino acid;
x) Leu428 substituted with Ile, or any aliphatic or non-polar residue;
xi) Leu58 substituted with Ser or Thr or any aliphatic polar amino acid;
xii) Gly154 substituted with Val or Ala or Ile or Thr or any aliphatic amino acid; and
xiii) Gly323 substituted with Val or Ala or Ile or any aliphatic non-polar amino acid.

2. The recombinant transaminase polypeptide as claimed in claim 1, wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 to 45.
3. The recombinant transaminase polypeptide as claimed in claim 1, wherein said polypeptide is represented amino acid sequences selected from SEQ ID No. 1 to 45.
4. A polynucleotide encoding the recombinant transaminase polypeptide as claimed in claims 1-3, wherein said polynucleotide is selected from SEQ ID Nos. 46-56.
5. A fusion protein comprising a recombinant transaminase polypeptide as claimed in claims 1-3 and 6X His-tag.
6. An expression vector comprising the polynucleotide as claimed in claim 4.
7. A host comprising the expression vector as claimed in claim 6, wherein said host cell is a bacterial cell.
8. A process of preparing a compound of structural formula I,

(I)
having the indicated stereochemical configuration at the stereogenic center marked with an *; in an enantiomeric excess of at least 70 % over the opposite enantiomer, wherein

X is OR2;

R1 is alkyl, aryl, heteroaryl, benzyl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, carbocyclic, heterocyclic, optionally being unsubstituted or substituted with one to five fluorine, or any halogen; and
R2 is alkyl, aryl, heteroaryl, benzyl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, optionally being unsubstituted or substituted; wherein the process comprising the step of contacting a prochiral ketone compound of structural Formula (II):

(II)
X is OR2;

R1 is alkyl, aryl, heteroaryl, benzyl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, carbocyclic, heterocyclic, optionally being unsubstituted or substituted with one to five fluorine, or any halogen; and
R2 is alkyl, aryl, heteroaryl, benzyl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, optionally being unsubstituted or substituted;
with the recombinant transaminase polypeptide as claimed in claim 1;
in the presence of an amino group donor; and wherein the conversion is such that the product obtained has enantiomeric form “R”.

9. The process as claimed in claim 8, wherein R1 in the compound of structural formula I is a benzyl group, and wherein the phenyl group of benzyl is unsubstituted or substituted with one to five fluorines.
10. The process as claimed in claim 8, wherein the compound of formula I is (R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid methyl ester.
11. The process as claimed in claim 8, wherein the compound of formula I is produced in the range of 60-100% enantiomeric excess.
12. The process as claimed in claim 8-11, wherein the amount of recombinant transaminases required for conversion of compound of structural formula II to compound of formula I is 1.5 mg/mL
13. The process as claimed in claim 8, wherein the rate of conversion of compound of formula II to compound of formula I ranges from 10% to 95%.

14. The process as claimed in claim 8, wherein the amino group donor is selected from o-Xylylenediamine or isopropyl amine.
15. The process as claimed in claim 8, wherein said process further comprises a step of protecting the amino group by an amino protecting group selected from formyl, acetyl, trifluoro acetyl, benzyl, benzyloxy carbonyl ("CBZ"), tert-butoxy carbonyl ("BOC"), trimethylsilyl ("TMS"), 2- trimethylsilyl-ethanesulfonyl (“SES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), and nitro- veratryloxycarbonyl (“NVOC”).
16. The process as claimed in claim 15, wherein the amino protecting group is a BOC protecting group.
17. The process as claimed in claim 16, wherein the Boc protected chiral amine is (R)-3-(tert-Butoxycarbonylamino)-4-(2,4,5-trifluorophenyl)butanoicacid.
18. The process as claimed in claim 8, wherein the organic solvent is selected from dimethylsulfoxide (DMSO), Dimethylformamide (DMF), methyl tert-butyl ether (MTBE), isopropyl acetate, methanol, ethanol or propanol.
19. The process as claimed in claim 18, wherein the solvent is a mixture of water and dimethylsulfoxide (DMSO).
20. The process as claimed in claim 8, wherein the prochiral ketone of structural formula II is 3-oxo-4-(2,4,5-trifluorophenyl)butyric acid methyl ester.
21. The process as claimed in claim 8, wherein the process of preparing the compound of formula I further comprises the step of reacting the compound of formula I with , 3- (trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazine, 3-(trifluoromethyl)-6,8-dihydro-5H-imidazo[1,5-a]pyrazine, and 3-[(2-methylpropan-2-yl)oxymethyl]piperazin-2-one to make Sitagliptin, Retagliptin, and Evogliptin, respectively.
22. A method of designing the recombinant transaminase polypeptide as claimed in claim 1, wherein said method is performed in silico using QZyme WorkbenchTM and comprising the steps of
- structural refinement and modelling;
- ligand docking;
- conformational sampling;
- estimating substrate binding affinity;
- modelling catalytic reaction;
- identifying mutable hotspots and further optimizing the hotspot, and wherein said method provides an efficient recombinant transaminase polypeptide as claimed in claims 1-3, having increased catalytic activity.