CN105018440B

CN105018440B - A kind of transaminase and its application in sitagliptin intermediate is synthesized

Info

Publication number: CN105018440B
Application number: CN201410169882.4A
Authority: CN
Inventors: 罗煜; 丁时澄; 瞿旭东
Original assignee: Shanghai Yi Ke Lai Biological Medicine Science And Technology Co Ltd
Current assignee: Yikelai Taizhou Pharmaceutical Co ltd
Priority date: 2014-04-24
Filing date: 2014-04-24
Publication date: 2018-06-05
Anticipated expiration: 2034-04-24
Also published as: CN105018440A

Abstract

The present invention provides a kind of new transaminase and its gene, recombinant expression carrier and recombinant expression transformants, the recombinase and preparation method thereof containing the gene and the transaminase are turning carbonyls asymmetry the application during ammonia prepares activity chiral amine.The transaminase of the present invention is from mycobacterium (Mycobacterium vanbaalenii) PYR 1, the preparation applied to (R) 3 amino 4 (2,4,5 trifluorophenyl) methyl butyrate.Compared with other preparation methods, production concentration prepared by this method catalysis is high, and enantioselectivity is strong, and reaction condition is mild, easy to operate, is easy to the advantages such as amplification, therefore has good prospects for commercial application in the production of Sitagliptin phosphate.

Description

Transaminase and application thereof in synthesis of sitagliptin intermediate

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to transaminase, a recombinant expression vector and a recombinant expression transformant containing an encoding gene of the transaminase, an expressed recombinase, a preparation method of the recombinase, and application of the transaminase as a catalyst in asymmetric synthesis of a sitagliptin intermediate.

Background

The diabetes mellitus is metabolic diseases which are caused by insulin deficiency and weakened action due to insulin secretion change, or insulin activity is reduced, or under the combined influence of the insulin secretion change and the insulin deficiency, or under the combined effect of the insulin secretion change and the insulin activity reduction, the metabolic diseases are characterized by hyperglycemia and are accompanied by metabolic disorders of protein, sugar and fat, diabetes mellitus and complications thereof are the third position after cardiovascular diseases and tumors and are important diseases which are harmful to human health, international diabetes mellitus union predicts that the total number of patients is more than 4.35 hundred million people by 2030, China becomes one of countries with the highest rate of increase of the world diabetes mellitus, about 4000 million diabetes mellitus patients are the second position after India at present, the second position in the world is the fourth type of diabetes mellitus, the II diabetes mellitus accounts for more than 90 percent, the middle-aged and old people are mostly more than 30 years old, the body is not sensitive to insulin, in the medicine for treating the II diabetes mellitus, sulfonylurea secretion promoters, biguanide insulin enhancers, α -glucosidase inhibitors, thiazolidinedione insulin inhibitors, non-insulin enhancers, GLP inhibitors and the like are not sensitive to insulin inhibitors, the world insulin secretion promoters are not sensitive inhibitors, the insulin inhibitors are clinically approved by 1-7 insulin-glucose oxidase, the insulin-glucose-oxidase inhibitors are not-oxidase inhibitors, the pharmaceutically acceptable drugs are the pharmaceutically acceptable drugs which have the effect of the clinically-insulin-.

The method is characterized in that a chiral α -amino acid is induced by adopting a chiral source, and then β -amino acid is generated through diazotization to construct a required chiral center.

International patent WO2004087650 discloses the synthesis route of sitagliptin by merck corporation, using a chiral ruthenium catalyst to perform asymmetric hydrogenation on a ketone to construct a chiral alcohol, which is then converted into a chiral amine. In the synthesis method, asymmetric hydrogenation catalyzed by ruthenium is needed, the catalyst is expensive, the total yield is only 52%, high-pressure hydrogen is used in the process, and the stereoselectivity is not high.

International patent publication WO2005003135 discloses the Merck companyThe developed synthetic method of sitagliptin takes S-phenylglycinamide as a chiral auxiliary agent to induce catalytic hydrogenation to synthesize chiral amine. The route requires two catalytic hydrogenations, the platinum catalyst used in the first is expensive, and a large amount of Pd (OH) is needed for the second deprotection₂the-C catalyst is high in cost and has an ee value of 96%, and further recrystallization is needed.

International patent WO2007050485 discloses a synthesis method of sitagliptin by merck corporation, which adopts asymmetric hydrogenation of enamine by chiral rhodium catalyst to synthesize chiral amine, the yield reaches 84%, the ee value reaches 94%, but the method needs expensive rhodium chiral catalyst, and the removal and recovery are difficult.

U.S. Pat. No. 6,829,3507 discloses that the biocatalyst obtained by modifying arthrobacter-derived transaminase by Codexis company replaces the rhodium catalyst in the above process, the ee value of the transamination product reaches 99%, and the substrate charge is 100 g/L. However, because the water solubility of the substrate is poor, DMSO (dimethyl sulfoxide) with the concentration of up to 50% needs to be added for assisting dissolution, so that the post-treatment loss of the product is large, the solvent residue of the DMSO is high, the recovery is difficult, and the cost is high.

Chinese patent CN102838511 discloses a production method of sitagliptin intermediate in Zhejiang Haixiang pharmaceutical industry, which adopts Grignard reagent to carry out nucleophilic substitution on chiral epichlorohydrin, and then uses cyanide to carry out substitution hydrolysis to synthesize β -hydroxy acid, wherein the total yield of the method is only 40%, and the application is limited due to the adoption of virulent cyanide.

Chinese patent CN102485718 discloses a route for sitagliptin synthesis in the pharmaceutical industry of the zhejiang sea, by using methionine as chiral source, but with a yield of only 14%, even 55% based on 2,4, 5-trifluorobromobenzene.

Chinese patent CN103014081 discloses the conversion of 3-carbonyl-4- (2,4, 5-trifluorophenyl) -methyl butyrate to R-3-amino-4- (2,4, 5-trifluorophenyl) -methyl butyrate using transaminase by suzhou han enzyme company, but does not disclose the sequence of the specific transaminase and the cloning method.

Disclosure of Invention

Aiming at the reported problems of low yield, poor stereoselectivity, high catalyst price, difficult solvent recovery and the like in the reaction for asymmetrically synthesizing sitagliptin and an intermediate thereof, the invention provides an enzyme-chemical synthesis method for further synthesizing the sitagliptin by carrying out enzyme catalysis synthesis on R-3-amino-4- (2,4, 5-trifluorophenyl) -methyl butyrate by using transaminase with high catalytic activity, strong enantioselectivity and good substrate tolerance and solvent tolerance. Also provides a gene of the transaminase, a recombinant expression vector containing the gene, a recombinant expression transformant and a high-efficiency preparation method thereof, and application of the transaminase in catalyzing asymmetric transamination of carbonyl substrates to synthesize sitagliptin intermediates.

The invention solves the technical problems through the following technical scheme:

in a first aspect, the present invention provides an isolated protein which is a protein of (a) or (b) below:

(a) consisting of SEQ ID No: 2. 4, 6, 8 or 10;

(b) proteins derived from (a) and having at least 90% identity to the amino acid sequence of (a) by substitution, deletion or addition of one or several amino acid residues in the amino acid sequence of (a) and having transaminase activity, in particular those amino acid substitutions mentioned hereinafter.

SEQ ID No: 2 is derived from mycobacterium (Mycobacterium vanbaaleni) PYR-1, has the function of transaminase, and is a novel transaminase.

SEQ ID No: 2 can be isolated from Mycobacterium PYR-1, or isolated from an expression transformant which recombinantly expresses the protein, or synthesized.

The identity between two amino acid sequences or between two nucleotide sequences can be obtained by algorithms commonly used in the art, preferably by calculation using NCBI Blastp and Blastn software based on default parameters.

SEQ ID No: 2 and ATA117 derived from arthrobacter, 52%. The amino acid sequence of the invention is shown as SEQ ID No: 2 has an amino acid sequence identity of less than 70% with known transaminases, with significant variability.

The protein (b) is a protein which is derived from (a) and has at least 90% identity to the amino acid sequence of (a) by substituting, deleting or adding one or several amino acids in the amino acid sequence of (a) and has transaminase activity. Wherein, the number of "several" means 2 to less than 100, preferably less than 30, and most preferably less than 10. Such as a fusion protein to which an exocrine signal peptide has been added, it has been found that such a fusion protein also has transaminase activity. That is, as long as the protein derived from (a) has transaminase activity in the manner described above, the object of the present invention can be achieved. According to the invention, in the sequence as shown in SEQ ID No: 2 to obtain the protein (a) with the amino acid sequence shown in SEQ ID No: 4.6, 8 and 10, and also belongs to the protein (b); or the nucleotide sequence shown in SEQ ID No: 2. 4, 6, 8, 10, or by mutation of 1 to 20 amino acid residues, the above protein (b) can be obtained while still maintaining the transaminase activity.

Obtaining the sequence shown in SEQ ID No: 2, the inventor also modifies the wild transaminase and mutates amino acid residues at some sites of an active center into other amino acid residues so as to further enhance the catalytic performance of the transaminase. The active center is defined as about the vicinity of the substrate binding siteThe spherical space of (a).

Preferably, the (b) protein is a protein derived from (a) by substitution at one or more positions among amino acid residues 56, 68, 69, 72, 76, 96, 129, 131, 143, 157, 206, 216, 222, 230, 243, 276, 289, 291, 295, 304 of the amino acid sequence of (a) and having transaminase activity.

In a particular embodiment, SEQ ID No: 2, wherein the leucine at position 72 is substituted by cysteine, the valine at position 76 is substituted by alanine, the phenylalanine at position 129 is substituted by valine, and the alanine at position 291 is substituted by glycine, and the amino acid sequence has the amino acid sequence shown in SEQ ID No: 4 has certain activity on the substrate.

In a particular embodiment, SEQ ID No: 2, the amino acid sequence has SEQ ID No: 6 has greatly improved activity on the substrate.

In a particular embodiment, SEQ ID No: 2, SEQ ID No: 8 has high activity to the substrate and greatly improved thermal stability.

In a particular embodiment, SEQ ID No: 2, the glutamic acid at position 56 is substituted by serine, the glycine at position 68 is substituted by tyrosine, the histidine at position 69 is substituted by leucine, the leucine at position 72 is substituted by cysteine, the valine at position 76 is substituted by alanine, the aspartic acid at position 96 is substituted by alanine, the phenylalanine at position 129 is substituted by valine, the asparagine at position 131 is substituted by threonine, the glycine at position 143 is substituted by isoleucine, the tyrosine at position 157 is substituted by threonine, the serine at position 206 is substituted by valine, the threonine at position 216 is substituted by isoleucine, the alanine at position 222 is substituted by cysteine, the proline at position 230 is substituted by alanine, the alanine at position 243 is substituted by arginine, the serine at position 276 is substituted by proline, the threonine at position 289 is substituted by alanine, the alanine at position 291 is substituted by glycine, a substitution of threonine at position 295 to tryptophan and a substitution of proline at position 304 to threonine, to obtain SEQ ID No: 10 has high activity to the substrate, and the thermal stability and the solvent stability are greatly improved.

The second aspect of the present invention provides an isolated nucleic acid which is a nucleic acid of the following (1) or (2):

(1) consisting of SEQ ID No: 1. 3, 5, 7 or 9;

(2) a nucleic acid encoding the following protein (a) or (b):

(a) consisting of SEQ ID No: 2. 4, 6, 8 or 10;

(b) a protein derived from (a) and having at least 90% identity to the amino acid sequence of (a) by substitution, deletion or addition of one or several amino acid residues in the amino acid sequence of (a) and having transaminase activity.

SEQ ID No: 1 is derived from mycobacterium PYR-1, which can be obtained by separating from mycobacterium PYR-1 genome, or from recombinant expression vector or recombinant transformant containing the nucleic acid, or artificially synthesizing from whole gene.

In the present invention, SEQ ID No: 1. the genes indicated by 3, 5, 7 and 9 are named MvAT, and the total length is 1014 bp. Wherein the coding sequence (CDS) is from 1 st base to 1011 th base, the initiation codon is ATG, and the termination codon is TGA. The sequence has No intron, and the amino acid sequences of the encoded proteins are respectively shown in SEQ ID No: 2. 4, 6, 8 and 10.

As known to those skilled in the art, due to the degeneracy of the codons, the nucleotide sequence encoding SEQ ID No: 2. 4, 6, 8, 10 is not limited to the nucleic acid sequence of SEQ ID No: 1. 3, 5, 7 and 9. The nucleic acid sequence of the transaminase gene of the invention can also be a nucleic acid sequence encoding the amino acid sequence of SEQ ID No: 2. 4, 6, 8, 10, or a pharmaceutically acceptable salt thereof. In addition, a polynucleotide homologue can also be provided by appropriately introducing substitutions, deletions or insertions. Homologs of the polynucleotides of the invention can be identified by comparison of the nucleic acid sequences SEQ ID Nos: 1. 3, 5, 7, 9 by substitution, deletion or addition within a range where the enzyme activity is maintained.

SEQ ID No: 1. 3, 5, 7, 9 homologues are also referred to as promoter variants. The promoter or signal sequence preceding the nucleic acid sequence may be altered by one or more nucleic acid substitutions, insertions or deletions without these alterations having a negative effect on the function of the promoter. Furthermore, the expression level of the target protein can be increased by changing the sequence of the promoter or even completely replacing it with a more efficient promoter from a different species of organism.

SEQ ID No: 1. 3, 5, 7, 9 also refers to a polypeptide having an amino acid sequence that hybridizes under standard conditions to the amino acid sequence of SEQ ID No: 1. 3, 5, 7, and 9, and a polynucleotide having a nucleotide sequence that hybridizes with the polynucleotide having the sequence shown in any one of 3, 5, 7, and 9. Hybridization under standard conditions can be carried out, for example, in the manner described in the molecular cloning guidelines: cold Spring Harbor laboratory Press, a general protocol in Molecular Biology (Current Protocols in Molecular Biology). Specifically, hybridization can be carried out by hybridizing a membrane carrying the transcribed DNA or RNA molecule to be detected with a labeled probe in a hybridization buffer. The hybridization buffer comprises 0.1 wt% SDS, 5 wt% dextran sulfate, a dilution inhibitor of 1/20, and 2-8 XSSC. 20 XSSC is a solution of 3M sodium chloride and 0.3M citric acid. The hybridization temperature is 50-70 ℃. After incubation for several hours or overnight, the membranes were washed with washing buffer. The washing temperature is room temperature, more preferably the hybridization temperature. The composition of the washing buffer is 6 XSSC +0.1 wt% SDS solution, more preferably 5 XSSC +0.1 wt% SDS. When the membrane is washed with such a washing buffer, the DNA or RNA molecule can be recognized by the label on the probe that hybridizes to the DNA or RNA molecule.

In a third aspect, the invention provides a recombinant expression vector comprising a nucleic acid sequence according to the invention. It can be constructed by ligating the transaminase gene of the present invention to various expression vectors by a method conventional in the art. The expression vector may be any vector conventionally used in the art, such as a commercially available plasmid, cosmid, phage or viral vector, and the like, and preferably plasmid pET21 a. Preferably, the recombinant expression vector of the present invention can be prepared by the following method: the nucleic acid product obtained by PCR amplification and expression vector pET21a or its mutant are double-digested with restriction enzymes Nde I and EcoRI respectively to form complementary cohesive ends, and the cohesive ends are connected by T4DNA ligase to form recombinant expression plasmid pET21a-MvAT expression plasmid containing transaminase gene of the present invention.

In a fourth aspect of the present invention, there is provided a recombinant expression transformant comprising the recombinant expression vector of the present invention. Can be produced by transforming the recombinant expression vector of the present invention into a host cell. The host cell may be a host cell conventional in the art, as long as it is sufficient that the recombinant expression vector can stably self-replicate and the carried transaminase gene of the present invention can be efficiently expressed. Coli (e.coli) is preferred in the present invention, and e.coli BL21(DE3) is more preferred. The preferred genetically engineered strain of the invention, i.e., E.coli BL21(DE3)/pET21a-MvAT, was obtained by transforming the aforementioned recombinant expression plasmid pET21a-MvAT into E.coli BL21(DE 3). The transformation method can be selected from the conventional methods in the field, such as electrotransformation method, heat shock method, etc., preferably the transformation method is selected from the heat shock method, and the heat shock condition is preferably: the mixture was heat-shocked at 45 ℃ for 90 seconds.

The fifth aspect of the present invention provides a method for preparing a recombinant transaminase, which comprises the following steps: culturing the recombinant expression transformant of the present invention, and obtaining the recombinant transaminase from the culture.

Wherein, the recombinant expression transformant is obtained by transforming the recombinant expression vector of the present invention into a host cell, as described above. The medium used for culturing the recombinant expression transformant may be any medium which is conventional in the art and which allows the transformant to grow and produce the transaminase of the present invention, and for the E.coli strain, LB medium (peptone 10g/L, yeast extract 5g/L, NaCl10g/L, pH7.0) is preferred. The culture method and culture conditions are not particularly limited, and may be varied depending on the type of host, the culture method and the likeAs appropriate according to the ordinary knowledge in the art, so long as it enables the transformant to grow and produce the transaminase of the present invention. Other specific procedures for culturing the transformant can be performed according to the routine procedures in the art. For E.coli strains, the following method is preferably used for producing the enzyme by shake flask fermentation: the recombinant Escherichia coli (preferably E.coli BL21(DE3)/pET21a-MvAT) related to the invention is inoculated into LB culture medium containing ampicillin for culture, and when the optical density OD of the culture solution₆₀₀When the concentration reaches 0.5 to 0.7 (preferably 0.6), isopropyl- β -D-thiogalactopyranoside (IPTG) with the final concentration of 0.05 to 1.0mmol/L (preferably 0.2mmol/L) is added for induction, and the recombinant transaminase can be efficiently expressed at the induction temperature of 10 to 40 ℃ (preferably 35 ℃).

The catalyst for catalyzing the asymmetric transamination of prochiral carbonyl compounds to form optically active chiral amines in the present invention may be a culture of a transformant of the recombinant transaminase produced as described above, or a transformant cell obtained by centrifuging a culture medium, or a product processed using the same. The "processed product" herein means an extract obtained from the transformant cells, an isolated product obtained by isolating and/or purifying transaminase in the extract, or an immobilized product obtained by immobilizing the transformant cells and an extract or isolated product of the extract.

In a sixth aspect, the invention provides a use of a protein according to the invention for catalyzing the asymmetric transamination of a prochiral carbonyl compound to form a chiral amine.

In the above applications, the conditions of the asymmetric transamination reaction can be selected according to the conditions customary in such reactions in the art, preferably as follows:

the protein is preferably a transaminase or a recombinant transaminase according to the invention. The prochiral carbonyl compound is preferably a 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyrate compound, namely a compound shown in the formula I:

wherein,

r is alkyl or benzyl.

Preferably, the first and second liquid crystal films are made of a polymer,

r is alkyl or benzyl with a carbon chain length of 1-8.

More preferably, ,

r is-CH₃、-CH₂CH₃、-CH(CH₃)₂、-CH₂CH₂CH₃、-C(CH₃)₃or-CH₂C₆H₅。

Most preferably, R is-CH₃I.e. formula I is methyl 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butanoate.

The conditions of the asymmetric transamination reaction according to the invention can be chosen according to the conditions customary in such reactions in the art, and preferably the application comprises the following steps: in an ethanol aqueous solution with the pH value of 7.0-10.0, under the existence of isopropylamine and pyridoxal phosphate (PLP), under the catalysis of the transaminase or the recombinant transaminase, a prochiral carbonyl compound is subjected to asymmetric transamination reaction to form optically active chiral amine.

Wherein the preferable concentration of the prochiral carbonyl compound in the reaction solution is 1-800 mmol/L. The amount of the transaminase is a catalytically effective amount, preferably 0.1 to 50 g/L. The amount of isopropylamine is preferably 1 to 60 g/L. The amount of PLP to be additionally added is preferably 0 to 1.0 mmol/L. The aqueous solution can be a buffer solution which is conventional in the field as long as the pH range is 7.0-10.0, and a phosphate buffer solution, such as a phosphate-sodium phosphate buffer solution, is preferred. The concentration of the phosphate buffer solution is preferably 0.05-0.1 mol/L, wherein the concentration refers to the total concentration of the conjugate acid and the conjugate base in the buffer solution. The concentration of the ethanol is preferably 5 to 50 percent. The asymmetric transamination reaction is preferably carried out under shaking or stirring conditions. The temperature of the asymmetric transamination reaction is preferably 20 to 55 ℃. The time of the asymmetric transamination reaction is preferably based on the time during which the product concentration no longer increases during the reaction. After the asymmetric transamination reaction is completed, the chiral amine product can be extracted from the reaction solution according to a conventional method in the art.

In the present invention, the crude enzyme solution is used as a catalyst, and PLP coenzyme is preferably added. If resting cells are used as the catalyst, no PLP coenzyme is needed, and only PLP coenzyme contained in the cells is needed.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: aiming at the reported problems of low yield, poor stereoselectivity, high catalyst price, difficult solvent recovery and the like in the reaction for asymmetrically synthesizing sitagliptin and an intermediate thereof, the method for synthesizing the R-3-amino-4- (2,4, 5-trifluorophenyl) -methyl butyrate by carrying out enzymatic synthesis on the novel transaminase with high catalytic activity, strong enantioselectivity and good substrate tolerance and solvent tolerance is provided. When the catalytic concentration is as high as 0.8mol/L (200g/L) of the substrate, the optical purity of the product is still as high as more than 99%. Compared with other preparation methods, the method has the advantages of high concentration of the product prepared by the method, high optical purity of the product, easy recovery of the solvent, mild reaction conditions, environmental friendliness, simple and convenient operation and easy industrial amplification, thereby having good industrial application prospect.

Drawings

FIG. 1 is an agarose gel electrophoresis of PCR products of the Mycobacterium PYR-1 transaminase gene.

FIG. 2 shows a PCR of a colony of a transformant in which Mycobacterium PYR-1 transaminase is recombinantly expressed, wherein M is a molecular weight standard, lane A is E.coli DH5 α/pET21a-MvAT, and lane B is E.coli BL21(DE3)/pET21 a-MvAT.

FIG. 3 is a polyacrylamide gel electrophoresis of the crude enzyme solution of recombinant expressed Mycobacterium PYR-1 transaminase.

M is the molecular weight standard, lane A is before induction, lane B is after induction.

FIG. 4 agarose gel electrophoresis of library 1 transaminase mutants. M is the molecular weight standard, and lane A is the agarose gel electrophoresis of the PCR fragment product obtained by site-directed mutagenesis. Lane B is an agarose gel electrophoresis of the product of the PCR of the mutant fragment using the gene cloning primers.

FIG. 5 agarose gel electrophoresis of library 2 transaminase mutants. M is the molecular weight standard, and lane A is the agarose gel electrophoresis of the PCR fragment product obtained by site-directed mutagenesis. Lane B is an agarose gel electrophoresis of the product of the PCR of the mutant fragment using the gene cloning primers.

Detailed Description

The present invention is further illustrated by the following examples, but is not limited thereto. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

The material sources in the following examples are:

genomic DNA of Mycobacterium (Mycobacterium vanbaaleni) PYR-1 was provided by doctor Hui Wu of the oral medical college of the university of Alabama State, USA.

Expression plasmids pET21a, E.coli DH5 α and E.coli BL21(DE3) competent cells, 2 XTaq PCRMasterMix, agarose gel DNA recovery kits were purchased from Changsheng biotechnology Limited liability company, Beijing ancient China.

EXAMPLE 1 cloning of Mycobacterium PYR-1 transaminase Gene

PCR primers were designed based on the gene sequence predicted to be Mycobacterium PYR-1 transaminase (NCBI accession number: YP-955297.1) as recorded by Genbank as upstream and downstream primers for gene cloning in Table 1. Wherein, the underlined part of the upstream primer is NdeI restriction site, and the underlined part of the downstream primer is EcoRI restriction site.

PCR amplification was performed using Mycobacterium PYR-1 genomic DNA as a template. The PCR system is as follows: 10 XKOD-Plus PCRbuffer 2. mu.L, 25mM MgSO₄1.2. mu.L, 2mM dNTP 2. mu.L, KOD-Plus PCR Hi-Fi enzyme 0.3. mu.L, DNA template 0.5. mu.L (containing DNA template 0.1. mu.g), ddH₂mu.L of O13, 0.5. mu.L (10mmol/L) of each of the gene clone forward primer and the gene clone reverse primer (SEQ ID NOS: 11 and 12) in Table 1 was used for PCR amplification. The PCR amplification step is as follows: (1) pre-denaturation at 95 ℃ for 3 min; (2) denaturation at 98 ℃ for 15 s; (3) annealing at 55 ℃ for 30 s; (4) extending for 1min at 72 ℃; repeating the steps (2) to (4) for 30 times; (5) extension was continued for 10min at 72 ℃ and cooled to 4 ℃. And (2) purifying the PCR product by agarose gel electrophoresis, recovering a target band (shown in figure 1) in a range of 900-1200 bp by using an agarose gel DNA recovery kit to obtain a complete mycobacterium PYR-1 transaminase full-length gene sequence, sequencing by DNA, wherein the full-length gene sequence is 1014bp and is named MvAT, and the nucleotide sequence and the coded amino acid sequence of the MvAT are respectively shown as SEQ ID No: 1 and 2.

TABLE 1PCR primer Table

EXAMPLE 2 construction of recombinant expression vectors

The transaminase gene DNA fragment obtained in example 1 was digested simultaneously with restriction enzymes NdeI and EcoRI at 37 ℃ for 8 hours, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The target fragment was ligated with plasmid pET21a, which was similarly digested with NdeI and EcoRI, with T4DNA ligase at 16 ℃ overnight to give recombinant expression plasmid pET21 a-MvAT.

EXAMPLE 3 preparation of recombinant expression transformants

Transforming the recombinant expression plasmid into an Escherichia coli (E.coli) DH5 α competent cell, carrying out heat shock for 90 seconds under the transformation condition of 45 ℃, screening a positive recombinant on an ampicillin-containing resistant plate, picking up a single clone, carrying out colony PCR (polymerase chain reaction) to verify the positive clone (shown as a lane in figure 2), culturing the recombinant bacterium, extracting the plasmid after the plasmid is amplified, re-transforming the plasmid into an E.coli BL21(DE3) competent cell, coating a transformation solution on an LB (LB) plate containing ampicillin, carrying out inversion culture at 37 ℃ overnight, and obtaining a positive recombinant transformant E.coli BL21(DE3)/pET21a-MvAT, and carrying out colony PCR to verify the positive clone (shown as a lane B in figure 2).

EXAMPLE 4 expression of recombinant transaminase

Recombinant E.coli BL21(DE3) obtained in example 3 was inoculated into ampicillin-containing LB medium (peptone 10g/L, yeast extract 5g/L, NaCl10g/L, pH7.0), cultured overnight with shaking at 37 ℃, inoculated in a 500ml Erlenmeyer flask containing 100ml of LB medium in an amount of 1% (v/v), cultured with shaking at 37 ℃ and 180rpm, IPTG was added to the culture medium to a final concentration of 0.2mmol/L as an inducer when OD600 of the culture medium reached 0.6, and after 12 hours of induction at 35 ℃, the culture medium was centrifuged, cells were collected, and washed twice with physiological saline to obtain resting cells. Suspending the obtained resting cells in a buffer solution with the pH value of 8.5, carrying out ultrasonic disruption in an ice bath, centrifuging and collecting supernatant fluid, namely the crude enzyme solution of the recombinant transaminase. Protein concentration was determined by the Bradford method. The crude enzyme solution was analyzed by polyacrylamide gel electrophoresis together with the precipitate (see FIG. 3), and the recombinant protein was present in a partially soluble form. And (3) freeze-drying the crude enzyme liquid by using a freeze dryer to obtain freeze-dried crude enzyme powder.

Example 5 recombinant transaminase catalysis of asymmetric transamination of carbonyl substrates

To 50ml of a sodium phosphate-isopropylamine buffer solution (100mmol/L, pH7.5), the crude enzyme solution of MvAT prepared in example 4 was added to a final protein concentration of 10g/L, 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid methyl ester was added to a final concentration of 10mmol/L and pyridoxal phosphate was added to a final concentration of 1mmol/L, 2.5ml of ethanol was added, and a nitrogen purge reaction was performed by inserting a syringe needle into the bottom at 20 ℃. After 48 hours of reaction, extraction was carried out twice with an equal volume of ethyl acetate, the extracts were combined, dried overnight with anhydrous sodium sulfate and analyzed to determine the substrate conversion and the ee value of the transaminated product. No transamination products were found to form.

The conversion rate determination method comprises the following steps: the reaction was diluted 100 fold with acetonitrile, centrifuged and 20. mu.L was analyzed for conversion on an Agilent1200HPLC column, Agilent Eclipse XAD-C18 reverse phase silica gel, mobile phase water: acetonitrile 40:60, 10mmol/L ammonium formate, flow rate of 1 mL/min, detection wavelength of 210nm and 260nm, retention time of transamination product of 4.6min, retention time of substrate of 7.4 min.

The ee value of the transaminated product was determined as follows: the product was checked for optical purity e.e on an Agilent1200HPLC and analyzed using a Daicel Chiralpak AD-H chiral chromatography column (4.6 × 150mm) with a mobile phase of ethanol, n-heptane, diethylamine, water 60:40:0.1:0.1, a flow rate of 1mL per minute, a column temperature of 35 ℃, and retention times of: substrate 5.6min, (S) -product: 7.9 min; (R) -product: 9.7 min. The detection wavelengths were 210nm and 260 nm.

Example 6 construction of transaminase mutation library 1

PCR amplification was carried out using the plasmid pET21a-MvAT constructed in example 3 as a template. The PCR system is as follows: 10 XKOD-Plus PCR buffer 2. mu.L, 25mM MgSO₄1.2. mu.L, 2mM dNTP 2. mu.L, KOD-Plus PCR Hi-Fi enzyme 0.3. mu.L, DNA template 0.5. mu.L (containing DNA template 0.1. mu.g), ddH₂O13 μ L, as shown in the table1, 0.5. mu.L (10mmol/L) of each of the upstream primer (SEQ ID No: 11) of the gene clone, the downstream primer (SEQ ID No: 14) of L72+ V76, the upstream primer (SEQ ID No: 13) of L72+ V76, the downstream primer (SEQ ID No: 16) of F129, the upstream primer (SEQ ID No: 15) of F129, the downstream primer (SEQ ID No: 18) of A291, the upstream primer (SEQ ID No: 17) of A291 and the downstream primer (SEQ ID No: 12) of the gene clone was subjected to PCR amplification. The PCR amplification step is as follows: (1) pre-denaturation at 95 ℃ for 3 min; (2) denaturation at 98 ℃ for 15 s; (3) annealing at 55 ℃ for 30 s; (4) extending for 1min at 72 ℃; repeating the steps (2) to (4) for 30 times; (5) extension was continued for 10min at 72 ℃ and cooled to 4 ℃. The PCR products were purified by agarose gel electrophoresis, and about 250, 150, 500, 180 target bands were recovered using agarose gel DNA recovery kit (see lane A in FIG. 4). Using these PCR products as templates, PCR amplification was carried out in the same manner as in example 1 and PCR products of about 1100bp were recovered (see lane B in FIG. 4), and after constructing plasmids in the same manner as in example 2, E.coli BL21(DE3) competent cells were transformed in the same manner as in example 3 to obtain transaminase mutation library 1(L72FSCVAG/V76CSAG/F129LMV/A291 AG).

Example 7 screening of transaminase mutation libraries

Mutant colonies were picked from the mutant library, inoculated into 200. mu.L of 2 XYT medium (containing 100. mu.g/mL of ampicillin) in a microtiter plate, and cultured at 37 ℃ for 24 hours. mu.L of the above culture was inoculated into a deep well plate containing 500. mu.L of an expression medium (2 XYT, 100. mu.g/ml ampicillin, 1mM IPTG), and cultured at 25 ℃ for 24 hours. 3500rpm was centrifuged for 15 minutes, 400. mu.L of lysis buffer (20mM phosphate buffer, pH7.5, containing 1mg/mL lysozyme) was added to resuspend the mixture, and the cells were disrupted by repeated freeze-thawing. Centrifugation was carried out at 4000rpm for 15 minutes, and 200. mu.L of supernatant was taken from each well to a new microtiter plate. The reaction mixture (200mM sodium phosphate, 200mM isopropylamine, 20mM 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid methyl ester, 2mM pyridoxal phosphate, 10% -80% (v/v) ethanol, pH 7.5-8.5) was added thereto, and the conversion was measured according to example 5 after shaking the mixture at 20 ℃ to 50 ℃ for 24 hours.

Example 8 screening of mutant library 1 to obtain transaminase mutant 1

The mutant library 1 constructed in example 6 was screened as in example 7 under the following conditions: 10% ethanol, pH7.5, 20 ℃, to obtain a mutant strain with a transformation rate of 11% (mutant 1). The mutant is sequenced, and is an L72C/V76A/F129V/A291G mutant, and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID No: 3 and 4.

Example 9 construction of transaminase mutation library 2

PCR amplification was performed using the plasmid constructed in example 3 as a template. The PCR system is as follows: 10 XKOD-Plus PCRbuffer 2. mu.L, 25mM MgSO₄1.2. mu.L, 2mM dNTP 2. mu.L, KOD-Plus PCR Hi-Fi enzyme 0.3. mu.L, DNA template 0.5. mu.L (containing DNA template 0.1. mu.g), ddH₂O13 μ L, PCR amplified with 0.5 μ L (10mmol/L) of each of the gene clone forward primer (SEQ ID No: 11) and H69 downstream primer (SEQ ID No: 20), H69 forward primer (SEQ ID No: 19) and G143 downstream primer (SEQ ID No: 22), G143 forward primer (SEQ ID No: 21) and S206 downstream primer (SEQ ID No: 24), S206 forward primer (SEQ ID No: 23) and T216 downstream primer (SEQ ID No: 26), T216 forward primer (SEQ ID No: 25) and P230 downstream primer (SEQ ID No: 28), P230 forward primer (SEQ ID No: 27) and gene clone downstream primer (SEQ ID No: 12) in Table 1, respectively. The PCR amplification step is as follows: (1) pre-denaturation at 95 ℃ for 3 min; (2) denaturation at 98 ℃ for 15 s; (3) annealing at 55 ℃ for 30 s; (4) extending for 1min at 72 ℃; repeating the steps (2) to (4) for 30 times; (5) extension was continued for 10min at 72 ℃ and cooled to 4 ℃. The PCR product was purified by agarose gel electrophoresis, and about 200, 50, 80, 300 target bands were recovered using an agarose gel DNA recovery kit (see lane A in FIG. 5). Using these PCR products as templates, PCR amplification was carried out in the same manner as in example 1 and PCR products of about 1100bp were recovered (see lane B in FIG. 5), and after constructing plasmids in the same manner as in example 2, E.coli BL21(DE3) competent cells were transformed in the same manner as in example 3 to obtain transaminase mutation library 2(H69FLV/G143ATIV/S206ATIV/T216IV/P230 LPAV).

Example 10 screening of mutant library 2 to obtain transaminase mutant 2

The mutant library 2 constructed in example 9 was screened as in example 7 under the following conditions: 10% ethanol, pH7.5, 20 ℃, get a mutant strain transformation rate up to 99% (mutant 2). The mutant is sequenced, and is H69L/L72C/V76A/F129V/G143I/S206V/T216I/P230A/T289A/A291G mutant, and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID No: 5 and 6.

Example 11 creation of random mutagenesis library

The transaminase mutant gene is taken as a template, a gene cloning upstream primer and a gene cloning downstream primer are taken as primers, and a random mutagenesis kit is used for carrying out PCR mutation. The PCR amplification step is as follows: (1) pre-denaturation at 95 ℃ for 3 min; (2) denaturation at 94 ℃ for 30 s; (3) extending for 1min at 68 ℃; repeating the steps (2) to (3) for 25 times; (5) extension was continued for 10min at 72 ℃ and cooled to 4 ℃. After plasmid construction as in example 2, E.coli BL21(DE3) competent cells were transformed as in example 3 to obtain a random mutagenesis library of transaminases.

Example 12 screening of random mutagenesis library to obtain mutant 3

Using the transaminase mutant 2 gene obtained in example 10 as a template, a random library was constructed in accordance with example 11, and screening was carried out in accordance with example 7 under the following conditions: 10% ethanol, pH7.5, 50 ℃, get a mutant strain transformation rate up to 99% (mutant 3). Sequencing the mutant, wherein the mutant is an E56S/H69L/L72C/V76A/F129V/G143I/S206V/T216I/P230A/A243R/S276P/T289A/A291G/T295W/P304T mutant, and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID No: 7 and 8.

Example 13 screening of random mutagenesis library to obtain mutant 4

Using the transaminase mutant 3 gene obtained in example 12 as a template, a random library was constructed in accordance with example 11, and screening was carried out in accordance with example 7 under the following conditions: 50% ethanol, pH8.5, 50 ℃, get a mutant strain transformation rate up to 99% (mutant 4). The mutant is sequenced, and is an E56S/G68Y/H69L/L72C/V76A/D96A/F129V/N131T/G143I/Y157T/S206V/T216I/A222C/P230A/A243R/S276P/T289A/A291G/T295W/P304T mutant, and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID No: 9 and 10.

Example 14 recombinant transaminase and mutants thereof catalyze asymmetric transamination of carbonyl substrates

The crude enzyme solution of MvAT or a mutant thereof prepared in example 4 was added to 50ml of a sodium phosphate-isopropylamine buffer solution (100mmol/L, pH7.0 to 8.5) to a final protein concentration of 10g/L, 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid methyl ester was added to a final concentration of 10mmol/L and pyridoxal phosphate was added to a final concentration of 1mmol/L, 2.5 to 50ml of ethanol was added, and nitrogen purging reaction was performed at 20 to 50 ℃ with a syringe needle inserted into the bottom. After 48 hours of reaction, extraction was carried out twice with an equal volume of ethyl acetate, the extracts were combined, dried overnight with anhydrous sodium sulfate and the substrate conversion and the ee value of the transaminated product were determined as in example 5. The results are shown in Table 2.

TABLE 2 results of MvAT and its mutants in catalyzing asymmetric transamination of substrates

MvAT enzyme

SEQ ID NO

pH

Reaction temperature (%)

Ethanol concentration (%)

Conversion (%)

ee value (%)

Wild type	2	7.5	25	5	<0.1	Not testing
							Mutant 1	4	7.5	25	5	16	99
Mutant 2	6	7.5	25	5	99	99
							Mutant 2	6	7.5	50	5	7	99
Mutant 3	8	7.5	50	5	99	99
							Mutant 3	8	8.5	50	50	13	99
Mutant 4	10	8.5	50	50	99	99

Example 15 transaminase mutant 4 catalyzes asymmetric transamination of carbonyl compounds

To 50mL of a sodium phosphate-isopropylamine buffer (100mmol/L, pH8.5) was added the crude enzyme solution of transaminase mutant 4 prepared according to example 4 to a final protein concentration of 10g/L, 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyrate substrate to a final concentration of 100mmol/L and pyridoxal phosphate to a final concentration of 1mmol/L, 50mL of ethanol was added, and a nitrogen purge reaction was performed by inserting a syringe needle into the bottom at 50 ℃. The reaction was stopped after the conversion was measured to 99% during the reaction or 24 hours after the reaction. After the reaction, the mixture was extracted twice with 100mL of ethyl acetate, and the extracts were combined, dried overnight with anhydrous sodium sulfate, and then the conversion of the substrate and the ee value of the transaminated product were determined as in example 5. The results are shown in Table 3.

TABLE 3 results of transaminase mutants 4 catalyzing asymmetric transamination of carbonyl compounds

Example 16 transaminase mutant 4 catalyzes asymmetric transamination of a methyl ester substrate

To 100mL of sodium phosphate-isopropylamine buffer (100mmol/L, pH8.5) was added the crude enzyme solution of transaminase mutant 4 prepared according to example 4 to a final protein concentration of 10g/L, 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyric acid methyl ester (19.7g) to a final concentration of 800mmol/L and pyridoxal phosphate to a final concentration of 1mmol/L, 100mL of ethanol was added, and nitrogen purging reaction was performed by inserting a syringe needle into the bottom at 50 ℃. The reaction was stopped after the conversion was determined to be 99% during the reaction. After the reaction, ethanol was removed by rotary evaporation at 80 ℃, proteins were removed by filtration, the pH was adjusted to 11.0, the mixture was extracted twice with 100mL of ethyl acetate, the extracts were combined, dried over anhydrous sodium sulfate overnight, the solvent was removed by rotary evaporation, and the mixture was distilled under reduced pressure to obtain 18.1g of (R) -3-amino-4- (2,4, 5-trifluorophenyl) -methyl butyrate with a yield of 92% and an ee value of the product of 99%.

EXAMPLE 17 Boc protection of the transaminated product

12.3 g of (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid methyl ester (0.05mol) obtained in example 16 was weighed, dissolved in a solution of 50mL of water and 50mL of tetrahydrofuran, 4g of NaOH (0.10mol) was added, and 12.0g of (Boc) was added under ice bath₂O (0.055mol), warmed to room temperature and reacted for 12 hours. Sodium carbonate was added to adjust the pH to 12, the mixture was extracted with 50mL of dichloromethane, the organic phase was discarded, the aqueous phase was adjusted to pH 2 with 1N hydrochloric acid, the combined organic phases were extracted with 50mL of dichloromethane, dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the mixture was recrystallized from methanol to give 14.8g of a white solid with a yield of 89%.

EXAMPLE 18Boc protected transamination product Synthesis Boc protected sitagliptin

3.32g of the product obtained in example 17 (0.01mol) and 2.28g of trifluoromethyltriazolopiperazine hydrochloride (0.01mol) are weighed into 20mL of dichloromethane, 1.62g of 1-hydroxybenzotriazole (0.012mol) and 2.29g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.012mol) are added under ice salt bath, 3g of triethylamine (0.03mol) are added dropwise, the reaction mixture is reacted at room temperature for 24 hours under stirring, the reaction mixture is washed 3 times with 20mL of water, the organic phase is dried over anhydrous magnesium sulfate, the solvent is removed by rotary evaporation to give 4.72g of a solid in 93% yield.

Example 19 deprotection of Boc protected sitagliptin

5.07g of the Boc protected sitagliptin (0.01mol) obtained in example 18 was weighed into 50mL of methanol, and 50mL of concentrated salt was addedAcid methanol solution 1:5, stirring at room temperature for 3 hours, rotary evaporating to remove methanol, and adding Na₂CO₃Neutralization, extraction with 50mL ethyl acetate 3 times, combining the organic phases, drying over anhydrous magnesium sulfate, and removal of the solvent to give an oil. Adding 60mL of ethanol and 10mL of water, heating to 80 ℃, adding 1.5g of concentrated phosphoric acid, stirring for 2 hours, cooling to room temperature, and stirring for 12 hours to precipitate 4.39g of solid, namely sitagliptin phosphate, wherein the yield is 87%.

It should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.

Claims

1. An isolated protein consisting of SEQ ID No: 4.6, 8 or 10.

2. An isolated nucleic acid consisting of SEQ ID No: 3. 5, 7 or 9.

3. A recombinant expression vector comprising the nucleic acid of claim 2.

4. The recombinant expression vector of claim 3, which is selected from the group consisting of a plasmid, a cosmid, a phage, and a viral vector.

5. The recombinant expression vector of claim 4, which is pET21 a.

6. A recombinant expression transformant comprising the recombinant expression vector of any one of claims 3 to 5.

7. The recombinant expression transformant according to claim 6, which is Escherichia coli.

8. The recombinant expression transformant according to claim 7, which is E.coli BL21(DE 3).

9. A method for preparing a recombinant transaminase, comprising the steps of: culturing the recombinant expression transformant of any one of claims 6 to 8, and obtaining the recombinant transaminase from the culture.

10. A catalyst for catalyzing asymmetric transamination of a prochiral carbonyl compound to form an optically active chiral amine, which is selected from the group consisting of a culture of the recombinant expression transformant according to any one of claims 4 to 6, cells of the transformant obtained by centrifuging a medium in the culture, or a product processed using the cells of the transformant; wherein the processed product is an extract obtained from the transformant cells, an isolated product obtained by isolating and/or purifying transaminase in the extract, or an immobilized product obtained by immobilizing the transformant cells and the extract or the isolated product of the extract; the prochiral carbonyl compound is selected from the following compounds:

wherein R is alkyl or benzyl.

11. Use of a protein according to claim 1, a recombinant transaminase obtainable by a process according to claim 9 or a catalyst according to claim 10 for catalyzing the asymmetric transamination of prochiral carbonyl compounds to optically active chiral amines; wherein the prochiral carbonyl compound is selected from the following compounds:

wherein R is alkyl or benzyl.

12. The use according to claim 11, wherein R is an alkyl group having a carbon chain length of 1 to 8 or a benzyl group.

13. The use of claim 11, wherein R is-CH₃、-CH₂CH₃、-CH(CH₃)₂、-CH₂CH₂CH₃、-C(CH₃)₃or-CH₂C₆H₅。

14. The use of claim 11, wherein R is-CH₃。

15. The use according to any one of claims 11-14, wherein the asymmetric transamination reaction is carried out in an aqueous ethanol solution containing isopropylamine at a ph of 7.0 to 10.0, optionally in addition to pyridoxal phosphate.

16. The use according to claim 15, wherein the prochiral carbonyl compound is present in the reaction solution at a concentration of 1-800 mmol/L, isopropylamine is present at a concentration of 1-60 g/L, pyridoxal phosphate is present at a concentration of 0-1.0 mmol/L, the aqueous solution is a phosphate buffer solution with a total conjugate acid-base concentration of 0.05-0.1 mol/L, the ethanol concentration is 5% (v/v) to 50% (v/v), and the reaction is carried out under shaking or stirring conditions at a reaction temperature of 20-55 ℃.

17. A method for synthesizing (R) -3-amino-4- (2,4, 5-trifluorophenyl) -methyl butyrate comprising the step of catalyzing asymmetric transamination of 3-carbonyl-4- (2,4, 5-trifluorophenyl) -methyl butyrate using the protein of claim 1, the recombinant transaminase obtained by the method of claim 9, or the catalyst of claim 10.

18. The method according to claim 17, wherein the reaction solution for the transamination reaction contains 10 to 800mmol/L of methyl 3-carbonyl-4- (2,4, 5-trifluorophenyl) -butyrate, 50 to 100mmol/L of sodium phosphate, 50 to 100mmol/L of isopropylamine, 0 to 1mmol/L of pyridoxal phosphate, 5 to 50% (v/v) of ethanol, the reaction pH is 7.5 to 8.5, and the reaction temperature is 25 to 50 ℃.

19. The method of claim 18, wherein said transamination reaction is performed by SEQ ID NO: 10, the reaction solution contains 800mmol/L of 3-carbonyl-4- (2,4, 5-trifluorophenyl) -methyl butyrate, 50mmol/L of sodium phosphate, 50mmol/L of isopropylamine, 1mmol/L of pyridoxal phosphate and 50% (v/v) of ethanol, the reaction pH is 8.5, and the reaction temperature is 50 ℃.

20. A process for the synthesis of sitagliptin, comprising the synthesis of the chiral amino intermediate (R) -3-amino-4- (2,4, 5-trifluorophenyl) -butyric acid methyl ester using the process of any one of claims 17 to 19.