CN116064449A - Transaminase mutant and application thereof - Google Patents

Abstract

The invention relates to a transaminase mutant and application thereof, wherein the transaminase mutant is formed by SEQ ID NO:1, wherein the mutation comprises at least one of the following mutation sites: the 60 th site is mutated from W to A, D or K, the 89 th site is mutated from F to A, D or K, the 121 th site is mutated from S to A, D or F, the 153 th site is mutated from Y to A, D or F, the 226 th site is mutated from E to A, R or F, the 261 rd site is mutated from V to A, D or Y, the 380 th site is mutated from Q to A, K or F, and the 417 th site is mutated from A to F, D or K. The aminotransferase mutant can be applied to green chemical synthesis of chiral amine compounds with large steric hindrance, and has high catalytic activity and good stability. Compared with the chemical synthesis method, the method has the advantages of simple and mild catalyzed reaction, no need of hydrogenation reaction, high reaction selectivity, low preparation cost and good application prospect.

Description

Transaminase mutant and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a transaminase mutant and application thereof.

Background

Aminotransferase catalyzes the stereoselective transfer of amino groups between an amino donor (amine donor) and a prochiral ketone substrate, and is an effective biocatalytic tool for the production of optically pure chiral amines. Transaminases (TAs) can be divided into two classes: alpha-aminotransferases (alpha-TAs) and omega-aminotransferases (omega-TAs), depending on the type of substrate being converted. alpha-TAs require the presence of a carboxyl group both at the alpha-position of the carbonyl function of the ketone substrate and on the amine donor. omega-TAs can accept aliphatic ketones and amines as substrates (i.e., not just alpha-keto acids and amino acids). omega-TAs can be further divided into two subgroups, beta-TAs and Amine Transaminases (ATAs), which are of industrial value because they are capable of reductive amination reactions using a wide range of amine donors and ketone acceptors. The ATAs catalyze the intermolecular exchange of amine donors and ketone acceptors, which has received considerable attention in recent years in the production of pharmaceutical intermediates.

Although the asymmetric transaminase-catalyzed transamination reaction provides an economic and green synthetic idea for synthesizing chiral amine compounds. However, some limitations and challenges of ATAs in large scale applications have also been identified, such as a fairly narrow substrate range, unfavorable thermodynamic reaction equilibrium, and substrate/product inhibition. In general, the catalytic effect of the transaminase is significantly reduced if the group next to the potentially chiral carbonyl group is larger than the methyl group. In order to overcome this limitation, a great deal of effort has been expended to evolve ATAs to improve their substrate range, especially in so-called "bulky" compounds. Different protein engineering methods are used to optimize the available ATAs.

CN112980810a discloses a transaminase mutant derived from chromobacterium violaceum (Chromobaterium violaceum) aimed at using this transaminase mutant to convert ketone compounds having a group next to a latent chiral carbonyl group greater than methyl into chiral amine compounds with high selectivity. However, the acquisition of the transaminase mutant needs to carry out saturation mutation and iterative mutation, the screening workload is large, and the operation is very complicated.

Disclosure of Invention

In order to solve the problems of low efficiency, low selectivity and high environmental impact in the chemical synthesis of chiral amine and low biological enzyme catalytic activity and poor stability in the biological synthesis process, an aspect of the invention is to provide a transaminase mutant which has high catalytic activity and good thermal stability and can catalyze and synthesize a chiral amine compound with large steric hindrance with high efficiency. In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

a transaminase mutant whose amino acid sequence consists of SEQ ID NO:1, wherein the mutation comprises at least one of the following mutation sites:

mutation from W to A, D or K at position 60 (W60A/D/K); mutation from F to A, D or K (F89A/D/K) at position 89; mutation from S to A, D or F at position 121 (S121A/F/D); mutation from Y to A, D or F (Y153A/D/F) at position 153; mutation from E to A, R or F at position 226 (E226A/R/F); mutation from V at position 261 to A, D or Y (V261A/D/Y); the amino acid sequence of the aminotransferase mutant has a mutation site in the mutated amino acid sequence and has an amino acid sequence having 80% or more homology with the mutated amino acid sequence, or the amino acid sequence of the aminotransferase mutant has a mutation at position 380 from Q to A, K or F (Q380A/F/K) and a mutation at position 417 from A to F, D or K (A417F/D/K).

Preferably, the mutation comprises at least one of the following mutation sites:

the amino acid sequence of the transaminase mutant has a mutation site in the amino acid sequence where mutation occurs, and has an amino acid sequence having a homology of 80% or more with the amino acid sequence where mutation from W to A (W60A), F to K (F89K), S to D (S121D), Y to A (Y153A) at position 60, E to R (E226R) at position 226, V to A (V261A), Q to F (Q380F) at position 380, A to F (A417F) at position 417, and the mutation from Y to A (Y153A) at position 226.

More preferably, the above transaminase mutants comprise the following mutation sites:

mutation from W to A at position 60, F to K at position 89, S to D at position 121, Y to A at position 153 and E to R at position 226, or,

mutation from W to A at position 60, F to K at position 89, S to D at position 121, Y to A at position 153, E to R and V to A at position 261, or,

mutation from W to A at position 60, F to K at position 89, S to D at position 121, Y to A at position 153, E to R at position 226, V to A at position 261 and Q to F at position 380, or,

the 60 th site is mutated from W to A, the 89 th site is mutated from F to K, the 121 th site is mutated from S to D, the 153 th site is mutated from Y to A, the 226 th site is mutated from E to R, the 261 rd site is mutated from V to A, the 380 th site is mutated from Q to F and the 417 th site is mutated from A to F.

More preferably, the aminotransferase mutant comprises a mutation site, wherein the 60 th site is mutated from W to A, the 89 th site is mutated from F to K, the 121 th site is mutated from S to D, the 153 th site is mutated from Y to A, the 226 th site is mutated from E to R, the 261 rd site is mutated from V to A, the 380 th site is mutated from Q to F and the 417 th site is mutated from A to F, and the amino acid sequence is shown in SEQ ID NO. 3.

It is an object of a further aspect of the present invention to provide genes encoding the above transaminase mutants.

Preferably, the nucleotide sequence of the above gene is shown as SEQ ID NO.4, or the nucleotide sequence of the above gene is a sequence with more than 95% homology with the sequence shown as SEQ ID NO. 4.

It is an object of a further aspect of the present invention to provide a recombinant expression vector comprising a gene encoding the above transaminase mutant.

Preferably, the recombinant expression vector is selected from pET-28a, pET-dute1, pRSF-dute1, more preferably pET-28a.

It is an object of a further aspect of the present invention to provide a genetically engineered bacterium for producing the above transaminase mutant, comprising the above recombinant expression vector.

Preferably, the genetically engineered bacterium is selected from the group consisting of E.coli MG1655 or E.coli BL21 (DE 3) pLysS.

More preferably, the genetically engineered bacterium is selected from E.coli BL21 (DE 3).

It is an object of a further aspect of the present invention to provide the use of the above transaminase mutants for the preparation of chiral amines by catalysis of carbonyl compounds or amino donors.

The object of a further aspect of the invention is a process for the production of chiral amines comprising the step of catalytic transamination of a ketone compound or an amino donor using the above transaminase mutants.

Preferably, the ketone compound is

Wherein R is ₁ And R is ₂ Each independently is selected from substituted or unsubstituted alkyl, unsaturated hydrocarbyl, aromatic hydrocarbyl or heterocyclic aromatic hydrocarbyl; r is R ₁ And R is ₂ May be singly or in combination with each other to form a substituted or unsubstituted ring.

The substitution means substitution with a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group.

In still another aspect, the present invention provides a method for preparing a compound of formula (II), comprising the step of catalytically converting a compound of formula (I) to a compound of formula (II) using the above transaminase mutant, wherein the reaction equation is as follows:

the aminotransferase mutant of the present invention is represented by SEQ ID NO:1, changing the amino acid sequence by a site-directed mutation method, realizing the change of the protein structure and function, and obtaining the transaminase with the mutation site by a directional screening method.

Drawings

FIG. 1 is a diagram of the recombinant expression vector for transaminase of example 1.

FIG. 2a shows the polyacrylamide gel electrophoresis results of the small-scale production of the aminotransferase mutants (i), (ii), (iii), (iv), (v) and (vi) proteins in shake flasks obtained in example 3, wherein lanes from left to right are Marker, blank, wild-type, mutant (i), mutant (ii), mutant (iii), mutant (iv), mutant (v) and mutant (vi), respectively.

FIG. 2b is a polyacrylamide gel electrophoresis of a small scale production of the transaminase mutant (vi) (SEQ ID NO: 3) in shake flask as obtained in example 3, wherein lanes from left to right are the polyacrylamide gel electrophoresis results of Marker and mutant (vi), respectively.

FIG. 3 shows a standard and the chiral amine product obtained in example 6 ¹ HNMR profile. The upper part being standard ¹ HNMR spectra, lower part of the product obtained in example 6 ¹ HNMR profile.

FIG. 4 shows the fluorine spectra of the chiral amine product obtained in example 6 and the standard, the upper half of the standard, and the lower half of the product obtained in example 6.

Detailed Description

Amino aminotransferase from Xenophilus sp.ap218f can selectively catalyze the conversion of carbonyl groups, but has lower activity and poorer stability. The inventors have improved the activity and stability of aminotransferase derived from Xenophilius sp.AP218F by rational design. Mutant sites are introduced into aminotransferase from Xenophilius sp.AP218F by way of whole plasmid PCR, and activity and stability of the mutants are detected, and mutants with improved activity and stability are selected.

The aminotransferase mutant gene provided by the invention is derived from a wild-type gene of Xenophilius sp.AP218F. The amino acid sequence of the wild-type gene is shown as SEQ ID NO.1, and the gene sequence after codon optimization for escherichia coli is shown as SEQ ID NO. 2. Wherein "wild type" refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism, can be isolated from a natural source and is not intentionally modified by human manipulation. The enzymes obtained after the expression of the genes have low catalytic activity and poor thermal stability on certain substrates.

According to the invention, the three-dimensional structure of aminotransferase from Xenophilius sp.AP218F is obtained through an online protein structure prediction tool, the three-dimensional structure (6S 4G, homology 87.58%) of aminotransferase with highest structural similarity is obtained through PDB for structural comparison, then the combination simulation of a substrate of the formula I and the aminotransferase protein three-dimensional structure is carried out through AutoDock, and finally the amino acid possibly related to the substrate combination is selected as the mutant amino acid through Pymol analysis. According to the analysis result of Pymol, a plurality of pairs of site-directed mutagenesis (W60A/D/K; F89A/D/K; S121A/F/D; Y153A/D/F, E226A/R/F, V261A/D/Y; Q380A/F/K; A417F/D/K) primers are designed, and a pET-28a is used as an expression vector by using a site-directed mutagenesis method to obtain a mutant plasmid with a target gene. Wherein, site-directed mutagenesis: refers to the introduction of a desired change (usually a change characterizing the favorable direction) into a target DNA fragment (which may be a genome or a plasmid) by a Polymerase Chain Reaction (PCR) method or the like, and includes addition, deletion, point mutation, etc. of a base. The site-directed mutagenesis can rapidly and efficiently improve the properties and characterization of target proteins expressed by DNA, and is a very useful means in gene research work.

The introduction of site-directed mutagenesis by whole plasmid PCR is currently a relatively large tool. The principle is that a pair of primers (forward and reverse) containing mutation sites and a template plasmid are annealed and then are ' circularly extended ' by polymerase, wherein the circularly extended refers to that the polymerase extends the primer according to the template, and returns to the 5' end of the primer to terminate after one circle, and then repeatedly carries out the cycle of heat annealing extension, and the reaction is distinguished from rolling circle amplification and does not form a plurality of tandem copies. The extension products of the forward and reverse primers are annealed and then paired into open-loop plasmids with nicks. The Dpn I enzyme digestion extension product is cut up by virtue of dam methylation modification of the original template plasmid from conventional escherichia coli, and the plasmid with a mutation sequence synthesized in vitro is not cut up due to no methylation, so that the plasmid can be successfully transformed in subsequent transformation, and thus the clone of the mutation plasmid can be obtained. The mutant plasmid was transformed into E.coli cells and overexpressed in E.coli. Then, wet cells were obtained by centrifugation, and crude enzyme was obtained by disrupting cells by sonication.

As a first aspect of the invention, the transaminase mutants provided by the invention consist of SEQ ID NO:1, wherein the mutation comprises at least one of the following mutation sites: mutation from W to A, D or K at position 60 (W60A/D/K); mutation from F to A, D or K (F89A/D/K) at position 89; mutation from S to A, D or F at position 121 (S121A/F/D); mutation from Y to A, D or F (Y153A/D/F) at position 153; mutation from E to A, R or F at position 226 (E226A/R/F); mutation from V at position 261 to A, D or Y (V261A/D/Y); the amino acid sequence of the aminotransferase mutant has a mutation site in the mutated amino acid sequence and has an amino acid sequence having 80% or more homology with the mutated amino acid sequence, or the amino acid sequence of the aminotransferase mutant has a mutation at position 380 from Q to A, K or F (Q380A/F/K) and a mutation at position 417 from A to F, D or K (A417F/D/K). In a preferred embodiment of the invention, the mutation comprises at least one of the following mutation sites: the amino acid sequence of the transaminase mutant has a mutation site in the amino acid sequence where mutation occurs, and has an amino acid sequence having a homology of 80% or more with the amino acid sequence where mutation from W to A (W60A), F to K (F89K), S to D (S121D), Y to A (Y153A) at position 60, E to R (E226R) at position 226, V to A (V261A), Q to F (Q380F) at position 380, A to F (A417F) at position 417, and the mutation from Y to A (Y153A) at position 226. Wherein "homology" refers to the identity between two amino acid sequences. The sequences defined according to the invention by different degrees of homology must also have an improved transaminase activity. One skilled in the art can, under the teachings of the present disclosure, obtain amino acid sequences of transaminase mutants that have a mutation site in the mutated amino acid sequence described above and that have more than 80% homology with the mutated amino acid sequence.

In a more preferred embodiment of the invention, the above transaminase mutants comprise a mutation site: W60A, F89K, S121D, Y153A and E226R. In a more preferred embodiment of the invention, the above transaminase mutants comprise a mutation site: W60A, F89K, S121D, Y153A, E226R and V261A. In a more preferred embodiment of the invention, the above transaminase mutants comprise a mutation site: W60A, F89K, S121D, Y153A, E226R, V261A and Q380F.

In a more preferred embodiment of the invention, the above transaminase mutants comprise a mutation site: W60A, F89K, S121D, Y153A, E226R, V261A, Q380F and A417F, the amino acid sequences of which are shown in SEQ ID NO. 3.

The aminotransferase mutant is characterized in that the aminotransferase mutant is shown in SEQ ID NO:1, changing the amino acid sequence by a site-directed mutation method, realizing the change of the protein structure and function, and obtaining the transaminase with the mutation site by a directed screening method, wherein the transaminase mutant has the advantage of greatly improving the enzyme activity, the conversion rate of a substrate is improved by 95 times relative to that of a transaminase female parent under the same condition, and the enzyme stability is correspondingly improved, thereby greatly reducing the cost in the industrial production of chiral amine.

As a second aspect of the present invention, the present invention provides a gene encoding the above transaminase mutant. The invention mutates the gene of the wild aminotransferase by rational design (site-directed mutagenesis or other methods for changing individual amino acids in protein molecules), overlap extension PCR, seamless cloning and other methods, thus obtaining the target gene of the aminotransferase mutant. In a preferred embodiment of the invention, the nucleotide sequence of the gene is shown as SEQ ID NO.4, or the nucleotide sequence of the gene is a sequence with more than 95% homology with the sequence shown as SEQ ID NO. 4. Wherein "homology" refers to the identity between two nucleotide sequences.

The transaminase obtained by encoding the gene of the invention improves the enzyme activity and the enzyme stability. The industrial production efficiency of chiral amine is higher and the cost is lower.

As a third aspect of the present invention, the present invention provides a recombinant expression vector. The recombinant expression vector contains a gene encoding the transaminase mutant of the invention. The gene is located at a proper position of the recombinant expression vector, so that the gene can be correctly and smoothly copied, transcribed or expressed. In order to meet the requirements of recombinant operation, the two ends of the gene sequence can be added with the enzyme cutting sites of proper restriction enzymes or additionally added with a start codon, a stop codon and the like. The recombinant expression vector can be a prokaryotic expression vector or a eukaryotic expression vector. In the present invention, the recombinant expression vector includes, but is not limited to, pET-28a, pET-dute1 or pRSF-dute1.

As a fourth aspect of the present invention, there is provided a genetically engineered bacterium for producing the above transaminase mutant, which comprises the above recombinant expression vector. In the present invention, the genetically engineered bacteria include, but are not limited to, E.coli MG1655 or E.coli BL21 (DE 3) pLysS.

The transaminase mutant can be prepared by fermenting and culturing the genetically engineered bacterium. For example, the above transaminase mutants can be industrially prepared under certain conditions of production tank fermentation. The fermentation conditions of the production tank are preferably as follows: DO is above 20% and the temperature is 20 ℃.

As a fifth aspect of the invention, the invention provides the use of the above transaminase mutants in the preparation of chiral amines by catalyzing carbonyl compounds.

As a sixth aspect of the present invention, there is provided a process for producing chiral amines, which comprises the step of catalytically transaminating ketones using the above transaminase mutants.

Preferably, the ketone compound is

Wherein R is ₁ And R is ₂ Each independently is a substituted or unsubstituted alkyl, unsaturated hydrocarbyl, aromatic hydrocarbyl, or heterocyclic aromatic hydrocarbyl; or R is ₁ And R is ₂ May be singly or in combination with each other to form a substituted or unsubstituted ring. The substitution means substitution with a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group.

More preferably, R ₁ And R is ₂ Each independently is a substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, an unsaturated hydrocarbon group, an aromatic hydrocarbon group or a heterocyclic aromatic hydrocarbon group, or R ₁ And R is ₂ Can be singly or mutually combined to form a substituted or unsubstituted ring with 1-20 carbon atoms.

More preferably, R ₁ And R is ₂ Each independently is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, an unsaturated hydrocarbon group, an aromatic hydrocarbon group or a heterocyclic aromatic hydrocarbon group, or R ₁ And R is ₂ Can be singly or twoWhich are combined with each other to form a substituted or unsubstituted ring having 1 to 10 carbon atoms.

The alkyl group may be selected from, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, tert-butyl, methoxy, ethoxy, tert-butoxy, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyl, allyl, cyclopentyl and cycloheptyl.

The above unsaturated hydrocarbon group may be selected from, for example, vinyl, 1-methylvinyl, 1-ethylvinyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-methyl-1-butenyl, 1, 3-butadienyl, 1-pentenyl, 2-pentenyl, 4-methyl-1-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1, 5-hexadienyl, 2-heptenyl, 2-octenyl, 2-nonenyl, and 2-decenyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-methyl-1-butynyl, 3-dimethyl-1-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 1-methyl-3-pentynyl, 1-methyl-3-hexynyl, 2-heptynyl, 2-decynyl, and the like.

The aromatic hydrocarbon group may be selected from phenyl and naphthyl, for example.

The heterocyclic aromatic hydrocarbon group may be selected from, for example, pyrimidinyl, furanyl, pyrrolyl, thienyl, imidazolyl, thiazolyl, pyridyl, benzofuranyl, indolyl.

The substitution means substitution with a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group.

More preferably, the above ketone compound is selected from compounds having the following structure:

in the step of catalyzing the transamination reaction of the ketone compound by using the transaminase mutant, the pH of a reaction system for catalyzing the transamination reaction is preferably 7-10, more preferably 8-9, and most preferably 8.5; the temperature of the reaction system is preferably 40℃to 60 ℃, more preferably 45℃to 55℃and most preferably 50 ℃.

As a seventh aspect of the present invention, there is provided a method for producing a compound represented by formula (II), comprising the step of catalytically converting a compound represented by formula (I) to a compound represented by formula (II) using the above transaminase mutant, wherein the reaction equation is as follows:

/>

preferably, in the above preparation method, the pH in the reaction system is preferably 7 to 10, more preferably 8 to 9, and most preferably 8.5; the temperature of the reaction system is preferably 40℃to 60 ℃, more preferably 45℃to 55℃and most preferably 50 ℃.

Compared with the prior art, the invention has the following advantages and effects:

1. the aminotransferase mutant of the amino acid sequence shown in SEQ ID NO.3 has higher catalytic activity for large steric hindrance substrates, so that the conversion rate of the substrates (the compounds shown in the formula (I)) in 12h is 60%, and the enzyme activity of wild type enzyme to the substrates is less than 1%. Furthermore, the aminotransferase mutants of the present invention have a half-life of more than 48 hours at 50℃while the wild-type enzyme has a half-life of only 12 hours under the same conditions.

2. The transaminase mutant provided by the invention has high catalytic activity and good heat resistance under alkaline conditions, and can be applied to biosynthesis for preparing chiral amine; compared with chemical synthesis method, the catalytic ammonia transfer reaction is simple and mild, the reaction selectivity is high, the preparation cost is low, and the method has better application prospect.

3. Compared with the wild type, the catalytic activity of the aminotransferase mutant on the ketone substrate is greatly improved, the chiral amine compound with large steric hindrance is synthesized by using aminotransferase catalysis, the green chemical synthesis is realized without using a heavy metal catalyst, and the aminotransferase with improved activity is used for carrying out the ammonia transfer reaction, so that the chiral amine compound with high selectivity is easy to obtain.

The present invention will be described in further detail with reference to the following examples, which are illustrative of the present invention and are not intended to limit the present invention thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1: establishment of wild transaminase gene engineering bacteria

The sequence of the wild type gene sequence of Xenophilius sp.AP218F aminotransferase (GenBank: OWY 40557.1) recorded by NCBI is optimized, then a whole gene fragment (the nucleotide sequence is shown as SEQ ID NO. 2) is artificially synthesized, the gene is inserted into a pET-28a plasmid by NdeI and XhoI endonucleases through a gene synthesis company (figure 1), the linked vector is transferred into escherichia coli BL21 (DE 3) to establish a wild type aminotransferase genetic engineering bacterium, and after kanamycin resistance screening, sequencing verification is carried out.

Example 2: transaminase mutant and acquisition of target gene

The three-dimensional structure of SEQ ID NO.1 is obtained through an online protein structure prediction tool, the three-dimensional structure (6S 4G, homology 84.97%) of aminotransferase with highest structural similarity is obtained through PDB for structural comparison, then the binding simulation of the substrate of the formula I and the aminotransferase protein three-dimensional structure is carried out through AutoDock, and finally the amino acid possibly related to the substrate binding is selected as the mutant amino acid through Pymol analysis.

According to the above-mentioned Pymol analysis result, mutation is made at least 1 site in the amino acid sequence of the wild-type aminotransferase shown in SEQ ID NO. 1: bit 60, bit 89, bit 121, bit 153, bit 226, bit 261, bit 380 and bit 417. And W at position 60 is mutated to A, D or K; f at position 89 is mutated to A, D or K; s at position 121 is mutated to A, D or F; y at position 153 is mutated to A, D or F; e at position 226 is mutated to A, R or F; v mutation at position 261 to A, D or Y; q at position 380 is mutated to A, K or F. Analyzing the distance between the substrate binding region and the substrate based on the docking result

A hydrophobic amino acid, preferably a mutation at least 1 position in the amino acid sequence of the wild-type transaminase shown in SEQ ID NO. 1: W60A, F89K, S121D, Y153A, E226R, V261A, Q380F, A417F, according to the Pymol analysis, the final design was made from the combined mutation sites: (i) W60A, F89K, S121D; (ii) W60A, F89K, S121D, Y153A; (iii) W60A, F89K, S121D, Y153A, E226R; (iv) W60A, F89K, S121D, Y153A, E226R, V261A; (v) W60A, F89K, S121D, Y153A, E226R, V261A, Q380F; (vi) The high activity mutant was selected from W60A, F89K, S121D, Y153A, E226R, V261A, Q380F, A417F. Wherein, when the aminotransferase group is mutated, a seamless cloning mode is adopted, and the primers on the pET28a plasmid are respectively positioned at the upstream and downstream of the aminotransferase gene. The mutation site is provided with a primer with 15bp homologous arms at two ends, and the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 53-60℃for 15s and extension at 72℃for 50s for 30 cycles; the extension was continued at 72℃for 10min and cooled to 4 ℃. />

The PCR amplified fragment is connected with pET28a plasmid vector by using a seamless cloning kit, the connected vector is transferred into escherichia coli BL21 (DE 3) to establish transaminase gene mutant, and the transaminase with the extension mutation is expressed by using escherichia coli BL21 (DE 3) as a host and pET28a plasmid as a vector.

The present invention screens high activity mutant strains by the enzyme activity detection method described in example 5 below, and identifies the high activity aminotransferase genes after mutation. The test results are shown in Table 1 below:

table 1.

Note that: in the above table, + + represents a substrate conversion of 0% or more and less than 20%, ++ represents a substrate conversion of 20% or more and less than 40%, ++ represents a large substrate conversion 40% or more and less than 80%, 40% or more less than 80 percent.

And (3) identifying the mutated gene of the high-activity transaminase to obtain a mutant with a combined mutation site (vi), wherein the amino acid sequence of the mutant is shown as SEQ ID NO.3, and the nucleotide sequence of the mutant is shown as SEQ ID NO. 4.

Example 3: small-scale production of transaminase mutant proteins in shake flasks

Recombinant cells of the individual E.coli aminotransferase mutants ((i), (ii), (iii), (iv), (v), (vi)) were cultured in LB liquid medium (100. Mu.g/ml kanamycin), at 37℃and 220rpm overnight; cultures were transferred at a 1:100 ratio into 50mL fresh LB medium (250 mL shake flask) and grown at 37 ℃. When the optical density at 600nm (OD 600) reached about 0.6, isopropyl thiogalactoside (IPTG) was added to a final concentration of 1mM and the cells were grown at 25℃for 16 hours. After centrifugation at 12000rpm at 4℃for 10min, the supernatant was discarded, and the cell pellet was resuspended at 200g/L in pre-chilled 100mM Tris-HCl buffer (pH 8.5), sonicated, and then centrifuged at 12000rpm at 4℃for 30min, the supernatant, i.e., crude enzyme solution, was collected and stored at-20 ℃. The crude enzyme solution was subjected to polyacrylamide gel electrophoresis, and the results are shown in FIG. 2a, wherein lanes from left to right in FIG. 2a are respectively Marker, blank, wild type, mutant (i), mutant (ii), mutant (iii), mutant (iv), mutant (v) and mutant (vi) polyacrylamide gel electrophoresis results. FIG. 2b shows the polyacrylamide gel electrophoresis results of mutant (vi), wherein lanes from left to right are Marker and mutant (vi), respectively.

Example 4: high density fermentation preparation of transaminase mutant proteins

The recombinant mutant strain obtained in example 2 was inoculated into 3mL of liquid LB medium, cultured overnight at 37℃under shaking at 220rpm, inoculated into 400mL of liquid LB medium at a ratio of about 1%, and cultured until the OD600 reached 4 as a seed solution, followed by high-density fermentation in 2L of fermentation medium. The initial temperature was 37℃and the stirring speed was 300rpm, the aeration rate was 1.5vvm/L/min, the pH was 6.8, and then the stirring speed was increased continuously up to 1000rpm. The fermentation culture nutrient is in two stages, after inoculation in the first stage, the culture is carried out for about 4 hours, the carbon source consumption in the culture medium is complete, and the feedback feeding is carried out according to DO. The temperature is reduced to 25 ℃ after feeding, dissolved oxygen is kept above 30%, isopropyl thiogalactoside (IPTG) is added for induction after feeding for 8 hours, and the mixture is placed in a tank after 12 hours of induction. The supernatant was centrifuged at 8000rpm for 10 minutes to obtain wet cells, and the wet cells were suspended in 100mM Tris-HCl buffer (pH 8.5) and crushed by a high-pressure homogenizing crusher to obtain a crude enzyme solution of high-density fermentation transaminase.

Example 5: whole-cell catalytic reaction system for transaminase

The substrates used in this example are compounds of the following formula (I): the substrate is reacted by the aminotransferase of the present invention to form a compound of formula (II).

48g of wet cells (solid after centrifugation of the fermentation broth) containing the above transaminase mutants (i), (ii), (iii), (iv), (v) and (vi), respectively, were added to a 250mL glass reactor at room temperature together with 236 mL 6M isopropylamine, 100mg pyridoxal phosphate and 72mL 100mM Tris-HCl buffer (pH 8.5). Stirring at 100rpm for 20min to mix all solids, adding 10% sodium hydroxide solution to adjust the pH to 8.5. Subsequently, the temperature was increased to 50℃using a circulating water bath. 3g of substrate (compound I described above) were then dissolved in 5mL of DMSO and slowly added to the stirred mixture in a 250mL reactor at 500 rpm. The pH was monitored during the reaction and controlled at 8.5, and samples of the mixture were analyzed by HPLC. After 12h, the conversion of the substrates under catalytic conditions (compound (I)) by mutants (I), (ii), (iii), (iv), (v) and (vi) was 4%, 9%, 12%, 16%, 45% and 85.9%, respectively; after 24h, the substrate conversions under catalytic conditions for mutants (i), (ii), (iii), (iv), (v) and (vi) were 17%, 19%, 26%, 31%, 60% and 99.9%, respectively. After completion of the reaction, the system containing mutant (vi) was adjusted to ph=2-3 with hydrochloric acid and filtered. After filtration the filtrate was extracted with 50mL of methyl tert-butyl ether. The aqueous phase was adjusted to ph=12 and extracted 2 times with 50mL of dichloromethane. The combined organic phases are dried over anhydrous sodium sulfate and concentrated at T < 40 ℃. The purity of the obtained target product is more than 98 percent, the de value is more than 99 percent, and the yield is obtained through HPLC detection90%. The product is ¹ HNMR image spectrogram 3, and standard substance ¹ HNMR maps are consistent.

Example 6: temperature stability and catalytic effects of transaminases and mutants

Under the catalytic reaction system of example 5, crude enzyme solutions of the transaminase enzymes of the wild type and mutant (vi) (the amino acid sequences of which are shown in SEQ ID NO: 3) were taken, respectively, incubated at different temperatures (30 to 50 ℃ C., temperature interval 10 ℃ C.) for 12, 24, 48 hours, and then the residual enzyme activities were determined in accordance with the following method (enzyme activities were defined as the amount of enzyme required to produce 1. Mu. Mol of the product (the aforementioned compound II) per minute under the aforementioned conditions were defined as 1 enzyme activity unit).

10.2ml of 100mM Tris-HCl,4.8ml of 6M isopropylamine, 20mg of pyridoxal phosphate, 0.4g of substrate (compound I described above) were dissolved in 1ml of DMSO,4ml of crude enzyme solution (1.6 g of wet cells). 45 ℃ C./200 rpm reaction for 3 hours, sampling and determination. The control was an enzyme which had not been subjected to temperature treatment and whose activity was set to 100%. The time for which the residual enzyme activity was reduced to about 50% of the original enzyme activity was taken as the half-life of the enzyme at that temperature, thereby determining the temperature stability of the transaminase.

Temperature (temperature)	Wild type transaminase	Mutant aminotransferase
				30℃	More than 48h	More than 48h
40℃	24h	More than 48h
			50℃	12h	More than 48h

Under the reaction system of example 5, crude enzyme solutions of wild transaminase and mutant transaminase were taken and reacted at 50℃respectively, and the conversion of the substrate was determined by liquid chromatography.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims (13)

1. A transaminase mutant, characterized in that the amino acid sequence of the transaminase mutant consists of SEQ ID NO:1, wherein the mutation comprises at least one of the following mutation sites:

the amino acid sequence of the transaminase mutant has a mutation site in the amino acid sequence where mutation occurs, and the amino acid sequence has more than 80% homology with the amino acid sequence where mutation at position 60 from W to A, D or K, mutation at position 89 from F to A, D or K, mutation at position 121 from S to A, D or F, mutation at position 153 from Y to A, D or F, mutation at position 226 from E to A, R or F, mutation at position 261 from V to A, D or Y, mutation at position 380 from Q to A, K or F, mutation at position 417 from A to F, D or K.

2. The transaminase mutant of claim 1, wherein the mutation comprises at least one of the following mutation sites:

the amino acid sequence of the aminotransferase mutant has a mutation site in the mutated amino acid sequence and has more than 80% of homology with the mutated amino acid sequence, wherein the mutation at the 60 th site is changed from W to A, the mutation at the 89 th site is changed from F to K, the mutation at the 121 th site is changed from S to D, the mutation at the 153 th site is changed from Y to A, the mutation at the 226 th site is changed from E to R, the mutation at the 261 rd site is changed from V to A, the mutation at the 380 th site is changed from Q to F, and the mutation at the 417 th site is changed from A to F.

3. The transaminase mutant of claim 2, characterized in that it comprises the following mutation sites:

mutation from W to A at position 60, F to K at position 89, S to D at position 121, Y to A at position 153 and E to R at position 226, or

Mutation from W to A at position 60, F to K at position 89, S to D at position 121, Y to A at position 153, E to R and V to A at position 261, or

The mutation from W to A at position 60, from F to K at position 89, from S to D at position 121, from Y to A at position 153, from E to R at position 226, from V to A at position 261 and from Q to F at position 380, or

The 60 th site is mutated from W to A, the 89 th site is mutated from F to K, the 121 th site is mutated from S to D, the 153 th site is mutated from Y to A, the 226 th site is mutated from E to R, the 261 rd site is mutated from V to A, the 380 th site is mutated from Q to F and the 417 th site is mutated from A to F.

4. A transaminase mutant according to claim 3, which comprises a mutation site: the amino acid sequence of the aminotransferase mutant is shown as SEQ ID NO.3, wherein the 60 th site is mutated from W to A, the 89 th site is mutated from F to K, the 121 th site is mutated from S to D, the 153 th site is mutated from Y to A, the 226 th site is mutated from E to R, the 261 rd site is mutated from V to A, the 380 th site is mutated from Q to F, the 417 th site is mutated from A to F.

5. A gene encoding the transaminase mutant of any one of claims 1 to 4.

6. The gene according to claim 5, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.4, or

The nucleotide sequence of the gene is a sequence with more than 95% of homology with the nucleotide sequence shown in SEQ ID NO. 4.

7. A recombinant expression vector comprising the gene of claim 5 or 6.

8. The recombinant expression vector according to claim 7, wherein the recombinant expression vector is selected from pET-28a, pET-duct 1 or pRSF-duct 1, more preferably pET-28a.

9. A genetically engineered bacterium for producing the transaminase mutant of any one of claims 1 to 4, comprising the recombinant expression vector of claim 7 or 8.

10. Genetically engineered bacterium according to claim 9, characterized in that the genetically engineered bacterium is selected from the group consisting of escherichia coli MG1655 or escherichia coli BL21 (DE 3) pLysS, more preferably escherichia coli BL21 (DE 3).

11. Use of a transaminase mutant according to any one of claims 1 to 4 for the preparation of chiral amines by catalyzing carbonyl compounds.

12. A process for the production of chiral amines, characterized in that it comprises the catalytic transamination of ketones using the transaminase mutants according to any of claims 1 to 4.

13. The method of claim 12, wherein the ketone compound is

Wherein R is ₁ And R is ₂ Each independently is selected from substituted or unsubstituted alkyl, unsaturated hydrocarbyl, aromatic hydrocarbyl or heterocyclic aromatic hydrocarbyl; or alternatively

R ₁ And R is ₂ May be singly or in combination with each other to form a substituted or unsubstituted ring,

the substitution means substitution with a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group.

Images