CN116064449A - A kind of transaminase mutant and its application - Google Patents

Abstract

The present invention relates to a transaminase mutant and its application. The transaminase mutant is obtained by mutation of the amino acid sequence shown in SEQID NO: 1, and the mutation includes at least one of the following mutation sites: the 60th position is formed by W Mutation 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, position 226 E is mutated to A, R or F, the 261st position is mutated from V to A, D or Y, the 380th position is mutated from Q to A, K or F, and the 417th position is mutated from A to F, D or K. The transaminase mutant can be applied to the green chemical synthesis of large-sterically hindered chiral amine compounds, and has high catalytic activity and good stability. Compared with the chemical synthesis method, the reaction catalyzed by it is simple and mild, does not require hydrogenation reaction, has high reaction selectivity, low preparation cost, and has good application prospect.

Description

A transaminase mutant and its application

Technical Field

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

Background Art

Transaminases catalyze the stereoselective transfer of amino groups between amino donors (amine donors) and prochiral keto substrates and are effective biocatalytic tools for the generation of optically pure chiral amines. Transaminases (TAs) can be divided into two categories: α-transaminases (α-TAs) and ω-transaminases (ω-TAs), depending on the type of substrate being transformed. α-TAs require the presence of a carboxyl group both at the α-position of the carbonyl functional group of the keto substrate and on the amine donor. ω-TAs can accept aliphatic ketones and amines as substrates (i.e., not just α-keto acids and amino acids). ω-TAs can be further divided into two subgroups, β-TAs and amine transaminases (ATAs), of which ATAs are of industrial interest because they are able to perform reductive amination reactions using a wide range of amine donors and ketone acceptors. ATAs catalyze the intermolecular exchange of amine donors and ketone acceptors and have received considerable attention in recent years in the production of pharmaceutical intermediates.

Although transaminase-catalyzed asymmetric transamination reactions provide an economical and green synthetic approach for the synthesis of chiral amine compounds. However, some limitations and challenges of ATAs in large-scale applications have also been identified, such as a rather narrow substrate range, unfavorable thermodynamic reaction equilibrium, and substrate/product inhibition. In general, if the group next to the potential chiral carbonyl is larger than the methyl group, the catalytic effect of the transaminase will be significantly reduced. To overcome this limitation, a lot of efforts have been made to evolve ATAs to improve their substrate range, especially in so-called "bulky" compounds. Different protein engineering approaches have been used to optimize the available ATAs.

CN112980810A discloses a transaminase mutant derived from Chromobaterium violaceum, which is intended to use the transaminase mutant to convert ketone compounds with a group larger than methyl next to the potential chiral carbonyl into chiral amine compounds with high selectivity. However, the acquisition of the transaminase mutant requires saturation mutation and iterative mutation, which requires a large amount of screening work and is very cumbersome.

Summary of the invention

In order to solve the problems of low efficiency, low selectivity and high environmental impact in the chemical synthesis of chiral amines, as well as low catalytic activity and poor stability of biological enzymes in the biosynthesis process, one aspect of the present invention is to provide a transaminase mutant with high catalytic activity, good thermal stability and high efficiency in catalyzing the synthesis of large sterically hindered chiral amine compounds. To achieve the purpose of the present invention, the present invention adopts the following technical solutions:

A transaminase mutant, the amino acid sequence of which is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation includes at least one of the following mutation sites:

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

Preferably, the above mutation includes at least one of the following mutation sites:

The 60th position mutates from W to A (W60A), the 89th position mutates from F to K (F89K), the 121st position mutates from S to D (S121D), the 153rd position mutates from Y to A (Y153A), the 226th position mutates from E to R (E226R), the 261st position mutates from V to A (V261A), the 380th position mutates from Q to F (Q380F), and the 417th position mutates from A to F (A417F), or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology to the mutated amino acid sequence.

More preferably, the above transaminase mutant comprises the following mutation sites:

The 60th position mutates from W to A, the 89th position mutates from F to K, the 121st position mutates from S to D, the 153rd position mutates from Y to A and the 226th position mutates from E to R, or,

The 60th position mutates from W to A, the 89th position mutates from F to K, the 121st position mutates from S to D, the 153rd position mutates from Y to A, the 226th position mutates from E to R and the 261st position mutates from V to A, or,

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

Position 60 mutated from W to A, position 89 mutated from F to K, position 121 mutated from S to D, position 153 mutated from Y to A, position 226 mutated from E to R, position 261 mutated from V to A, position 380 mutated from Q to F and position 417 mutated from A to F.

More preferably, the above-mentioned transaminase mutant includes mutation sites 60 from W to A, 89 from F to K, 121 from S to D, 153 from Y to A, 226 from E to R, 261 from V to A, 380 from Q to F and 417 from A to F, and its amino acid sequence is shown in SEQ ID NO.3.

Another object of the present invention is to provide a gene encoding the above-mentioned aminotransferase mutant.

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

Another aspect of the present invention is to provide a recombinant expression vector comprising a gene encoding the above-mentioned transaminase mutant.

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

Another aspect of the present invention is to provide a genetically engineered bacterium for producing the above-mentioned aminotransferase mutant, which comprises the above-mentioned recombinant expression vector.

Preferably, the genetically engineered bacteria is selected from Escherichia coli MG1655, Escherichia coli BL21 (DE3), or Escherichia coli BL21 (DE3) pLysS.

More preferably, the genetically engineered bacteria is selected from Escherichia coli BL21 (DE3).

Another aspect of the present invention is to provide the use of the above transaminase mutant in catalyzing the preparation of chiral amines from carbonyl compounds or amino donors.

Another aspect of the present invention is a method for producing chiral amines, which comprises the step of using the above-mentioned aminotransferase mutant to catalyze the transamination reaction of a ketone compound or an amino donor.

其中R₁和R₂各自独立地为选取代或未被取代的烷基、不饱和烃基、芳烃基或杂环芳烃基；R₁和R₂可单独或两者互相结合形成取代或未被取代的环。Preferably, the ketone compound is

Wherein R ₁ and R ₂ are each independently a substituted or unsubstituted alkyl, unsaturated hydrocarbon, aromatic hydrocarbon or heterocyclic aromatic hydrocarbon group; R ₁ and R ₂ can be alone or in combination with each other to form a substituted or unsubstituted ring.

The substitution refers to 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.

Another aspect of the present invention is a method for preparing a compound of formula (II), which comprises the step of catalytically converting the compound of formula (I) using the above-mentioned aminotransferase mutant to form a compound of formula (II), and the reaction equation is as follows:

The above-mentioned transaminase mutant of the present invention is mutated on the basis of the transaminase shown in SEQ ID NO: 1 by a site-directed mutagenesis method, thereby changing its amino acid sequence and realizing changes in protein structure and function, and then a directed screening method is used to obtain a transaminase having the above-mentioned mutation site. The transaminase mutant of the present invention has the advantage of greatly improved enzyme activity, and its enzyme activity is increased by several times compared with the transaminase parent, and its stability is also greatly improved. When used for the production of chiral amines, the cost of industrial production of chiral amines is greatly reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

Figure 2b is a polyacrylamide gel electrophoresis diagram of the small-scale production of the transaminase mutant (vi) (whose amino acid sequence is SEQ ID NO: 3) in a shake flask obtained in Example 3, wherein the lanes from left to right are the polyacrylamide gel electrophoresis results of the Marker and the mutant (vi), respectively.

3 is the ¹ HNMR spectrum of the standard product and the chiral amine product obtained in Example 6. The upper part is the ¹ HNMR spectrum of the standard product, and the lower part is the ¹ HNMR spectrum of the product obtained in Example 6.

FIG4 is the fluorine spectrum of the standard product and the chiral amine product obtained in Example 6, the upper half is the fluorine spectrum of the standard product, and the lower half is the fluorine spectrum of the product obtained in Example 6.

DETAILED DESCRIPTION

The aminotransferase derived from Xenophilus sp.AP218F can selectively catalyze the conversion of carbonyl groups, but its activity is low and its stability is poor. The inventors improved the activity and stability of the aminotransferase derived from Xenophilus sp.AP218F by a rationally designed method. Mutation sites were introduced into the aminotransferase derived from Xenophilus sp.AP218F by means of whole plasmid PCR, and the activity and stability of the mutants were tested to select mutants with improved activity and stability.

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

The invention obtains the three-dimensional structure of the aminotransferase derived from Xenophilus sp.AP218F through an online protein structure prediction tool, obtains the three-dimensional structure of the transaminase with the highest structural similarity (6S4G, homology 87.58%) through PDB for structural comparison, then performs binding simulation of the substrate of formula I and the three-dimensional structure of the transaminase protein through AutoDock, and finally selects the amino acid that may be related to the substrate binding as the mutant amino acid through Pymol analysis. According to the Pymol analysis results, multiple pairs of site-directed mutation (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 mutant plasmid with a target gene is obtained by using a site-directed mutation method and taking pET-28a as an expression vector. Among them, site-directed mutagenesis refers to the introduction of desired changes (usually changes that characterize favorable directions) into target DNA fragments (which can be genomes or plasmids) through methods such as polymerase chain reaction (PCR), including addition, deletion, and point mutation of bases. Site-directed mutagenesis can quickly and efficiently improve the properties and characterization of target proteins expressed by DNA, and is a very useful method in genetic research.

The introduction of site-directed mutations using whole plasmid PCR is a method that is currently used more frequently. The principle is that a pair of primers (forward and reverse) containing the mutation site are annealed with the template plasmid and then "circularly extended" by the polymerase. The so-called circular extension refers to the polymerase extending the primer according to the template, returning to the 5' end of the primer after one circle to terminate, and then undergoing repeated heating annealing and extension cycles. This reaction is different from rolling circle amplification and will not form multiple tandem copies. After annealing, the extension products of the forward and reverse primers are paired to form an open-circular plasmid with a nick. The extension product is cut by Dpn I. Since the original template plasmid is derived from conventional Escherichia coli and is modified by dam methylation, it is sensitive to Dpn I and is chopped up. The plasmid with a mutation sequence synthesized in vitro is not methylated and is not cut open. Therefore, it can be successfully transformed in the subsequent transformation, and the mutant plasmid clone can be obtained. The mutant plasmid is transformed into Escherichia coli cells and overexpressed in Escherichia coli. Then, the wet cells are obtained by centrifugation, and the crude enzyme is obtained by ultrasonic cell disruption.

As a first aspect of the present invention, the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 is mutated, and the mutation includes at least one of the following mutation sites: the 60th position is mutated from W to A, D or K (W60A/D/K); the 89th position is mutated from F to A, D or K (F89A/D/K); the 121st position is mutated from S to A, D or F (S121A/F/D); the 153rd position is mutated from Y to A, D or F (Y153A/D/F); the 226th position is mutated from E ... R or F (E226A/R/F); position 261 mutates from V to A, D or Y (V261A/D/Y); position 380 mutates from Q to A, K or F (Q380A/F/K), position 417 mutates from A to F, D or K (A417F/D/K), or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology to the mutated amino acid sequence. In a preferred embodiment of the present invention, the mutation includes at least one of the following mutation sites: position 60 mutates from W to A (W60A), position 89 mutates from F to K (F89K), position 121 mutates from S to D (S121D), position 153 mutates from Y to A (Y153A), position 226 mutates from E to R (E226R), position 261 mutates from V to A (V261A), position 380 mutates from Q to F (Q380F), position 417 mutates from A to F (A417F), or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology with the mutated amino acid sequence. Wherein "homology" refers to the identity between two amino acid sequences. The sequences defined by different degrees of homology in the present invention must also have improved transaminase activity at the same time. Under the guidance of the disclosure of the present application, a person skilled in the art can obtain an amino acid sequence of a transaminase mutant having the mutation site in the above-mentioned mutated amino acid sequence and having an amino acid sequence with more than 80% homology with the mutated amino acid sequence.

In a more preferred embodiment of the present invention, the aminotransferase mutant comprises the mutation sites: W60A, F89K, S121D, Y153A and E226R. In a more preferred embodiment of the present invention, the aminotransferase mutant comprises the mutation sites: W60A, F89K, S121D, Y153A, E226R and V261A. In a more preferred embodiment of the present invention, the aminotransferase mutant comprises the mutation sites: W60A, F89K, S121D, Y153A, E226R, V261A and Q380F.

In a more preferred embodiment of the present invention, the above-mentioned transaminase mutant includes mutation sites: W60A, F89K, S121D, Y153A, E226R, V261A, Q380F and A417F, and its amino acid sequence is shown in SEQ ID NO.3.

The above-mentioned transaminase mutant of the present invention is mutated by site-directed mutagenesis on the basis of the transaminase sequence shown in SEQ ID NO: 1, thereby changing its amino acid sequence and realizing changes in protein structure and function, and then obtaining a transaminase having the above-mentioned mutation site by a directed screening method. The transaminase mutant of the present invention has the advantage of greatly improved enzyme activity. Its conversion rate of the substrate is increased by 95 times compared with the transaminase parent under the same conditions, and the enzyme stability is also correspondingly improved, thereby greatly reducing the cost in the industrial production of chiral amines.

As a second aspect of the present invention, the present invention provides a gene encoding the above-mentioned aminotransferase mutant. The present invention mutates the gene of the wild-type aminotransferase by rational design (site-directed mutagenesis or other methods to change individual amino acids in the protein molecule) and overlap extension PCR, seamless cloning and other methods to obtain the target gene of the above-mentioned aminotransferase mutant. In a preferred embodiment of the present invention, 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% homology with the sequence shown in SEQ ID NO.4. Wherein "homology" refers to the degree of identity between two nucleotide sequences.

The transaminase obtained by the gene encoding of the present invention improves the enzyme activity and enzyme stability, making the industrial production efficiency of chiral amines higher and the cost 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 present invention. The gene is located at an appropriate position in the recombinant expression vector so that the above gene can be correctly and smoothly replicated, transcribed or expressed. In order to meet the requirements of the recombination operation, in the recombinant expression vector, both ends of the above gene sequence can be added with suitable restriction endonuclease sites, or additional start codons, stop codons, etc. can be added. 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, the present invention provides a genetically engineered bacterium for producing the above-mentioned aminotransferase mutant, which comprises the above-mentioned recombinant expression vector. In the present invention, the genetically engineered bacterium includes but is not limited to Escherichia coli MG1655 or Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS.

The above transaminase mutant can be prepared by fermenting and culturing the genetically engineered bacteria. For example, the above transaminase mutant can be prepared industrially under certain production tank fermentation conditions. The production tank fermentation conditions are preferably: DO 20% or more, temperature 20°C.

As a fifth aspect of the present invention, the present invention provides the use of the above-mentioned aminotransferase mutant in catalyzing the preparation of chiral amines from carbonyl compounds.

As a sixth aspect of the present invention, the present invention provides a method for producing chiral amines, which comprises the step of utilizing the above-mentioned aminotransferase mutant to catalyze the transamination reaction of a ketone compound.

其中R₁和R₂各自独立地为取代或未被取代的烷基、不饱和烃基、芳烃基或杂环芳烃基；或R₁和R₂可单独或两者互相结合形成取代或未被取代的环。所述取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。Preferably, the ketone compound is

Wherein _R1 and _R2 are each independently a substituted or unsubstituted alkyl, unsaturated hydrocarbon, aromatic hydrocarbon or heterocyclic aromatic hydrocarbon; or _R1 and _R2 may be alone or in combination with each other to form a substituted or unsubstituted ring. The substitution refers to 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, _R1 and _R2 are each independently a substituted or unsubstituted alkyl group, an unsaturated hydrocarbon group, an aromatic hydrocarbon group or a heteroaromatic hydrocarbon group having 1 to 20 carbon atoms, or _R1 and _R2 may be combined alone or in combination to form a substituted or unsubstituted ring having 1 to 20 carbon atoms.

More preferably, _R1 and _R2 are each independently a substituted or unsubstituted alkyl group, an unsaturated hydrocarbon group, an aromatic hydrocarbon group or a heteroaromatic hydrocarbon group having 1 to 10 carbon atoms, or _R1 and _R2 may be combined alone or in combination to form a substituted or unsubstituted ring having 1 to 10 carbon atoms.

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

The unsaturated hydrocarbon group may be selected from vinyl, 1-methylvinyl, 1-ethylvinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-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, 3,3-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-octynyl, 2-nonynyl and 2-decynyl, etc.

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

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

The substitution refers to 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 ketone compound is selected from compounds having the following structures:

In the step of using the above-mentioned transaminase mutant to catalyze the transamination reaction of ketone compounds, the pH of the reaction system for the catalytic transamination reaction is preferably 7-10, more preferably 8-9, and most preferably 8.5; the temperature of the reaction system is preferably 40°C to 60°C, more preferably 45-55°C, and most preferably 50°C.

As a seventh aspect of the present invention, the present invention provides a method for preparing a compound represented by formula (II), the preparation method comprising the step of catalytically converting the compound represented by formula (I) using the above-mentioned aminotransferase mutant to form a compound represented by formula (II), and the reaction equation is as follows:

Preferably, in the above preparation method, in the reaction system, the pH is preferably 7-10, more preferably 8-9, most preferably 8.5; the temperature of the reaction system is preferably 40°C-60°C, more preferably 45-55°C, most preferably 50°C.

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

1. The aminotransferase mutant of the present invention, as shown in SEQ ID NO.3, has a higher catalytic activity for a large sterically hindered substrate, so that the conversion rate of the substrate (the compound shown in formula (I)) is 60% in 12 hours, while the enzyme activity of the wild-type enzyme for such substrate is less than 1%. In addition, the aminotransferase mutant of the present invention has a half-life of more than 48 hours at 50°C, while the half-life of the wild-type enzyme under the same conditions is only 12 hours.

2. The transaminase mutant provided by the present invention has high catalytic activity and good heat resistance under alkaline conditions, and can be used in the biosynthesis of chiral amines; compared with chemical synthesis methods, the transamination reaction catalyzed by it is simple and mild, has high reaction selectivity, low preparation cost, and has good application prospects.

3. The catalytic activity of the transaminase mutant of the present invention towards ketone substrates is greatly improved compared to the wild type, and the synthesis of large sterically hindered chiral amine compounds by transaminase catalysis is realized without the need for heavy metal catalysts, thereby achieving green chemical synthesis. Moreover, the use of transaminase with enhanced activity for transamination reactions can easily obtain highly selective chiral amine compounds.

The present invention is further described in detail below in conjunction with the examples, which are explanations of the present invention and are not limited to the following examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

Example 1: Establishment of wild transaminase genetically engineered bacteria

The whole gene fragment (nucleotide sequence as shown in SEQ ID NO.2) was artificially synthesized after sequence optimization based on the wild-type gene sequence of Xenophilus sp.AP218F transaminase included in NCBI (GenBank: OWY40557.1), and the gene was inserted into the pET-28a plasmid (Figure 1) by a gene synthesis company through NdeI and XhoI endonucleases. The ligated vector was transferred into Escherichia coli BL21 (DE3) to establish the wild-type transaminase gene engineering bacteria, and the kanamycin resistance was screened and sequenced for verification.

Example 2: Obtaining transaminase mutants and target genes

The three-dimensional structure of SEQ ID NO.1 was obtained through an online protein structure prediction tool, and the three-dimensional structure of the transaminase with the highest structural similarity (6S4G, homology 84.97%) was obtained through PDB for structural comparison. Then, the binding simulation of the substrate of formula I and the three-dimensional structure of the transaminase protein was performed through AutoDock. Finally, amino acids that may be related to substrate binding were selected as mutant amino acids through Pymol analysis.

疏水氨基酸，优选使SEQ ID NO.1所示野生型转氨酶氨基酸序列中的至少1个位点发生突变：W60A，F89K，S121D，Y153A，E226R，V261A，Q380F，A417F，根据Pymol分析，最终设计从组合突变位点：(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)W60A，F89K，S121D，Y153A，E226R，V261A，Q380F，A417F中筛选高活性突变株。其中，在对转氨酶基进行突变时，采用的是无缝克隆的方式，pET28a质粒上的引物分别位于转氨酶基因的上下游。突变位点处设置两端同源臂15bp的引物，PCR反应条件为：95℃预变性5min；94℃变性30s，53-60℃退火15s及72℃延伸50s进行30个循环；于72℃下继续延伸10min，冷却至4℃。According to the above Pymol analysis results, at least one site in the wild-type aminotransferase amino acid sequence shown in SEQ ID NO.1 is mutated: position 60, position 89, position 121, position 153, position 226, position 261, position 380 and position 417. And the W at position 60 is mutated to A, D or K; the F at position 89 is mutated to A, D or K; the S at position 121 is mutated to A, D or F; the Y at position 153 is mutated to A, D or F; the E at position 226 is mutated to A, R or F; the V at position 261 is mutated to A, D or Y; the Q at position 380 is mutated to A, K or F. According to the docking results, the distance between the substrate binding region and the substrate is analyzed.

Hydrophobic amino acids, preferably mutating at least one site in the wild-type aminotransferase amino acid sequence shown in SEQ ID NO.1: W60A, F89K, S121D, Y153A, E226R, V261A, Q380F, A417F. According to Pymol analysis, the final design is from the combination of 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) W60A, F89K, S121D, Y153A, E226R, V261A, Q380F, A417F were selected for high-activity mutants. When the aminotransferase base was mutated, a seamless cloning method was used, and the primers on the pET28a plasmid were located at the upstream and downstream of the aminotransferase gene, respectively. Primers with 15 bp homology arms at both ends were set at the mutation site, and the PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 53-60°C for 15 s, and extension at 72°C for 50 s for 30 cycles; extension was continued at 72°C for 10 min and cooled to 4°C.

The fragment after PCR amplification according to the above method was connected with the pET28a plasmid vector using a seamless cloning kit, and the connected vector was transferred into Escherichia coli BL21 (DE3) to establish the transaminase gene mutant. Escherichia coli BL21 (DE3) was used as the host and the pET28a plasmid was used as the vector to express the extended mutant transaminase.

The present invention uses the enzyme activity detection method described in Example 5 below to screen high-activity mutants and identify the high-activity transaminase gene after mutation. The detection results are shown in Table 1 below:

Table 1.

Note: In the above table, + represents that the substrate conversion rate is greater than or equal to 0% and less than 20%, ++ represents that the substrate conversion rate is greater than or equal to 20% and less than 40%, +++ represents that the substrate conversion rate is greater than or equal to 40% and less than 80%, and ++++ represents that the substrate conversion rate is greater than or equal to 80%.

The gene of the mutated high-activity transaminase was identified, and the nucleotide sequence of the mutant having the combined mutation site (vi) (whose amino acid sequence is shown in SEQ ID NO.3) was obtained as shown in SEQ ID NO.4.

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

The recombinant cells of the single E. coli aminotransferase mutants ((i), (ii), (iii), (iv), (v), (vi)) were cultured in LB liquid medium (100 μg/ml kanamycin) at 37°C and 220 rpm overnight; the culture was transferred into 50 mL of fresh LB medium (250 mL shake flask) at a ratio of 1:100 and grown at 37°C. When the optical density at 600 nm (OD600) reached about 0.6, isopropylthiogalactoside (IPTG) was added to a final concentration of 1 mM, and the cells were grown at 25°C for 16 hours. After centrifugation at 12000 rpm and 4°C for 10 min, the supernatant was discarded, and the cell pellet was resuspended in pre-cooled 100 mM Tris-HCl buffer (pH 8.5) at 200 g/L, ultrasonically disrupted, and then centrifuged at 12000 rpm and 4°C for 30 min. The supernatant, i.e., the crude enzyme solution, was collected and stored at -20°C. The crude enzyme solution was subjected to polyacrylamide gel electrophoresis, and the results are shown in Figure 2a, wherein the lanes from left to right in Figure 2a are the polyacrylamide gel electrophoresis results of Marker, blank, wild type, mutant (i), mutant (ii), mutant (iii), mutant (iv), mutant (v) and mutant (vi). Figure 2b is the polyacrylamide gel electrophoresis result of mutant (vi), wherein the lanes from left to right are the polyacrylamide gel electrophoresis results of Marker and mutant (vi).

Example 4: High-density fermentation preparation of transaminase mutant protein

The recombinant mutant strain obtained in Example 2 was inoculated into 3mL liquid LB medium, and after overnight culture at 37°C and 220rpm, it was inoculated into 400mL liquid LB medium at a ratio of about 1%, and cultured until OD600 reached 4 as seed liquid, and then accessed into 2L fermentation medium for high-density fermentation. The initial temperature was 37°C, the stirring speed was 300rpm, the ventilation volume was 1.5vvm/L/min, and the pH was 6.8, and then the stirring speed was continuously increased to a maximum of 1000rpm. The fermentation culture was divided into two stages. After the first stage was inoculated, it was cultured for about 4 hours, the carbon source in the culture medium was completely consumed, and the feeding was fed back according to DO. After feeding, the temperature dropped to 25°C, the dissolved oxygen was maintained at more than 30%, and isopropylthiogalactoside (IPTG) was added for induction after 8 hours of feeding, and the tank was released after 12 hours of induction. The wet cells were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the cells were suspended in 100 mM Tris-HCl buffer (pH 8.5). The suspended cells were crushed by a high-pressure homogenizer to obtain a crude enzyme solution of high-density fermentation transaminase.

Example 5: Whole-cell catalytic reaction system of transaminase

The substrate used in this example is a compound represented by the following formula (I): the substrate is catalyzed by the transaminase of the present invention to form a compound represented by formula (II).

At room temperature, 48 g of wet cells (solids after centrifugation of the fermentation broth) containing the above-mentioned transaminase mutants (i), (ii), (iii), (iv), (v) and (vi) were added to a 250 mL glass reactor with 23 mL of 6 M isopropylamine, 100 mg of pyridoxal phosphate and 72 mL of 100 mM Tris-HCl buffer (pH 8.5). Stir at 100 rpm for 20 min to mix all the solids, and add 10% sodium hydroxide solution to adjust the pH to 8.5. Subsequently, a circulating water bath was used to increase the temperature to 50° C. Then 3 g of substrate (the aforementioned compound I) was dissolved in 5 mL of DMSO and slowly added to the stirred mixture at 500 rpm in a 250 mL reactor. The pH was monitored during the reaction and controlled at 8.5, and the mixture was sampled for HPLC analysis. After 12 hours, the conversion rates of the substrate (compound (I)) under the catalytic conditions of mutants (i), (ii), (iii), (iv), (v) and (vi) were 4%, 9%, 12%, 16%, 45% and 85.9%, respectively; after 24 hours, the conversion rates of the substrate under the catalytic conditions of mutants (i), (ii), (iii), (iv), (v) and (vi) were 17%, 19%, 26%, 31%, 60% and 99.9%, respectively. After the reaction was complete, the system containing mutant (vi) was adjusted to pH = 2-3 with hydrochloric acid and filtered. After filtration, the filtrate was extracted with 50 mL of methyl tert-butyl ether. The aqueous phase was adjusted to pH = 12 and then extracted twice with 50 mL of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and concentrated at T < 40 ° C. The target product was obtained and tested by HPLC, with a purity of > 98%, a de value of > 99%, and a yield of 90%. The ¹ H NMR spectrum of the product is shown in Figure 3, which is consistent with the ¹ H NMR spectrum of the standard product.

Example 6: Temperature stability and catalytic effect of transaminases and mutants

In the catalytic reaction system of Example 5, crude enzyme solutions of wild-type and mutant (vi) (whose amino acid sequence is shown in SEQ ID NO: 3) were taken respectively, incubated at different temperatures (30-50° C., with a temperature interval of 10° C.) for 12, 24, and 48 hours, and then the residual enzyme activity was determined according to the following method (enzyme activity is defined as: under the above conditions, the amount of enzyme required to generate 1 μmol of product (the aforementioned compound II) per minute is defined as 1 enzyme activity unit).

10.2ml100mMTris-Hcl, 4.8ml6Misopropylamine, 20mgpyridoxal phosphate, 0.4gsubstrate (the aforementioned compound I) dissolved in 1mlDMSO, 4mlcrude enzyme solution (1.6gwet cells). 45℃/200rpm for 3 hours and then sampled for determination. The control is the enzyme without temperature treatment, and its activity is set as 100%. The time when the residual enzyme activity drops to about 50% of the original enzyme activity is the half-life of the enzyme at this temperature, which is used to determine the temperature stability of the transaminase.

temperature Wild-type transaminase Mutant transaminase 30℃ More than 48 hours More than 48 hours 40℃ 24h More than 48 hours 50℃ 12h More than 48 hours

In the reaction system of Example 5, crude enzyme solutions of wild-type and mutant transaminase were respectively taken and reacted at 50° C. The substrate conversion rate was determined by liquid chromatography analysis.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as falling within the scope of protection of the present invention.

Claims (13)

1. A transaminase mutant, characterized in that the amino acid sequence of the transaminase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, and the mutation includes at least one of the following mutation sites: The 60th position mutates from W to A, D or K, the 89th position mutates from F to A, D or K, the 121st position mutates from S to A, D or F, the 153rd position mutates from Y to A, D or F, the 226th position mutates from E to A, R or F, the 261st position mutates from V to A, D or Y, the 380th position mutates from Q to A, K or F, and the 417th position mutates from A to F, D or K, or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology to the mutated amino acid sequence. 2. The aminotransferase mutant according to claim 1, characterized in that the mutation comprises at least one of the following mutation sites: Position 60 mutates from W to A, position 89 mutates from F to K, position 121 mutates from S to D, position 153 mutates from Y to A, position 226 mutates from E to R, position 261 mutates from V to A, position 380 mutates from Q to F, and position 417 mutates from A to F, or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence, and has an amino acid sequence with more than 80% homology to the mutated amino acid sequence. 3. The transaminase mutant according to claim 2, characterized in that the transaminase mutant comprises the following mutation sites: The 60th position mutates from W to A, the 89th position mutates from F to K, the 121st position mutates from S to D, the 153rd position mutates from Y to A and the 226th position mutates from E to R, or The 60th position mutates from W to A, the 89th position mutates from F to K, the 121st position mutates from S to D, the 153rd position mutates from Y to A, the 226th position mutates from E to R and the 261st position mutates from V to A, or position 60 from W to A, position 89 from F to K, position 121 from S to D, position 153 from Y to A, position 226 from E to R, position 261 from V to A and position 380 from Q to F, or Position 60 mutated from W to A, position 89 mutated from F to K, position 121 mutated from S to D, position 153 mutated from Y to A, position 226 mutated from E to R, position 261 mutated from V to A, position 380 mutated from Q to F and position 417 mutated from A to F. 4. The transaminase mutant according to claim 3, characterized in that the transaminase mutant includes mutation sites: position 60 mutates from W to A, position 89 mutates from F to K, position 121 mutates from S to D, position 153 mutates from Y to A, position 226 mutates from E to R, position 261 mutates from V to A, position 380 mutates from Q to F, and position 417 mutates from A to F, and the amino acid sequence of the transaminase mutant is shown in SEQ ID NO.3. 5. A gene encoding the transaminase mutant according to any one of claims 1 to 4. 6. The gene according to claim 5, characterized in that the nucleotide sequence of the gene is as shown in SEQ ID NO.4, or The nucleotide sequence of the gene is a sequence having more than 95% homology with the nucleotide sequence shown in SEQ ID NO.4. 7. A recombinant expression vector, characterized in that the recombinant expression vector comprises the gene according to claim 5 or 6. 8 . The recombinant expression vector according to claim 7 , characterized in that the recombinant expression vector is selected from pET-28a, pET-dute1 or pRSF-dute1, more preferably pET-28a. 9. A genetically engineered bacterium, characterized in that the genetically engineered bacterium is used to produce the transaminase mutant according to any one of claims 1 to 4, and comprises the recombinant expression vector according to claim 7 or 8. 10. The genetically engineered bacterium according to claim 9, characterized in that the genetically engineered bacterium is selected from Escherichia coli MG1655, Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS, more preferably Escherichia coli BL21 (DE3). 11. Use of the aminotransferase mutant according to any one of claims 1 to 4 in catalyzing the preparation of chiral amines from carbonyl compounds. 12. A method for producing chiral amines, characterized in that the method comprises catalyzing the transamination of ketone compounds using the transaminase mutant according to any one of claims 1 to 4. 13. The method according to claim 12, characterized in that the ketone compound is

wherein _R1 and _R2 are each independently a substituted or unsubstituted alkyl, unsaturated hydrocarbon, aromatic hydrocarbon or heterocyclic aromatic hydrocarbon group; or _R1 and _R2 may be combined alone or together to form a substituted or unsubstituted ring. The substitution refers to 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.

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