CN118291419A

CN118291419A - Mutant of thermostable aminotransferase and application thereof

Info

Publication number: CN118291419A
Application number: CN202410538927.4A
Authority: CN
Inventors: 于慧敏; 魏宇文; 李付龙
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2024-04-30
Filing date: 2024-04-30
Publication date: 2024-07-05

Abstract

The invention discloses a mutant of thermostable aminotransferase and application thereof. The invention provides a protein, which is A) or B) as follows: a) The protein is obtained by mutating at least one, at least 2, at least 3 or at least 4 of 136 th, 142 th, 228 th and 249 th amino acid residues in wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase to obtain protein with aminotransferase activity; b) The protein shown is a protein which is derived from the protein A) and has transaminase activity by adding a tag sequence to the tail end of the amino acid sequence of the protein shown in the protein A). The thermal stability of the recombinant aminotransferase mutant catalysts obtained by the invention is obviously improved compared with that of wild type catalysts. Double improvement of heat stability and enzyme activity is realized, and high stereoselectivity of the catalyst is maintained.

Description

Mutant of thermostable aminotransferase and application thereof

Technical Field

The invention belongs to the fields of protein engineering and genetic engineering, and relates to a mutant of heat stable aminotransferase and application thereof.

Background

Chiral amine compounds are important in the pharmaceutical industry. It is estimated that over 40% of the active pharmaceutical ingredients contain these critical functional groups, including the free-flowing antidiabetic drugs SITAGLIPTIN, hypoglycemic drugs Alogliptin, trelagliptin and LINAGLLIPTIN, anti-HIV/AIDS drug Dolutegravir and Janus kinase inhibitor Tofacitinib et al (C.K.Savi l e,et al.,"Science"2010,329,305-309.L.Frodsham,et al.,"Org.Process Res.Dev."2013,17,1123-1130.M.Pollicita,et al.,"J.Anti-microb.Chemother."2014,69,2412–2419). however, traditional chemical syntheses require complex reaction conditions and expensive transition metal catalysts. With the advent of sustainable concepts and policies such as two-carbon strategy, biocatalyst-mediated synthesis of chiral compounds such as chiral amines has become a promising alternative to chemical synthesis. Transaminases are widely used as biocatalysts for chiral amine synthesis due to their ability to catalyze asymmetric ammonification of a variety of compounds.

Aminotransferases (TRANSAMINASE, TA for short, EC 2.6.1. X), also known as aminotransferases, are a class of pyridoxal phosphate (pyridoxal-5' -phosphoate, PLP) dependent transferases that can specifically transfer an amino group to a substrate ketone in the presence of an amino donor to yield the corresponding chiral amine (Simon, R.C.et al., "ACS Catal.," 2014,4 (1), 129-143.). The transaminase has wide sources, widely exists in the nature, does not need to add coenzyme and metal ions in the reaction process, has simple reaction and easy operation, and is widely applied to the synthesis of chiral drugs and intermediates thereof as a green catalyst. Compared with the traditional chemical synthesis method, the transaminase has obvious advantages in synthesizing chiral amine. Despite its wide application potential in the pharmaceutical industry, one major challenge facing the use of aminotransferase in industrial scale applications is the problem of thermal stability. Many wild-type aminotransferases, especially (R) -selective aminotransferases, used in the synthesis of optically pure chiral amines have been reported to have low thermal stability and can only be used under mild conditions, such as aminotransferase ATA-117 from Arthrobacter sp. And AtATA from Aspergillus terreus are only able to withstand reaction temperatures below 30℃ (C.K. Savile, et al Science 2010,329,305-309.Andrzej et al, plos One 2014). At high temperatures, many aminotransferases lose activity, leading to a decrease in catalytic efficiency and irreversible inactivation of the enzyme. Therefore, improving the thermal stability of transaminases is a key goal for industrial applications. Li et al have obtained a novel aminotransferase RbTA derived from photosynthetic bacteria (Rhodobater sp.) in their studies by genetic excavation and engineering, and can synthesize a variety of chiral amino alcohols and chiral N-heterocyclic amines using isopropylamine as an amino donor. However, the heat stability of this transaminase also needs to be improved for better application in large-scale industrial production (Li, F.et al., ACS catalyst.2023, 13,422-432.Li, F.et al., "adv. Synth. Catalyst.," 2021,363 (19), 4582-4589.).

The aminotransferase RbTA has the potential of synthesizing chiral amine of various types as a multifunctional catalyst, the reaction process is green and efficient, the application prospect is wide, and more attention is paid. In addition, molecular transformation is carried out on the biocatalyst by adopting rational design, so that the thermal stability of RbTA is improved, and the method has important significance in promoting the industrial application process of the biocatalyst.

Disclosure of Invention

The invention aims at (R) -selective aminotransferase RbTA with great application prospect, designs a high-heat-stability enzyme mutant by utilizing protein engineering and molecular transformation, meets the enzyme stability requirement under the industrial catalysis condition, and applies the enzyme mutant to the catalytic synthesis of a plurality of chiral amino compounds with high added values.

In a first aspect, the invention provides a protein (also known as transaminase mutant), which is a) or B) as follows:

A) The protein is obtained by mutating at least one, at least 2, at least 3 or at least 4 of 136 th, 142 th, 228 th and 249 th amino acid residues in wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase to obtain protein with aminotransferase activity;

b) The protein shown is a protein which is derived from the protein A) and has transaminase activity by adding a tag sequence to the tail end of the amino acid sequence of the protein shown in the protein A).

The term "corresponding to" as used herein has a meaning commonly understood by one of ordinary skill in the art. Specifically, "corresponding to" means that two sequences are aligned by homology or sequence identity, and that one sequence corresponds to a specified position in the other sequence. Thus, for example, in the case of "amino acid residue corresponding to position 136 of the protein shown in sequence 1", if a6 XHis tag is added to one end of the protein shown in sequence 1, position 136 corresponding to the amino acid sequence shown in sequence 1 may be position 142 in the resulting mutant.

Among the proteins described above, the protein shown in A) is any one of the following:

1) The protein is obtained by mutating 136 th amino acid residue in wild type aminotransferase or amino acid sequence corresponding to the aminotransferase, so as to obtain protein with aminotransferase activity;

2) The protein shown is obtained by mutating any one or any 2 or any 3 amino acid residues of 142 th, 228 th and 249 th amino acid residues of the wild type aminotransferase or the amino acid sequence corresponding to the wild type aminotransferase on the basis of the protein shown in the 1), so as to obtain the protein with aminotransferase activity;

3) The protein is obtained by mutating 228 th amino acid residue in the amino acid sequence of wild type aminotransferase;

4) The protein shown is obtained by mutating any one or any 2 or any 3 amino acid residues in 136 th, 142 th and 249 th amino acid residues of the wild type aminotransferase or the amino acid sequence corresponding to the wild type aminotransferase based on the protein shown in the 3), so as to obtain the protein with aminotransferase activity;

5) The protein is obtained by mutating the 142 th amino acid residue in the amino acid sequence of wild type aminotransferase;

6) The protein shown is obtained by mutating any one or any 2 or any 3 amino acid residues in 136 th, 228 th and 249 th amino acid residues of the wild type aminotransferase or the amino acid sequence corresponding to the wild type aminotransferase on the basis of the protein shown in the step 5), so as to obtain the protein with aminotransferase activity;

7) The protein is obtained by mutating the 249 th amino acid residue in the amino acid sequence of wild type aminotransferase;

8) The protein shown is obtained by mutating any one or any 2 or any 3 amino acid residues in 136 th, 142 th and 228 th amino acid residues in the wild type transaminase or the amino acid sequence corresponding to the wild type transaminase based on the protein shown in 7), and the protein with transaminase activity is obtained.

In the above protein, the mutation modes of the 136 th, 142 th, 228 th and 249 th amino acid residues are as follows:

Mutation of R at position 136 to P, G, A, F, S or K;

t at position 142 is mutated to P, G, L, R, M, K, Q, W, Y or F;

F at position 228 is mutated to Y, Q, N, T, S, I, L, W, V or A;

the 249I mutation is Y, Q, N, T, S, V, M or L.

1) Mutating F at 228 th position in the wild type aminotransferase or an amino acid sequence corresponding to the aminotransferase into Y to obtain a protein with aminotransferase activity;

2) Mutating the 249 th I of the wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase to Q to obtain a protein with aminotransferase activity;

3) Mutating the 136 th R in the wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase into P to obtain protein with aminotransferase activity;

4) Mutating the T at position 142 in the amino acid sequence of the wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase into P to obtain protein with aminotransferase activity;

5) Mutating the 136 th R in the wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase into P, and mutating the 142 th T into P to obtain protein with aminotransferase activity;

6) Mutating the 136 th R in the wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase into P, and mutating the 228 th F into Y to obtain a protein with aminotransferase activity;

7) Mutating the 142 th T of the wild type aminotransferase or an amino acid sequence corresponding to the aminotransferase into P, and mutating the 228 th F into Y to obtain a protein with aminotransferase activity;

8) Mutating the 136 th R in the wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase into P, and mutating the 249 th I into Q to obtain protein with aminotransferase activity;

9) Mutating the 142 th T of the wild type aminotransferase or an amino acid sequence corresponding to the aminotransferase into P, and mutating the 249 th I into Q to obtain a protein with aminotransferase activity;

10 Mutating F at 228 th position to Y and I at 249 th position to Q in the wild type aminotransferase or an amino acid sequence corresponding to the aminotransferase to obtain a protein with aminotransferase activity;

11 Mutating the 136 th R to P, the 142 th T to P and the 228 th F to Y in the wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase to obtain a protein with aminotransferase activity;

12 Mutating the 136 th R to P, the 142 th T to P and the 249 th I to Q in the wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase to obtain a protein with aminotransferase activity;

13 Mutating the 136 th R to P, the 228 th F to Y and the 249 th I to Q in the wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase to obtain a protein with aminotransferase activity;

14 Mutating the 142 th T to P, the 228 th F to Y and the 249 th I to Q in the wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase to obtain a protein with aminotransferase activity;

15 The 136 th R is mutated to P, the 142 th T is mutated to P, the 228 th F is mutated to Y, and the 249 th I is mutated to Q in the wild type aminotransferase or the amino acid sequence corresponding thereto, to obtain a protein with aminotransferase activity.

In the above protein, the wild-type transaminase is an (R) -selective transaminase;

and/or the number of the groups of groups,

The wild-type transaminase is any one of the following:

(a) SEQ ID NO: 1;

(b) And SEQ ID NO:1, a protein having an identity of 90% or more;

(c) And SEQ ID NO:1, and the protein has more than 95% identity.

In the above-described proteins, the protein has a higher stability and/or higher enzymatic activity than the wild-type transaminase;

and/or, the stability is thermal stability.

In a second aspect, the present invention provides a nucleic acid molecule encoding a protein according to the first aspect;

or an expression cassette, a recombinant vector or a recombinant bacterium containing said nucleic acid molecule.

The recombinant bacteria are obtained by transferring a recombinant vector containing the nucleic acid molecules into host cells;

The recombinant vector containing the nucleic acid molecule is obtained by inserting the nucleic acid molecule into an expression vector.

The expression vector selected in the present invention can exist stably and autonomously replicate in various hosts of prokaryotic or eukaryotic cells, such as conventional plasmids (pET series), shuttle vectors PNV18.1, phage or viral vectors, etc., preferably pET-28a (+). The nucleotide sequence in the first aspect of the invention is inserted into the pET-28a (+) plasmid skeleton by molecular biological operations such as PCR, DNA ligation and the like on a preferable vector to construct a recombinant vector, which is named pET28a-RbTAstable.

The host cell is selected from any one of E.coli (ESCHERICHIA COLI), rhodococcus erythropolis (Rhodococcus ruber), rhodococcus turbidi (Rhodococcus opacus), bacillus subtilis (Bacillus subtilis), corynebacterium glutamicum (Corynebacterium glutamicum) and yeast. Coli E.coli BL21 (DE 3) is preferred in the present invention. And (3) converting the recombinant vector pET28a-RbTAstable into E.coli BL21 (DE 3) to obtain the corresponding genetically engineered bacterium E.coli BL21 (DE 3) pET28a-RbTAstable.

In a third aspect, the invention provides the use of a protein according to the first aspect or a nucleic acid molecule, expression cassette, recombinant vector or recombinant bacterium according to the second aspect in any of the following:

1) Preparing a chiral amine synthesis catalyst;

2) Chiral amines are prepared or synthesized.

In the application, the chiral amine catalyst is used for catalyzing prochiral ketone compounds to generate chiral amine in the presence of an amino donor;

wherein the chiral amine is selected from at least one of the following or an intermediate or analogue thereof: (R) -3-amino-1-butanol, (R) -2-amino-1-propanol, (R) -2-amino-2-phenylethanol, (R) -1-methoxy-2-propylamine, (R) -2-pentylamine, (R) -2-hexylamine, (R) -2-heptylamine, (R) -4-methyl-2-pentylamine, (R) -5-methyl-2-hexylamine, (R) -3-aminopiperidine, (R) -N-t-butoxycarbonyl-3-aminopyrrole, (R) -N-benzyl-3-aminopiperidine, (R) -N-t-butoxycarbonyl-3-aminopiperidine, (R) -N-benzyloxycarbonyl-3-aminopiperidine and (R) -3-aminoazepan-1-carboxylic acid tert-butyl ester and the like;

wherein the prochiral ketone can be prochiral ketone substrate shown in 1 a-10 a.

In a fourth aspect, the present invention provides a chiral amine catalyst comprising the protein of the first aspect, the recombinant bacterium of the second aspect or whole cells after induction culture thereof, the whole cell lysate, immobilized enzymes prepared from the protein or the whole cell lysate, immobilized cells prepared from the recombinant bacterium or the whole cells.

The catalyst may include three forms of whole cell catalyst, free protein catalyst and immobilized cell catalyst. The whole cell catalyst is whole cells obtained by enrichment culture and induced expression of target proteins of the recombinant bacteria constructed in the second aspect of the invention; the free protein catalyst is crude enzyme liquid obtained by ultrasonic crushing or high-pressure homogenizing crushing of the whole cells and centrifugation, and also comprises pure enzyme obtained by protein purification means; the immobilized cell catalyst is prepared by immobilizing whole cell catalyst or free protein catalyst on a carrier.

In a fifth aspect, the present invention provides a method of synthesizing a chiral amine comprising the steps of: catalyzing a prochiral ketone to produce a chiral amine in the presence of an amino donor using the chiral amine catalyst of the fourth aspect;

Or the invention also provides a method for improving the stability of transaminase, which comprises the following steps: mutating amino acid residues 136, 142, 228 and 249 of the amino acid sequence of the wild-type transaminase in accordance with the mutation mode of any one of claims 1 to 6), to obtain a protein having transaminase activity;

the protein having transaminase activity has a higher thermostability than the wild-type transaminase.

In the above-mentioned context,

The catalytic temperature is 30-40 ℃, preferably 30 ℃.

The catalyst is in the form of a whole cell catalyst, a free protein catalyst or an immobilized cell catalyst;

the immobilized cell catalyst is obtained by immobilizing the protein of the first aspect or the recombinant bacterium of the second aspect on a carrier.

In an embodiment of the present invention, the prochiral ketone is any one of prochiral ketones 1a to 10 a.

Compared with the prior art, the experiment proves that the invention has the following beneficial effects: the thermal stability of the recombinant aminotransferase mutant catalysts obtained by the invention is obviously improved compared with that of wild type catalysts. Taking mutant R136P/T142P/F228Y/I249Q with most obvious heat stability improvement as an example, the half-life of enzyme activity at 40 DEG CThe evaluation index is improved by 20 times compared with the wild female parent, and the evaluation index is improved from 2.98min to 62.8min; half-aging temperature of heat treatment for 10minAs an evaluation index, compared with a wild female parent, the temperature is raised from 31.0 ℃ to 47.5 ℃, the temperature is raised by 16.5 ℃, and the temperature application range of the enzyme is remarkably enlarged. Meanwhile, the enzyme activity of the constructed thermostable mutant is also superior to that of a wild-type female parent, wherein the activity of the optimal mutant is improved by 3 times compared with that of the wild-type mutant, and the ee value of the product is more than 99%. Double improvement of heat stability and enzyme activity is realized, and high stereoselectivity of the catalyst is maintained.

Drawings

FIG. 1 is RbTA expression vectors.

FIG. 2 is an SDS-PAGE electrophoresis of RbTA thermostable mutant expression and purification.

FIG. 3 is a standard curve of acetophenone.

FIG. 4 shows the results of the enzyme activity half-life at 40℃and the half-life temperature of heat treatment for 10min of RbTA thermostable mutants R136P/T142P/F228Y/I249Q.

FIG. 5 shows the results of a RbTA thermostable mutant on various chiral amine catalytic activities.

Detailed Description

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 design of transaminase mutants

Based on analysis of the aminotransferase RbTA structure (PDB: 7 DBE), modification of arginine at position 136 (R136), threonine at position 142 (T142), and phenylalanine at position 228 (F228) and isoleucine at position 249 (I249) near the substrate binding pocket on the protein surface as mutation hotspots was locked based on the interaction, the degree of structural flexibility, and the sequence evolution information. For amino acid sites on the surface of the protein, the aim is to improve the thermal stability by reducing the flexibility degree of the structure and reducing the thermal disturbance of the structure, and the designed mutants are R136P and T142P; for the amino acid sites near the substrate binding pocket, the goal is to achieve an improvement in thermal stability by strengthening the protein core hydrogen bond network, replacing selected amino acids with "hydrogen bond strengthening codons" (tyrosine Y/glutamine Q/asparagine N/threonine T/serine S), and obtaining designed mutants F228Y and I249Q through computer simulation screening.

The amino acid sequence of wild-type aminotransferase RbTA is SEQ ID NO:1, a step of;

the nucleotide sequence of the encoding gene of the wild-type aminotransferase RbTA is SEQ ID NO:2.

Vector pET28a-RbTA (fig. 1) expressing wild-type aminotransferase RbTA is a vector which encodes the amino acid sequence of SEQ ID NO:2, and the gene encoding the wild-type aminotransferase RbTA replaces a fragment between NcoI and EcoRI of the pET-28a (+) plasmid.

For wild-type aminotransferase RbTA, either of R136P, T142P, F Y and I249Q mutations can be made in its amino acid sequence (SEQ ID NO: 1), resulting in a single point mutant, designated aminotransferase RbTA mutant R136P, T P, F228Y or I249Q; the combination mutations shown in Table 1 can also be performed on these sites to obtain a combination mutant of multiple mutations of the aminotransferase RbTA mutant.

Table 1 shows a list of combined mutants

R136P/T142P	R136P/T142P/F228Y
		R136P/F228Y	R136P/T142P/I249Q
R136P/I249Q	R136P/F228Y/I249Q
		T142P/F228Y	T142P/F228Y/I249Q
T142P/I249Q	R136P/T142P/F228Y/I249Q
		F228Y/I249Q

Each mutant shown in table 1 represents a sequence set forth in SEQ ID NO:1, and a mutant obtained after a specific mutation is generated in the corresponding amino acid position in the amino acid sequence shown in the formula 1.

EXAMPLE 2 construction of transaminase mutant library and genetically engineered bacterium

Selecting pET-28a (+) plasmid as an expression vector, performing enzyme digestion for 0.5-2h at 37 ℃ by using Nco I and EcoRI, and separating by agarose gel electrophoresis to obtain an enzyme-digested pET-28a (+) linear DNA product. Using the wild-type transaminase recombinant expression vector pET28a-RbTA constructed in example 1 as a template, rbTA upstream and downstream primers containing plasmid restriction sites (Nco I or EcoRI) and forward and reverse primers containing different mutation sites were designed according to the desired introduced mutation, PCR amplification was performed using PHANTA DNA polymerase (TAKARA Co.) to obtain a DNA fragment containing the selected backbone restriction site at one end and the mutation site and homology arm at the other end, and also separation by gel electrophoresis.

The DNA polymerase, buffer and restriction enzyme used for PCR amplification were all purchased from TAKARA company. The PCR amplification system is as follows: template 1. Mu.L, forward primer 2. Mu.L, reverse primer 2. Mu.L, 2X Phanta Mix. Mu.L, and ddH ₂ O20. Mu.L. The PCR cycle conditions were:

pre-denaturation at 95℃for 5min;

Denaturation at 95℃for 15sec, annealing at 56℃for 15sec, extension at 72℃for 1kbp/min (34 cycle);

supplementing and extending for 5min at 72 ℃;

Preserving at 4 ℃ forever;

The RbTA upstream and downstream primers containing plasmid restriction sites (NcoI or EcoRI) comprise NcoI-RbTA-F and EcoRI-RbTA-R, and the forward and reverse primers containing mutation sites are specific primers used for introducing different single-point mutations, and are specifically shown in Table 2.

Table 2 is a list of primers used to construct a library of transaminase mutants

Taking the construction of R136P as an example, an upstream fragment of R136 was cloned using NcoI-RbTA-F and a reverse primer R136P-R, and a downstream fragment of R136 was cloned using forward R136P-F and EcoRI-RbTA-R, to obtain 2 DNA fragments containing the mutation of R136P in total.

The isolated pET-28a (+) linear DNA product was then DNA purified with the 2 DNA fragments described above using the Gel Extraction Kit kit from OMEGA bio-tek, mixed in a certain ratio and ligated for 30min at 50℃using the Gibson ligation kit (Clone Smarter Technologies). The Gibson ligation product was used to obtain 10. Mu.L of competent cells of E.coli Trans10 (from Transgene) by heat shock transformation, the competent cells were spread on LB solid medium containing 50. Mu.g/mL kanamycin, colonies were grown overnight at 37℃and were subjected to PCR and electrophoresis verification by using primers T7 and T7-ter, and colonies with correct electrophoresis bands were selected and sequenced (designated Azenta Co.) to obtain E.coli containing recombinant expression vector pET28a-RbTAmut of transaminase mutant with correct sequencing. And (3) constructing a RbTA mutant expression vector successfully.

The LB solid culture medium comprises the following components: 15g/L of agar powder, 10g/L of peptone, 10g/L of sodium chloride, 5g/L of yeast powder and the balance of water.

The combined mutant is constructed by carrying out multiple rounds of mutation after one round on the basis of single-point mutant, and the introduction of mutation sites also uses specific primers.

Expression of the respective RbTA mutant vectors (pET 28a-RbTAstable, rbTAstable represents a different aminotransferase RbTA mutant) was a vector obtained by substituting the coding gene of each aminotransferase RbTA mutant for a fragment between NcoI and EcoRI of the pET-28a (+) plasmid.

The aminotransferase RbTA mutant is obtained by mutating a wild aminotransferase RbTA amino acid sequence (SEQ ID NO: 1) with any one, any two, any three or any four of R136P, T142P, F Y and I249Q to obtain a aminotransferase RbTA mutant.

The method comprises the following steps:

The aminotransferase RbTA mutant R136P is the amino acid sequence of wild type aminotransferase RbTA of SEQ ID NO:1 st 136 th site R is mutated into P, other amino acid sequences are unchanged, and the obtained mutant;

The aminotransferase RbTA mutant T142P is the amino acid sequence of wild type aminotransferase RbTA of SEQ ID NO:1 st 142 th T is mutated into P, other amino acid sequences are unchanged, and the obtained mutant;

the aminotransferase RbTA mutant F228Y is the amino acid sequence of wild-type aminotransferase RbTA of SEQ ID NO: f at position 228 of 1 is mutated into Y, other amino acid sequences are unchanged, and the obtained mutant;

the aminotransferase RbTA mutant I249Q is the wild-type aminotransferase RbTA amino acid sequence of SEQ ID NO:1 st 249 th is mutated into Q, other amino acid sequences are unchanged, and the obtained mutant;

The aminotransferase RbTA mutant R136P/T142P is the amino acid sequence of the wild type aminotransferase RbTA as shown in SEQ ID NO:1 st 136 th R is mutated to P, 142 th T is mutated to P, and other amino acid sequences are unchanged, thus obtaining a mutant;

The aminotransferase RbTA mutant R136P/F228Y is a mutant with the amino acid sequence of the wild aminotransferase RbTA as shown in SEQ ID NO:1, wherein the R at 136 th position is mutated into P, the F at 228 th position is mutated into Y, and other amino acid sequences are unchanged, thus obtaining a mutant;

the aminotransferase RbTA mutant R136P/I249Q is a mutant with the amino acid sequence of wild aminotransferase RbTA as shown in SEQ ID NO:1 st 136 th position R is mutated into P, 249 th position I is mutated into Q, and other amino acid sequences are unchanged, thus obtaining the mutant.

The aminotransferase RbTA mutant T142P/F228Y is the amino acid sequence of the wild type aminotransferase RbTA of SEQ ID NO:1 st 142 th is mutated to P, 228 nd F is mutated to Y, and other amino acid sequences are unchanged, thus obtaining the mutant.

The aminotransferase RbTA mutant T142P/I249Q is a mutant with the amino acid sequence of wild type aminotransferase RbTA as shown in SEQ ID NO:1 st position 142T mutation to P, 249I mutation to Q, other amino acid sequence unchanged, the mutant.

The aminotransferase RbTA mutant F228Y/I249Q is a mutant which has the amino acid sequence of wild type aminotransferase RbTA as shown in SEQ ID NO:1, the F at position 228 is mutated into Y, the I at position 249 is mutated into Q, and other amino acid sequences are unchanged, thus obtaining the mutant.

The aminotransferase RbTA mutant R136P/T142P/F228Y is a mutant which has the amino acid sequence of wild aminotransferase RbTA as shown in SEQ ID NO:1, wherein the 136 th R is mutated to P, the 142 th T is mutated to P, the 228 th F is mutated to Y, and other amino acid sequences are unchanged, thus obtaining a mutant;

The aminotransferase RbTA mutant R136P/T142P/I249Q is a mutant which has the amino acid sequence of wild aminotransferase RbTA as shown in SEQ ID NO:1, wherein the 136 th R is mutated to P, the 142 th T is mutated to P, the 249 th I is mutated to Q, and other amino acid sequences are unchanged, thus obtaining a mutant;

The aminotransferase RbTA mutant R136P/F228Y/I249Q is a mutant which has the amino acid sequence of wild aminotransferase RbTA as shown in SEQ ID NO:1, wherein the 136 th R is mutated to P, the 228 th F is mutated to Y, the 249 th I is mutated to Q, and other amino acid sequences are unchanged, thus obtaining a mutant;

The aminotransferase RbTA mutant T142P/F228Y/I249Q is a mutant which has the amino acid sequence of wild type aminotransferase RbTA as shown in SEQ ID NO:1 st position 142 is mutated to P, 228 nd position F is mutated to Y, 249 th position I is mutated to Q, and other amino acid sequences are unchanged, thus obtaining mutants;

The aminotransferase RbTA mutant R136P/T142P/F228Y/I249Q is a mutant which has the amino acid sequence of wild type aminotransferase RbTA as shown in SEQ ID NO:1, wherein the 136 th R is mutated to P, the 142 th T is mutated to P, the 228 th F is mutated to Y, the 249 th I is mutated to Q, and other amino acid sequences are unchanged, thus obtaining the mutant.

The coding gene of the aminotransferase RbTA mutant is obtained by mutating any one, any two, any three or any four of codons corresponding to R136P, T P, F Y and I249Q of a nucleotide sequence (SEQ ID NO: 2) of a coding gene of a wild aminotransferase RbTA to obtain the coding gene of the aminotransferase RbTA mutant.

The E.coli expressing aminotransferase RbTA is obtained by transferring the vector pET28a-RbTA expressing the wild type aminotransferase RbTA into E.coli to obtain E.coli expressing aminotransferase RbTA.

The colibacillus expressing the aminotransferase RbTA mutant is obtained by transferring the above vectors expressing each RbTA mutant into colibacillus respectively to obtain colibacillus expressing the aminotransferase RbTA mutant.

Example 3 preparation of cell and free enzyme catalyst

1. Preparation of cell catalysts for transaminases

The E.coli expressing aminotransferase RbTA and each E.coli expressing aminotransferase RbTA mutant obtained in example 2 were inoculated into 5mL of LB liquid medium (peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, deionized water as solvent, pH=7.0) containing kanamycin (50. Mu.g/mL), and cultured overnight at 37 ℃. The bacterial liquid is harvested and the expression plasmid is extracted by using PLASMID MINI KIT I plasmid extraction kit of OMEGA bio-tek. Transformation into competent cells E.coli BL21 (DE 3) by heat shock method, coating LB plate containing kanamycin, culturing overnight at 37℃and then picking single colony for gene sequencing (attuned Azenta).

Single colony with correct sequence is inoculated in LB liquid medium containing kanamycin for 12h at 37 ℃ and 200rpm to obtain seed liquid for expressing aminotransferase or mutant thereof. The seed solution was inoculated into a shake flask containing 1000mL of LB liquid medium (containing 50. Mu.g/mL of kanamycin) at a ratio of 1% (v/v), cultured at 37℃for 3 hours at 200rpm, added with IPTG at a final concentration of 0.5mM, and cultured at 16℃for 16 hours at 200rpm to induce protein expression, thereby obtaining a fermentation broth. Centrifuging the fermentation broth at 8000 Xg for 20min, and collecting cells to obtain a cell catalyst of aminotransferase RbTA and a cell catalyst of aminotransferase RbTA mutant respectively.

The strain is preserved by glycerol with the final concentration of 25 percent, and is preserved in a refrigerator at the temperature of minus 80 ℃.

2. Free enzyme catalyst acquisition

1) Preparation of crude enzyme solution

Weighing a proper amount of cell catalyst (0.1 g/mL), re-suspending by using purified A solution (25 mM Tris-HCl,300mM NaCl, 10mM imidazole, the balance of water, pH 7.5), crushing by using a high-pressure homogenizer (the pressure is controlled at 700-900 bar), centrifuging at 13000 Xg for 30min, and collecting the crushed supernatant, wherein the obtained crushed supernatant is crude enzyme solution.

The expression of transaminase and its mutants in crude enzyme solutions was characterized by SDS-PAGE, specifically by SDS-PAGE detection of crude enzyme solutions derived from E.coli expressing transaminase RbTA (indicated as WT SO in the figure) and E.coli expressing transaminase RbTA mutant R136P/T142P/F228Y/I249Q (indicated as RbTA-stab l e SO in the figure).

As shown in SO of FIG. 2, it can be seen that the aminotransferase RbTA and the aminotransferase RbTA mutant R136P/T142P/F228Y/I249Q in the crude enzyme solution both have higher expression levels (about 40KD target protein).

The crude enzyme solution derived from E.coli expressing other aminotransferase RbTA mutants can also obtain the target protein of about 40 KD.

2) Preparation of pure enzymes

After the crude enzyme solutions were placed in a-70 ℃ refrigerator and frozen overnight, freeze-dried crude enzyme powder was prepared by a freeze dryer. In addition, the target crude enzyme is purified by an affinity chromatography method in one step, crude enzyme liquid obtained by crushing is uploaded to a nickel column, then purified A liquid and purified B liquid (25 mM Tris-HCl,300mM NaCl,500mM imidazole, 1mM DTT and the balance of water) are used for mixing to obtain eluent with different imidazole concentrations (10-500 mM), then target protein is eluted, and the eluent under different gradients is collected. The purity of the target protein is determined by SDS-PAGE, and for transaminase and mutants thereof, the target protein is washed off in a low concentration imidazole solution (corresponding to 0% -15% by volume of buffer B), only a small amount of the target protein is eluted, which indicates that the target protein can be well combined with a nickel column through His tag, and separation and purification of the target protein can be realized through affinity chromatography. Along with the increase of the imidazole concentration (corresponding to the volume percentage of the buffer solution B being 20-60 percent respectively), the target protein can be eluted, and finally the protein band is single, the purity is higher, and the aminotransferase RbTA pure enzyme and the aminotransferase RbTA mutant pure enzyme are obtained.

The eluates containing the target proteins were pooled and concentrated by centrifugation using 10-30kDa ultrafiltration tubes at 4℃and 4000 rpm. The replacement wash was then performed with buffer C (25 mM Tris-HCl, pH 8.0;150mM NaCl,1mM DTT, balance water) and repeated 2-3 times to remove imidazole from the solution, and when concentrated to less than 1mL, pure protein was collected. Protein concentration was determined using Nandrop2000,2000 and placed in a-80℃refrigerator after liquid nitrogen flash freezing.

The protein content of each component during purification was analyzed by SDS-PAGE.

As a result, as shown in FIG. 2, S0 is a crude enzyme solution after crushing, S1 is a crude enzyme solution remaining after Ni column adsorption, W1 is a solution obtained by washing impurities with a purified A solution, W2 is a solution obtained by washing impurities with a low-concentration imidazole solution (corresponding to 0 to 15% by volume of buffer B), E1 is a target protein-containing solution obtained by eluting with a high-concentration imidazole solution, and E2 is a target protein-containing solution obtained by eluting again with a purified B solution, and it can be seen that the target pure enzyme solution is obtained.

Example 4 preparation of immobilized enzyme and immobilized cell

An appropriate amount of the immobilized carrier was weighed, equilibrated with potassium phosphate buffer (100 mM, pH 7.0), and the treated tree carrier (epoxy resin, tianjin Nankai and Chemie Co., ltd.) was added to potassium phosphate buffer at a carrier to buffer solution ratio of 1/5 (w/v; g: ml), followed by glutaraldehyde solution (50%) to a final concentration of 2% v/v. After activation in an shaker (16 ℃,200 rpm) for 2-3 hours, the activated support was rinsed with deionized water to remove residual glutaraldehyde. An appropriate amount of the activated immobilized carrier was weighed and placed in a buffer, and an enzyme solution (1 mg/mL of the pure enzyme prepared in example 3, 50mM Tris-HCl solution) was added, the ratio of carrier to enzyme solution still being 1/5 (w/v). The mixture was immobilized in a constant temperature shaker or shaker (16 ℃ C., 200 rpm) for 8h. And then washing the immobilized enzyme with a buffer solution to remove residual enzyme solution, and then placing the immobilized enzyme in a refrigerator at 4 ℃ for standby to obtain the immobilized enzyme.

An appropriate amount of the immobilized carrier was weighed, equilibrated with potassium phosphate buffer (100 mM, pH 7.0), and the treated tree carrier was added to the buffer, and the cell catalyst prepared in example 3 was added thereto, with the ratio of carrier to cell catalyst solution (0.1 g/mL) still being 1/5 (w/v). The mixture was immobilized in a constant temperature shaker or shaker (16 ℃ C., 200 rpm) for 8h. Then washing the immobilized cells with a buffer solution to remove the residual cell catalyst, and then placing the immobilized cells in a refrigerator at 4 ℃ for standby to obtain the immobilized cells.

Example 5 determination of specific Activity of transaminase and mutant thereof

The pure enzyme stored in a refrigerator at-80℃as described in example 3 was taken and dissolved in ice, and then diluted with a buffer (25 mM Tris-HCl, pH 7.5) to give an enzyme dilution with a final concentration of 0.2 mg/mL. Taking the substrate 4-hydroxy-2-butanone (prochiral ketone 1 a) as an amino acceptor as an example, 20. Mu.L of an enzyme dilution with a final concentration of 0.2mg/mL was added to 180. Mu.L of the reaction system (0.1M Tris-HCl,1mM pyridoxal phosphate PLP,10mM alpha-phenylethylamine, 1% v/v DMSO,100mM 4-hydroxy-2-butanone, the balance being water, pH=8.0). The reaction system is placed at 30 ℃ for shaking, and the light absorption at 280nm is measured by using a TECAN INFINITE EPLEX enzyme-labeled instrument every 5min, and the total measurement is 30min. The collected 280nm light absorption data was curve-fitted to the reaction time, the product formation rate was converted from the standard curve of the product acetophenone (FIG. 3), and the specific activity of transaminase was calculated.

RbTA and method for calculating mutant activity:

Wherein DeltaA _280,RbTA is the change of absorbance at 280nm in the measurement reaction time of a transaminase measurement sample, deltaA _{280, Control} is the change of absorbance at 280nm in the measurement reaction time of a blank sample, t is the measurement reaction time (5 min or 30 min), k is the slope of an acetophenone standard curve, V is the volume of a reaction system, and m is the mass of enzyme added in the reaction.

The results are shown in Table 3.

Example 6 determination of the thermal stability of recombinant transaminase mutations

The pure enzyme stored in a refrigerator at-80℃as described in example 3 was taken and dissolved in ice, and then diluted with a buffer (25 mM Tris-HCl, pH 7.5) to give an enzyme dilution with a final concentration of 0.2 mg/mL.

The enzyme activities were measured as in example 5 by heat-treating 100. Mu.L of enzyme dilutions having a final concentration of 0.2mg/mL in a water bath at 40℃for 0, 1, 2, 3, 5, 7.5, 10, 15, 20, 30, 40, 50min, respectively. Calculating relative activity from the measurement result, fitting with exponential inactivation curve to obtain half-life of enzyme activity at 40deg.C

Meanwhile, 100. Mu.L of the same enzyme dilution with a final concentration of 0.2mg/mL was subjected to heat treatment at 10, 20, 25, 30, 35, 40, 50, 60℃for 10min, and the enzyme activity of the treated sample was measured. After calculating the relative vitality, fitting by using a Sigmoid function to obtain the half-decay temperature of heat treatment for 10min

The results are shown in Table 3.

EXAMPLE 7 determination of recombinant transaminase mutant Activity and thermostability

A total of 15 transaminase mutants were obtained by site-directed mutagenesis and combinatorial mutagenesis in example 1, and the mutants were subjected to activity screening using 4-hydroxy-2-butanone as substrate.

The enzyme activity was measured by the method described in example 5, and the thermostability was evaluated by the method described in example 6.

Enzyme activity on substrate 4-hydroxy-2-butanone: 0.216+/-0.016U/mg, and the definition of the enzyme activity unit 1U is as follows: under the reaction conditions, the amount of enzyme required to catalyze 1. Mu. Mol of substrate per minute is one enzyme activity unit, denoted by U.

The activity and thermostability of the above transaminase mutants are shown in Table 3.

Table 3 shows the results of the measurement of the activity and the thermostability of the transaminase mutants

In table 3, each mutant shown represents a sequence set forth in SEQ ID NO:1, and a mutant obtained after a specific mutation is generated in the corresponding amino acid position in the amino acid sequence of the wild-type transaminase. In the activity column, a plus sign "+" indicates that the mutant protein activity is improved by 0-1 times compared with the wild type aminotransferase activity; two plus signs "++" indicate that the mutant has an increased activity of 1-2 times compared to the wild-type transaminase, and three plus signs "++" indicate that the mutant has an increased activity of 2-3 times compared to the wild-type transaminase. 0-1 fold indicates an increase but <100%, but >0%; in the thermostability columns, a plus sign "+" indicates the ratio of mutant protein to wild-type transaminaseIncrease by 0 to 2 times orThe temperature is increased by 0 to 4 ℃; two plus signs "++" indicate that mutant proteins are compared to wild-typeIncrease by 2 to 5 times orThe temperature is increased by 4 to 8 ℃; three plus signs' ++ + + + is shown mutant proteins are compared to wild-typeThe improvement of 5 to 10 times orThe temperature is increased by 8 to 12 ℃; four plus signs' ++ is represented by mutant proteins are compared to wild-typeThe improvement of 10 to 20 times orThe temperature is increased by 12 to 16 ℃; five plus signs "+: ++ is represented by mutant proteins are compared to wild-typeThe improvement of more than 20 times orThe temperature is increased by more than 16 ℃.

As can be seen from the above results, the wild-type transaminase has a thermostability determined by viability2.52Min, thermal stability31.9 ℃. A total of 15 mutants with improved thermostability were obtained, which were compared with the wild typeThe improvement of 0 to 20 times,The temperature is increased by 0-16 ℃. In addition, the heat stability modification does not reduce the activity of the enzyme, and the activity of the mutant is also improved by 0-3 times. Wherein the mutant R136P/T142P/F228Y/I249Q has an enzyme activity half-life at 40 DEG CCompared with a wild transaminase female parent, the transaminase female parent is improved by 20.1 times, and the transaminase female parent is improved from 2.98min to 62.8min; half-aging temperature of heat treatment for 10minAs an evaluation index, compared with a wild-type transaminase female parent, the temperature is increased from 31 ℃ to 47 ℃, the temperature is increased by 16 ℃, the applicable range of the enzyme is remarkably enlarged, and meanwhile, the activity is improved by 3 times at the highest, so that the transaminase female parent is the optimal mutant (shown as RbTAstable in a graph of R136P/T142P/F228Y/I249Q).

Example 8 determination of the catalytic Activity of recombinant aminotransferase mutants on various chiral amines

Through activity measurement, the aminotransferase mutant constructed based on the wild-type aminotransferase RbTA has activity on various prochiral ketone substrates (1 a-10 a, figure 5), and the aminotransferase mutant is specifically as follows:

The reaction mode is as follows: the substrate (final concentration: 500 mM), prosthetic group (1 mM), amino donor (1M), catalyst (0.1 g/mL) were sequentially added to the reaction buffer to obtain a reaction system, which was placed in a constant temperature shaker to perform a reaction at 30℃and a rotational speed of 1000rpm for 3 hours. After the reaction was completed, the reaction conversion was measured using gas chromatography, and the selectivity of the product was measured using liquid chromatography.

The gas chromatography detection conditions are as follows: constant pressure sample injection, split ratio 1/33, sample injection amount 1. Mu.L, sample inlet and detector temperature 250℃and column HP-5 (30 m,0.25 μm, agilent J & W SCIENTIFIC, USA). The specific activity of transaminase was calculated from the reaction conversion and from the reaction time and the amount of enzyme added. The conditions for the selective detection of the liquid chromatographic product are as follows: using 10. Mu.L of the reaction sample, 30. Mu. L Marfey reagent (20mM,Thermo Scientific,USA), 36. Mu.L NaHCO3 (1M, shanghai microphone) and 100. Mu.L DMSO (Shanghai microphone) were added and the derivatization was performed at 40℃for 2h. The derivatized samples were subjected to liquid chromatography using CP (25m,0.25,Agilent J&WScientific,USA) with water and methanol as mobile phases at a flow rate of 0.6mL/min.

The conversion was calculated as follows:

Wherein a ₁ is the peak area of the product after the reaction, and a ₂ is the peak area of the substrate after the reaction.

The specific enzyme activity calculation formula is as follows:

The results of the amidase mutant R136P/T142P/F228Y/I249Q (RbTAstable in the figure) are shown in FIG. 5, wt is wild type aminotransferase, and the thermostable aminotransferase mutant R136P/T142P/F228Y/I249Q constructed on the basis of wild type RbTA is measured to have activity on 10 substrates in the figure, and the ee value of the product is more than 99%.

The reaction buffer is Tris-HCl (pH 8.0) buffer, the substrate is prochiral ketone 1 a-10 a, the prosthetic group is pyridoxal phosphate, the amino donor is isopropylamine, the catalyst is immobilized enzyme of the pure enzyme obtained in example 3 or the transaminase mutant prepared in example 4 or immobilized cell (the catalyst in FIG. 5 is R136P/T142P/F228Y/I249Q pure enzyme).

The enzyme activity (U) is defined as: under the above reaction conditions, the amount of enzyme required to catalyze 1. Mu. Mol of substrate per minute is one enzyme activity unit, denoted by U.

Claims

1. A protein, which is A) or B) as follows:

2. The protein of claim 1, wherein:

The protein shown in the A) is any one of the following:

3) The protein is obtained by mutating 228 th amino acid residue in wild type aminotransferase or corresponding amino acid sequence to obtain protein with aminotransferase activity;

5) The protein is obtained by mutating amino acid residue at position 142 in wild type transaminase or corresponding amino acid sequence to obtain protein with transaminase activity;

7) The protein is obtained by mutating the 249 th amino acid residue in wild type aminotransferase or the amino acid sequence corresponding to the aminotransferase, so as to obtain the protein with aminotransferase activity;

3. The protein according to claim 1 or 2, characterized in that:

the mutation modes of the 136 th, 142 th, 228 th and 249 th amino acid residues are as follows:

Mutation of R at position 136 to P, G, A, F, S or K;

t at position 142 is mutated to P, G, L, R, M, K, Q, W, Y or F;

F at position 228 is mutated to Y, Q, N, T, S, I, L, W, V or A;

the 249I mutation is Y, Q, N, T, S, V, M or L.

4. A protein according to any one of claims 1-3, characterized in that:

A) The protein shown is any one of the following:

5. The protein according to any one of claims 1 to 4, wherein:

The wild-type transaminase is any one of the following:

(a) SEQ ID NO: 1;

(b) And SEQ ID NO:1, a protein having an identity of 90% or more;

(c) And SEQ ID NO:1, and the protein has more than 95% identity.

6. The protein of any one of claims 1-5, wherein: the protein has a higher stability and/or higher enzymatic activity than the wild-type transaminase;

and/or, the stability is thermal stability.

7. A nucleic acid molecule encoding the protein of any one of claims 1-6;

8. Use of a protein according to any one of claims 1 to 6 or a nucleic acid molecule, expression cassette, recombinant vector, or recombinant bacterium according to claim 7 in any one of the following:

1) Preparing a chiral amine synthesis catalyst;

2) Chiral amines are prepared or synthesized.

9. A chiral amine catalyst comprising the protein of any one of claims 1-6, the recombinant bacterium of claim 7 or whole cells after induction culture thereof, the whole cell lysate, an immobilized enzyme prepared from the protein or the whole cell lysate, an immobilized cell prepared from the recombinant bacterium or the whole cell.

10. A method of synthesizing a chiral amine comprising the steps of: catalyzing a prochiral ketone to produce a chiral amine in the presence of an amino donor using the chiral amine catalyst of claim 9;

or a method for improving the stability of transaminase, comprising the steps of: mutating the amino acid residues 136, 142, 228 and 249 of the wild-type transaminase or the amino acid sequence corresponding thereto in accordance with the mutation mode of any one of claims 1 to 6) to obtain a protein having transaminase activity;