CN116042560A - Mutant of recombinant aminotransferase and application thereof - Google Patents
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Abstract
The invention relates to a mutant of recombinant aminotransferase and application thereof. A series of these transaminase mutants were obtained by constructing a library of mutants of the transaminases and the optimal mutants were obtained by combining the mutations. The recombinant aminotransferase mutant catalyst obtained by the invention can asymmetrically catalyze and synthesize a plurality of N-heterocyclic chiral amines with high added value, the reaction conversion rate can reach 99%, and the optical purity of the product can reach more than 99%. Wherein the catalytic effect of the combined mutant is greatly improved compared with the activity of the wild type parent and the single-point mutant.
Description
Technical Field
The invention relates to the fields of protein engineering and genetic engineering, in particular to design and application of a mutant of recombinant aminotransferase.
Background
Optically pure N-heterocyclic amines are important biologically active molecules of interest for use in the pharmaceutical industry. They are useful for the preparation of a wide variety of biologically active drugs including the hypoglycemic drugs alogliptin, trelagliptin and linagliptin, the fourth fluoroquinolone antibiotics besifloxacin and the epidermal growth factor receptor inhibitor nazatinib, and therefore have very wide applications in the chiral pharmaceutical synthesis field (Eckhardt, M.et al, J.Med. Chem.,2007,50,6450-6453; seganish, W.M. et al, ACS Med. Chem. Lett.,2015,6,942-947; lim, J.et al, bioorg. Med. Chem. Lett.,2015,25,5384-5388; lelais, G.et al, J.Med. Chem.,2016,59,6671-6689; feng, Y.et al, org. Process Res. Dev.,2017,21 (4), 648-654. In view of the great value of chiral N-heterocyclic amine in the synthesis of drug molecules, the research and development of the synthesis technology of the chiral N-heterocyclic amine has become a research hotspot in the field of new drug development.
In the synthetic route of chiral N-heterocyclic amines, the introduction of chiral amino groups is the most critical. Much research is devoted to exploring efficient synthetic methods for these compounds, which currently include chemical synthesis and biocatalysis. Chemical synthetic routes reported in the literature include kinetic resolution of racemic amines, asymmetric synthesis of prochiral ketones and hydrogenation of chiral precursors. The conventional chiral amine chemical synthesis method has complicated reaction steps, poor optical purity, etc. (Hennessy, e.j. Etc., bioorg. Med. Chem. Lett.,2012,22,1690-1694; ji, j. Etc., org. Biomol. Chem.,2015,13,7624-7627; kumar, a. Etc., der.pharma. Chemica.,2015,7,297-300; gundersen, m.t. etc., org. Process res. Dev.,2016,20,602-608).
The biocatalysis method uses cells or enzyme proteins as catalysts to catalyze and convert low-value compounds into high-added-value target products, and has the advantages of high stereoselectivity, environmental protection and the like. In recent years, enzymatic strategies for chiral amine synthesis have also been rapidly developed. For example, asymmetric synthesis of chiral N-heterocyclic amines can be achieved with the aid of reductive amination enzymes (AspredAm) under the participation of coenzymes, but naturally derived amine dehydrogenases are less in source and generally have low catalytic activity, and cannot meet industrial application indexes (Aleku, G.A. et al, nat Chem,2017,9 (10), 961-969).
Aminotransferase (TA) is also known as aminotransferase, a type of pyridoxal phosphate (PLP) dependent transferase that can specifically and selectively transfer an amino group to a substrate ketone in the presence of an amino donor to produce 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. However, few studies have been reported for the catalytic synthesis of chiral N-heterocyclic amines using aminotransferase, which has been reported to include only ATA-025-IMB CbTA, rbTA (CN 103865964; CN108384767; petri, A. Et al, beilstein J.Org.chem.,2019,15,60-66; wang, C. Et al, ACS Omega,2021,6 (26), 17058-17070; li, F. Et al, adv. Synth. Catalyst, 2021,363 (19), 4582-4589) and naturally derived aminotransferase with low catalytic activity. For example, aminotransferase derived from photosynthetic bacteria (Rhodobacter sp.) can catalyze N-t-butoxycarbonyl-3-piperidone to generate (R) -N-t-butoxycarbonyl-3-aminopiperidine, and the catalytic activity is only 0.034U/mg, so that the aminotransferase cannot be applied to large-scale industrial production.
The chiral N-heterocyclic amine synthesized by using aminotransferase as a catalyst is taken as a green and environment-friendly synthesis route, has very broad application prospect and is also receiving more and more attention. In addition, the molecular transformation is carried out on the biocatalyst by adopting rational design, so that the catalytic performance of the natural enzyme is improved, and the method has important significance for promoting the industrialized application process of the natural enzyme. However, the catalytic activity of the aminotransferase is known to be low, so that a more efficient enzyme catalyst needs to be developed to reduce the reaction cost and promote industrial application.
Disclosure of Invention
Specifically, the invention solves the technical problems existing in the prior art through the following technical schemes.
1. A transaminase mutant having an amino acid sequence obtained by mutating one or more sites of the wild-type amino acid sequence shown in SEQ ID No. 1 selected from the group consisting of: amino acid I at position 6, amino acid L at position 7, and amino acid L at position 158.
2. The transaminase mutant of item 1, wherein amino acid I at position 6 in SEQ ID NO. 1 is substituted with a non-polar amino acid selected from the group consisting of: A. v, G, L, P, F, W and M; wherein amino acid L at position 7 in SEQ ID NO. 1 is substituted with a non-polar amino acid selected from the group consisting of: A. v, G, I, P, F, W and M; wherein amino acid L at position 158 in SEQ ID NO. 1 is substituted with a non-polar amino acid selected from the group consisting of: A. v, G, I, P, F, W and M.
3. The transaminase mutant according to item 1 or 2, which is further mutated at amino acid Y at position 125 in SEQ ID NO. 1.
4. The transaminase mutant of item 3, wherein amino acid Tyr at position 125 in SEQ ID No. 1 is substituted with a non-polar amino acid selected from the group consisting of: A. v, G, L, I, P, F, W and M.
5. The transaminase mutant of any one of claims 1 to 4, wherein the mutation is any one selected from the following groups: I6V, I6A, L7V, L7A, L158V, L158A, I A/L7A, I6A/L7V, I A/L158V, L A/L158A, L V/L158V, L A/L158V, I A/Y125A, I6V/Y125A, L V/Y125A, I6V/L7V/L158A, I A/L7A/L158V, I A/L7A/Y125A, I V/L7V/L158V/Y125A and I6A/L7A/L158V/Y125A.
6. The transaminase mutant of item 5, wherein the mutation is I6A/L7A/L158V/Y125A.
7. The transaminase mutant of claim 6, wherein the amino acid sequence of the transaminase mutant is shown in SEQ ID NO. 2.
8. A nucleotide sequence encoding the transaminase mutant of any one of claims 1-7.
9. The nucleotide sequence according to item 8, wherein the nucleotide sequence is shown as SEQ ID NO. 3.
10. A recombinant expression vector, wherein the recombinant expression vector comprises the nucleotide sequence of item 8 or 9.
11. The recombinant expression vector according to item 10, wherein the recombinant expression vector is constructed by a vector selected from the group consisting of: expression vectors suitable for E.coli, expression vectors suitable for Rhodococcus, shuttle vectors, phages and viral vectors; preferably, the escherichia coli applicable expression vector is pET-28a, and the rhodococcus applicable expression vector is pNV18.1.
12. A recombinant host cell comprising the nucleotide sequence of item 8 or 9 or the recombinant expression vector of item 10 or 11; preferably the recombinant host cell is selected from the group consisting of: rhodococcus, escherichia coli, bacillus and yeast.
13. An enzyme composition comprising the transaminase mutant of any one of claims 1-7; preferably, the enzyme composition is in a form selected from the group consisting of: free enzyme, free cell, immobilized enzyme and immobilized cell.
14. Use of the transaminase mutant of any one of claims 1 to 7, the nucleotide sequence of claim 8 or 9, the recombinant expression vector of claim 10 or 11, the recombinant host cell of claim 12, or the enzyme composition of claim 13 to catalyze a transamination reaction of an N-heterocyclic ketone compound to prepare a chiral N-heterocyclic amine.
15. The use according to item 15, wherein the chiral N-heterocyclic amine is (R) -N-heterocyclic amine.
16. The use according to item 14 or 15, wherein the N-heterocyclic ketone compound is selected from the group consisting of: piperidones, pyrrolidinones, azepinones, and derivatives thereof.
17. The use according to any one of claims 14-16, wherein the N-heterocyclic ketone compound is selected from the group consisting of: 3-piperidone, N-t-butoxycarbonyl-3-pyrrolidone, N-benzyl-3-piperidone, N-t-butoxycarbonyl-3-piperidone, N-benzyloxycarbonyl-3-piperidone, and N-t-butoxycarbonyl-3-azepinone; the chiral N-heterocyclic amine is correspondingly selected from the group consisting of: (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.
In order to make the technical scheme of the invention clearer, the invention is further described below with reference to the attached drawings and the specific embodiments.
Drawings
FIG. 1 shows a reaction scheme for synthesizing chiral N-heterocyclic amines using a transaminase as a catalyst;
FIG. 2 shows a model of the crystal structure of the aminotransferase RbTA and the amino acid mutation sites around its pocket;
FIG. 3 shows the reaction sequence for the catalytic synthesis of (R) -N-t-butoxycarbonyl-3-aminopiperidine by the recombinant aminotransferase RbTA mutant aminotransferase; and
FIG. 4 shows the protein expression of recombinant aminotransferase RbTA mutants. Wherein S is supernatant; c is a control.
Detailed Description
1. Definition of the definition
The term "transaminase" as used in the present invention is a class of enzymes that catalyze the transfer of an amino acid from a keto acid. Specifically, the amino acid contains an amine group (-NH) 2 ) Keto acids contain a ketone group (=o). In the ammonia transfer, the-NH-group on one molecule 2 The group is exchanged with an = O group on another molecule, and after the exchange the amino acid becomes a keto acid, which becomes an amino acid.
The 20 essential amino acids can be classified according to the different nature of the groups they carry. Amino acids can be classified into nonpolar amino acids, polar uncharged amino acids, positively charged (basic) amino acids and negatively charged (acidic) amino acids according to the polarity of the groups carried or the tendency to interact with water at physiological pH, etc. The term "nonpolar amino acid" as used in the present invention includes alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W) and methionine (Met, M).
The term "N-heterocycle" as used herein refers to an organic cyclic compound containing nitrogen, common N-heterocycles include: ternary N-heterocycles, quaternary N-heterocycles, five-membered N-heterocycles, six-membered N-heterocycles, seven-membered N-heterocycles, eight-membered N-heterocycles, fused N-heterocycles, N-heterobridged rings and macrocyclic N-heterocycles. Substances further containing a ketone group in the "N-heterocyclic" compound are referred to as "N-heterocyclic ketones" and include, but are not limited to, piperidones, pyrrolidinones, azepinones, or derivatives thereof.
The terms "mutation/mutant", "wild-type", "amino acid", "nucleotide", "expression (of a protein)", "substitution", "recombination", "expression vector", "host cell", "chiral", etc. as used in the present invention are conventional terms used in the field of molecular biology and/or biochemistry and have the same meaning as understood by the skilled person.
2. Transaminase mutants of the invention
In a first aspect, the present invention provides a transaminase mutant having an amino acid sequence obtained by mutating one or more sites of the wild-type amino acid sequence shown in SEQ ID NO. 1 selected from the group consisting of: amino acid I at position 6, amino acid L at position 7, and amino acid L at position 158. In one embodiment, the transaminase mutant has amino acid I at position 6 in SEQ ID NO. 1 replaced with a non-polar amino acid selected from the group consisting of: A. v, G, L, P, F, W and M. In one embodiment, the transaminase mutant has amino acid L at position 7 in SEQ ID NO. 1 replaced with a non-polar amino acid selected from the group consisting of: A. v, G, I, P, F, W and M. In one embodiment, the transaminase mutant has amino acid L at position 158 of SEQ ID NO. 1 replaced with a non-polar amino acid selected from the group consisting of: A. v, G, I, P, F, W and M. In one embodiment, the transaminase mutant is further mutated at amino acid Y at position 125 in SEQ ID NO. 1. In one embodiment, the aminotransferase mutant has amino acid Tyr at position 125 in SEQ ID No. 1 replaced with a non-polar amino acid selected from the group consisting of: A. v, G, L, I, P, F, W and M. In one embodiment, the transaminase mutant is a mutation selected from the group consisting of SEQ ID No. 1: I6V, I6A, L7V, L7A, L158V, L158A, I A/L7A, I6A/L7V, I A/L158V, L A/L158A, L V/L158V, L A/L158V, I A/Y125A, I6V/Y125A, L V/Y125A, I6V/L7V/L158A, I A/L7A/L158V, I A/L7A/Y125A, I V/L7V/L158V/Y125A and I6A/L7A/L158V/Y125A. In one embodiment, the transaminase mutant is a mutation based on SEQ ID NO. 1 as follows: I6A/L7A/L158V/Y125A. In one embodiment, the amino acid sequence of the transaminase mutant is shown in SEQ ID NO. 2.
In a second aspect, the invention provides a gene sequence or nucleotide sequence encoding a transaminase mutant according to the invention. In one embodiment, the gene sequence or nucleotide sequence is set forth in SEQ ID NO. 3.
In a third aspect, the invention provides a recombinant expression vector comprising a nucleotide sequence according to the invention. In one embodiment, the recombinant expression vectors of the invention can be stably present and autonomously replicating in a variety of hosts in prokaryotic or eukaryotic cells. In one embodiment, the recombinant expression vector of the invention is constructed by a vector selected from the group consisting of: coli-suitable expression vectors, rhodococcus-suitable expression vectors, shuttle vectors, phages and viral vectors. In one embodiment, the recombinant expression vector of the invention is prepared using a vector selected from the group consisting of: pET series vectors (preferably pET-28 a), E.coli-Rhodococcus shuttle vector (preferably PNV 18.1). The nucleotide sequence of the present invention is inserted into the above vector (e.g., pET-28 a) by molecular biological operations such as cleavage, ligation, etc., based on the above vector, to construct the recombinant expression plasmid (e.g., pET28 a-RbTAvariant) of the present invention. In one embodiment, the pET series vectors include, but are not limited to, pET-22b, pET-3a, pET-3d, pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-23b, pET-24a, pET-25b, pET-26b, pET-27b, pET-28a, pET-29a, pET-30a, pET-31b, pET-32a, pET-35b, pET-38b, pET-39b, pET-40b, pET-41a, pET-41b, pET-42a, pET-43a, pET-44a and pET-49b.
In a fourth aspect, the invention provides a recombinant host cell comprising a nucleotide sequence according to the invention or a recombinant expression vector according to the invention. In one embodiment, the recombinant host cell of the invention is selected from the group consisting of E.coli, rhodococcus erythropolis (also known as Rhodococcus erythropolis (Rhodococcus ruber)), bacillus subtilis (Bacillus subtilis), or yeast (Saccharomyces). In one embodiment, the recombinant host cell of the invention is E.coli BL21 (DE 3). In one embodiment, the recombinant expression plasmid (such as pET28 a-RbTAvariant) is transformed into escherichia coli BL21 (DE 3) to obtain corresponding genetically engineered bacteria (such as E.coli BL21 (DE 3) pET28 a-RbTAvariant).
In a fifth aspect, the invention provides an enzyme composition comprising a transaminase mutant according to the invention. In one embodiment, the enzyme composition of the invention is in a form selected from the group consisting of: free enzyme, free cell, immobilized enzyme and immobilized cell. In one embodiment, the enzyme composition of the invention is a free cell. In one embodiment, the episomal cells are whole cells obtained by subjecting the recombinant host cells of the invention to enrichment culture and/or induced expression of a protein of interest. In one embodiment, the enzyme composition of the invention is a free enzyme. In one embodiment, the free enzyme is a crude enzyme solution obtained by subjecting whole cells to ultrasonication or high pressure homogenization and centrifugation, or an enzyme obtained by further protein purification means. In one embodiment, the enzyme composition of the invention is an immobilized enzyme and/or an immobilized cell. In one embodiment, the immobilized cells and/or immobilized enzymes are prepared by immobilizing free cells and/or free enzymes to a carrier.
3. Use of the transaminase mutants of the invention
In a sixth aspect, the invention provides the use of a transaminase mutant according to the invention. In one embodiment, the aminotransferase mutants, nucleotide sequences, recombinant expression vectors, recombinant host cells, or enzyme compositions of the present invention are used to catalyze the transamination of N-heterocyclic ketones to produce chiral N-heterocyclic amines. In one embodiment, the chiral N-heterocyclic amine is an (R) -N-heterocyclic amine. In one embodiment, the N-heterocyclic ketone compound is selected from the group consisting of: piperidones, pyrrolidinones, azepinones, and derivatives thereof. In one embodiment, the N-heterocyclic ketone compound is selected from the group consisting of: 3-piperidone, N-t-butoxycarbonyl-3-pyrrolidone, N-benzyl-3-piperidone, N-t-butoxycarbonyl-3-piperidone, N-benzyloxycarbonyl-3-piperidone and N-t-butoxycarbonyl-3-azepinone. In one embodiment, the resulting chiral N-heterocyclic amine is selected from the group consisting of: (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.
In one embodiment, the aminotransferase mutant of the present invention is capable of asymmetrically catalyzing and synthesizing a variety of chiral aminopiperidines with high added values, and the reaction conversion rate can be more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, and the optical purity of the obtained chiral product can be more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%. In one embodiment, the transaminase mutants of the invention have significantly improved transaminase activity compared to the wild-type parent. In one embodiment, the transaminase activity of the transaminase mutant of the present invention is 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 25-fold or more, 30-fold or more, 35-fold or more, 40-fold or more, 45-fold or more, 50-fold or more, 55-fold or more, 60-fold or more, 65-fold or more, 70-fold or more, 72-fold or more, 75-fold or more, 78-fold or more, 80-fold or more, 90-fold or more, 95-fold or more, 100-fold or more, 110-fold or more, 120-fold or more, 130-fold or more, 140-fold or more, 150-fold or more, 160-fold or more, 170-fold or more, 180-fold or more, 190-fold or more, 195-fold or more, 200-fold or more than. In one embodiment, the transaminase mutants of the present invention are used to catalyze chiral products having ee (percent enantiomeric excess) of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more while maintaining the above-described high activity.
4. Advantages of the invention
The invention has the following technical advantages:
(1) The recombinant aminotransferase mutant catalyst obtained by the invention can asymmetrically catalyze and synthesize various chiral aminopiperidines with high added values, the reaction conversion rate is high (up to 99%), and the optical purity of the obtained chiral product is high (up to more than 99%).
(2) The activity of the transaminase mutant obtained through protein engineering is obviously improved compared with that of a wild female parent, wherein the activity of the optimal mutant is improved by 200 times compared with that of the wild female parent, and the ee of the product is more than 99%;
(3) The transaminase mutant obtained by protein engineering is utilized for preparation reaction, the separation yield is more than 80%, and the space-time yield is up to 240g/L/d;
(4) The synthesis of various chiral aminopiperidines through transaminase catalysis can be used for synthesizing hypoglycemic drugs including alogliptin, trogliptin, linagliptin, a fourth fluoroquinolone antibiotic besifloxacin and an epidermal growth factor receptor inhibitor of nazatinib;
(5) The recombinant aminotransferase mutant catalyst obtained by the invention can catalyze aliphatic ketone, aromatic ketone and other heterocyclic ketone compounds as well, the reaction conversion rate is high (up to 99%), and the optical purity of the obtained chiral product is high (up to more than 99%).
(6) The rhodococcus recombinant aminotransferase mutant catalyst obtained by the invention has obviously improved stability and organic solvent tolerance (up to 50%).
Detailed Description
The specific embodiments of the present invention are listed only as examples of the present invention, and the present invention is not limited to the specific embodiments described below. Any equivalent modifications and substitutions of the embodiments described below will be apparent to those skilled in the art, and are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other embodiments, methods, means, apparatus and steps well known to those skilled in the art have not been described in detail in order to not obscure the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, all units used in this specification are units of international standard, and the numerical values and numerical ranges appearing in the present invention are understood to include systematic errors unavoidable in industrial production.
Example 1 design of transaminase mutants
On the basis of analyzing the aminotransferase protein structure, molecular dynamics simulation is utilized to explore the binding mechanism of substrate molecules and enzyme molecules, and steric hindrance effect exists in the substrate binding process through interaction analysis, so that the method is key to influencing the enzyme catalytic activity. Subsequently, we locked the isoleucine at position 6, leucine at position 7, tyrosine at position 125 and/or leucine at position 158 (abbreviated as I6, L7, Y125 and L158, respectively) of the amino acid sites surrounding the substrate binding site, as viewed by structure. In order to reduce steric effects, it is desirable to mutate selected amino acids to smaller side chain amino acids such as valine or alanine, single point mutants were designed as I6V, I6A, L7V, L A, L158V, L158A, Y a and Y125V. And on this basis, these sites were subjected to combinatorial mutation, thereby designing combinatorial mutants as shown in Table 1 below.
TABLE 1 list of mutants
| I6V | I6A/L7A | L7V/L158A | L158A/Y125A | I6A/L7V/L158V/125A |
| I6A | I6A/L7V | L7V/L158V | L158A/Y125V | I6A/L7V/L158V/125V |
| L7V | I6A/L158V | L7V/125A | L158V/Y125A | I6A/L7A/L158V/125A |
| L7A | I6A/Y125A | L7V/125V | L158V/Y125V | I6A/L7A/L158V/125V |
| L158V | I6A/Y125V | L7A/L158V | I6V/L7V/L158A | I6A/L7V/L158A/125A |
| L158A | I6A/L158A | L7A/L158A | I6V/L7V/L158V | I6A/L7V/L158A/125V |
| Y125A | I6V/L158V | I6A/L7A/L158A | I6A/L7V/L158A | I6A/L7A/L158A/125A |
| Y125V | I6V/L158A | I6A/L7A/L158V | I6A/L7V/L158V | I6A/L7A/L158A/125V |
EXAMPLE 2 construction of a library of transaminase mutants
The recombinant expression vector pET28a-RbTA constructed in example 1 was selected as a template, and forward and reverse primers containing a mutation site (see Table 2 in particular) were designed for full plasmid amplification using PrimeSTAR HS DNA Polymerase (Takara).
The 20. Mu.L PCR reaction system comprises:
1. Mu.L pET28a-RbTA plasmid template (about 100 ng/. Mu.L);
10 μL of 2 XPrimeSTAR HS DNA polymerase;
1.5. Mu.L of forward primer (10. Mu.M);
1.5. Mu.L of reverse primer (10. Mu.M);
6μL ddH 2 O。
the forward primers were specific primers used in the construction process for the different mutants as shown in table 2. Taking L6V construction as an example, the forward primer in the amplification system is L6V-up. The reverse primer was a specific primer used in the construction process for the different mutants as shown in table 2. Taking L6V construction as an example, the reverse primer in the amplification system is L6V-Down.
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.
The conditions for the PCR reaction were as follows:
(1) Pre-denaturation at 98 ℃ for 1 min;
(2) Denaturation at 98℃for 30 sec;
(3) (Tm-5) of the primer for 10 seconds;
(4) Extending at 72 ℃ for 7 minutes;
the steps (2) - (4) were carried out for 30 cycles in total and finally extended at 72℃for 10 minutes.
The recombinant plasmid obtained by PCR amplification was digested with DpnI enzyme, transformed into E.coli DH 5. Alpha. Competent cells, plated on LB plate containing kanamycin (50. Mu.g/mL), and placed in an incubator at 37℃for inversion culture for about 12 hours. And (3) selecting the monoclonal seed for sequencing verification, and preserving the bacteria by using 20% glycerol after sequencing correctly, and storing in a refrigerator at the temperature of-70 ℃. The recombinant expression vector containing the mutation was designated pET28a-RbTAvariant.
TABLE 2 Forward primer (up) and reaction primer (Down) sequences used in constructing mutant libraries
EXAMPLE 3 construction of transaminase mutant genetically engineered bacteria (E.coli)
The recombinant expression vector RbTAvariant prepared in example 2 was transformed into competent cells E.coli BL21 (DE 3) by a heat shock method, coated with LB plate containing kanamycin (50. Mu.g/mL), cultured overnight at 37℃and then picked up as a single colony to transfer into LB liquid culture containing 50. Mu.g/mL kanamycin, cultured for 12 hours at 37℃and subjected to sample transfer for sequencing, and the correct clone was stored in a refrigerator at-70℃to obtain a genetically engineered bacterium hosting E.coli. The high-efficiency soluble expression can be realized in an escherichia coli system through fermentation culture and induced expression of aminotransferase.
EXAMPLE 4 construction of transaminase mutant genetically engineered bacteria (Rhodococcus)
The aminotransferase mutant gene obtained in example 2 was constructed on suicide plasmid pYsacB, and the aminotransferase mutant gene was constructed on the genome of Rhodococcus by the strategy of homologous recombination double crossover. Transforming the recombinant plasmid into competent cells Rhodococcus ruber by electrotransformation, coating a solid plate containing spectinomycin (50 mug/mL), resuscitating, culturing, coating on a plate culture medium containing 50 mug/mL spectinomycin, and culturing at 20-37 ℃; then picking single colony, and verifying colony PCR to verify that suicide plasmid is integrated into correct position in rhodococcus genome; inoculating colony with successful single exchange into seed culture medium without antibiotics, culturing at 20-37deg.C for 12 hr, diluting 100 times, spreading 200 μL on plate containing 100g/L sucrose, culturing at 28deg.C, performing colony PCR after single colony is grown, verifying nitrile hydratase gene sequence integration onto genome, delivering to sequencing company, and storing the strain with correct sequencing in a refrigerator at-70deg.C for use. The high-efficiency soluble expression can be realized in a rhodococcus system by fermenting culture and induced expression of aminotransferase.
EXAMPLE 5 construction of secretion expression System of transaminase mutant Gene engineering bacteria
The aminotransferase mutant gene obtained in example 2 was constructed on a secretory expression vector pNV-18.1-Pa2-SP suitable for use in a rhodococcus system, and then the recombinant plasmid was electrotransferred into rhodococcus competent cells, at 28℃and resuscitated at 200rpm for 2.5 hours. After resuscitating, the supernatant was centrifuged at 13000rpm at high speed and 800. Mu.L was removed, the centrifuged cells were resuspended and plated on rhodococcus solid medium (kanamycin 25. Mu.g/mL) and placed in a 28℃incubator for culturing in inversion for 48-60 hours. Selecting positive clone to obtain recombinant rhodococcus capable of secreting and expressing transaminase. Secretion soluble expression can be achieved in rhodococcus systems by fermentation culture and induction of the expression of aminotransferase.
EXAMPLE 6 preparation of cell catalyst and free enzyme catalyst
Inoculating genetically engineered bacteria containing the coding sequence of the aminotransferase mutant into a seed solution culture medium containing antibiotics, and culturing at 200rpm at an optimal temperature (the escherichia coli is 37 ℃ and the rhodococcus is 28 ℃), thereby obtaining seed solution. The seed solution in the test tube is transferred to the fermentation medium in an ultra clean bench. For Escherichia coli, the fermentation medium containing the seed solution is placed in a shaking table at 37℃and cultured at 200rpm for 2 to 3 hours. OD of the culture solution to be cultured 600 When the value reaches 0.6-0.8, IPTG with the final concentration of 0.1-0.8 mM is added for induction expression, and the induction temperature is 16-37 ℃. The preferred IPTG concentration is 0.2mM and the preferred induction temperature is 16 ℃. After induction for 24 hours under preferred conditions, the cells were collected by centrifugation to obtain an E.coli cell catalyst. For rhodococcus, after seed solution is inoculated into fermentation medium, co with final concentration of 0.08mM is added into fermentation medium 2+ After 48 hours of induction expression, cells were collected by centrifugation to obtain a rhodococcus cell catalyst of nitrile hydratase.
An appropriate amount of the cell catalyst (10% w/v) was weighed, resuspended in solution A (25 mM Tris-HCl, pH 7.5), broken with a high pressure refiner, and the broken supernatant was collected by centrifugation to obtain a broken supernatant as a crude enzyme solution. The crude enzyme solution is placed in a refrigerator at the temperature of-70 ℃ for freezing overnight, and freeze-dried crude enzyme powder is prepared by a freeze dryer. Alternatively, the crude enzyme of interest may be purified in one step by affinity chromatography, the crude enzyme solution is loaded onto a nickel column, then eluents of different imidazole concentrations (10-500 mM) are used, then the protein of interest is eluted, and the eluents under different gradients are 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 of 20-60 percent respectively), the target protein can be eluted, and finally the obtained protein has single strip and higher purity. 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) and the solution was repeated 2-3 times to remove imidazole and when concentrated to less than 1mL, pure protein was collected. Protein concentration was determined using Nandrop2000 and placed in a-70 ℃ freezer after liquid nitrogen flash freezing.
EXAMPLE 7 preparation of immobilized enzyme and immobilized cell
An appropriate amount of immobilized carrier was weighed, equilibrated with potassium phosphate buffer (100 mM, pH 7.0), the treated tree carrier was added to the buffer at a carrier to solution ratio of 1/5 (w/V), and glutaraldehyde solution (50%) was then added 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. Weighing a proper amount of activated immobilized carrier, placing the immobilized carrier in a buffer solution, and adding an enzyme solution, wherein the ratio of the carrier to the enzyme solution is still 1/5 (w/v). The mixture was immobilized on a constant temperature shaker or shaker (16 ℃ C., 200 rpm) for 8 hours. The immobilized enzyme was then washed with buffer to remove residual enzyme solution, which was then stored in a refrigerator at 4℃for further use.
Weighing an appropriate amount of immobilized carrier, balancing with potassium phosphate buffer (100 mM, pH 7.0), adding the treated tree carrier into the buffer, adding cell catalyst, and keeping the ratio of carrier to cell catalyst at 1/5 (w/v). The mixture was immobilized on a constant temperature shaker or shaker (16 ℃ C., 200 rpm) for 8 hours. The immobilized cells were then washed with buffer to remove residual cell catalyst, and then stored in a refrigerator at 4℃for further use.
EXAMPLE 8 determination of specific Activity of transaminase and its mutants
The pure enzyme stored in a refrigerator at-70℃as described in example 4 was taken and dissolved in ice, diluted with buffer (25 mM Tris-HCl, pH 7.5) to a final concentration of 2mg/mL, 50. Mu.L of the diluted solution was added to 300. Mu.L of Tris-HCl buffer (pH 8.0,0.1M), and then 50. Mu.L of pyridoxal phosphate PLP (10 mM), 50 were added in this ordermu.L of isopropylamine (2M, pH 8.0) was preheated on a shaker (30 ℃ C., 1000 rpm) for 2 minutes, then 50. Mu.L of substrate (100 mM, v/v) was added to react for 15 minutes, then 100. Mu.L of NaOH (10M) was added to quench, then an equal volume of dichloromethane was added to extract, and the organic phase was collected by centrifugation and dried. The amounts of substrate and product were detected by gas phase under the following conditions: constant pressure sample injection, split ratio of 1/30, sample injection amount of 1. Mu.L, sample inlet and detector temperature of 250deg.C, chromatographic column HP-5 (30 m,
0.25μm,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 product selectivity was determined using CP (25 m, -/->
0.25,Agilent J&W Scientific,USA)。
The conversion rate is calculated as follows:
conversion rate
Wherein A is 1 As the peak area of the product after the reaction, A 2 The area of the substrate peak after the reaction. />
The specific activity of the aminotransferase is calculated as follows:
EXAMPLE 9 recombinant transaminase mutant Activity assay
By site-directed mutagenesis and combined mutagenesis, we obtained a total of 40 recombinant aminotransferase mutants, which were screened for viability using N-t-butoxycarbonyl-3-piperidone as substrate.
The enzyme activity of the transaminase mutants was calculated by detecting the formation of the product or the decrease in the substrate by gas chromatography, and the specific measurement method was carried out in accordance with the method described in example 6. Table 3 provides the results of the activity determination of the aminotransferase mutants of the invention having the active specific sequences.
TABLE 3 determination of the Activity of recombinant aminotransferase mutants
In table 3, each mutant shown represents a sequence set forth in SEQ ID NO:2, and a specific mutation is generated in the corresponding amino acid position in the amino acid sequence shown in the formula 2. In the activity column, a plus sign "+" indicates that mutant proteins have 1-10 times higher activity than wild type; two plus signs "++" indicate that the mutant has 10-20 times higher activity than wild type; 3 plus signs "++ + +" indicate that the mutant has 20-30 times higher activity than the wild type, four plus signs "+++ +". "means mutant ratio the activity of the wild type is improved by 30-40 times; five plus signs "+++" the + "indicates the mutant ratio the activity of the wild type is improved by 40-50 times; six plus signs "+++" ++ indicates mutant the activity is improved by 50-60 times compared with the wild type activity; 7 plus signs "+++". ++ indicates mutant the activity is improved by 60-70 times compared with the wild type activity; 8 plus signs "+++". ++ indicates mutant the activity is improved by 70-80 times compared with the wild type activity; ++ + + and ++ + ++ ++ ++++++++) ++ or indicating that the mutant has 200 times higher activity than the wild type.
Conclusion: through vitality determination, 40 mutants with improved vitality are obtained in total, and compared with wild type vitality, the vitality of the mutants is improved by 1-200 times. Wherein the mutant I6A/L7A/L158V/125A activity is improved by up to 200 times, and is the optimal mutant. According to activity measurement, the activity of the aminotransferase mutant provided by the invention on the substrate (1 a-6 a) is improved compared with that of a wild type, and the activity of the mutant obtained by combining the mutations is improved most remarkably, and the catalytic result of the mutant on the substrate N-t-butoxycarbonyl-3-piperidone shown in Table 2 is specifically referred. 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 hour is one enzyme activity unit, denoted by U.
EXAMPLE 10 catalytic Synthesis of (R) -3-aminopiperidine by recombinant aminotransferase RbTA mutant
A substrate (final concentration: 500 mM), a prosthetic group (1 mM), an amino donor (1M), and a catalyst (25 g/L) were sequentially added to the reaction system. The reaction was carried out in a constant temperature shaker at 30℃and 1000rpm for 12 hours. After the reaction, the conversion and the selectivity of the product were calculated as in example 5, and the conversion was found to be 99% and the ee value of the product was greater than 99%.
The preferred reaction buffer is Tris-HCl (pH 9.0) buffer, the substrate is 3-piperidone, the prosthetic group is pyridoxal phosphate, the amino donor is isopropylamine, and the catalyst is the transaminase mutant prepared in example 4 or example 5.
EXAMPLE 11 catalytic Synthesis of (R) -N-t-butoxycarbonyl-3-aminopyrrole from recombinant aminotransferase RbTA mutant
A substrate (final concentration: 500 mM), a prosthetic group (1 mM), an amino donor (1M), and a catalyst (25 g/L) were sequentially added to the reaction system. The reaction was carried out in a constant temperature shaker at 30℃and 1000rpm for 12 hours. After the reaction, the conversion and the selectivity of the product were calculated as in example 5, and the conversion was found to be 99% and the ee value of the product was greater than 99%.
The preferable reaction buffer is Tris-HCl (pH 9.0) buffer, the substrate is N-t-butoxycarbonyl-3-pyrrolidone, the prosthetic group is pyridoxal phosphate, the amino donor is isopropylamine, and the catalyst is the transaminase mutant prepared in example 4 or example 5.
EXAMPLE 12 catalytic Synthesis of (R) -N-benzyl-3-aminopiperidine by recombinant aminotransferase RbTA mutant
A substrate (final concentration: 500 mM), a prosthetic group (1 mM), an amino donor (1M), and a catalyst (25 g/L) were sequentially added to the reaction system. The reaction was carried out in a constant temperature shaker at 30℃and 1000rpm for 12 hours. After the reaction, the reaction conversion rate and the selectivity of the product are calculated according to the method in the example 5, the reaction conversion rate of the mutant is higher than that of the wild type, the conversion rate of the optimal mutant can reach 99%, and the ee value of the product is higher than 99%.
The preferred reaction buffer is Tris-HCl (pH 9.0) buffer, the substrate is N-benzyl-3-piperidone, the prosthetic group is pyridoxal phosphate, the amino donor is isopropylamine, and the catalyst is the transaminase mutant prepared in example 4 or example 5.
TABLE 4 catalytic synthesis of (R) -N-benzyl-3-aminopiperidines by recombinant aminotransferase mutants
EXAMPLE 13 catalytic Synthesis of (R) -N-t-butoxycarbonyl-3-aminopiperidine by recombinant aminotransferase RbTA mutant
A substrate (final concentration: 500 mM), a prosthetic group (1 mM), an amino donor (1M), and a catalyst (25 g/L) were sequentially added to the reaction system. The reaction was carried out in a constant temperature shaker at 30℃and 1000rpm for 12 hours. After the reaction, the reaction conversion rate and the selectivity of the product are calculated according to the method in the example 5, the reaction conversion rate of the mutant is higher than that of the wild type, the conversion rate of the optimal mutant can reach 99%, and the ee value of the product is higher than 99%.
The preferable reaction buffer is Tris-HCl (pH 9.0) buffer, the substrate is N-t-butoxycarbonyl-3-piperidone, the prosthetic group is pyridoxal phosphate, the amino donor is isopropylamine, and the catalyst is the transaminase mutant prepared in example 4 or example 5.
TABLE 5 catalytic synthesis of (R) -N-t-Butoxycarbonyl-3-aminopiperidine by recombinant aminotransferase mutants
EXAMPLE 14 catalytic Synthesis of (R) -N-benzyloxycarbonyl-3-aminopiperidine by recombinant aminotransferase RbTA mutant
A substrate (final concentration: 500 mM), a prosthetic group (1 mM), an amino donor (1M), and a catalyst (25 g/L) were sequentially added to the reaction system. The reaction was carried out in a constant temperature shaker at 30℃and 1000rpm for 12 hours. After the reaction, the reaction conversion rate and the selectivity of the product are calculated according to the method in the example 5, the reaction conversion rate of the mutant is higher than that of the wild type, the conversion rate of the optimal mutant can reach 99%, and the ee value of the product is higher than 99%.
The preferred reaction buffer is Tris-HCl (pH 9.0) buffer, the substrate is N-benzyloxycarbonyl-3-piperidone, the prosthetic group is pyridoxal phosphate, the amino donor is isopropylamine, and the catalyst is the transaminase mutant prepared in example 4 or example 5.
TABLE 6 catalytic synthesis of (R) -N-benzyloxycarbonyl-3-aminopiperidines from recombinant aminotransferase mutants
| Mutant | Conversion rate | Ee value of the product | Mutant | Conversion rate | Ee value of the product |
| Wild type | 7％ | >99％ | L7A/L158V | 75％ | >99％ |
| I6V | 36％ | >99％ | L7A/L158A | 88％ | >99％ |
| I6A | 39％ | >99％ | I6A/L7A/L158A | 79％ | >99％ |
| L7V | 36％ | >99％ | I6A/L7A/L158V | 95％ | >99％ |
| L7A | 27％ | >99％ | L158A/Y125A | 90％ | >99％ |
| L158V | 48％ | >99％ | L158A/Y125V | 91％ | >99％ |
| L158A | 49％ | >99％ | L158V/Y125A | 89％ | >99％ |
| Y125A | 89％ | >99％ | L158V/Y125V | 89％ | >99％ |
| Y125V | 79％ | >99％ | I6V/L7V/L158A | 82％ | >99％ |
| I6A/L7A | 46％ | >99％ | I6V/L7V/L158V | 78％ | >99％ |
| I6A/L7V | 39％ | >99％ | I6A/L7V/L158A | 86％ | >99％ |
| I6A/L158V | 39％ | >99％ | I6A/L7V/L158V | 88％ | >99％ |
| I6A/Y125A | 93％ | >99％ | I6A/L7V/L158V/125A | 99％ | >99％ |
| I6A/Y125V | 81％ | >99％ | I6A/L7V/L158V/125V | 99％ | >99％ |
| I6A/L158A | 49％ | >99％ | I6A/L7A/L158A/125V | 99％ | >99％ |
| I6V/L158V | 59％ | >99％ | I6A/L7A/L158V/125V | 99％ | >99％ |
| I6V/L158A | 38％ | >99％ | I6A/L7V/L158A/125A | 99％ | >99％ |
| L7V/L158A | 49％ | >99％ | I6A/L7V/L158A/125V | 99％ | >99％ |
| L7V/L158V | 65％ | >99％ | I6A/L7A/L158A/125A | 99％ | >99％ |
| L7V/125A | 82％ | >99％ | I6A/L7A/L158V/125A | 99％ | >99％ |
| L7V/125V | 83％ | -- | -- |
EXAMPLE 15 catalytic Synthesis of (R) -3-amino-azepane-1-carboxylic acid tert-butyl ester by recombinant aminotransferase RbTA mutant
A substrate (final concentration: 500 mM), a prosthetic group (1 mM), an amino donor (1M), and a catalyst (25 g/L) were sequentially added to the reaction system. The reaction was carried out in a constant temperature shaker at 30℃and 1000rpm for 12 hours. After the reaction, the reaction conversion rate and the selectivity of the product are calculated according to the method in the example 5, the reaction conversion rate of the mutant is higher than that of the wild type, the conversion rate of the optimal mutant can reach 99%, and the ee value of the product is higher than 99%.
The preferred reaction buffer is Tris-HCl (pH 9.0) buffer, the substrate is N-t-butoxycarbonyl-3-azepinone, the prosthetic group is pyridoxal phosphate, the amino donor is isopropylamine, and the catalyst is the transaminase mutant prepared in example 4 or example 5.
TABLE 7 catalytic synthesis of (R) -3-amino azepane-1-carboxylic acid tert-butyl ester by recombinant aminotransferase mutant
| Mutant | Conversion rate | Ee value of the product | Mutant | Conversion rate | Ee value of the product |
| Wild typeA kind of electronic device with a display unit | 12％ | >99％ | L7A/L158V | 85％ | >99％ |
| I6V | 46％ | >99％ | L7A/L158A | 78％ | >99％ |
| I6A | 53％ | >99％ | I6A/L7A/L158A | 99％ | >99％ |
| L7V | 46％ | >99％ | I6A/L7A/L158V | 95％ | >99％ |
| L7A | 47％ | >99％ | L158A/Y125A | 99％ | >99％ |
| L158V | 68％ | >99％ | L158A/Y125V | 99％ | >99％ |
| L158A | 79％ | >99％ | L158V/Y125A | 99％ | >99％ |
| Y125A | 99％ | >99％ | L158V/Y125V | 99％ | >99％ |
| Y125V | 99％ | >99％ | I6V/L7V/L158A | 92％ | >99％ |
| I6A/L7A | 76％ | >99％ | I6V/L7V/L158V | 98％ | >99％ |
| I6A/L7V | 79％ | >99％ | I6A/L7V/L158A | 96％ | >99％ |
| I6A/L158V | 69％ | >99％ | I6A/L7V/L158V | 98％ | >99％ |
| I6A/Y125A | 99％ | >99％ | I6A/L7V/L158V/125A | 99％ | >99％ |
| I6A/Y125V | 99％ | >99％ | I6A/L7V/L158V/125V | 99％ | >99％ |
| I6A/L158A | 59％ | >99％ | I6A/L7A/L158A/125V | 99％ | >99％ |
| I6V/L158V | 89％ | >99％ | I6A/L7A/L158V/125V | 99％ | >99％ |
| I6V/L158A | 78％ | >99％ | I6A/L7V/L158A/125A | 99％ | >99％ |
| L7V/L158A | 69％ | >99％ | I6A/L7V/L158A/125V | 99％ | >99％ |
| L7V/L158V | 85％ | >99％ | I6A/L7A/L158A/125A | 99％ | >99％ |
| L7V/125A | 99％ | >99％ | I6A/L7A/L158V/125A | 99％ | >99％ |
| L7V/125V | 99％ | -- | -- |
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention in any way. While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that this is by way of example only, and the scope of the invention is defined by the appended claims. Various changes and modifications may be made to the above-described disclosure by those skilled in the art without departing from the scope of the invention, which is to be accorded the full scope of the invention.
Sequence listing
Seq1: amino acid sequence of wild-type transaminase
MNQLTILEAGLDEIICETVPGEAIQYSRYSLDRTSPLAGGCAWIEGAFVPAAAARISIFD
AGFGHSDVTYTVAHVWHGNFFRLEDHVERFLAGAEKMRIPMPATKAEIMDLMRGCV
SKSGLREAYVNVCVTRGYGRKPGEKTLEALESQLYVYAIPYLWVFSPIRQIEGIDAVIA
QSVRRSPANVMDPWIKNYQWGDLVRATFEAQERGARTAFLLDSDGFVTEGPGFNVL
MVKDGTVFTAARNVLPGITRRTALEIARDFGLQTVIGDVTPEMLRGADEIFAATTAGG
VTPVVALDGAPVGAGVPGDWTRKIRTRYWQMMDEPSDLIEPVSYI
Seq2: amino acid sequence of transaminase mutants
MNQLTAAEAGLDEIICETVPGEAIQYSRYSLDRTSPLAGGCAWIEGAFVPAAAARISIF
DAGFGHSDVTYTVAHVWHGNFFRLEDHVERFLAGAEKMRIPMPATKAEIMDLMRGC
VSKSGLREAYVNVCVTRGYGRKPGEKTLEALESQLYVYAIPYVWVFSPIRQIEGIDAVI
AQSVRRSPANVMDPWIKNYQWGDLVRATFEAQERGARTAFLLDSDGFVTEGPGFNV
LMVKDGTVFTAARNVLPGITRRTALEIARDFGLQTVIGDVTPEMLRGADEIFAATTAG
GVTPVVALDGAPVGAGVPGDWTRKIRTRYWQMMDEPSDLIEPVSYI
Seq3: nucleotide sequences encoding transaminase mutants
ATGAACCAGCTGACCGCCGCCGAGGCGGGCCTGGACGAGATCATCTGCGAGACCG
TCCCGGGCGAGGCCATCCAGTACTCCCGCTACTCCCTGGACCGCACCTCGCCGCTC
GCCGGTGGCTGCGCGTGGATCGAGGGCGCCTTCGTGCCGGCGGCCGCGGCCCGCA
TCTCGATCTTCGACGCCGGCTTCGGCCACTCGGACGTCACCTACACCGTGGCCCAC
GTCTGGCACGGCAACTTCTTCCGCCTGGAGGACCACGTGGAGCGGTTCCTCGCGGG
CGCCGAGAAGATGCGCATCCCGATGCCGGCCACCAAGGCCGAGATCATGGACCTG
ATGCGGGGCTGCGTGTCCAAGTCGGGCCTCCGGGAGGCCTACGTCAACGTGTGCG
TCACCCGGGGCTACGGCCGGAAGCCGGGCGAGAAGACCCTGGAGGCCCTCGAGTC
GCAGCTGTACGTGTACGCCATCCCGTACGTCTGGGTCTTCTCCCCGATCCGCCAGA
TCGAGGGCATCGACGCGGTGATCGCCCAGTCCGTGCGTCGCTCGCCGGCCAACGT
GATGGACCCGTGGATCAAGAACTACCAGTGGGGCGACCTGGTCCGGGCCACCTTC
GAGGCCCAGGAGCGCGGTGCCCGCACCGCGTTCCTGCTGGACTCGGACGGCTTCG
TCACCGAGGGCCCGGGCTTCAACGTGCTGATGGTGAAGGACGGCACCGTGTTCAC
CGCGGCCCGCAACGTCCTCCCGGGCATCACCCGTCGCACCGCCCTGGAGATCGCCC
GGGACTTCGGCCTCCAGACCGTGATCGGCGACGTCACCCCGGAGATGCTCCGCGG
TGCCGACGAGATCTTCGCGGCCACCACCGCCGGCGGCGTCACCCCGGTCGTGGCC
CTCGACGGCGCCCCGGTGGGCGCGGGCGTGCCGGGCGACTGGACCCGGAAGATCC
GCACCCGGTACTGGCAGATGATGGACGAGCCGTCGGACCTCATCGAGCCGGTGTC
CTACATCTGA
Claims (17)
1. A transaminase mutant having an amino acid sequence obtained by mutating one or more sites of the wild-type amino acid sequence shown in SEQ ID No. 1 selected from the group consisting of: amino acid I at position 6, amino acid L at position 7, and amino acid L at position 158.
2. The transaminase mutant according to claim 1, wherein amino acid I at position 6 in SEQ ID No. 1 is substituted with a non-polar amino acid selected from the group consisting of: A. v, G, L, P, F, W and M; wherein amino acid L at position 7 in SEQ ID NO. 1 is substituted with a non-polar amino acid selected from the group consisting of: A. v, G, I, P, F, W and M; wherein amino acid L at position 158 in SEQ ID NO. 1 is substituted with a non-polar amino acid selected from the group consisting of: A. v, G, I, P, F, W and M.
3. The transaminase mutant according to claim 1 or 2, which is further mutated at amino acid Y at position 125 in SEQ ID No. 1.
4. A transaminase mutant according to claim 3, in which amino acid Tyr at position 125 in SEQ ID No. 1 is substituted with a non-polar amino acid selected from the group consisting of: A. v, G, L, I, P, F, W and M.
5. The transaminase mutant of any one of claims 1 to 4, wherein the mutation is any one selected from the following groups: I6V, I6A, L7V, L7A, L158V, L158A, I A/L7A, I6A/L7V, I A/L158V, L A/L158A, L V/L158V, L A/L158V, I A/Y125A, I6V/Y125A, L V/Y125A, I6V/L7V/L158A, I A/L7A/L158V, I A/L7A/Y125A, I V/L7V/L158V/Y125A and I6A/L7A/L158V/Y125A.
6. The transaminase mutant of claim 5, wherein the mutation is I6A/L7A/L158V/Y125A.
7. The transaminase mutant according to claim 6, wherein the amino acid sequence of the transaminase mutant is shown in SEQ ID NO. 2.
8. A nucleotide sequence encoding the transaminase mutant of any one of claims 1 to 7.
9. The nucleotide sequence according to claim 8, wherein the nucleotide sequence is shown as SEQ ID NO. 3.
10. A recombinant expression vector, wherein the recombinant expression vector comprises the nucleotide sequence of claim 8 or 9.
11. The recombinant expression vector of claim 10, wherein the recombinant expression vector is constructed from a vector selected from the group consisting of: expression vectors suitable for E.coli, expression vectors suitable for Rhodococcus, shuttle vectors, phages and viral vectors; preferably, the E.coli suitable expression vector is pET-28a, and the Rhodococcus suitable expression vector is pNV18.1.
12. A recombinant host cell comprising the nucleotide sequence of claim 8 or 9 or the recombinant expression vector of claim 10 or 11; preferably the recombinant host cell is selected from the group consisting of: rhodococcus, escherichia coli, bacillus and yeast.
13. An enzyme composition comprising the transaminase mutant of any one of claims 1-7; preferably, the enzyme composition is in a form selected from the group consisting of: free enzyme, free cell, immobilized enzyme and immobilized cell.
14. Use of a transaminase mutant according to any one of claims 1 to 7, a nucleotide sequence according to claim 8 or 9, a recombinant expression vector according to claim 10 or 11, a recombinant host cell according to claim 12, or an enzyme composition according to claim 13 for catalyzing the transamination of an N-heterocyclic ketone compound to prepare a chiral N-heterocyclic amine.
15. The use of claim 15, wherein the chiral N-heterocyclic amine is (R) -N-heterocyclic amine.
16. The use according to claim 14 or 15, wherein the N-heterocyclic ketone compound is selected from the group consisting of: piperidones, pyrrolidinones, azepinones, and derivatives thereof.
17. The use according to any one of claims 14-16, wherein the N-heterocyclic ketone compound is selected from the group consisting of: 3-piperidone, N-t-butoxycarbonyl-3-pyrrolidone, N-benzyl-3-piperidone, N-t-butoxycarbonyl-3-piperidone, N-benzyloxycarbonyl-3-piperidone, and N-t-butoxycarbonyl-3-azepinone; the chiral N-heterocyclic amine is correspondingly selected from the group consisting of: (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.
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