CN118374468A - Transaminase mutant and application thereof in preparation of (S) -chroman-4-amine - Google Patents
Transaminase mutant and application thereof in preparation of (S) -chroman-4-amine Download PDFInfo
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Abstract
The invention discloses a transaminase mutant and application thereof in preparation of (S) -chroman-4-amine, wherein the transaminase mutant is obtained by directed evolution of wild-type transaminase and comprises any one mutation or combination of mutations in C60W, V242A, L272M. The aminotransferase mutant disclosed by the invention can specifically recognize chroman-4-one by a substrate, and can efficiently synthesize (S) -chroman-4-amine, so that the stereoselectivity and the yield are obviously improved; the invention discloses a product for preparing (S) -chroman-4-amine, which comprises a transaminase mutant or a coding gene thereof or a corresponding expression vector or a corresponding recombinant cell with high yield and high stereoselectivity, wherein the temperature condition and the pH value condition of the application of the product are easy to realize, and the product has an industrialized application basis; the invention also discloses a preparation method and application of the mutant, which are beneficial to industrial production and application of (S) -chroman-4-amine.
Description
Technical Field
The invention relates to an enzyme mutant, in particular to a transaminase mutant and application thereof in preparation of (S) -chroman-4-amine, and belongs to the technical fields of genetic engineering and enzyme engineering.
Background
Aminotransferase (TRANSAMINASE) is a class of enzymes that catalyze the transfer of amino acids from keto acids and is ubiquitous in animals, plant tissues and microorganisms. Transaminase is widely used in regioselective and stereoselective transamination reactions of substrates such as aliphatic and aromatic keto acids, aldehydes, ketones and ketoses, for the synthesis of chiral amines, amino acids and derivatives thereof. In industrial production, many drugs have developed a process for preparing a drug substance or an intermediate thereof using transaminase, for example, sitagliptin, a migraine drug for treating diabetes type ii, and zizanpam and ubenimpam. Transaminase is a key biotechnological enzyme in the asymmetric synthesis of chiral amine compounds and resolution of racemates of amine compounds.
Chiral amine is a compound with amino group connected to chiral center, and plays important role in biological genetic, metabolic and other physiological processes. As a structural unit widely existing in the nature, chiral amine has various biological activities and is applied to synthesis of a plurality of medicines and intermediates such as nerve medicines, antihypertensive medicines, anti-infective medicines and the like. It is counted that about 40% of the new drugs approved in recent years by the U.S. food and drug administration contain chiral amine structures. Because chiral amine medicines with different chiralities have remarkable differences in medicine safety, human metabolism and the like, how to efficiently and stereoselectively synthesize a single chiral amine medicine has important research significance in the fields of medicine innovation and production.
(S) -chroman-4-amines (US 2004/157739) are chiral amines which are key intermediates involved in the synthetic production of various drugs, for example the antihypertensive drug chroman-Carlin (Cromakalim) as a potassium channel agonist for the treatment of hypertension, cardiovascular and cerebrovascular diseases. The (S) -chroman-4-amine is a colorless solid with molecular weight of 149.19, and can be dissolved in solvents such as methanol, dimethyl sulfoxide, etc. The synthesis of (S) -chroman-4-amine can adopt a kinetic resolution method, and the racemic chroman-4-amine is hydrolyzed after being derived to obtain chiral pure (S) -chroman-4-amine, but the method has complicated steps and the actual yield is not more than 50%, so that the industrial application value is low. The biosynthesis method takes chroman-4-ketone as a raw material, and generates chiral pure (S) -chroman-4-amine under the catalysis of aminotransferase, and the method has the advantages of mild reaction conditions, high yield, high stereoselectivity and the like. The reaction can transfer the amino group of the cheap amino donor (D/L-alanine and isopropylamine) to the chroman-4-ketone to generate byproducts (pyruvic acid and acetone) which are easy to separate, and has good industrial application prospect. However, the wild-type transaminase is limited in the preparation of (S) -chroman-4-amines by substrate specificity, stereoselectivity and catalytic efficiency, thus limiting the industrial production applications of (S) -chroman-4-amines.
Disclosure of Invention
The invention aims to: the invention aims to provide a transaminase mutant with high substrate specificity, stereoselectivity and conversion efficiency, and application of the transaminase mutant in preparation of (S) -chroman-4-amine.
The technical scheme is as follows: in a first aspect, the invention provides a transaminase mutant, the amino acid sequence of which is obtained by mutation of at least one of the sequences shown in SEQ ID NO. 1 and C60W, V242A, L M.
In a second aspect, the invention provides a nucleic acid molecule encoding the transaminase mutant according to the first aspect, the nucleotide sequence of which is obtained by base mutation of the sequence shown in SEQ ID NO. 2.
In a third aspect, the present invention provides an expression vector comprising a nucleotide sequence as set forth in the second aspect. The expression vector may be a PET series expression vector.
In a fourth aspect, the present invention provides a recombinant cell comprising an expression vector according to the third aspect, and a method of constructing the same. The construction method of the recombinant cell comprises the following steps:
(1) Constructing an expression vector: ligating the nucleic acid molecule according to the second aspect to the digested plasmid to obtain the expression vector according to the third aspect;
(2) Construction of recombinant cells: transferring the constructed expression vector into competent cells of escherichia coli, culturing and screening to obtain recombinant cells.
In a fifth aspect, the present invention provides a product comprising a transaminase mutant according to the first aspect or a nucleic acid molecule according to the second aspect or an expression vector according to the third aspect or a recombinant cell according to the fourth aspect, and uses thereof. Such applications include the catalysis of the conversion of chroman-4-ones to (S) -chroman-4-amines. The reaction conditions used include a reaction at a temperature of 25 to 45℃and a pH of 9.0 to 11.5 for 12 hours.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
1. the invention provides a transaminase mutant, which can specifically recognize chroman-4-one by a substrate, and can efficiently synthesize (S) -chroman-4-amine, so that the stereoselectivity and the yield are obviously improved;
2. the invention provides a product for preparing (S) -chroman-4-amine, which comprises aminotransferase mutants with high substrate specificity, stereoselectivity and conversion efficiency, or encoding genes thereof, corresponding expression vectors or corresponding recombinant cells, and the temperature condition and pH value condition of the product application are easy to realize, so that the product has an industrialized application basis;
3. the invention provides a preparation method and application of a transaminase mutant, which have wide application prospects in industrial production and application of (S) -chroman-4-amine.
Drawings
FIG. 1 is a graph showing the effect of the yields and stereoselectivity of wild-type transaminases and mutants.
FIG. 2 is a diagram showing the reaction of a transaminase to catalyze the conversion of chroman-4-one to (S) -chroman-4-amine.
FIG. 3 is a graph showing the effect of the yield and stereoselective relative activity of transaminase mutants (C60deg.W+V240A+L272M) at different temperatures.
FIG. 4 is a graph showing the effect of the productivity and stereoselectivity of transaminase mutants (C60deg.W+V240A+L272M) at different pH values.
Detailed Description
Features and exemplary embodiments of various aspects of the invention are described in detail below. 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 application. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present application 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 application. It will be understood by those skilled in the art that the present application 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 application.
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 should be understood to include systematic errors unavoidable in industrial production.
In all discussions herein, standard single letter codes for amino acids are used. Standard substitution notation is also used, i.e. C60W means that the cysteine (C) at position 60 at the N-terminus is replaced by tryptophan (W). C60deg W+V1204A means that cysteine (C) at position 60 at the N-terminus is replaced by tryptophan (W), and valine (V) at position 242 at the N-terminus is replaced by alanine (A).
The term "wild-type" refers to a gene or gene product that is isolated from a naturally occurring source. Wild-type genes are the most commonly observed genes in a population and are therefore arbitrarily designed as "normal" or "wild-type" forms of the genes. Conversely, the term "modified," "mutant" or "variant" refers to a gene or gene product that exhibits a modification (e.g., substitution, truncation, or insertion) of a sequence, post-translational modification, and/or functional property (e.g., altered property) as compared to the wild-type gene or gene product. Note that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered properties compared to the wild-type gene or gene product. Methods for introducing or substituting naturally, non-naturally occurring amino acids are well known in the art.
Wild type transaminase
Transaminases catalyze the transfer of amino groups between amino acids and keto acids, including the conversion of chroman-4-ones to (S) -chroman-4-amines. The invention obtains wild aminotransferase (PDB: 5G2P_A. Nucleotide sequence is shown in a sequence table SEQ ID NO: 1) by amplifying and expressing aminotransferase gene AsTA (the nucleotide sequence is shown in the sequence table SEQ ID NO: 2) from Arthrobacter Arthrobactersp in vitro.
Transaminase mutants
The transaminase gene is subjected to directed evolution transformation by utilizing rational design, so that a transaminase mutant with high substrate specificity, stereoselectivity and conversion efficiency in preparation of (S) -chroman-4-amine is finally obtained, and the transaminase mutant can be used for synthesizing (S) -chroman-4-amine by a biological enzyme method. The invention utilizes the reported sequence and structure information of aminotransferase to select some potential enzyme genes by non-redundant search in NCBI and other databases according to the principles of protein structural similarity, conserved site analysis, host source diversity and the like. These genes are functionally expressed in E.coli expression systems and subsequently purified to give transaminase mutants.
Both the wild-type transaminase and the mutant of the present invention have the ability to catalyze the conversion of chroman-4-one to (S) -chroman-4-amine, the reaction process is shown in FIG. 2.
Examples
Example 1 sequence of transaminase mutants
In the present invention, mutation of a specific site was performed on the amino acid sequence (SEQ ID NO: 1) of a wild-type aminotransferase (WT) to obtain a plurality of mutants having remarkably improved stereoselectivity and productivity. Any one mutation or random combination of C60W, V242A, L M is selected, and the constructed mutant is C60W, V242A, L272M, C W+V242A, C W+L272M, V242A+L272M, C W+V242A+L272M.
EXAMPLE 2 construction of transaminase mutant plasmids
1. Acquisition of pET28a-AsTA
The aminotransferase wild-type gene from Arthrobactersp was synthesized by Jin Weizhi (su zhou) and constructed on pET28a vector, and the vector was transformed into e.coli dh5α strain, and recombinant e.coli dh5α/pET28a-AsTA was inoculated into a medium-filled 5mL tube and cultured with shaking at 37 ℃ at 220rpm for 12 hours. After the culture, the cells were centrifuged at 12,000rpm for 1min and the cells were collected, and plasmid was extracted from E.coli DH 5. Alpha./pET 28a-AsTA using a high purity plasmid miniprep kit as a template for iterative mutation, to construct plasmid of pET28a-AsTA mutant.
2. Construction of recombinant E.coli BL21 (DE 3)/pET 28a-AsTA mutant
The gene mutation adopts a full plasmid PCR method to obtain the target mutant gene, a C60W is taken as an example to specifically design a required primer, and other mutants are designed by adopting the principle and carry out single-point iterative mutation.
C60W upstream primer: CAATCAGCTGTGGTGTGTGAATCTGGG A
C60W downstream primer: CCCAGATTCACACACCACAGCTGATTG A
The PCR system is shown in Table 1:
TABLE 1PCR reaction System
The PCR reaction conditions are shown in Table 2:
TABLE 2PCR reaction conditions
After the PCR amplification is finished, the amplified products are detected by 0.9% agarose gel electrophoresis, and the result shows that the amplified products are single bands with the sizes of about 6000 bp. And purifying and recovering the amplified product by using a DNA recovery and purification kit.
The purified gene fragment was digested with dpnl to remove the template and then recombined with recombinase. The recombinant product is transformed into E.collDH5α competent cells, coated on the surface of LB solid medium containing 30 μg/mL kanamycin sulfate, cultured for 12h at 37 ℃, single colony is picked up to LB liquid culture, positive transformants successfully constructed are identified by PCR method, and the correctness of mutation sites is verified by sequencing. After verification, part of the sterile glycerol with the final concentration of 25% is added, numbering is carried out, the sterile glycerol is preserved at-80 ℃ for standby, and part of the bacteria are extracted with a plasmid extraction kit, and the recombinant plasmid is preserved in a refrigerator at-20 ℃.
And transferring the recombinant expression plasmid pET28a-AsTA which is sequenced successfully into E.coliBL21 (DE 3) as an expression host to construct a recombinant mutant expression strain E.coliBL21 (DE 3)/pET 28a-AsTA.
EXAMPLE 3 cultivation of transaminase mutants and preparation of crude enzyme solutions
The successfully constructed recombinant mutant expression strain E.coliBL21 (DE 3)/pET 28a-AsTA is coated on a kanamycin sulfate plate with a final concentration of 30 mug/mL, single colonies are selected to be inoculated in 5mL of resistant LB medium and cultured at 37 ℃ for overnight at 200rpm/min, 1% of the inoculum size is transferred into 500mL of resistant LB medium, IPTG with a final concentration of 0.5mM is added when OD 600 reaches about 0.6, and induction is carried out for about 14 hours at 18 ℃.
After centrifugation to obtain cells, the cells were resuspended in buffer, sonicated in an ice bath (2 s apart, 5s apart, 30min on time) and centrifuged at 12,000rpm/min for 20min at 4 ℃. Collecting supernatant, and filtering with 0.22 μm water filter head to obtain crude enzyme solution.
EXAMPLE 4 catalytic preparation of (S) -chroman-4-amines by transaminases and mutants thereof
The corresponding engineering bacteria expressing transaminase and mutants thereof were cultured as constructed in examples 1-3 and the crude enzyme solution obtained was used as catalyst.
The reaction system is as follows: crude enzyme with OD 600 =40, 40mM chroman-4-one, 1M isopropyl amine (IPA) 0.5mM pyridoxal phosphate (PLP), 100mM ph=9.0 Tris-HCl buffer. The reaction temperature was controlled to 30℃by water bath, and the reaction was carried out under magnetic stirring for 12 hours, and the yield and stereoselectivity of the (S) -chroman-4-amine synthesized from the transaminase and its mutant were examined by liquid chromatograph, and the results are shown in Table 3 and FIG. 1.
TABLE 3 catalytic preparation of (S) -chroman-4-amines by aminotransferase and mutants thereof
| Strain | Cell concentration | Yield rate | Stereoselectivity of |
| WT | OD600＝40 | 9％ | 89％ |
| C60W | OD600＝40 | 51％ | 94％ |
| V242A | OD600＝40 | 16％ | 92％ |
| L272M | OD600＝40 | 27％ | 93％ |
| C60W+V242A | OD600＝40 | 59％ | 95％ |
| C60W+L272M | OD600＝40 | 65％ | 96％ |
| V242A+L272M | OD600＝40 | 38％ | 95％ |
| C60W+V242A+L272M | OD600＝40 | 94％ | 99％ |
EXAMPLE 5 optimal temperature for the catalytic preparation of (S) -chroman-4-amine by the transaminase mutant (C60deg.W+V240A+L272M)
The corresponding engineering bacteria expressing transaminase and mutants thereof were cultured as constructed in examples 1-3 and the crude enzyme solution obtained was used as catalyst.
The reaction system is as follows: crude enzyme with OD 600 =40, 40mM chroman-4-one, 1M isopropyl amine (IPA) 0.5mM pyridoxal phosphate (PLP), 100mM ph=9.0 Tris-HCl buffer. The reaction temperature was controlled to 25℃at 30℃at 35℃at 40℃at 45℃by water bath, and the magnetic stirring was carried out for 12 hours, and the yield and stereoselectivity of (S) -chroman-4-amine synthesized from transaminase and its mutant were examined by liquid chromatograph, and the results are shown in FIG. 3.
Experiments prove that different temperatures have a remarkable effect on the catalytic action of the transaminase mutant, and the reactivity of the transaminase mutant shows a tendency to be in a normal state along with the change of the temperature. At 30℃the best catalytic effect is observed, whereas below or above this temperature the enzyme activity is relatively low.
EXAMPLE 6 catalytic preparation of (S) -chroman-4-amine by transaminase mutant (C60deg.W+V240A+L272M)
The corresponding engineering bacteria expressing transaminase and mutants thereof were cultured as constructed in examples 1-3 and the crude enzyme solution obtained was used as catalyst.
The reaction system is as follows: crude enzyme with OD 600 =40, 40mM chroman-4-one, 1M Isopropylamine (IPA) 0.5mM pyridoxal phosphate (PLP), tris-HCl buffer with 100mM ph=9.0, tris-HCl buffer with 100mM ph=10, tris-HCl buffer with 100mM ph=10.5, tris-HCl buffer with 100mM ph=11, boric acid buffer with 100mM ph=11.5. The reaction temperature was controlled to 30℃by water bath, and the reaction was carried out under magnetic stirring for 12 hours, and the yield and stereoselectivity of the (S) -chroman-4-amine synthesized from the transaminase and its mutant were examined by liquid chromatograph, and the results are shown in FIG. 4.
Experiments prove that different pH values have a remarkable influence on the catalysis of the transaminase mutants, and the reactivity of the transaminase mutants shows a tendency to be in a normal state along with the change of the pH values. At a pH of 9.0, the best catalytic effect was observed and the enzyme activity decreased under alkaline conditions.
Claims (10)
1. A transaminase mutant is characterized in that the amino acid sequence of the transaminase mutant is obtained by mutating at least one of the sequences shown in SEQ ID NO. 1 and C60W, V242A, L272M.
2. A nucleic acid molecule encoding the transaminase mutant of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule is obtained by base mutation of the sequence shown in SEQ ID NO. 2.
4. An expression vector comprising the nucleotide sequence of claim 3.
5. The expression vector of claim 4, wherein the expression vector is a PET series expression vector.
6. A recombinant cell comprising the expression vector of claim 4.
7. A method of constructing a recombinant cell according to claim 6, comprising the steps of:
(1) Constructing an expression vector: ligating the nucleic acid molecule of claim 2 to the digested plasmid to obtain the expression vector of claim 4;
(2) Construction of recombinant cells: transferring the constructed expression vector into competent cells of escherichia coli, culturing and screening to obtain the recombinant cells of claim 6.
8. A product comprising the transaminase mutant of claim 1 or the nucleic acid molecule of any one of claims 2 to 3 or the expression vector of claim 4 or the recombinant cell of claim 6.
9. Use of the product of claim 8 for catalyzing the conversion of chroman-4-one to (S) -chroman-4-amine.
10. The use according to claim 9, wherein the reaction conditions of the use comprise a reaction at a temperature of 25 to 45 ℃ and a pH of 9.0 to 11.5 of 12 h.
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