A transaminase mutant and its application in the preparation of (S)-chroman-4-amine

Abstract

The invention discloses a transaminase mutant and its application in the preparation of (S)-chroman-4-amine, the transaminase mutant is obtained by directed evolution of wild-type transaminase, including any one of C60W, V242A, and L272M mutations or a combination thereof. The transaminase mutant disclosed in the present invention can substrate-specifically identify chroman-4-one, efficiently synthesize (S)-chroman-4-amine, and achieve a significant increase in stereoselectivity and yield; the invention discloses a product for preparing (S)-chroman-4-amine, the product including a transaminase mutant or its encoding gene or corresponding expression vector or corresponding recombinant cell with high yield and high stereoselectivity, the temperature conditions and pH conditions of the product application are easy to achieve, with an industrial application basis; the invention also discloses a preparation method of the mutant and its application, which contributes to the industrial production and application of (S)-chroman-4-amine.

Description

Technical Field

[0001] The present invention relates to an enzyme mutant, in particular to a transaminase mutant and application thereof in the preparation of (S)-chroman-4-amine, belonging to the technical fields of genetic engineering and enzyme engineering. Background Art

[0002] Transaminases are a class of enzymes that catalyze the transfer of amino groups between amino acids and keto acids and are commonly found in animals, plant tissues, and microorganisms. Transaminases are widely used in regioselective and stereoselective transamination reactions of substrates such as aliphatic and aromatic keto acids, aldehydes, ketones, and ketoses to synthesize chiral amines, amino acids, and their derivatives. In industrial production, many pharmaceuticals have developed processes using transaminases to prepare APIs or their intermediates, such as the anti-type 2 diabetes drug sitagliptin and the migraine drugs remegipam and ubegiritam. Transaminases are key biotechnological enzymes in the asymmetric synthesis of chiral amines and the resolution of racemic amines.

[0003] Chiral amines are compounds with amino groups attached to their chiral centers. They play important roles in a variety of biological processes, including genetics and metabolism. As widely occurring structural units in nature, chiral amines possess diverse biological activities and are used in the synthesis of numerous drugs and intermediates, including neurological drugs, antihypertensive drugs, and anti-infective drugs. According to statistics, approximately 40% of new drugs approved by the US Food and Drug Administration in recent years contain chiral amine structures. Because chiral amine drugs of different chiralities exhibit significant differences in drug safety and human metabolism, the efficient and stereoselective synthesis of single chiral amine drugs is of great research significance in pharmaceutical innovation and production.

[0004] (S)-4-chromanamine (US2004 / 157739) is a chiral amine that serves as a key intermediate in the synthesis and production of various drugs, such as the antihypertensive drug Cromakalim, a potassium channel agonist used to treat hypertension and cardiovascular and cerebrovascular diseases. (S)-4-chromanamine is a colorless solid at room temperature and pressure with a molecular weight of 149.19 and is soluble in solvents such as methanol and dimethyl sulfoxide. (S)-4-chromanamine can be synthesized using a kinetic resolution method, where racemic 4-chromanamine is derivatized and then hydrolyzed to obtain chirally pure (S)-4-chromanamine. However, this method is cumbersome and the actual yield does not exceed 50%, making it of low industrial application value. The biosynthetic method, using chroman-4-one as a starting material and catalyzed by transaminases, produces chirally pure (S)-chroman-4-amine. This method offers advantages such as mild reaction conditions, high yield, and high stereoselectivity. This reaction can transfer the amino group of inexpensive amino donors (D / L-alanine and isopropylamine) to chroman-4-one, producing easily separable byproducts (pyruvate and acetone), promising promising industrial applications. However, the production of (S)-chroman-4-amine using wild-type transaminases is limited by substrate specificity, stereoselectivity, and catalytic efficiency, thus restricting its industrial production and application. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a transaminase mutant with high substrate specificity, stereoselectivity and conversion efficiency, and to provide the application of the transaminase mutant in the preparation of (S)-chroman-4-amine.

[0006] Technical solution: In the first aspect, the present invention provides a transaminase mutant, wherein the amino acid sequence of the transaminase mutant is obtained by subjecting the sequence shown in SEQ ID NO: 1 to at least one mutation among C60W, V242A, and L272M.

[0007] In a second aspect, the present invention provides a nucleic acid molecule encoding the transaminase mutant as described in the first aspect, wherein the nucleotide sequence of the nucleic acid molecule is obtained by base mutation of the sequence shown in SEQ ID NO: 2.

[0008] In a third aspect, the present invention provides an expression vector comprising the nucleotide sequence described in the second aspect. The expression vector may be a PET series expression vector.

[0009] In a fourth aspect, the present invention provides a recombinant cell and a method for constructing the same, wherein the recombinant cell comprises the expression vector described in the third aspect. The method for constructing the recombinant cell comprises the following steps:

[0010] (1) constructing an expression vector: ligating the nucleic acid molecule described in the second aspect with the enzyme-digested plasmid to obtain the expression vector described in the third aspect;

[0011] (2) Construction of recombinant cells: The constructed expression vector is transferred into competent Escherichia coli cells, and the recombinant cells are cultured and screened to obtain the recombinant cells.

[0012] In a fifth aspect, the present invention provides a product and its use, comprising the transaminase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. The use comprises catalyzing the conversion of chroman-4-one to (S)-chroman-4-amine. The reaction conditions for the use comprise a temperature of 25-45° C. and a pH of 9.0-11.5, and a reaction time of 12 hours.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0014] 1. The present invention provides a transaminase mutant that can substrate-specifically recognize chroman-4-one and efficiently synthesize (S)-chroman-4-amine, achieving significantly improved stereoselectivity and yield;

[0015] 2. The present invention provides a product for preparing (S)-chroman-4-amine, comprising a transaminase mutant or its encoding gene, or a corresponding expression vector, or a corresponding recombinant cell having high substrate specificity, stereoselectivity, and conversion efficiency. The temperature and pH conditions for application of the product are easy to achieve, and the product has a basis for industrial application.

[0016] 3. The present invention provides a method for preparing a transaminase mutant and its application, which has broad application prospects in the industrial production and application of (S)-chroman-4-amine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The graph shows the effect of yield and stereoselectivity of wild-type transaminase and mutants.

[0018] Figure 2 The invention relates to the reaction process of converting chroman-4-one into (S)-chroman-4-amine by transaminase.

[0019] Figure 3 This is a graph showing the yield and stereoselectivity of the transaminase mutant (C60W+V242A+L272M) at different temperatures.

[0020] Figure 4This is a diagram showing the yield and stereoselectivity of the transaminase mutant (C60W+V242A+L272M) at different pH values. DETAILED DESCRIPTION

[0021] The features and exemplary embodiments of various aspects of the present invention will be described in detail below. The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below.

[0022] For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should all be encompassed within the scope of the present application. Where specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can be implemented equally without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main idea of ​​the present application.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in this invention should be understood to include the inevitable systematic errors in industrial production.

[0024] In all discussions herein, the 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). C60W+V242A means that the cysteine ​​(C) at position 60 at the N-terminus is replaced by tryptophan (W), and the valine (V) at position 242 at the N-terminus is replaced by alanine (A).

[0025] The term "wild-type" refers to a gene or gene product that has been isolated from a naturally occurring source. A wild-type gene is the most commonly observed gene in a population and is therefore arbitrarily designed to be the "normal" or "wild-type" form of a gene. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, truncations, or insertions), post-translational modifications, and / or functional properties (e.g., altered properties) 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 natural, non-naturally occurring amino acids are well known in the art.

[0026] Wild-type transaminase

[0027] Transaminases catalyze the transfer of amino groups between amino acids and keto acids, including the conversion of chroman-4-one to (S)-chroman-4-amine. The present invention obtains a wild-type transaminase (PDB: 5G2P_A; amino acid sequence: SEQ ID NO: 1) by in vitro amplification and expression of the transaminase gene AsTA from Arthrobacter sp (nucleotide sequence: SEQ ID NO: 2).

[0028] transaminase mutants

[0029] Using rational design, directed evolution of the transaminase gene was performed, ultimately yielding a transaminase mutant with high substrate specificity, stereoselectivity, and conversion efficiency in the preparation of (S)-chroman-4-amine. This mutant can be used for the enzymatic synthesis of (S)-chroman-4-amine. The present invention utilizes publicly available transaminase sequence and structural information, screening potential enzyme genes through non-redundant searches in databases such as NCBI. These genes were then functionally expressed in an Escherichia coli expression system and subsequently purified to yield the transaminase mutants.

[0030] 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. Figure 2 .

[0031] Example

[0032] Example 1 Sequences of transaminase mutants

[0033] In the present invention, specific site mutations were performed on the wild-type transaminase (WT) amino acid sequence (SEQ ID NO: 1) to obtain multiple mutants with significantly improved stereoselectivity and yield. The mutants constructed by selecting any one of the mutations C60W, V242A, and L272M, or a random combination thereof, were C60W, V242A, L272M, C60W+V242A, C60W+L272M, V242A+L272M, and C60W+V242A+L272M.

[0034] Example 2 Construction of transaminase mutant plasmid

[0035] 1. Obtaining pET28a-AsTA

[0036] The wild-type transaminase gene from Arthrobacter sp was synthesized by GenWeiZhi (Suzhou) and constructed on the pET28a vector. The vector was then transformed into the E. coli DH5α strain. The recombinant E. coli DH5α / pET28a-AsTA was inoculated into a 5 mL test tube of culture medium and cultured at 37°C, 220 rpm, and shaken for 12 hours. After the culture, the cells were centrifuged at 12,000 rpm for 1 minute and harvested. The plasmid was extracted from the E. coli DH5α / pET28a-AsTA using a high-purity plasmid miniprep kit and used as a template for iterative mutagenesis to construct the pET28a-AsTA mutant plasmid.

[0037] 2. Construction of recombinant E. coli BL21(DE3) / pET28a-AsTA mutant

[0038] The target mutant gene was obtained by whole-plasmid PCR. The primers required were specifically designed using C60W as an example. Other mutants were designed using this principle and single-point iterative mutagenesis was performed.

[0039] C60W upstream primer: CAATCAGCTG TGG TGTGTGAATCTGGG

[0040] C60W downstream primer: CCCAGATTCACACA CCA CAGCTGATTG

[0041] PCR system is shown in Table 1:

[0042] Table 1 PCR reaction system

[0043]

[0044]

[0045] PCR reaction conditions are shown in Table 2:

[0046] Table 2 PCR reaction conditions

[0047]

[0048] After the PCR amplification was completed, the amplified product was detected by 0.9% agarose gel electrophoresis, and the results showed that the amplified product was a single band with a size of about 6000 bp. The amplified product was purified and recovered using a DNA recovery and purification kit.

[0049] The purified gene fragments were digested with DpnI to remove the template and then recombined using a recombinase. The recombinant product was transformed into E. coli DH5α competent cells and plated onto the surface of LB solid medium containing 30 μg / mL kanamycin sulfate. The cells were incubated at 37°C for 12 hours, and single colonies were picked and transferred to LB liquid culture. Successful transformants were identified by PCR, and the correctness of the mutation site was verified by sequencing. After verification, a portion of the cells was added with sterile glycerol to a final concentration of 25%, numbered, and stored at -80°C until further use. A portion of the cells was used to extract the plasmid using a plasmid extraction kit, and the recombinant plasmids were stored at -20°C.

[0050] The recombinant expression plasmid pET28a-AsTA that was successfully sequenced was transformed into E. coli BL21 (DE3) as the expression host to construct the recombinant mutant expression strain E. coli BL21 (DE3) / pET28a-AsTA.

[0051] Example 3 Cultivation of transaminase mutants and preparation of crude enzyme solution

[0052] The recombinant mutant expression strain E. coli BL21 (DE3) / pET28a-AsTA was successfully constructed and spread onto a plate containing kanamycin sulfate at a final concentration of 30 μg / mL. A single colony was picked and inoculated into 5 mL of LB medium containing resistance and cultured overnight at 37°C at 200 rpm / min. 1% of the inoculum was transferred to 500 mL of LB medium containing resistance and the OD was 0. 600 When the concentration reaches about 0.6, add IPTG with a final concentration of 0.5 mM and induce at 18°C ​​for about 14 hours.

[0053] After centrifugation, resuspend the cells in buffer and disrupt them by ultrasonication in an ice bath (2-second on-time, 5-second interval, 30-minute on-time). Centrifuge at 12,000 rpm for 20 minutes at 4°C. Collect the supernatant and filter through a 0.22 μm aqueous filter to obtain the crude enzyme solution.

[0054] Example 4 Preparation of (S)-chroman-4-amine catalyzed by transaminase and its mutants

[0055] The corresponding engineered bacteria expressing transaminase and their mutants were cultured according to the construction of Examples 1-3 and the crude enzyme liquid obtained was used as a catalyst.

[0056] The reaction system is: OD 600 =40 crude enzyme solution, 40mM chroman-4-one, 1M isopropylamine (IPA), 0.5mM pyridoxal phosphate (PLP), and 100mM Tris-HCl buffer (pH = 9.0). The reaction temperature was controlled at 30°C in a water bath with magnetic stirring and the reaction was carried out for 12 hours. The yield and stereoselectivity of the transaminase and its mutants in synthesizing (S)-chroman-4-amine were determined by liquid chromatography. The results are shown in Table 3 and Figure 1 .

[0057] Table 3 Preparation of (S)-chroman-4-amine catalyzed by transaminase and its mutants

[0058] strain Cell concentration Yield Stereoselectivity WT <![CDATA[OD 600 =40]]> 9％ 89％ C60W <![CDATA[OD 600 =40]]> 51％ 94％ V242A <![CDATA[OD 600 =40]]> 16％ 92％ L272M <![CDATA[OD 600 =40]]> 27％ 93％ C60W+V242A <![CDATA[OD 600 =40]]> 59％ 95％ C60W+L272M <![CDATA[OD 600 =40]]> 65％ 96％ V242A+L272M <![CDATA[OD 600 =40]]> 38％ 95％ C60W+V242A+L272M <![CDATA[OD 600 =40]]> 94％ 99％

[0059] Example 5 Optimum Temperature for Preparation of (S)-chroman-4-amine Catalyzed by Transaminase Mutant (C60W+V242A+L272M)

[0060] The corresponding engineered bacteria expressing transaminase and their mutants were cultured according to the construction of Examples 1-3 and the crude enzyme liquid obtained was used as a catalyst.

[0061] The reaction system is: OD 600 =40 crude enzyme solution, 40mM chroman-4-one, 1M isopropylamine (IPA), 0.5mM pyridoxal phosphate (PLP), and 100mM Tris-HCl buffer (pH = 9.0). The reaction temperature was controlled at 25°C, 30°C, 35°C, 40°C, and 45°C in a water bath with magnetic stirring. The reaction was carried out for 12 hours. The yield and stereoselectivity of the transaminase and its mutants in synthesizing (S)-chroman-4-amine were determined by liquid chromatography. The results are shown in FIG. Figure 3 .

[0062] Experiments have shown that different temperatures significantly affect the catalytic activity of the transaminase mutant, with its reaction activity showing a slightly normal trend with temperature. The optimal catalytic effect was observed at 30°C, while enzyme activity was relatively low at temperatures below or above this temperature.

[0063] Example 6 Optimal pH for the preparation of (S)-chroman-4-amine catalyzed by the transaminase mutant (C60W+V242A+L272M)

[0064] The corresponding engineered bacteria expressing transaminase and their mutants were cultured according to the construction of Examples 1-3 and the crude enzyme liquid obtained was used as a catalyst.

[0065] The reaction system is: OD 600 =40 crude enzyme solution, 40mM chroman-4-one, 1M isopropylamine (IPA) and 0.5mM pyridoxal phosphate (PLP). The reaction buffers were 100mM Tris-HCl buffer at pH = 9.0, 100mM Tris-HCl buffer at pH = 10, 100mM Tris-HCl buffer at pH = 10.5, 100mM Tris-HCl buffer at pH = 11, and 100mM boric acid buffer at pH = 11.5. The reaction temperature was controlled at 30°C in a water bath with magnetic stirring. The reaction was allowed to proceed for 12 hours. The yield and stereoselectivity of the transaminase and its mutants in synthesizing (S)-chroman-4-amine were determined by liquid chromatography. The results are shown in Table 1. Figure 4 .

[0066] Experiments have shown that different pH levels significantly affect the catalytic activity of the transaminase mutants, with their reactivity exhibiting a positive trend with changes in pH. The optimal catalytic effect was observed at pH 9.0, while enzyme activity decreased under alkaline conditions.

Claims

1. A transaminase mutant, characterized in that: The amino acid sequence of the transaminase mutant is obtained by mutation of SEQ ID NO: 1, and the mutation is C60W+V242A+L272M.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the transaminase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that 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, characterized in that The expression vector comprises the nucleotide sequence of claim 3.

5. The expression vector according to claim 4, characterized in that The expression vector is a PET series expression vector.

6. A recombinant cell, characterized in that The recombinant cell comprises the expression vector according to claim 4.

7. A method for constructing the recombinant cell according to claim 6, characterized in that: The steps include: (1) Construction of an expression vector: ligating the nucleic acid molecule of claim 2 with the enzyme-digested plasmid to obtain the expression vector of claim 4; (2) Construction of recombinant cells: The constructed expression vector is transferred into competent Escherichia coli cells, and the recombinant cells according to claim 6 are obtained by culture and screening.

8. A product catalyzing the conversion of chroman-4-one to (S)-chroman-4-amine, characterized in that: The product comprises the transaminase mutant according to claim 1 or the nucleic acid molecule according to any one of claims 2 to 3 or the expression vector according to claim 4 or the recombinant cell according to claim 6.

9. Use of the product according to claim 8 in catalyzing the conversion of chroman-4-one into (S)-chroman-4-amine.

10. The use according to claim 9, characterized in that The applied reaction conditions include reacting for 12 hours at a temperature of 25 to 45° C. and a pH of 9.0 to 11.5.

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