Transaminase mutants and uses thereof

Abstract

Provided is a transaminase mutant and its use, which has a sequence in which amino acid mutations have occurred in the sequence shown in SEQ ID NO: 1, and the site of amino acid mutation includes the V242W site. The transaminase mutant is based on the transaminase shown in SEQ ID NO: 1, and is mutated by a site-directed mutagenesis method to change the amino acid sequence and protein structure and function, and then a transaminase having the above mutation site is obtained by a directed screening method, and the obtained transaminase has high catalytic activity, specific selectivity, and a wide substrate spectrum, and is expected to be industrially applicable.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and in particular to transaminase mutants and uses thereof. [Background technology]

[0002] Chiral amine compounds are important intermediates for chiral drugs and are widely used in pharmaceuticals and other fields. Their industrial production primarily relies on transition-metal-catalyzed asymmetric synthesis. However, this process requires expensive transition-metal complexes as catalysts, and these transition metals are limited and expensive, making this approach unsustainable. Bioenzyme-catalyzed methods have attracted considerable attention due to the readily available ketone substrates, mild reaction conditions, and high product selectivity.

[0003] Transaminases (TA, EC2.6.1.X), also known as aminotransferases, can reversibly catalyze the transamination reaction between ketone and amino groups. Transaminases, in particular, have excellent stereoselectivity, reproducible cofactors, strong reactivity, and environmentally friendly properties, making them useful for the biocatalytic production of chiral amines and widely used in the synthesis of pharmaceutical and agrochemical intermediates.

[0004] Transaminase reactions typically require pyridoxal phosphate as a coenzyme, covalently bound to the ε-amino group of the lysine residue at the active center of the transaminase, but also require an amino donor to participate in the reaction; commonly used amino donors include isopropylamine and phenylethylamine.

[0005] Although the use of transaminases for the production of chiral amines has attracted considerable attention, enzymatic methods have many problems for scale-up production. For example, existing transaminases have low selectivity and low conversion rates due to the existence of reversible reactions, which are disadvantageous for industrial production. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide transaminase mutants and uses thereof that improve the selectivity of transaminases. [Means for solving the problem]

[0007] To achieve the above object, one aspect of the present invention provides a transaminase mutant, which has a sequence in which an amino acid mutation has occurred in the sequence shown in SEQ ID NO: 1, and the site of the amino acid mutation includes the V242W site, or the transaminase mutant has an amino acid sequence that includes the above mutation site and has 80% or more, preferably 90% or more, and more preferably 95% or more identity with the amino acid sequence having the mutation site, or the amino acid sequence of the transaminase mutant has 90% or more identity with the sequence shown in SEQ ID NO: 1 and has transaminase catalytic activity.

[0008] Furthermore, the sites where amino acid mutations occurred were V242W+L59Q, V242W+F164C, V242W+F164Q, V242W+F164W, V242W+F164Y, V242W+L272G, V242W+L272I, V242W+L272K, V242W+L272M, V242W+L272P, V242W+L272V, V242W+L272Y, V242W+V328C, V242W+V328I, V242W+V328L, V242W+V328M, V242W+V328Q, V242W+V328S, and V242W+V328T. , V242W+V328W, V242W+T330F, V242W+T330I, V242W+T330S, V242W+A436H, V242W+A436K, V242W+A436L, V242W+A436N, V242W+A436P, V242W+A436Q, V2 42W+A436S, V242W+A436Y, V242W+R442A, V242W+R442C, V242W+R442F, V242 W+R442G, V242W+R442H, V242W+R442N, V242W+R442Q, V242W+R442S, V242W+ R442T, V242W+F164Q+V328A, V242W+F164Q+V328C, V242W+F164Q+V328D, V 242W+F164Q+V328E, V242W+F164Q+V328F, V242W+F164Q+V328G, V242W+F16 4Q+V328H, V242W+F164Q+V328I, V242W+F164Q+V328L, V242W+F164Q+V328M , V242W+F164Q+V328P, V242W+F164Q+V328Q, V242W+F164Q+V328R, V242W+F 164Q+V328S, V242W+F164Q+V328W, V242W+F164Q+V328T, V242W+F164Q+V3 28Y, V242W+F164Q+R442T, V242W+F164Q+V328I+G2S, V242W+F164Q+V328I+ T46M, V242W+F164Q+V328I+G48D, V242W+F164Q+V328I+C185Y, V242W+F164 Q+V328I+S186N, V242W+F164Q+V328I+S194P, V242W+F164Q+V328I+T197M,V242W+F164Q+V328I+N202D, V242W+F164Q+V328I+Y205L, V242W+F164Q+V328I+T24 5A, V242W+F164Q+V328I+V252I, V242W+F164Q+V328I+S268N, V242W+F164Q+V328I+ L353F, V242W+F164Q+V328I+N359D, V242W+F164Q+V328I+ R409T, V242W+F164Q+V328I+E424K, V242W+F164Q+V328I+A436V, V242W+F164Q+V328I+R442T, V242W+F164Q+V328I+R442T+G48D, V 242W+F164Q+V328I+R442T+S194P, V242W+F164Q+V328I+R442T+V252I, V242W+F164Q+V328I+R442T+S194P+V252I, V242W+F164Q+V The transaminase mutant may contain any of the mutation sites in the combinations of 328I+R442T+V252I+G48D, V242W+F164Q+V328I+R442T+S194P+G48D, or V242W+F164Q+V328I+R442T+S194P+V252I+G48D, or the transaminase mutant may contain the above mutation site and have an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more identity to the amino acid sequence having the mutation site.

[0009] According to another aspect of the present invention, there is provided a DNA molecule encoding any of the transaminase mutants described above.

[0010] According to a further aspect of the present invention there is provided a recombinant plasmid which contains any of the DNA molecules described above.

[0011] Furthermore, the recombinant plasmids are pET-22a(+), 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-23a(+), 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(+), pE T-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, p QE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18, or pUC-19.

[0012] According to another aspect of the present invention, there is provided a host cell, which contains any of the recombinant plasmids described above.

[0013] Furthermore, the host cell includes a prokaryotic cell or a eukaryotic cell, preferably the prokaryotic cell is a BL21-DE3 cell or an E. coli Rosetta-DE3 cell, and the eukaryotic cell is a yeast cell.

[0014] According to a further aspect of the present invention, there is provided a method for producing a chiral amine, the method comprising catalyzing a transamination reaction of a ketone compound and an amino donor with a transaminase, wherein the transaminase is any of the transaminase mutants described above.

[0015] Furthermore, ketone compounds include [ka] (n=0, 1, 2 or 3, X=C, N, O or S, R=H, F, Cl, Br, CH3 or CH2CH3), and preferably, the ketone compound is [ka] is.

[0016] Further, the amino donor is isopropylamine or alanine, preferably isopropylamine.

[0017] Furthermore, in a reaction system in which the transamination reaction of a ketone compound and an amino donor is catalyzed by a transaminase, the amount of the enzyme used is 1.5 to 6.6 mg / mL, preferably 1.7 mg / mL, and the temperature of the reaction system in which the transamination reaction of a ketone compound and an amino donor is catalyzed by a transaminase is preferably 20°C to 45°C, more preferably 30°C. [Effects of the Invention]

[0018] The above transaminase mutants of the present invention are based on the transaminase shown in SEQ ID NO: 1, and are mutated by site-directed mutagenesis to change the amino acid sequence, protein structure, and function. Then, transaminases having the above mutation sites are obtained by directed screening. The transaminases obtained in the present invention have high catalytic activity, specific selectivity, and a wide substrate spectrum, and are expected to be industrially applicable. DETAILED DESCRIPTION OF THE INVENTION

[0019] It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without contradiction. The present invention will be described in detail below with reference to the embodiments.

[0020] Transaminases are biocatalysts whose main component is protein, and the reactions they catalyze may be represented by the following reaction scheme: [ka] (n=0, 1, 2 or 3, X=C, N, O or S, R=H, F, Cl, Br, CH3 or CH2CH3) Conventional transaminases have poor selectivity and reversibility, which reduces the conversion rate, making them unsuitable for industrial production. In view of this technical problem, the present invention aims to improve the selectivity and activity of transaminase based on the transaminase shown in SEQ ID NO: 1 through directed evolution, thereby obtaining a transaminase with high catalytic activity and specific selectivity.

[0021] First, mutation sites are introduced into the transaminase by site-directed mutagenesis, and the selectivity of the mutants is detected. Mutants with improved selectivity are selected. Here, mutant V242W has approximately two-fold higher selectivity than the starting template, but its activity is inferior. Subsequently, mutations are continued using V242W as a template to obtain mutants with even more significant improvements in selectivity and activity.

[0022] Site-directed mutagenesis refers to the introduction of base changes or fragment insertions or deletions at specific sites in a DNA fragment (genome or plasmid) using methods such as polymerase chain reaction (PCR). Site-directed mutagenesis can rapidly and efficiently improve the properties and characterization of target proteins expressed by DNA, making it a very useful tool in genetic research.

[0023] The use of whole-plasmid PCR to introduce single or multiple site-specific mutations has the advantages of simplicity and efficiency. The principle is as follows: a pair of primers (forward and reverse) containing the mutation site is annealed to the template plasmid, followed by "cycle extension" using a polymerase. This cycle extension refers to the polymerase extending the primer along the template, returning to the 5' end of the primer after one cycle, and then repeating the cycle of heating, annealing, and extension. Unlike rolling circle amplification, this reaction does not produce multiple tandem copies. The extension products of the forward and reverse primers are annealed and paired to form a nicked open-circular plasmid. When the extension product is digested with Dpn I, the original template plasmid, derived from conventional E. coli and modified by Dam methylation, is sensitive to Dpn I and cleaved. However, the in vitro-synthesized plasmid containing the mutated sequence is unmethylated and therefore uncleaved. Subsequent transformation can then be successfully performed to obtain a clone of the mutant plasmid.

[0024] The mutant plasmid was transformed into E. coli host cells, and then the cells were disrupted by sonication to obtain crude enzyme for reaction verification. The optimal conditions for transaminase induction were 20°C, 0.06 mM IPTG, 16 h.

[0025] According to one exemplary embodiment of the present invention, there is provided a transaminase mutant, which has a sequence in which an amino acid mutation has occurred in the sequence shown in SEQ ID NO: 1, where the site of the amino acid mutation includes the V242W site, or the amino acid sequence of the transaminase mutant has the site of the amino acid mutation and has 90%, 95%, or 99% or more identity with the sequence shown in SEQ ID NO: 1, and has transaminase catalytic activity.

[0026] The term "homology" as used herein has the meaning generally known in the art, and the rules and criteria for determining homology between different sequences are also well known to those skilled in the art. Such variant sequences can be obtained by those skilled in the art based on the teachings of the present disclosure.

[0027] Preferably, the sites at which amino acid mutations occur include any of the following combination mutation sites: V242W+L59Q, V242W+F164C, V242W+F164Q, V242W+F164W, V242W+F164Y , V242W+L272G, V242W+L272I, V242W+L272K, V242W+L272M, V242W+L272 P, V242W+L272V, V242W+L272Y, V242W+V328C, V242W+V328I, V242W+V32 8L, V242W+V328M, V242W+V328Q, V242W+V328S, V242W+V328T, V242W+V3 28W, V242W+T330F, V242W+T330I, V242W+T330S, V242W+A436H, V242W+A 436K, V242W+A436L, V242W+A436N, V242W+A436P, V242W+A436Q, V242W+ A436S, V242W+A436Y, V242W+R442A, V242W+R442C, V242W+R442F, V242W +R442G, V242W+R442H, V242W+R442N, V242W+R442Q, V242W+R442S, V242 W+R442T, V242W+F164Q+V328A, V242W+F164Q+V328C, V242W+F164Q+V32 8D, V242W+F164Q+V328E, V242W+F164Q+V328F, V242W+F164Q+V328G, V2 42W+F164Q+V328H, V242W+F164Q+V328I, V242W+F164Q+V328L, V242W+F 164Q+V328M, V242W+F164Q+V328P, V242W+F164Q+V328Q, V242W+F164Q+ V328R, V242W+F164Q+V328S, V242W+F164Q+V328W, V242W+F164Q+V328T , V242W+F164Q+V328Y, V242W+F164Q+R442T, V242W+F164Q+V328I+G2S, V242W+F164Q+V328I+T46M, V242W+F164Q+V328I+G48D, V242W+F164Q+V 328I+C185Y, V242W+F164Q+V328I+S186N, V242W+F164Q+V328I+S194P,V242W+F164Q+V328I+T197M, V242W+F164Q+V328I+N202D, V242W+F164Q+V328I+Y205L, V242W+F164Q+V328I+T245A, V242W+F 164Q+V328I+V252I, V242W+F164Q+V328I+S268N, V242W+F164Q+V328I+L353F, V242W+F164Q+V328I+N359D, V242W+F164Q+V3 28I+R409T, V242W+F164Q+V328I+E424K, V242W+F164Q+V328I+A436V, V242W+F164Q+V328I+R442T, V242W+F164Q+V328I+R44 2T+G48D, V242W+F164Q+V328I+R442T+S194P, V242W+F164Q+V328I+R442T+V252I, V242W+F164Q+V328I+R442T+S194P+V252E V242W+F164Q+V328I+R442T+V252I+G48D, V242W+F164Q+V328I+R442T+S194P+G48D, or V242W+F164Q+V328I+R442T+S194P+V252I+G48D, The above transaminase mutants of the present invention are based on the transaminase shown in SEQ ID NO: 1, and are mutated by site-directed mutagenesis to change the amino acid sequence, protein structure, and function. Then, transaminases having the above mutation sites are obtained by directed screening. The transaminases obtained in the present invention have high catalytic activity, specific selectivity, and a wide substrate spectrum, and are expected to be industrially applicable.

[0028] According to one exemplary embodiment of the present invention, a DNA molecule is provided, which encodes a transaminase mutant with improved selectivity and activity, thereby reducing the amount of enzyme added and easing the difficulty of post-processing in industrial amino acid production.

[0029] The above-mentioned DNA molecules of the present invention may exist in the form of an "expression cassette." An "expression cassette" refers to a linear or circular nucleic acid molecule, including DNA and RNA sequences, capable of directing the expression of a specific nucleotide sequence in an appropriate host cell. It generally includes a promoter operatively linked to the target nucleotide, optionally linked to a terminator signal and / or other regulatory elements. An expression cassette may also include sequences necessary for the accurate translation of the nucleotide sequence. The coding region typically encodes a protein of interest, but may also encode a functional RNA of interest, such as an antisense or non-translated RNA, in either a sense or antisense orientation. An expression cassette containing a polynucleotide sequence of interest may be chimeric, meaning that at least one component thereof is heterologous to at least one other component. Expression cassettes may be naturally occurring or obtained by efficient recombinant formation for heterologous expression.

[0030] According to one exemplary embodiment of the present invention, there is provided a recombinant plasmid, which contains any of the above-mentioned DNA molecules, and the DNA molecules of the recombinant plasmid are positioned in appropriate positions within the recombinant plasmid so that the DNA molecules can be accurately and smoothly copied, transcribed, or expressed.

[0031] Although the term "containing" is used to define the DNA molecule of the present invention, it does not mean that other sequences unrelated to its function can be added to both ends of the DNA sequence. Those skilled in the art know that to meet the requirements of recombinant manipulation, it is necessary to add appropriate restriction enzyme cleavage sites to both ends of the DNA sequence, or to add additional initiation codons, termination codons, etc. Therefore, limiting the DNA molecule in a closed manner cannot realistically cover these situations.

[0032] The term "plasmid" as used herein includes any double-stranded or single-stranded, linear or circular, plasmid, cosmid, phage or Agrobacterium binary nucleic acid molecule, preferably a recombinant expression plasmid, which may be a prokaryotic or eukaryotic expression plasmid, with prokaryotic expression plasmids being preferred. In some embodiments, the vector used in the recombinant plasmid is selected from the following: pET-22a(+), 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-22b(+), pET-22c(+), pET-22d ... ), pET-23a(+), 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-4 3a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQ E40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18, or pUC-19 According to one exemplary embodiment of the present invention, a host cell containing any of the above-described recombinant plasmids is provided. Suitable host cells for the present invention include, but are not limited to, prokaryotic or eukaryotic cells. Preferably, the prokaryotic cell is a BL21-DE3 cell or an E. coli Rosetta-DE3 cell, and the eukaryotic cell is a yeast.

[0033] According to one exemplary embodiment of the present invention, there is provided a method for producing chiral amines, comprising catalyzing a transamination reaction of a ketone compound and an amino donor with a transaminase, wherein the transaminase is a transaminase mutant of the present invention. Preferably, the ketone compound is [ka] (n=0, 1, 2 or 3, X=C, N, O or S, R=H, F, Cl, Br, CH3 or CH2CH3).

[0034] In a reaction system in which the transamination reaction of a ketone compound and an amino donor is catalyzed by the transaminase of the present invention, the pH is 7 to 11, preferably 7.5. The temperature of the reaction system in which the transamination reaction of a ketone compound and an amino donor is catalyzed by the transaminase is 20 to 45°C, more preferably 30°C. In other words, the temperature may be any value within the range of 20 to 45°C, such as 20, 21, 22, 25, 27, 28, 29, 20, 31, 32, 35, 37, 38, 39, 40, 42, or 45. In a reaction system in which a transamination reaction between a ketone compound and an amino donor is catalyzed by a transaminase, the amount of the enzyme used is 1.5 to 6.6 mg / mL, preferably 1.7 mg / mL. In other words, the amount of the enzyme may be any value within the range of 1.5 to 6.6 mg / mL, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.6, 3.8, 4.0, 4.4, 4.5, 4.9, 5.1, 5.5, or 6.6.

[0035] In one embodiment of the present invention, the substrate for the transaminase mutant of the present invention is:

[0036] Substrate 1: [ka] Tetr ahydrofuran-3-one Tetrahydrofuran-3-one Substrate 2: [ka] 2-Methylthiolan-3-one 2-Methylthiolan-3-one Substrate 3: [ka] 2-Chlorocyclopentanone 2-Chlorocyclopentanone Substrate 4: [ka] Cyclopentanone Cyclopentanone Substrate 5: [ka] 3-Methylcyclobutan-l-one 3-Methylcyclobutan-1-one According to one exemplary embodiment of the present invention, the method for verifying the reactions of Substrate 1, Substrate 2, Substrate 3, Substrate 4, and Substrate 5 is as follows.

[0037] 10 mg of substrate, 1 mg of enzyme, 0.1 mg of pyridoxal phosphate, and 20 mg of 6 M isopropylamine hydrochloride were added to a 5 mL centrifuge tube, and the mixture was supplemented with 0.1 M phosphate buffer (pH 7.5) to a total volume of 0.5 mL. The mixture was reacted at 45°C and 200 rpm for 16 hours.

[0038] After the reaction is complete, the selectivity of substrates 1, 2, and 3 is detected. To 0.06 mL of the reaction mixture, 0.04 mL of 0.1 M phosphate buffer (pH 7.5) was added. To 0.1 mL of the diluted mixture, 0.3 mL of an equal volume solution of acetonitrile, water, and sodium bicarbonate was added and mixed uniformly. A 0.1 mL aliquot was taken, 0.9 mL of 5 mg / mL Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide reagent was added, and the mixture was left in a metal bath at 50 °C for 3 hours. The induced mixture was removed and centrifuged at 12,000 rpm for 5 minutes. A 0.5 mL aliquot was taken, 0.5 mL of an equal volume solution of acetonitrile and water was added, mixed uniformly, and subjected to ee detection. In this study, the activity (expressed as substrate conversion rate) was detected as follows: After catalyzing the substrate reaction with the enzyme, 100 μL of the reaction mixture was taken, 900 μL of methanol was added, and the mixture was shaken and centrifuged. The supernatant was collected to obtain a sample. The relative peak area of ​​the product, detected by HPLC (high-performance liquid chromatography), indicates enzyme activity.

[0039] In one exemplary embodiment of the present invention, the amino donor is isopropylamine or alanine, preferably isopropylamine.

[0040] The beneficial effects of the present invention will be further explained below with reference to examples.

[0041] Example 1

[0042] Reaction verification was carried out for Substrate 1. The reaction system was composed of 10 mg of Substrate 1, 0.1 mg of pyridoxal phosphate, 20 mg of 6M isopropylamine hydrochloride, 1 mg of enzyme, and 0.1M phosphate buffer pH 7.5 (made up to a total volume of 0.5 mL). The reaction conditions were 45 °C, 200 rpm, and 16 h. Here, for the mutants L59Q, L59W, H149D, H149I, H149R, F164W, V242W, V242H, V242Q, V328I, improvement was obvious. Among them, for V242W, the e.e. was the highest, about twice that of the Template, but the activity (substrate conversion rate) decreased to some extent. The results are shown in Table 1.

[0043]

Table 1

[0044] Note: * indicates an activity of 1 - 30%, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** indicates 80 - 90%, ****** indicates 90 - 95%, ******* indicates over 95%.

[0045] Example 2 Using V242W as a template, mutations were sustained at six sites of L59, F164, L272, V328, T330, A436, and R442, and a total of 40 mutants were obtained. Reaction verification was performed on Substrate 1. The reaction system consisted of 10 mg of substrate, 0.1 mg of pyridoxal phosphate, 20 mg of 6 M isopropylamine hydrochloride, 1 mg of enzyme, and 0.1 M phosphate buffer pH 7.5 (supplemented to a total volume of 0.5 mL). The reaction conditions were 45 °C, 200 rpm, and 16 h. From the results shown in Table 2, for V242W+F164Q, V242W+L272K, V242W+V328M, V242W+V328I, and V242W+R442T, etc., the e.e. was significantly improved compared to V242W, and the activity (conversion rate) and selectivity of V242W+F164Q were significantly improved compared to the template V242W. Therefore, V242W+F164Q was used as the subsequent template.

[0046]

Table 2

[0047] Note: * indicates an activity of 1 - 30%, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** indicates 80 - 90%, ****** indicates 90 - 95%, and ******* indicates > 95%.

[0048] Example 3 Using V242W+F164Q as a template, a total of 18 single-point mutation sites were constructed, including 17 amino acid sites of V328 and R442T. Reaction verification was carried out for Substrate 1. The reaction system consisted of 10 mg of substrate, 0.1 mg of pyridoxal phosphate, 20 mg of 6 M isopropylamine hydrochloride, 1 mg of enzyme, and 0.1 M phosphate buffer pH 7.5 (made up to a total volume of 0.5 mL). The reaction conditions were 45 °C, 200 rpm, and 16 h. From the results shown in Table 3, two mutants screened as having significantly improved activity (expressed as conversion rate) were V242W+F164Q+V328I and V242W+F164Q+R442T, respectively. V242W+F164Q+V328I was used as the template for the next step.

[0049]

Table 3

[0050] Note: * indicates activity of 1 - 30%, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** indicates 80 - 90%, ****** indicates 90 - 95%, and ******* indicates > 95%.

[0051] Example 4 Using V242W+F164Q+V328I as a template, 18 single-point mutation sites of G2S, T46M, G48D, C185Y, S186N, S194P, T197M, N202D, Y205L, T245A, V252I, S268N, L353F, N359D, R409T, E424K, A436V, and R442 were constructed. Reaction verification was performed on Substrate 1. The reaction conditions were as follows: 10 mg of substrate, 0.1 mg of pyridoxal phosphate, 20 mg of 6 M isopropylamine hydrochloride, 0.25 mg of enzyme, 0.1 M phosphate buffer pH 7.5 (supplemented to a total volume of 0.5 mL), 45 °C, 200 rpm, and 16 h. Since V242W+F164Q+V328I+R442T had a greatly improved activity, it was used as the template for the next step. Table 4 shows the corresponding e.e. and activity results.

[0052]

Table 4

[0053] Note: * indicates an activity of 1 - 30%, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** indicates 80 - 90%, ****** indicates 90 - 95%, and ******* indicates > 95%.

[0054] Example 5 Using V242W+F164Q+V328I+R442T as a template, single-point mutations were performed, and G48D, S194P, and V252T were used as primers. Reaction verification was carried out for Substrate 1. The reaction system consisted of 10 mg of Substrate 1, 0.1 mg of pyridoxal phosphate, 20 mg of 6 M isopropylamine hydrochloride, 0.25 mg of enzyme, and 0.1 M phosphate buffer pH 7.5 (supplemented to a total volume of 0.5 mL). The reaction conditions were 45°C, 200 rpm, and 16 h. V242W+F164Q+V328I+R442T+S194P and V242W+F164Q+V328I+R442T+V252I had the highest activity, and according to the results of protein electrophoresis, the protein expression of mutants was revealed in the supernatant.

[0055]

Table 5

[0056] Note: * indicates activity of 1 - 30%, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** indicates 80 - 90%, ****** indicates 90 - 95%, and ******* indicates > 95%.

[0057] # indicates that the percentage of the SDS-PAGE band is between 10 - 25%, ## indicates that the percentage of the SDS-PAGE band is between 25% - 40%, indicates that the percentage of the SDS-PAGE band is between 40% - 55%, # indicates that the percentage of the SDS-PAGE band is between 55% - 70%, ## indicates that the percentage of the SDS-PAGE band is between 70% - 85%, and indicates that the percentage of the SDS-PAGE band is between 85% - 90%.

[0058] Example 6 Single-point mutations were performed using V242W+F164Q+V328I+R442T+S194P as a template, and the mutation sites were V252I and G48D. Using V242W+F164Q+V328I+R442T+V252I as a template, the mutation site was G48D. Reaction verification was performed for Substrate 1. The reaction system consisted of 10 mg of substrate, 0.1 mg of pyridoxal phosphate, 20 mg of 6 M isopropylamine hydrochloride, 0.25 mg of enzyme, and 0.1 M phosphate buffer pH 7.5 (made up to a total volume of 0.5 mL), and the reaction conditions were 45°C, 200 rpm, and 16 h. From the results shown in Table 6, the mutant V242W+F164Q+V328I+R442T+V252I+G48D had improved activity, and protein expression in the supernatant became clear.

[0059]

Table 6

[0060] Note: * indicates activity of 1 - 30%, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** indicates 80 - 90%, ****** indicates 90 - 95%, and ******* indicates > 95%.

[0061] # indicates that the percentage of the SDS-PAGE band is between 10 - 25%, ## indicates that the percentage of the SDS-PAGE band is between 25% - 40%, indicates that the percentage of the SDS-PAGE band is between 40% - 55%, # indicates that the percentage of the SDS-PAGE band is between 55% - 70%, ## indicates that the percentage of the SDS-PAGE band is between 70% - 85%, and indicates that the percentage of the SDS-PAGE band is between 85% - 90%.

[0062] Example 7 Using V242W+F164Q+V328I+R442T+S194P+V252I as a template, single-point mutations were carried out, and the mutation site was set as G48D. Reaction verification was performed on Substrate 1. The reaction system consisted of 10 mg of Substrate, 0.1 mg of pyridoxal phosphate, 20 mg of 6 M isopropylamine hydrochloride, 0.25 mg of enzyme, and 0.1 M phosphate buffer pH 7.5 (supplemented to a total volume of 0.5 mL). The reaction conditions were 45 °C, 200 rpm, and 40 h. From the results shown in Table 7, the mutant V242W+F164Q+V328I+R442T+S194P+V252I+G48D had the highest activity.

[0063]

Table 7

[0064] Note: * indicates 1 - 30% activity, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** indicates 80 - 90%, ****** indicates 90 - 95%, and ******* indicates > 95%.

[0065] Example 8 Under the condition that the enzyme amount was set to 1 mg, verification was carried out for various substrate concentrations of Substrate 1. Reaction conditions: 0.1 mg of pyridoxal phosphate, various concentrations of 6 M isopropylamine hydrochloride, total volume 2 mL, 0.1 M phosphate buffer pH 7.5, 45 °C, 200 rpm, 40 h. From the results shown in Table 8, the e.e. increased with the increase in substrate concentration, but the substrate conversion rate decreased with the increase in substrate concentration. Preferably, the substrate concentration was 30 mg / mL.

[0066]

Table 8

[0067] Note: * indicates activity of 1 - 30%, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** indicates 80 - 90%, ****** indicates 90 - 95%, ******* indicates more than 95%.

[0068] Example 9 Under the condition that the concentration of Substrate 1 was set to 30 mg / mL, verification was carried out for the usage amounts of various enzymes. The reaction system was composed of 0.5 mg of pyridoxal phosphate, 90 mg of 6 M isopropylamine hydrochloride, 0.1 M phosphate buffer pH 7.5, and the total volume was 1.5 mL. The reaction conditions were 45 °C, 200 rpm, and 40 h. From the results shown in Table 9, the e.e. slightly improved in response to the decrease in the enzyme amount, but no significant change was observed in the activity of the mutant. Preferably, the enzyme amount is 1.7 mg / mL.

[0069]

Table 9

[0070] Note: * indicates activity of 1 - 30%, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** represents 80 - 90%, ****** indicates 90 - 95%, ******* indicates more than 95%.

[0071] Example 10 The reaction temperature was optimized for Substrate 1. The reaction system consisted of 60 mg of substrate, 3 mg of enzyme, 0.6 mg of pyridoxal phosphate, 120 mg of 6M isopropylamine hydrochloride, and 0.1M phosphate buffer pH 7.5 (made up to a total volume of 1.8 mL). The reaction conditions were 45 °C, 200 rpm, and 40 h. From the results shown in Table 10, no obvious change in e.e. was observed with decreasing temperature, but the activity clearly decreased at 20 °C, and it was preferred to carry out the reaction at 30 °C.

[0072]

Table 10

[0073] Note: * indicates 1 - 30% activity, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** represents 80 - 90%, ****** indicates 90 - 95%, and ******* indicates > 95%.

[0074] Example 11 The reaction of Substrate 1 was scaled up. The reaction system consisted of 1 g of substrate, 0.05 g of enzyme, 10 mg of pyridoxal phosphate, 2 g of 6M isopropylamine hydrochloride, and 0.1M phosphate buffer pH 7.5 (made up to a total volume of 30 mL). The reaction conditions were 30 °C, 200 rpm, and 60 h. The reaction was carried out in a 250 mL Erlenmeyer flask. From the results shown in Table 11, the mutant had significantly improved e.e. and activity compared to the e.e. and activity of the parent.

[0075]

Table 11

[0076] Note: * indicates activity 1-30%, ** indicates 30-60%, *** indicates 60-70%, **** indicates 70-80%, ***** indicates 80-90%, ****** indicates 90-95%, and ******* indicates >95%.

[0077] After the reaction was completed, the system was treated according to the following specific steps: After removing impurities, the system was transferred to a 50 mL centrifuge tube; half the system volume of ethyl acetate was added, followed by shaking, and then the mixture was divided into two 50 mL centrifuge tubes and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded, and half the system volume of ethyl acetate was added to the lower system, followed by shaking and centrifuging at 4000 rpm for 10 minutes. This process was repeated once. Half the system volume of ethyl acetate was added to the lower system, followed by shaking and centrifuging at 4000 rpm for 10 minutes. This process was repeated once. The lower system was transferred to a 250 mL round-bottom flask and weighed. To remove the substrate, dry sodium hydroxide powder was added to the system in the round-bottom flask in an amount equivalent to half the weight of the remaining system, and the system was maintained below 40°C throughout this process. 0.5 times the system volume (half the system volume) of methyl tert-butyl ether was added, transferred to two 50 mL centrifuge tubes, and centrifuged at 4000 rpm for 10 minutes. The supernatant was added with 0.5 times the volume (half the volume of the supernatant) of methyl tert-butyl ether, shaken, and centrifuged at 4000 rpm for 10 minutes. This procedure was repeated once. The supernatant was transferred to a round-bottom flask and rotary evaporated. The yield of the product was 53%.

[0078] Example 12 The optimized reaction conditions were tested for substrates 2, 3, 4, and 5. The reaction mixture consisted of 10 mg of substrate, 0.1 mg of pyridoxal phosphate, 20 mg of 6 M isopropylamine hydrochloride, 0.5 mg of enzyme, and 0.1 M phosphate buffer, pH 7.5 (refilled to a total volume of 0.3 mL). The reaction conditions were 30°C, 200 rpm, and 60 h. The results shown in Table 12 indicate that the mutant activity improved with each substrate.

[0079] [Table 12] TIFF0007763350000025.tif203163TIFF0007763350000026.tif74162Note: - indicates -50% < e.e. < 1%, + indicates 1 - 30%, ++ indicates 30 - 60%, +++ indicates 60 - 80%, +++++ indicates 80 - 90%, ++++++ indicates 90 - 95%, and +++++++ indicates over 95%.

[0080] Note: * indicates activity of 1 - 30%, ** indicates 30 - 60%, *** indicates 60 - 70%, **** indicates 70 - 80%, ***** indicates 80 - 90%, ****** indicates 90 - 95%, and ******* indicates over 95%.

[0081] In addition, when the three-dimensional structure of the transaminase was analyzed using computer simulation analysis software, it was found that many of the mutation sites were located near the active center, and it was revealed that this mutation might enhance the binding between the substrate and the enzyme. Therefore, the selectivity and catalytic efficiency were improved.

[0082] The above are merely preferred embodiments of the present invention and do not limit the present invention. For those skilled in the art, various modifications and changes are possible for the present invention. Any corrections, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A transaminase mutant, A transaminase mutant having a sequence in which amino acid mutations have occurred in the sequence shown in SEQ ID NO: 1, wherein the amino acid mutations are at the following combinations of mutation sites: V242W+L59Q, V242W+F164C, V242W+F164Q, V242W+F164W, V242W+F164Y, V242W+L272G, V242W+L272I, V242W+L272K, V242W+L272M, V242W+L272P, V242W+L272V, V242W+L272Y, V242W+V328C, V242W+V328K V242W+V328L, V242W+V328M, V242W+V328Q, V242W+V328S, V242W+V328T, V242W+V3 28W, V242W+T330F, V242W+T330I, V242W+T330S, V242W+A436H, V242W+A436K, V242W +A436L, V242W+A436N, V242W+A436P, V242W+A436Q, V242W+A436S, V242W+A436Y, V2 42W+R442A, V242W+R442C, V242W+R442F, V242W+R442G, V242W+R442H, V242W+R442N 、V242W+R442Q、V242W+R442S、V242W+R442T、V242W+F164Q+V328A、V242W+F164Q+V328C、V242W+F164Q+V328D、V242W+F164Q+V328E、V242W+F164Q+V328F、V242W+F164Q+V328G、V242W+F164Q+V328H、V242W+F164Q+V328I、V242W+F164Q+V328L、V242W+F164Q+V328M、V242W+F164Q+V328P、V242W+F164Q+V328Q、V242W+F164Q+V328R、V242W+F164Q+V328S、V242W+F164Q+V328W、V242W+F164Q+V328T、V242W+F164Q+V328Y、V242W+F164Q+R442T、V242W+F164Q+V328I+G2S、V242W+F164Q+V328I+T46M、V242W+F164Q+V328I+G48D、V242W+F164Q+V328I+C185Y、V242W+F164Q+V328I+S186N、V242W+F164Q+V328I+S194P、V242W+F164Q+V328I+T197M、V242W+F164Q+V328I+N202D、V242W+F164Q+V328I+Y205L、V242W+F164Q+V328I+T245A、V242W+F164Q+V328I+V252I、V242W+F164Q+V328I+S268N、V242W+F164Q+V328I+L353F、V242W+F164Q+V328I+N359D、V242W+F164Q+V328I+R409T、V242W+F164Q+V328I+E424K、V242W+F164Q+V328I+A436V、V242W+F164Q+V328I+R442T、V242W+F164Q+V328I+R442T+G48D、V242W+F164Q+V328I+R442T+S194P、V242W+F164Q+V328I+R442T+V252I、V242W+F164Q+V328I+R442T+S194P+V252I、V242W+F164Q+V328I+R442T+V252I+G48D、V242W+F164Q+V328I+R442T+S194P+G48D、or V242W+F164Q+V328I+R442T+S194P+V252I+G48D,

2. A DNA molecule encoding the transaminase mutant of claim 1.

3. A recombinant plasmid comprising the DNA molecule of claim 2.

4. The recombinant plasmids are pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-2 1a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pE T-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-43b (+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pR The recombinant plasmid of claim 3, which is SET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18, or pUC-19.

5. A host cell comprising the recombinant plasmid of claim 3 or 4.

6. The host cell of claim 5, comprising a prokaryotic or eukaryotic cell.

7. The host cell according to claim 6, wherein the prokaryotic cell is a BL21-DE3 cell or an E. coli Rosetta-DE3 cell, and the eukaryotic cell is a yeast cell.

8. 1. A method for producing a chiral amine, comprising the step of catalyzing a transamination reaction of a ketone compound and an amino donor with a transaminase, 2. A method for producing a chiral amine, wherein the transaminase is the transaminase mutant of claim 1.

9. The ketone compound is 【Chemistry 1】 (n=0, 1, 2 or 3, X=C, N, O or S, R=H, F, Cl, Br, CH ３ or CH ２ CH ３ 9. The method of claim 8, wherein

10. The ketone compound is 【Chemistry 2】 10. The method of claim 9, wherein:

11. 9. The method of claim 8, wherein the amino donor is isopropylamine or alanine.

12. 12. The method of claim 11, wherein the amino donor is isopropylamine.

13. 9. The method according to claim 8, wherein the amount of the enzyme used in the reaction system in which the transamination reaction of the ketone compound and the amino donor is catalyzed by the transaminase is 1.5 to 6.6 mg / mL.

14. 14. The method according to claim 13, wherein the amount of the enzyme used in the reaction system in which the transamination reaction of the ketone compound and the amino donor is catalyzed by a transaminase is 1.7 mg / mL.

15. 14. The method according to claim 13, wherein the temperature of the reaction system in which the transamination reaction of the ketone compound and the amino donor is catalyzed by the transaminase is 20°C to 45°C.

16. 16. The method according to claim 15, wherein the temperature of the reaction system in which the transamination reaction of the ketone compound and the amino donor is catalyzed by the transaminase is 30°C.

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