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, specifically 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 industrialized production mainly relies on transition metal catalyzed asymmetric synthesis, but this process requires expensive transition metal complexes as catalysts, and the resources of these transition metals are limited and expensive, making this approach difficult to achieve sustainability. Bioenzyme catalysis has attracted much attention due to the easy availability of substrate ketones, 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. Among them, transaminases have excellent stereoselectivity, reproducible cofactors, strong reactivity and environmentally friendly properties, and are widely used in the biocatalytic production of chiral amines and in the synthesis of pharmaceutical and agrochemical intermediates.

[0004] Transaminase reactions usually require pyridoxal phosphate as a coenzyme, which is 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, phenylethylamine, etc.

[0005] Although the progress of using transaminases for the production of chiral amines has attracted much attention, the enzymatic method has many problems in the application of scale-up production. For example, existing transaminases have low selectivity and low conversion rate 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] In order 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, 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 the site of the amino acid mutation and has 90% or more homology 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 includes any of the combinations of mutation sites: 328I+R442T+V252I+G48D, V242W+F164Q+V328I+R442T+S194P+G48D, or V242W+F164Q+V328I+R442T+S194P+V252I+G48D, or the transaminase mutant includes the above mutation sites and has an amino acid sequence having 80% or more, preferably 90% or more, and more preferably 95% or more identity to the amino acid sequence having the mutation sites.

[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 comprising 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, the host cell containing any of the recombinant plasmids described above.

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

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

[0015] Furthermore, the ketone compounds are [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] It 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. Effect of the Invention

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

[0019] In addition, the embodiments and features of the embodiments in the present application may be combined with each other without causing any contradiction. MODE FOR CARRYING OUT THE PRESENT DISCLOSURE The present invention will be described in detail below with reference to the embodiments.

[0020] Transaminases are biocatalysts whose main component is a protein, and the reaction 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 that reduces the conversion rate, which makes them disadvantageous for industrial production. In view of this technical problem, the present invention is directed to improving the selectivity and activity of a transaminase based on the transaminase shown in SEQ ID NO: 1 through directed evolution to obtain a transaminase with high catalytic activity and specific selectivity.

[0021] First, a mutation site is introduced into the transaminase by site-directed mutagenesis, and the selectivity of the mutants is detected, and a mutant with improved selectivity is selected. Here, the mutant V242W has about 2-fold higher selectivity than the starting template, but has inferior activity. Then, mutations are continued using V242W as a template to obtain mutants with 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) by 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] Using 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 and the template plasmid are annealed, and then "cycle extension" is performed using polymerase. The so-called cycle extension refers to the polymerase extending the primer according to the template, returning to the 5' end of the primer after one cycle, and repeating the cycle of heating, annealing, and extension. This reaction does not form multiple tandem copies, unlike rolling circle amplification. The extension products of the forward primer and the reverse primer are annealed and paired to form a nicked open circular plasmid. When digesting the extension product with Dpn I, since the original template plasmid is derived from conventional E. coli and modified by dam methylation, it is sensitive to Dpn I and is cut, but the plasmid with the in vitro synthesized mutant sequence is not methylated and therefore not cut, so the subsequent transformation can be successfully performed and a clone of the mutant plasmid is obtained.

[0024] The mutant plasmid was transformed into E. coli host cells, and then the cells were disrupted by sonication to obtain the crude enzyme for reaction validation. The optimal conditions for transaminase inducible expression 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 having a sequence in which an amino acid mutation has occurred in the sequence shown in SEQ ID NO:1, the site of the amino acid mutation comprising a V242W site, or an amino acid sequence of the transaminase mutant having the site of the amino acid mutation and having 90%, 95% or 99% or more homology to the sequence shown in SEQ ID NO:1, and having 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 are made 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 mutant of the present invention is based on the transaminase shown in SEQ ID NO: 1, and is mutated by site-directed mutagenesis to change its amino acid sequence, protein structure and function. Then, a transaminase having the above mutation site is obtained by directed screening. The transaminase obtained in the present invention has high catalytic activity, specific selectivity and a wide substrate spectrum, and is expected to be industrially applicable.

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

[0029] The above DNA molecules of the present invention may be present 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 particular nucleotide sequence in a suitable host cell. It generally includes a promoter operatively linked to a nucleotide sequence of interest, which is optionally operatively linked to a terminator signal and / or other regulatory elements. An expression cassette may also include sequences necessary for accurate translation of the nucleotide sequence. The coding region usually codes for a protein of interest, but also for a functional RNA of interest, such as an antisense RNA or a non-translated RNA, in a sense or antisense orientation. An expression cassette containing a polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one other component. An expression cassette may be naturally occurring, but is obtained by efficient recombination formation for heterologous expression.

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

[0031] In the present invention, the term "containing" is used to define the above DNA molecule, but 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 in order 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, if the term is defined in a closed manner, it cannot realistically cover these situations.

[0032] The term "plasmid" as used in the present invention includes any plasmid, cosmid, phage or Agrobacterium binary nucleic acid molecule, double-stranded or single-stranded, linear or circular, preferably a recombinant expression plasmid, which may be a prokaryotic expression plasmid or a eukaryotic expression plasmid, but is preferably a prokaryotic expression plasmid. In some embodiments, the vector used for 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-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 is provided that contains any of the above recombinant plasmids. 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-based compound and an amino donor with a transaminase, the transaminase being a mutant transaminase of the present invention. Preferably, the ketone-based 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 the 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., that is, the temperature value may be any value within the range of 20 to 45° C., for example, 20, 21, 22, 25, 27, 28, 29, 20, 31, 32, 35, 37, 38, 39, 40, 42, 45, etc. 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, that is, the amount of the enzyme may be any value in the range of 1.5 to 6.6 mg / mL, for example, 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, 6.6, etc.

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

[0036] Substrate 1: [ka] Tetrahydrofuran-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 tube 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. 0.04 mL of 0.1 M phosphate buffer pH 7.5 was added to 0.06 mL of the reaction system, 0.3 mL of an equal volume solution of acetonitrile, water, and sodium bicarbonate was added to 0.1 mL of the dilution system, mixed uniformly, 0.1 mL was collected, 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 h. The induction system was removed and centrifuged at 12000 rpm for 5 min, 0.5 mL was collected, 0.5 mL of an equal volume solution of acetonitrile and water was added, mixed uniformly, and subjected to ee detection. In this application, the activity (expressed as substrate conversion rate) detection method is as follows. 100 μL was collected from the reaction system after the substrate reaction was catalyzed by the enzyme, 900 μL of methanol was added, shaken and centrifuged, and the supernatant was collected to obtain a sample. The relative peak area occupied by the product detected by HPLC (high performance liquid chromatography) represents the 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 now be further described with reference to the following examples.

[0041] Example 1

[0042] 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 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, and V328I, the 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%, and ******* indicates > 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 for Substrate 1. The reaction system was 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 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 (represented by the 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 carried out for Substrate 1. The reaction conditions were: Substrate 10 mg, pyridoxal phosphate 0.1 mg, 6 M isopropylamine hydrochloride 20 mg, enzyme 0.25 mg, 0.1 M phosphate buffer pH 7.5 (made up to a total volume of 0.5 mL), 45 °C, 200 rpm, 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 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 (made up 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 the mutants was revealed in the supernatant.

[0055]

Table 5

[0056] Note: * indicates 1 - 30% activity, ** 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, with the mutation sites being V252I and G48D. Using V242W+F164Q+V328I+R442T+V252I as a template, the mutation site was G48D. 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, 0.25 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 6, the mutant V242W+F164Q+V328I+R442T+V252I+G48D had improved activity, and protein expression in the supernatant became apparent.

[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 performed, and the mutation site was set as G48D. Reaction verification was carried out for Substrate 1. The reaction system was 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). 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 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 over 95%.

[0068] Example 9 Under the condition that the concentration of Substrate 1 was 30 mg / mL, verification was carried out for the usage amounts of various enzymes. The reaction system consisted of 0.5 mg of pyridoxal phosphate, 90 mg of 6 M isopropylamine hydrochloride, 0.1 M phosphate buffer at 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 with the decrease in the enzyme amount, but no significant change was observed in the activity of the mutant. Preferably, the enzyme amount was 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 over 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 preferable to carry out the reaction at 30 °C.

[0072]

Table 10

[0073] Note: * indicates activity of 1 - 30%, ** 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 in the following specific steps. Impurities were removed, and the system was transferred to a 50 mL centrifuge tube. Half the volume of the system was added with ethyl acetate, and the mixture was shaken, then divided into two 50 mL centrifuge tubes and centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and half the volume of the system was added with ethyl acetate to the lower system, and the mixture was shaken and centrifuged at 4000 rpm for 10 min. This was repeated once. Half the volume of the system was added with ethyl acetate to the lower system, and the mixture was shaken and centrifuged at 4000 rpm for 10 min. This was repeated once. The lower system was transferred to a 250 mL round-bottom flask and weighed. To remove the substrate, sodium hydroxide dry 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 (half the volume of the system) of methyl tertiary butyl ether was added, transferred to two 50 mL centrifuge tubes, and centrifuged at 4000 rpm for 10 min. The supernatant was added with 0.5 times the volume (half the volume of the supernatant) of methyl tertiary butyl ether, shaken, and centrifuged at 4000 rpm for 10 min. This was repeated once. The supernatant was transferred to a round-bottom flask and rotary evaporated. The product yield was 53%.

[0078] Example 12 The optimized reaction conditions were examined for substrates 2, 3, 4, and 5, and the reaction system 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 (total volume was replenished to 0.3 mL), and the reaction conditions were 30°C, 200 rpm, and 60 h. From the results shown in Table 12, the mutant activity was improved for all substrates.

[0079] [Table 12] TIFF2025501264000025.tif203163TIFF2025501264000026.tif74162Note: - indicates -50% < e.e. < 1%, + indicates 1 - 30%, ++ indicates 30 - 60%, +++ indicates 60 - 80%, +++++ indicates 80 - 90%, ++++++ indicates 90 - 95%, +++++++ 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%, ******* 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 strengthen the binding between the substrate and the enzyme. Therefore, the selectivity and catalytic efficiency were improved.

[0082] The above are only 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 comprising: SEQ ID NO: 1, wherein the amino acid mutation occurs at the site of the mutation, which includes the V242W site; or The amino acid sequence of the transaminase mutant has a site where the amino acid mutation occurs, has 90% or more homology with the sequence shown in SEQ ID NO: 1, and has transaminase catalytic activity.

2. The transaminase mutant of claim 1, characterized in that the site at which the amino acid mutation occurs includes any of 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+V328W、V242W+T330F、V242W+T330I、V242W+T330S、V242W+A436H、V242W+A436K、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、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,

3. A DNA molecule, characterized in that it codes for a transaminase mutant according to claim 1 or 2.

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

5. 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 4, 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.

6. A host cell comprising the recombinant plasmid according to claim 4 or 5.

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

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

9. 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, 3. A method for producing a chiral amine, characterized in that the transaminase is a transaminase mutant according to claim 1 or 2.

10. 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 10. The method of claim 9, wherein

11. The ketone compound is 【Chemistry 2】 11. The method of claim 10, wherein:

12. 10. The method of claim 9, wherein the amino donor is isopropylamine or alanine.

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

14. 10. The method according to claim 9, wherein 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.

15. The method according to claim 14, characterized in that 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 1.7 mg / mL.

16. The method according to claim 14, 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.

17. The method according to claim 16, characterized in that 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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