Recombinant omega-transaminase mutant and application thereof

Abstract

The invention discloses a recombinant omega-transaminase mutant and application thereof, relates to the technical field of biochemistry, and discloses plasmids and recombinant cells of the recombinant omega-transaminase mutant, and the nucleotide sequence of a coding gene of the mutant is shown as SEQ ID No.4. According to the application of the mutant of the recombinant omega-transaminase in synthesis of the chiral N-heterocyclic amine intermediate, a buffer solution, a substrate, a cofactor, an amino donor and a catalyst are sequentially added into a reaction system for reaction, and the chiral N-heterocyclic amine intermediate is generated. The activity and stereoselectivity of the omega-transaminase mutant obtained through enzyme engineering modification are obviously improved. The recombinant omega-transaminase catalyst can catalytically synthesize five chiral N-heterocyclic amines with high added values, the reaction conversion rate can reach 99% at most, and the optical purity of the product can reach 99% or above.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry, and particularly relates to a recombinant omega-transaminase mutant and application thereof. BACKGROUND

[0002] Chiral amines play an important role in medicinal chemistry as key intermediates for the synthesis of chiral drugs. Statistics show that more than 40% of chiral drug molecules contain chiral amine units. In particular, N-heterocyclic chiral amines, due to their structural characteristics and reaction properties of N-heterocyclic compounds and chiral amines, are often used as intermediates in the synthesis of various chiral drugs. For example, (R)-1-Boc-3-aminopiperidine is a key intermediate for the dipeptidyl peptidase IV (DPP-4) inhibitor alogliptin and linagliptin, R (R)-1-Boc-3-aminopyrrolidine is an intermediate for FGFR inhibitor fobaitini and phosphoinositide-3-kinase delta (PI3Kδ) inhibitor Leniolisib, S (R)-1-Boc-3-aminopyrrolidine is an intermediate for FGFR inhibitor fobaitini and phosphoinositide-3-kinase delta (PI3Kδ) inhibitor Leniolisib, S (R)-1-Boc-3-aminopyrrolidine is an intermediate for FGFR inhibitor fobaitini and phosphoinositide-3-kinase delta (PI3Kδ) inhibitor Leniolisib, R (R)-1-Boc-3-aminopyrrolidine is an intermediate for FGFR inhibitor fobaitini and phosphoinositide-3-kinase delta (PI3Kδ) inhibitor Leniolisib. Compared with traditional chemical methods, enzyme catalysis has the advantages of mild reaction conditions, environmental friendliness, and high stereoselectivity, and has become an important means for the synthesis of chiral amine drugs and key intermediates in recent years, showing significant application potential in the fields of drug research and production. At present, a variety of biological catalysts have been successfully applied to the synthesis of N-heterocyclic chiral amines, including transaminases, imine reductases and amine dehydrogenases. However, although imine reductases and amine dehydrogenases after enzyme engineering can achieve asymmetric synthesis of N-heterocyclic chiral amines, both types of enzymes need to rely on expensive NAD(P)H as a consumable cofactor, which makes it impossible to meet the economic requirements of industrial production.

[0003] Omega-transaminases are a class of pyridoxal-5'-phosphate (PLP)-dependent transferases that catalyze the transfer of an amino group between donor and acceptor compounds, and can accept aliphatic and aromatic ketones as substrates to generate stereospecific amines. The cofactor PLP of transamination reaction is inexpensive and recyclable, and the reaction operation is simple. In 2010, Savile et al. improved the catalytic activity of omega-transaminase ATA117 by computer design and in vitro evolution, which increased by about 25000 times, realized the asymmetric synthesis of sitagliptin with an enantioselectivity of 99% ee, and the reaction scale reached 200 g / L, and was successfully applied to industrial production. In 2021, Novick et al. used engineered transaminase CDX-043 to synthesize the chiral amine precursor of sacubitril at a substrate concentration of 75 g / L and an enzyme loading of 1%, and the conversion rate reached 90% within 24 h with a stereoselectivity of >99.9:0.1 dr. In summary, omega-transaminases show significant application advantages and development prospects in the field of chiral amine synthesis.

[0004] At present, the research on the synthesis of N-heterocyclic chiral amines by transaminase is relatively limited, and the existing research mainly focuses on the synthesis of N-heterocyclic amines with a single chiral center. The synthesis of double-chiral N-heterocyclic amines with important application value is still blank. Therefore, it is of key significance to develop efficient omega-transaminases as biocatalysts for the synthesis of N-heterocyclic amines with a single or double chiral center to accelerate the application process of omega-transaminases in industrial production. Wild-type omega-transaminases have low activity on non-natural substrates, which are difficult to be applied in industrial production. Chiral N-heterocyclic amines, especially N-heterocyclic amines with two non-adjacent chiral centers, are important pharmaceutical intermediates, but there is no literature report on the efficient and green synthesis method of such compounds.

[0005] Therefore, the skilled person in the art is committed to developing a high-activity and high-stereoselectivity omega-transaminase mutant for industrial production of N-heterocyclic chiral amine compounds, especially developing an efficient and environmentally friendly method for asymmetric synthesis of chiral N-heterocyclic amines by using engineered omega-transaminases. SUMMARY

[0006] In view of the above defects of the prior art, the technical problem to be solved by the present application is to develop a high-activity and high-stereoselectivity omega-transaminase mutant for industrial production of N-heterocyclic chiral amine compounds, especially to develop an efficient and environmentally friendly method for asymmetric synthesis of chiral N-heterocyclic amines by using engineered omega-transaminases.

[0007] To achieve the above-mentioned purpose, the present application provides a recombinant omega-transaminase mutant, and the coding gene nucleotide sequence thereof is shown in SEQ ID No. 4.

[0008] The application further provides a plasmid of the recombinant omega-transaminase mutant.

[0009] The application further provides a recombinant cell of the recombinant omega-transaminase mutant.

[0010] The application further provides an application of the mutant of the recombinant omega-transaminase in synthesis of an N-heterocyclic amine intermediate: sequentially adding a buffer, a substrate, a coenzyme, an amino donor and a catalyst in a reaction system, and performing reaction to generate the N-heterocyclic amine intermediate; the catalyst is the mutant of the recombinant omega-transaminase.

[0011] Further, the buffer is Tris-HCl, the coenzyme is pyridoxal phosphate, and the amino donor is isopropylamine.

[0012] Further, the substrate has a chemical formula as shown in Formula 1, Formula 2, Formula 3, Formula 4 or Formula 5: Formula 1, Formula 2, Formula 3, Formula 4, Formula 5.

[0013] Further, the N-heterocyclic amine intermediate is ( R )-1-Boc-3-aminopyrrolidine, (2 S ,4 R )-4-amino-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester, ( R )-1-Boc-3-aminopiperidine, (3 S ,5 R )-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester or (2 S ,5 R )-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester.

[0014] Further, the Tris-HCl has a pH of 7.5 and a concentration of 100 mM, the pyridoxal phosphate has a concentration of 0.2 mM, and the isopropylamine has a concentration of 200 mM.

[0015] Further, the catalyst is a whole-cell catalyst, a free enzyme catalyst or an immobilized enzyme catalyst of the mutant of the recombinant omega-transaminase.

[0016] Further, the reaction has a temperature of 37 ℃ and a reaction time of 4 h.

[0017] Further, the omega-transaminase AlTA from Ancylobacter lacus Alcaligenes latus has an amino acid sequence as shown in SEQ ID No. 1.

[0018] Further, the ω-transaminase AlTA is modified by semi-rational design to obtain a mutant thereof, wherein the mutant is obtained by single-point mutation and / or iterative saturation mutation of the 113th, 124th, 147th and 189th amino acids in the amino acid sequence of the ω-transaminase AlTA (i.e., SEQ ID No. 1).

[0019] Further, one of the key mutants is obtained by mutating the 113th phenylalanine to tyrosine, the 124th glutamine to glutamic acid, the 147th valine to phenylalanine and the 189th leucine to threonine in the amino acid sequence shown in SEQ ID No. 1, and the mutated amino acid sequence is shown in SEQ ID No. 3.

[0020] Further, the gene sequence encoding the ω-transaminase AlTA. By converting the amino acid sequence into a nucleotide sequence and optimizing the codon, the nucleotide sequence encoding the ω-transaminase AlTA is obtained by using gene synthesis technology, and the nucleotide sequence is shown in SEQ ID No. 2; the coding gene of the ω-transaminase mutant, and the nucleotide sequence is shown in SEQ ID No. 4.

[0021] Further, the recombinant cells of the recombinant ω-transaminase mutant, wherein the host cells are selected from prokaryotic hosts such as Escherichia coli, Bacillus subtilis and / or eukaryotic hosts such as Saccharomyces cerevisiae, Pichia pastoris, insect cells, mammalian cells.

[0022] Further, the host cells are Escherichia coli BL21(DE3). E. coli Further, the recombinant expression plasmid pET28a-AlTA is transformed into Escherichia coli BL21(DE3) to obtain a genetically engineered bacterium expressing the ω-transaminase AlTA; the recombinant expression plasmid pET28a-AlTAmut containing the mutant gene is transformed into Escherichia coli BL21(DE3) to obtain a genetically engineered bacterium containing the corresponding mutant. E. coli Further, the recombinant expression plasmid pET28a-AlTA is transformed into Escherichia coli BL21(DE3) to obtain a genetically engineered bacterium expressing the ω-transaminase AlTA; the recombinant expression plasmid pET28a-AlTAmut containing the mutant gene is transformed into Escherichia coli BL21(DE3) to obtain a genetically engineered bacterium containing the corresponding mutant. E. coli Further, the recombinant expression plasmid pET28a-AlTA is transformed into Escherichia coli BL21(DE3) to obtain a genetically engineered bacterium expressing the ω-transaminase AlTA; the recombinant expression plasmid pET28a-AlTAmut containing the mutant gene is transformed into Escherichia coli BL21(DE3) to obtain a genetically engineered bacterium containing the corresponding mutant.

[0023] Further, the preparation method of the catalyst of the ω-transaminase and the mutant thereof. The catalyst includes three forms of free enzyme catalyst, immobilized enzyme catalyst and whole cell catalyst. The whole cell catalyst refers to the whole cell obtained after enrichment culture and induction expression of the target protein of the constructed genetically engineered bacterium; the free enzyme catalyst is the cell-free extract obtained by biological enzymolysis, high-pressure crushing or ultrasonic crushing and centrifugation, and the pure enzyme obtained by Ni affinity chromatography purification; the immobilized enzyme catalyst is the immobilized enzyme obtained by immobilizing the free enzyme catalyst in activated carbon, silica gel, quartz sand, sodium alginate or ion exchange resin.

[0024] Furthermore, methods for producing chiral amino compounds from ω-transaminases and their mutants include those for ( R )-1-Boc-3-aminopyrrolidine, (2 S 4 R 4-Amino-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester, ( R )-1-Boc-3-aminopiperidine, (3 S 5 R 5-Amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester, (2 S 5 R Biocatalytic synthesis of key pharmaceutical intermediates such as 5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester; among which, ( R )-1-Boc-3-aminopiperidine is a key intermediate in alogliptin, a drug used to treat type 2 diabetes. (2) S 5 R 5-Amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester is an intermediate of litexitinib, a drug used to treat severe alopecia areata.

[0025] Furthermore, the ω-transaminase mutants obtained through enzyme engineering showed significantly improved activity and stereoselectivity. The optimal mutant exhibited a 4-fold increase in activity and a more than 13-fold increase in diastereoselectivity compared to the wild type. The recombinant ω-transaminase catalyst can catalyze the synthesis of five high-value-added chiral N-heterocyclic amines, with a maximum conversion rate of 99% and a product optical purity exceeding 99%, demonstrating significant potential for industrial application.

[0026] In a preferred embodiment 1 of the present invention, the preparation process of ω-transaminase AlTA is described in detail; In another preferred embodiment 2 of the present invention, the construction process of the ω-transaminase mutant is described in detail; In another preferred embodiment 3 of the present invention, the screening process for ω-transaminase AlTA mutants is described in detail; In another preferred embodiment 4 of the present invention, the synthesis catalyzed by the ω-transaminase AlTA mutant M4 is described in detail. R The process of )-1-Boc-3-aminopyrrolidine; In another preferred embodiment 5 of the present invention, the synthesis of ((2) catalyzed by the ω-transaminase AlTA mutant M4 is described in detail. S 4 R The process of 4-amino-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester; In another preferred embodiment 6 of the present invention, the synthesis catalyzed by the ω-transaminase AlTA mutant M4 is described in detail. R The process of )-1-Boc-3-aminopiperidine; In another preferred embodiment 7 of the present application, the process of synthesizing (3 S ,5 R )-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester catalyzed by the omega-transaminase AlTA mutant M4 is described in detail. In another preferred embodiment 8 of the present application, the process of synthesizing (2 S ,5 R )-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester catalyzed by the omega-transaminase AlTA mutant M4 is described in detail.

[0027] The present application has the following beneficial technical effects: The present application evolves omega-transaminase through semi-rational design of protein active pocket guided by structure. The activity and stereoselectivity of the omega-transaminase mutant obtained through enzyme engineering are obviously improved, wherein the activity of the optimal mutant is 4 times higher than that of the wild type, and the diastereoselectivity is more than 13 times higher.

[0028] The omega-transaminase can catalyze the production of chiral amine compounds from ketone substrates. The modified omega-transaminase has high catalytic efficiency and stereoselectivity, and can efficiently synthesize optically pure chiral N-heterocyclic amines, especially key pharmaceutical intermediates such as (3 S ,4 R )-4-amino-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester, (3 S ,5 R )-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester, (2 S ,5 R )-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester, etc.

[0029] The engineered omega-transaminase catalyst provided by the present application can catalyze the synthesis of five kinds of high-value chiral N-heterocyclic amines, and the reaction conversion rate can be up to 99%, and the optical purity of the product can be up to 99% or more.

[0030] The concept, specific structure and technical effects of the present application will be further described below with reference to the accompanying drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the conversion rate and stereoselectivity experimental results of the omega-transaminase AlTA wild type and mutant M4 catalyzing the substrate 1-5 of a preferred embodiment of the present application. DETAILED DESCRIPTION

[0032] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0033] Example 1: Preparation of ω-transaminase AlTA

[0034] The gene for ω-transaminase AlTA was codon-optimized, synthesized by a commercial company, and ligated between the NedⅠ / XhoⅠ restriction sites of the pET28a plasmid, which was named pET28a-AlTA.

[0035] 1 μL of plasmid pET28a-AlTA (approximately 50 ng) was thermally transformed into 100 μL E.coli BL21(DE3) competent cells were diluted and plated onto LB agar plates containing 50 μg / mL kanamycin sulfate, and incubated upside down at 37 °C for 12 h until single colonies grew.

[0036] Single clones were selected and cultured in LB liquid medium containing kanamycin (50 μg / L) for 12 h. They were then transferred to fresh LB liquid medium containing kanamycin (50 μg / L) at a 1% (v / v) inoculation rate and cultured at 37℃ and 220 rpm until the OD600 reached 0.6–0.8. Recombinant protein expression was induced for 16 h at 20℃ by adding 0.2 mM isopropyl-β-D-thiogalactoside (IPTG). Cells were collected by centrifugation at 6,000 rpm for 10 min. The cells were washed twice with Buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl) and resuspended in Buffer A to obtain the AlTA whole-cell catalyst. The cell suspension was lysed under 700 bar pressure for 3 min until the bacterial suspension became clear. The suspension was centrifuged at 10,000 rpm for 45 min, and the supernatant was the crude AlTA enzyme solution.

[0037] The protein was purified by nickel column Ni-NTA Beads affinity chromatography. The specific steps were as follows: the crude enzyme solution was loaded onto the nickel column pre-equilibrated with Buffer A, and was loaded twice, eluted with Buffer B (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 20 mM imidazole) for 10 column volumes, then eluted with Buffer C (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 50 mM imidazole) for 10 column volumes, and finally eluted with Buffer D (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 250 mM imidazole) to completely elute the target protein. The eluate was collected into an ultrafiltration tube, concentrated to 2.5 mL by centrifugation at 4000 rpm, loaded onto a PD-10 desalting column, completely flowed through, and eluted with 3.5 mL Buffer E (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% glycerol) to obtain the AlTA pure enzyme.

[0038] Example 2: Construction of ω-transaminase mutants

[0039] The construction method of the ω-transaminase mutant was illustrated by taking the construction of saturated mutation at F113 as an example. The recombinant expression vector pET28a-AlTA constructed in Example 1 was selected as a template, and F113-F and F113-R in Table 1 were selected as primers. The nucleotide sequence of primer F113-F is shown in SEQ ID No. 5, and the nucleotide sequence of primer F113-R is shown in SEQ ID No. 6. Overlap PCR amplification was performed by using PhantaFlash DNA polymerase (Nanjing Novozyme Biotech Co., Ltd.) with the following PCR system: 10 μL 2 × Phanta Flash Master Mix, 1 μL pET28a-AlTA plasmid, 1 μL primer (10 μM), 7 μL ddH2O. The PCR conditions were as follows: 98 °C for 30 s; 98 °C for 10 s, 65 °C for 5 s, 72 °C for 1 min, 30 cycles; 72 °C for 1 min. After the PCR, 1% agarose gel electrophoresis was used to check whether the amplified fragment was reasonable.

[0040] Table 1 Primer sequences used for constructing the ω-transaminase mutant library

[0041] The PCR product was treated with Dpn I enzyme and then transformed into E.coliThe BL21(DE3) competent cells were cultured, and the mutant library was obtained after sequencing verification. The recombinant expression vector containing the mutant was named pET28a-AlTAmut. On this basis, the plasmid was extracted, and the mutation was iterated in sequence until the mutant with multiple mutation sites was prepared.

[0042] Example 3: Screening of ω-transaminase AlTA mutant

[0043] The mutant strain containing pET28a-AlTAmut was inoculated into a 48-well plate containing 2 mL of LB liquid medium (containing 50 μg / mL of kanamycin sulfate), and cultured at 37°C and 220 rpm until the OD600 reached 0.6-0.8. Then, 0.2 mM IPTG was added for induction, and the culture was induced at 20°C and 220 rpm for 16 h. After the culture, the bacteria were collected by centrifugation at 6,000 rpm for 10 min, and the bacteria were resuspended with 200 μL of cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% TieChui™ E. coli Lysis Buffer) for 10 min at 4°C. The supernatant was obtained by centrifugation at 4,000 rpm for 30 min at 4°C, and the following reaction was performed: The 100 μL reaction contained 10 mM substrate 1, 200 mM isopropylamine, 0.2 mM PLP, 100 mM Tris-HCl buffer (pH 7.5), 20% v / v mutant crude enzyme solution, and the reaction was performed at 37°C for 30 min. Then, 200 μL of methanol was added to quench the reaction, and the reaction mixture was centrifuged at 12,000 rpm for 20 min. The supernatant was used to determine the conversion rate, relative activity, and stereoselectivity of the mutant. The conversion rate was determined by OPA-precolumn derivative HPLC, and the conversion rate was calculated using the following formula: Conversion rate c (%) = product concentration / substrate concentration x 100% The activity of the wild-type ω-transaminase AlTA was defined as 100%. The relative activity of the mutant was calculated using the following formula: Relative activity = mutant conversion rate / wild-type conversion rate x 100% The stereoselectivity ee value, de value, and E value were calculated using the following formula:

[0044]

[0045]

[0046] Table 2 Activity and stereoselectivity of ω-transaminase AlTA and its mutants

[0047] Example 4: ω-transaminase AlTA mutant M4 catalyzes synthesis of R )-1-Boc-3-aminopyrrolidine

[0048] In a 1 mL reaction system, buffer, substrate 1, co-factor, amino donor, and enzyme catalyst were added in sequence, and reacted at 37°C for 4 h. After the reaction, the conversion rate and selectivity of the product were calculated according to the method in Example 2. It was determined that the conversion rate was 62%, and the ee value of the product was greater than 99%.

[0049] The preferred substrate 1 concentration is 20 mM, the reaction system is 100 mL, 10 mL, or 1 mL, the reaction buffer is Tris-HCl (pH 7.5) buffer, the buffer concentration is 100 mM, the co-factor is pyridoxal phosphate, the co-factor concentration is 0.2 mM, the amino donor is isopropylamine, and the amino donor concentration is 200 mM. The preferred catalyst is a crude enzyme solution, and the concentration is 0.2 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, or 10 mg / mL.

[0050] Example 5: ω-transaminase AlTA mutant M4 catalyzes synthesis of ((2 S ,4 R )-4-amino-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester

[0051] In a 1 mL reaction system, buffer, substrate 2, co-factor, amino donor, and enzyme catalyst were added in sequence, and reacted at 37°C for 4 h. After the reaction, the conversion rate and selectivity of the product were calculated according to the method in Example 2. It was determined that the conversion rate was 42%, the de value of the product was 95%, and the ee value was greater than 99%.

[0052] The preferred substrate 2 concentration is 20 mM, the reaction system is 100 mL, 10 mL, or 1 mL, the reaction buffer is Tris-HCl (pH 7.5) buffer, the buffer concentration is 100 mM, the co-factor is pyridoxal phosphate, the co-factor concentration is 0.2 mM, the amino donor is isopropylamine, and the amino donor concentration is 200 mM. The preferred catalyst is a crude enzyme solution, and the concentration is 0.2 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, or 10 mg / mL.

[0053] Example 6: ω-transaminase AlTA mutant M4 catalyzes synthesis of R )-1-Boc-3-aminopyrrolidine

[0054] The buffer, substrate 3, co-factor, amino donor, and enzyme catalyst were sequentially added into a reaction system of 1 mL, and reacted at 37°C for 4 h. After the reaction, the conversion rate and the selectivity of the product were calculated according to the method in Example 2. The conversion rate was determined to be 99%, and the ee value of the product was greater than 99%.

[0055] The preferred substrate 3 concentration was 20 mM, the reaction system was 100 mL, 10 mL, or 1 mL, the reaction buffer was Tris-HCl (pH 7.5) buffer, the buffer concentration was 100 mM, the co-factor was pyridoxal phosphate, the co-factor concentration was 0.2 mM, the amino donor was isopropylamine, and the amino donor concentration was 200 mM. The preferred catalyst was a crude enzyme solution, and the concentration was 0.2 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, or 10 mg / mL.

[0056] Example 7: ω-transaminase AlTA mutant M4 catalyzed synthesis of (3 S ,5 R )-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester

[0057] The buffer, substrate 4, co-factor, amino donor, and enzyme catalyst were sequentially added into a reaction system, and reacted at 37°C for 4 h. After the reaction, the conversion rate and the selectivity of the product were calculated according to the method in Example 2. The conversion rate was determined to be 36%, the de value of the product was 95%, and the ee value was greater than 99%.

[0058] The preferred substrate 4 concentration was 20 mM, the reaction system was 100 mL, 10 mL, or 1 mL, the reaction buffer was Tris-HCl (pH 7.5) buffer, the buffer concentration was 100 mM, the co-factor was pyridoxal phosphate, the co-factor concentration was 0.2 mM, the amino donor was isopropylamine, and the amino donor concentration was 200 mM. The preferred catalyst was a crude enzyme solution, and the concentration was 0.2 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, or 10 mg / mL.

[0059] Example 8: ω-transaminase AlTA mutant M4 catalyzed synthesis of (2 S ,5 R )-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester

[0060] The buffer, substrate 5, co-factor, amino donor, and enzyme catalyst were sequentially added into a reaction system, and reacted at 37°C for 4 h. After the reaction, the conversion rate and the selectivity of the product were calculated according to the method in Example 2. The conversion rate was determined to be 45%, the de value of the product was 96%, and the ee value was greater than 99%.

[0061] The concentration of the above-mentioned preferred substrate 5 is 20 mM, the reaction system is 100 mL, 10 mL, 1 mL, the reaction buffer is Tris-HCl (pH 7.5) buffer, the buffer concentration is 100 mM, the co-factor is pyridoxal phosphate, the co-factor concentration is 0.2 mM, the amino donor is isopropylamine, and the amino donor concentration is 200 mM. The preferred catalyst is a crude enzyme solution, and the concentration is 0.2 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, or 10 mg / mL.

[0062] The experimental results of ω-transaminase AlTA wild type and mutant M4 catalyzing substrates 1-5 are shown in Table 1, wherein part A is the conversion rate and stereoselectivity of the catalysis of substrates 1-5, respectively, and part B is the chemical reaction equation of the key intermediate of the diabetes treatment drug alogliptin generated by the catalysis of substrate 1. Figure 1 R The chemical reaction equation of the intermediate of the severe alopecia areata treatment drug litixitin generated by the catalysis of substrate 1. S R The chemical reaction equation of the intermediate (2,5)-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester of the severe alopecia areata treatment drug litixitin generated by the catalysis of substrate 1.

[0063] The above detailed the preferred embodiments of the present application. It should be understood that those of ordinary skill in the art can make many modifications and variations without creative labor based on the concept of the present application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiments based on the prior art according to the concept of the present application shall be within the protection scope defined by the claims.​​

Claims

1. A recombinant ω-transaminase mutant, characterized in that, The nucleotide sequence of the gene encoding the mutant is shown in SEQ ID No.

4.

2. A plasmid of the recombinant ω-transaminase mutant as described in claim 1.

3. A recombinant cell of the recombinant ω-transaminase mutant as described in claim 1.

4. The application of a mutant of the recombinant ω-transaminase as described in claim 1 in the synthesis of N-heterocyclic amine intermediates, characterized in that, The application involves sequentially adding a buffer solution, substrate, cofactor, amino donor, and catalyst to a reaction system to generate the N-heterocyclic amine intermediate; the catalyst is a mutant of the recombinant ω-transaminase.

5. The application as described in claim 4, characterized in that, The buffer solution is Tris-HCl, the cofactor is pyridoxal phosphate, and the amino donor is isopropylamine.

6. The application as described in claim 4, characterized in that, The chemical formula of the substrate is shown in Formula 1, Formula 2, Formula 3, Formula 4 or Formula 5: Formula 1, Equation 2, Formula 3, Equation 4, Formula 5.

7. The application as described in claim 4, characterized in that, The N-heterocyclic amine intermediate is ( R )-1-Boc-3-aminopyrrolidine, (2 S 4 R 4-Amino-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester, ( R )-1-Boc-3-aminopiperidine, (3 S 5 R 5-Amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester or (2 S 5 R 5-Amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester.

8. The application as described in claim 5, characterized in that, The Tris-HCl has a pH of 7.5 and a concentration of 100 mM, the pyridoxal phosphate has a concentration of 0.2 mM, and the isopropylamine has a concentration of 200 mM.

9. The application as described in claim 3, characterized in that, The catalyst is the whole-cell catalyst of the recombinant ω-transaminase mutant, the free enzyme catalyst, or the immobilized enzyme catalyst.

10. The application as described in claim 4, characterized in that, The reaction was carried out at a temperature of 37 °C for 4 h.

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