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CN-122012445-A - Transaminase mutant and application thereof

CN122012445ACN 122012445 ACN122012445 ACN 122012445ACN-122012445-A

Abstract

The invention relates to the technical field of biology, in particular to the technical field of enzyme engineering, and specifically relates to a transaminase mutant and application thereof. Specifically, the transaminase mutant is mutated by a site-directed mutation method based on the wild transaminase shown in SEQ ID NO. 1 (particularly, the 121 th site is mutated from E to N, the 152 th site is mutated from Y to A, the 260 th site is mutated from V to A and/or the 324 th site is mutated from T to S), so that the amino acid sequence is changed, the change of the structure and the function of the protein is realized, and the transaminase with the mutation site is obtained by a directional screening method.

Inventors

  • Pu Zhongji
  • LIU XUEWEI
  • FU WANYUE

Assignees

  • 杭州百恩科思生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260407

Claims (10)

  1. 1. A transaminase mutant, which is obtained by mutating the amino acid sequence of a wild-type transaminase shown in SEQ ID No. 1: (1)Y152A; (2)V260A; (3)T324S; (4) E121N+Y152a+v260A; or (b) (5)E121N+Y152A+V260A+T324S。
  2. 2. A polynucleotide encoding the transaminase mutant of claim 1.
  3. 3. A recombinant vector comprising the polynucleotide of claim 2.
  4. 4. A host cell comprising the polynucleotide of claim 2 or the recombinant vector of claim 3.
  5. 5. The host cell of claim 4, wherein the host cell is a fungal cell, a bacterial cell or a plant cell, preferably the host cell is a bacterial cell, more preferably the bacterial cell is an e.
  6. 6. Use of the transaminase mutant according to claim 1 or the host cell according to claim 4 or 5 for the preparation of chiral amines by catalyzing carbonyl compounds.
  7. 7. The use according to claim 6, wherein the carbonyl compound is 1 Imidazo [1,2 A ] pyridine 6 The chiral amine is (1S) 1 Imidazo [1,2 A ] pyridine 6 And (3) ethylamine.
  8. 8. The use according to claim 7, wherein the route of the use comprises reacting 1 in the presence of an amine source Imidazo [1,2 A ] pyridine 6 The ethanone is contacted with the transaminase, preferably the amine source is isopropylamine.
  9. 9. A process for preparing cermetini, comprising the step of combining a transaminase mutant according to claim 1 or a host cell according to claim 4 or 5 with 1 Imidazo [1,2 A ] pyridine 6 Mixing the ethyl ketone and the amine source under proper conditions to generate (1S) 1 Imidazo [1,2 A ] pyridine 6 And (3) ethylamine.
  10. 10. A method for producing a transaminase mutant according to claim 1, characterized in that it comprises: (a) Culturing the host cell according to claim 4 or 5 under conditions suitable for expression of the transaminase mutant, and (B) Recovering the transaminase mutants.

Description

Transaminase mutant and application thereof Technical Field The invention relates to the technical field of biology, in particular to the technical field of enzyme engineering, and specifically relates to a transaminase mutant and application thereof. Background The small molecule MET inhibitor sivoratinib (savolitinib), developed in 2021, month 6, 22, and yellow medicine, was approved in china, which means that china was the first to be a selective MET inhibitor of the first lot, which is also the MET inhibitor of type 3 worldwide. The approved indication for sivoratinib is locally advanced or metastatic non-small cell lung cancer with a transition in the mesenchyme-epithelial transforming factor (MET) exon 14. Cerrotini or 3- [ (1S) -1-imidazo [1,2-a ] pyridin-6-ylethyl ] -5- (1-methylpyrazol-4-yl) triazolo [4,5-b ] pyrazine (1) is a small molecule drug developed by Hutchison MEDIPHARMA LIMITED and Astrazeneca. It is a potent c-Met kinase inhibitor, being tested in combination with octenib (Osimertinib) to treat non-small cell lung cancer and advanced or metastatic papillary renal cell carcinoma patients. The current chemical synthesis methods for producing sivoratib involve five steps, the first of which involves the transamination of the substrate ketone compound (2) to produce the enantioselective amine product compound (3) (WO 2020053198 A1). The engineered (S) -selective aminotransferase can be used for such conversion under industrial process conditions. While selectivity is suitable, there is a need to improve enzyme activity to accept higher substrate loadings to optimize industrial production. Aminotransferase catalyzed stereoselective transfer of amino groups between an amino donor and a carbonyl substrate is an effective biocatalytic tool for the formation of chiral amines. Transaminases (TAs) can be divided into two classes, alpha-transaminases (alpha-TAs) and omega-transaminases (omega-TAs), depending on the type of substrate being converted. Unlike traditional alpha-aminotransferases (acting on the alpha-amino group), omega-aminotransferases act on an amino group in a non-alpha position (e.g., beta, gamma, or more distal) and are capable of transferring the amino group from an amino donor (e.g., an amino acid) to a keto acid acceptor to form the corresponding chiral amine and keto acid. Omega-aminotransferase has shown great potential in chiral amine synthesis as a star tool in the biocatalysis field, and is expected to play a more central role in green pharmacy and sustainable chemistry in the future. Although the asymmetric transaminase-catalyzed transamination reaction provides an economic and green synthetic idea for synthesizing chiral amine compounds. However, the presence of aminotransferases in large scale applications has limitations and challenges such as a rather narrow substrate range, unfavorable thermodynamic reaction equilibrium, and substrate/product inhibition. CN112930348B discloses a process for preparing sivoratinib, using the commercial enzyme ATA-436 derived from Codexis, which is very expensive, resulting in high production costs. CN116209754a disclosure provides engineered S-selective aminotransferase mutants with improved activity and substrate tolerance. Engineered transaminases were evolved to further improve the activity and substrate tolerance in the asymmetric enantioselective transamination of the substrate ketone 1-imidazo [1,2a ] pyridin-6-yl ethanone to the product (1S) -1-imidazo [1,2a ] pyridin-6-yl ethylamine. However, the transaminase mutant needs tens of mutations, the screening workload is large, the operation is very complicated, and the sources and the sequences of the detailed reporter enzyme genes are not available. Thus, there remains a need in the art for transaminases that are both highly selective and highly active. Disclosure of Invention In order to solve the problems of low efficiency and high cost in the process of synthesizing the cermetini, low substrate feeding amount, poor enzyme stability and high cost in the process of biosynthesis, the invention provides a aminotransferase mutant which has high catalytic activity, good thermal stability, large feeding amount and low cost and can catalyze and synthesize the cermetini chiral amine compound intermediate with high efficiency. Specifically, the transaminase mutant is obtained by changing the amino acid sequence of the transaminase mutant on the basis of wild transaminase shown in SEQ ID NO.1 through a site-directed mutagenesis method (particularly, mutation from E to N at 121 to Y to A at 152 and mutation from V to A to S at 260 and mutation from T to S at 324), so as to change the structure and function of the protein, and then through a directional screening method, the transaminase mutant has the advantage of greatly improving the enzyme activity, the enzyme activity is greatly improved by multiple times compared with that of a transaminase female parent, and the stability is greatly i