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CN-121975762-A - Transaminase mutant and preparation method and application thereof

CN121975762ACN 121975762 ACN121975762 ACN 121975762ACN-121975762-A

Abstract

The invention discloses a transaminase mutant, a preparation method thereof and application thereof in synthesizing (R) -1-Boc-3-aminopiperidine. The mutant is a protein variant obtained by site-directed mutagenesis based on the amino acid sequence shown in SEQ ID NO. 1. The mutant provided by the invention shows remarkably improved catalytic activity and stereoselectivity when catalyzing asymmetric amination of 1-Boc-3-piperidone to synthesize (R) -1-Boc-3-aminopiperidine, and the conversion rate and the enantiomeric excess value of the product are both more than 99%. The mutant has definite coding gene, is easy to prepare by a recombinant expression system, has simple process and mild condition, and is suitable for industrial production of (R) -1-Boc-3-aminopiperidine.

Inventors

  • WANG LIUZHU
  • ZHU SHUANGQUAN
  • WU SILIN
  • GU GUOQING
  • QIU YI

Assignees

  • 上海泓博尚奕药物技术有限公司

Dates

Publication Date
20260505
Application Date
20260210

Claims (10)

  1. 1. A transaminase mutant, which is characterized in that the mutant at least comprises Y122A and can further comprise one or more of V69T, Q155V, D A on the basis of the amino acid sequence shown in SEQ ID NO. 1.
  2. 2. The transaminase mutant of claim 1, characterized in that the mutant comprises V69T and Y122A substitutions.
  3. 3. The transaminase mutant of claim 1, which comprises V69T, Y a and Q155V substitutions.
  4. 4. The transaminase mutant of claim 1, which comprises D49A, V69T, Y a and Q155V substitutions.
  5. 5. The transaminase mutant according to any one of claims 2 to 4, characterized in that it has an amino acid sequence as shown in SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7 or SEQ ID NO. 9.
  6. 6. A polynucleotide encoding the transaminase mutant of any one of claims 2 to 4; preferably, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 or SEQ ID NO. 10.
  7. 7. A recombinant vector or host cell comprising the polynucleotide of claim 6.
  8. 8. A process for preparing the transaminase mutants of claim 2 to 4, which comprises culturing the host cell of claim 7 such that the transaminase mutants are expressed and recovering the transaminase mutants.
  9. 9. Use of the transaminase mutants of any of claims 2 to 4 for the preparation of (R) -1-Boc-3-aminopiperidine.
  10. 10. The use according to claim 9, characterized in that it comprises reacting 1-Boc-3-piperidone with an amino donor under the catalysis of the transaminase mutant; Wherein the pH of the reaction is 7.5 to 8.0, The temperature of the reaction is 30 to 40 ℃, The amino donor is isopropylamine and the amino donor is isopropyl amine, The reaction comprises the cofactor pyridoxal phosphate.

Description

Transaminase mutant and preparation method and application thereof Technical Field The invention belongs to the technical field of biocatalysis, and particularly relates to a transaminase mutant and a preparation method and application thereof. Background Chiral amine is an indispensable chiral structural unit in medicines, pesticides and fine chemicals, and has important industrial value in efficient and high-selectivity synthesis. Traditional chemical synthesis methods often rely on harsh reaction conditions, toxic reagents or chiral auxiliary agents, and require additional resolution steps to improve optical purity, are tedious in process and low in efficiency, and are difficult to meet green production requirements. In contrast, biocatalysis, in particular to asymmetric amination reaction catalyzed by omega-aminotransferase, has the advantages of mild condition, high stereoselectivity, environmental friendliness and the like, and has become an important way for synthesizing chiral amine. The (R) -1-Boc-3-aminopiperidine is an important chiral amine, is a direct component of the core skeleton of the dipeptidyl peptidase IV inhibitor linagliptin, and is also a key chiral intermediate for synthesizing other similar medicaments (such as sitagliptin, troglitazone, alogliptin and the like). At present, the asymmetric amination of 1-Boc-3-piperidone by using aminotransferase to catalyze the synthesis of (R) -1-Boc-3-aminopiperidine has been reported. However, the wild-type or engineering transaminase used in the prior art has the common problems of insufficient catalytic activity, unsatisfactory stereoselectivity and the like, so that the reaction conversion rate is low, the optical purity of the product cannot meet the requirement of a high-purity drug intermediate, and the industrialized application potential of the route is severely limited. Thus, there is an urgent need in the art to develop a transaminase mutant with higher activity and stereoselectivity to achieve efficient, green synthesis of (R) -N-Boc-3-aminopiperidine. Disclosure of Invention In the prior art, the catalytic synthesis of (R) -1-Boc-3-aminopiperidine by aminotransferase has insufficient activity and selectivity, so that the reaction efficiency and the chiral purity of the product are not ideal. The present invention aims to provide a novel transaminase mutant and its application, which overcome the above-mentioned drawbacks. In order to achieve the aim of the invention, the invention adopts the following technical scheme: In a first aspect of the invention, a transaminase mutant is provided. The mutant at least comprises Y122A based on the amino acid sequence shown in SEQ ID NO.1, and can further comprise one or more of V69T, Q155, 155V, D49A. "Transaminase mutant" refers to a protein variant obtained by genetic engineering means by one or more changes to the amino acid sequence of the original wild-type transaminase, which generally retains or alters some or all of the catalytic function of the original enzyme. "SEQ ID NO. 1" is the amino acid sequence of the wild-type template enzyme used according to the invention, which is derived from the ω -transaminase (NoATA) of Nocardioides sp.JS 614, which serves as starting point and reference for all modifications according to the invention. "Substitution" refers to the replacement of one amino acid with another at a particular position in a protein. The invention is described by adopting a general expression method of 'original amino acid single letter code-position-new amino acid single letter code', and the specific meaning is as follows: "D49A" means that at position 49 of the amino acid sequence, aspartic acid (ASPARTIC ACID, single letter code D) is replaced by alanine (Alanine, single letter code A). "V69T" means that Valine (Valine, one-letter code V) is substituted with threonine (Threonine, one-letter code T) at position 69 of the amino acid sequence. "Y122A" means that at position 122 of the amino acid sequence, tyrosine (single letter code Y) is replaced with alanine (Alanine, single letter code A). "Q155V" means that glutamine (Glutamine, one-letter code Q) is substituted with Valine (Valine, one-letter code V) at position 155 of the amino acid sequence. Preferably, the mutant comprises V69T and Y122A substitutions, i.e. comprises a substitution of valine at position 69 with threonine and a substitution of tyrosine at position 122 with alanine. Preferably, the mutant comprises V69T, Y a and Q155V substitutions, i.e. comprises valine at position 69 substituted by threonine, tyrosine at position 122 substituted by alanine and glutamine at position 155 substituted by valine. Preferably, the mutant comprises D49A, V69T, Y a and Q155V substitutions, i.e. comprises substitution of aspartic acid at position 49 with alanine, substitution of valine at position 69 with threonine, substitution of tyrosine at position 122 with alanine and substitution of glutamine at position 155 with vali