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CN-121975760-A - R-type transaminase mutant and application thereof

CN121975760ACN 121975760 ACN121975760 ACN 121975760ACN-121975760-A

Abstract

The invention provides an R-type transaminase mutant and application thereof, wherein the mutant has mutation of 62 th site, 152 th site and 155 th site compared with the amino acid sequence of wild-type transaminase shown as SEQ ID NO. 1. The mutant has high catalytic activity and excellent stereoselectivity on heterocyclic ketone substrates, and can be used for efficiently preparing R-configuration heterocyclic chiral amine.

Inventors

  • CAO MINGFENG
  • YU DONGJIE
  • GE RAN
  • HE NING

Assignees

  • 厦门大学

Dates

Publication Date
20260505
Application Date
20251225

Claims (10)

  1. 1. An R-type transaminase mutant having mutations at positions 62, 152 and 155 compared to the amino acid sequence of a wild-type transaminase as set forth in SEQ ID No. 1.
  2. 2. The mutant R-type transaminase of claim 1, which has mutations of T62F, C V and Q155R compared to the amino acid sequence of the wild-type transaminase shown in SEQ ID No. 1.
  3. 3. The mutant R-type transaminase of claim 1, which has an amino acid sequence shown in SEQ ID No. 5.
  4. 4. A nucleic acid molecule encoding the mutant R-type transaminase of any one of claims 1 to 3.
  5. 5. An expression vector comprising the nucleic acid molecule of claim 4.
  6. 6. A recombinant cell carrying the expression vector of claim 5.
  7. 7. Use of a mutant of the R-type transaminase according to any of claims 1 to 3 for the catalytic preparation of (R) -heterocyclic amines.
  8. 8. The use according to claim 7, wherein the (R) -heterocyclic amine is (R) -1-Boc-3-aminopyrrolidine.
  9. 9. A method for preparing (R) -1-Boc-3-aminopyrrolidine by catalysis, which is characterized in that 1-Boc-3-pyrrolidone is used as a substrate, the R-type transaminase mutant of any one of claims 1-3 is used as a catalyst, and the catalyst is reacted in a buffer system in which an amino donor exists.
  10. 10. The method of claim 9, wherein the reaction conditions are pH 7.0 to 8.0 and the temperature is 25 to 35 ℃.

Description

R-type transaminase mutant and application thereof Technical Field The invention belongs to the technical field of bioengineering, and particularly relates to an R-type transaminase mutant and application thereof. Background Chiral amine is used as a core skeleton of modern drug design, and the efficient asymmetric synthesis technology of chiral amine directly relates to the development efficiency of innovative drugs. According to FDA statistics, the drugs containing chiral amine structures account for more than 70% of the small molecular drugs obtained by FDA in recent ten years, wherein N-heterocyclic chiral amine has become a core intermediate of key varieties such as DPP-4 inhibitors, antiviral drugs and the like due to unique spatial configuration and biocompatibility, and has good market prospect. However, in the related art, the synthesis of heterocyclic chiral amines has the following drawbacks: 1. Chemical synthesis method, which relies on noble metal catalytic hydrogenation and multi-step resolution purification, leads to complex process and poor enantioselectivity (ee value is usually less than 95%), and generates a large amount of organic waste liquid with high environmental protection pressure. 2. The enzyme catalysis method is characterized in that the steric hindrance of the heterocyclic amine substrate is larger, the problems of low conversion rate (< 40 percent), uncontrolled stereoselectivity and the like commonly exist in a conventional enzyme catalysis system, and aminotransferase is an important pyridoxal phosphate (PLP) dependent enzyme for catalyzing amino transfer from an amino donor to a substrate to produce various chiral amines. Natural transaminases have a certain chiral preference, most transaminases prefer to produce (S) -chiral products, and for (R) -type products, (R) -transaminases have low catalytic activity, which severely limits their application in pharmaceutical industry. Although new enzyme transformation strategies such as molecular dynamics and the like appear in recent years, certain progress is made in the aspects of expanding the spectrum of a transaminase substrate and improving the thermal stability, aiming at the specific catalysis requirement of heterocyclic ketone substrates, R-type transaminase mutants capable of simultaneously solving three core problems of steric hindrance adaptation, stereoselectivity control and catalysis efficiency improvement are still lacking, so that the development of the R-type transaminase mutants with excellent performance becomes a key for breaking through the green synthesis technical bottleneck of heterocyclic chiral amines. Disclosure of Invention The present invention aims to solve at least to some extent one of the technical problems in the above-described technology. Therefore, the invention provides an R-type transaminase mutant and application thereof, wherein the mutant has high catalytic activity and excellent stereoselectivity on heterocyclic ketone substrates, and can be used for efficiently preparing R-configuration heterocyclic chiral amine. To this end, in a first aspect of the invention, there is provided a mutant of an R-type transaminase having mutations at positions 62, 152 and 155 compared to the amino acid sequence of the wild-type transaminase as shown in SEQ ID No. 1. According to the embodiment of the invention, the mutant has high substrate affinity and Boc group tolerance, can realize the efficient preparation of single-configuration products (e.e. >99% and conversion rate > 90%), has obviously improved catalytic activity on the substrates, can efficiently catalyze and synthesize (R) -1-Boc-3-aminopyrrolidine, has a conversion rate up to 93% compared with wild enzyme, has mild reaction conditions, is suitable for industrial production, and provides a new way for green synthesis of heterocyclic chiral amine. Alternatively, the mutant has mutations of T62F, C V and Q155R compared to the amino acid sequence of the wild-type aminotransferase as set forth in SEQ ID NO. 1. Further, the mutant has an amino acid sequence shown as SEQ ID NO. 5. In a second aspect of the invention, a nucleic acid molecule is provided, which encodes the above-described mutant R-aminotransferase. In a third aspect of the invention there is provided an expression vector comprising a nucleic acid molecule as described above. In a fourth aspect of the invention, there is provided a recombinant cell carrying an expression vector as described above. In a fifth aspect of the invention, there is provided the use of a mutant of the above-described R-aminotransferase in the catalytic preparation of (R) -heterocyclic amines. Alternatively, the (R) -heterocyclic amine is (R) -1-Boc-3-aminopyrrolidine. In a sixth aspect of the invention, there is provided a method for the catalytic preparation of (R) -1-Boc-3-aminopyrrolidine, which is obtained by reacting 1-Boc-3-pyrrolidone as substrate with the above-mentioned mutant R-aminotransferase as c