Omega-transaminase mutant obtained through DNA synthesis shuffling combination mutation and application

Abstract

The invention discloses an omega-transaminase mutant obtained by DNA synthesis shuffling combination mutation, the omega-transaminase mutant is obtained by point mutation of wild type omega-transaminase from Aspergillus terreus, the amino acid sequence of the wild type omega-transaminase is shown as SEQ ID No.1, and the mutation site of the omega-transaminase mutant is any one of the following: (1) F115L-H210N-M150C-M280C; (2) F115L-H210N; (3) F115L-H210N-E253A-I295V; (4) I77L-F115L-E133A-H210N-N245D; (5) I77L-Q97E-F115L-L118T-E253A-G292D; (6) I77L-E133A-N245D-G292D; and (7) H210N-N245D-E253A-G292D. The forward mutation obtained in the earlier stage is randomly combined through the DNA synthesis shuffling combination mutation method, and experimental verification shows that the method caneffectively improve the forward mutation probability and improve the experimental efficiency and feasibility, and mutant enzymes with thermodynamic stability obviously superior to that of wild enzymesare obtained through screening.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an omega-transaminase mutant obtained by DNA synthesis shuffling combination mutation and its application. Background technique [0002] Chiral amines are key intermediates or chiral building blocks for the synthesis of many important drugs. At present, the production methods of these important chiral amine compounds are mainly chemical synthesis methods, and generally require expensive transition metal complex catalysts such as ruthenium and rhodium or extreme reaction conditions such as high pressure, and have poor selectivity, low yield, and high emissions. Wait for congenital deficiency. Therefore, whether the industrial production of chiral amines can be realized through asymmetric biocatalysis has become the focus of widespread attention of researchers. Two kinds of enzymes are mainly used in the asymmetric synthesis of chiral amine compounds by enzymatic catalysis, one is am...

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Problems solved by technology

However, various means for improving the thermal stability of transaminases have their own advantages and disadvantages. Although the mutants obtained in the above experiments can improve the half-inactivation temperature and prolong the half-life compared with the wild-type ω-transaminase, if they are to be used in actual production For better application, the half-inactivation temperature and half-life of the above mutants still need to be further improved

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