ω-transaminase mutant based on ancestral sequence reconstruction
The amino acid sequence of a w-transaminase mutant derived from mutation of ω-transaminase from Aspergillus terreus , is as shown in SEQ ID NO. 4 or SEQ ID NO. 6. Compared with the wild-type enzyme, the half-lives of the ω-transaminase mutants are all above 24 h, while the half-life of the wild-type is only 6.90 min. The half-inactivation temperature of the mutants are 49.00° C. and 49.03° C., respectively, which are about 11° C. higher than that of the wild-type (37.89° C.), such that the thermal stability is significantly improved.
1 . An ω-transaminase mutant based on ancestral sequence reconstruction, wherein the ω-transaminase mutant is derived from mutation of ω-transaminase from Aspergillus terreus , and the ω-transaminase mutant has the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 6.
2 . A gene encoding the ω-transaminase mutant of claim 1 .
3 . The gene of claim 2 , wherein the ω-transaminase mutant has the gene sequence of SEQ ID NO. 3 or SEQ ID NO. 5.
4 . A recombinant expression plasmid, comprising the gene of claim 3 .
5 . A genetically engineered bacterium, comprising the recombinant expression plasmid of claim 4 .
6 . A method for catalyzing generation of acetophenone from (R)-(+)-α-methylbenzylamine, wherein acetophenone is generated by a transamination reaction, using (R)-(+)-α-methylbenzylamine and pyruvic acid as substrates and catalyzed by the ω-transaminase mutant of claim 1 .
7 . A method for catalyzing generation of acetophenone from (R)-(+)-α-methylbenzylamine, wherein acetophenone is generated by a transamination reaction, using (R)-(+)-α-methylbenzylamine and pyruvic acid as substrates and catalyzed by the genetically engineered bacterium of claim 5 .