Improving The Thermostability Of (R)-?-transaminase From Bacillus Altitudinis W3 By Site-directed Mutation

?-transaminase,as a green biocatalyst,can be more efficiently applied to the synthesis of a variety of pharmaceutical intermediates.The?-transaminase in nature has poor thermostability,so it is more meaningful to develop?-transaminase which can adapt to complex industrial production conditions with better thermostability.The(R)-?-transaminase used in this study is derived from Bacillus altitudinis W3,but its thermostability expressed in Escherichia coli BL21(DE3)was poor and its industrial application was limited,so it has certain theoretical significance and potential application value to use molecular modification strategy to improve its thermostability.The main research results of this paper are as follows:(1)By multiple amino acid sequence alignment with four amino acids of thermophilic?-transaminase,five potential sites related to thermostability were identified for site-directed mutagenesis,all of which were mutated to proline which may enhance protein rigidity.The single mutants A191P,D192P,E225P,T237P and G270P were obtained respectively.Through the primary screening of thermostability,two single mutants(D192P and T237P)and combined mutation(D192P/T237P)which with better thermostability than wild-type were obtained.(2)The results of thermostability test showed that the half-inactivation temperature and half-life of wild-type enzyme were 39.3°C and 12.7 min,respectively,single mutant D192P and T237P were higher than those of wild-type enzyme.The half-inactivation temperature and half-life of the combined mutant D192P/T237P were obviously increased to 45.6°C and 31.7 min,respectively,which were 16%and 150%higher than those of wild-type enzyme.The optimum reaction temperature of wild-type enzyme and single mutants D192P and T237P was 45°C,while that of combined mutant D192P/T237P was increased to 50°C.(3)Other enzymatic properties of?-transaminase were determined and analyzed.The optimum reaction p H of wild-type enzyme and mutant enzymes was 7.0,and there was no significant difference in p H stability.In terms of tolerance to organic solvents,both wild-type enzyme and mutant enzymes did not have good tolerance to methanol,but mutant enzyme was slightly better than wild-type enzyme,and methanol could be used as termination reagent for enzyme catalytic reaction.Both wild-type enzyme and mutant enzymes had good tolerance to DMSO and mutant enzymes had better tolerance than wild-type enzyme,so it could be considered to add appropriate amount of DMSO as cosolvent to promote the reaction more efficiently.Although the combined mutant D192P/T237P showed the highest improvement in thermostability and relative activity,its catalytic efficiency was also significantly improved.Its k_cat/K_m value was 0.45 L·mmol^-1·min^-1,while the k_cat/K_m value of wild enzyme was 0.38L·mmol^-1·min^-1.(4)Through the determination of circular dichroism spectra of wild-type enzyme and mutant enzymes,it was found that the secondary structure of?-transaminase did not change much before and after mutagenesis.The molecular force within the protein shows that the newly formed hydrogen bonds and hydrophobic interactions may be the main reason for the improvement of the thermostability of wild-type enzyme.