科研之友
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摘要
Bacillus subtilis esterase (BSE) exhibits high activity, extraordinary substrate/product tolerance and excellent enantioselectivity in the production of l-menthol through enantioselective hydrolysis of dl-menthyl acetate. However, rapid inactivation of wild-type BSE at elevated temperatures often hampers its applications. In this work, directed evolution was used to create thermostable mutants of BSE. After screening and recombination of beneficial mutations, BSEV4 was chose for the best mutant. The BSEV4 had half-lives of 462, 248 and 0.34 h at 30,40 and 50 degrees C, respectively, which were 5.6, 4.1 and 2.0 folds longer than those of BSEWT. Moreover, BSEV4 showed an increase of 4.5 degrees C in T-50(15) and a higher temperature optimum compared with the wild-type enzyme. In the kinetic resolution of dl-menthyl acetate at 1.0 M substrate loading, BSEV4 displayed improvements in operational stability than BSEWT, leading to a 1.5-fold higher total turnover number at 45 degrees C. The model structure of BSEV4 with four mutations, built with a highly homologous p-nitrobenzyl esterase (PDB ID: 1QE3) as the template, revealed that the newly formed hydrogen bonds and ionic bonds were beneficial for enhancing the thermostability of BSE.
- 出版日期2014-11
- 单位华东理工大学; 生物反应器工程国家重点实验室
