文献类型：期刊文献

英文题名：Characterization and rational design for substrate specificity of a prolyl endopeptidase from Stenotrophomonas maltophilia

作者：Yu, Junjie[1];Wu, Junjie[1];Xie, Dewei[1];Du, Lei[1];Tang, Ya-Jie[2];Xie, Jingli[1,3];Wei, Dongzhi[1,3]

机构：[1]East China Univ Sci & Technol, Sch Biotechnol, Dept Food Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shandong Univ, State Key Lab Microbial Technol, Qingdao 266237, Peoples R China;[3]Shanghai Collaborat Innovat Ctr Biomfg SCICB, Shanghai 200237, Peoples R China

年份：2020

卷号：138

外文期刊名：ENZYME AND MICROBIAL TECHNOLOGY

收录：;EI(收录号：20201508407201);WOS:【SCI-EXPANDED(收录号:WOS:000540161300002)】；

基金：This work was supported by the Open Funding Project of the Key Laboratory of Fermentation Engineering (Ministry of Education) of China, the Opening Project of Shanghai Key Laboratory of New Drug Design (Grant No. 17DZ2271000), China and the National Natural Science Foundation of China (31801489).

语种：英文

外文关键词：Prolyl endopeptidase; substrate specificity; rational design; ACE inhibition; casein; bioactive peptide

摘要：A novel prolyl endopeptidase from Stenotrophomonas maltophifia, SmPEP, was discovered and characterized. The specific activity of the recombinant SmPEP expressed by Escherichia coli BL21 (DE3), was 68.3 U/mg at pH 8.0 and 37 degrees C. In order to improve the substrate specificity for long-chain peptide, rational design was applied based on the structure constructed by homology modeling. Inter-domain sites within the 13-propeller domain were chosen for the mutation to weaken the inter-domain interaction and form an open conformation for long-chain substrate entering into the active site. The substrate specificity on a designed long-chain substrate, PQPQLPY-PQPQLP, of the mutants F263A and E184 G increased 8.77 and 5.75 times respectively versus wild-type. After the saturated mutation of the both sites, the reactive rate of mutant F263 V on 13-mer peptide was 10.2 times higher than that of the wild-type. Then the mutant F263 V was used in the hydrolysis of casein, and the ACE inhibitory activity of the hydrolysate was significantly improved compared with wild type enzyme, which verified the efficiency of the design strategy.