文献类型：期刊文献

英文题名：Characterization, Semirational Design for pH Robustness, and the Application in Bioactive Peptide Production of a X-Prolyl Dipeptidyl Aminopeptidase from Lactococcus lactis MY-3

作者：Gu, Shengdi[1];Yu, Junjie[1,2];Du, Lei[1];Zhang, Daihui[3];Zhao, Li[1];Xie, Jingli[1,4]

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]Shanghai Inst Supervis & Inspect Food Prod & Cosme, Shanghai 200233, Peoples R China;[3]Chinese Acad Forestry, Inst Chem Ind Forest Prod, Nanjing 210042, Jiangsu, Peoples R China;[4]Shanghai Collaborat Innovat Ctr Biomfg SCICB, Shanghai 200237, Peoples R China

年份：2024

卷号：72

期号：13

起止页码：7279

外文期刊名：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY

收录：;EI(收录号：20241315802831);WOS:【SCI-EXPANDED(收录号:WOS:001189965400001)】；

基金：This work was supported by the National Key Research and Development Program of China(nos. 2020YFA0907800 and 2021YFC2100300);the Natural Science Foundation of Shanghai(no. 21ZR1416200),China.

语种：英文

外文关键词：X-prolyl-dipeptidylaminopeptidase; pH robustness; catalytic mechanism; semirational design; ACEinhibitory activity

摘要：PepXLcMY-3, an X-prolyl dipeptidyl aminopeptidase derived from Lactobacillus lactis MY-3, was screened and recombinantly expressed in Escherichia coli. The enzyme could exhibit about 40% activity within the pH range of 6.0-10. To further improve the pH robustness, site E396 located in the active pocket was discovered through alanine scanning. The mutant E396I displayed both developed activity and k(cat)/K-m. The optimal pH of E396I shifted from 6.0 to 10 compared to WT, with the relative activity within the pH range of 6.0-10 significantly increased. The site K648 was then proposed by semirational design. The activity of mutant E396I/K648D reached 4.03 U/mg. The optimal pH was restored to 6.0, and the pH stability was further improved. E396I/K648D could totally hydrolyze beta-casomorphin 7 within 30 min. The hydrolysate showed 64.5% inhibition on angiotensin I converting enzyme, which was more efficient than those produced by E396I and WT, 23.2 and 44.7%, respectively.