文献类型：期刊文献

英文题名：Effect of K225 residue to the catalytic efficiency of Kex2 protease

作者：Yang, Fan[1];Liu, Li[1];Liu, Yingying[1];Li, Suxia[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2020

卷号：176

外文期刊名：PROTEIN EXPRESSION AND PURIFICATION

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000573472000007)】；

基金：This study was partial financially supported by the State Key Laboratory of Bioreactor Engineering (No. 2060204), East China University of Science and Technology.

语种：英文

外文关键词：Kex2 protease; Pichia pastoris; Kex2-K225L mutant; Stability; Steady-state kinetics

摘要：The gene encoding S. cerevisiae Kex2 protease derivative Kex2-667 (encoding the N-terminal 20th to 667th amino acid residues of Kex2 protease, containing the propeptide, catalytic domain, P domain and Ser/Thr enrichment region) and its 225th amino acid residue mutant K225L were overexpressed in Pichia pastoris. Proteases were purified by dialysis and anion exchange chromatography (Q-FF). Their properties were further investigated. For catalysis efficiency, the value of Kcat/Km of Kex2-667-K225L was 3 folds higher than that of Kex2-667. Both were quite stable at 25 degrees C and 37 degrees C after 8 h of incubation at pH5.6, while Kex2-667 remained nearly 90% of the total activity while Kex2-667-K225L remained only 80%. The stability of Kex2-667-K225L was lower than that of Kex2-667 from pH4.0 to pH9.0. Due to the mutation site K225 was located at one of the calcium ion binding sites, it resulted in a tighter calcium ion binding region, which may be the reason why the catalytic efficiency of Kex2-667-K225L was improved while the stability was a little decreased.