文献类型：期刊文献

英文题名：Preparation and Irreversible Inhibition Mechanism Insight into a Recombinant Kunitz Trypsin Inhibitor from Glycine max L. Seeds

作者：Xu, Yanji[1];Zhang, Panpan[1];Liu, Xiao[2];Wang, Zhike[2];Li, Suxia[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shanghai Yaxin Biotechnol Ltd Co, Shanghai 200231, Peoples R China

年份：2020

卷号：191

期号：3

起止页码：1207

外文期刊名：APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY

收录：;EI(收录号：20200608135110);WOS:【SCI-EXPANDED(收录号:WOS:000515718900001)】；

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：Soybean Kunitz trypsin inhibitor; Protein refolding; Biochemical property; Inhibition kinetic assay; Molecular modeling

摘要：Soybean Kunitz trypsin inhibitor (SKTI), extracted from soybean (Glycine max L.) seeds, possesses insect resistance and anti-tumor properties. But its specific mechanisms of action are not yet known. This article reports an efficient method to produce recombinant SKTI (rSKTI) in Escherichia coli, reveals some biochemical properties of rSKTI, and discusses the inhibition mechanism of SKTI. The rSKTI was expressed as inclusion body in E. coli BL21 (DE3). After refolding, the active rSKTI was obtained and was further purified with anion-exchange chromatography (DEAE-FF) efficiently. There were similar biochemical properties between SKTI and rSKTI. The optimum pH and the optimum temperature were pH 8.0 and 35 degrees C, respectively, being stable during pH 7.0-11.0 and below 37 degrees C. The activity against trypsin was inhibited by Co2+, Mn2+, Fe3+, Al3+, and epoxy chloropropane. Inhibition kinetic assay of SKTI against trypsin as Lineweaver-Burk plots analysis both showed an unchanged K-m and a decreased V-max with N-benzoyl-l-arginine ethyl ester (BAEE) as substrate. Molecular modeling showed Arg63 of SKTI (active residue of SKTI) that interacts with four residues of trypsin, including three catalytic site (His57, Asp102, and Ser195) and one binding site (Asp189), forming five interactions. These provide reference for understanding the inhibition mechanism of such kind of Kunitz trypsin inhibitors.