文献类型：期刊文献

中文题名：重组Bowman-Birk型大豆胰蛋白酶抑制剂的性质及抑制机理

英文题名：Properties and Inhibition Kinetics of Recombinant Bowman-Birk Trypsin Inhibitor

作者：刘利[1];杨帆[1];李素霞[1];郭奥[1];刘晓[2];王之可[2]

机构：[1]华东理工大学生物反应器国家重点实验室,上海200237;[2]上海雅心生物技术有限公司,上海200231

年份：2021

卷号：47

期号：3

起止页码：292

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2020】；CSCD:【CSCD_E2021_2022】；

语种：中文

中文关键词：Bowman-Birk型大豆胰蛋白酶抑制剂;重组表达;抑制动力学;酶学性质

外文关键词：Bowman-Birk soybean trypsin inhibitor;recombination expression;inhibition kinetics;enzymatic properties

摘要：利用大肠杆菌表达系统,成功表达并纯化获得了重组Bowman-Birk型大豆胰蛋白酶抑制剂(rBBTI)。对比研究了天然Bowman-Birk型胰蛋白酶抑制剂(BBTI)和rBBTI的酶学性质和稳定性,以及分别抑制胰蛋白酶和糜蛋白酶的抑制动力学。结果表明:rBBTI和BBTI都有较好的热稳定性,pH对rBBTI的活性影响较大;rBBTI和BBTI抑制胰蛋白酶和糜蛋白酶的最适条件分别为pH 8、25℃和pH 9、16℃;二者的抑制机理相同,抑制胰蛋白酶时表现为典型的反竞争性抑制作用,而抑制糜蛋白酶时表现为典型的非竞争性抑制作用;rBBTI和BBTI对胰蛋白酶的抑制效率均高于对糜蛋白酶的抑制效率,但rBBTI抑制胰蛋白酶和糜蛋白酶的效率略低于BBTI。
Bowman-Birk soybean trypsin inhibitor(BBTI),extracted from soybean(Glycine max L.)seeds,possesses insect resistance and anti-tumor properties.However,its specific mechanisms of action are still unknown.An efficient method to produce recombinant BBTI(rBBTI)in E.coli was reported.Some biochemical properties of rBBTI were revealed and the inhibition mechanism of BBTI was discussed.The rBBTI was successfully expressed with E.coli(BL21)expression system,and was further purified by Ni affinity chromatography and DEAE-FF column efficiently.The BBTI and rBBTI showed similar biochemical properties.The optimum conditions for inhibiting trypsin were pH 8 and 25℃,and the optimum conditions for inhibiting chymotrypsin were pH 9 and 16℃.BBTI and rBBTI were stable below 37℃.The inhibition kinetics assay of BBTI and rBBTI against trypsin as Lineweaver-Burk plots analysis showed an increased Michaelis constant(Km)and a decreased maximum reaction rate of enzyme(Vmax)with N-benzoyl-L-arginine ethyl ester(BAEE)as substrate.It suggested that BBTI and rBBTI were anti-competitive inhibitors interacted with trypsin.Both the inhibition kinetics assay of BBTI and rBBTI against chymotrypsin as Lineweaver-Burk plots analysis showed an unchanged Km and a decreased Vmax with N-acetyl-L-tyrosine ethyl ester(ATEE)as substrate.It suggested that BBTI and rBBTI were anti-competitive inhibitors interacted with chymotrypsin.Molecular modeling showed that LYS-16 of BBTI(trypsin active site of BBTI)interacted with residues of trypsin,forming effective hydrogen bonding interactions.However,there were hydrophobic residues at chymotrypsin activity domain of BBTI,and forming hydrophobic interactions with residues of chymotrypsin.These provide a reference for understanding the inhibition mechanism of BBTI,and the different inhibition rate of BBTI against trypsin or chymotrypsin.