重组Bowman-Birk型大豆胰蛋白酶抑制剂的性质及抑制机理  

Properties and Inhibition Kinetics of Recombinant Bowman-Birk Trypsin Inhibitor

作　　者：刘利 杨帆[1] 李素霞[1] 郭奥 刘晓 王之可 LIU Li;YANG Fan;LI Suxia;GUO Ao;LIU Xiao;WANG Zhike(State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China;Shanghai Yaxin Biotechnology Co.Ltd,Shanghai 200231,China)

机构地区：[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

出　　处：《华东理工大学学报（自然科学版）》2021年第3期292-299,共8页Journal of East China University of Science and Technology

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.

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 

分 类 号：Q78[生物学—分子生物学]