Thermobifida fusca角质酶突变体D204C/E253C高效可溶表达及其应用  

Highly Soluble Expression of Thermobifida fusca Cutinase Mutant D204C/E253C and Its Application

作　　者：朱方剑 王蕾[1,2,3] 吴敬 陈晟[1,2,3] Zhu Fangjian;Wang Lei;Wu Jing;Chen Sheng(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi,214122;Key Lab of Ministry of Education for Industrial Biotechnology,School of Biotechnology,Jiangnan University,Wuxi,214122;International Joint Laboratory on Food Safety,Jiangnan University,Wuxi,214122)

机构地区：[1]江南大学食品科学与技术国家重点实验室,无锡214122 [2]江南大学生物工程学院工业生物技术教育部重点实验室,无锡214122 [3]江南大学教育部食品安全国际合作联合实验室,无锡214122

出　　处：《基因组学与应用生物学》2021年第2期678-685,共8页Genomics and Applied Biology

基　　金：国家重点研发计划项目(2019YFA0706900);国家自然科学基金项目(31571776);国家杰出青年基金项目(31425020)共同资助。

摘　　要：Thermobifida fusca角质酶突变体D204C/E253C是通过在野生型角质酶中引入二硫键获得的热稳定性突变体。由于二硫键的引入,导致突变体D204C/E253C在表达过程中,易错误折叠形成大量包涵体,可溶表达比率极低。这极大地限制了其发酵制备以及在PET纤维改性中的应用。为了加强突变体D204C/E253C的可溶表达,本研究将突变体D204C/E253C和周质蛋白二硫键氧化还原酶DsbC在大肠杆菌突变株Escherichia coli Origami B(DE3)中共表达,来异构化异常的二硫键。实验结果表明,共表达的角质酶突变体D204C/E253C正常折叠表达,酶活为61 U/m L,是非共表达的6.8倍;共表达的突变体D204C/E253C在80℃的半衰期可达16 h,能够在80℃对PET纤维进行改性。本研究通过与分子伴侣蛋白DsbC在大肠杆菌突变株E.coli Origami B(DE3)中的共表达,实现角质酶突变体D204C/E253C的高效可溶表达,为大规模工业应用提供了一定的理论基础。Thermobifida fusca cutinase mutant D204 C/E253 C is thermostable mutation obtained by introducing a disulfide bond in the previous study.Due to the inducing of disulfide bond,the mutant D204 C/E253 C was easily misfolded to form a large number of aggregates during the expression process.Only a small part of the mutant D204 C/E253 C was expressed in soluble form.This greatly restricts its fermentation preparation and application in PET fiber modification.In order to enhance the soluble expression,mutant D204 C/E253 C was co-expressed with the disulfide oxidoreductase in Escherichia coli Origami B(DE3)to isomerize the abnormal disulfide bond.The experimental results showed that the co-expressed cutinase mutant D204 C/E253 C was normally folded and expressed.In the case of coexpression,enzyme activity of cutinase mutant D204 C/E253 C was 61 U/mL,which was6.8 times that of the normal expression.The co-expressed mutant D204 C/E253 C had a half-life of 16 h at 80℃which can be used in the PET fiber modified at 80℃.In this study,efficient soluble expression of the cutinase mutant D204 C/E253 C was achieved through co-expression with molecular chaperone protein DsbC inE.coli Origami B(DE3),which provided a certain theoretical basis for large-scale industrial applications.

关 键 词：角质酶 二硫键 可溶性表达 DSBC PET纤维改性 

分 类 号：TQ342.2[化学工程—化纤工业] TQ920.1[轻工技术与工程—发酵工程]