C-端设计定点突变提高 Streptomyces Avermitilis壳聚糖酶稳定性及催化活性  

C-Terminal Design Site-Directed Mutation Improves the Stability of Streptomyces Avermitilis Chitosanase

作　　者：郭静[1] 高文君 朱小梅 赵晗 张欣 王怡 王芯楺 赵希岳[1] 蔡志强[1] GUO Jing;GAO Wenjun;ZHU Xiaomei;ZHAO Han;ZHANG Xin;WANG Yi;WANG Xinrou;ZHAO Xiyue;CAI Zhiqiang(School of Pharmacy,Changzhou University,Changzhou 213164,China)

机构地区：[1]常州大学药学院,江苏常州213164

出　　处：《常州大学学报（自然科学版）》2022年第5期82-92,共11页Journal of Changzhou University：Natural Science Edition

基　　金：江苏高等教育自然科学资助项目(19KJB180001);山东省重大科技创新工程资助项目(2019JZZY020605);常州大学科研启动资助项目(ZMF17020115)。

摘　　要：为提高阿维链霉菌(Streptomyces avermitilis)SaCsn46A壳聚糖酶的温度稳定性,利用SWISS-MODEL在线服务器,以链霉菌SirexAA-E壳聚糖酶(PDB登录号:4ily.1A)为模板对SaCsn46A的蛋白结构进行同源模拟,将预测获得的SaCsn46A蛋白结构提交至在线预测软件PoPMuSiC-2.1,寻找对SaCsn46A稳定性有显著影响的氨基酸残基,通过定点突变构建突变体,在大肠杆菌(Escherichia coli)Rosetta中异源表达野生型壳聚糖酶及突变体。经Ni-NTA亲和层析获得电泳纯重组酶蛋白,测定野生型壳聚糖酶及突变体G237A的酶学性质,发现突变体最适温度提高5℃,并且温度稳定性及pH稳定性显著提升。为进一步提高突变体的表达效率,对突变体表达菌株进行诱导条件优化,确定最优条件为IPTG浓度0.7 mmol/L,诱导温度20℃,诱导时间8 h。In order to improve the thermostability of Streptomyces avermitilis chitosanase SaCsn46A,the protein structure of SaCsn46A was simulated by using SWISS-MODEL online server using Streptomyces sp.SirexAA-E chitosanase(PDB accession number:4ilily.1A)as template.The predicted protein structure of SaCsn46A was submitted to online prediction software PoPMuSiC-2.1 to search for amino acid residues that had significant influence on the stability of SaCsn46A,and mutants were constructed by site-directed mutation.The wild-type chitosanase and its mutant were heterologous expressed in Escherichia coli Rosetta,respectively.The recombinant protein were purified by Ni-NTA affinity chromatography.Enzymatic properties of wild-type chitosanase and the mutant G237A were determined and the optimum temperature of G237A was increased by 5℃.And temperature stability and pH stability significantly improved.In order to further improve the expression efficiency of the mutant,the inducement conditions of the mutant expressing strain were optimized.The optimal conditions were determined as follows:IPTG concentration 0.7 mmol/L,induction temperature 20℃,induction time 8 h.

关 键 词：壳聚糖酶 定点突变 理性设计 PoPMuSiC 表达优化 

分 类 号：Q816[生物学—生物工程]