[1]郭静,高文君,朱小梅,等.壳聚糖酶稳定性及催化活性[J].常州大学学报(自然科学版),2022,34(05):82-92.[doi:10.3969/j.issn.2095-0411.2022.05.011]
 WANG Xinrou,ZHAO Xiyue,CAI Zhiqiang.GUO Jing, GAO Wenjun, ZHU Xiaomei, ZHAO Han, ZHANG Xin, WANG Yi,[J].Journal of Changzhou University(Natural Science Edition),2022,34(05):82-92.[doi:10.3969/j.issn.2095-0411.2022.05.011]
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壳聚糖酶稳定性及催化活性()
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常州大学学报(自然科学版)[ISSN:2095-0411/CN:32-1822/N]

卷:
第34卷
期数:
2022年05期
页码:
82-92
栏目:
生物医药工程
出版日期:
2022-09-28

文章信息/Info

Title:
GUO Jing, GAO Wenjun, ZHU Xiaomei, ZHAO Han, ZHANG Xin, WANG Yi,
文章编号:
2095-0411(2022)05-0082-11
作者:
郭静高文君朱小梅赵晗张欣王怡王芯楺赵希岳蔡志强
(常州大学药学院,江苏常州213164)
Author(s):
WANG Xinrou ZHAO Xiyue CAI Zhiqiang
(School of Pharmacy, Changzhou University, Changzhou 213164, China)
关键词:
壳聚糖酶 定点突变 理性设计 PoPMuSiC 表达优化
Keywords:
chitosanase site-directed mutation rational design PoPMuSiC expression condition optimization
分类号:
Q 816
DOI:
10.3969/j.issn.2095-0411.2022.05.011
文献标志码:
A
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。
Abstract:
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.

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(责任编辑:李艳,周安迪)

备注/Memo

备注/Memo:
收稿日期: 2022-04-28。
基金项目: 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)。
作者简介: 郭静(1987—), 女, 辽宁本溪人, 博士, 讲师。E-mail: 00002655@cczu.edu.cn
更新日期/Last Update: 1900-01-01