构巢曲霉几丁质酶ChiB的结构分析与重组表达  

Structure analysis and recombinant expression of chitinase ChiB from Aspergillus nidulans

作　　者：赵静 郭小利 金盛宇 王宇 刘中华 ZHAO Jing;GUO Xiaoli;JIN Shengyu;WANG Yu;LIU Zhonghua(Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, College of Life Science, Nanjing Normal University, Nanjing 210000, China)

机构地区：[1]南京师范大学,生命科学学院,江苏省微生物与功能基因组学重点实验室,南京210000

出　　处：《重庆理工大学学报（自然科学）》2022年第4期268-273,共6页Journal of Chongqing University of Technology：Natural Science

基　　金：国家自然科学基金青年科学基金项目(31600053)。

摘　　要：真菌的细胞壁由几丁质、葡聚糖等成分构成,几丁质酶在真菌细胞壁的重构过程起到重要作用。分析显示,人类条件致病真菌构巢曲霉的几丁质酶B(Aspergillus nidulans Chitinase,AnChiB)属于糖基水解酶家族18(glycosyl hydrolases family 18)二型(type II)几丁质水解酶。结构分析显示,AnChiB的N端18个氨基酸残基为信号肽,蛋白质三维结构为由8个α-螺旋和8个β-折叠组成的(β/α)8 TIM桶状结构,催化位点位于第4个β-折叠上;在第7个α-螺旋和β-折叠间存在一个插入序列,与桶状结构形成一条狭长的底物结合沟,底物结构位点延底物结合沟分布。本研究利用大肠杆菌(Escherichia coli)重组表达系统重组表达并纯化了AnChiB。酶学性质分析显示AnChiB能够水解晶体几丁质,最适pH为5。同时表达量分析显示AnChiB在构巢曲霉生长早期表达量升高,并在48 h达到最高峰。本研究表明AnChiB可能参与构巢曲霉生长过程中的细胞壁重构。The cell wall of fungi is composed of chitin,glucan and other components.Chitinase plays an important role in the process of fungal cell wall reconstruction.The Aspergillus nidulans chitinase B(AnChiB)of the human conditional pathogenic fungus Aspergillus nidulans belongs to the glycosyl hydrolases family 18(type II)chitinase.Structural analysis showes that the N-terminal 18 amino acid residues of AnChiB are signal peptides,and the three-dimensional structure of the protein is a(β/α)8 TIM barrel structure composed of 8α-helices and 8β-sheets.The catalytic site of AnChiB is located on the fourthβ-sheet.There is an insert sequence between the seventhα-helix andβ-sheet,which forms a long and narrow substrate binding groove with the barrel structure.The substrate binding sites are distributed along the substrate binding groove.AnChiB is recombinantly expressed and purified using Escherichia coli recombinant expression system.The analysis of enzymatic properties shows that AnChiB can hydrolyze insoluble crystal chitin powder,and the optimum pH is 5.At the same time,the expression level analysis shows that the expression level of AnChiB increases in the early growth stage of A.nidulans,and reachs the highest peak at 48 hours.It indicates that AnChiB may be involved in the cell wall remodeling during the growth of A.nidulans.

关 键 词：几丁质酶 结构分析 重组表达 

分 类 号：Q939[生物学—微生物学]