淀粉酶链霉菌几丁质酶克隆表达及催化功能分析  

Cloning,expression and catalytic function analysis of chitinase from Streptomyces diastaticus

作　　者：李芹 王立梅[2] 齐斌[1,2] LI Qin;WANG Li-mei;QI Bin(College of Pharmaceutical Science,Soochow University,Suzhou,Jiangsu 215123,China;Research Center of Fermentation Engineering,Changshu Institute of Technology,Changshu,Jiangsu 215500,China)

机构地区：[1]苏州大学医学部药学院,江苏苏州215123 [2]常熟理工学院苏州市食品生物技术重点实验室,江苏常熟215500

出　　处：《食品与机械》2022年第5期1-7,70,共8页Food and Machinery

基　　金：江苏省科技厅重点研发计划(重点)项目(编号:BE2020386);苏州市科技计划项目(编号:SS202120,SS202124)。

摘　　要：目的:提高几丁质酶降解几丁质生产几丁寡糖产量。方法:利用基因工程方法对淀粉酶链霉菌几丁质酶进行克隆、原核表达、酶学性质探究,并通过同源建模、催化域氨基酸比对、定点突变等方法探究几丁质酶活性位点。结果:克隆和原核表达后,产酶周期由7 d缩短至24 h,酶活可达132 U/L,较原始菌酶活性(100 U/L)提高了32%。决定几丁质酶活性的氨基酸为催化域的128,130位的天冬氨酸和132位的谷氨酸。结论:对几丁质酶基因克隆表达后,其产酶周期缩短、酶活性提高。Objective:This study focused on improving the production of chitosan oligosaccharides by chitinase degradation of chitin.Methods:Using genetic engineering methods to clone,prokaryotic expressing,and investigating enzymatic properties of the chitinase from Streptomyces diastaticus,and the chitinase activity was explored by homology modeling,amino acid comparison of the catalytic domain,and site-directed mutagenesis.Results:After cloning and prokaryotic expression,the enzyme production cycle was shortened from 7 d to 24 h,and the enzyme activity reached 132 U/L,which was 32%higher than the original bacterial enzyme activity(100 U/L).The amino acids that determine the activity of chitinase was Asp at position 128,130,and Glu at position 132 of the catalytic domain.Conclusion:Clonal expression of the chitinase gene resulted in a shorter enzyme production cycle and increased enzyme activity.

关 键 词：淀粉酶链霉菌 几丁质酶 克隆表达 同源建模 定点突变 

分 类 号：Q78[生物学—分子生物学] TQ426.97[化学工程]