C端结构域截短提高苏云金芽孢杆菌来源几丁质酶的活力  

C-terminal domain truncation improves the activity of chitinase derived from Bacillus thuringiensis

作　　者：黎欣宇 邓旭 蒋思婧[1] 周玉玲[1] 欧阳玉茹 贺妮莎[1] 张桂敏[1] LI Xinyu;DENG Xu;JIANG Sijing;ZHOU Yuling;OUYANG Yuru;HE Nisha;ZHANG Guimin(State Key Laboratory of Biocatalysis and Enzyme Engineering,School of Life Sciences,Hubei University,Wuhan 430062,Hubei,China)

机构地区：[1]湖北大学生命科学学院,湖北省生物资源绿色转化协同创新中心,省部共建生物催化与酶工程国家重点实验室,湖北武汉430062

出　　处：《微生物学报》2022年第4期1379-1389,共11页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31970059);湖北省教育厅中青年项目(202010701301003)。

摘　　要：【目的】尝试利用蛋白质结构域工程的手段优化苏云金芽胞杆菌(Bacillus thuringiensis BMB 171)来源的几丁质酶BtChiA的酶活力,并对水解胶体几丁质的产物进行定性和定量分析。【方法】根据BtChiA的序列分析其结构域的组成,构建BtChiA及部分结构域缺失的突变体蛋白在大肠杆菌中的表达菌株并进行蛋白的表达纯化;利用HPLC分析几丁质酶水解产物的类型及含量。【结果】异源表达纯化获得了纯度90%以上,大小正确的BtChiA (全长蛋白)和BtChiA△50 (截短几丁质结合结构域CBD和纤连蛋白结构域FnⅢ)。BtChiA△50水解胶体几丁质的活力是BtChiA的1.64倍。两者水解胶体几丁质的产物均为几丁单糖GlcNAc和几丁二糖(GlcNAc)2,全长蛋白BtChiA的水解产物中(GlcNAc)2的含量是GlcNAc的0.97倍,BtChiA△50水解产物中(GlcNAc)2的含量是GlcNAc的1.79倍。【结论】利用蛋白质结构域工程构建了水解活性提高的BtChiA△50突变体、优化了水解产物的组成。[Objective]Using protein domain engineering to construct and optimize the enzymatic activity of chitinase BtChiA derived from Bacillus thuringiensis BMB 171, and constructing qualitative and quantitative analysis of the product of the enzyme hydrolyzing colloidal chitin. [Methods]According to the sequence of BtChiA, the composition of its domain was analyzed. The expression strain of BtChiA and mutant protein with partial domain deletion in Escherichia coli was constructed and then the protein was expressed and purified. Analyzing the type and content of chitinase hydrolysates by HPLC. [Results]BtChiA(full-length protein) and BtChiA△50(truncated chitin binding domain CBD and fibronectin domain FnⅢ) were obtained by heterologous expression and purification. The vitality of BtChiA△50 to hydrolyze colloidal chitin is 1.64 times higher than that of BtChiA. The hydrolyzed products of both are chitin monosaccharide GlcNAc and chitobiose(GlcNAc)2. The content of(GlcNAc)2is 0.97 times lower than GlcNAc in the product of BtChiA, the content of(GlcNAc)2 is 1.79 times higher than GlcNAc in the product of BtChiA△50. [Conclusion]Protein domain engineering is a feasible strategy to improve the activity of chitinase BtChiA and optimize the composition of the hydrolysates.

关 键 词：苏云金芽胞杆菌 几丁质酶 蛋白质结构域工程 酶活力 

分 类 号：S476[农业科学—农业昆虫与害虫防治]