生孢噬纤维菌内切葡聚糖酶的异源表达及CBM6对其酶学性质的影响  

Heterologous expression of endoglucanase from Sporocytophaga sp.CX11 and the effect of CBM6 on its enzymatic properties

作　　者：盛钧美 荆晓凤 王继伟 陈晓艺[1] 李宪臻[1] SHENG Junmei;JING Xiaofeng;WANG Jiwei;CHEN Xiaoyi;LI Xianzhen(School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《大连工业大学学报》2024年第1期8-15,共8页Journal of Dalian Polytechnic University

基　　金：辽宁省自然科学基金项目(2020-MS-276)。

摘　　要：内切葡聚糖酶是纤维素酶系的重要组分之一,但大部分内切葡聚糖酶酸碱耐受性较差。为挖掘新型内切葡聚糖酶,本研究从生孢噬纤维菌CX11基因组中克隆了一个内切葡聚糖酶基因Sm_1350。该基因大小为2 196 bp,编码732个氨基酸,编码蛋白包含一个分泌信号肽、GH5催化结构域、6家族碳水化合物结合模块(CBM6)和一个C末端结构域(CTD)。利用实时荧光定量PCR技术对Sm_1350在不同碳源条件下的相对表达水平进行了分析,结果表明,Sm_1350在纤维二糖和滤纸培养基中的表达水平均显著高于在葡萄糖培养基中的表达水平,推测其在CX11降解纤维素过程中发挥一定作用。本研究构建了Sm_1350及其3种截短突变体,分别为Sm_1350、Sm_1350-GH5-CBM6、Sm_1350-GH5和Sm_1350-CBM6,并在大肠杆菌中进行异源表达。结果表明,去除CTD结构域明显提高了目的蛋白的可溶性表达水平。以CMC为底物时,Sm_1350-GH5-CBM6与Sm_1350-GH5酶活力分别为(7 000.00±50.00)U/g和(2 542.73±35.03)U/g,表明去除CBM6降低了Sm_1350-GH5对可溶性多糖的水解能力。酶学性质分析结果显示,Sm_1350-GH5-CBM6与Sm_1350-GH5均有较好的pH稳定性,去除CBM6结构域只改变了Sm_1350-GH5的最适温度,对最适pH以及温度稳定性没有产生影响。此外,Sm_1350-CBM6具有较强的几丁质结合能力,在蛋白纯化方面具有良好的应用前景。Endoglucanase is one of the important components of cellulase,but most endoglucanases have poor acid-base tolerance.In order to excavate novel endoglucanase,an endoglucanase gene Sm_1350 was cloned from the genome of Sporocytophaga sp.CX11.The sequence of Sm_1350 was 2196 bp in length and encoding 732 amino acids.The coding protein was consisted with a signal peptide,GH5 catalytic domain,6 family carbohydrate binding module(CBM6)and a C-terminal domain(CTD).The relative expression levels of Sm_1350 under different carbon source culture conditions were determined by real-time qPCR.The expression level of Sm_1350 in cellobiose and filter paper medium was higher than that in glucose medium,suggesting that it may play a role in the degradation of cellulose by the strain CX11.Sm_1350 and its truncated mutants(Sm_1350,Sm_1350-GH5-CBM6,Sm_1350-GH5 and Sm_1350-CBM6)were constructed and heterologous expressed in E.coli,respectively.The deletion of CTD domain had an effective effect on soluble expression level.When CMC was used as substrate,the enzyme activities of Sm_1350-GH5-CBM6 and Sm_1350-GH5 were(7000.00±50.00)U/g and(2542.73±35.03)U/g,respectively,indicating that the deletion of CBM6 weakened the hydrolysis capacity of Sm_1350-GH5 to soluble polysaccharides.The enzymatic properties showed that both Sm_1350-GH5-CBM6 and Sm_1350-GH5 had wider pH tolerant range.The deletion of CBM6 only changed the optimal temperature,and had no effect on the optimal pH and temperature stability.In addition,Sm_1350-CBM6 exhibites strong ability to bind chitin,which has potential applications in protein purification.

关 键 词：内切葡聚糖酶 生孢噬纤维菌 CBM6结构域 酶学性质 

分 类 号：Q816[生物学—生物工程]