生孢噬纤维菌β-葡萄糖苷酶的异源表达与酶学性质表征  

Heterologous expression and characterization ofβ-glucosidase from Sporocytophaga myxococcoides

作　　者：赵鑫 费嵩禹 宋祥和 陈晓艺[1] ZHAO Xin;FEI Songyu;SONG Xianghe;CHEN Xiaoyi(School of Biological and 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》2022年第5期332-338,共7页Journal of Dalian Polytechnic University

基　　金：辽宁省自然科学基金项目(2020-MS-276)。

摘　　要：β-葡萄糖苷酶是木质纤维素生物降解途径的关键酶,广泛应用于纤维素酶制剂中以提高底物的转化效率。为了挖掘和开发新型β-葡萄糖苷酶,从生孢噬纤维菌CX11基因组中克隆了4个β-葡萄糖苷酶基因Sm_0268、Sm_0864、Sm_3000和Sm_5379,并在大肠杆菌中进行异源表达。重组蛋白Sm_0268、Sm_0864、Sm_3000和Sm_5379分子质量分别为81.7、108.4、83.5和82.1 ku,酶活力分别为(24.2±0.4)U/g、(125.9±0.6)U/g、(32.4±0.6)U/g和(76.1±0.5)U/g。Sm_0268、Sm_3000和Sm_5379的最适反应温度为50℃,Sm_0864的最适反应温度为45℃。Sm_3000在温度达到60℃时仍能保留40%的活性,具有一定的高温催化能力。Sm_0268、Sm_0864和Sm_3000的最适反应pH为7.5,Sm_5379的最适反应pH为7.0,Sm_0864和Sm_3000具有一定的碱耐受性。利用实时荧光定量PCR对编码基因在不同碳源培养条件下的相对表达水平进行分析,结果表明,在以纤维二糖为唯一碳源培养菌株CX11时,4个β-葡萄糖苷酶基因的表达水平均上调,推测它们均不同程度参与CX11降解纤维素的过程。β-glucosidase is a key enzyme in the biodegradation pathway of lignocellulose and widely used in cellulase preparations to improve the conversion efficiency of substrates.To excavate and develop new types ofβ-glucosidase,four genes ofβ-glucosidase(Sm_0268,Sm_0864,Sm_3000,and Sm_5379)were cloned from the genome of Sporocytophaga sp.CX11,and were expressed heterologously in E.coli.The molecular weight of the recombinant proteins Sm_0268,Sm_0864,Sm_3000 and Sm_5379 were 81.7,108.4,83.5 and 82.1 ku,respectively.The enzyme activities were(24.2±0.4)U/g,(125.9±0.6)U/g,(32.4±0.6)U/g and(76.1±0.5)U/g,respectively.The results showed that the optimal temperature of Sm_0268,Sm_3000 and Sm_5379 was 50℃,and the optimal reaction temperature of Sm_0864 was 45℃.Sm_3000 could retain 40%activity when the temperature reached 60℃,showing a certain high-temperature catalytic ability.The optimal reaction pH of Sm_0268,Sm_0864 and Sm_3000 was 7.5,while the optimal reaction pH of Sm_5379 was 7.0.Sm_0864 and Sm_3000 showed certain alkali tolerance.The relative expression levels of the above-mentioned coding genes under different carbon source culture conditions were analyzed by q-RT PCR.The results showed that when cellobiose was used as the sole carbon source,the relative expression levels of the four genes were all up-regulated,suggesting that they were all involved in the process of CX11 degrading cellulose in a certain extent.

关 键 词：Β-葡萄糖苷酶 生孢噬纤维菌 异源表达 酶学性质 

分 类 号：Q816[生物学—生物工程]