来源于生孢噬纤维菌的新型β-葡萄糖苷酶SmBgl3A的性质研究  

Characterization of a Novelβ-glucosidase SmBgl3A from Sporocytophaga sp.CX11

作　　者：费嵩禹 王梓懿 荆晓凤 杨帆[1] 陈晓艺[1] 李宪臻[1] FEI Songyu;WANG Ziyi;JING Xiaofeng;YANG Fan;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：《食品与生物技术学报》2023年第1期83-92,共10页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金青年基金项目(31801469);辽宁省自然科学基金项目(2020-MS-276)。

摘　　要：通过表达水平分析比较,从生孢噬纤维菌Sporocytophaga sp.CX11基因组中筛选出在菌株降解纤维二糖过程中起关键作用的β-葡萄糖苷酶基因Sm Bgl3A。该基因含有2283 bp碱基对,编码760个氨基酸,其编码蛋白质与来源于Bacteroides ovatus的β-葡萄糖苷酶Bo GH3B同源性为46.76%。将该基因在大肠杆菌中进行异源表达,粗酶液经Ni-NTA亲和层析一步纯化得到电泳级纯度的酶蛋白Sm Bgl3A,重组蛋白质相对分子质量与理论值(8.12×10^(4))一致。酶学性质表征结果表明,Sm Bgl3A最适温度和p H分别为35℃和6.5;p H在5.0~7.0、温度在40℃以下时,稳定性较好,同时有着较好的Na Cl耐受性。Sm Bgl3A的最适底物为p NPG,以p NPG为底物时的比活力高达14.74 U/mg;K_(m)值为10.88 mmol/L,k_(cat)值为46.72 s^(-1)。Sm Bgl3A作为一种新型的、酶学性质优良的β-葡萄糖苷酶,进一步扩展了β-葡萄糖苷酶的种类,为该酶在纤维素降解、食品工业等领域的应用奠定基础。Aβ-glucosidase gene Sm Bgl3A,which might play a key role in cellobiose degradation and utilization,was screened from Sporocytophaga sp.CX11 genome through the analysis and comparison of expression level.The gene contains 2283 bp and encodes 760 amino acids.The encoded protein of Sm Bgl3A has 46.76%homology with theβ-glucosidase Bo GH3B from Bacteroides ovatus.The gene Sm Bgl3A was heterologously expressed in Escherichia coli.The electrophoretically pure recombinant protein Sm Bgl3A was achieved by the purification of crude enzymes using Ni-NTA affinity chromatography,and the relative molecular weight of recombinant protein was consistent with the theoretical value(8.12×10~4).Characterization of enzymatic properties showed that the optimum temperature and p H of Sm Bgl3A were 35℃and 6.5,respectively.When the p H value was 5.0~7.0 and the temperature was below 40℃,Sm Bgl3A was stable.In addition,Sm Bgl3A showed good Na Cl tolerance.The optimal substrate for Sm Bgl3A was p NPG,and the specific enzyme activity with p NPG as substrate was 14.74 U/mg.K_(m)and k_(cat)of Sm Bgl3A towards p NPG were 10.88 mmol/L and 46.72 s^(-1),respectively.Sm Bgl3A is a novel enzyme with excellent enzymatic properties,further expanding the kinds ofβ-glucosidase and laying a foundation for the enzyme application in cellulose degradation,food industry and other fields.

关 键 词：葡萄糖苷酶 GH3家族 生孢噬纤维菌CX11 异源表达 酶学性质 

分 类 号：Q814[生物学—生物工程]