白色瘤胃球菌双功能糖苷水解酶IDSGH5-23的表达与性质分析  

Expression and characterization of a bifunctional glycoside hydrolase IDSGH5-23 from Ruminococcus albus

作　　者：韩俊彦 丁钰杰 尉俏女 徐晓锋 章成昌 张骥 王佳堃[2] 尹尚军[1] 徐洁皓 王谦 HAN Junyan;DING Yujie;WEI Qiaonü;XU Xiaofeng;ZHANG Chengchang;ZHANG Ji;WANG Jiakun;YIN Shangjun;XU Jiehao;WANG Qian(College of Biological and Environmental Sciences,Zhejiang Wanli University,Ningbo 315100,Zhejiang,China;Institute of Dairy Science,College of Animal Sciences,Zhejiang University,Hangzhou 310058,Zhejiang,China;Start Bioservices Co.,Ltd.,Hangzhou 310018,Zhejiang,China)

机构地区：[1]浙江万里学院生物与环境学院,浙江宁波315100 [2]浙江大学动物科学学院奶业科学研究所,浙江杭州310058 [3]杭州云心质力生物科技有限公司,浙江杭州310018

出　　处：《浙江大学学报（农业与生命科学版）》2024年第6期963-972,共10页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：浙江省“尖兵”“领雁”研发攻关计划项目(2022C02043);浙江省“生物工程”一流学科自设课题项目(ZS2023008)。

摘　　要：葡聚糖酶和木聚糖酶等糖苷水解酶(glycoside hydrolase,GH)是能水解、消减饲料中非淀粉多糖类抗营养因子酶制剂的重要组分。本研究基于前期构建的湖羊瘤胃微生物宏转录组文库信息,从瘤胃微生物c DNA中克隆了一种兼具葡聚糖酶和木聚糖酶双功能酶的基因IDSGH5-23,将其在大肠埃希菌中进行异源表达,并研究了重组IDSGH5-23蛋白的催化性质。结果表明:IDSGH5-23的理论分子量和等电点分别为45.8 kDa和4.44,与来自白色瘤胃球菌(Ruminococcus albus)的GH5蛋白(GenBank登录号:WP_013499238.1)的氨基酸序列相似度达99.51%。在本试验条件下,IDSGH5-23的最适反应温度和pH值分别为30℃和6.0,在pH 4.0~7.0范围内较为稳定;IDSGH5-23能高效催化大麦β-葡聚糖、地衣多糖、魔芋胶和榉木木聚糖,其中,水解地衣多糖主要生成纤维三糖,水解大麦β-葡聚糖生成聚合度大于3的纤维寡糖,水解榉木木聚糖生成木三糖至木六糖。综上所述,本研究获取了一个来自瘤胃微生物的兼具葡聚糖酶和木聚糖酶双功能酶的IDSGH5-23,为研发饲用酶制剂和制备寡糖奠定了基础。Glycoside hydrolases such as glucanase and xylanase are important components of enzyme preparations that can hydrolyze and ablate non-starch polysaccharide antinutritional factors in feed.According to the metatranscriptomic data obtained previously from rumen microbiota in Hu sheep,a novel bifunctional glucanase/xylanase gene,IDSGH5-23,was cloned from cDNA of rumen microbiota.The gene was heterologously expressed in Escherichia coli,and the recombinant enzyme was functionally characterized.The results showed that the theoretical molecular weight and isoelectric point of IDSGH5-23 were 45.8 kDa and 4.44,exhibiting 99.51%similarity to amino acid sequence of GH5 protein(GenBank accession number:WP_013499238.1)from Ruminococcus albus.The IDSGH5-23 had the optimum temperature of 30℃and the optimum pH of 6.0,and remained stable ranging of pH 4.0-7.0.Moreover,the IDSGH5-23 was robust in catalyzing barleyβ-glucan,lichenan,konjac gum and beechwood xylan.The enzyme IDSGH5-23 mainly released cellotriose from lichenan or cellooligosaccharides with a degree of polymerization beyond 3 from barleyβ-glucan.Besides,the enzyme IDSGH5-23 was capable of generating xylotriose,xylotetraose,xylopentaose and xylohexaose from beechwood xylan.In summary,a bifunctional glucanase/xylanase protein derived from rumen microbes,IDSGH5-23,is obtained and characterized in this study,laying a foundation for feed enzyme development and oligosaccharides preparation.

关 键 词：瘤胃微生物 葡聚糖酶 木聚糖酶 催化性质 底物水解 

分 类 号：Q814.4[生物学—生物工程]