湖羊瘤胃微生物来源的低温葡聚糖酶的表达与功能表征  

Expression and functional characterization of a low-temperature glucanase derived from rumen microbiota of Hu sheep

作　　者：丁钰杰 徐晓锋 韩俊彦[1,2] 周叶波 麻子莹 沈桢祎 钱国英 王佳堃[2] 徐洁皓[1] 王谦 DING Yujie;XU Xiaofeng;HAN Junyan;ZHOU Yebo;MA Ziying;SHEN Zhenyi;QIAN Guoying;WANG Jiakun;XU Jiehao;WANG Qian(College of Biological and Environmental Sciences,Zhejiang Wanli University,Ningbo 315100,Zhejiang,China;The Institute of Dairy Science,Zhejiang University,Hangzhou 310058,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]浙江大学奶业科学研究所,浙江杭州310058

出　　处：《浙江大学学报（农业与生命科学版）》2024年第4期531-541,共11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);浙江省教育厅一般科研项目(Y202353501);浙江省“生物工程”一流学科(A类)学生创新计划项目(CX2023001)。

摘　　要：葡聚糖酶作为添加剂在饲料、食品和纺织工业中具有重要的应用价值。本研究从湖羊瘤胃微生物c DNA中扩增IDSGH5-50基因,通过大肠埃希菌BL21(DE3)进行异源表达,研究重组蛋白rIDSGH5-50的酶学性质和水解产物。结果表明:IDSGH5-50编码690个氨基酸,蛋白质分子量为75.78 kDa,等电点为4.75;重组蛋白rIDSGH5-50的最适反应条件为温度30℃、pH 6.0,且能在4~20℃下保持较高活性(>70%),在pH 5.0~8.0范围内稳定性较高。rIDSGH5-50的活性底物谱分析显示,rIDSGH5-50能催化大麦β-葡聚糖、地衣多糖、罗望子木葡聚糖和魔芋胶,对应的比活性分别为(10.42±0.16)、(7.12±0.08)、(7.03±0.38)、(5.94±0.65) U/mg。薄层色谱分析表明,在rIDSGH5-50催化下,大麦β-葡聚糖主要降解成纤维四糖和纤维五糖,地衣多糖主要降解成纤维三糖和纤维五糖,魔芋胶主要降解成纤维五糖,罗望子木葡聚糖主要降解成聚合度>5的寡糖。本研究报道了一种来自瘤胃球菌属的低温内切β-1,4-葡聚糖酶IDSGH5-50,为饲料、食品酶制剂开发奠定了基础。Glucanase is an important additive with significant application value in feed,food,and textile industries.In this study,the IDSGH5-50 gene was amplified from the rumen microbiota cDNA of Hu sheep and heterologously expressed in Escherichia coli BL21(DE3)to investigate its enzymatic properties and hydrolysis products.The results revealed that IDSGH5-50 encoded 690 amino acids,with a molecular weight of 75.78 kDa and an isoelectric point of 4.75.The optimal reaction conditions for rIDSGH5-50 were 30℃and pH 6.0,and it maintained high activity(>70%)at temperatures of 4-20℃.It exhibited stable activity at pH 5.0-8.0.The active substrate spectrum of rIDSGH5-50 showed that rIDSGH5-50 could catalyze barleyβ-glucan,lichenin,tamarind xyloglucan,and konjac gum,and the specific activities of them were(10.42±0.16),(7.12±0.08),(7.03±0.38),and(5.94±0.65)U/mg,respectively.Thin layer chromatography analysis showed that barleyβ-glucan was mainly degraded to cellotetrose and cellopentose,that lichenin was mainly degraded to cellotriose and cellopentose,that konjac gum was mainly degraded to cellopentose,and that tamarind xyloglucan was mainly degraded to oligosaccharides with a degree of polymerization>5,which was catalyzed by rIDSGH5-50.This study reported a cold-adaptedβ-1,4-glucanase IDSGH5-50 from the genus Ruminococcus,establishing the foundation for enzyme preparations used in feed and food enzyme preparation.

关 键 词：湖羊瘤胃 葡聚糖酶 低温 冷适应 底物水解 

分 类 号：Q814.4[生物学—生物工程]