摘要
【目的】葡聚糖酶是饲用添加剂的重要成分,本研究旨在从湖羊消化道微生物中挖掘性质优良的GH9家族葡聚糖酶基因,用于研发新型饲用酶制剂。【方法】从湖羊瘤胃微生物cDNA中扩增IDSGLUC9-25基因,在大肠杆菌中进行异源表达,对重组蛋白进行诱导表达和纯化,研究重组蛋白的酶学性质和底物水解模式。【结果】IDSGLUC9-25基因编码527个氨基酸,包含一个CelD_N结构和一个GH9家族催化结构域;重组蛋白rIDSGLUC9-25分子量约为62.7 kDa,最适反应温度和pH分别为40℃和6.0,在30–50℃下活性较高,在pH 4.0–8.0范围内能够保持较高的稳定性,经pH4.0–8.0缓冲液处理1h后残余活性均大于90%;底物谱分析表明,rIDSGLUC9-25能催化大麦β-葡聚糖、苔藓地衣多糖、魔芋胶和木葡聚糖,比活性分别为(443.55±24.48)、(65.56±5.98)、(122.37±2.85)和(159.16±7.73)U/mg;利用薄层色谱法(thinlayer chromatography,TLC)和高效液相色谱法(high performance liquid chromatography,HPLC)分析水解产物发现,rIDSGLUC9-25降解大麦葡聚糖主要生成纤维三糖(占总还原糖64.19%±1.19%)和纤维四糖(占总还原糖26.24%±0.12%),催化地衣多糖主要生成纤维三糖(占总还原糖78.46%±0.89%)。【结论】本研究报道了一种来自密螺旋体属细菌的内切β-1,4-葡聚糖酶IDSGLUC9-25(EC3.2.1.4),能高效催化多糖底物生成纤维三糖和纤维四糖,为研发饲用酶制剂和制备低聚寡糖建立基础。
[Objective]Glucanases serve as one of the main components in feed additives.This study identified and characterized a novel GH9 glucanase gene derived from rumen microbiota in herbivores,aiming to provide a reference for the research and development of feed enzymes.[Methods]We obtained the IDSGLUC9-25 gene from the rumen fluid cDNA of Hu sheep and heterologously expressed it in Escherichia coli.The recombinant protein was induced for expression by isopropylβ-D-thiogalactopyranoside,purified,and then subjected to functional characterization.[Results]IDSGLUC9-25 encoded a protein consisting of 527 amino acid residues,which included a CelD_N domain and a GH9 family catalytic domain.The recombinant rIDSGLUC9-25 protein exhibited a molecular weight of approximately 62.7 kDa and the highest enzymatic activity at 40°C and pH 6.0.The enzyme displayed robust catalytic activity within the temperature range of 30–50°C.After preincubation at pH 4.0–8.0 for 1 h,rIDSGLUC9-25 retained the relative activity over 90%.The substrate spectrum analysis revealed that rIDSGLUC9-25 exhibited specific activities against barleyβ-glucan,moss lichenan,konjac gum,and xyloglucan,with the activities of(443.55±24.48),(65.56±5.98),(122.37±2.85),and(159.16±7.73)U/mg,respectively.The hydrolysis assay showed that rIDSGLUC9-25 primarily catalyzed the hydrolysis ofβ-glucan into cellotriose(representing 64.19%±1.19%of total reducing sugars)and cellotetraose(representing 26.24%±0.12%of total reducing sugars).Additionally,the enzyme predominantly generated cellotriose from the hydrolysis of lichenan(representing 78.46%±0.89%of total reducing sugars).[Conclusion]This study characterizes IDSGLUC9-25,an endo-β-1,4-glucanase(EC 3.2.1.4)derived from Treponema sp.The enzyme exhibited robust activity in the conversion of polysaccharides into cellotriose and cellotetraose,establishing a foundation for the development of feed enzymes and functional oligosaccharides preparation.
作者
徐晓锋
韩俊彦
丁钰杰
廖静
高德英
张骥
王佳堃
尹尚军
王谦
徐洁皓
XU Xiaofeng;HAN Junyan;DING Yujie;LIAO Jing;GAO Deying;ZHANG Ji;WANG Jiakun;YIN Shangjun;WANG Qian;XU Jiehao(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)
出处
《微生物学报》
CAS
CSCD
北大核心
2024年第3期755-766,共12页
Acta Microbiologica Sinica
基金
浙江省研发攻关计划(2022C02043)
浙江省“生物工程”一流学科自设课题(ZS2023008)。
