疏绵状嗜热丝孢菌β-葡萄糖苷酶的基因组挖掘与表达  

The Genome Mining and Expression for β-Glucosidase of Thermomyces lanuginosus

作　　者：黎小军[1] 杨丽英[1] 彭琛琛[1] 郑建永 LI Xiaojun;YANG Liying;PENG Chenchen;ZHENG Jianyong(Department of Foundamental Medicine,Xinyu University,Xinyu Jiangxi 338004,China;College of Biotechnology and Bioengineering,Zhejiang University of Technology,Hangzhou Zhejiang 310032,China)

机构地区：[1]新余学院基础医学教研室,江西新余338004 [2]浙江工业大学生物工程学院,浙江杭州310032

出　　处：《江西师范大学学报（自然科学版）》2024年第3期247-252,共6页Journal of Jiangxi Normal University(Natural Science Edition)

基　　金：国家自然科学基金(31660247);江西省教育厅科学技术研究课题(GJJ202305)资助项目。

摘　　要：基因组挖掘是发现生物催化剂的高效工具,开发热稳定性β-葡萄糖苷酶具有重要的应用价值.该文采用基因组挖掘策略,从嗜热真菌疏绵状嗜热丝孢菌的基因组中挖掘出7个注释为β-葡萄糖苷酶基因或β-葡萄糖苷酶预测蛋白基因,其中TLG-1、TLG-2、TLG-4、TLG-5、TLG-6属于GH3家族,TLG-3和TLG-7分别属于GH1家族和GH17家族,且TLG-7与GenBank数据库已有序列的同源性很低,只有48.43%.选择同源性低的、同源序列被研究少的TLG-2、TLG-5、TLG-6和TLG-7进行基因克隆和毕赤酵母异源表达,重组毕赤酵母经甲醇诱导表达后,用发酵液上清进行SDS-PAGE分析,结果表明TLG-2、TLG-5、TLG-6和TLG-7均成功分泌表达.对发酵液进行β-葡萄糖苷酶活力测定结果表明:TLG-2的活力最高,达到166.6 U·mL^(-1);其次是TLG-5的活力,其值为79.8 U·mL^(-1),TLG-6和TLG-7的活力较低,分别为9.9 U·mL^(-1)和9.5 U·mL^(-1).研究结果为疏绵状嗜热丝孢菌β-葡萄糖苷酶的应用奠定了基础.Genome mining is an efficient tool for discovering biocatalysts,and developing thermal stabilityβ-glucosidase has important application value.Seven genes,annotated asβ-glucosidase gene orβ-glucosidase predicts protein,are excavated using genome mining technology from thermophilic fungus Thermomyces lanuginosus genomic data.Among them,TLG-1,TLG-2,TLG-4,TLG-5,and TLG-6 belong to the GH3 family,while TLG-3 and TLG-7 belong to the GH1 family and GH17 family,respectively.TLG-7 has low homology(48.43%)with the existing sequences in the GenBank database.Fourβ-glucosidase(TLG-2,TLG-5,TLG-6,and TLG-7),with low homology and limited research on homologous sequences,are selected for gene cloning and heterologous expression in Pichia pastoris.After the recombinant Pichia pastoris is induced by methanol for expression,the supernatant of the fermentation broth is taken for SDS-PAGE analysis andβ-glucosidase activity testing.The results show that TLG-2,TLG-5,TLG-6,and TLG-7 are successfully secretory expressed.TLG-2 has the highest activity,reaching 166.6 U·mL^(-1),followed by TLG-5 with 79.8 U·mL^(-1),while TLG-6 and TLG-7 have lower activity,only 9.9 U·mL^(-1) and 9.5 U·mL^(-1),respectively.The above results provid some references for the application ofβ-glucosidase from T.lanuginosus.

关 键 词：基因组挖掘 Β-葡萄糖苷酶 疏绵状嗜热丝孢菌 异源表达 

分 类 号：Q786[生物学—分子生物学]