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.
Abstract
Genome mining is an efficient tool for discovering biocatalysts,and developing thermal stability β-glucosi-dase has important application value.Seven genes,annotated as β-glucosidase gene or β-glucosidase predicts pro-tein,are excavated using genome mining technology from thermophilic fungus Thermomyces lanuginosus genomic da-ta.Among them,TLG-1,TLG-2,TLG-4,TLG-5,and TLG-6 belong to the GH3 family,while TLG-3 and TLG-7 be-long to the GH1 family and GH17 family,respectively.TLG-7 has low homology(48.43％)with the existing se-quences in the GenBank database.Four β-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 pas-toris.After the recombinant Pichia pastoris is induced by methanol for expression,the supernatant of the fermentation broth is taken for SDS-PAGE analysis and β-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,fol-lowed 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.
基金项目
国家自然科学基金(31660247)
江西省教育厅科学技术研究课题(GJJ202305)
维普
万方数据