[Background] Only a small proportion of microbes can be cultured in laboratories,thereby hampering the discovery of novel β-glucosidases from microbes.Novel microbial β-glucosidases can be discovered using culture-independent metagenomic approach.[Objective] Discovery of new β-glucosidase from soil microbes by metagenomic technology.[Methods] Screening a metagenomic library on esculin-containing Luria-Bertani agar plates and the putative β-glucosidase gene derived from the active clone was heterologously expressed for characterization.[Results] We found a β-glucosidase active clone by screening 620 000 clones in the library and an ORF (YNBG3) whose product is homologous with the third family of β-glucosidase was identified from the active clone.YNBG3 was heterologously expressed and characterized.Its optimum pH is 5.2,and optimal temperature is 53 ℃.YNBG3 showed good tolerance to organic solvents including dimethyl sulphoxide,acetone and ethanol,and the activity of YNBG3 was enhanced in presence of EDTA and urea.[Conclusion] A new 3-glucosidase,with thermostability and stability in the presence of organic solvents and urea,was discovery in this study.%[背景]通过培养微生物来获得新p-葡萄糖苷酶因只有少部分微生物可以被培养而受到限制,但宏基因组学技术可以挖掘非培养微生物来源的β-葡萄糖苷酶资源.[目的]利用宏基因组学技术挖掘土壤微生物来源的新型p-葡萄糖苷酶,并对其酶学性质进行初步分析.[方法]构建土壤微生物的宏基因组文库,利用七叶苷平板显色法对所构建的文库进行筛选获得阳性克隆,并对阳性克隆所含的p-葡萄糖苷酶基因进行异源表达和生物化学特性分析.[结果]通过筛选文库中的62万个克隆,获得一个具有β-葡萄糖苷酶活性的克隆,其插入片段中包含一个2 301 bp的ORF (YNBG3),蛋白同源性分析显示其属于β-葡萄糖苷酶第三家族.对YNBG3酶的生化分析确定其最佳反应温度为53℃,最适pH为5.2,有较好的热稳定性,对一定浓度范围内的DMSO、丙酮、乙醇等有机试剂有较好的耐受性,EDTA和尿素可增加该酶的活性.[结论]利用宏基因组学技术获得了一个有较好热稳定性及耐受一定浓度有机试剂和尿素的新β-葡萄糖苷酶.
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