[目的]利用宏基因组学技术挖掘土壤微生物来源的新型酯酶.[方法]构建土壤微生物宏基因组文库,利用三丁酸甘油酯平板法对所构建的文库进行筛选,并对阳性克隆中鉴定出的酯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12万个克隆,获得了一个阳性克隆,对克隆中的DNA片段进行序列分析,发现了一个可能的酯酶基因,通过研究其表达产物,确定其最适pH为9.0,最适反应温度为56℃,在90℃下仍可保持20％的酶活性;能专一性水解短链脂类,对长链脂类无水解作用;对一定浓度范围内的有机试剂如二甲基亚砜、甲醇、乙醇有较好的耐受性,尤其当二甲基亚砜含量为10％(体积比)时,相对酶活可提高44％.[结论]不依赖于微生物可培养性的宏基因组学技术可以发现新的活性酶,本研究获得的对高温、有机试剂有较好耐受性的酯酶ESTYN1具有在工业生产中应用的潜力.%[Objective] To discover novel esterase from soil microbes by metagenomic approach.[Methods] Tributyrin-containing LB agar plates were used to screen a metagenomic library and the predictive esterase gene on the positive clone was heterologously expressed for biochemical analysis.[Results] We successfully found a positive clone from a soil microbial metagenomic library that comprised of 120 000 clones by function-based screening.Then the putative esterase gene (estyn1) of this clone was heterologously expressed and its properties including optimum pH,optimal temperature,and tolerance to organic solvents and metal ions were studied.Its optimum pH was 9.0 and its optimal temperature was 56 ℃.The esterase activity was enhanced in low concentration of organic solvents,especially in DMSO (dimethylsulfoxide,activity enhanced to 144％ at 10％ DMSO).[Conclusion] Novel enzymes could be discovered by metagenomic approach.ESTYN1,with good tolerance to organic solvents and high temperature,would be a promising enzyme for industrial production.
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