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【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°C,在90°C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】 The purpose of this study was to explore new esterases from soil microorganisms using metagenomic techniques. 【Method】 The library of soil microbial metagenome was constructed. The constructed library was screened by glycerol tributyrin assay. The esterase gene identified in positive clones was analyzed for its heterologous expression and biochemical characteristics. [Results] A total of 120,000 clones in the library were screened and a positive clone was obtained. Sequence analysis of the DNA fragment in the clone revealed one possible esterase gene. The optimal pH was determined by studying its expression product 9.0, the optimum reaction temperature is 56 ° C, at 90 ° C can still maintain 20% of the enzyme activity; specific hydrolysis of short-chain lipids, no hydrolysis of long-chain lipids; to a certain concentration range Organic reagents such as dimethylsulfoxide, methanol, ethanol have better tolerance, especially when the content of dimethyl sulfoxide is 10% (volume ratio), the relative activity can be increased by 44%. 【Conclusion】 The results showed that ESTYN1, a novel esterase with good resistance to high temperature and organic reagents, could be used to discover novel active enzymes. It has the potential of industrial application.