【作者】 金鹏；

【导师】 王秋岩；

【作者基本信息】 杭州师范大学 ， 遗传学， 2012， 硕士

【摘要】 近年来,随着宏基因组技术的出现和广泛使用,显著拓展了人们对环境微生物多样性的认识。宏基因组技术是指不经过微生物培养,直接提取环境微生物的总基因组的技术。使用该技术,对原本在环境中超过99%的不可培养微生物基因的操作和研究成为了现实,这促使人们发现了大量具有价值的新的蛋白和活性小分子物质。本课题研究采用宏基因组技术,针对来源于贵州铜仁梵净山的土壤微生物样本,构建了一个库容克隆数量达到5×104的宏基因组文库。基于三丁酸甘油酯水解生成透明圈的功能筛选方法,从文库中分离出新酯酶全长基因1条(命名为EstC23)。进化关系分析表明,该酶隶属于脂肪酶第Ⅳ家族(Hormo ne-sens itive lipase family, HSL)。对该基因的异源过表达研究表明,该基因在T7强启动子作用下,产生的可溶性蛋白占细胞总蛋白量的30%左右。纯酶蛋白的酶学性质表征显示,EstC23在25℃pH8.0条件下,对pNP-butyrat的酶活达254U mg1,该酶的最适反应温度为40℃,特别是在5-10℃低温下仍能维持最大活性的50%。EstC23在浓度达50%(v/v)的非极性有机溶剂(高logP)中显现出极强的稳定性,尤其在50%(v/v)的苯类和烷烃类有机溶剂中,其活力能提升至92%至274%之间,孵育一周后酶活性不会降低。同时,酯酶EstC23能水解叔醇酯底物,这是通常的酯酶或脂肪酶不具备的底物活性。这些结果表明,EstC23在酯合成、手性化合物的拆分、不稳定中间体化合物合成、肽合成等方面具有潜在应用价值。后续我们以EstC23核酸序列为基础设计特异性引物,基于宏基因组特异性PCR技术(Metagenomic Gene Specific PCR, MGS-PCR)和截断宏基因组特异性PCR技术(Truncated Metagenomic Gene Specific PCR, TMGS-PCR),对来自全国不同地域环境的65组宏基因组DNA进行序列筛选,分别获得了同源家族全长功能基因7条和核心片段15条,序列分析比对,这些基因及基因片段表现出了高度的序列多样性,其核酸相同百分比在50-97%之间,这些序列构成了一个新的酯酶基因亚家族。这为后续以酯酶亚家族为对象的功能研究,以及DNA-family-shuffling为方法的体外功能进化,提供了有价值的实验材料。更多还原

【Abstract】 In the past decade, advances in the field of metagenomics have dramatically revised our view of biodiversity. Considering the estimation that more than99%of microorganisms in most environments are not amenable to culturing, very little knowledge is known about their genomes. The isolation, archiving, and analysis of environmental DNA (or so-called metagenomes) have enabled us to mine microbial diversity, allowing us to access their genomes. In which researchers have found a large number of valuable new active substances.In this study, An environmental DNA library approximately50,000metagenomic clones was constructed using soil sample from Fanjingshan of Tongren, Guizhou. Function-based metagenomics screening, a new esterase designated EstC23was isolated, which evolutionary relationship analysis showed that this enzyme belongs to the lipase IV family (Hormone-sensitive lipase family, HSL). The protein was amenable to overexpression in Escherichia coli under control of the T7promoter, resulting in expression of the active, soluble protein that constituted30%of the total cell protein content. Characterization of enzymatic properties show that the specific activity of EstC23on p-nitrophenyl butyrate was254U mg-1at25℃and pH8.0, EstC23showed optimal activity at40℃and retained about50%maximal activity at5-10℃. EstC23showed remarkable stability in up to50%(v/v) benzene and alkanes (high logP solvents). When incubated for7days in the presence of50%benzene or alkanes, the enzyme maintained its92%-274%elevated activity. The purified enzyme also cleaved sterically hindered esters of tertiary alcohols. These results indicate that EstC23has potential for using in ester synthesis, chiral compounds separation, unstable intermediate compound synthesis and peptide synthesis.In subsequent research work, based on EstC23specific primers, we employed Metagenomic Gene Specific PCR (MGS-PCR) and Truncated Metagenomic Gene Specific PCR (TMGS-PCR) to screen homologous family genes of EstC23with65metagenomics DNA samples derived from domestic different geographies. We identified seven lipolytic activity full-length genes and fifteen homologous family core fragments, sequences analysis showed a broad sequence diversity and a wild rang of sequence identity (50-97%), which constitute a new esterase subfamily.This study provides a valuable experimental material for the following work such as function of research on esterase subfamily or evolution in vitro DNA family Shuffling.更多还原