深海沉积物微生物元基因组文库来源的新的酯酶基因的克隆、表达及酶学性质  

Cloning,expression and characterization of a novel esterase from marine sediment microbial metagenomic library

作　　者：徐士庆[1] 胡永飞[1] 袁爱花[1] 朱宝利[1] 

机构地区：[1]中国科学院微生物研究所,中国科学院病原微生物与免疫学重点实验室,北京100101

出　　处：《微生物学报》2010年第7期891-896,共6页Acta Microbiologica Sinica

基　　金：国家"863计划"(2007AA09Z443;2007AA021301);中国科学院知识创新工程项目(KSCX2-YW-G-022);国家自然科学基金(30770060)~~

摘　　要：【目的】从深海沉积物微生物元基因组文库中克隆新的酯酶基因,并进行酶学性质研究。【方法】利用含有三丁酸甘油酯的酯酶选择性筛选培养基,从深海沉积物微生物元基因组文库中筛选得到酯酶阳性Fosmid克隆。对筛选得到的fosmid FL10进行部分酶切构建亚克隆文库,筛选得到酯酶阳性亚克隆pFLS10。PCR扩增目的片段后与pET28a连接构建酯酶基因原核表达质粒,转化大肠杆菌(Escherichia coli)BL21。纯化表达产物并对其进行活性测定及酶学性质研究。【结果】序列分析显示该pFLS10亚克隆质粒含有一段924bp的ORF(Open Reading Frame),与一海洋元基因组文库中筛选出的酯酶ADA70030序列一致性为71%。该酶为一新的低温酯酶,对C4底物(对硝基苯丁酸酯)水解能力最强。该酶最适作用温度为20℃,最适作用pH为7.5,20℃时较为稳定,pH8-10的范围内有良好的pH稳定性,K+、Mg2+对该酶具有一定的激活作用,Mn2+等对其具有不同程度的抑制作用。【结论】应用元基因组技术筛选到了新的酯酶基因fls10并进行了克隆表达,该酶在低温及碱性条件下较为稳定且活力较高,对于工业化生产具有一定的应用潜力。关键词:深海沉积物;元基因组文库;低温酯酶;[Objective]To clone,express and characterize a novel esterase from marine sediment microbial metagenomic library.[Methods]Using esterase segregation agar containing tributyrin,we obtained esterase positive fosmid clone FL10 from marine sediment microbial metagenomic library.This fosmid was partially digested with Sau3A I to construct the sublibrary,from which the esterase positive subclone pFLS10 was obtained.The full length of the esterase gene was amplified and cloned into the expressing vector pET28a,and the recombinant plasmid was transformed into E.coli BL21 cells.We analyse the enzyme activity and study the characterization of the esterase after its expression and purification.[Results]An ORF （Open Reading Frame） of 924 bp was identified from the subclone pFLS10.Sequence analysis indicated that it showed 71% amino acid identity to esterase （ADA70030） from a marine sediment metagenomic library.The esterase is a novel low-temperature-active esterase and had highest lipolytic activity to the substrate of 4-nitrophenyl butyrate （C4）.The optimum temperature of the esterase was 20 ℃,the optimum pH was 7.5.The esterase in this study had good thermostability at 20 ℃ and good pH stability at pH8-10.Significant increase in lipolytic activity was observed with addition of K ^＋ and Mg^2 ＋,while decrease with Mn^2 ＋ etc.[Conclusion]We obtained the novel esterase gene fls10 from the marine sediment microbial metagenomic library.The esterase had good thermostability and high lipolytic activity at low temperature and under basic conditions,which laid a basis for industrial application.

关 键 词：深海沉积物 元基因组文库 低温酯酶 酶学特征 

分 类 号：Q93[生物学—微生物学] Q78