A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea.
A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea.
复制标题
DOI：
10.1186/1475-2859-10-95
复制
发表时间：
2011-11-09
影响因子：
6.4
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Peng Q;Zhang X;Shang M;Wang X;Wang G;Li B;Guan G;Li Y;Wang Y
海洋微生物是一个庞大而多样的群体，它们暴露在各种压力、温度、盐度、营养物质供应和其他环境条件下。它们提供了具有独特性质的新型酶的巨大潜在来源，这些酶可能在工业和生物技术中有用。为探索南海的脂解遗传资源，在水深< 100 m的海区采集了23个沉积物样品。以质粒载体为载体，构建了南海沉积物宏基因组文库，文库中含有约194 Mb的群落DNA。筛选未扩增文库的一部分，分离出15个具有水解三丁酸甘油酯能力的独特脂解克隆。在大肠杆菌中成功表达了一个含有新型酯酶（Est_p1）的阳性重组克隆（pNLE 1）。coli中进行纯化。在一系列测定中，Est_p1在pH 8.57，40°C下显示最大活性，对硝基苯丁酸酯（C4）作为底物。与其它宏基因组酯酶相比，Est_p1对底物C4具有显著的专一性（kcat/Km值为11，500 S-1 m M-1），且不受苯甲基磺酰氟的抑制，表明该酶的底物结合口袋适合底物C4，丝氨酸活性位点残基被掩埋在底物结合口袋的底部，被盖子结构所遮蔽。酯酶对短链脂肪酸，特别是丁酸具有专一性，是一种重要的调味工具。由于Est_p1对底物C4具有良好的活性和专一性，因此在需要水解短链酯类的香料工业中具有潜在的应用价值。
Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth < 100 m marine areas. A metagenomic library of South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters.
登录
查看更多内容
影响因子：
64.8
作者：
Bowler, Chris;Karl, David M.;Colwell, Rita R.
通讯作者：
Colwell, Rita R.
源自元基因组的新型拟除虫菊酯 - 氢化酯酶的分子克隆和表征。
影响因子：
6.4
作者：
Li G;Wang K;Liu YH
通讯作者：
Liu YH
影响因子：
14.9
作者：
Fischer, M;Pleiss, J
通讯作者：
Pleiss, J
影响因子：
4.4
作者：
Lee, Mi-Hwa;Lee, Choong-Hwan;Yoon, Jung-Hoon
通讯作者：
Yoon, Jung-Hoon
影响因子：
4.4
作者：
Heath, Caroline;Hu, Xiao Ping;Cowan, Donald
通讯作者：
Cowan, Donald