New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library
New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library
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DOI：
10.1128/aem.71.2.817-825.2005
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发表时间：
2005-02-01
影响因子：
4.4
通讯作者：
Oh, JW
中科院分区：
文献类型：
--
作者：
Rhee, JK;Ahn, DG;Oh, JW
利用热环境样品中的元基因组构建的融合环境DNA文库，对转化的大肠杆菌细胞进行功能筛选，获得了耐热酯酶基因。该基因全长936个碱基，对应311个氨基酸残基，相对分子质量为34 kDa。该酶与嗜热古细菌的酶有显著的氨基酸相似性(%)。与其他酯酶和脂肪酶的氨基酸序列比较表明，该酶应被归类为激素敏感脂肪酶家族的一个新成员。从大肠杆菌中高效表达和纯化的重组酯酶活性在30℃以上，最高可达95℃，并具有较高的热稳定性。在pH为5.5~7.5的范围内表现出较高的活性，最适pH约为6.0。在所考察的对硝基苯酯(C-4~C-16)中，酶的最适底物为对硝基苯己酸酯(C-6)，含有大于10个碳原子的酰基链长的酯不具有脂肪分解活性，表明该酶是一种酯酶，而不是脂肪酶。
A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30degreesC up to 95degreesC and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C-4 to C-16) examined was p-nitrophenyl caproate (C-6), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.