Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People's Republic of China.
Appl Microbiol Biotechnol. 2011 May;90(3):961-70. doi: 10.1007/s00253-010-3079-0. Epub 2011 Feb 19.
A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K(m) of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0-60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0-10.0 showing the highest activity at pH 9.0.
从南海海洋沉积物样本构建的 fosmid 文库中鉴定出一种具有低温脂肪酶活性的克隆。负责该基因的 estF 由 1080bp 组成，编码 359 个氨基酸残基，具有典型的 28 个氨基酸残基的 N 端信号肽。与其他脂肪酶的氨基酸序列系统发育分析表明，EstF 和七个密切相关的假定脂肪酶在系统发育树中组成了一个独特的分支。此外，这些假定的酯酶在氨基酸序列中表现出独特的保守位点。重组 EstF 被过表达和纯化，并部分表征了其生化特性。EstF 水解一系列对硝基苯酚酯（C2 到 C16）的最佳底物是对硝基苯酚丁酸酯（C4），其 K(m)为 0.46mM。活性随着酰基链长超过 10 个碳原子的底物迅速下降。我们发现 EstF 在 0-60°C 的温度范围内具有活性，在 50°C 时表现出最佳活性，但在 60°C 时不稳定。它在 pH 值为 7.0-10.0 的范围内表现出高水平的活性，在 pH 值为 9.0 时表现出最高的活性。
