Department of Agricultural Chemistry, Sunchon National University, Suncheon 540-742, Republic of Korea.
Biochem Biophys Res Commun. 2010 Feb 26;393(1):45-9. doi: 10.1016/j.bbrc.2010.01.070. Epub 2010 Jan 25.
A metagenomic library was constructed from completely fermented compost using a fosmid vector. From a total of 23,400 clones, 19 esterase-positive clones were selected on LB plates containing 1% glyceryl tributyrate as the substrate. The esterase gene of an esterase-positive clone, est2K, was on an ORF of 1299 bp and encoded a protein of 432 amino acids. Est2K had a SMTK motif and was a family VIII esterase. Unlike most family VIII esterases, Est2K had a signal peptide of 27 amino acids. The molecular mass and pI of the mature Est2K was calculated to be 44,668 Da and 4.48, respectively. The amino acid sequence of Est2K showed 72% identity with that of EstC, an esterase of an uncultured bacterium from leachate. The purified Est2K was optimally active at pH 10.0 and 50 degrees C. Est2K was stable in the presence of 30% methanol and exhibited a 2.4-fold higher activity in the presence of 5% methanol than in the presence of 1% isopropanol. Est2K preferred short to medium length p-nitrophenyl esters, especially p-nitrophenyl butyrate, as the substrate. Est2K did not hydrolyze beta-lactam antibiotics ampicillin and nitrocefin, even though Est2K showed the highest similarity to EstC.
使用黏粒载体从完全发酵的堆肥中构建了宏基因组文库。在含有 1%甘油三丁酸作为底物的 LB 平板上，从总共 23400 个克隆中选择了 19 个酯酶阳性克隆。酯酶阳性克隆 est2K 的酯酶基因位于一个 1299bp 的 ORF 上，编码一个 432 个氨基酸的蛋白质。Est2K 具有 SMTK 基序，是家族 VIII 酯酶。与大多数家族 VIII 酯酶不同，Est2K 具有 27 个氨基酸的信号肽。成熟的 Est2K 的分子量和等电点分别计算为 44668 Da 和 4.48。Est2K 的氨基酸序列与未培养的渗滤液细菌 EstC 的酯酶显示出 72%的同一性。纯化的 Est2K 在 pH 10.0 和 50°C 下具有最佳活性。Est2K 在 30%甲醇存在下稳定，并在 5%甲醇存在下表现出比 1%异丙醇存在下高 2.4 倍的活性。Est2K 优先选择短至中长的对硝基苯酯作为底物，特别是对硝基苯丁酸。即使 Est2K 与 EstC 显示出最高的相似性，它也不能水解β-内酰胺抗生素氨苄西林和硝基头孢菌素。
