Lee Yong-Suk
Department of Biotechnology, Dong-A University Busan, South Korea.
Front Microbiol. 2016 Mar 2;7:218. doi: 10.3389/fmicb.2016.00218. eCollection 2016.
A novel esterase, MtEst45, was isolated from a fosmid genomic library of Microbulbifer thermotolerans DAU221. The encoding gene is predicted to have a mass of 45,564 Da and encodes 495 amino acids, excluding a 21 amino acid signal peptide. MtEst45 showed a low amino acid identity (approximately 23-24%) compared with other lipolytic enzymes belonging to Family III, a closely related bacterial lipolytic enzyme family. MtEst45 also showed a conserved GXSXG motif, G131IS133YG135, which was reported as active site of known lipolytic enzymes, and the putative catalytic triad composed of D237 and H265. Because these mutants of MtEst45, which was S133A, D237N, and H265L, had no activity, these catalytic triad is deemed essential for the enzyme catalysis. MtEst45 was overexpressed in Escherichia coli BL21 (DE3) and purified via His-tag affinity chromatography. The optimal pH and temperature of MtEst45 were estimated to be 8.17 and 46.27°C by response surface methodology, respectively. Additionally, MtEst45 was also active between 1 and 15°C. The optimal hydrolysis substrate for MtEst45 among p-nitrophenyl esters (C2-C18) was p-nitrophenyl butyrate, and the K m and V max values were 0.0998 mM and 550 μmol/min/mg of protein, respectively. MtEst45 was strongly inhibited by Hg(2+), Zn(2+), and Cu(2+) ions; by phenylmethanesulfonyl fluoride; and by β-mercaptoethanol. Ca(2+) did not affect the enzyme's activity. These biochemical properties, sequence identity, and phylogenetic analysis suggest that MtEst45 represents a novel and valuable bacterial lipolytic enzyme family and is useful for biotechnological applications.
从嗜热微小杆菌DAU221的fosmid基因组文库中分离出一种新型酯酶MtEst45。预测该编码基因的质量为45,564 Da，编码495个氨基酸，不包括一个21个氨基酸的信号肽。与属于III族的其他脂解酶（一个密切相关的细菌脂解酶家族）相比，MtEst45显示出较低的氨基酸同一性（约23-24%）。MtEst45还显示出保守的GXSXG基序G131IS133YG135，据报道这是已知脂解酶的活性位点，以及由D237和H265组成的假定催化三联体。由于MtEst45的这些突变体（即S133A、D237N和H265L）没有活性，因此这些催化三联体被认为是酶催化所必需的。MtEst45在大肠杆菌BL21(DE3)中过表达，并通过His标签亲和层析进行纯化。通过响应面法估计，MtEst45的最佳pH值和温度分别为8.17和46.27°C。此外，MtEst45在1至15°C之间也具有活性。在对硝基苯酯（C2-C18）中，MtEst45的最佳水解底物是对硝基苯丁酸，其K m和V max值分别为0.0998 mM和550 μmol/min/mg蛋白质。MtEst45受到Hg(2+)、Zn(2+)和Cu(2+)离子、苯甲基磺酰氟和β-巯基乙醇的强烈抑制。Ca(2+)不影响该酶的活性。这些生化特性、序列同一性和系统发育分析表明，MtEst45代表了一个新型且有价值的细菌脂解酶家族，可用于生物技术应用。
