Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021 India.
Division of Microbial Diseases, UCL Eastman Dental Institute, London, UK.
Indian J Microbiol. 2014 Sep;54(3):284-92. doi: 10.1007/s12088-014-0455-y. Epub 2014 Feb 13.
The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6-7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.
人类口腔宏基因组 DNA 被克隆到质粒 pUC19 中，用于在大肠杆菌中构建 DNA 文库。对 40000 个宏基因组克隆进行功能筛选，鉴定出一个在丁酸甘油酯琼脂平板上显示出晕圈的克隆 LIP2。LIP2 插入 DNA 的序列分析揭示了一个 939bp 的 ORF（omlip1），它与不动杆菌属 junii SH205 的脂肪酶 1 具有同源性。omlip1 ORF 被克隆并使用 pET 表达系统在大肠杆菌 BL21(DE3)中表达。重组酶被纯化至均一性，并研究了其生化特性。纯化的 OMLip1 水解 p-硝基苯酯和中长链脂肪酸的三酰基甘油酯，表明该酶是一种真正的脂肪酶。纯化的蛋白质在 pH7 和 37°C 时表现出最佳活性。该脂肪酶在 pH6-7 的范围内和低于 40°C 的温度下稳定。重要的是，酶活性不受许多二价阳离子的存在或不存在的影响。OMLip1 的金属离子不敏感性为其在工业过程中的潜在应用提供了可能。
