Microbial Genomics Research Unit, Department of Bioinformatics,Bharathidasan University, Tiruchirapalli, India.
Microb Cell Fact. 2012 Jun 1;11:72. doi: 10.1186/1475-2859-11-72.
Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome.
A metagenomic library was constructed from the marine sponge Haliclona simulans in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene lpc53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C β-lactamases. Heterologous expression of lpc53E1 in E. coli and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with p- nitrophenyl palmitate (C16) at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3-12) and temperature (4 -60°C) ranges and high levels of stability in the presence of various solvents at NaCl concentrations as high as 5 M and at temperatures ranging from 10 to 80°C. A maximum lipase activity of 2,700 U/mg was observed with 10 mM p-nitrophenyl palmitate as substrate, in the presence of 5 mM Ca2+ and 5 M NaCl, and a reaction time of 15 min at pH 7 and 40°C; while KM and Vmax values were calculated to be 1.093 mM-1 and 50 μmol/min, respectively.
We have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.
脂肪酶（EC 3.1.1.3）催化三酰基甘油水解为甘油，并参与短链和长链酰基甘油的合成。它们在各种应用中被广泛用于工业，例如烘焙、洗衣洗涤剂以及作为替代能源策略中的生物催化剂。海洋生态系统被认为是具有工业有用酶的生物多样性的重要储备库。然而，这些生态系统中的绝大多数微生物不易培养。基于功能宏基因组的方法通过促进鉴定新型酶（如本研究中从海洋海绵宏基因组中鉴定出的耐卤脂肪酶）来解决这个问题。
从海洋海绵 Haliclona simulans 中构建了一个 pCC1fos 载体的宏基因组文库，包含约 48000 个fosmid 克隆。在 1%三丁酸琼脂平板上进行高通量筛选，鉴定出 58 个阳性脂肪酶克隆。对 10 个最具活性的 fosmid 克隆进行序列分析后，发现 pCC1fos53E1 克隆含有一个推定的脂肪酶基因 lpc53E1，由 387 个氨基酸编码，预测分子量为 41.87 kDa。对 Lpc53E1 预测氨基酸序列的序列分析表明，它是脂肪酶家族 VIII 的成员，具有 SXTK 基序，与 C 型β-内酰胺酶有关。在大肠杆菌中异源表达 lpc53E1 并随后对重组蛋白进行生化特性分析表明，该酶对长链脂肪酸酯具有最高的底物特异性。在 40°C、5 M NaCl 存在下、pH 7 时，用 p-硝基苯棕榈酸酯（C16）观察到最佳活性；此外，重组酶在宽 pH（3-12）和温度（4-60°C）范围内具有活性，并且在高达 5 M NaCl 和 10-80°C 的温度下，在各种溶剂存在下具有高水平的稳定性。在存在 10 mM p-硝基苯棕榈酸酯、5 mM Ca2+和 5 M NaCl 的情况下，在 pH 7 和 40°C 下反应 15 分钟，观察到最大脂肪酶活性为 2700 U/mg；而 KM 和 Vmax 值分别计算为 1.093 mM-1和 50 μmol/min。
我们通过对海洋海绵 fosmid 宏基因组文库进行功能筛选，分离出一种新型耐卤脂肪酶。重组酶在广泛的盐度、pH 和温度范围内的活性和稳定性；以及在有机溶剂和金属离子存在下，表明该酶在各种工业应用中具有实用性。
