State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China; E-Mails:
Int J Mol Sci. 2011;12(10):6765-80. doi: 10.3390/ijms12106765. Epub 2011 Oct 13.
Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (≤C(10)), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the K(m) values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening.
纤维素分解粘细菌索冈氏菌基因组测序揭示了许多开放阅读框（ORFs），它们编码各种降解酶，与已报道的降解酶的序列相似性较低，但没有一种得到过表征。本文从索冈氏菌 So0157-2 菌株中克隆了一个预测的脂肪酶基因（lipA）并进行了表征。lipA 大小为 981bp，编码一个由 326 个氨基酸组成的多肽，其中含有五肽（GHSMG）和催化三联体残基（Ser114、Asp250 和 His284）。在 GenBank 数据库中搜索表明，LipA 蛋白与人单甘油脂酶的最大同一性只有 30%。新型 lipA 基因在大肠杆菌 BL21 中表达，并用 Ni-NTA 亲和层析法纯化重组蛋白（r-LipA）。该酶水解短链或中链脂肪酸（≤C(10)）的 p-硝基苯酯，最大活性在 pNP 醋酸盐上。r-LipA 是一种冷适应脂肪酶，在较宽的温度和 pH 值范围内具有较高的酶活性。在 4°C 和 30°C 下，r-LipA 对 pNP 醋酸盐的 K(m)值分别为 0.037 ± 0.001 和 0.174 ± 0.006mM。较高的 pH 和温度条件促进了长链脂肪酸的 pNP 酯的水解活性。值得注意的是，这种脂肪酶在存在商业洗涤剂和有机溶剂的情况下保留了大部分活性。结果表明，r-LipA 蛋白具有一些新的特性，可能有希望用于工业应用，而索冈氏菌是脂肪酶筛选的一个有趣资源。
