School of Food Science and Engineering, Guangdong Research Center of Lipid Science and Applied Engineering Technology, State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510641, China.
School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China.
Int J Mol Sci. 2019 Feb 12;20(3):780. doi: 10.3390/ijms20030780.
Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The k/K value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.
具有独特底物特异性的脂肪酶在生物技术应用中受到高度追捧。在本研究中，通过基因组挖掘策略鉴定了一种假定的海洋 sp. 单酰基甘油脂肪酶（GMGL）编码基因。该基因在 中作为 His 标签融合蛋白表达，并通过亲和层析进行纯化，发酵液的得率为每升 264 毫克。重组 GMGL 在 60°C 和 pH8.0 下表现出最高的水解活性，半衰期在 70°C 下为 60 分钟。GMGL 对单酰基甘油（MAG）底物有活性，但对二酰基甘油（DAG）或三酰基甘油（TAG）没有活性，并且在酯化反应中产生 MAG 作为单一产物。建模结构分析表明，催化三联体由 Ser97、Asp196 和 His226 形成，柔性帽区域由 Ala120 到 Thr160 的残基构成。位于底物结合隧道中的 Leu142、Ile145 和 Ile170 残基的突变研究表明，这些残基与酶的底物特异性有关。突变体 Leu142Ala、Ile145Ala 和 Ile170Phe 对 pNP-C6 底物的 k/K 值与野生型相比分别增加了 2.3 倍、1.4 倍和 2.2 倍。
