A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study

Wei Tang; Dongming Lan; Zexin Zhao; Shuang Li; Xiuting Li; Yonghua Wang

doi:10.3390/ijms20030780

A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study

Int J Mol Sci. 2019 Feb 12;20(3):780. doi: 10.3390/ijms20030780.

Authors

Wei Tang¹, Dongming Lan², Zexin Zhao³, Shuang Li⁴, Xiuting Li⁵, Yonghua Wang⁶

Affiliations

¹ 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. 201620119718@mail.scut.edu.cn.
² 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. dmlan@scut.edu.cn.
³ School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China. bizexin-zhao@mail.scut.edu.cn.
⁴ 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. lishuang6991@163.com.
⁵ Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Beijing 100048, China. lixt@btbu.edu.cn.
⁶ 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. yonghw@scut.edu.cn.

Abstract

Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli 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_cat/K_m 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.

Keywords: marine Geobacillus sp.; monoacylglycerol lipase; mutagenesis study; substrate selectivity; thermostability.

MeSH terms

Amino Acid Sequence
Catalysis
Cloning, Molecular / methods
Diglycerides / genetics
Escherichia coli / genetics
Esterification / genetics
Geobacillus / genetics*
Hydrogen-Ion Concentration
Hydrolysis
Monoacylglycerol Lipases / genetics*
Mutagenesis / genetics*
Recombinant Proteins / genetics
Sequence Alignment
Substrate Specificity / genetics
Triglycerides / genetics

Substances

Diglycerides
Recombinant Proteins
Triglycerides
Monoacylglycerol Lipases