Improving the position specificity of <i>Themomyces lanuginosus</i> lipase based on semi-rational design.

Yadi MA; Cuiping You; Guoqiang Zhang; Jianghua LI; Guocheng DU

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Improving the position specificity of Themomyces lanuginosus lipase based on semi-rational design.

Author: Yadi MA ¹ ; Cuiping YOU ² ; Guoqiang ZHANG ¹ ; Jianghua LI ¹ ; Guocheng DU ¹
Author Information

1. Science Center for Future Foods, Jiangnan University, Wuxi 214122, Jiangsu, China.
2. School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Publication Type:Journal Article
Keywords: diglycerol; lipase; position isomerism; semi-rational design; supercritical fluid chromatography
MeSH: Humans; Diglycerides; Molecular Docking Simulation; Binding Sites; Catalysis; Lipase/genetics*
From: Chinese Journal of Biotechnology 2023;39(8):3481-3493
CountryChina
Language:Chinese
Abstract: Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.