基于半理性设计提高疏棉状嗜热丝孢菌脂肪酶催化特异性

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

摘要:

甘油二酯(diacylglycerol,DAG)是油脂代谢的中间产物，在人体中具有重要的生理功能，主要通过脂肪酶水解油脂制备。但对1,2-甘油二酯(1,2-diacylglycerol,1,2-DAG)、1,3-甘油二酯(1,3-diacylglycerol,1,3-DAG)的检测分离方法及脂肪酶的催化特异性的研究较少，限制了其广泛应用。基于以上问题，本研究首先通过超临界流体色谱与蒸发光散射检测器联用，优化检测分析参数，建立了1,2-DAG (0.025–0.200 g/L)和1,3-DAG (0.025–0.150 g/L)的高效定量检测方法。进一步基于疏棉状嗜热丝孢菌脂肪酶(Thermomyces lanuginosus lipase,TLL)与三油酸甘油酯的分子对接，挑选了5个潜在底物结合位点并通过定点饱和突变构建了突变体库，利用超临界流体色谱方法对突变体催化特异性进行筛选。突变文库中I202V表现出最高的1,3-位置选择特异性，较野生型TLL提高了11.7%。本文基于半理性设计方法实现了对TLL位置催化特异性的改造，并建立了一种能够高效分离检测DAG位置异构体的方法，为脂肪酶催化特异性研究提供了参考。

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.