CAJ | 학술논문

脂肪酶是一种应用广泛的重要生物催化剂，它对手性底物具有良好的立体选择性，而且能适应包括疏水有机溶剂在内的多种非天然反应介质。提高脂肪酶在非天然环境中的立体选择性已经成为关于脂肪酶的研究热点。然而已有研究在提高选择性的同时，往往伴随催化活力的降低。本文提出一种基于计算机模拟的理性改造方法，能够在不降低脂肪酶活力的情况下大幅提高其在有机介质中的立体选择性。以洋葱假单胞菌脂肪酶（Pseudomonas cepacia lipase，PcL）在正己烷中催化手性仲醇的转酯化反应为模型，借助分子动力学模拟，预测N-乙酰咪唑（NAI）修饰PcL的效果。蛋白质谱证实产生主要影响的是位于PcL活性口袋内的 Tyr29残基。进一步通过实验验证，发现经 NAI修饰酪氨酸残基（Tyr）后PcL催化拆分手性仲醇的对映体选择性最高从E=12.6提高到48.1，同时催化活力也有所提高。
지방매시일충응용엄범적중요생물최화제，타대수성저물구유량호적입체선택성，이차능괄응포괄소수유궤용제재내적다충비천연반응개질。제고지방매재비천연배경중적입체선택성이경성위관우지방매적연구열점。연이이유연구재제고선택성적동시，왕왕반수최화활력적강저。본문제출일충기우계산궤모의적이성개조방법，능구재불강저지방매활력적정황하대폭제고기재유궤개질중적입체선택성。이양총가단포균지방매（Pseudomonas cepacia lipase，PcL）재정기완중최화수성중순적전지화반응위모형，차조분자동역학모의，예측N-을선미서（NAI）수식PcL적효과。단백질보증실산생주요영향적시위우PcL활성구대내적 Tyr29잔기。진일보통과실험험증，발현경 NAI수식락안산잔기（Tyr）후PcL최화탁분수성중순적대영체선택성최고종E=12.6제고도48.1，동시최화활력야유소제고。
Lipase is one of the most important biocatalysts because of its stereoselectivity towards chiral substrates and reasonable adaptiveness of non-conventional reaction media,including hydrophobic organic solvents. Increasing the selectivity of lipase in such non-conventional environments has been a continuous hotspot. However,current researches on improving lipase selectivity usually cause critical decrease of catalytic activity. In this work,a novel computation- directed approach to enhance lipase enantioselectivity in organic media without losing activity was introduced. Stereoselective acylation of chiral secondary alcohols catalyzed byPcL(Pseudomonas cepacia lipase) inn-hexane was taken as the model reaction. According to the results from molecular dynamics simulation,when the Tyr29 inside thePcL catalytic cavity was modified by N-acetylimidazole (NAI),the enantioselectivity of the lipase was significantly increased. The simulation result was agreed with the experimental data. MALDI-TOF-MS confirmed the Tyr29 modified position,and kinetic resolution of chiral secondary alcohols by NAI-modifiedPcL displayed an increase of enantioselectivity ratio from 12.6 to 48.1 and higher activity.