利用月桂酸和山茶油的酸解反应分析脂肪酶的位置专一性

摘要

Regioselectivity of lipases played a crucial role in structured lipids synthesis and oil modification.Because of differences between aqueous and non-aqueous systems,regioselectivity analyzed by the conventional hydrolysis method might not always match that in synthesis reactions.In this paper,acidolysis of lauric acid and camellia oil was developed to directly evaluate regioselectivity of lipases in solvent-free system.Fatty acid composition and positional distribution of reaction products were detected by gas chromatography.Through this protocol,Lipozyme RM IM,L02,L03 and L04 were identified as sn-1 (3) regioselectivity,L01 was partially selective,and Novozym 435 was nearly non-selective.Predictability of the model reaction had been verified by replacing substrates of lipases.According to the comparative analysis of results of the acidolysis method and hydrolysis one,regioselectivity of lipases under two situations was generally the same,except selectivity of Novozym 435 which was susceptible to solvent systems.Therefore the present method avoided limits of hydrolysis determination results in synthesis reactions.And moreover it lowered the requirement on purity of reactants,which means it can be used to screen numerous lipases at low cost.%脂肪酶的位置专一性在结构酯的合成和油脂改性中具有重要意义.由于水体系和非水体系的差异,传统的水解判定法得出的专一性与该酶在合成反应中的表现可能并不一致.本研究利用月桂酸和山茶油的酸解反应来达到直接评估酶在无溶剂体系中位置专一性的目的.反应产物的脂肪酸组成和位置分布由气相色谱测定.通过此方法,Lipozyme RM IM、L02、L03和L04被鉴定为sn-l(3)位专一性,L01为弱专一性,Novozym 435近似无专一性.通过替换酶的底物,模型反应的可预测性得到验证.根据酸解法和水解法结果的对比分析,2种条件下酶的位置专一性通常是相同的,除了易受到溶剂体系影响的Novozym 435.因此,新方法能够避免水解判定结果在合成反应中应用的局限性.此外,它还降低了对底物纯度的要求,这意味着其能在低成本下用于脂肪酶的大规模筛选.