不同疏水参数有机溶剂中木瓜蛋白酶及其固定化酶活性的变化

Enzyme activity changes of papain and immobilized papain in organic solvents with different logP values

【目的】探寻疏水参数（10gP）不同的有机溶剂对木瓜蛋白酶及其固定化酶活性的影响。【方法】制备固定化木瓜蛋白酶，在疏水参数不同（logP=-1．30，-1．10，-0．76，-0．33，-0．24，0．28，0．49，0．68，0．80，1．31，1，80，2．0，2．5，3．5和5．0）的15种有机溶剂及其不同体积分数（ψ-5％，10％，15％，20％，25％，30％，35％，40％，50％，60％和70％）的溶液中，测定木瓜蛋白酶及其固定化酶的活性，以不含相应有机溶剂的0．1mol／LpH7．2磷酸缓冲液或Tris-盐酸缓冲液为对照，分析不同有机溶剂及其体积分数对酶活性的影响。【结果】在低疏水参数（10gP≤-0．24）的有机溶剂中，有机溶剂二甲基亚砜、1，4-二氧六环和乙腈具有在低体积分数（妒≤10％）下，激活木瓜蛋白酶及其固定化酶活性的特性，有机溶剂甲醇、乙醇虽然在低体积分数（p≤10％）下不能激活木瓜蛋白酶及其固定化酶活性，但其活性与对照（So—O）相当；低疏水参数有机溶剂在高体积分数（9≥30％）下抑制酶活。当有机溶剂的疏水参数为0．28～1．31时，其对木瓜蛋白酶及其固定化酶活性有显著抑制作用。在有机溶剂疏水参数较高（logP≥1．80）条件下，低体积分数（9≤10％）时酶活性下降较慢，超过-定体积分数（P≥30％）后,酶活性开始升高。【结论】低logP（10gP≤0．24）有机溶剂在低体积分数（ψ≤10％）时可激活或稳定木瓜蛋白酶及其固定化酶活性，而在高体积分数（ψ≥30％）时对酶活有抑制作用；中logP（0．28≤logP≤1．31）有机溶剂对木瓜蛋白酶及其固定化酶活性具有显著抑制作用；高logP（10gP≥1．80）有机溶剂在低体积分数（ψ≤10％）时抑制木瓜蛋白酶及其固定化酶活性，但在高体积分数（ψ≥30％）时酶活性升高。

[Objective] The aim of this paper was to study the effect of organic solvents with different logP values on enzyme activity papain and immobilized papain. [Method] Immobilization of papain on sodi- um alginate-chitosan and nylon cloth were prepared. Enzyme activity of papain and immobilized papain were determined in 15 kinds of organic solvents with different log P values（logP= --1.30, --1.10, --0. 76, --0.33,--0.24,0.28,0.49,0.68,0.80,1.31,1.80,2.0,2.5,3.5 and 5.0）and volume fractions（5%,10%, 15%,20%,25%,30%,35%,40%,50%,60% and 70%）,the effect of enzyme activity in different organic solvents and volume fractions were analyzed by taking pH 7.2 Tris-hydroehloric acid buffer or pH 7.2 phosphate buffer without corresponding organic solvents as comparison. [Result] The results showed that in lower logP values（logP≤-0.24） and lower volume fractions（ψ≤10 %）of the added organic solvents, enzyme activity of papain and immobilized papain was activated by some organic solvents（e, g. dimethyl sulfoxide, 1,4-dioxane and acetonitrile）,and it was observed in higher volume fractions（ψ≥30%） of or- ganic solvents（logP≤-0.24） that the enzyme retained their activity in some organic solvents（e, g. metha- nol and ethanol）, but decreased in their activity. In organic solvents with logP values between 0.28 and 1.31,a dramatic reduction in their activity was observed. In organic solvents with higher logP values （logP≥1. 80）,it was observed at higher volume fractions （ψ≥30%） enzyme activity decreased slowly in lower volume fractions（ψ≤10%）, but increased in the activity of the enzyme. [Conclusion] Enzyme activity of papain and immobilized papain were activated or stabilized in lower logP values（logP≤-0.24）and low- er volume fraetions（ψ≤10%） of the added organic solvents, but a reduction in their activity was observed in higher volume fraetions（ψ≥30%） of organic solvents. In organic solvents with middle logP values （0.28≤logP≤1.31） enzyme activity decreased remarkably. In organic solvents with higher logP values （logP≥1. 80）,it was observed at higher volume fractions（ψ≥30%） enzyme activity decreased in lower volume fractions（ψ≤10%）,but increased in the activity of the enzyme.