Montero S, Blanco A, Virto M D, Landeta L C, Agud I, Solozabal R, Lascaray J M, de Renobales M, Llama M J, Serra J L
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad del País Vasco, Bilbao, Spain.
Enzyme Microb Technol. 1993 Mar;15(3):239-47. doi: 10.1016/0141-0229(93)90144-q.
Lipase (triacylglycerol ester hydrolase, E.C.3.1.1.3) from Candida rugosa has been immobilized on commercially available microporous polypropylene. The enzyme was rapidly adsorbed on the support, and more than 60% of the soluble activity disappeared from the medium after 1 min of incubation at room temperature. A recovery of immobilized activity of 21% was obtained when the wet preparation was immediately assayed with olive oil at the end of the immobilization protocol. The activity of the immobilized enzyme drastically decreased with the loss of water of the preparation. Pretreatment of the support with organic solvents significantly increased the recovered immobilized activity. Our results strongly suggest that the soluble lipase could exist in different aggregation forms depending on the pH of the medium. At acidic pH, the relative proportion of high-molecular-weight forms of the enzyme is higher than at pH 7.0, suggesting that the lipase would be also immobilized in different aggregation forms depending on the pH used in the immobilization procedure. Crosslinking of the adsorbed enzyme with glutaraldehyde diminished its activity but increased the stability of the lipase against the washing-out effect of Triton X-100. Data on the most relevant catalytic properties of the soluble and immobilized enzyme, such as optimum pH and temperature as well as ranges of stability, kinetic parameters, and activation energy for the hydrolysis of olive oil and p-nitrophenyl acetate, are reported.
来自皱褶假丝酵母的脂肪酶（三酰基甘油酯水解酶，E.C.3.1.1.3）已被固定在市售的微孔聚丙烯上。该酶迅速吸附在载体上，在室温下孵育1分钟后，培养基中超过60%的可溶性活性消失。在固定化操作结束时，当用橄榄油立即检测湿制剂时，获得了21%的固定化活性回收率。固定化酶的活性随着制剂水分的流失而急剧下降。用有机溶剂对载体进行预处理显著提高了回收的固定化活性。我们的结果强烈表明，可溶性脂肪酶可能根据培养基的pH值以不同的聚集形式存在。在酸性pH值下，该酶高分子量形式的相对比例高于pH 7.0时，这表明脂肪酶也会根据固定化过程中使用的pH值以不同的聚集形式固定。用戊二醛对吸附的酶进行交联会降低其活性，但会提高脂肪酶对Triton X-100洗脱作用的稳定性。报告了关于可溶性和固定化酶最相关催化特性的数据，如最佳pH值和温度以及稳定性范围、动力学参数以及橄榄油和对硝基苯乙酸水解的活化能。
