溶胶凝胶法固定化黑曲霉脂肪酶的性质研究

近年来溶胶-凝胶法固定脂肪酶已成为研究热点。选用TMOS、MTMS、ETMS和PTMS 4种硅烷试剂对黑曲霉脂肪酶进行了固定化研究。固定化的最佳配方为ETMS/TMOS=5：1、水与硅烷试剂分子比为8；固定化脂肪酶的固定率为80.2%、相对活性为136.3%；以乳化橄榄油作为底物，在50℃和pH4.0的条件下，固定化脂肪酶与游离脂肪酶K_m分别为1.899×10^-4M和2.789×10^-4M；最适反应pH均为pH4.0，固定化脂肪酶在pH4.0~pH5.5之间其活性能保持95%以上；固定化脂肪酶最适反应温度为60℃，较游离脂肪酶提高了10℃；固定化脂肪酶的酸碱稳定性和热稳定性较非固定化酶有显著的提高。固定化脂肪酶的使用寿命和保存稳定性良好，使用12次后仍能够保留71.7%活性，在室温避光条件下保存180天后仍可保留79.2%活性。

Immobilization and Properties of Lipase from Aspergillus niger on Sol-gels,Hydrophobic Supports

The study on immobilization of lipase by sol-gel has become research hotspot in recent years.The aim is to find the optimum conditions for the immobilization of lipase from Aspergillus niger and also to select the most suitable support maximizing the immobilized enzyme yield under these optimum conditions.TMOS,MTMS,ETMS,PTMS were used as the precursors in the preparation of encapsulated lipase from Aspergillus niger. The 80.2% of immobilized degree and the 136.3% of relative activity were obtained under optimum conditions, ETMS/TOMS=5:1 and molar ratio of water/silane = 8.With emulsified olive oil as substrate,the kinetics properties for immobilized enzyme were assessed as follows. The K_m of immobilized lipase and free lipase in pH 4.0 and 50℃ conditions were detected to be 1.899×10^-4 mol/L and 2.789×10^-4mol/L by double reciprocal plot,Lineweaver-Burk plot, respectively. The optimum pH of immobilized lipase and free lipase were analyzed to be 4.0 in optimum temperature. The relative activity of immobilized lipase was found above 95% in the range of pH4.0 to pH5.5. The optimum temperature of immobilized lipase was observed at 60℃ which was 10℃ higher than free lipase. The pH stability and thermostability of immobilized lipase were obviously improved in comparison with free enzyme. When immobilized lipase was cyclically used for twelve of reactions, it still retained 71.7% of original activity. When the immobilized lipase was storaged in the dark at room temperature for 180 days, it maintained 79.2% of the initial activity.