脂肪酶在尼龙网上的固定化及其酶学性质研究

Immobilization of Candida sp. Lipase on Nylon Net and Its Enzymatic Characteristics

以尼龙网为载体,戊二醛为交联剂制备固定化脂肪酶(Candida sp.99-125),并对固定化条件及固定化酶的酶学性质进行研究。结果表明,最适固定化条件为:戊二醛体积分数3%、交联时间60min、脂肪酶质量浓度10mg/mL、固定化时间6h。与游离酶相比,固定化脂肪酶的最适温度从45℃提高至50℃,最适pH值相同,均为7.0,并在pH5.0～7.0范围内均有非常高的活力。固定化脂肪酶相对于游离酶具有更好的热稳定性和pH值稳定性,并具有良好的操作稳定性,其重复操作5次后相对酶活力仍保持在80%左右。固定化脂肪酶的Km值(0.57mol/L)和最大反应速率Vmax(0.29×10-3mol/(L.s))显著高于游离酶,对底物的亲和力较游离酶弱。

Lipase（Candida sp.99-125） was immobilized on nylon net by glutaraldehyde as cross-linking agent.Effects of various parameters on the activity of immobilized lipase and characteristics of immobilized lipase were investigated.The maximum enzyme activity achieved at the following conditions： glutaraldehyde concentration of 3%,cross-linking time of 60 min,enzyme concentration of 10 mg/mL,and immobilized time of 6 h.The optimal temperature of immobilized lipase shifted from 45 ℃ to 50 ℃,compared with free lipase.The immobilized lipase maintained high activity in a broad pH range of 5.0 to 7.0,with optimum pH at 7.0,which was identical to that of the free lipase.Thermal,pH,and operational stabilities of lipase were greatly improved after immobilization onto nylon net.Immobilized lipase retained about 80% of the initial activity after fifth repeated use.The Km（0.57 mol/L） and Vmax（0.29 × 10-3mol/（L.s）） of immobilized lipase using olive oil as substrate was significantly higher as compared to free lipase.The immobilization of lipase decreased its affinity to substrate as compared to free lipase.