表面复合修饰磁性颗粒固定化脂肪酶效能研究

摘要

The immobilized enzyme is recyclable and easy to separate,which has high catalytic performance and better environment adaptability compared with the free enzyme.The application performance of immobilized and free lipase were investigated in the present study.Lipase was immobilized by the method of covalent linkage with the composite-modified nano-superparamagnetic Fe3O4 particle aggregates as the carrier.The optimum temperature,pH value,the operation stability and the basic kinetics of the afforded immobilized lipase were compared with the free lipase respectively.The results showed that the maximum loading amount of immobilized enzyme was 0.72 mg/10 mg when the lipase concentration was 4.01 mg/mL at room temperature,and the optimum pH and temperature were 6 and 35 ℃.respectively.Compared with free lipase,immobilized lipase was more sensitive to the pH in acid environment,but had a wider range of temperature tolerance.The Lineweaver-Burk curve of immobilized lipase catalyzed hydrolysis was linear correlation.Km value of immobilized lipase was 9.65 g/mL,which was much higher than that of free lipase,and the affinity was weaker than free lipase.The relative activity of immobilized enzyme (lipase)was still remaining 51.6％ after 7 times recycle use,the half-life period was 6.2 times and the repetitive operation was stable.%以表面复合修饰的纳米超顺磁性Fe3O4颗粒聚集体为载体固定胰脂肪酶,比较分析固定化脂肪酶的固载效能及酶学特性,研究了固定化及游离脂肪酶的最适温度和pH值、不同环境下的操作稳定性等应用特性及基本动力学特征.结果表明:在室温下脂肪酶质量浓度为4.01 mg/mL时具有最大的固载酶量0.72 mg/10 mg,固定化脂肪酶最适作用pH为6,最适温度35℃,与游离脂肪酶相比,对酸性环境pH的变化更敏感,但具有更广泛的温度耐受性.固定化脂肪酶催化水解Lineweaver-Burk曲线线性相关,米氏常数Km值为9.65 g/mL,远高于游离脂肪酶,亲和性弱于游离脂肪酶.固定化脂肪酶重复催化反应7次,相对酶活力仍剩余51.6％,半衰期为6.2次,重复操作较稳定.