一种复合酶的固定化及应用研究

Immobilization and Application of a Composite Enzyme

摘要:

以壳聚糖为原料制备了壳聚糖微球，并通过扫描电镜对微球的形态和结构进行了表征。以自制的壳聚糖微球为载体固定化复合酶，分别通过单因素试验和正交试验，对影响酶固定化效果的因素及固定化酶的酶学性质进行了考察，并采用HPLC对其应用于催化京尼平苷制备京尼平的效果行了研究。结果表明：壳聚糖微球平均粒径约为2 mm，表面呈多孔结构。酶固定化最优化条件为：吸附时间4 h，交联时间1 h，交联剂质量分数0.2%。固定化酶的最适温度为60 ℃，游离酶最适温度55 ℃，且固定化酶温度稳定性比游离酶高；固定化酶与游离态酶的最适pH 5.0，且固定化酶pH稳定性比游离酶高；固定化酶在使用8次以后剩余酶活力仍在50%以上；固定化酶湿态储存适合短期内使用和储存，干态储存适合长期储存。采用此固定化方法制得的固定化酶催化京尼平苷水解制备京尼平效果良好。

Abstract:

Making chitosan as raw material, chitosan microspheres were prepared. The morphology and structure of the microspheres were characterized by SEM. Composite enzyme was immobilized by using chitosan microspheres as carrier. The influence of factors affecting the effect of immobilization enzyme was investigated via single factor test and orthogonal test. The enzymatic properties of immobilized enzyme were investigated also. The application effect of using the immobilized enzyme to hydrolysis geniposide to genipin was researched via HPLC method.The results indicated that, the average diameter of chitosan microsphere was about 2 mm and has a porous surface. The optimal conditions for enzyme immobilization were: adsorption time 4 h, crosslink time 1 h, concentration of crosslinking agent 0.2%. The optimum applying temperature for immobilized enzyme was 60 ℃, and 55 ℃ for free enzyme. Both temperature stability and pH stability of immobilized enzyme were higher than that of free enzyme. Both of the optimum pH for immobilized enzyme and free enzyme was 5.0. The residual enzyme activity of immobilized enzyme was more than 50% after being used for 8 times. For immobilized enzyme, wet storage was suitable for short-term application and storage, however, dry storage was suitable for long-term storage.The effect was perfect using the immobilized enzyme to hydrolysis geniposide to genipin.