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本研究利用载体材料纤维素纳米晶(CNC)、木瓜蛋白酶(papain,PA)以及非离子型聚丙烯酰胺(non-ionic polyacrylamide,n PAM)的表面电荷特性,制备了新型的固定化木瓜蛋白酶,利用Zeta电位分析,探寻固定化过程中的静电作用机制;并通过傅立叶变换红外光谱(FT-IR)分析固定化PA的组成成分和各物质的相互作用。通过对固定化过程中的各因素进行研究,得到了制备该固定化酶的最优条件,分别为p H 5.0,CNC和PA质量比为36:1(CNC/PA),n PAM(5%质量分数)的添加量为0.3 m L,酶层自组装时间30 min;在此条件下酶活回收率达85.8%。随后,对游离酶和固定化酶的动力学参数研究显示固定化酶的Vmax/Km值(1.40 min-1)明显高于游离酶(0.80 min-1),表明所制备的固定化PA具有更高的催化效率、更广的最适p H范围和温度范围以及操作稳定性,在重复使用5批次之后,相对酶活仍在96.5%以上。可见双层自组装技术是一种进行固定化酶的新途径。
In this study, a new type of immobilized papain was prepared by using surface charge characteristics of cellulose nanocrystals (CNC), papain (PA) and non-ionic polyacrylamide (n PAM) Zeta potential analysis was used to explore the mechanism of electrostatic interaction during immobilization. The composition of immobilized PA and the interaction of each substance were analyzed by Fourier transform infrared spectroscopy (FT-IR). The optimal conditions for the preparation of the immobilized enzyme were obtained by studying the various factors in the immobilization process. The optimal conditions for preparing the immobilized enzyme were: p H 5.0, mass ratio of CNC to PA of 36: 1 (CNC / PA), n PAM of 5% Mass fraction) was 0.3 m L, and enzyme layer self-assembly time was 30 min. Under this condition, the enzyme activity recovery rate reached 85.8%. Subsequently, the kinetic parameters of the free enzyme and the immobilized enzyme showed that the Vmax / Km of the immobilized enzyme (1.40 min-1) was significantly higher than that of the free enzyme (0.80 min-1), indicating that the prepared immobilized PA had more High catalytic efficiency, wider optimum p H range and temperature range, and operational stability, the relative enzyme activity is still above 96.5% after 5 batches are used repeatedly. Visible double-layer self-assembly technology is a new way to immobilize enzymes.