采用双层静电自组装技术制备nPAM-木瓜蛋白酶-CNC催化剂

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。

Preparation of nPAM-papain-cellulose Nanocrystal (CNC) Biocatalyst by Double-layer Electrostatic Self-assembly Technique

A novel, immobilized papain (PA) was prepared by double layers electrostatic self-assembly using the surface charge characteristics of support material cellulose nanocrystal (CNC), papain (PA), and non-ionic polyacrylamide (nPAM). The electrostatic interaction mechanism of the immobilization process was explored via zeta potential (ZP) analysis and Fourier transform infrared spectroscopy (FTIR) was used to analyze the composition of immobilized PA and the interactions between each substance. The optimal immobilization conditions were obtained through a study of each factor in the immobilization process, as follows: pH: 5.0; CNC/PA mass ratio: 36:1; dosage of 5% (by mass) nPAM: 0.3 mL; the enzyme layer assembly time: 30 min. Under these conditions, the enzyme activity recovery was as high as 85.8%. Subsequently, the kinetic parameters of the free and immobilized enzymes were studied, and the results showed that the Vmax/Km of immobilized enzyme (1.40 min-1) was significantly higher than that of free enzyme (0.80 min-1), indicating that the prepared immobilized enzyme had higher catalytic efficiency as well as wider optimal pH and temperature range. Moreover, the immobilized PA still remained > 96.5% of its initial enzyme activity after the repeated use for five cycles. Therefore, double-layer electrostatic self-assembly technique is a novel and convenient method for enzyme immobilization.