摘要
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。
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(n PAM). The electrostatic interaction mechanism of the immobilization process was explored via zeta potential(ZP) analysis and Fourier transform infrared spectroscopy(FT-IR) 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: p H: 5.0; CNC/PA mass ratio: 36:1; dosage of 5%(by mass) n PAM: 0.3 m L; 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 p H 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.
出处
《现代食品科技》
EI
CAS
北大核心
2015年第8期192-198,共7页
Modern Food Science and Technology
基金
国家自然科学基金资助项目(21222606
21376096)
广东省自然科学基金重点项目(S2013020013049)
华南理工大学中央高校基本科研业务费(2013ZG0003)
全国百篇优秀博士论文作者资助项目(201504)
