Abstract: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.
