A tri-enzyme co-immobilized magnetic complex: Process details, kinetics, thermodynamics and applications

A tri-enzyme mixture of cellulase, pectinase and xylanase present in fermentation broth of Aspergillus niger culture was directly recovered by solvent precipitation and co-immobilized on magnetic nanoparticles (MNPs) by cross linking with glutaraldehyde. A 2:1 ratio of isopropanol:total protein in the fermentation broth gave a precipitate with maximal activity recovery of the three enzymes. The co-immobilization on MNPs was optimized at 30 mM glutaraldehyde for 3 h incubation, and 3:1 MNPs:enzyme ratio, yielding a maximum activity recovery of cellulase (80.25 ± 1.03%), pectinase (84.76 ± 1.71%) and xylanase (75.62 ± 0.76%), respectively. The tri-enzyme co-immobilized MNPs were characterized by particle size analysis, FTIR, XRD, VSM and SEM studies, and showed enhanced thermal stability as evident from lower k_d, higher t_1/2 and D-value in the range of 55-75 °C. The tri-enzyme co-immobilized MNPs showed a shift in optimum temperature from 55 to 60 °C with improved pH tolerance along with a slender rise in kinetic constants and enhanced thermodynamic parameters. It was stable for 36 days at 5 °C, and retained >90% activity up to 4 cycles. The tri-enzyme co-immobilized MNPs were successfully utilized for extraction of piperine from black pepper, preparation of sugarcane cell protoplasts, and clarification of papaya juice with prominent yield and reusability vis-à-vis traditional methods.