PAPAIN IMMOBILIZATION ONTO POROUS POLY(GAMMA-METHYL L-GLUTAMATE) BEADS

Water-insoluble papain was prepared by immobilizing papain onto the surface of porous poly(gamma-methyl L-glutamate) (PMLG) beads with and without spacer. The mode of the immobilization between papain and porous PMLG beads was covalent fixation. The relative activity and the stability of the immobilized papain was investigated. The retained activity of the papain covalently immobilized by the azide method was found to be excellent toward a small ester substrate, N-benzyl L-arginine ethyl ester (BAEE), compared with that of the peptide binding method. The values of the Michaelis constant K(m) and the maximum reaction velocity V(m) for free and immobilized papain on the PMLG beads were estimated. The apparent K(m) was larger for immobilized papain than for the free enzyme, while V(m) was smaller for the immobilized papain. The thermal stability of the covalently immobilized papain was higher than that of the free papain. The initial enzymatic activity of the covalently immobilized papain remained approximately unchanged with storage time, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.