MECHANICAL AND KINETIC-PROPERTIES OF PVA HYDROGEL IMMOBILIZING BETA-GALACTOSIDASE

The mechanical and kinetic properties of PVA (polyvinyl alcohol) hydrogel prepared by the iterative freezing and thawing (IFT) method were studied in order to assess its applicability as an immobilizing support. The PVA hydrogel showed rubber-like elasticity and the Young's modulus of the gel increased with increasing polymer concentration. The gel strength greatly improved with the number of freezing and thawing iterations (n). Thermal treatment of 10% gel with n=2 for 10 min at 45-degrees-C caused a significant loss of strength, but at n=7 no change in gel strength was observed. A beta-galactosidase-producing recombinant E. coli was permeabilized by toluene and the kinetic characteristics of immobilized whole cells in the PVA hydrogel were investigated using ONPG (2-nitrophenyl-beta-D-galactopyranoside) as a substrate under negligible intraparticle diffusion resistance. Compared with free cells, the Michaelis constant of the beta-galactosidase was increased by the entrapment in PVA hydrogel, although that of the free cells decreased with the addition of 3% PVA into the reaction mixture. The kinetic parameters of both free and immobilized cells were not influenced by n until it reached 7 times, and the PVA concentration did not affect the kinetic parameters of the immobilized cells. The optimal pH of the enzyme activity was not changed by immobilization, although the activity profile was broader than that of the free cells.