Activity of urea amidohydrolase immobilized within poly[di(methoxyethoxyethoxy)phosphazene] hydrogels

Abstract

Urea amidohydrolase (urease) was immobilized within poly[di(methoxyethoxyethoxy)phosphazene] (MEEP) hydrogels. This was accomplished by mixing an aqueous solution (pH 7) of the soluble polymer with the enzyme. Films of the conjugate were cast and the solvent removed to yield an MEEP/enzyme composite. The conjugate films were dried in a vacuum and were then cross-linked by exposure to 0.2 or 0.5 Mrad of 60Co gamma-radiation to give an MEEP network with the enzyme entrapped within its matrix. The cross-linked films were sectioned into strips and were washed with pH 7 buffer to remove enzyme adhering to the surface. The films were then allowed to swell to form a hydrogel in pH 7 buffer to which was added a 1.0 M aqueous urea solution. The increase in pH from the conversion of urea to ammonia was monitored over a 24 h period. The immobilized enzyme could be recycled at least five times without significant loss of activity. Several control experiments were also performed by monitoring the pH of buffer solutions that contained hydrogels devoid of entrapped urease, and by monitoring the pH of solutions of the free, non-irradiated and free, irradiated urease after the addition of the urea solution. The polymer-free, irradiated urease lost little to no activity compared with its non-irradiated counterpart. The MEEP gel-immobilized enzyme retained approximately 80% of the activity of the non-irradiated, polymer-free urease.