OPTIMIZATION OF COVALENTLY COUPLING ENZYMES TO POLYMERIC MEMBRANES - EPR STUDIES OF PAPAIN

In an effort to better understand bioreactor systems, papain (EC 3.4.22.2) was covalently immobilized onto vinyl alcohol/vinyl butyral copolymer (PMB) membrane by means of glutaraldehyde (GA), 1,1'-carbonyldiimidazole (CDI), or 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP). Various kinetic and performance characteristics of the immobilized papain were evaluated. It was found that the characteristics of the membrane-bound papain depended on the immobilization methods. The CDI- and FMP-immobilized papain bioreactors showed better storage and thermal stability than did the GA-immobilized papain bioreactor, although the apparent Michaelis constant, K(m), of the GA-immobilized papain was closer to the free enzyme than to the corresponding CDI- and FMP-immobilized enzymes. In separate experiments, a 6-carbon spacer was inserted between the membrane surface and the covalently bound enzyme. It was found that the insertion of a spacer reduced the disturbance of the enzyme systems, resulting in K(m) values intermediate between the free and directly bound enzymes for all three immobilization methods. Electron paramagnetic resonance spectroscopy was also used to investigate the conformational change and the active site structure of papain. It was found that the active site SH group of papain immobilized with a 6-carbon spacer had faster motion than that of directly bound enzyme, but slower motion than that of the free enzyme. With both direct-coupling and with a spacer, the SH group motion at the active site of papain by CDI and FMP immobilizations was similar, but slower than the corresponding GA immobilization. The conformational changes of the active site of papain upon immobilization with and without a spacer were in agreement with the functional properties of the enzyme. There was a good correlation between the motion of spin-labeled cysteine in the active site of papain and kinetic properties of this protease: As motion slowed, K(m) increased and V(max) decreased. Of the immobilization procedures used, GA immobilization with a spacer yielded kinetic and structural characteristics most similar to the free enzyme while providing increased stability and reusability relative to the latter.