Structural and enzymatic characterizations of papain immobilized onto vinyl alcohol/vinyl butyral copolymer membrane

In an effort to better understand membrane-bound bioreactors, the protease papain (EC 3.4.22.2) was immobilized into vinyl alcohol/vinyl butyral copolymer (PMB) membrane by means of glutaraldehyde. Various kinetic and performance characteristics of the immobilized papain were evaluated. The immobilized papain showed good storage and thermal stability and reusability. pH, temperature, and denaturant dependencies of the bound enzyme were compared to those of the free enzyme. The apparent Michaelis constant, K_m, of the membrane-immobilized papain was larger than that for the free enzyme, although the use of a spacer to place the enzyme at a distance from the polymer surface resulted in a K_m value intermediate between the free and directly-bound values. Electron paramagnetic resonance spectroscopy was also used to investigate the active site structure of papain. It was found that the conformational changes of the active site of papain upon immobilization were in agreement with the enzymatic properties of the enzyme. These results are discussed with reference to fundamental investigation of membrane-immobilized enzyme systems.