J. of Chemical Technology and Biotechnology, 64, 157-164 (1995)

Sowmya Ganapathi 1,3, D. Allan Butterfield 2,3 and Dibakar Bhattacharyya 1.3*

Dept. of 1 Chemical Engineering, 2 Chemistry and 3 Center of Membrane Sciences

University of Kentucky, Lexington, KY 40506-0046

ABSTRACT

Papain, a sulfhydryl protease has been immobilized on flat-sheet modified polysulfone (MPS) membranes and hydroxyethyl cellulose (HEC) coated polyethersulfone hollow fibers. Amidase activity of the enzyme in solution and on the membranes has been assayed. Immobilized papain on the MPS membrane and the hollow fibers retains 12% and 25% of its activity (with 1 mM substrate) in solution, respectively. Loading experiments revealed decreased activity on the MPS membrane with increased enzyme loading. Adsorption experiments for the reaction product, p-nitroaniline (PNA) have been performed and an attempt has been made to correct for this in activity calculations. Apparent Michaelis-Menten parameters were determined for the MPS and hollow fibers, with both Km and Vmax being lower in the immobilized cases. Electron paramagnetic resonance (EPR) study of the changes in active site conformation of an enzyme on a hollow fiber membrane are reported for the first time. These experiments using the sulfhydryl-specific, (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanethiolsulfonate (MTS) spin label depicted the presence of two subpopulations of immobilized papain on the hollow fibers, one of them active and one denatured. This research was supported by the National Science Foundation.

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