SIMULTANEOUS ISOLATION AND IMMOBILIZATION OF STREPTAVIDIN-BETA-GALACTOSIDASE - SOME KINETIC CHARACTERISTICS OF THE IMMOBILIZED ENZYME AND REGENERATION OF BIOREACTORS

A streptavidin-beta-galactosidase fusion protein was expressed in Esch erichia coli and bioselectively adsorbed from crude cell lysates to bi otin covalently immobilized on controlled pore glass. Michaelis consta nts for the immobilized enzyme with o-nitrophenyl-beta-D-galactopyrano side and lactose were 0.28 and 0.4 mM, respectively. Very similar valu es were obtained with a commercial preparation of soluble enzyme, indi cating that neither folding of the fusion protein nor interaction of t he streptavidin domain,vith immobilized biotin altered the structure o f the substrate binding site. As compared to soluble enzyme, the appar ent optimum pH for activity was shifted 0.5 units to the acid region a nd the optimum temperature was 5 degrees C lower. Similar bioreactor a ctivities were regenerated five times by desorption of the fusion enzy me protein with 6 M guanidinium chloride followed by readsorption from cell lysates.