IMMOBILIZATION OF LINAMARASE ON NONPOROUS GLASS-BEADS

The immobilization of purified linamarase [beta-D-glucohydrolase, EC:3 .2.1.21] onto non-porous glass beads involved the silanization of the HF-treated glass beads with 2% gamma-aminopropyl-triethoxysilane in ac etone, covalent coupling of the alkyl amine to glutaraldehyde and subs equent attachment of the enzyme molecule to glutaraldehyde via a Schif f's base linkage. The immobilized linamarase catalyzed the hydrolysis of its natural substrate, linamarin 2-hydroxyisobutyro-nitrile-beta-D- glucopyranoside) and the synthetic substrate analog, p-nitrophenyl-bet a-D-glucopyranoside (pNP-beta-D-glucopyranoside). Glucono-1,5-lactone inhibited the immobilized enzyme competitively irrespective of which o f the two substrates was used, while imidazole showed competitive inhi bition with linamarin as substrate but non-competitive inhibition with pNP-beta-D-glucopyranoside. The Ki values obtained for glucono-1,5-la ctone were 2.04 mM and 0.97 mM with linamarin and pNP-beta-D-glucopyra noside as substrates, respectively. The Ki values for imidazole as inh ibitor were 6.00 mM and 38.20 mM with these two substrates, respective ly. The energies of activation, E-11, for the reaction catalyzed by th e immobilized enzyme with linamarin and pNP-beta-D-glucopyranoside as substrates were 4.00 kcal/mol and 5.7 kcal/mol, respectively. Determin ation of the operational stability of the immobilized enzyme gave a ha lf-life of 14 days at 27 degrees C assuming continual use of the immob ilized enzyme in a fixed-bed reactor for the hydrolysis of cyanogenic glycosides. (C) 1998 Elsevier Science Ltd. All rights reserved.