CONTROL OF THE ASSOCIATION STATE OF TETRAMERIC GLUCOSE-FRUCTOSE OXIDOREDUCTASE FROM ZYMOMONAS-MOBILIS AS THE RATIONALE FOR STABILIZATION OFTHE ENZYME IN BIOCHEMICAL REACTORS

Tetrameric, NADP-containing glucose-fructose oxidoreductase (GFOR)from Zymomonas mobilis catalyzes the oxidation of glucose into glucono-del ta-lactone coupled to the reduction of fructose to sorbitol. GFOR is i nactivated during substrate turnover in, vitro, the long-term stabilit y of the enzyme during conversions in biochemical reactors thereby bei ng drastically reduced, The process of inactivation is triggered by st ructural transitions that ape induced by the lactone product and invol ves aggregation as the ultimate cause of irreversible inactivation. Gu anidinium hydrochloride-induced changes in the conformation of GFOR se em to be similar to those observed in the presence of lactone, and are manifested by incubation time-dependent increases in protein fluoresc ence and the solvent-exposed hydrophobic surface. The formation of hig h-order protein associates in solution in the presence of this denatur ant proceeds from the native tetramer to a reversibly inactivated octa mer and then to a dodecameric form that cannot be reactivated through spontaneous or assisted refolding, Therefore, stabilization of GFOR du ring turnover requires that the marked tendency of the enzyme to form aggregates is prevented efficiently, This goal has been accomplished i n the presence of low urea concentrations (1.0 M), which led to a 10-f old increase in the half-life of GFOR under operational conditions.