EFFICIENT PROTECTION OF GLUCOSE-FRUCTOSE OXIDOREDUCTASE FROM ZYMOMONAS-MOBILIS AGAINST IRREVERSIBLE INACTIVATION DURING ITS CATALYTIC ACTION

Use of cell-free glucose-fructose oxidoreductase (GFOR)from Zymomonas mobilis in a continuous process for the simultaneous production of sor bitol and gluconic acid requires efficient stabilization of the enzyme . Whereas GFOR was found to be stable in the presence or absence of an y of its substrates or produce at 25 degrees C, the enzyme was rapidly inactivated during the time course of its own catalytic action. The l oss of activity was demonstrated to be strictly linked to catalysis, a nd consequently, partially inactivated GFOR remained stable when a tot al conversion of the substrates had been attained. The oxidation of cy steine residues seems to be involved in this unusual mechanism of enzy me inactivation, because dithiothreitol, reduced glutathione, cysteine , and thioglycolate were identified as being efficient protecting agen ts of GFOR. Inter- or intramolecular disulfide bond formation apparent ly does not occur during the inactivation process because, once inacti vated, no GFOR activity could be recovered by means of a subsequent ad dition of reductive chemicals. When 5 mM dithiothreitol was used as a stabilizer during continuous operation with GFOR retained in an ultraf iltration membrane reactor, an equilibrium of glucose and fructose fed and converted could be maintained for >100 h at a productivity of 0.6 mmol IU GFOR h(-1).