Simultaneous enzymatic synthesis of mannitol and gluconic acid .1. Characterization of the enzyme system

During a screening for the enzyme mannitol dehydrogenase (MDH; EC 1.1.1.67), which is to be employed in the enzymatic conversion of fructose to mannitol, the bacterium Pseudomonas fluorescens, the fungus Schizophyllum commune and the yeast Torulaspora delbruckii were identified as promising producers of NAD C-dependent MDH In P. fluorescens the formation of this enzyme activity could be induced by mannitol, whereas in T. delbruckii synthesis was provoked by fructose. The enzyme was constitutively formed during growth of S. commune on easily metabolizable sugars. Mannitol dehydrogenase from P. fluorescens and T. delbruckii were purified 65-fold and 280-fold, respectively, to apparent homogeneity, while only a partially purified preparation of the enzyme from S. commune was obtained. The dehydrogenases were further characterized in consideration of a possible coenzyme-dependent process in which the reduction of fructose is coupled to the enzymatic oxidation of glucose to gluconate, thus regenerating the reduced form of the nicotinamide coenzyme. The pH-optima for the reduction fructose --> mannitol for the enzymes from P. fluorescens, S. commune and T. delbruckii were found to be 7.3, 7.2 and 6.3, respectively, whereas for the reverse oxidation mannitol --> fructose the pH-optima were 10.0, 9.8, and 10.0, respectively. MDH from S. commune was rather unstable, losing 50% of its activity within 60 h at 4 degrees C. In contrast, the other two enzymes were stable at room temperature and retained 50% of their activity after 42 h (P. fluorescens) or 166 h (T. delbruckii. Furthermore, both enzymes could be efficiently stabilized so that their half-life time exceeded well over 1500 h at ambient temperature. The enzymes of the latter organisms were found to be rather specific for both their coenzyme and their substrates. A glucose dehydrogenase CEC 1.1.1.47) from Bacillus megaterium, which is necessary for the continuous regeneration of the coenzyme and catalyzes the NAD(+)-dependent oxidation of glucose to glucono-delta-lactone, was characterized with respect to its thermal stability.