Gluconate Oxidizing Polyol Dehydrogenase Membrane Bound

D-Gluconate ^ 5-keto-D-gluconate

Gluconobacter species oxidize sugars and sugar acids and uniquely accumulate two different keto-D-gluconates, 2-keto-D-gluconate, and 5-keto-D-gluconate, in the culture medium by the oxidation of D-gluconate [51, 60, 61]. Recently, PQQ-dependent ARDH and SLDH have been purified from G. suboxydans, both of which have similar broad substrate specificity towards several different polyols. ARDH and SLDH were shown to be identical based on their immunocross-reactivity and also on gene disruption and were suggested to be the same as the previously isolated glycerol dehydrogenase (GLDH) (EC 1.1.99.22). Thus, GLDH

is the major polyol dehydrogenase involved in the oxidation of almost all sugar alcohols in Gluconobacter sp.

In addition, the so-called quinoprotein GLDH was also uniquely shown to oxidize D-gluconate, which was completely different from flavoprotein D-gluconate dehydrogenase (GADH) (EC 1.1.99.3), that is the direct catalyst for the production of 2-keto-D-gluconate. During the investigation of ARDH, it was found that D-arabitol oxidation was always parallel to D-gluconate oxidation. The gene disruption experiment and the reconstitution of the purified enzyme clearly showed that the production of 5-keto-D-gluconate in G. suboxydans is solely dependent on the quinoprotein GLDH (Fig. 1.6).

Production of 5-keto-D-gluconate is important, allowing the Gray's method for vitamin C production via L-idonate and 2-keto-L-gulonate to be practical. This pathway looks stable unlike the other well-known routes involving L-sorbosone or 2,5-diketo-D-gluconate as the intermediates. 5-Keto-D-gluconate is a stable compound while L-sorbosone and 2,5-diketo-D-gluconate are labile compounds (Fig. 1.7).

Exclusive 5-keto-D-gluconate production would be possible using a mutant in which GADH yielding 2-keto-D-gluconate is deleted. As new information on 5-keto-D-gluconate production accumulates, 5-keto-D-gluconate production should become practical, which will allow us to utilize the new route for vitamin C production proposed by Gray [62, 63]. According to this, half of the 5-keto-D-gluconate is converted to L-idonate and then to 2-keto-L-gulonate by another oxidative fermentation before finally being converted to L-ascorbate, as shown in Fig. 1.7.

G. suboxydans IFO 3255 G. suboxydans IFO 3255

Wild type GLDH::Km

Fig. 1.6 Comparison of keto-D-gluconate accumulation with a wild strain (left frame) and a mutant lacking polyol dehydrogenase (right frame). G. suboxydans IFO 3255 wildtype strain (left frame) and GLDH-defective mutant 3255s/dA::Km (right frame) were cultured in 100 mL of D-glucose-D-gluconate medium. During cultivation, 2KGA and 5KGA were analyzed periodically as indicated.

Incubation period (h)

Fig. 1.6 Comparison of keto-D-gluconate accumulation with a wild strain (left frame) and a mutant lacking polyol dehydrogenase (right frame). G. suboxydans IFO 3255 wildtype strain (left frame) and GLDH-defective mutant 3255s/dA::Km (right frame) were cultured in 100 mL of D-glucose-D-gluconate medium. During cultivation, 2KGA and 5KGA were analyzed periodically as indicated.

Fig. 1.7 Different routes for vitamin C.
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