474
VERNON H. CHELDELIN, CHIH H. WANG, AND TSOO E. KING
reach a maximum until after more than 15 minutes because the electron
transfer from glucose to cytochrome is much slower than at pH 5.5, but
the autoxidation of the reduced cytochrome is even slower. All these
observations are in line with the fact that the oxidation by molecular
oxygen in this organism shows optimal pH values of 5 to 6 (267); the
pH optimum for growth is likewise 5.5 to 6 (271). In contrast, the
pyridine nucleotide-dependent enzymes (both the nonparticulate glucose dehydrogenase and all the oxidative enzymes in the soluble extracts
required for the pentose cycle), show maximal activities at much higher
pH values than 7. In the region near pH 5.5, their actions are virtually
nil. At the same time, the nonparticulate soluble extract has very limited
ability to use molecular oxygen; in fact it contains practically no cytochromes. It seems that a concept of an intracellular pH gradient or
compartmentation must be proposed in order to accommodate the
observation. However, in resting-cell experiments at pH 6.0 when molecular oxygen is used as the final acceptor, the oxidation of glucose exhibits
a diphasic curve. The initial rapid rate is evidently due to the first
reaction in this formation of gluconate, and later the slow reaction is
from further oxidation. These facts lead us to believe that in resting
cells the pyridine nucleotide independent particulate glucose oxidase
plays the predominant role in the oxidation of glucose. Thus in fermentation media, rapid aeration is needed for the maximal growth,
and high yields of gluconic acid can be attained at pH 5.5. On either
side of the optimal value, the gluconic acid formed is greatly decreased.
C. FURTHER OXIDATION OF GLUCONIC ACID
Studies on the further oxidation of D-gluconic acid have been almost
exclusively made in microorganisms. The 2-keto acid is formed in good
yield from not only D-gluconate but also other aldonic acids such as
L-idonate and D-galactonate by Pseudomonas (286a, 287) and by Acetobacter species (155, 156, 275, 276, 288-290). Kulhanek has generalized
that those aldonic acids have the same configuration as gluconate in the
first 3 carbon atoms are oxidizable at position 2 (291, 292). However,
Khesghi et al. (293) have found that D-gluconate is oxidized only to
5-ketogluconate in Acetobacter suboxydans whereas Claridge et al. have
reported that both glucose and gluconate are oxidized to 2-ketogluconate
(294, 295). On the other hand, Kulka and Walker (296) have tested
many species of Acetobacter and found that eight species can oxidize
gluconate to both 2- and 5-ketogluconate. Although 2,5-diketogluconate
is a product of gluconate oxidation (297, 298), 5-ketogluconate is not
the precursor of the diketo acid (299).
A more conclusive study has been reported by DeLey and co-
VERNON H. CHELDELIN, CHIH H. WANG, AND TSOO E. KING
reach a maximum until after more than 15 minutes because the electron
transfer from glucose to cytochrome is much slower than at pH 5.5, but
the autoxidation of the reduced cytochrome is even slower. All these
observations are in line with the fact that the oxidation by molecular
oxygen in this organism shows optimal pH values of 5 to 6 (267); the
pH optimum for growth is likewise 5.5 to 6 (271). In contrast, the
pyridine nucleotide-dependent enzymes (both the nonparticulate glucose dehydrogenase and all the oxidative enzymes in the soluble extracts
required for the pentose cycle), show maximal activities at much higher
pH values than 7. In the region near pH 5.5, their actions are virtually
nil. At the same time, the nonparticulate soluble extract has very limited
ability to use molecular oxygen; in fact it contains practically no cytochromes. It seems that a concept of an intracellular pH gradient or
compartmentation must be proposed in order to accommodate the
observation. However, in resting-cell experiments at pH 6.0 when molecular oxygen is used as the final acceptor, the oxidation of glucose exhibits
a diphasic curve. The initial rapid rate is evidently due to the first
reaction in this formation of gluconate, and later the slow reaction is
from further oxidation. These facts lead us to believe that in resting
cells the pyridine nucleotide independent particulate glucose oxidase
plays the predominant role in the oxidation of glucose. Thus in fermentation media, rapid aeration is needed for the maximal growth,
and high yields of gluconic acid can be attained at pH 5.5. On either
side of the optimal value, the gluconic acid formed is greatly decreased.
C. FURTHER OXIDATION OF GLUCONIC ACID
Studies on the further oxidation of D-gluconic acid have been almost
exclusively made in microorganisms. The 2-keto acid is formed in good
yield from not only D-gluconate but also other aldonic acids such as
L-idonate and D-galactonate by Pseudomonas (286a, 287) and by Acetobacter species (155, 156, 275, 276, 288-290). Kulhanek has generalized
that those aldonic acids have the same configuration as gluconate in the
first 3 carbon atoms are oxidizable at position 2 (291, 292). However,
Khesghi et al. (293) have found that D-gluconate is oxidized only to
5-ketogluconate in Acetobacter suboxydans whereas Claridge et al. have
reported that both glucose and gluconate are oxidized to 2-ketogluconate
(294, 295). On the other hand, Kulka and Walker (296) have tested
many species of Acetobacter and found that eight species can oxidize
gluconate to both 2- and 5-ketogluconate. Although 2,5-diketogluconate
is a product of gluconate oxidation (297, 298), 5-ketogluconate is not
the precursor of the diketo acid (299).
A more conclusive study has been reported by DeLey and co-