Biosci. Biotechnol. Biochem., 75 (12), 2418–2420, 2011
Note
Enzymatic Synthesis of 4-Pentulosonate (4-Keto-D-pentonate)
from D-Aldopentose and D-Pentonate by Two Different Pathways
*
Using Membrane Enzymes of Acetic Acid Bacteria
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Osao ADACHI,1; Roque A. HOURS,2 Emiko SHINAGAWA,3 Yoshihiko AKAKABE,1
1
Toshiharu YAKUSHI,1 and Kazunobu MATSUSHITA
1Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
2Research and Development Center for Industrial Fermentation (CINDEFI; UNLP, CONICET La Plata),
School of Science, La Plata National University, 47 y 115 (B1900ASH), La Plata, Argentina
3Department of Chemical and Biological Engineering, Ube National College of Technology,
Tokiwadai, Ube 755-8555, Japan
Received August 5, 2011; Accepted September 20, 2011; Online Publication, December 7, 2011
4-Keto-D-arabonate (D-threo-pent-4-ulosonate) and
4-keto-D-ribonate (D-erythro-pent-4-ulosonate) were pre-
pared from D-arabinose and D-ribose by two successive
reactions of membrane-bound enzymes, D-aldopentose
4-dehydrogenase and 4-keto-D-aldopentose 1-dehydro-
genase of Gluconobacter suboxydans IFO 12528. Alter-
natively, they were prepared from D-arabonate and
D-ribonate with another membrane-bound enzyme, D-
pentonate 4-dehydrogenase. Analytical data confirmed
the chemical structures of the 4-pentulosonates pre-
pared. This is the first report of successful enzymatic
synthesis of 4-pentulosonates.
enzyme, D-pentonate 4-dehydrogenase, in G. suboxy-
dans IFO 12528. To confirm these observations, in
this study, we attempted enzymatic synthesis of 4KAB
from D-arabinose and D-arabonate, and of 4KRN from
D-ribose and D-ribonate.
G. suboxydans IFO 12528 was grown on a medium
containing 0.5% D-glucose, 2% Na-D-gluconate, 0.3%
glycerol, 0.3% yeast extract (Oriental Yeast, Tokyo),
and 0.2% polypeptone until microbial growth reached
the early stationary phase. Preparation of the membrane
fraction was done as described previously.2) A wet paste
of precipitated membrane fraction was lyophilized
(Taitec, VD-800F, Tokyo) and stored at ꢀ20 ꢁC until
use. The reaction mixture contained 1 g of dried
membrane fraction of G. suboxydans IFO 12528 and
300 mg of substrate in 100 mL of 10 mM acetate buffer.
Acetate buffer pH 5.0 was used for D-aldopentose
oxidation and acetate buffer pH 4.0 was used for D-
pentonate oxidation. The reaction was carried out
overnight at 30 ꢁC under stirring. The reaction mixture
was centrifuged at 105 ꢂ g for 60 min to precipitate the
membrane fraction. The clear supernatant was applied to
a column of Dowex 1 ꢂ 4 (1 ꢂ 10 cm, acetate form), and
the non-adsorbed materials were passed through the
column by washing the column with water. Elution of
the column was done by a linear gradient concentration
of acetic acid formed by 300 mL of water and 300 mL of
0.2 M acetic acid, and 10 mL fractions were collected.
Elution was checked by TLC chromatography under
conditions reported previously.1) Preliminary detection
of a keto-compound that formed in the reaction mixture
was done by TLC-chromatography by spraying an
alkaline-ethanol solution of 2,3,5-triphenyltetrazolium
chloride (TTC) over the TLC plate as described
previously.1,2) The acetic acid in the pooled fractions
was removed by evaporation under reduced pressure at
45 ꢁC. The resulting viscous liquid was neutralized with
finely powdered CaCO3. After the brown color was
removed by the addition of activated charcoal, the
solution was evaporated until crystals appeared.
Key words: D-pentonate 4-dehydrogenase; D-aldopentose
4-dehydrogenase;
4-keto-D-aldopentose
4-pentulosonate
4-keto-D-aldopentose;
1-dehydrogenase;
In a previous study,1) 4-keto-D-arabonate (D-threo-
pent-4-ulosonate, 4KAB) accumulation in a culture
medium of Gluconacetobacter liquefaciens RCTMR
10 was confirmed as one of the metabolites derived from
2,5-diketo-D-gluconate (25DKA) in the D-glucose oxi-
dizing system of acetic acid bacteria. Two sequential
enzymes, 25DKA decarboxylase and 4-keto-D-arabinose
1-dehydrogenase, were necessary for 4KAB formation.
Identification of 4KAB was the first finding of a novel
sugar acid in carbohydrate chemistry. In our recent
study,2) 4KAB was formed after C4-position specific
oxidation of D-arabonate by a membrane-bound D-
pentonate 4-dehydrogenase. As an alternative route for
4KAB formation, D-arabinose was oxidized to 4-keto-
D-arabinose (4KAR) with
a membrane-bound D-
aldopentose 4-dehydrogenase. The putative 4KAR was
further oxidized to 4KAB by 4-keto-D-aldopentose
1-dehydrogenase. Likewise, D-ribose was oxidized to
4-keto-D-ribonate (D-erythro-pent-4-ulosonate, 4KRN)
via 4-keto-D-ribose (4KRB), similarly to the case of
D-arabinose to 4KAB. Furthermore, D-ribonate was
oxidized to 4KRN by the same membrane-bound
*
Part of this paper was presented at the 13th International Congress of Bacteriology and Applied Microbiology held in Sapporo, Japan, September
6–10, 2011, and at the 16th Japanese-German Workshop on Enzyme Technology held in Toyama, Japan, September 14–15, 2011.
To whom correspondence should be addressed. Tel: +81-83-933-5857; Fax: +81-83-933-5859; E-mail: osao@yamaguchi-u.ac.jp
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Abbreviations: 4KAB, 4-keto-D-arabonate; 4KAR, 4-keto-D-arabinose; 4KRB, 4-keto-D-ribose; 4KRN, 4-keto-D-ribonate
Adachi, Osao
