Thermotolerant
Gluconobacter frateurii CHM 43 was selected for
L-erythrulose production from
meso-erythritol at higher temperatures. Growing cells and the membrane fraction of the strain rapidly oxidized
meso-erythritol to
L-erythrulose irreversibly with almost 100% of recovery at 37°C.
L-Erythrulose was also produced efficiently by the resting cells at 37°C with 85% recovery. The enzyme responsible for
meso-erythritol oxidation was found to be located in the cytoplasmic membrane of the organism. The EDTA-resolved enzyme required PQQ and Ca
2+ for
L- erythrulose formation, suggesting that the enzyme catalyzing
meso-erythritol oxidation was a quinoprotein. Quinoprotein membrane-bound
meso-erythritol dehydrogenase (Q
MEDH) was solubilized and purified to homogeneity. The purified enzyme showed a single band in SDS-PAGE of which the molecular mass corresponded to 80 kDa. The optimum pH of Q
MEDH was found at pH 5.0. The Michaelis constant of the enzyme was found to be 25 m
M for
meso-erythritol as the substrate. Q
MEDH showed a broad substrate specificity toward C3-C6 sugar alcohols in which the erythro form of two hydroxy groups existed adjacent to a primary alcohol group. On the other hand, the cytosolic NAD-denpendent
meso-erythritol dehydrogenase (C
MEDH) of the same organism was purified to a crystalline state. C
MEDH showed a molecular mass of 60 kDa composed of two identical subunits, and an apparent sedimentation constant was 3.6
s. C
MEDH catalyzed oxidoreduction between
meso-erythritol and
L-erythrulose. The oxidation reaction was observed to be reversible in the presence of NAD at alkaline pHs such as 9.0–10.5.
L-Erythrulose reduction was found at pH 6.0 with NADH as coenzyme. Judging from the catalytic properties, the NAD-dependent enzyme in the cytosolic fraction was regarded as a typical pentitol dehydrogenase of NAD-dependent and the enzyme was independent of the oxidative fermentation of
L-erythrulose production.
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