Gluconobacter oxydans NAD-dependent,
DD
-fructose reducing, polyol
dehydrogenases activity: screening, medium optimisation and application
for enzymatic polyol production
Sofie Parmentier
1
, Joeri Beauprez
1
, Filip Arnaut
2
, Wim Soetaert
1
& Erick J. Vandamme
1,
*
1
Department of Biochemical and Microbial technology, Ghent University, Coupure links 653, B-9000 Ghent,
Belgium
2
Puratos N.V., Industrialaan 25, B-1702 Groot-Bijgaarden, Belgium
*Author for correspondence (Fax: +32-9-264-62-31; E-mail: erick.vandamme@ugent.be)
Received 7 November 2004; Revisions requested 3 December 2004; Revisions received 4 January 2005; Accepted 5 January 2005
Key words: coenzyme regeneration, Gluconobacter oxydans, polyol dehydrogenase
Abstract
Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehy-
drogenase production. The highest enzyme activities were obtained when this strain was cultivated on a
medium consisting of 30 g glycerol l
)1
, 7.2 g peptone l
)1
and 1.8 g yeast extract l
)1
. Two
DD
-fructose
reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol
dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6),
while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol
dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used
to produce
DD
-mannitol and
DD
-sorbitol enzymatically from
DD
-fructose. Efficient coenzyme regeneration was
accomplished by formate dehydrogenase-mediated oxidation of formate into CO
2
.
Introduction
Various unique sugar-metabolising enzymes occur
in acetic acid bacteria (Adachi et al. 1999b). The
enzymes of Gluconobacter (oxydans) have been
divided into two groups based on their location
and function. One group consists of particulate
enzymes tightly bound to the membrane and
linked to the cytochrome systems. The second
group consists of enzymes located in the cytoplasm
catalysing the intracellular metabolism of carbo-
hydrates. The cytosolic soluble dehydrogenases are
NAD(P)-dependent (Gupta et al. 2001). Acetic
acid bacteria are able to oxidise various substrates,
producing useful oxidation products, such as
LL
-
sorbose,
DD
-gluconate, keto-
DD
-gluconates,
LL
-ery-
thrulose, and dihydroxyacetone. Such oxidation
reactions are called ‘oxidative fermentation’, since
they involve incomplete substrate oxidation
accompanying accumulation of the corresponding
oxidation product in huge amounts in the growth
medium. The membrane-bound dehydrogenases
can be divided into quinoproteins and flavopro-
teins having either a quinone coenzyme like pyr-
roloquinoline quinone (PQQ) or covalently bound
flavin adenine dinucleotide (FAD). The membrane
bound dehydrogenases do not require NAD
+
nor
NADP
+
as electron acceptor. In the cytosolic
fraction various kinds of NAD(P)-dependent de-
hydrogenases predominate, most of which show
the same reaction as the dehydrogenases in the
cytoplasmic membranes under different reaction
conditions (Adachi et al. 1999a). NAD(P)-depen-
dent enzymes have no evidence of bioenergy pro-
duction during the growth of acetic acid bacteria.
In acetic acid bacteria different NAD(P)-depen-
Biotechnology Letters (2005) 27: 305–311 Ó Springer 2005
DOI 10.1007/s10529-005-0684-6