Novel enzymatic method for the production of xylitol from D-arabitol by Gluconobacter oxydans.

Article abstract of DOI:10.1271/bbb.66.2614

Microorganisms capable of producing xylitol from D-arabitol were screened for. Of the 420 strains tested, three bacteria, belonging to the genera Acetobacter and Gluconobacter, produced xylitol from D-arabitol when intact cells were used as the enzyme source. Among them, Gluconobacter oxydans ATCC 621 produced 29.2 g/l xylitol from 52.4 g/l D-arabitol after incubation for 27 h. The production of xylitol was increased by the addition of 5% (v/v) ethanol and 5 g/l D-glucose to the reaction mixture. Under these conditions, 51.4 g/l xylitol was obtained from 52.4 g/l D-arabitol, a yield of 98%, after incubation for 27 h. This conversion consisted of two successive reactions, conversion of D-arabitol to D-xylulose by a membrane-bound D-arabitol dehydrogenase, and conversion of D-xylulose to xylitol by a soluble NAD-dependent xylitol dehydrogenase. Use of disruptants of the membrane-bound alcohol dehydrogenase genes suggested that NADH was generated via NAD-dependent soluble alcohol dehydrogenase.

Full text of DOI:10.1271/bbb.66.2614

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Novel Enzymatic Method for the Production of Xylitol
from D-Arabitol by Gluconobacter oxydans
Shun-ichi SUZUKIa, Masakazu SUGIYAMAa, Yasuhiro MIHARAa, Ken-ichi HASHIGUCHIa &
Kenzo YOKOZEKIa
a AminoScience Laboratories, Ajinomoto Co., Inc. Suzuki-cho, Kawasaki-ku, Kawasaki-shi
210-8681, Japan
Published online: 22 May 2014.
To cite this article: Shun-ichi SUZUKI, Masakazu SUGIYAMA, Yasuhiro MIHARA, Ken-ichi HASHIGUCHI & Kenzo YOKOZEKI
(2002) Novel Enzymatic Method for the Production of Xylitol from D-Arabitol by Gluconobacter oxydans , Bioscience,
Biotechnology, and Biochemistry, 66:12, 2614-2620, DOI: 10.1271/bbb.66.2614
To link to this article: http://dx.doi.org/10.1271/bbb.66.2614
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Biosci. Biotechnol. Biochem., 66 (12), 2614–2620, 2002
Novel Enzymatic Method for the Production of Xylitol
from -Arabitol by Gluconobacter oxydans
D
Shun-ichi SUZUKI, Masakazu SUGIYAMA,† Yasuhiro MIHARA, Ken-ichi HASHIGUCHI
and Kenzo YOKOZEKI
,
AminoScience Laboratories, Ajinomoto Co., Inc., Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan
Received June 14, 2002; Accepted August 23, 2002
Microorganisms capable of producing xylitol from
arabitol were screened for. Of the 420 strains tested,
three bacteria, belonging to the genera Acetobacter and
D
-
an alternative process that is independent from
lose, involving oxidation of -arabitol (the 2-epimer
of xylitol) to -xylulose by -arabitol dehydrogenase,
followed by reduction of -xylulose to xylitol by
xylitol dehydrogenase (Fig. 1). -Arabitol can be pro-
duced e‹ciently from -glucose by fermentation with
D
-xy-
D
D
D
Gluconobacter, produced xylitol from
D
-arabitol when
D
intact cells were used as the enzyme source. Among
them, Gluconobacter oxydans ATCC 621 produced
29.2 g l xylitol from 52.4 g l -arabitol after incubation
D
D
WD
osmophilic yeasts, such as Pichia, Candida, or De-
W
baryomyces species.10–16) Additionally, there have
been a few reports on the combination of the two
for 27 h. The production of xylitol was increased by the
addition of 5 (v v) ethanol and 5 g l -glucose to the
W
WD
reaction mixture. Under these conditions, 51.4 g l
%
reactions: -arabitol oxidation to -xylulose by
D
D
W
xylitol was obtained from 52.4 g l -arabitol, a yield of
WD
Gluconobacter suboxydans and
D
-xylulose reduction
98
%
, after incubation for 27 h. This conversion consist-
ed of two successive reactions, conversion of -arabitol
to -xylulose by a membrane-bound -arabitol de-
hydrogenase, and conversion of -xylulose to xylitol by
to xylitol by yeasts such as Saccharomyces
17,18)
D
cerevisiae
.
However, these processes are compli-
D
D
cated and the productivity was low because the two
reactions were conducted separately. Simple conver-
D
a soluble NAD-dependent xylitol dehydrogenase. Use of
disruptants of the membrane-bound alcohol de-
hydrogenase genes suggested that NADH was generated
via NAD-dependent soluble alcohol dehydrogenase.
sion of -arabitol to xylitol could therefore be a use-
D
ful method for xylitol production.
In this paper, we report the discovery of microor-
ganisms that possess two enzymes enabling the
production of xylitol from
of acetic acid bacteria belonging to the genus
Gluconobacter had a membrane-bound -arabitol
D
-arabitol. Some strains
Key words: xylitol; NADH regeneration; Glucono-
bacter; membrane-bound dehydrogenase
D
dehydrogenase (AraDH) and a soluble xylitol de-
hydrogenase (XDH), and to e‹ciently produce
Xylitol is a naturally occurring pentahydroxy sugar
alcohol. It has a similar quality of sweetness to su-
crose, and is used as an alternative natural sweetener.
Xylitol also has the useful property of caries preven-
tion and is used for oral health care. On the basis of
these properties, xylitol has numerous applications in
the ˆelds of food and pharmaceuticals.1–5)
xylitol directly from
D
-arabitol. The mechanism of
xylitol production was also investigated.
Materials and Methods
Screening of microorganisms for xylitol produc-
Xylitol is produced commercially by chemical
tion from -arabitol with cell extracts. The screening
D
reduction (hydrogenation) of
D
-xylose derived from
medium contained (NH4)2SO4 5 g l, K2HPO4 3 g l,
W
W
・
hemicellulose-xylan hydrolysates of substrates such
KH2PO4 1 g l, MgSO4 7H2O 0.5 g l, Bacto yeast
W
W
as birchwood or corn.1) Considerable eŠorts have
also been focused on microbial reduction of
extract 1 g l,
D
-arabitol 10 g l,
D
-xylose 10 g l, and
W
W
W
D
-xylose
xylitol 10 g l (pH 6.5). The microorganisms were in-
W
with yeasts such as Candida or Saccharomyces spe-
cies.6–9) However, these processes are not competitive
because of their low productivity compared with the
chemical process.
oculated into 4 ml of the screening medium in 20-ml
9
test tubes and cultured with shaking at 30 C for 24 h.
Cells thus obtained were collected by centrifugation,
washed with 100 m potassium phosphate buŠer
(KPB), pH 7.0, and suspended in 1 ml of KPB. Cells
M
The microbial conversion of -arabitol to xylitol is
D
†
To whom correspondence should be addressed. Fax: +81-44-244-6581; E-mail: masakazu
ä
sugiyama
＠
ajinomoto.com
Abbreviations: AraDH,
D-arabitol dehydrogenase; XDH, xylitol dehydrogenase; ADH, alcohol dehydrogenase; KPB, potassium phos-
phate buŠer

Production of Xylitol from
D
-Arabitol by Gluconobacter oxydans
2615
were disrupted with a glass beads beater (Multi Beads
Shocker; Yasui-Kikai Co., Japan). After removal of
from the reported sequence,20) and cloned into the
PstI-XbaI site in pHSG298 (Takara Shuzo, Japan).
Then, the ampR gene of pUC18 (Takara Shuzo,
×
g for 10 min,
cell debris by centrifugation at 10,000
the resulting supernatant was used as a cell-free ex-
tract. Reactions for xylitol production were per-
Japan) was ampliˆed with the primers 5
AGATCTCATTCAAATATGTATCCGCTC-3
-AGTGGATCCGAGTAAACTTGGTCTGACA-
GTTACC-3 , digested with BamHI and BglII, and
inserted into the BamHI site in the adhA gene of the
plasmid. The 5 region of the adhB gene was ampli-
ˆed with the primers -GGCGAGCTCGAAC-
GCATTAACTCGGGACAG-3 and 5 -AGGGAT-
CCAGTTGTCGATCGGTGCACC-3 , and the 3
region was ampliˆed with the primers 5 -ACTGG-
ATCCCACCGAGCCTGCGCAGC-3 and 5 -GGC-
TCTAGATTATTGTGCGTCGTCCTCGCC-3
?-TTC-
?
and
formed at 30
containing 100 m
arabitol, 10 m NAD or NADP, and 100
free extract. Xylitol, -xylulose, and -arabitol were
9
C for 18 h in 1 ml of reaction mixture
5?
M
Tris-HCl (pH 8.0), 50 g l
D
-
?
W
M
ml of cell-
D
D
?
measured by HPLC on a Shodex Sugar SC1211
column (Showa Denko, Japan). The column temper-
5?
?
?
ature was 60
9
C, and 50
z
acetonitrile in 50 mg l Ca-
?
?
W
EDTA solution was used as the mobile phase at a
?
‰ow rate of 0.8 ml min. Xylitol was also measured
by an enzymatic method: reaction mixtures contain-
?
?
W
?.
ing 100 m
1.5 U ml
M
CAPS-NaOH (pH 10.0), 1 m
M
NADP,
These ampliˆed fragments were digested with SacI-
BamHI and XbaI-BamHI, respectively, and cloned
into the SacI-XbaI site of pHSG298. The ampR gene
was inserted into the BamHI site in the adhB gene of
the plasmid. Each of the resulting plasmids was linea-
rized by XbaI digestion and introduced into G.
oxydans ATCC 621 cells by electoroporation.19)
Transformants were selected on YPG agar medium
L
-xylulose reductase (Sigma, USA) and
C for 2 h and
W
1 20 (v v) sample were incubated at 25
9
W
W
the absorbance at 340 nm was observed to measure
xylitol.
Screening of microorganisms for xylitol produc-
tion using intact cells. The ability to produce xylitol
by intact cells was also examined. One-tenth of a
gram of each strain was added to 1 ml of reaction
containing 50
mg ml ampicillin. Disruptants were
W
selected onto YPG agar plates with ampicillin and
mixture (50 g l -arabitol, 10 g l glucose, 100 m
W D
M
0.005
z
allyl alcohol,21) and disruption of adh was
conˆrmed by measuring ADH activity.
W
KPB; pH 7.5) in a test tube. Reactions with resting
cells were performed at 30 C for 24 h with shaking.
9
Enzyme assays. Cells were suspended in 50 m
M
Xylitol production by G. oxydans ATCC 621.
PDM medium (containing Bacto yeast extract 30 g l,
KPB (pH 6.5) from an overnight culture in YPG
medium at 30 C. Cell-free extracts were prepared by
9
W
meat extract 5 g l, potato dextrose 24 g l, glycerol
ultrasonic treatment followed by fractionation of
membrane and soluble fractions by ultracentrifuga-
W
W
15 g l, -arabitol 10 g l) and YPG medium (contain-
W D
W
×
g for 30 min at 49C. The super-
ing Bacto yeast extract 5 g l, Bacto peptone 3 g l,
tion at 100,000
natants were used as the soluble fractions. The pellets
were resuspended in 50 m KPB (pH 6.5) and used as
the membrane fraction. The membrane-bound de-
W
W
glucose 30 g l) were used for the cultivation of
W
Gluconobacter
ed into a 500-ml ‰ask containing 50 ml of PDM
medium and cultured at 30 C for 3 days with shak-
ing. Cells were collected by centrifugation at 10,000
for 10 min and washed with KPB. One-tenth of a
gram of washed cells was inoculated into 1 ml of
reaction mixture (52.4 g l -arabitol, 10 g l glucose,
.
G. oxydans ATCC 621 was inoculat-
M
9
9
hydrogenase activities were measured at 30 C by the
ferricyanide method of Ameyama and Adachi.22)
Membrane-bound ADH activity was measured at pH
×
g
5.5 in a reaction mixture containing 100 m
M
ethanol
WD
KPB (pH 6.0), and 20 g l CaCO3) in a 10-ml
W
and 10 m potassium ferricyanide. Membrane-
M
100 m
M
bound AraDH activity was measured at pH 5.0 in a
reaction mixture containing 100 mM D-arabitol and
W
test tube. Resting cell reactions were performed at
30
or
9
D
C for 27 h with shaking. After 6 h, ethanol and
10 m
dehydrogenase activities were measured spec-
trophotometrically at 30 C by following the sub-
strate-dependent formation of NADH at 340 nm.
The reaction mixture contained 100 m glycine-
NaOH buŠer (pH 9.5), 2 m NAD, and 100 m sub-
strate (xylitol, -arabitol, or ethanol). One enzyme
unit was deˆned as the amount of the enzyme oxidiz-
ing 1 mol of substrate per min.
M
potassium ferricyanide. The cytoplasmic
W
-glucose were added to the reaction mixture at
ˆnal concentrations of 5
tively.
z
(v v) and 5 g l, respec-
9
W
W
M
Disruption of adh genes of G. oxydans. The adhA
gene and adhB gene were disrupted separately by
homologous recombination in G. oxydans ATCC
M
M
D
621. Chromosomal DNA was prepared as described
m
19)
by Okumura et al
.
Plasmids for disruption were
constructed as follows. The adhA gene was ampliˆed
by PCR with primers (5 -GGCCTGCAGGGTCTA-
CTGACGCCGATCAAG-3 and 5 -GCCTCTAG-
AGCAGCCTCAGGGGTGATCCGC-3 designed
Xylitol production using gene disruptants of mem-
brane-bound ADH. The sugar-rich (SR) medium
used for the cultivation of G. oxydans strains con-
?
?
?
?
)
tained
D
-arabitol 50 g l, glycerol 50 g l, sodium
W
W

2616
S. SUZUKI et al.
Table 1. Xylitol Production from 50 g l -Arabitol with Cell-free Extract and Intact Cells
WD
Xylitol (g l)
W
Strain
Cell-free extracta)
Intact cellsb)
Acetobacter xylinum ATCC 14851
Achromobacter viscosus ATCC 12448
Agrobacterium tumefaciens ATCC 2778
Agrobacterium radiobacter ATCC 4718
Arthrobacter para‹neus ATCC 15591
Arthrobacter hydrocarboglutamicus ATCC 15583
Azotobacter indicus ATCC 9037
Brevibacterium ketoglutamicum ATCC 15588
Corynebacterium fasciens IAM 1079
Erwinia amylovora IFO 12687
Flavobacterium peregrinum CCM 1080-A
Flavobacterium fucatum FERM P-9133
Gluconobacter oxydans ATCC 621
Gluconobacter suboxydans NRRLB-755
Micrococcus sp. strain CCM 825
Morganella morganii AJ 2771
Nocardia opaca NCIB 9409
Planococcus eucinatus FERM-P9133
Pseudomonas synxantha ATCC 796
Rhodococcus erythropolis ATCC 11048
Streptomyces coelicolor AJ 9010
0.1
0.1
0.3
0.6
4.4
4.0
0.3
3.4
1.6
4.3
0.3
1.2
0.3
0.3
0.2
1.7
3.8
0.5
0.2
5.5
0.5
0.4
0.9
11
—
—
—
—
—
—
—
—
—
—
—
17
5.8
—
—
—
—
—
—
—
—
—
Streptomyces lividans IFO 13385
Streptomyces tanishiensis ATCC 15238
a)
Xylitol produced with cell-free extracts in the presence of 10 m
Xylitol produced with intact cells.
M NAD.
b)
—, Not detected.
gluconate 10 g l, (NH4)2SO4 5 g l, K2HPO4 3 g l,
Table 2. Enzyme Activities for Xylitol Production in G. oxydans
ATCC 621
W
W
W
KH2PO4 1 g l, MgSO4 7H2O 0.5 g l, Bacto yeast ex-
・
W
W
tract 5 g l, peptone 5 g l, and CaCO3 20 g l. G.
AraDHa)
AraDHb)
XDHc)
W
W
W
W
Enzyme
oxydans ATCC 621 and the ADH disruptants were
(U mg)
(U mg)
(U mg)
W
W
inoculated into 50 ml of SR medium in a 500-ml ‰ask
Soluble fraction
Membrane fraction
0.003
N.D.
0.03
1.36
0.20
N.D.
and cultured at 309C for 24 h with shaking. Cells
were harvested by centrifugation and washed with
KPB. One-tenth of a gram of washed cells was added
a)
AraDH activities measured by monitoring of the formation of NADH
at pH 9.5.
to 10 ml of reaction mixture (
D
-arabitol 100 g l,
b)
c)
AraDH activities measured by the ferricyanide method at pH 5.0.22)
XDH activities measured by monitoring of the formation of NADH at
pH 9.5.
W
(NH4)2SO4 5 g l, K2HPO4 3 g l, KH2PO4 1 g l,
W
W
W
・
MgSO4 7H2O 0.5 g l, Bacto yeast extract 5 g l, pep-
W
W
N.D., Not detected.
tone 5 g l, and CaCO3 20 g l) in a 500-ml ‰ask.
W
W
Reactions with resting cells were performed at 30
9
C
for 48 h with shaking. After 16 h, ethanol was added
Enzymes catalyzing xylitol production from
arabitol
D
-
to the reaction mixture at a ˆnal concentration of 2
z
(v v).
W
G. oxydans ATCC 621 was selected as the
representative strain, and used for further experi-
ments.
D
-Arabitol dehydrogenase (AraDH) activity
Results
was detected in the membrane fraction with ferric-
yanide as an electron acceptor. Furthermore,
NAD-dependent xylitol dehydrogenase activity was
detected in the soluble fraction (Table 2). These ob-
Screening of microorganisms producing xylitol
from -arabitol
D
A total of 304 bacterial strains and 116 yeast
strains were examined. In the ˆrst screening, using
cell-free extracts, 23 bacterial strains were found to
servations suggest that
D
-arabitol was oxidized to -
D
xylulose by the membrane-bound AraDH, and then
produce xylitol from
D
-arabitol (Table 1). A second
D
-xylulose was reduced to xylitol by NAD-dependent
screening was conducted with intact cells. Only three
bacterial strains were found to produce xylitol from
xylitol dehydrogenase in the soluble fraction (Fig. 1).
D
-arabitol in intact cells; all of these strains belonged
Xylitol production by G. oxydans ATCC 621
to the acetic acid bacteria
Gluconobacter).
(
Acetobacter and
Using intact cells at 10
z
(w v) concentration, -
D
W
arabitol (52.4 g l) was stoichiometrically oxidized to
W

Production of Xylitol from
D
-Arabitol by Gluconobacter oxydans
2617
D
-xylulose within 6 h, and 29.2 g l xylitol was pro-
Xylitol production by gene disruptants of mem-
brane-bound ADH
W
duced at 27 h (Fig. 2). -Xylulose reduction by XDH
D
requires NADH as a reducing cofactor. In order to
increase xylitol production, the eŠects of ethanol and
Gluconobacter has both a membrane-bound alco-
hol dehydrogenase (ADH) and a soluble ADH.23–25)
In order to ˆnd which ADH is responsible for genera-
tion of NADH, an adhA disruptant strain was con-
structed. The adhA disruptant was conˆrmed to lack
ADH activity in the membrane fractions, whereas it
was found to still possess cytoplasmic ADH activity
(Table 3).
D
-glucose on xylitol production were investigated.
The addition of 5 (v v) ethanol to the reaction in-
creased the yield of xylitol to 36.1 g l. Although the
z
W
W
eŠect of the addition of 5 g l glucose alone was
W
equivocal, the combination of 5
glucose led to a further increase in xylitol production
to 51.4 g l (molar yield 98 ). It is likely that ethanol
z ethanol and 5 g l
W
z
The xylitol productivities with the wild-type strain
and adh disruptants were also tested. The eŠects of
ethanol on the xylitol production were also observed
W
aŠects xylitol production by increasing the supply of
NADH.
by addition of 2
z ethanol using 1z (w v) cells.
W
With 1 (w v) cells, the wild-type strain produced
z
W
Table 3. ADH Activities and Xylitol Production of Membrane-
bound ADH Disruptants
ADH activity (U mg)
Membrane-bounda) Solubleb) „EtOH
Xylitol (g l)
W
W
Strain
Wild
+EtOH
± ±
9.2( 2.9) 31.9( 7.3)
6.0
N.D.
N.D.
1.0
1.0
1.2
± ±
7.7( 3.2) 24.9( 6.9)
DadhA
adhB
±
±
D
12.5( 3.3) 47.1( 6.6)
Fig. 1. Proposed Enzymatic Scheme for Xylitol Production
a)
b)
ADH activities measured by the ferricyanide method at pH 5.5.22)
ADH activities measured by monitoring of the formation of NADH at
pH 9.5.
from
D
-Arabitol by G. oxydans
.
AraDH, membrane-bound
D
-arabitol dehydrogenase; XDH,
N.D., Not detected.
NAD-dependent soluble xylitol dehydrogenase.
Fig. 2. Xylitol Production from D-Arabitol by G. oxydans.
A, control; B, addition of 5 g l -glucose at 6 h; C, addition of 5
z
(v v) ethanol; D, addition of 5 g l -glucose and 5
WD
z ethanol. ,

WD
W
#
D
-arabitol;

,
D
-xylulose; , xylitol.

2618
S. SUZUKI et al.
9.2 g l of xylitol from 100 g l of
D
-arabitol in the ab-
membrane-bound AraDH of G. oxydans ATCC 621
might be identical to that of G. suboxydans IFO
3257. The NAD-dependent xylitol dehydrogenase ac-
tivity was located in the soluble fraction. Several
XDHs have been characterized and cloned from the
xylose-utilizing yeasts.29–33) For enzymes of bacterial
W
W
sence of ethanol, and 31.9 g l of xylitol in the
W
presence of 2
z
ethanol, respectively. The adhA dis-
ruptant also produced increased amount of xylitol af-
ter the addition of ethanol. These results raise the
possibility that the NAD-dependent soluble ADH is
responsible for the increase of xylitol production in
the presence of ethanol. Furthermore, another dis-
ruptant of the adh gene, adhB, which encodes the
origin, there are only a few reports on XDHs in en-
34)
teric bacteria such as Morganella morganii
.
All of
these XDHs were also NAD-dependent enzymes. It
has been reported that the NAD-dependent -sor-
bitol dehydrogenase in G. suboxydans IFO 3257 also
reduces
-xylulose to xylitol.35)
With intact cells of G. oxydans
almost completely converted to -xylulose, but the
yield of xylitol was low and -xylulose still remained.
Since the XDH in G. oxydans ATCC 621 requires
NADH to reduce -xylulose to xylitol, we examined
the eŠects on xylitol yield of adding substrates for
NADH generation. Ethanol and -glucose were
found to be eŠective in increasing the xylitol yield.
A molar yield of 98 conversion of -arabitol
(52.4 g l) to xylitol (51.4 g l) was reached by addi-
cytochrome
ADH, was constructed. Increased xylitol production
in the presence of 2 (v v) ethanol was also ob-
c
-553 subunit of membrane-bound
D
z
D
W
served with the adhB disruptant, which produced
,
D
-arabitol was
47.1 g l xylitol.
D
W
D
Discussion
D
In this study, we found that cell-free extracts from
several bacterial strains have the ability to produce
D
xylitol from
D
-arabitol in the presence of NAD as a
coenzyme. To our knowledge, this is the ˆrst report
z
D
describing the production of xylitol from -arabitol
D
W
W
with a single strain of microorganism as catalyst.
tion of 5
z
(v v) ethanol and 5 g l -glucose to the
WD
W
Among the strains identiˆed, G. oxydans was found
incubation mixture. In the absence of ethanol, the
to be most suitable for producing xylitol from
D
-
amount of xylitol plus
D
-xylulose was decreased, sug-
-xylulose or -arabitol was
arabitol because it has the ability to produce xylitol
with intact cells.
gesting that a fraction of
D
D
degraded by G. oxydans
.
Many strains other than Gluconobacter and
Acetobacter were also discovered in the ˆrst screen-
ing, but none of those strains could produce xylitol
The mechanism of the eŠect of ethanol on the
production of xylitol was investigated. The mem-
brane-bound alcohol dehydrogenase is a well-known
enzyme in acetic acid bacteria, and an alternative
NAD-dependent alcohol dehydrogenase also exists in
the cytoplasm. Disruption of the adhA gene, encod-
ing the membrane-bound alcohol dehydrogenase
subunit, suggested that NADH was generated via the
NAD-dependent soluble ADH, because this dis-
ruptant had no membrane-bound ADH activity
(Table 3). Furthermore, a disruptant of adhB, encod-
from
D
-arabitol by intact cells. The decrease of
D
-
arabitol during the reaction was observed (data not
shown), indicating that these strains consumed
arabitol rather than converting it to xylitol.
D
-
-
G. oxydans produced xylitol from
D
-arabitol via
D
xylulose. -Xylulose production from
D
D
-arabitol by
Gluconobacter is well known as an oxidative fermen-
tation reaction.17,26) Gluconobacter has a membrane-
bound
irreversible oxidation of
resulting in the accumulation of
D
-arabitol dehydrogenase which catalyze
-arabitol to -xylulose,
-xylulose from
ing cytochrome c-553, produced an increased amount
D
D
of xylitol in the presence of ethanol compared with
D
D
-
the wild-type strain and the adhA disruptant.
arabitol. In contrast, other bacterial strains have only
the soluble NAD-dependent AraDH which catalyze
Cytochrome c-553 has been reported to be involved
in the cyanide-insensitive, non-energy-generating by-
36–38)
reversible oxidation of
xylulose. Thus, it might be possible that the metabol-
ic pathway of -arabitol in Gluconobacter is diŠerent
D
-arabitol and reduction of
D
-
pass in the respiratory chain of Gluconobacter,
and the increase in xylitol yield may be a result of loss
of unfavorable consumption of NADH via this path-
D
from those of other strains. The diŠerence of
metabolism may cause the diŠerence of the xylitol
way. Since many studies have shown that
D
-arabitol
-glucose by fermentation
can be produced from
D
productivity from
D
-arabitol by intact cells.
-arabitol dehydrogenase activ-
with certain osmophilic yeasts, the results of this
study raise the possibility of e‹cient production of
Membrane-bound
D
ity has been observed in G. oxydans ATCC 621, and
it has been reported that a membrane-bound glycerol
dehydrogenase of G. industrius IFO 3260 and a
xylitol from -glucose.
D
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