Purification and properties of membrane-bound D-sorbitol dehydrogenase from Gluconobacter suboxydans IFO 3255.

Article abstract of DOI:10.1271/bbb.66.57

D-Sorbitol dehydrogenase was solubilized from the membrane fraction of Gluconobacter suboxydans IFO 3255 with Triton X-100 in the presence of D-sorbitol. Purification of the enzyme was done by fractionation with column chromatographies of DEAE-Cellulose, DEAE-Sepharose, hydroxylapatite, and Sephacryl HR300 in the presence of Triton X-100. The molecular mass of the enzyme was 800 kDa, consisting of homologous subunits of 80 kDa. The optimum pH of the enzyme activity was 6.0, and the optimum temperature was 30 degrees C. Western blot analysis suggested the occurrence of the enzyme in all the Gluconobacter strains tested.

Full text of DOI:10.1271/bbb.66.57

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Purification and Properties of Membrane-bound
D-Sorbitol Dehydrogenase from Gluconobacter
suboxydans IFO 3255
a
a
Teruhide SUGISAWA & Tatsuo HOSHINO
a
Department of Applied Microbiology, Nippon Roche Research Center 200 Kajiwara,
Kamakura, Kanagawa 247-8530, Japan
Published online: 22 May 2014.
To cite this article: Teruhide SUGISAWA & Tatsuo HOSHINO (2002) Purification and Properties of Membrane-bound D-
Sorbitol Dehydrogenase from Gluconobacter suboxydans IFO 3255, Bioscience, Biotechnology, and Biochemistry, 66:1,
57-64, DOI: 10.1271/bbb.66.57
To link to this article: http://dx.doi.org/10.1271/bbb.66.57
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Biosci. Biotechnol. Biochem., 66 (1), 57–64, 2002
Puriˆcation and Properties of Membrane-bound -Sorbitol Dehydrogenase
D
from Gluconobacter suboxydans IFO 3255*
Teruhide SUGISAWA† and Tatsuo HOSHINO
Department of Applied Microbiology, Nippon Roche Research Center, 200 Kajiwara, Kamakura,
Kanagawa 247-8530, Japan
Received July 5, 2001; Accepted September 11, 2001
5
)
D
-Sorbitol dehydrogenase was solubilized from the
membrane fraction of Gluconobacter suboxydans IFO
255 with Triton X-100 in the presence of -sorbitol.
the enzyme isolated by Shinagawa et al
volved in -sorbose production from -sorbitol.
However, -sorbitol was converted to -sorbose
.
was in-
L
D
3
D
D
L
Puriˆcation of the enzyme was done by fractionation
with column chromatographies of DEAE-Cellulose,
DEAE-Sepharose, hydroxylapatite, and Sephacryl
HR300 in the presence of Triton X-100.
The molecular mass of the enzyme was 800 kDa,
consisting of homologous subunits of 80 kDa. The
optimum pH of the enzyme activity was 6.0, and the
optimum temperature was 30
Western blot analysis suggested the occurrence of the
enzyme in all the Gluconobacter strains tested.
e‹ciently by Gluconobacter suboxydans IFO 3255 at
pH 7.0, suggesting occurrence of another type of
membrane-bound
D
-sorbitol dehydrogenase fun-
ctioning at a pH of around 7.0 in the membrane frac-
tion of the organism.
Here, we describe the puriˆcation and characteri-
zation of a new type of membrane-bound -sorbitol
D
9C.
dehydrogenase from Gluconobacter suboxydans IFO
3255, and discuss distribution of the enzyme in
Gluconobacter strains.
Key words: acetic acid bacteria; Gluconobacter
Materials and Methods
strains; -sorbitol dehydrogenase
D
Chemicals.
D
-Sorbitol and 2,6-dichlorophenolin-
Related to studies on the enzymes participating in
the formation of -sorbose from -sorbitol,
dophenol (DCIP) were purchased from Nakarai
Tesque. Inc. and E. Merck, respectively. Triton
X-100 and hydroxylapatite (Bio-Gel HTP) were pur-
chased from Bio-Rad Laboratories. DEAE-cellulose
DE-52 was from Whatman, Ltd. DEAE-Sepharose
CL-6B and Sephacryl S300 were from Pharmacia
Fine Chemicals. All other chemicals were reagent
grade.
L
D
1
)
2)
Widmer and Cummins et al. have reported that the
cell-free extract of Acetobacter suboxydans (syn.
Gluconobacter suboxydans) contains three kinds of
enzymes participating in -sorbitol oxidation. In ad-
D
dition to a dehydrogenase in the membrane fraction,
there are two enzymes in the soluble fraction; one
catalyzes
NAD and another catalyzes
the presence of NADP. The NAD-dependent de-
D
-fructose formation in the presence of
L
-sorbose formation in
Microorganism and cultivation. G. suboxydans
IFO 3255, and other Gluconobacter, Acetobacter,
3
)
hydrogenase has been partially puriˆed, and the
NADP-dependent dehydrogenase has been isolated
and Frateuria strains were purchased from the In-
stitute for Fermentation, Osaka (IFO). The medium
4
)
as an NADPH-linked
membrane-bound -sorbitol dehydrogenase of G.
suboxydans var. IFO 3254 has been puriˆed and
L
-sorbose reductase. The
consisted of 20 g of -sorbitol, 3 g of yeast extract,
D
D
3 g of beef extract, 3 g of corn steep liquor, 10 g of
polypeptone, 1 g of urea, 1 g of KH PO , 0.2 g of
MgSO 7H O, and 1 g of CaCO in 1 liter of
a
2
4
5
)
characterized by Shinagawa et al. and had an opti-
mum pH of 4.5. However, its product is not men-
tioned in the report. It is known that acetic acid bac-
4
・
2
3
deionized water. The pH was adjusted to 7.0 with so-
dium hydroxide before sterilization. The cultivation
in a ‰ask was done aerobically with rotary shaking
for one day; the cultivation in a 30-liter jar fermentor
teria produce
L
-sorbose from -sorbitol at a low pH
D
range of 3.5 to 5.0. Therefore, it was considered that
*
A poster of this paper was presented at the workshop at the annual meeting of the Japan Society for Bioscience, Biotechnology, and
Agrochemistry held in Fukuoka from March 30 to April 1, 1999. An abstract of the paper appears in the Nippon Nougeikagaku Kaishi
3, 272 (1999).
To whom correspondence should be addressed. Teruhide SUGISAWA, Tel: +81-467-47-2224; Fax: +81-467-45-6812; E-mail:
teruhide.sugisawa roche.com
,
7
†
＠

5
8
T. SUGISAWA and T. HOSHINO
was done with an aeration rate of 15 L min and an
temperature, agitation speed, and aeration speed,
respectively. During the fermentation, pH was con-
W
agitation speed of 500 rpm at 30
The broth was centrifuged at 400
to remove calcium carbonate, and then at 10,000
9
C for 21.5 hours.
×
g
for 10 minutes
trolled at 7.0 with Na
was converted to -sorbose within 18.5 hours cultiva-
tion and 89.3 g l of -sorbose from the initial
sorbitol concentration of 98.9 g l were produced.
2
CO
3
. As a result,
D
-sorbitol
×
g
L
for pelleting the cells. The cell cake was washed once
with physiological saline solution. The cells (200 g
wet weight from 20 liters of the culture) were frozen
L
D
-
W
W
The average productivity of
L
-sorbose was
at „20
9
C until use.
4.83 g l h.
WW
Preparation of membrane fraction. The cells
Solubilization
(
100 g wet weight) were suspended in 200 ml of
We tried to solubilize the membrane-bound
D
-
5
0 m
M
phosphate buŠer (pH 7.0) and passed through
sorbitol dehydrogenase of G. suboxydans IFO 3255.
a French pressure cell press at 20,000 psi. After cen-
At ˆrst, we tried to apply the method reported by
5)
trifugation to remove intact cells, the supernatant
Shinagawa et al., in which the membrane-bound -
D
(
cell free extract) was centrifuged at 80,000
×
g
for
sorbitol dehydrogenase of G. suboxydans var
. a IFO
one hour. We designated this precipitate as the mem-
brane fraction, which was resuspended into the
above buŠer (100 ml), and the protein concentration
3254 was solubilized. However, the -sorbitol de-
D
hydrogenase activity of G. suboxydans IFO 3255 was
lost in the supernatant and residual membrane
fractions after the solubilization of the membrane in
was 22.8 mg ml.
W
0
.01
Triton X-100, 0.1
10 mg ml of the membrane protein for 2 h at 59
M
sodium acetate buŠer (pH 5.0) containing 1
KCl, 0.1 M D-sorbitol and about
C. It
-sorbitol
dehydrogenase of G. suboxydans IFO 3255 has
diŠerent properties from that of G. suboxydans var
IFO 3254.
Therefore, we modiˆed this method for enzyme
z
Enzyme assay. The basal reaction mixture for
M
assaying
of 50 m
D
-sorbitol dehydrogenase activity consisted
potassium phosphate buŠer (pH 6.0),
W
M
was suggested that the membrane-bound
D
0
.25 m
M
DCIP, and 0.325 m
M
phenazine methosul-
fate (PMS), which was prepared just before the as-
say. A cuvette had a 1-cm light path and contained
.
a
0
D
.4 ml of the basal reaction mixture, 0.1 ml of 0.4
-sorbitol and the enzyme solution, and water with a
M
solubilization. The eŠects of pH and the concentra-
tions of buŠers, detergents, and KCl on the solubili-
zation were studied. When the membrane fraction
total volume of 0.51 ml. The reference cuvette con-
tained all components without the substrate. The
reaction was started at 25
9
C with
D
-sorbitol, and the
was mixed in 0.05
M
potassium phosphate buŠer (pH
Triton X-100 and 0.04
C, 74 of -sorbitol de-
enzyme activity was measured at the initial reduction
rate of DCIP at 600 nm. One enzyme unit is deˆned
as the amount of the enzyme that catalyzes the reduc-
7.0) containing 1
z
M
D
-
sorbitol for 2 h at 5
9
z
D
hydrogenase activity was recovered into the solubi-
lized supernatant from the membrane fraction as
shown in Table 1. The enzyme was not solubilized
tion of 1
mmol of DCIP per min. The extinction
coe‹cient of DCIP at pH 5.5, 6.0, 6.5, and 7.0 to
„
1
1
0.0 was 9.45, 10.8, 13.0, and 15.0 m
ly.
M
, respective-
with
n
-octyl-
b
-
D
-glucopyranoside and the activity
KCl. -Sorbitol had
was lost by the addition of 0.1
M
D
a stabilizing eŠect on the enzyme solubilization.
Measurement of protein concentration. BCA pro-
tein assay reagent (Pierce, Rockford, IL) was used
for the measurement of protein concentrations with
bovine serum albumin as the standard.
Puriˆcation
All steps were done between 4 and 8
9
C with the
buŠer 0.01
stated.
M
potassium phosphate unless otherwise
Results
Step 1. Solubilization. The membrane fraction
was suspended in the buŠer (pH 7.0) to give about
Conversion of
xydans IFO 3255
D
-sorbitol to
L
-sorbose by G. subo-
10 mg ml of protein, and then 1z Triton X-100 and
W
0.1
shaken at 180 rpm for 2 h and centrifuged at
80,000 for 60 min for the removal of the
precipitate. The
M
D
-sorbitol were added. The suspension was
G. suboxydans IFO 3255 was cultivated in glass jar
fermentor, type MB-C2000 (Iwashiya, Tokyo,
Japan) which had a total volume of 3 l with a top
drive system and temperature, pH, D.O., and
exhaust gas monitors, containing the medium
×
g
D
-sorbitol dehydrogenase activity
was recovered in the supernatant (200 ml).
Step 2. DEAE-Cellulose column chromato-
graphy. The supernatant (200 ml) was put on a
column of DEAE-cellulose
equilibrated with the buŠer (pH 7.0) containing
0.05 M D-sorbitol and 0.1
Triton X-100. After it
was washed with the same buŠer, elution of the en-
(
2
0
weight volume; 10
z
D
-sorbitol, 0.5
corn steep liquor, 0.0086
urea, 0.086 KH PO , and 0.15
z yeast extract,
W
.5
z
z
MgSO
4
・
7H
2
O,
an-
(q2.5 by 30 cm)
.086
z
z
2
4
z
tifoam). Its working volume was 2 liters and the fer-
mentation was done at 30 C, 700 rpm, 0.5 vvm for
z
9

Membrane-bound
D
-Sorbitol Dehydrogenase
-Sorbitol Dehydrogenase
59
Table 1. Solubilization of Membrane-bound
D
Relative activity of D-sorbitol dehydrogenase after solubilization
(
z)
Conditions (1) to (4) for solubilization
Activity in supernatant
—
Activity in membrane fraction
100
(
(
1) Membrane fraction before solubilization
2) Solubilization in 0.01
M
sodium acetate
KCl and
buŠer (pH 5.0) containing 0.1
M
0
0
1
0
0
.1 M D-sorbitol with:
.5
.0
.2
.3
z
z
z
z
Triton X-100
Triton X-100
0.0
0.0
6.0
8.0
0.0
0.0
0.0
0.0
n
n
-octyl-
-octyl-
b
-glucopyranoside
-glucopyranoside
b
(
(
3) Solubilization in various concentrations
of potassium phosphate buŠer (pH 7.0)
containing 1
z Triton X-100 without KCl:
0
0
0
0
.01
.05
.1
M
buŠer+0.04 M D-sorbitol
buŠer+0.04 M D-sorbitol
buŠer+0.04 M D-sorbitol
23.0
74.0
38.5
65.5
2.5
6.0
3.5
5.0
M
M
.05
M
buŠer without
D
-sorbitol
4) Solubilization in 0.05
M
potassium
phosphate buŠer (pH 7.0) containing
.04 M D-sorbitol
with 0.5 -octyl-
0
2.0
119
z
n
b-glucopyranoside
Table 2. Puriˆcation of Membrane-bound D-Sorbitol Dehydrogenase from Gluconobacter suboxydans IFO 3255
Step
Volume
ml)
Total activity
(units)
Total protein
(mg)
Speciˆc activity
(
(units mg-protein)
W
Cell-free extract
Membrane fraction
Solubilized fraction
DEAE-cellulose (DE52)
DEAE-Sepharose (CL6B)
Hydroxylapatite (Bio-Gel HTP)
Sephacryl HR300
320
100
200
125
40.0
52.0
13.0
9,939
6,894
2,878
1,812
924.0
415.4
173.5
9,024
2,280
1,480
250
1.10
3.02
1.94
7.25
16.4
30.9
45.3
56.5
13.5
3.83
zyme was done with the same buŠer containing 0.1
NaCl. The fractions having enzyme activity were col-
lected.
Step 3. DEAE-Sepharose column chromato-
graphy. The pooled enzyme fractions (125 ml)
from the previous step were dialyzed against two
batches, each of one liter of the buŠer containing
M
equilibrated with the same buŠer. The enzyme activi-
ty was eluted during the washing of the column. The
above fractionation step was repeated and the frac-
tions having the enzyme activity were combined. The
total volume was 52 ml after the active fraction was
dialyzed against the buŠer containing 0.05
M
D
-
sorbitol and 0.1 Triton X-100. Then, the fraction
z
0
.05 M D-sorbitol and 0.1
lyzed enzyme fractions were put on a DEAE-
Sepharose column ( 1.5 by 50 cm) equilibrated with
the same buŠer. After it was washed with the same
buŠer, -sorbitol dehydrogenase was eluted with a
linear gradient of NaCl (0 to 0.2 ). The major en-
zyme activity was eluted at the NaCl concentration of
.16 to 0.18
z
Triton X-100. The dia-
was concentrated to 10 ml by ultraˆltration (PM 10,
Amicon).
q
Step 5. Sephacryl HR300 column chromato-
graphy. A portion of the enzyme fraction (2 ml)
D
was put on Sephacryl HR300 column (
120 cm) equilibrated with the buŠer (pH 7.0) con-
taining 0.05 NaCl, 0.05 M D-sorbitol and 0.1 Tri-
q1.0 by
M
M
z
0
M
.
ton X-100, and then developed. This fractionation
step was repeated and the active fractions were com-
bined. The active fraction (13 ml) dialyzed against
Step 4. Hydroxylapatite column
chromato-
graphy. The pooled enzyme fractions (40 ml) were
dialyzed against two batches, each of 500 ml of the
the buŠer containing 0.05 M D-sorbitol and 0.1
ton X-100 was pooled and stored at „80 C.
Puriˆcation of the enzyme is summarized in
Table 2.
z Tri-
buŠer containing 0.05 M D-sorbitol and 0.1
z
Triton
9
X-100. A portion of the enzyme sample (5 ml) was
put on a hydroxylapatite column ( 2.5 by 20 cm) and
q

6
0
T. SUGISAWA and T. HOSHINO
Molecular structure of puriˆed membrane-bound
D
-sorbitol dehydrogenase by HPLC and SDS-gel
electrophoresis
The puriˆed enzyme with a speciˆc activity of
4
5.45 units per mg of protein (0.2 mg ml) that was
W
used for the analysis was dissolved in 0.01
M
potassi-
um phosphate buŠer (pH 7.0) containing 0.05 M D
-
sorbitol and 0.1
z
Triton X-100.
The molecular weight of the native
D
-sorbitol
dehydrogenase was measured by HPLC using a size
exclusion gel column (TSK gel G3000 SWXL
column,
potassium phosphate buŠer (pH 7.0) containing
.3 NaCl. The molecular standards were
cyanocobalamin (1.35 kDa), myoglobin (17 kDa),
ovalbumin (44 kDa), -globulin (158 kDa), and
thyroglobulin (670 kDa). The puriˆed enzyme
showed a single peak and the molecular mass was
estimated to be 800 kDa, however its peak was out of
the range of molecular standards of 1.35 kDa to
q
7.8 by 300 mm) equilibrated with a 0.1
M
0
M
g
Fig. 1. SDS-Gel Electropholesis of Puriˆed
D-Sorbitol De-
hydrogenase.
Puriˆed
5.3 was used; 3 or 5
staining with Coomassie brilliant blue
of -sorbitol dehydrogenase was measured. Lane (1), Standard
marker proteins (kDa); Lane (2), 3 g of puriˆed -sorbitol de-
of puriˆed -sorbitol de-
D
-sorbitol dehydrogenase with a speciˆc activity of
g of protein was added. After the protein
-250, the molecular mass
4
m
R
6
70 kDa.
D
m
D
In the SDS-PAGE, the enzyme showed a single
hydrogenase; Lane (3),
hydrogenase.
5
m
g
D
band with a molecular mass of 80 kDa
±
1.0 kDa on
(
Fig. 1, lanes 2 and 3). It was therefore estimated the
puriˆed
D
-sorbitol dehydrogenase consists of ten
homologous subunits.
Table 3. Substrate Speciˆcity of Puriˆed Enzyme and Identiˆca-
tion of its Product
Catalytic properties of membrane-bound
sorbitol dehydrogenase
D
-
a)
Relative activity
Substrate
Product
(
z)
Substrate speciˆcity. Substrate speciˆcities of the
puriˆed enzyme for sugars, sugar alcohols, and or-
ganic acid were surveyed as the catalytic activity and
the product from each sugar alcohol was identiˆed
D
D
D
-Sorbitol
-Mannitol
-Arabitol
100
49.9
175
172
66.6
117
0
0
0
0
0
L
-Sorbose
-Fructose
-Xylulose
D
D
meso-Erythritol
-Adonitol
Glycerol
Erythrulose
-Ribulose
(
Table 3). Among the substances tested,
-arabitol, erythritol, and glycerol were highly oxi-
dized, and -mannitol and -adonitol were also oxi-
dized at 49.9 and 66.6 of the reaction rate with
-sorbitol, respectively. -Sorbose, -fructose,
xylulose, erythrulose, -ribulose, and dihydroxyace-
tone were produced from -sorbitol, -mannitol,
-arabitol, erythritol, -adonitol, and glycerol,
D
-sorbitol,
D
D
Dihydroxyacetone
D
b)
D
-Glucose
-Fructose
—
D
D
D
—
—
—
—
—
z
L-Sorbose
D
L
D
D
-
Methanol
Ethanol
Sucrose
D
0
6.65
D
D
D
-Gluconate
not done
D
D
respectively. The puriˆed membrane-bound enzyme
is thought to be a sugar alcohol dehydrogenase. The
To identify the product from each substrate, 2 units of puriˆed enzyme
were incubated in 1.0 ml of reaction mixture (8 m PMS and 0.2 potassium
phosphate buŠer pH 7.0) with 0.04 -sorbitol, -mannitol, -arabitol,
erythritol, -adonitol, or glycerol for 4 h at 30 C. The reaction product was
M
M
D
D
D
enzyme had the activity on
D
-gluconate, however, the
D
9
product analysis was not done because of its low ac-
analyzed by HPLC and thin layer chromatography.
6)
a)
Relative activity is expressed as percentage of the reaction rate obtained
tivity (6.65
z
).
with the substrate
not tested.
D-sorbitol.
EŠects of substrate concentration of reaction rate.
b)
The velocity of the oxidizing reaction using varying
the concentrations of
D
-sorbitol from 0.5 to 80 m
M
was measured to calculate the Km value for
D
-
than 60z of the initial activity remained when the
reaction was done in the pH range between 7.0 and
9.0.
EŠects of temperature on the activity and the
stability. As shown in Fig. 3A, the enzyme showed
the highest activity at 30 9C . Decreases of 60 and 70z
sorbitol. It was calculated to be 18 m
M
when DCIP
was used as the electron acceptor for the reaction.
EŠects of pH on the activity and the stability. The
correlation between the reaction rates of
D
-sorbitol
dehydrogenase and pH values of the reaction mixture
was measured, with all other assay conditions being
kept the same. The enzyme showed the highest activi-
ty at pH 6.0 (Fig. 2A). As shown in Fig. 2B, more
in its activity were observed at 45 and 50
tively, and no activity was detected at 60
9
9C.
C, respec-
9
The enzyme was stable up to 35 C, and lost about

Membrane-bound
D
-Sorbitol Dehydrogenase
61
Fig. 2. EŠects of pH on the Activity and the Stability of Puriˆed D-Sorbitol Dehydrogenase.
The activity was measured under standard assay conditions with the following buŠers: 0.1
M
McIlvaine buŠer (pH 5.5
Na CO –NaHCO
Triton X-100 of diŠerent pH for 16 h at 4
¿
8, ――);
$
―
0
―
.1
M
Phosphate buŠer (pH 6
¿
7.5,
―
); 0.2
M
Tris buŠer (pH 7
¿
9, ――); and 0.2
M
2
3
3
buŠer (pH 9
¿
10,
C,
―). The enzyme was left standing in buŠers containing 0.05 M D-sorbitol and 0.1
z
9
and then the residual activity was measured.
Fig. 3. EŠects of temperature on the Activity and the Stability of Puriˆed
D
-Sorbitol Dehydrogenase.
C for stability (B). Thermostability was tested by
potassium phosphate buŠer (pH 7.0) containing 0.05 M D-sorbitol
Triton X-100. The residual activity was measured after the treated enzyme was immediately cooled in iced water.
Data are expressed as a percentage of the activity at 30
incubating the enzyme for 5 min at various temperatures in 0.01
and 0.1
9 9
C for activity (A) and and 25
M
z
2
0, 70, or 90
at 40, 50, or 60
EŠects of metal ions and inhibitors. As shown in
Table 4, 17 of the activity was stimulated by the
addition of 0.91 m
either 0.91 m of Cu or 0.91 m
z
of its activity after by the incubation
Distribution of membrane-bound
dehydrogenase in other acetic acid bacteria
Distribution of membrane-bound -sorbitol de-
D
-sorbitol
9
C, respectively (Fig. 3B).
D
z
hydrogenase in various acetic acid bacteria was sur-
veyed with Western blot analysis using the antibody
for the enzyme. Cell homogenates containing 3 to
2
+
M
Co . However, the addition of
2+ 2+
M
M
of Fe was
strongly inhibitory, and the addition of 1.79 m
M
of
5 mg of protein treated with 2z sodium dodecyl sul-
2+
Zn was inhibitory by 68
z
. The eŠects of various
fate (SDS) were prepared, and then the SDS-PAGE
was done. As shown in Table 5, all of the tested
Gluconobacter strains showed a positive band at the
position of molecular mass of 80 kDa, indicating the
inhibitors on the activity were investigated. Quinine
and monoiodoacetate was inhibitory by 25 and 75
respectively.
z,
D
-sorbitol dehydrogenase is present in these microor-
ganisms. On the other hand, the critical positive band

6
2
T. SUGISAWA and T. HOSHINO
Table 4. EŠects of Metal Ions and Inhibitors on
hydrogenase
D
-Sorbitol De-
Table 5. Distribution of the
Acetic Acid Bacteria
D
-Sorbitol Dehydrogenase in Other
Relative activity (
z
)
Immunological
blotting
Compounds added
Strain tested
Concentration in reaction mixture
〈
CaCl
CoCl
CuCl
Fe(SO
MgCl
MnCl
ZnCl
ZnSO
〈
EDTA
Metals
〉
0.91 m
M
1.79 m
M
Gluconobacter albidus IFO 3250
G. albidus IFO 3251
G. albidus IFO 3253
G. capsulatus IFO 3462
G. cerinus IFO 3263
G. cerinus IFO 3264
G. cerinus IFO 3265
G. cerinus IFO 3267
G. cerinus IFO 3270
G. dioxyacetonicus IFO 3271
G. dioxyacetonicus IFO 3274
G. gluconicus IFO 3171
G. gluconicus IFO 3285
G. gluconicus IFO 3286
G. industrius IFO 3260
G. melanogenus IFO 3292
G. melanogenus IFO 3293
G. melanogenus IFO 3294
G. nonoxygluconicus IFO 3276
G. oxydans IFO 3189
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
2
96 96
・
6H
O
O
H
117
0
0
88
80
56
44
0.96 m
96.7
108
0
0
92
88
32
40
1.89 m
100
75
113
25
2
2
2
・
2
2H
)
・
x
2
O
4
・
6H
O
O
2
2
・
4H
2
2
2
・
7H
O
4
2
Inhibitors
〉
M
M
Quinine
NaN
Monoiodoacetate
・
HCl
79.2
91.7
75.0
3
Relative activity is expressed as percentage of the reaction rate obtained
without metals and inhibitors. The eŠects of metal ions and inhibitors on
sorbitol dehydrogenase activity were investigated by measuring the activity
with a standard assay. After the addition of the enzyme solution to the basal
reaction mixture as described in Materials and Methods, a solution of each
metal ion source was stirred in and the reaction started with the addition of
D
-
D
-sorbitol.
G. oxydans subsp. sphaericus IFO 12467
G. roseus IFO 3990
G. rubiginosus IFO 3244
G. suboxydans IFO 3130
G. suboxydans IFO 3172
G. suboxydans IFO 3254
G. suboxydans IFO 3255
G. suboxydans IFO 3256
G. suboxydans IFO 3257
G. suboxydans IFO 3258
G. suboxydans IFO 3289
G. suboxydans IFO 3290
G. suboxydans IFO 3291
was not observed in Acetobacter strains, with the
exception of A. aceti subsp. orleansis IFO 3259, A.
aceti subsp. xylinum IFO 3288 and A. aceti xylinum
IFO 13722. The cell homogenate of A. aceti subsp.
aceti IFO 3281, A. liquefaciens IFO 12388 and F.
aurantia IFO 3245 showed a slight positive band at
the position of molecular mass 80 kDa.
Discussion
Acetobacter aceti subsp. aceti IFO 3281
A. aceti subsp. aceti IFO 3284
A. aceti subsp. orleansis IFO 3259
A. aceti subsp. xylinum IFO 3288
A. aceti subsp. xylinum IFO 13772
A. acetigenus IFO 3277
A. acetigenus IFO 3280
A. acetosus IFO 3129
A. ascendens IFO 3188
A. liquefaciens IFO 12257
weakly positive
d
+
+
+
d
d
d
d
5
)
Shinagawa et al. solubilized and puriˆed a
bitol dehydrogenase from the membrane fraction of
G. suboxydans var IFO 3254 which had sig-
niˆcantly high activity at a low pH of 4.5, and more
than 90 of its activity was lost at a pH of 7.0. They
suggested that the enzyme participating in -sorbose
fermentation is a membrane-bound enzyme and not
an NADP-dependent enzyme since -sorbose rapidly
accumulates in the culture medium. Baker and
D
-sor-
.
a
z
L
7
)
d
L
A. liquefaciens IFO 12388
weakly positive
A. pasteurianus subsp. lovaniensis IFO 13753
A. pasterianus subsp. paradoxus IFO 13754
A. pasterianus subsp. pasteurianus IFO 3222
A. peroxydans IFO 13755
A.rancens IFO 3298
A. sp. IFO 3248
—
d
—
—
d
8
)
Claus studied the solubilization of NAD(P)-
independent -sorbitol dehydrogenase of G. oxydans
subsp. suboxydans ATCC 621 using octyl-glucoside
and reported that -sorbitol dehydrogenase from the
D
D
—
strain ATCC 621 was substantially diŠerent from the
5
)
Frateuria aurantia IFO 3245
F. aurantia IFO 13328
weakly positive
—
enzyme obtained by Shinagawa et al. However, the
enzyme was not isolated nor characterized except for
9
)
its solubilization study. Recently, Choi et al. isolat-
+: The positive band was strongly detected at the 80 kDa position on
SDS-PAGE.
ed a PQQ-dependent membrane-bound
dehydrogenase from G. suboxydans KCTC 2111
equivalent to ATCC 621), which consisted of three
subunits having molecular masses of 75, 50, and
4 kDa. This enzyme is thought to be the same en-
D
-sorbitol
„
: A crossing band was not detected at any position on SDS-PAGE.
d: A crossing band was detected at a position diŠerent from the 80 kDa
position on SDS-PAGE.
(
1
8
)
zyme as that described by Baker and Claus. The
prosthetic group of the Shinagawa's enzyme is FAD
and heme
and heme
c
, whereas that of the Choi's one is PQQ
c
. Both membrane-bound enzymes catalyz-

Membrane-bound
D
-Sorbitol Dehydrogenase
63
Table 6. Properties of Related Membrane-bound Enzymes
Enzyme
Reference
D
-Sorbitol dehydrogenase
Glycerol dehydrogenase
5
)
7)
10)
This study
Shinagawa et al
.
Choi et al
.
Adachi et al.
Microorganism
G. suboxydans IFO 3255 G. suboxydans IFO 3254 G. suboxydans ATCC 621
G. industrius IFO 3260
Molecular weight
800 kDa
131 kDa
(63, 51, 17 kDa)
(139 kDa?)
(75, 50, 14 kDa)
Huge aggregation
(80 kDa)
11)
(
Subunit)
(80 kDa
6.0–7.0
7.5–8.5
×
10)
Optimum pH
4.5
(5.0?)
7.5–8.0
pH stability
4.5–5.5
no data
8.5–10.0
Substrate speciˆcity
D
D
D
-Sorbitol
-Mannitol
-Arabitol
100
49.9
175
172
117
0
100
5
0
0
no data
0
no data
no data
100
68
no data
no data
no data
no data
70
45
26
136
111
100
0
meso-Erythritol
Glycerol
D
-Xylitol
-Adonitol
D
66.6
0
42
0
Ethanol
20
Km with
D
-sorbitol
18 m
M
at pH 6.0
30 m
M
at pH 4.5
no data
34 m
0.5
M
with glycerol
Solubilization
1
z
Triton X-100
1
z
Triton X-100
1.5
z
n
-octyl-
z
dimethyldodecyl-
with 0.04 M D-sorbitol
with 0.1 M D-sorbitol
and 0.1 KCl
glucoside
amineoxide
M
Prosthetic group
not determine
FAD and heme
c
PQQ and heme c
PQQ
ing the oxidation of
D
-sorbitol were puriˆed and their
IFO 3260, we think the
D
-sorbitol dehydrogenase of
properties were studied, and their results seemed to
G. suboxydans IFO3255 is similar to the glycerol de-
11)
give the product
L
-sorbose from
D
-sorbitol.
hydrogenase because of the similarities in molecu-
lar mass and substrate speciˆcity. In addition, it is
However, the product is not mentioned in the above
5
,9)
11)
reports.
The membrane-bound
described how the glycerol dehydrogenase formed
D
-sorbitol dehydrogenase
huge aggregation in the puriˆcation step. Our en-
zyme seemed to be an aggregation form because the
from G. suboxydans IFO 3255 in this paper has quite
diŠerent properties from those of the enzymes
non-denaturing -sorbitol dehydrogenase had a rela-
D
described above. The present membrane-bound
D
-
tively high molecular mass of 800 kDa consisting of
10 homologous subunits (80 kDa). The comparison
of the properties of membrane-bound sugar alcohol
dehydrogenases discussed here is summarized in
Table 6.
sorbitol dehydrogenase had an optimal pH range
around neutral. We tried to measure absorption spec-
tra of the puriˆed enzyme to investigate the prosthet-
ic group of the enzyme; however, peaks correspond-
ing to cytochrome
(
c
and PQQ were not observed
Interestingly, it was suggested by Western blot
analysis that our enzyme is located in all of
Gluconobacter strains tested (33 strains). The enzyme
isolated here is thought to have an important role
relating to the ˆrst reaction of sugar alcohol oxida-
tion for Gluconobacter strains in a neutral pH range.
data not shown). Generally, the activity of PQQ
2+
enzyme is stimulated by the addition of Ca and
PQQ when these co-factors of the enzyme are free,
and inhibited by the addition of EDTA. The isolated
enzyme, however, did not show the properties men-
tioned above and the enzyme activity was not stimu-
2
+
lated by the addition of PQQ or Ca and was not in-
hibited by the addition of EDTA. The study of the
prosthetic group of this enzyme is insu‹cient at this
time.
summarized the properties of vari-
ous membrane-bound sugar alcohol dehydrogenases
in acetic acid bacteria and quoted that the mem-
brane-bound glycerol dehydrogenase of G. indus-
rius IFO 3260 consists of one subunit having the
molecular mass of 80 kDa and its prosthetic group is
PQQ. Although the optimum pH is diŠerent between
those of G. suboxydans IFO3255 and G. indusrius
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1
2
3
)
)
)
Widmer, C., King, T. E., and Cheldelin, V. H., Par-
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.
,
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1
1)
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D
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