A D-sorbitol dehydrogenase in homogenous form, which catalyzes the oxidation of D-sorbitol to L-sorbose, has a molecular structure consisting of homologous subunits of molecular weight 79,000.+-.5,000 each, a substrate specificity for polyols and an optimum pH of 6.0-7.0. Said D-sorbitol dehydrogenase is producible by a process comprising cultivating a microorganism belonging to the genus Gluconobacter or Acetobacter, or a mutant or variant thereof, which is capable of producing the D-sorbitol dehydrogenase in the cells and isolating it from the cells, e.g. by disrupting the cells and isolation from cell-free extract of the disrupted cells. The so-isolated D-sorbitol dehydrogenase is useful for catalyzing the oxidation of D-sorbitol to L-sorbose, the latter being an important intermediate for the production of vitamin C.

BACKGROUND OF THE INVENTION 
Enzymes which catalyze the oxidation of D-sorbitol to L-sorbose are known. 
J. T. Cummins, T. E. King and V. H. Cheldelin (J. Biol. Chem., 
224,323-329, 1957) reported that the cell free extract of Acetobacter 
suboxydans (syn. Gluconobacter suboxydans) contains three enzymes 
participating in pathways of D-sorbitol oxidation. Two of these enzymes 
catalyzing the oxidation of D-sorbitol to L-sorbose have been purified. 
One was isolated as nicotine amide adenine dinucleotide phosphate 
(hereinafter referred to as NADP)-dependent L-sorbose reductase from the 
soluble fraction of Gluconobacter melanogenus IFO 3293 by T. Sugisawa, T. 
Hoshino and A. Fujiwara (Agric. Biol. Chem., 55,2043-2049, 1991), and 
another was isolated as membrane-bound D-sorbitol dehydrogenase from 
Gluconobacter suboxydans var. .alpha. IFO 3254 by E. Shinagawa, K. 
Matsushita, O. Adachi and M. Ameyama (Agric. Biol. Chem., 46, 135-141, 
1982). 
SUMMARY OF THE INVENTION 
The present invention concerns a novel D-sorbitol dehydrogenase, a process 
for producing the same and a process for producing ketoses, especially 
L-sorbose utilizing said enzyme. 
The novel D-sorbitol dehydrogenase (hereinafter referred to as SLDH) 
provided by the present invention catalyzes the oxidation of D-sorbitol to 
L-sorbose. L-Sorbose is an important intermediate for the production of 
vitamin C. 
DETAILED DESCRIPTION OF THE INVENTION 
The SLDH provided by the present invention is clearly distinct from the 
enzymes of Cummins et al. and Shinagawa et al. in the subunit structure of 
the enzyme, the molecular weight, substrate specificity and optimum pH. 
The molecular weight of the NADP-dependent L-sorbose reductase of Sugisawa 
et al. is 60,000. The membrane-bound D-sorbitol dehydrogenase isolated by 
E. Shinagawa et al. consisted of three kinds of subunits with molecular 
weights of 63,000, 51,000 and 17,000. This enzyme oxidized D-sorbitol 
specifically, and also oxidized D-mannitol at 5% of the reaction rate with 
D-sorbitol. However, the Shinagawa et al. enzyme did not oxidize 
D-arabitol or erythritol. Furthermore, Shinagawa et al. showed that the 
optimum pH of the enzyme was 4.5, and that the enzyme activity was stable 
at pH 5.0. However, 92% of the activity was lost at pH 7.0. 
In distinction from the enzymes of Sugisawa et al. and Shinagawa et al., 
the molecular weight of the SLDH provided by the present invention is 
about 800,000.+-.50,000 and it consists of ten homologous subunits having 
a molecular weight of 79,000.+-.5,000, each. Further, the SLDH of the 
present invention does not require NADP to catalyze the reaction. 
Moreover, the SLDH of the present invention oxidizes not only D-sorbitol 
and D-mannitol, but also D-arabitol and erythritol. The optimum pH of the 
SLDH of the present invention is 6.0, and its activity is stable even at 
pH 8.0. 
The microorganisms used for the present invention are microorganisms 
belonging to genus Gluconobacter and Acetobacter. Mutants and variants of 
said microorganism can be also used in the present invention. Thus, the 
present invention comprises an SLDH enzyme in homogenous form from 
microorganisms of the genus Gluconobacter and Acetobacter which acts on 
D-sorbitol to produce L-sorbose (i.e., catalyzes the oxidation of 
D-sorbitol to L-sorbose), and which has the following physico-chemical 
properties: 
a) Molecular structure: A molecular weight of 800,000.+-.50,000 consisting 
of ten homologous subunits having a molecular weight of 79,000.+-.5,000; 
b) Substrate specificity: Active on polyols; 
c) Optimum pH for oxidizing D-sorbitol to L-sorbose: 6.0-7.0; 
d) Oxidizes D-sorbitol to L-sorbose at a pH in the range from pH 5.5-8.0. 
Preferred microorganisms of the genus Gluconobacter and Acetobacter are 
described herein. The most preferred microorganism, Gluconobacter 
suboxydans IFO 3255, has been deposited at the Deutsche Sammlung von 
Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg lb, D-38124 
Braunschweig, Germany on Feb. 13, 1995 under the Budapest Treaty. The 
allotted deposit number is DSM 9715. 
The invention also comprises a method for producing L-sorbose from 
D-sorbitol which comprises reacting the D-sorbitol in an aqueous medium 
under aerobic conditions in the presence of a catalytically effective 
amount of the SLDH of the present invention and an effective amount of an 
electron acceptor whereby the D-sorbitol is oxidized to the L-sorbose. Any 
conventional compound which has the ability to act as an electron acceptor 
can be utilized in conjunction with the enzyme of this invention. As an 
electron acceptor, 2,6-dichlorophenolindophenol (hereinafter referred to 
as DCIP), phenazine methosulfate (hereinafter referred to as PMS), 
ferricyanide or cytochrome c are preferred. 
The process of the invention may be carried out under any conventional 
conditions whereby the SLDH of the invention oxidizes D-sorbitol to 
L-sorbose. The reaction is preferably carried out at a pH in the range 
from about 5.5 to about 8.0 and at a temperature in the range from about 
20.degree. to about 50.degree. C. More preferably, the process of the 
invention is carried out at a pH in the range from about 6.0 to 7.0 and at 
a temperature in the range from about 20.degree. to 40.degree. C. 
It is another object of the present invention to provide a process for 
producing the novel enzyme SLDH by cultivation of a microorganism 
belonging to the genus Gluconobacter or Acetobacter, or a mutant thereof, 
which is capable of producing the SLDH in the cells, disruption of the 
cells, isolation and purification of the enzyme from cell-free extract of 
disrupted cells, preferably from the membrane fraction of the 
microorganism. A still further object of the present invention is to 
provide a process for producing L-sorbose utilizing said enzyme, SLDH. 
The further physico-chemical properties of the purified sample of the novel 
SLDH prepared according to the Examples herein after are as follows: 
1) Enzyme activity 
The novel SLDH of the present invention catalyzes the oxidation of 
D-sorbitol to L-sorbose in the presence of an electron acceptor according 
to the following reaction formula: 
EQU D-Sorbitol+Electron acceptor.fwdarw.L-Sorbose+Reduced electron acceptor 
The enzyme does not utilize oxygen as an electron acceptor. This was 
affirmed by the lack of catalytic activity of the enzyme in an attempt to 
convert D-sorbitol to L-sorbose using oxygen as a possible electron 
acceptor. Furthermore, no oxygen consumption was detected in the reaction 
mixture as detected by a dissolved oxygen probe. However, any conventional 
compound which has the ability to act as an electron acceptor can be 
utilized in conjunction with the enzyme of this invention. As an electron 
acceptor, 2,6-dichlorophenolindophenol (hereinafter referred to as DCIP), 
phenazine methosulfate (hereinafter referred to as PMS), ferricyanide or 
cytochrome c are preferred. 
The enzyme assay was performed as follows. The basal reaction mixture for 
assaying D-sorbitol dehydrogenase activity consisted of 50 mM potassium 
phosphate buffer (pH 6.0), 0.25 mM DCIP and 0.325 mM PMS, which was 
prepared just before the assay. A cuvette with a 1-cm light path contained 
0.4 ml of the basal reaction mixture, 0.1 ml of 0.4M D-sorbitol and enzyme 
solution with a total volume of 0.51 ml. The reference cuvette contained 
all components except for the substrate. The reaction was started at 
25.degree. C. with D-sorbitol and the enzyme activity was measured as the 
initial reduction rate of DCIP at 600 nm. One enzyme unit is defined as 
the amount of the enzyme that catalyzes the reduction of 1 .mu.mol of DCIP 
per minute. The extinction coefficient of DCIP at pH 6.0 was 10.8 
mM.sup.-1. 
2) Substrate specificity 
Substrate specificity of the enzyme was determined using the same enzyme 
assay method as described above in 1) except that various substrate 
solutions (100 mM) were used instead of D-sorbitol. The results of the 
measurement are shown in Table 1. Among the tested substances, D-sorbitol, 
D-arabitol, erythritol and glycerol were highly oxidized, and D-mannitol 
and D-adonitol were also oxidized, at 49.9 and 66.6% of the reaction rate 
of D-sorbitol. 
TABLE 1 
______________________________________ 
Substrate specificity of the purified D-sorbitol dehydrogenase 
Relative activity.sup.a) 
Substrate (%) 
______________________________________ 
D-Sorbitol 100 
D-Mannitol 49.9 
D-Arabitol 175 
meso-Erythritol 
172 
D-Adonitol 66.6 
Glycerol 117 
D-Glucose 0 
D-Fructose 0 
L-Sorbose 0 
D-Xylitol 0 
Methanol 0 
Ethanol 0 
Sucrose 0 
______________________________________ 
.sup.a) Relative activity is expressed as the percent of the reaction rat 
obtained with the substrate Dsorbitol. 
3) Optimum pH 
The correlation between the reaction rate of the SLDH and pH was determined 
using the same enzyme assay method as described above in 1) except that 
various pH's and buffers were used. The results are shown in Table 2. The 
enzyme showed optimum pH values of 6.0-7.0 and showed the highest activity 
at pH 6.0. 
TABLE 2 
______________________________________ 
Optimum pH for the SLDH activity 
Relative activity (%).sup.a) 
Buffers (0.1M) 
Potassium 
pH value McIlvain phosphate 
Tris-HCl 
______________________________________ 
4.0 -- -- -- 
4.5 -- -- -- 
5.0 -- -- -- 
5.5 22.2 -- -- 
6.0 82.2 100.0 -- 
6.5 48.9 73.3 -- 
7.0 40.0 57.8 60.0 
7.5 33.3 51.1 31.1 
8.0 31.1 -- 11.1 
8.5 -- -- 0 
9.0 -- -- 0 
______________________________________ 
.sup.a) Data are expressed as a percentage of the activity at pH 6.0 of 
potassium phosphate buffer. 
4) pH stability 
The enzyme was kept standing in buffers of various pHs for 16 hours at 
4.degree. C., and then the residual activity was measured using the same 
enzyme assay method as described above in 1). The results of the 
measurement are shown in Table 3. Over 60% of the activity remained at pHs 
between 7.0 and 9.0. 
TABLE 3 
______________________________________ 
pH Stability for the SLDH activity 
Relative activity (%).sup.a) 
Buffers (0.1M) 
pH value McIlvain Potassium phosphate 
Tris-HCl 
______________________________________ 
4.0 -- -- 
4.5 0 -- -- 
5.0 0 -- -- 
5.5 0 -- -- 
6.0 0 0 -- 
6.5 0 10.0 -- 
7.0 50.0 60.0 75.0 
7.5 60.0 70.0 80.0 
8.0 100.0 -- 80.0 
8.5 -- -- 95.0 
9.0 -- -- 85.0 
______________________________________ 
.sup.a) Data are expressed as a percentage of the activity at pH 8.0 of 
McIlvain buffer. 
5) Thermostability 
Thermostability was tested by incubating the enzyme for 5 minutes at 
various temperatures in 0.01M potassium phosphate buffer (pH 7.0). The 
residual activity was measured using the same enzyme assay method as 
described above in 1), after which the treated enzyme was cooled 
immediately in ice water. The results are shown in Table 4. The enzyme was 
stable up to 35.degree. C., and lost about 20, 70 and 90% of its activity 
after it had been incubated at 40.degree., 50.degree. and 60.degree. C., 
respectively. 
TABLE 4 
______________________________________ 
Temperature stability for the SLDH activity 
Temperature Relative activity.sup.a) 
(.degree.C.) (%) 
______________________________________ 
0 100 
20 100 
25 106 
30 106 
35 106 
40 82.4 
50 29.4 
60 11.8 
______________________________________ 
.sup.a) Data are expressed as a percentage of the activity at 20.degree. 
C. 
6) Optimum temperature 
The enzyme activities were measured at temperatures from 20.degree. to 
60.degree. C. in the same assay method as described above in 1). The 
results are shown in Table 5. The enzyme showed optimum temperature at 
from 20.degree. to 40.degree. C. and showed the highest activity at 
30.degree. C. The decrease of 60 and 70% in its activity was observed at 
45.degree. and 50.degree. C., respectively, and no more activity was 
detected at 60.degree. C. 
TABLE 5 
______________________________________ 
Optimum temperature for the SLDH activity 
Temperature Relative activity.sup.a) 
(.degree.C.) (%) 
______________________________________ 
20 82.1 
28 83.2 
30 100 
35 91.7 
37 77.0 
40 74.8 
45 39.9 
50 28.9 
55 4.5 
60 0 
______________________________________ 
.sup.a) Data are expressed as a percentage of the activity at 30.degree. 
C. 
7) Effects of metal ions and inhibitors 
The effects of metal ions and inhibitors on the SLDH activity were examined 
by measuring the activity using the same assay method as described above 
in 1). After the addition of enzyme solution to the basal reaction mixture 
each metal solution was stirred in and the reaction was started with the 
addition of D-sorbitol. As shown in Table 6, 8 to 17% of the activity was 
stimulated by the addition of 0.91 and 1.79 mM Co.sup.2+. However, the 
addition of 0.91 mM Cu.sup.2+ and Fe.sup.3+ in each case was strongly 
inhibitory, and the addition of Zn.sup.2+ was inhibitory by 44 to 68%. The 
effects of various inhibitors on the activity were investigated. As shown 
in Table 7, quinine hydrochloride and monoiodoacetate were inhibitory by 
25% and 75%, respectively. 
TABLE 6 
______________________________________ 
Effects of various metals on the activity of D-sorbitol dehydrogenase 
Relative activity (%) 
Concentration (mM) 
Metal 0.91 1.79 
______________________________________ 
Ca(NO.sub.3).sub.2.4H.sub.2 O 
96 96 
CaCl.sub.2 96 96 
CoCl.sub.2.6H.sub.2 O 
117 108 
CuSO.sub.4 0 0 
Cu(NO.sub.3).sub.2.3H.sub.2 O 
0 0 
CuCl.sub.2.2H.sub.2 O 
0 0 
Fe.sub.2 (SO.sub.4).sub.3.xH.sub.2 O 
0 0 
MgCl.sub.2.6H.sub.2 O 
88 92 
MnCl.sub.2.4H.sub.2 O 
80 88 
MnSO.sub.4.4.about.6H.sub.2 O 
88 88 
Na.sub.2 MoO.sub.4.2H.sub.2 O 
68 96 
TiCl.sub.4 96 88 
ZnCl.sub.2 56 32 
ZnSO.sub.4.7H.sub.2 O 
44 40 
NiSO.sub.4.6H.sub.2 O 
88 108 
______________________________________ 
Relative activity is expressed as the percentage of the reaction rate 
obtained without metal compounds shown in the table. 
TABLE 7 
______________________________________ 
Effects of inhibitors on the activity of D-sorbitol dehydrogenase 
Relative activity (%) 
Concentration (mM) 
Metal 0.96 1.89 
______________________________________ 
EDTA 96.7 100 
Quinine-HCl 79.2 75.0 
N-Ethylmaleimide 116.7 100 
NaN.sub.3 91.7 112.5 
Monoiodoacetate 75.0 25.0 
Sodium fluoroacetate 
104.2 108.3 
Sodium fluoride 100 100 
Na.sub.2 HAsO.sub.4.7H.sub.2 O 
116.7 120.0 
______________________________________ 
Relative activity is expressed as the percentage of the reaction rate 
obtained without any inhibitors shown in the table. 
8) Effects of substrate concentration on reaction rate 
The velocity of the oxidizing reaction on varying the concentration of 
D-sorbitol from 0.5 to 80 mM was measured to determine the Km value for 
D-sorbitol. The apparent Michaelis constant was calculated to be 18 mM 
with DCIP as an electron acceptor for the reaction. 
9) Molecular weight 
The molecular weight of the native SLDH was determined by HPLC using a size 
exclusion gel column at 280 nm and a flow rate of 1.0 ml per minute. The 
purified SLDH consisted of an homologous subunit with a molecular weight 
of 79,000.+-.5,000 in the presence of sodium dodecyl sulfate (SDS). 
10) Purification procedure 
Purification of the SLDH is effected by the combination of known 
purification methods such as ion exchange chromatography, liquid 
chromatography, adsorption chromatography, gel-filtration chromatography, 
gel-electrophoresis, salting out and dialysis. 
The SLDH provided by the present invention can be prepared by cultivating 
an appropriate microorganism, disrupting the cells and isolating and 
purifying it from cell free extract of disrupted cells, preferably from 
the membrane fraction of the microorganism. 
Examples of the strains most preferably used in the present invention are 
Gluconobacter albidus IFO 3250, Gluconobacter albidus IFO 3251, 
Gluconobacter albidus IFO 3253, Gluconobacter capsulatus IFO 3462, 
Gluconobacter cerinus IFO 3263, Gluconobacter cerinus IFO 3264, 
Gluconobacter cerinus IFO 3265, Gluconobacter cerinus IFO 3267, 
Gluconobacter cerinus IFO 3270, Gluconobacter dioxyacetonicus IFO 3271, 
Gluconobacter dioxyacetonicus IFO 3274, Gluconobacter gluconicus IFO 3171, 
Gluconobacter gluconicus IFO 3285, Gluconobacter gluconicus IFO 3286, 
Gluconobacter industrius IFO 3260, Gluconobacter melanogenus IFO 3292, 
Gluconobacter melanogenus IFO 3293, Gluconobacter melanogenus IFO 3294, 
Gluconobacter nonoxygluconicus IFO 3276, Gluconobacter oxydans IFO 3189, 
Gluconobacter oxydans subsp. sphaericus IFO 12467, Gluconobacter roseus 
IFO 3990, Gluconobacter rubiginosus IFO 3244, Gluconobacter suboxydans IFO 
3130, Gluconobacter suboxydans IFO 3172, Gluconobacter suboxydans IFO 
3254, Gluconobacter suboxydans IFO 3255, Gluconobacter suboxydans IFO 
3256, Gluconobacter suboxydans IFO 3257, Gluconobacter suboxydans IFO 
3258, Gluconobacter suboxydans IFO 3289, Gluconobacter suboxydans IFO 
3290, Gluconobacter suboxydans IFO 3291, Acetobacter aceti subsp. aceti 
IFO 3281, Acetobacter aceti subsp. orleansis IFO 3259, Acetobacter aceti 
subsp. xylinum IFO 3288, Acetobacter aceti subsp. xylinum IFO 13772 and 
Acetobacter liquefaciens IPO 12388. 
These microorganisms, which can be employed in the process of the present 
invention, include those which are being preserved in a public depository 
(culture collection) for delivery to any one upon request such as the 
Institute of Fermentation Osaka, Japan (IFO). Of these, a specific and 
preferred microorganism, Gluconobacter suboxydans IFO 3255, has been 
deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen 
GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig, Germany on Feb. 13, 
1995 under the Budapest Treaty. The allotted deposit number is DSM 9715. 
The microorganism may be cultured in an aqueous medium supplemented with 
appropriate nutrients under aerobic conditions. The cultivation may be 
conducted at a pH of 3.5 to 8.0, preferably 5.0 to 7.5. The cultivation 
period varies depending upon the microorganism and the nutrient medium to 
be used, and is preferably about 6 to 100 hours. A preferred temperature 
range for carrying out the cultivation is from about 20.degree. C. to 
about 40.degree. C., preferably from about 25.degree. C. to about 
35.degree. C. 
It is usually required that the culture medium contains such nutrients as: 
assimilable carbon sources, such as glycerol, D-mannitol, D-sorbitol, 
erythritol, ribitol, xylitol, arabitol, inositol, dulcitol, D-ribose, 
D-fructose, D-glucose and sucrose, preferably D-sorbitol, D-mannitol or 
glycerol; digestible nitrogen sources such as organic substances, for 
example, peptone, yeast extract, baker's yeast and corn steep liquor, and 
inorganic substances, for example, ammonium sulfate, ammonium chloride and 
potassium nitrite; vitamins; and trace elements. 
In the following, an embodiment for isolation and purification of the SLDH 
from the microorganisms after the cultivation is briefly described. 
(1) Cells are harvested from the liquid culture broth by centrifugation or 
filtration. 
(2) The harvested cells are washed with water, physiological saline or a 
buffer solution having an appropriate pH. 
(3) The washed cells are suspended in the buffer solution and disrupted by 
means of a homogenizer, sonicator, French press or treatment with lysozyme 
and the like to give a solution of disrupted cells. 
(4) The SLDH is isolated and purified from the cell free extract of 
disrupted cells, preferably from the membrane fraction of the 
microorganism. 
The SLDH provided by the present invention is useful as a catalyst for the 
production of L-sorbose from D-sorbitol. The reaction should be conducted 
at pH values of about 5.5 to about 8.0, preferably from 6.0 to 7.0, in the 
presence of an electron acceptor, for example, DCIP, PMS, ferricyanide, 
cytochrome c and the like in a solvent such as phosphate buffer, tris 
buffer, citrate buffer and the like. A preferred temperature range for 
carrying out the reaction is from about 20.degree. C. to about 50.degree. 
C., preferably from 20.degree. C. to 40.degree. C. When the pH and 
temperature are set at about 6.5 to 7.5 and 30.degree. C., respectively, 
the reaction usually gives particularly good results. Depending upon other 
reaction conditions such as the amount of enzyme, D-sorbitol, electon 
acceptor, pH, temperature or aeration speed, the D-sorbitol is completely 
converted to L-sorbose within about 2 to 24 hours, preferably within about 
12 hours. The concentration of D-sorbitol in a solvent varies depending 
upon other reaction conditions, but in general is suitably from about 10 
to about 300 g/L, most preferably from about 10 to about 200 g/L. 
In addition to the above, the cultured cells are also useful for the 
production of ketoses from polyols, especially for the production of 
L-sorbose from D-sorbitol. L-Sorbose produced from D-sorbitol in a solvent 
is isolated by a combination of such conventional methods as 
centrifugation and concentration. However, the solvent containing 
L-sorbose without the isolation step can also be used as the starting 
material in the industrial production of vitamin C by the Reichstein 
method. 
In the reaction, the enzyme may also be used in an immobilized state with 
an appropriate carrier. Any means of immobilizing an enzyme generally 
known in the art may be used. For instance, the enzyme may be bound 
directly to a membrane, granules or the like of a resin having functional 
group(s), or it may be bound to the resin through bridging compounds 
having bifunctional group(s), for example, glutaraldehyde.

The following Examples illustrate the present invention. 
EXAMPLE 1 
Preparation of D-sorbitol dehydrogenase 
(1) Cultivation of Gluconobacter suboxydans IFO 3255 
Gluconobacter suboxydans IFO 3255 (DSM 9715) was supplied by the Institute 
for Fermentation, Osaka (IFO), and used throughout this study. The medium 
consisted of 20 g of D-sorbitol, 3 g of yeast extract, 3 g of beef 
extract, 3 g of corn steep liquor, 10 g of polypeptone, 1 g of urea, 1 g 
of KH.sub.2 PO.sub.4, 0.2 g of MgSO.sub.4 .multidot.7H.sub.2 O, and 1 g of 
CaCO.sub.3 in 1 liter of deionized water. The pH was adjusted at 7.0 with 
sodium hydroxide before the addition of CaCO.sub.3. The cultivation in a 
flask was carried out aerobically with rotary shaking for one day, or that 
in a 30-liter jar fermentor was carried out for 21.5 hours at 30.degree. 
C., 500 rpm for agitation and 15 L/min. for aeration. The broth was 
centrifuged at 400.times.g for 10 minutes to remove calcium carbonate, and 
then at 10,000.times.g to pellet the cells. The cell cake was washed once 
with physiological saline. Thus, the intact cells (200 g wet weight) were 
obtained from 20 liters of culture. The cells were frozen at -20.degree. 
C. until used. 
(2) Preparation of membrane fraction 
The cells (100 g wet weight) were suspended in 200 ml of 50 mM phosphate 
buffer (pH 7.0) and passed through a French pressure cell press at 20,000 
psi. After centrifugation to remove intact cells, the supernatant (cell 
free extract) was centrifuged at 80,000.times.g for one hour and this 
precipitate was designated as the membrane fraction (2.28 g wet weight). 
(3) Solubilization 
The SLDH was isolated from the membrane fraction of Gluconobacter 
suboxydans IFO 3255 (DSM 9715). At first, the method reported by E. 
Shinagawa, K. Matsushita, O. Adachi and M. Ameyama, (Agric. Biol. Chem., 
46, 135-141, 1982), was used to solubilize the SLDH. The solubilization 
was performed by treating the membrane with 0.01M sodium acetate buffer 
(pH 5.0) containing 1% Triton X-100, 0.1M KCl, 0.1M D-sorbitol and about 
10 mg/ml of the membrane protein for 2 hours at 5.degree. C. However, the 
SLDH activity was not recovered from the membrane fraction and the whole 
activities in both solubilized supernatant and residual membrane fraction 
were lost under the above conditions. 
Therefore, the effects of the solubilization conditions such as pH value, 
the concentration of buffer, detergents and KCl on the SLDH activity were 
studied. The recovery of SLDH activity was 74% into solubilized 
supernatant from the membrane fraction, when the membrane fraction was 
mixed in 0.05M potassium phosphate buffer (pH 7.0) containing 1% Triton 
X-100 and 0.04M D-sorbitol for 2 hours at 5.degree. C. as shown in Table 
8. The enzyme was not solubilized with n-octyl-.beta.-D-glucopyranoside 
and the activity was lost by the addition of 0.1M KCl. 
TABLE 8 
______________________________________ 
Solubilization of membrane-bound D-sorbitol dehydrogenase 
Relative activity of 
D-sorbitol dehydrogenase 
after solubilization 
(%) 
in the in the 
supernatant 
membrane 
Reaction mixture for the solubilization 
solubilized 
remained 
______________________________________ 
(1) Membrane fraction before solubilization 
100 
(2) Solubilization in 0.01M sodium acetate 
buffer (pH 5.0) containing 0.1M KCl and 
0.1M D-sorbitol. 
with 0.5% Triton X-100 
0 0 
1.0% Triton X-100 0 0 
0.2% n-octyl-.beta.-glucopyranoside 
6 0 
0.3% n-octyl-.beta.-glucopyranoside 
8 0 
(3) Solubilization at various concentrations 
of potassium phosphate buffer (pH 7.0) 
containing 1% Triton X-100 without KCl. 
0.01M buffer + 0.04M D-sorbitol 
23 2.5 
0.05M buffer + 0.04M D-sorbitol 
74 6 
0.1M buffer + 0.04M D-sorbitol 
38.5 3.5 
0.05M buffer without D-sorbitol 
65.5 5 
(4) Solubilization in 0.05M potassium 
2 119 
phosphate buffer (pH 7.0) containing 
0.04M D-sorbitol. 
with 0.5% n-octyl-.beta.-glucopyranoside 
______________________________________ 
To give the active fraction of SLDH for the isolation step {Example 1-(4)} 
from the membrane fraction of Gluconobacter suboxydans IFO 3255 (DSM 
9715), frozen membranes were thawed and suspended in the buffer (pH 7.0) 
to give about 10 mg/ml of protein, and then 1% Triton X-100 and 0.1M 
D-sorbitol were added. The suspension was shaken at 180 rpm for 2 hours, 
and then centrifuged at 80,000.times.g for 60 minutes to remove the 
precipitate. The SLDH activity was recovered in the solubilized 
supernatant (200 ml). 
(4) Diethylaminoethyl (hereinafter referred to as DEAE) -cellulose column 
chromatography 
The solubilized supernatant (200 ml) obtained Example 1-(3) was put on a 
column of DEAE-cellulose (2.5.times.30 cm) equilibrated and washed with 
the buffer (pH 7.0) containing 0.05M D-sorbitol and 0.1% Triton X-100. 
Elution of the enzyme was performed with 0.1M NaCl in the same buffer. The 
fractions having enzyme activity were collected. 
(5) DEAE-Sepharose column chromatography 
The pooled enzyme fractions (125 ml) from the previous step were dialyzed 
against two batches of one liter of the buffer containing 0.05M D-sorbitol 
and 0.1% Triton X-100, and put on a DEAE-Sepharose column (1.5.times.50 
cm) equilibrated and washed with the same buffer, and the SLDH activity 
was eluted with a linear gradient of NaCl (0 to 0.2M). Major enzyme 
activity was eluted at NaCl concentration ranging from 0.16 to 0.18M. 
6) Hydroxylapatite column chromatography 
The pooled active fraction (40 ml) from the previous step was dialyzed 
against two batches of 500 ml of the buffer containing 0.05M D-sorbitol 
and 0.1% Triton X-100. A part of the enzyme (5 ml) was put on a 
hydroxylapatite column (2.5.times.20 cm) equilibrated. The enzyme activity 
was eluted during the washing of the column. After the same preparation 
had been repeated, fractions having enzyme activity were collected. The 
total volume was 52 ml after the active fraction was dialyzed against the 
buffer. Then the fraction was concentrated to 10 ml by ultra-filtration 
(PM10, Amicon). 
(7) Sephacryl HR300 column chromatography 
A portion of the enzyme fraction (2 ml) from the previous step was put on a 
Sephacryl HR300 column (1.times.120 cm) equilibrated with the buffer (pH 
7.0) containing 0.05M NaCl, 0.05M D-sorbitol and 0.1% Triton X-100 and 
developed. This fractionation step was repeated and the active fraction 
was combined. The active fraction dialyzed against the buffer (13 ml) was 
pooled and stored at -80.degree. C. 
Summary of the purification steps of the enzyme is shown in Table 9. 
TABLE 9 
______________________________________ 
Purification of membrane-bound D-sorbitol dehydrogenase 
from Gluconobacter suboxydans IFO3255 (DSM 9715) 
Specific 
Total Total activity 
Volume activity 
protein 
(units/ Recovery 
Step (ml) (units) (mg) mg-protein) 
(%) 
______________________________________ 
Cell free extract 
320 9,939 9,024 1.10 100 
Membrane 100 6,894 2,280 3.02 69.4 
fraction 
Solubilized 
200 2,878 1,480 1.94 29.0 
fraction 
DEAE-Cellulose 
125 1,812 250 7.25 18.2 
(DE52) 
DEAE-Sepharose 
40 924.0 56.5 16.4 9.29 
(CL6B) 
Hydroxylapatite 
52 415.4 13.46 
30.9 4.18 
(BIO-GEL HTP) 
Sephacryl S300 
13 173.5 3.83 45.3 1.75 
______________________________________ 
(8) Purity of the isolated enzyme 
The purified enzyme with a specific activity of 45.43 units per mg of 
protein (0.2 mg/ml) was used for the following analysis. 
The molecular weight of the native D-sorbitol dehydrogenase was determined 
by HPLC (detection, 254 .mu.m; flow rate, 1 ml/min) using a size exclusion 
gel column (TSK gel G3000 SWXL column, 7.8 by 300 mm) equilibrated with 
0.1M potassium phosphate buffer (pH 7.0) containing 0.3M NaCl. The 
molecular weight standards cyanocobalamin (1.35K), myoglobin (17K), 
ovalbumin (44K), .gamma.-globulin (158K) and thyroglobulin (670K) were 
used. The purified enzyme showed a single peak and the molecular weight 
was determined to be about 800,000.+-.50,000. 
In the presence of sodium dodecyl sulfate (SDS), the enzyme showed a single 
band with a molecular weight of about 79,000.+-.5,000. From these results, 
the purified SLDH consisted of ten homologous subunits. 
(9) Identification of the reaction product 
To identify the product converted from each substance, the reaction mixture 
(1 ml) containing 0.04M each of D-sorbitol, D-mannitol, D-arabitol, 
erythritol, D-adonitol and glycerol, and 8 mM PMS was incubated for 4 
hours at 30.degree. C. in 0.2M potassium phosphate buffer (pH 7.0) with 
2.0 units of the purified enzyme. The reaction product was analyzed by 
HPLC and thin layer chromatography. L-Sorbose, D-fructose, D-xylulose, 
erythrulose, D-ribulose and dihydroxyacetone were produced from 
D-sorbitol, D-mannitol, D-arabitol, erythritol, D-adonitol and glycerol, 
respectively. 
EXAMPLE 2 
L-Sorbose production by purified SLDH 
A reaction mixture (total volume 1.04 ml) containing 0.2 ml of purified 
SLDH (0.04 mg protein), 0.04 ml of 0.2M PMS, 0.1 ml of 0.4M D-sorbitol, 
0.4 ml of 0.5M potassium phosphate buffer (pH 7.0) and 0.3 ml of water was 
incubated at 30.degree. C. with gentle shaking. As a result, L-sorbose was 
formed at the rate of about 1.3 mg/hour. 
EXAMPLE 3 
Distribution of membrane-bound D-sorbitol dehydrogenase 
Distribution of membrane-bound D-sorbitol dehydrogenase in various acetic 
acid bacteria was surveyed by the immunological blotting assay using the 
antibody against the SLDH provided in the present invention. Each cell 
homogenate of various acetic acid bacteria was treated with SDS to put 
each 20 .mu.l of the solution containing 3 to 5 .mu.g of protein on SDS 
polyacrylamide gel, and then the electrophoresis was carried out. Protein 
bands developed in the gel were electrophoretically transferred to the 
nitrocellulose membrane and reacted with the antibody. Then the 
nitrocellulose membrane was treated by using the Bio-Rad Immun-Blot kit 
for Goat Anti-Rabbit, and it was investigated as to which microorganism 
showed the positive band at the position of molecular weight (MW) 
79,000.+-.1,000. As shown in Table 10, all of the tested Gluconobacter 
strains and Acetobacter aceti subsp. orleansis IFO 3259, Acetobacter aceti 
subsp. xylinum IFO 3288 and Acetobacter aceti xylinum IFO 13772 showed the 
positive band. The cell homogenate of Acetobacter aceti subsp. aceti IFO 
3281 and Acetobacter liquefaciens IFO 12388 showed weakly positive band at 
the position of MW 79,000.+-.1,000. 
TABLE 10 
______________________________________ 
Immunoblotting analysis by using the antibody of SLDH 
Strain Immunoblotting analysis 
______________________________________ 
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 + 
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 (DSM 9715) 
+ 
G. suboxydans IFO 3256 
+ 
G. suboxydans IFO 3257 
+ 
G. suboxydans IFO 3258 
+ 
G. suboxydans IFO 3289 
+ 
G. suboxydans IFO 3290 
+ 
G. suboxydans IFO 3291 
+ 
Acetobacter aceti subsp. aceti IFO 3281 
weakly positive 
A. aceti subsp. orleansis IFO 3259 
+ 
A. aceti subsp. xylinum IFO 3288 
+ 
A. aceti subsp. xylinum IFO 13772 
+ 
A. liquefaciens IFO 12388 
weakly positive 
______________________________________ 
+: The crossing band was strongly detected at the position of MW 79,000 
.+-. 1,000.