JP2003159093A - Method for producing d-fructose - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for producing D-fructose differing in fundamental principle from the conventional glucose isomerization method, comprising oxidative fermentation of D-sorbitol into D-fructose in nearly 100% yield using aerobic bacteria, especially acetic acid bacteria, through predicting the possibility of the oxidative fermentation using an enzyme not in accordance with the Bertrand-Hudson rule based on a study for the fundamental analysis of oxidative fermentation for many years. <P>SOLUTION: This method for producing D-fructose from D-sorbitol involves the oxidative fermentation utilizing FAD-SLDH (a specific sorbitol dehydrogenase). This new method is greatly superior to the conventional glucose isomerization method having many shortcomings in terms of D-fructose yield, production efficiency and production cost. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the use of aerobic bacteria, especially acetic acid bacteria, to convert D-sorbitol to D-fructose (fructose).
To a cytoplasmic membrane-bound sorbitol dehydrogenase (FAD-SLDH) using covalently bound FAD as a coenzyme.

[0002]

2. Description of the Related Art A method for producing D-fructose has been made into a US patent by Kiyoki Ueda et al. (US patent 3,306,7
52). The strain used here is Bacillus fructo
sus (now B. megaterium) 15450, ATCC 15451, Psuedmo
nas boreopolis ATCC 15452, and Psuedmonas fluores
cens ATCC 15453 (Basic paper: Ueda K., et al.,
J. Ferment. Technol., 46, 541-549, 1967). Although Kiyoki Ueda et al. Have not clarified the characteristics of the enzyme involved in D-fructose production, it is considered to be a NAD-dependent sorbitol dehydrogenase from the viewpoint of the paper and US patent documents. The present inventors
Purchasable from C, a follow-up test was conducted according to the same paper and US patent document, but D-fructose was not produced at all. Since NAD-dependent sorbitol dehydrogenase is an enzyme localized in the cytoplasm, any of the above strains must have a mechanism for taking sorbitol into the cytoplasm, oxidizing it, and then excreting it out of the cell again. To do this, the cells had to waste wasted energy, but no such mechanism existed.

In the conventional glucose isomerization method, due to the reaction equilibrium of the glucose isomerase, the reaction reaches equilibrium when the D-fructose yield is 42% and 55% at the maximum.
Therefore, the product is prepared by increasing the D-fructose concentration in the reaction solution by an ion exchange column method or the like.
It is expensive.

[0004]

The Bertrand-Hudson law has been regarded as the most important law of oxidative fermentation. The present inventors have predicted the possibility of oxidative fermentation by an enzyme that does not follow the Bertrand-Hudson rule from many years of studies on the basic analysis of oxidative fermentation, using aerobic bacteria, especially acetic acid bacteria, to convert D-sorbitol to D -A search was made for a strain capable of oxidatively fermenting fructose at a yield close to 100%, and development of a method for producing D-fructose using the strain was an issue.

[0005]

[Means for Solving the Problems] D-fructose is produced by the glucose isomerization method in the world, both in Japan and abroad. Glucose isomerase is used in the current method for producing D-fructose, but since this isomerization reaction is a reversible enzyme reaction and is accompanied by a reaction equilibrium, the yield of the product is seriously affected. The reaction reaches equilibrium with a D-fructose yield of 42% and a maximum of 55% (Fig. 1). Therefore, to make a high-concentration fructose solution,
A purification operation such as an ion exchange column method is required to remove unreacted glucose, and the product is expensive. On the other hand, oxidative fermentation is an irreversible oxidation reaction,
The greatest advantage is that all the substrate is converted to product (Figure 1). When the D-fructose production is carried out by the oxidative fermentation of the present invention, the D-fructose production method by the glucose isomerization method, which has many bottlenecks, can be used at a low D-fructose yield, production efficiency and production cost. It is possible to achieve a D-fructose production method capable of surpassing the above.

D-fructose is important as a sweetener,
It is said that the demand for soft drinks will exceed 10 million tons worldwide (see Fig. 2). Also, D-
Since fructose becomes sweeter at lower temperatures (cooling), it is superior to sucrose and glucose as a sweetener for soft drinks (Fig. 3). Innovative technology for D-fructose production that can serve both to prevent obesity resulting from excessive sugar intake in civilized countries and promote self-sufficient production of cheap natural sweeteners in underdeveloped countries The present invention has great global significance such as development.

About a century ago, G. Bertrand stated that in the oxidation of sugar alcohols, the two alcoholic OHs adjacent to the primary alcohol group were preferentially oxidized only in the erythro coordination. − Announced the Hudson Rule (G. Bertrand, Ann. Chim. Phys., 3, 181, 195,
227, (1904)). Oxidation of D-sorbitol to L-sorbose (right flow in FIG. 4 (A)), D-mannitol to D-fructose (FIG. 4 (B)), glycerin to dioxyacetone, erythritol to erythrulose, and arabitol to xylose. This rule has been followed without exception, and has been regarded as the most important law of oxidative fermentation.

The inventors of the present invention have learned from many years of research on the basic analysis of oxidative fermentation that the Bertrand-Hudson rule is a rule that applies only to enzymes having PQQ as a coenzyme. Therefore, when an enzyme that uses FAD as a coenzyme is predicted to be an enzyme that does not follow this law and aerobic bacteria are searched for, FAD that catalyzes the production of D-fructose from D-sorbitol is found as a result of extensive screening. A strain having an enzyme that acts as a coenzyme was discovered. The demonstration of this prediction is highly original, groundbreaking, and academically unique. In the oxidative fermentation by the aerobic bacterium of the present invention, there is no particular limitation as long as it is an aerobic bacterium, but a gram-negative aerobic bacterium is preferable, and acetic acid bacterium is particularly useful.

In the present invention, starch is hydrolyzed to obtain glucose, which is catalytically hydrogenated to be converted to sorbitol, and then a one-step incomplete oxidation reaction is performed to give a stoichiometric yield of 100%. Gives D-fructose. on the other hand,
In the conventional glucose isomerization method, the reaction is stopped at a yield of about 50% stoichiometrically due to the reaction equilibrium. In the production method of the present invention, the production yield of D-fructose from D-sorbitol by oxidative fermentation is close to 100%, even if it is necessary to convert glucose into sorbitol by chemical reduction. If you consider
In all, it is an innovative technological development that cannot be predicted by the current glucose isomerization method. The conversion of glucose to sorbitol is already an existing technology in L-sorbose fermentation, and its cost burden does not hinder considering D-fructose production efficiency.

The enzyme involved in the production of D-fructose from D-sorbitol of the present invention is a cytoplasmic membrane-bound sorbitol dehydrogenase (FAD-SLDH) which uses covalent FAD as a coenzyme. Plasma-bound sorbitol dehydrogenase (PQQ) using PQQ as a coenzyme that catalyzes L-sorbose fermentation that has been widely studied
-SLDH) is basically different. It was confirmed that the genes encoding both enzymes, the reaction products, and the enzyme structures and coenzymes involved were different.

As an example of the acetic acid bacterium, the enzyme in acetic acid bacterium which catalyzes the reaction according to the Bertrand-Hudson rule uses PQQ as a coenzyme.
PQQ-SLDH that produces L-sorbose is EDTA
Is significantly inhibited by. This is because the metal ion connecting PQQ and the enzyme is chelated and PQQ is released from the enzyme, and the enzyme activity is lost. When PQQ and a divalent metal ion are added to the enzyme inactivated in this way, PQQ-SLDH restores the enzyme activity again. On the other hand, the FAD-SLDH of the present invention has a high concentration of ED.
Addition of TA to the enzyme solution does not inhibit the enzyme activity.

Therefore, when examining the oxidation of D-sorbitol, high-concentration EDTA was added to the enzyme solution in advance to completely inactivate PQQ-SLDH, and then the remaining D-
When the oxidative activity of sorbitol was measured, it was FAD-SLD.
It is possible to investigate only the enzymatic activity of H. In this way, the presence of FAD-SLDH, which has strong and weak enzyme activities but was not inhibited by EDTA, was found for the first time in some strains. Thus, the enzyme from which PQQ-SLDH was removed oxidizes D-sorbitol to 100%.
D-fructose was produced in near yield. On the other hand, Gluconobacter oxydans ATCC621 which is famous for L-sorbose fermentation
The sorbitol oxidative activity of E. coli is completely occupied by PQQ-SLDH and thus completely inhibited by EDTA. Therefore, no D-fructose is produced in this strain. FAD-SLDH and PQQ-SLDH were uniformly purified from the cell membrane of acetic acid bacteria, and the results of comparing various properties were summarized as a control table (Table 1). In addition, the inventors of the present invention have found that D-fructose and L- which are necessary for the present invention
A specific method for the determination of sorbose has already been established (Am
eyama, M., et al., J. Bacteriol., 145,814,1981; Ad
achi, O., et al., Biosci. Biothecnol. Biochem., 63,2
137,1999).

[0013]

[Table 1]

The method for producing D-fructose according to the present invention will be described below. Using purified cell membranes containing FAD-SLDH or FAD-SLDH alone, pH of 5
When reacted under the conditions of ~ 6, D-sorbitol is 100
React until it is converted to D-fructose with a yield close to%. Even if cells or cell membranes containing only FAD-SLDH are immobilized with an immobilizing agent such as calcium alginate, D-fructose can be similarly obtained in high yield. When aerobic bacteria containing only FAD-SLDH are cultured, D-fructose is accumulated in the culture solution in high yield. In particular, the use of thermostable acetic acid bacteria further improves the D-fructose production efficiency. When using cells or cell membranes containing PQQ-SLDH, EDTA should be used in advance.
After inactivating PQQ-SLDH with a chelating agent such as the above, it may be similarly used for the production of D-fructose. Preferably by mutation PQQ-SL
Bacteria that have lost DH activity are good. Bacteria that have lost PQQ-SLDH activity can be obtained by artificial mutation treatment, or can be obtained by a genetic engineering method.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of the present invention will be shown below, but the present invention is not limited to these examples.

[0016]

Example 1 D- due to ordinary acetic acid bacteria and other aerobic bacteria
When investigating the oxidation of sorbitol, high concentration EDTA (50 mM) was added to the enzyme solution in advance and left overnight,
After completely inactivating the enzyme PQQ-SLDH having PQQ as a coenzyme, the oxidative activity of D-sorbitol was measured (Table 2). For some strains, the existence of the cytoplasmic membrane-bound sorbitol dehydrogenase FAD-SLDH, which was not significantly inhibited by EDTA, although the enzyme activity was strong or weak, could be revealed for the first time. In this way, PQQ-SLDH
The enzyme from which D was removed oxidizes D-sorbitol to 100
D-fructose was produced in a yield close to%. The D-sorbitol oxidative activity of the ATCC621 strain, which is famous for L-sorbose fermentation, is entirely occupied by PQQ-SLDH, so that it is completely inhibited by EDTA.
It became clear from the inhibition rate of 0%. This ATCC6
21 strains could not produce D-fructose at all.

[0017]

[Table 2]

[0018]

[Example 2] 0.5% of acetic acid bacteria and other aerobic bacteria
Grow the cells in a nutrient medium containing D-sorbitol, 0.1% yeast extract, 0.1% peptone, etc., collect the cells, and adjust the pH to 5 to 7 such as Tris-hydrochloride buffer or acetate buffer.
Then, the cells were suspended in 10 to 50 mM or so and the cells were broken by a French press or an ultrasonic oscillator. After removing unbroken cells as a precipitate by low speed centrifugation,
Centrifugation was carried out at 00 × g for 1 hour to separate the cytoplasmic component of the supernatant from the precipitated membrane component. When normal ultracentrifugation is performed, FAD-SLDH and PQQ-SLDH are also present in the supernatant.
Did not exist either. The cell membrane fraction was suspended in a buffer solution, and the buffer solution was adjusted so that the protein concentration of the cytoplasmic membrane fraction was 10 mg / ml, 50 mM EDTA was added, and the mixture was left standing overnight in a refrigerator. FAD-SLDH is 1
It was solubilized from the cytosolic fraction with a concentration of the detergent Triton X-100 (Table 3). The optimum solubilization condition is 1% Tri
The conditions were ton X-100 + 0.1M D-sorbitol (Table 3).

[0019]

[Table 3]

[0020]

Example 3 Gluconobacter suboxydans var. Α IFO 3
The purification method of FAD-SLDH of 258 strains is as follows. Apply the cytoplasmic membrane fraction (10 mg / ml) to 10 mM Tris-HCl.
(PH7.4) 0.1 M D-sor after suspending in buffer
1% Triton X-100 containing bitol was added and stirred for 2 hours to solubilize the cytoplasmic membrane fraction. 120,000 ×
The solubilized enzyme solution was recovered by centrifugation at 60 g for 60 minutes.
10 m containing 25 mM D-sorbitol and 1% Triton X-100
After dialyzing for 6 hours in M Tris-HCl (pH 7.4) buffer, purification by column chromatography was performed.

The solution after dialysis was treated with 10 mM Tris-HCl (pH 7.4 containing 25 mM D-sorbitol and 1% Triton X-100).
) FAD-SLDH was adsorbed by passing through a DEAE-cellulose column equilibrated with buffer. next,
The active fraction was collected by elution with a 0-0.3 M KCl concentration gradient. The active fraction was added to 25 mM D-sorbitol and 1% Triton X.
7 in 10 mM acetate buffer (pH 5.5) containing -100
After dialysis for 25 hours, 25 mM D-sorbitol and 1% Triton X
FAD- was passed through a CM-cellulose column equilibrated with 10 mM acetate buffer (pH 5.5) containing -100.
SLDH was adsorbed. Next, the active fraction was recovered by elution with a 0-0.3 M KCl concentration gradient to obtain a purified enzyme (Table 4). As a result, two active fractions were recovered, but from peak I, the specific activity of FAD-SL was 0.34 (units / mg).
2% recovery of DH, specific activity 1.25 from peak II
(Units / mg) FAD-SLDH was obtained with a recovery rate of 5%. The obtained purified FAD-SLDH was an enzyme containing no heme c component (Subunit II).

[0022]

[Table 4]

[0023]

Example 4 Gluconobacter suboxydans var. Α IFO 3
The properties of FAD-SLDH purified from 258 strains were examined. The enzyme activity of the purified FAD-SLDH solution was measured by the potassium ferricyanide method while changing the pH of the enzyme solution. As a result, it was revealed that the optimum pH was found in the range of 4.5 to 6.0 (Fig. 5). In addition, purified FAD-S
It was shown that the amount of D-fructose produced by LDH was proportional to the reaction time (FIG. 6), and the reaction was proportional to the D-sorbitol concentration of the substrate and was saturated at about 200 (μmol / ml) (FIG. 7). ). From this data, the Michaelis constant for the enzyme substrate D-sorbitol is 20-30 mM.
Can be estimated.

The amount of D-fructose produced from D-sorbitol was measured by changing the amount of purified FAD-SLDH added during the reaction. The amount of D-fructose produced was proportional to the amount of purified FAD-SLDH added. The production amount increased. The results are shown on the horizontal axis of the amount of purified FAD-SLDH added and on the vertical axis of the enzyme activity in the reaction solution (FIG. 8).

The optimum pH of purified FAD-SLDH is 4.
It is in the range of 5 to 6.0 (Fig. 5). Substrate specificity is D-
It reacts only specifically with sorbitol and no other carbohydrates are oxidized by FAD-SLDH (Table 5). The reaction product is D-fructose and no L-sorbose is produced. Known PQQ-SLDH is D
-Oxidizing sorbitol to produce L-sorbose, glycerin to dioxyacetone, erythritol erythrulose, arabitol xylose, glucone 5
-An enzyme with broad substrate specificity that also catalyzes reactions such as ketogluconic acid. As described above, also in terms of these substrate specificities, the FAD-SLDH of the present invention has the PQQ-SLD
Notably different from H. Also, while PQQ-SLDH follows the Bertrand-Hudson rule, FAD-S
It could be clearly defined that LDH is an enzyme that does not follow the Bertrand-Hudson rule.

[0026]

[Table 5]

[0027]

Example 5 Gluconobacter suboxydans var. Α IFO 3
The purification method of FAD-SLDH of 254 strains is as follows. The cytoplasmic membrane fraction (10 mg / ml) was suspended in 10 mM acetate buffer (pH 5.0) buffer, and then 0.1 M
1% Triton X-100 with D-sorbitol and 0.1 M KCl
Was added and stirred for 2 hours to solubilize the cytoplasmic membrane fraction. The solubilized enzyme solution was recovered by centrifugation at 80,000 xg for 90 minutes. The recovered solubilized enzyme solution was stored overnight,
After fractionation with 20% PEG 6000 solution, the precipitate was collected and suspended in 10 mM acetate buffer (pH 5.0) containing 0.1% Triton X-100, and then purified by column chromatography. .

The solution after dialysis was equilibrated with 10 mM acetate buffer (pH 5.0) containing 0.1% Triton X-100.
The flow-through fraction was collected by passing through an EAE-cellulose column. Then, concentrate with 20% PEG 6000 solution and
10 mM acetate buffer containing 1% Triton X-100 (pH 5.
8) After dialysis in buffer, 0.1% Triton X-10
Pass through a CM-cellulose column equilibrated with 10 mM acetate buffer (pH 5.8) containing 0
FAD-SLDH was adsorbed. Then 0-0.15 M KCl
The active fraction was collected by elution with a concentration gradient (Table 6).
As a result, FAD-SLD with a specific activity of 433 (units / mg)
H was obtained with a recovery of 19%.

[0029]

[Table 6]

The obtained purified FAD-SLDH was added to SDS-
As a result of development by gel electrophoresis, it was found to be composed of three subunits corresponding to molecular masses of 63, 51 and 17 kDa. In addition, the absorption spectrum shows 417, 522 and 551 nm peculiar to reduced heme c.
It was found that this enzyme is an enzyme containing a heme c component. Comparing with the results of Example 2, due to the conditions of column chromatography, heme c
It was revealed that a purified enzyme containing a component may be obtained and a purified enzyme containing no heme c component may be obtained.

When a separately prepared subunit II of the heme c component is added to the purified enzyme containing no heme c component,
It was shown that the production of D-fructose from D-sorbitol was promoted (Fig. 10). The separately prepared heme c component alone did not retain the D-fructose-forming activity from D-sorbitol. From this, the enzyme that does not contain the heme c component described in Example 3 and the enzyme of this example are enzymes that catalyze the same reaction, and that the heme c component is lost due to the difference in the purification conditions. Indicates that there is something to do. Therefore, when D-fructose is produced from D-sorbitol using an enzyme, it is necessary to confirm that the purified FAD-SLDH has a heme c component and use it for production. However, when manufacturing using cells or membrane preparations, heme c
There is no need to worry that the components will fall off and the reaction efficiency will decrease.

[0032]

Example 6 Gluconobacter suboxydans var. Α IFO 3
258 strains were cultured and the progress of the culture was examined. Gluconobacter suboxy dans var. Α IFO was added to a medium (100 ml / 500 ml shaking culture flask) containing 4.0% D-sorbitol, 0.1% yeast extract and 0.1% polypeptone.
3258 was inoculated and cultured with shaking at 30 ° C. Even if you use a jar fermenter for large-scale preparation, D-
Fructose production followed a similar course. Gluconobacter suboxydans var.α IFO 325 used in this example
Since 8 contains PQQ-SLDH in nearly 15% of the total SLDH, the maximum yield of D-fructose was 75-8.
It is estimated to be 0%. The horizontal axis shows the culture time, the left vertical axis shows the value obtained by measuring the growth of bacteria with a Klett photometer, and the right vertical axis shows the amounts of residual D-sorbitol and produced D-fructose (FIG. 10). The pH of the medium is also shown on the left vertical axis. The amount of D-sorbitol remaining is NAD-dependent D
-Sorbitol dehydrogenase (Adachi, O., et al.,
Biosci. Biotech., Biochem., 63, 2137, 1999), the amount of D-fructose produced was D-fructose dehydrogenase (Ameyama, M., et al., J. Bacterio).
L., 145, 814, 1981).

[0033]

Example 7 PQQ-SLDH-free cell membrane (2 un
its of FAD-SLDH) was added to the reaction solution, and ferricyanide as an electron acceptor was 0.15 μmol or 0.3 μm.
was reacted with D-sorbitol, and the change with time in the amount of D-fructose produced was measured by the absorbance at 660 nm. The absorbance was about 0.6 under 0.15 μmol D-sorbitol and about 1.2 under 0.3 μmol D-sorbitol, respectively, and D-sorbitol was oxidized to D-fructose at a conversion rate of 100%. Corresponding to the given value. Therefore, it was shown that this reaction system produced D-fructose from D-sorbitol at a conversion rate close to the theoretical value (FIG. 11). In this reaction system, it was confirmed that L-sorbose was not produced from D-sorbitol.

When the time course was examined using the purified enzyme under the same conditions, the production rate of D-fructose was almost 100%. In addition, PQQ-SLDH-free cells were used to react with stirring at 30 ° C, and the reaction solution was withdrawn over time to remove D-fructose and L in the reaction solution.
-As a result of quantifying sorbose, it was similarly revealed that only D-fructose was produced.

[0035]

INDUSTRIAL APPLICABILITY According to the present invention, the carbon at the 2-position of D-sorbitol is oxidized by a one-step incomplete oxidation reaction by aerobic bacteria, especially acetic acid bacteria, and D-fructose is produced at a yield close to 100%. Can be manufactured.

[Brief description of drawings]

FIG. 1 is a diagram showing a fructose conversion pathway by glucose isomerase and a conversion pathway of the present invention by sugar alcohol dehydrogenase in acetic acid bacteria, which are used on an industrial scale.

FIG. 2 is a diagram showing a production profile of isomerized sugar in North America (Masao Nomoto, Enzyme Engineering, Academic Publishing Center, 1993).

FIG. 3 is a diagram showing a comparison of sweetness of three kinds of sugars (Takahisa Ohta, Enzymes in daily life, Tokyo Kagaku Dojin, 1994) and changes in sweetness with temperature.

FIG. 4 is a schematic diagram of sugar alcohol oxidation. (A) shows an oxidation site for producing D-fructose and L-sorbose from D-sorbitol, and (B) shows an oxidation site for producing D-fructose from D-mannitol. FIG.

FIG. 5: Optimal p of purified D-sorbitol dehydrogenase
It is a figure which shows H.

FIG. 6 shows the effect of incubation time on purified D-sorbitol dehydrogenase activity.

FIG. 7 shows the effect of substrate concentration on the purified D-sorbitol dehydrogenase activity.

FIG. 8 is a graph showing the influence of the amount of enzyme on the purified D-sorbitol dehydrogenase activity.

FIG. 9 is a diagram showing that the enzyme is activated by adding the subunit II.

Fig. 10 Gluconobacter suboxidans var. Α IFO 325
PH, D-fructose concentration and D-
It is a figure which shows the change of the sorbitol concentration.

FIG. 11 is a diagram showing the time course of the amount of D-fructose produced from D-sorbitol.

Continued front page    (72) Inventor Rei Adachi             1677-1 Yoshida, Yamaguchi City, Yamaguchi Prefecture Yamaguchi University             Faculty of Agriculture (72) Inventor Kazunobu Matsushita             1677-1 Yoshida, Yamaguchi City, Yamaguchi Prefecture Yamaguchi University             Faculty of Agriculture (72) Inventor Hirohide Toyama             1677-1 Yoshida, Yamaguchi City, Yamaguchi Prefecture Yamaguchi University             Faculty of Agriculture (72) Inventor Emiko Shinagawa             2-14-1, Tokiwadai, Ube City, Yamaguchi Prefecture Ube             National College of Technology (72) Inventor Ganjana Teragur             Kingdom of Thailand Bangkok 10900 Kasetsart Large             Study (72) Inventor Zuangtip Moonman Me             Kingdom of Thailand Bangkok 10140             Institute of Technology Thonburi F-term (reference) 4B050 CC01 DD02 EE02 FF03E                       FF05E FF11E LL01 LL02                       LL05                 4B064 AF02 CA02 CA21 CB13 CC01                       CD10 CE03 CE06 CE11 DA01                       DA10 DA13

Claims (8)

[Claims] 1. A method for producing D-fructose from D-sorbitol by an aerobic bacterium. 2. A method for producing D-fructose from D-sorbitol by Gram-negative aerobic bacteria. 3. D from sorbitol due to acetic acid bacteria
-Fructose production method. 4. D- by an acetic acid bacterium of the genus Gluconobacter
A method for producing D-fructose from sorbitol. 5. A method for producing D-fructose from D-sorbitol with Gluconobacter suboxidans. 6. The method for producing D-fructose from D-sorbitol according to claim 1, wherein any one of cells, membrane fractions and purified enzyme is used. 7. The method for producing D-fructose from D-sorbitol according to claim 1, wherein any one of the cells after EDTA treatment, the membrane fraction and the purified enzyme is used. 8. A cytoplasmic membrane-bound sorbitol dehydrogenase using covalent FAD as a coenzyme.