JP3056295B2 - Alcohol dehydrogenase and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel NAD-independent alcohol dehydrogenase having a wide substrate specificity and a method for producing the same.

[0002]

2. Description of the Related Art Coenzyme-independent dehydrogenases such as NAD and NADPH, which catalyze the oxidation of a microorganism-derived hydroxymethyl group to a carboxyl group, include, for example,
Bacteria belonging to the genus Acetobacter [Agricultural Biological Chemistry (Agric. Biol. Che)
m. 42, 2331 (1978)], a bacterium belonging to the genus Gluconobacter [Agricultural Biological Chemistry (Agric. Biol. Che).
m. 42, 2045 (1978)] or alcohol dehydrogenases such as Pseudomonas bacteria [for example, Biochemical Journal (Biochem.
J. , 223, 921 (1984)], methanol dehydrogenase of methanol bacteria [Agricultural Biological Chemistry (Agric. Bi)
ol. Chem. 54, 3123 (199)
0)] are known, but none of them have limited substrate specificity, and there is no report that they act on other than alcohols such as methanol and ethanol. On the other hand, D-
Dehydrogenases that act on saccharides such as sorbitol and L-sorbose (hereinafter sometimes simply referred to as sorbose) to catalyze the oxidation of their hydroxymethyl groups to aldehyde groups include those of the genus Gluconobacter. D-sorbitol dehydrogenase [Agricultural Biological Chemistry (Agric. Biol. Che)
m. 46, 135 (1982)], sorbose dehydrogenase [Agricultural Biological.
Chemistry (Agric. Biol. Chem.),
55, paragraph 363, (1991)], glucose dehydrogenase [Agricultural Biological Chemistry, Agric. Biol. Chem.
Vol. 1505 (1980)], etc., but there is no report that dehydrogenases acting on these saccharides act on alcohols such as methanol and ethanol. All of these have limited substrate specificity and are insufficient from the viewpoint of versatility. In the past, there have been known several examples in which an enzyme is induced by a metal, but there has been no report on an enzyme specifically induced and synthesized by a rare earth element.

On the other hand, the present inventors have proposed the Reichstein method [Helvetica Chimica Acta].
ta), vol. 17, 311, 1934], an industrially advantageous method for producing L-ascorbic acid was studied, and a soil-isolated bacterium already named Pseudogluconobacter saccharoketogenes was identified as L-ascorbic acid. It has been found that sorbose produces a significant amount of 2-keto-L-gulonic acid (hereinafter sometimes referred to as 2KGA) (see JP-A-62-228287). Furthermore, during the improvement study of this method, if the present bacterium was cultured by adding a rare earth element to the medium, the 2-keto L from L-sorbose was inadvertently cultivated.
-It has been found that the production yield of gulonic acid is remarkably improved (see JP-A-64-85088).

[0004]

As described above, there have been known various examples in which an enzyme is induced and produced by a metal element, but there has been no report on a rare earth element. Thus, an object of the present invention is to provide a novel alcohol-dehydrogenase derived from rare earth elements which has industrially useful broad substrate specificity and a method for producing the same.

[0005]

Means for Solving the Problems The present inventors have made intensive studies on the addition of rare earth elements to a culture medium to achieve the above object, and as a result, the above pseudogluconobacter saccharoketogenes has a novel alcohol dehydration. Production of the enzyme, the enzyme plays a central role in 2KGA fermentation by the present strain, and in culturing the present strain, the enzyme is significantly induced and produced by the presence of rare earth elements in the medium. The present inventors have found that the present invention has been further studied and completed the present invention. That is, the present invention provides (1) a NAD-independent alcohol dehydrogenase containing a rare earth element in an enzyme molecule that catalyzes the oxidation of a compound having a hydroxymethyl group to an aldehyde group; Alcohol dehydrogenase according to the above (1) having physicochemical properties, optimal pH 3.5-6.5 optimal temperature 40-50 ° C isoelectric point 4.2 ± 0.5 molecular structure 64000 by SDS-PAGE ± 50
A polypeptide of molecular weight 00 or this polypeptide 2
Dimer consisting of molecules (3) Pseudogluconobacte
r) the alcohol dehydrogenase according to the above (1), which is produced by the microorganism belonging to r), (4) the alcohol dehydrogenase according to the above (2), which is induced and produced by a rare earth element, and (5) a compound having a hydroxymethyl group. A microorganism belonging to Pseudogluconobacter having a NAD-independent alcohol dehydrogenase-producing ability containing a rare earth element in an enzyme molecule that catalyzes oxidation of a hydroxymethyl group to an aldehyde group, and a mutant thereof are cultured and produced. (1) characterized in that the alcohol dehydrogenase is isolated and purified.
(6) The method for producing an alcohol dehydrogenase according to the above (6), wherein the rare earth element is present in a medium.

Examples of the compound having a hydroxymethyl group serving as a substrate for the alcohol dehydrogenase of the present invention include L-sorbose, D-glucose, D-mannose, sucrose, α-methyl-β-D-glucoside, and n-octyl. −
Various sugars including monosaccharides such as β-D-glucoside, maltotriose, glyceraldehyde, xylose and starch, oligosaccharides and polysaccharides, D-sorbitol, xylitol, D-mannitol, maltitol and galactitol Sugar alcohols, such as methanol, ethanol,
Alcohols such as propanol, glycols such as ethylene glycol, D-gluconic acid, 2-keto-D
-Gluconic acid (hereinafter referred to as 2KGL), 2-keto-L
-Gulonic acid (hereinafter referred to as 2KGA), 5-keto-D-
Gluconic acid (hereinafter referred to as 5KGL), sugar acids such as D-glucuronic acid, D-guluronic acid and salts thereof, tris (hydroxymethyl) aminomethane, tris (hydroxymethyl) nitromethane, retinol, 3-dehydroretinol And the like.

[0007] The alcohol dehydrogenase is independent of NAD as well as NADP, and is 2,6-dichlorophenolindophenol (hereinafter abbreviated as DCIP), potassium ferricyanide, phenazine metasulfate, Ulsters Blue, catalyzes the oxidation of the hydroxymethyl group of the substrate to an aldehyde group in the presence of an electron acceptor such as a tetrazolium salt. Oxygen cannot be a direct electron acceptor.

[0008] Optimum pH of the alcohol dehydrogenase of the present invention
Is generally in the range of about 3.5 to 6.5, though it varies somewhat depending on the substrate and the rare earth elements contained. Particularly, in the case of the enzyme derived from La, it is in the range of 4.0 to 5.5, and in the case of the enzyme derived from Ce, it is in the range of 4.0 to 6.0, as shown in the following Examples. In general, when the temperature of the enzyme is higher, the reaction itself is faster, and the inactivation of the enzyme is also remarkable. However, under the measurement conditions described in 1) of Example 2 below, the optimum temperature of the enzyme of the present invention is 40 ° C. ~ 50 ° C. The isoelectric point is usually in the range of 4.2 ± 0.5, though it varies somewhat depending on the strain from which it is derived. The enzyme has a molecular weight of 64
000 ± 5000 (by SDS-PAGE), and two molecules of this polypeptide may form a dimer.

The activity of the enzyme is inhibited by various metal ions. For example, Zn ²⁺ and Hg ²⁺ have a strong inhibitory effect, and Cu ²⁺ , Co ²⁺ , Ni ²⁺ and Mn ²⁺ also have an inhibitory effect. Antimycin A and rotenone also inhibit the activity of the enzyme.

The rare earth elements contained in the enzyme molecule include scandium (Sc), yttrium (Y), lanthanum (La), cerium (Ce), praseodymium (P
r), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (H
o), erbium (Er), ytterbium (Yb),
Lutetium (Lu) and the like.

[0011] The alcohol dehydrogenase of the present invention is produced by induction from various microorganisms belonging to the genus Pseudogluconobacter with the ability to produce the enzyme, using various rare earth elements as described above. Here, the induced production means that when an enzyme-producing bacterium is cultured by adding a rare earth element to a medium, protein production, that is, production of the enzyme is performed. In particular, the specific activity is highest when induced with lanthanum and cerium.

In order to produce the alcohol dehydrogenase of the present invention, a microorganism belonging to the genus Pseudogluconobacter having the enzyme-producing ability is cultured. The enzyme is produced in the cells,
Since it is accumulated, it can be produced by collecting cells after culturing, crushing the cells, and separating and purifying the cells from a cell-free extract, preferably a cytoplasmic fraction.

The microorganism to be used can be obtained by a microorganism belonging to the genus Pseudogluconobacterium having the enzyme-producing ability and a usual mutagenesis operation, for example, a treatment with a mutagen such as nitrosoguanidine, an ultraviolet irradiation treatment, or a genetic recombination. These mutants include, for example, the following strains described in EP-A-221,707. Pseudogluconobacter Saccharoketogenes K5
91s strain FERM BP-1130, IFO14464 Pseudogluconobacter saccharoketogenes 12
-5 strain FERM BP-1129, IFO14465 Pseudogluconobacter saccharoketogenes TH
14-86 strain FERM BP-1128, IFO14466 Pseudogluconobacter saccharoketogenes 12
-15 strain FERM BP-1132, IFO14482 Pseudogluconobacter saccharoketogenes 12
-4 strain FERM BP-1131, IFO14483 Pseudogluconobacter saccharoketogenes 22
-3 strains FERM BP-1133, IFO14484

Under aerobic conditions, the microorganism is a nutrient source that can be used by the strain, ie, a carbon source (a carbohydrate such as glucose, sucrose, starch or an organic substance such as peptone or yeast extract), a nitrogen source (ammonium salt, urea). And corn steep liquor, inorganic and organic nitrogen-containing substances such as heptone), inorganic salts (potassium, sodium, calcium,
Magnesium, iron, manganese, cobalt, copper, phosphoric acid,
Salts such as thiosulfate) and micronutrients (CoA, pantothenic acid, biotin, thiamine, riboflavin, FMN
And L-cysteine, amino acids such as L-glutamic acid, or a liquid medium containing a natural product containing them. Culture pH4 ~
9, preferably at pH 6-8. The culturing time varies depending on the microorganism used, the composition of the medium, and the like, but is preferably 10 to 100 hours. A suitable temperature range for culturing is 10 to 40 ° C, preferably 25 to 35 ° C.

The above-mentioned rare earth element is added to the medium. The rare earth element may be added as a metal powder or a metal powder, or may be used as a carbonate, a sulfate, a nitrate, a chloride, an oxide, or an oxalate. Each of them may be used alone, or two or more rare earth elements may be used simultaneously. Further, a crude product obtained in the process of separating and purifying various elements can also be used. The amount of the rare earth element added to the medium may be selected within a range that does not significantly suppress the growth of the microorganism to be used.
1.0% by weight, preferably 0.0001 to 0.1% by weight
It is. As a method of adding to the medium, the medium may be added in advance to the medium, but may be added intermittently during the culture or continuously. It was confirmed that the enzyme was not induced even when an alkali metal such as calcium or strontium was added to the medium.

After completion of the culture, the alcohol dehydrogenase can be separated and purified by a method generally used for enzyme purification, for example, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, dialysis, or the like.
Affinity chromatography or the like is used.

The novel alcohol dehydrogenase of the present invention only catalyzes the oxidation of L-sorbose to L-sorbosone in the production of 2-keto-L-gulonic acid, which is important as an ascorbic acid production intermediate. Instead, it can be applied to the stereospecific oxidation of compounds having various hydroxymethyl groups by utilizing its wide substrate specificity. For example, novel compounds can be produced by acting on various sugar alcohols, sugars, glycols, and alcohols.

[0018]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 Preparation of alcohol dehydrogenase (1) Culture of pseudogluconobacter saccharoketogenes Pseudogluconobacter saccharoketogenes K5
91s (IFO14464, FERM BP-113
The agar slant culture of (0) was subjected to seed medium 20 consisting of 2.5% sorbitol, 1.0% yeast extract and 1.0% peptone.
One platinum loop was inoculated into a 200 ml Erlenmeyer flask containing milliliters, and cultured at 30 ° C. for 2 days on a rotary shaker. The resulting seed culture was inoculated in its entirety into a Sakaguchi flask containing 500 ml of the above seed medium,
Culture was performed for 2 days on a reciprocating shaker. Culture 5 obtained
100 ml of yeast extract 1%, peptone 1
50 liters of fermenter containing 30 liters of medium consisting of 0.001% lanthanum chloride 0.001% and actocol 0.02%, 30 ° C., aeration 15 liter / min, stirring 250r
The cells were cultured for 3 days under pm conditions. 8000 rpm
m, and centrifuged for 20 minutes to obtain bacterial cells. The obtained bacterial cells are at 0.
Washed twice with 85% saline. In this way, 3
Approximately 100 g of wet bacterial cells were obtained from 0 liter of the culture solution.

(2) Preparation of Cytoplasmic Fraction 100 g of the wet cells of Pseudogluconobacter saccharoketogenes obtained in the above step were suspended in 300 ml of 10 mM phosphate buffer (pH 6.7). The suspension was crushed in 50 ml aliquots at 1.5 A,
The cells were treated at 00 W for 30 minutes to crush the cells. 3000 rp
After centrifugation for 10 minutes to remove viable cells, 35,000 r
Centrifugation at pm for 90 minutes. 250 ml of the obtained centrifugal supernatant was collected as a cytoplasmic fraction.

(3) DEAE Toyopearl column chromatography 100 ml of the cytoplasmic fraction obtained in the above step is equilibrated with 100 mM glycerol, 10 mM phosphate buffer (hereinafter sometimes simply referred to as buffer). DEAE Toyopearl column (3.0φx30c
m). The enzyme was eluted with a linear gradient of sodium chloride up to 0.3M.

(4) Butyl Toyopearl column chromatography 63.5 g of ammonium sulfate was added to and dissolved in 100 ml of the enzyme fraction obtained in the above step. After allowing to stand at 4 ° C. for 2 hours, the mixture was centrifuged at 15000 rpm for 20 minutes, and the obtained precipitate was dissolved by adding 20 ml of a buffer containing 1.5 M ammonium sulfate. Centrifugation was performed at 10,000 rpm for 20 minutes to remove insolubles. 20 ml of the centrifuged supernatant obtained is
A butyl toyopearl column (3.0 φ × 15 cm) equilibrated with a buffer containing 1.5 M ammonium sulfate was applied. The enzyme was eluted with a linear gradient of ammonium sulfate up to 0M.

(5) Gel Filtration Column Chromatography 31.2 g of ammonium sulfate was added to and dissolved in 50 ml of the enzyme fraction obtained in the above step. After leaving still at 4 ° C. for 2 hours, centrifugation was performed at 15000 rpm for 20 minutes, and 5 ml of a buffer solution was added to the obtained precipitate to dissolve it.
The solution was applied to a gel filtration column (3.0 φ × 100 cm) equilibrated with a buffer containing M sodium chloride. 4 active fractions
Stored at ° C. The specific activity of the obtained enzyme is about 50 u / mg.
It was protein. The protein amount was calculated using a BCA total protein quantification kit (Pierce) and a standard curve prepared with bovine serum globulin.

(6) Purity of isolated enzyme In order to examine the purity of the purified enzyme, SDS-polyacrylamide gel electrophoresis and agarose isoelectric focusing were performed. In each case, the enzyme shows a single-band,
Further, after the gel subjected to agarose isoelectric focusing, the gel was subjected to 100 mM phosphate buffer containing 100 mM ethanol and 10 mM potassium ferricyanide (pH 7.0).
0) at room temperature for 5 minutes, followed by washing with water and a reaction terminating solution (F
Color was developed in 5 g of e ₂ (SO ₄ ) ₃ , 95 ml of 85% phosphoric acid made up to 1 liter with distilled water) to confirm that this protein had enzymatic activity. This enzyme is
For SDS-polyacrylamide gel electrophoresis, 640
G-3000SWXL having a molecular weight of 00 ± 5000.
Gel filtration HPLC using a column (Tosoh Corp.) showed a molecular weight of 125,000 ± 15000, which is considered to be a dimer consisting of a single subunit having a molecular weight of 64000 ± 5000 under ordinary conditions.

(7) Confirmation of the reaction product 5 microliters of a basic reaction solution consisting of 100 microliters of McIlvaine buffer (pH 5.0), 5 microliters of 0.1 M potassium ferricyanide, and 20 microliters of distilled water was added. 0.2 M sorbose and 5 microliters of an enzyme solution were added and reacted at 30 ° C. for 24 hours. The reaction product was analyzed by thin-layer chromatography and high-performance liquid chromatography. As a result, the reaction product was identical to the standard L-sorbosone. Similarly, 0.2 M ethanol and 5 μl of the enzyme solution were added to the basic reaction solution and reacted at 30 ° C. for 24 hours, and the reaction product was analyzed by high performance liquid chromatography. As a result, the reaction product coincided with the standard acetaldehyde. .

Example 2 1) Method for Measuring Enzyme Activity In this example, the enzyme activity is expressed in terms of the amount of enzyme required to reduce 2 μM potassium ferricyanide per minute at 30 ° C. in the presence of a substrate as one unit. The basic composition of the reaction solution is as shown below. Composition of basic reaction solution: McIlvaine buffer (pH 5.0) 0.5 ml Substrate aqueous solution, 0.25 ml 100 mM potassium ferricyanide aqueous solution 0.2 ml After preheating this basic reaction solution at 30 ° C. for 5 minutes, 0.05
The reaction was started by adding milliliter of an appropriately diluted enzyme solution, and after 10 minutes, 0.5 ml of a reaction stopping solution [5 g of Fe ₂ (SO ₄ ) ₃ , ₃ g of SDS, 85% phosphoric acid
Was dissolved in distilled water to make 1 liter], the reaction was stopped, and the mixture was allowed to stand in a dark place at room temperature for 30 minutes, and the absorbance at 660 nm was measured. As a control, the same amount of distilled water was used instead of the substrate solution.

2) Specific Activity and Substrate Specificity According to the activity measurement method described in 1), the specific activity of the alcohol dehydrogenase induced by lanthanum with respect to the ethanol substrate when various substrates were used was examined. Was. Table 1 shows the results.

[Table 1]

3) Optimum pH The effect of pH on the enzyme activity was examined according to the activity measurement method described in 1). The optimum pH is 4.
It is around 5-5.5.

4) PH Stability According to the activity measurement method described in 1), the residual activity at various pHs was examined using McKilvane buffer. The stable pH range is approximately 4.5 to 9 as shown in FIG.
It is.

5) Optimum temperature The effect of temperature on the enzyme activity was examined according to the activity measurement method described in 1). Under the measurement conditions described in 1), the optimum temperature is around 45 ° C. as shown in FIG.

6) Thermal stability The residual activity after heat treatment for 1 hour at various temperatures was examined. The results are shown in FIG. From these results, the residual activity after heat treatment at 50 ° C. for 1 hour was about 70%.

7) Influence of metal ions In accordance with the activity measurement method described in 1), the effects of various metal ions on the enzyme activity were examined. The results are shown in Table 2. From the results, it was found that Zn ²⁺ and Hg ²⁺ had strong inhibitory effects, and that Cu ²⁺ , Co ²⁺ , Ni ²⁺ and Mn ²⁺ also inhibited the reaction.

[Table 2]

8) Effect of Inhibitor The effect of various inhibitors on the enzyme activity was examined according to the method described in 1). Table 3 shows the results. For this enzyme, a very strong inhibitory activity was observed for antimycin A, and a strong inhibitory effect was also observed for rotenone.

[Table 3]

9) Michaelis constant (Km) The activity at various substrate concentrations was measured according to the method described in 1). Under these measurement conditions, the Michaelis equation is not necessarily followed depending on the substrate concentration range, but as shown in FIG. 5, when measured in a concentration range of 100 mM to 1 M, the Michaelis constant was 225 mM.

Example 3 Alcohol dehydrogenase was purified similarly using cerium chloride instead of lanthanum chloride used in Example 1.
The specific activity of the obtained enzyme was about 20 units / mg protein.

Example 4 Neodymium chloride was used in place of lanthanum chloride used in Example 1 to purify an alcohol dehydrogenase in the same manner. The specific activity of the obtained enzyme was about 4 units / mg protein.

Example 5 Pseudogluconobacter saccharoketogenes K5
91s (IFO14464, FERM BP-113
The agar slant culture of 0) was inoculated in a 200 ml Erlenmeyer flask containing 20 ml of a seed medium containing 1.0% yeast extract and 1.0% peptone, and one loopful was inoculated at 30 ° C. for 2 days. Cultured on a mill. The obtained seed culture was mixed with 200 ml of the seed medium described above.
The whole volume was inoculated into a liter Erlenmeyer flask and cultured at 30 ° C. for 2 days on a rotary shaker. Yeast extract 1.0
5 ml of this culture was added to a 1-liter Erlenmeyer flask containing 200 ml of a medium consisting of 1.0% and 1.0% peptone, and cultured at 30 ° C. for 2 days. Under these conditions, 0.001% of yttrium, lanthanum, cerium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, ytterbium chloride or praseodymium, scandium oxide was added as the rare earth element compound. The culture was performed at 30 ° C. for 2 days. The cells were collected from the resulting culture by centrifugation, suspended in 200 ml of physiological saline, collected again by centrifugation, and the cells were collected in 5 ml of 10 mM sodium phosphate buffer (p
H6.7), suspended in a dilution buffer consisting of 100 mM NaCl. After the cell suspension was completely crushed by an ultrasonic crusher, the cell residue was removed by low-speed centrifugation to obtain a crude extract. Table 4 shows the alcohol dehydrogenase activity of this crude extract.

[0037]

[Table 4]

Example 6 The crude extract obtained in Example 5 was subjected to agarose isoelectric focusing. The crude extract obtained from cells cultured with a rare earth compound added thereto was simultaneously electrophoresed as a control. A protein band corresponding to the purified alcohol dehydrogenase obtained in Example 2 was observed, and the gel that had been similarly electrophoresed was subjected to activity staining using ethanol as a substrate. Showed activity.

Yeast extract 1.0%, peptone 1.0
% In a 1 liter Erlenmeyer flask containing 200 ml of medium consisting of Pseudogluconobacter
Saccharoketogenes strain K591s (FERM BP-
1130, IFO14464), Pseudogluconobacter saccharoketogenes strain 12-5 (FERMBP)
-1129, IFO14465), Pseudogluconobacter saccharoketogenes TH14-86 strain (FE
RM BP-1128, IFO14466), Pseudogluconobacter saccharoketogenes 12-15 strain (FERMBP-1132, IFO14482), Pseudogluconobacter saccharoketogenes 12-4
Strains (FERM BP-1131, IFO14483),
Pseudogluconobacter Saccharoketogenes 22
-3 strains (FERM BP-1133, IFO1448
4) Inoculate one platinum loop of the agar culture at 30 ° C. for 3 days.
A crude extract was prepared in the same manner as in Example 5 from the cells obtained by subjecting the culture solution obtained by performing a rotary shaking culture to collect and wash the culture solution. The same experiment was carried out using a medium obtained by adding 0.001% of lanthanum chloride to the above medium to prepare a crude extract. When the alcohol dehydrogenase activity of these crude extracts was measured, the activity was detected only in the cultivated lanthanum chloride-added culture of all the strains used.

Example 8 Using the purified alcohol dehydrogenase obtained in Example 2 as an antigen, antiserum was prepared from rabbits. The antiserum was examined by the Ouchterlony double immunodiffusion method. As a result, the crude extract of the cultured bacterial cells supplemented with rare earth elements obtained in Examples 5 and 7 contained Pseudogluconobacter saccharoketo. It was shown that there was a protein serologically identical or very similar to the alcohol dehydrogenase of the Genes K591s strain.

Example 9 44 mg of La-ADH obtained in Example 2 was ashed by wet ashing and the La concentration was determined by atomic absorption spectroscopy. The result showed that it contained 0.23% (W / W). Was.

[0042]

According to the present invention, a novel NAD-independent alcohol dehydrogenase having a wide substrate specificity and a method for producing the same are provided.

[Brief description of the drawings]

FIG. 1 shows the effect of pH on the activity of the enzyme of the present invention.

FIG. 2 shows the pH stability of the enzyme of the present invention.

FIG. 3 shows the effect of temperature on the activity of the enzyme of the present invention.

FIG. 4 shows the thermostability of the enzyme of the present invention.

FIG. 5 shows a Hane-Woolfplot of the enzyme of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ identification code FI C12R 1:01) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 9/00-9/99 C12N 1 / 00-1/38 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. An NAD-independent alcohol dehydrogenase comprising a rare earth element in an enzyme molecule which catalyzes the oxidation of a hydroxymethyl group to an aldehyde group of a compound having a hydroxymethyl group, and having the following physicochemical properties. Optimum pH 3.5-6.5 Optimum temperature 40-50 ° C Isoelectric point 4.2 ± 0.5 Molecular structure 64000 ± 500 by SDS-PAGE
A polypeptide having a molecular weight of 0 or a dimer comprising two molecules of the polypeptide 2. A pseudogluconobacter (Pseudogluc)
The NAD-independent alcohol dehydrogenase according to claim 1, which is produced by a microorganism belonging to the genus onobacter. 3. The NAD-independent alcohol dehydrogenase according to claim 2, which is induced and produced by a rare earth element. 4. A genus of Pseudogluconobacter having an ability to produce an NAD-independent alcohol dehydrogenase containing a rare earth element in an enzyme molecule that catalyzes the oxidation of a compound having a hydroxymethyl group to an aldehyde group. The method for producing a NAD-independent alcohol dehydrogenase according to claim 1, wherein the microorganism belonging to or a mutant thereof is cultured, and the produced alcohol dehydrogenase is isolated and purified. 5. The method for producing an NAD-independent alcohol dehydrogenase according to claim 4, wherein a rare earth element is present in the medium.