JPH0568542A - Alcohol dehydrogenase and its production - Google Patents

Abstract

PURPOSE:To obtain a new subject enzyme of NAD nondependence type having wide substrate specificity by culturing a bacterium belonging to the genus Pseudogluconobacter, capable of producing a specific alcohol dehydrogenase of a specific NAD nondependence type, ad isolating and collecting. CONSTITUTION:A bacterium belonging to the genus Pseudogluconobacter, capable of producing an alcohol dehydrogenase of an NAD nondependence type which contains a rare earth element in the enzyme molecule as a catalyst to oxidize hydroxymethyl of a hydroxymethyl-containing compound into aldehyde or its variant is cultured in a medium containing the rare earth element (preferably lanthanum or cesium) and the cells are collected, ground, and its cell-free extract is separated and purified to give a polypeptide having optimum pH at 3.5-6.5, optimum temperature of 40-50 deg.C, isoelectric point of 4.2+ or -0.5, molecular structure in terms of molecular weight of 64,000+ or -5,000 by SDS-PAGE or this enzyme comprises two molecules of the polypeptide.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel NAD-independent alcohol dehydrogenase having a broad 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 hydroxymethyl group derived from a microorganism to a carboxyl group are described in, for example,
Bacteria of the genus Acetobacter [Agricultural Biological Chemistry (Agric. Biol. Che
m. ), Vol. 42, Item 2331 (1978)], Gluconobacter bacterium [Agricultural Biological Chemistry (Agric. Biol. Che.
m. ), Vol. 42, Item 2045 (1978)] or alcohol dehydrogenases such as Pseudomonas bacteria [eg, Biochemical Journal (Biochem.
J. ), 223, Item 921, (1984)], methanol dehydrogenase of methanol bacteria [Agricultural Biological Chemistry (Agric. Bi.
ol. Chem. ), Volume 54, Item 3123 (199
0)] and the like are known, but all 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-
Examples of dehydrogenases that act on sugars such as sorbitol and L-sorbose (hereinafter, may be simply referred to as sorbose) to catalyze the oxidation of the hydroxymethyl group to an aldehyde group include Gluconobacter bacteria. D-sorbitol dehydrogenase [Agricultural Biological Chemistry (Agric. Biol. Che
m. ), Vol. 46, Item 135 (1982)], sorbose dehydrogenase [Agricultural Biological.
Chemistry (Agric. Biol. Chem.),
Volume 55, Item 363, (1991)], Glucose Dehydrogenase [Agricultural Biological Chemistry (Agric. Biol. Chem.), 44th.
Vol. 1505 (1980)] and the like are known, but there is no report that dehydrogenases acting on these sugars act on alcohols such as methanol and ethanol. All of these have limited substrate specificity, and are insufficient in terms of versatility. In addition, although there have been known some cases in which an enzyme is induced by a metal, no report has been made on an enzyme specifically induced and synthesized by a rare earth element.

On the other hand, the present inventors have found that the Reichstein method [Helvetica Chimica Ac
ta), Vol. 17, 311, 1934], and various studies have been conducted on an industrially advantageous L-ascorbic acid production method, and a soil-separating bacterium already named Pseudogluconobacter saccharoketgenes is L-. 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 rare earth elements were added to the medium and the present bacterium was cultivated, 2-keto L from L-sorbose was inadvertently added.
-It has been found that the production yield of gulonic acid is significantly improved (see JP-A-64-85088).

[0004]

As described above, various examples of conventional enzyme-induced production of a metal element are known, but no report has been made on rare earth elements. Thus, it is an object of the present invention to provide a novel rare earth element-derived alcohol dehydrogenase having industrially useful broad substrate specificity and a method for producing the same.

[0005]

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

Examples of the compound having a hydroxymethyl group which is a substrate of the alcohol dehydrogenase of the present invention include L-sorbose, D-glucose, D-mannose, sucrose, α-methyl-β-D-glucoside and n-octyl. −
of β-D-glucoside, maltotriose, glyceraldehyde, xylose, various sugars including oligosaccharides, polysaccharides such as starch, 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. Etc.

The alcohol dehydrogenase is independent not only of NAD but also of NADP, and is 2,6-dichlorophenolindophenol (hereinafter abbreviated as DCIP), potassium ferricyanide, phenazine metasulfate, wolsters. It catalyzes the oxidation of hydroxymethyl groups of the above-mentioned substrates to aldehyde groups in the presence of electron acceptors such as blue and tetrazolium salts. Oxygen cannot be the direct electron acceptor.

Optimum pH of the alcohol dehydrogenase of the present invention
Is somewhat in the range of about 3.5 to 6.5, though it varies somewhat depending on the substrate and the contained rare earth element. In particular, the La-induced enzyme has a range of 4.0 to 5.5, and the Ce-induced enzyme has a range of 4.0 to 6.0, as shown in the Examples below. Generally, the higher the temperature is, the faster the reaction itself becomes, but the inactivation of the enzyme also becomes remarkable. However, under the measurement conditions described in Example 2 1) below, the optimum temperature of the enzyme of the present invention is 40%. In the range of -50 ° C. The isoelectric point is usually in the range of 4.2 ± 0.5, though it varies depending on the strain of origin. The enzyme has a molecular weight of 64
000 ± 5000 (by SDS-PAGE) polypeptide, 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) etc. are mentioned.

The alcohol dehydrogenase of the present invention is produced by inducing various rare earth elements as described above from a microorganism belonging to the genus Pseudogluconobacter having the ability to produce the enzyme. Here, inducible production means that protein is produced, that is, the enzyme is produced when an enzyme-producing bacterium is cultured by adding a rare earth element to a medium. In particular, lanthanum and cerium induce the highest specific activity.

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

The microorganism to be used is obtained by a microorganism belonging to the genus Pseudogluconobacter having the ability to produce the enzyme and a usual mutagenesis operation, for example, treatment with a mutagen such as nitrosoguanidine, ultraviolet irradiation treatment or genetic recombination. These mutants include, for example, the following strains described in European Patent Publication No. 221,707. Pseudo-Gluconobacter Saccharoketgenes K5
91s strain: FERM BP-1130, IFO1446
4 Pseudogluconobacter Saccharoketgenes 12
-5 strain: FERM BP-1129, IFO14465 Pseudogluconobacter saccharoketgenes T
H14-86 strain: FERM BP-1128, IFO1
4466 Pseudogluconobacter Saccharoketgenes 1
2-15 strain: FERM BP-1132, IFO144
82 Pseudogluconobacter Saccharoketgenes 1
Strain 2-4: FERMBP-1131, IFO14483 Pseudogluconobacter saccharoketgenes 2
2-3 strain: FERMBP-1133, IFO11484

The microorganism is a nutrient source that can be utilized by the strain under aerobic conditions, that is, a carbon source (a carbohydrate such as glucose, sucrose, starch or an organic substance such as peptone, yeast extract), a nitrogen source (ammonium salts, urea). Inorganic and organic nitrogen-containing substances such as corn steep liquor and 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
It is possible to culture in a liquid medium containing vitamins / coenzymes such as or the like, amino acids such as L-cysteine and L-glutamic acid, or a natural product containing them. Culture pH 4 ~
It can be performed at pH 9, preferably pH 6-8. The culturing time varies depending on the microorganisms used, the composition of the medium, etc., 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 kinds of rare earth elements may be used at the same time. Further, a crude product obtained in the process of separating and refining 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 inhibit the growth of the microorganism used, and usually 0.000001 to
1.0% by weight, preferably 0.0001 to 0.1% by weight
Is. As a method for addition to the medium, it may be added to the medium in advance, but it 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 the completion of the culture, the alcohol dehydrogenase can be separated and purified by a method commonly used for enzyme purification, for example, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, dialysis,
Affinity chromatography or the like is used.

The novel alcohol dehydrogenase of the present invention only catalyzes the oxidation of L-sorbose to L-sorbose during the production of 2-keto-L-gulonic acid, which is important as an intermediate for producing ascorbic acid. However, by utilizing its broad substrate specificity, it can be applied to stereospecific oxidation of various compounds having a hydroxymethyl group. For example, a novel compound 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 thereto. Example 1 Preparation of alcohol dehydrogenase (1) Culturing of Pseudogluconobacter saccharoketgenes Pseudogluconobacter saccharoketgenes K5
91s (IFO14464, FERM BP-113
Seed medium consisting of 2.5% sorbitol, 1.0% yeast extract and 1.0% peptone was added to the agar slant culture of 0).
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 into a Sakaguchi flask containing 500 ml of the above seed medium at 28 ° C.
The culture was performed for 2 days on a reciprocal shaker. The obtained culture 5
00 ml of yeast extract 1%, peptone 1
% Lanthanum chloride 0.001%, Actol 0.02% inoculate into a 50 liter fermenter containing 30 liter medium, 30 ℃, aeration 15 liter / min, stirring 250r
It was cultured for 3 days under the condition of pm. The culture solution is 8000 rp
The cells were centrifuged for 20 minutes to obtain bacterial cells. The bacterial cells obtained were 0.
It was washed twice with 85% saline. In this way, 3
About 100 g of wet bacterial cells was obtained from 0 liter of culture solution.

(2) Preparation of cytoplasmic fraction 100 g of wet bacterium of Pseudogluconobacter saccharoketgenes obtained in the above step was suspended in 300 ml of 10 mM phosphate buffer (pH 6.7) and suspended. 50 milliliters of turbid solution with ultrasonic crusher 1.5A, 2
The cells were crushed by treating with 00W for 30 minutes. 3000 rp
35,000 r after centrifuging for 10 minutes to remove viable cells
It was centrifuged 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 was equilibrated with 100 mM glycerol, 10 mM phosphate buffer (hereinafter, this may be simply referred to as a 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 and dissolved in 100 ml of the enzyme fraction obtained in the above step. After standing at 4 ° C. for 2 hours, the mixture was centrifuged at 15,000 rpm for 20 minutes, and 20 ml of a buffer containing 1.5 M ammonium sulfate was added to and dissolved in the obtained precipitate. Insoluble matter was removed by centrifugation at 10,000 rpm for 20 minutes. 20 ml of the obtained centrifugal supernatant is
It was applied to a Butyl Toyopearl column (3.0φ × 15 cm) equilibrated with a buffer containing 1.5 M ammonium sulfate. The enzyme was eluted with a linear ammonium sulfate gradient to 0M.

(5) Gel filtration column chromatography 31.2 g of ammonium sulfate was added and dissolved in 50 ml of the enzyme fraction obtained in the above step. After standing at 4 ° C. for 2 hours, the mixture was centrifuged at 15,000 rpm for 20 minutes, and 5 ml of a buffer solution was added to the resulting precipitate to dissolve it,
It was applied to a gel filtration column (3.0φ × 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 50u / mg
It was protein. The protein amount was calculated using a standard curve prepared with bovine serum globulin using a BCA total protein quantification kit (Pierce).

(6) Purity of the isolated enzyme To examine the purity of the purified enzyme, SDS-polyacrylamide gel electrophoresis and agarose isoelectric focusing were performed. In both cases the enzyme shows a single band,
Further, the gel subjected to agarose isoelectric focusing was subjected to gel electrophoresis in 100 mM phosphate buffer (pH 7.mM) containing 100 mM ethanol and 10 mM potassium ferricyanide.
0) at room temperature for 5 minutes and then washed with water to stop the reaction (F
It was confirmed that the protein had an enzymatic activity by developing the color in 5 g of e ₂ (SO ₄ ) _{3 and} 95 ml of 85% phosphoric acid adjusted to 1 liter with distilled water. This enzyme is
640 by SDS-polyacrylamide gel electrophoresis
Showing a molecular weight of 00 ± 5000, G-3000SWXL
A gel filtration HPLC using a column (Tosoh Corp.) showed a molecular weight of 125,000 ± 15,000. Therefore, it is considered to be a dimer consisting of a single subunit having a molecular weight of 64,000 ± 5000 under normal conditions.

(7) Confirmation of reaction product 100 μl of McKilvane buffer (pH 5.0), 5 μl of 0.1 M potassium ferricyanide, and 20 μl of distilled water were added to 5 μl of a basic reaction solution. 0.2M sorbose and 5 microliters of 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, and as a result, the reaction product was in agreement with the authentic L-sorbosone. Similarly, 0.2 M ethanol and 5 microliters of 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 was found to be a standard acetaldehyde. ..

Example 2 1) Method for measuring enzyme activity In this example, the enzyme activity is expressed as 1 unit of the amount of enzyme required to reduce 2 μM potassium ferricyanide in the presence of a substrate at 30 ° C. for 1 minute. The basic reaction solution composition is as shown below. Basic reaction solution composition: McKilvane 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 milliliters of an appropriately diluted enzyme solution, and 10 minutes later, 0.5 milliliter of a reaction stop solution [Fe ₂ (SO ₄ ) ₃ 5 g, SDS 3 g, 85% phosphoric acid was added.
The reaction was stopped by adding 95 ml of distilled water to 1 liter], and the mixture was allowed to stand in the dark at room temperature for 30 minutes, and then 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 measuring method described in 1), the specific activity of the alcohol dehydrogenase of Example 1 induced by lanthanum with respect to the ethanol substrate when various substrates were used was examined. It was The results are shown in Table 1.

[Table 1]

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

4) PH stability According to the activity measuring method described in 1), the residual activity at various pHs was examined using McIlvane buffer. The stable pH range is about 4.5-9 as shown in FIG.
Is.

5) Optimum temperature The effect of temperature on the enzyme activity was examined according to the activity measuring 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 at various temperatures for 1 hour was investigated. The results are shown in Fig. 4. From this result, the residual activity after heat treatment at 50 ° C. for 1 hour was about 70%.

7) Effect of Metal Ions The effect of various metal ions on the enzyme activity was examined according to the activity measuring method described in 1), and the results are shown in Table 2. From these results, it was found that Zn ²⁺ and Hg ²⁺ strongly inhibit the reaction, and Cu ²⁺ , Co ²⁺ , Ni ²⁺ and Mn ²⁺ also inhibit the reaction.

[Table 2]

8) Effects of inhibitors The effects of various inhibitors on the enzyme activity were examined according to the method described in 1). The results are shown in Table 3. Regarding this enzyme, a very strong inhibitory activity against antimycin A and a strong inhibitory effect against rotenone were observed.

[Table 3]

9) Michaelis constant (Km) The activity at various substrate concentrations was measured according to the method described in 1). Under this measurement condition, although it cannot be said that the Michaelis equation is always obeyed depending on the substrate concentration range, the Michaelis constant was 225 mM when measured in the concentration range of 100 mM to 1 M as shown in FIG.

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

Example 4 The same alcohol dehydrogenase was purified by using neodymium chloride instead of the lanthanum chloride used in Example 1. The specific activity of the obtained enzyme was about 4 units / mg protein.

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

[0037]

[Table 4]

Example 6 When 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 was electrophoresed at the same time as a control. A protein band corresponding to the purified alcohol dehydrogenase obtained in Example 2 was observed, and the gel subjected to the same electrophoresis was subjected to activity staining using ethanol as a substrate, and the position corresponding to the purified alcohol dehydrogenase was found. Activity was observed.

Example 7 Pseudogluconobacter saccharoketgenes strain K591s (FERM BP-1130) was placed in a 1-liter Erlenmeyer flask containing 200 ml of a medium containing 1.0% yeast extract and 1.0% peptone, respectively. , IF
O14464), Pseudogluconobacter saccharoketgenes 12-5 strain (FERM BP-1129,
IFO14465), Pseudogluconobacter Saccharoketgenes TH14-86 strain (FERM BP-
1128, IFO14466), Pseudogluconobacter saccharoketgenes 12-15 strain (FERM
BP-1132, IFO14482), Pseudogluconobacter saccharoketgenes 12-4 strain (FER
MBP-1131, IFO14483), Pseudogluconobacter saccharoketgenes 22-3 strain (F
ERM BP-1133, IFO11484) agar culture 1 platinum loop was inoculated, and the culture was subjected to rotary shaking culture at 30 ° C. for 3 days, and the culture solution was collected and washed.
A crude extract was prepared in the same manner as in Example 5. The same experiment was carried out using the above-mentioned medium containing 0.0001% lanthanum chloride to prepare a crude extract. When the alcohol dehydrogenase activity of these crude extracts was measured, the activity was detected only in the lanthanum chloride-added cultures of all the strains used.

Example 8 Using the purified alcohol dehydrogenase obtained in Example 2 as an antigen, antiserum was prepared from rabbits. Using this antiserum, the result of examination by an octalony double immunodiffusion method showed that in the crude extract of the rare earth element-added cultured bacterial cells obtained in Example 5 and Example 7, pseudogluconobacter saccharoket was found. It was shown that there is a serologically similar or very similar protein to the alcohol dehydrogenase of Genes K591s strain.

Example 9 44 mg of La-ADH obtained in Example 2 was ashed by the wet ashing method and the La concentration was quantified by the atomic absorption method. As a result, it was found to contain 0.23% (W / W). It was

[0042]

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

[Brief description of drawings]

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.

Claims (6)

[Claims] 1. A NAD-independent alcohol dehydrogenase containing a rare earth element in an enzyme molecule which catalyzes the oxidation of a compound having a hydroxymethyl group to an aldehyde group. 2. The alcohol dehydrogenase according to claim 1, which has the following physicochemical properties. Optimum pH 3.5-6.5 Optimum temperature 40-50 ° C Isoelectric point 4.2 ± 0.5 Molecular structure SDS-PAGE 64000 ± 50
00 molecular weight polypeptide or this polypeptide 2
Dimer consisting of molecules 3. A pseudogluconobacter (Pseudogluc)
The alcohol dehydrogenase according to claim 1, which is produced by a microorganism belonging to the genus onobacter). 4. The alcohol dehydrogenase according to claim 2, which is produced by induction with a rare earth element. 5. A Pseudogluconobacter genus capable of producing a NAD-independent alcohol dehydrogenase containing a rare earth element in an enzyme molecule which catalyzes the oxidation of a compound having a hydroxymethyl group to an aldehyde group. The method for producing an alcohol dehydrogenase according to claim 1, wherein the microorganism belonging to the strain or a mutant strain thereof is cultured, and the produced alcohol dehydrogenase is isolated and purified. 6. The production method according to claim 5, wherein a rare earth element is present in the medium.