JP3102543B2 - Glutamate dehydrogenase and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel glutamate dehydrogenase and a method for producing the same, and more particularly to a thermostable and stable glutamate dehydrogenase which is suitable for measuring the amount of glutamic acid and ammonia in a sample and is stable in a reagent solution. It is about the law.

[0002]

BACKGROUND OF THE INVENTION Glutamate dehydrogenase is used in animals,
Although widely present in plants, microorganisms and the like, they are classified into three types according to coenzyme specificity. An enzyme that requires NAD ⁺ as a coenzyme (EC 1.4.1.2) is present in higher plants, bacteria, molds, and yeasts and is involved in the degradation of glutamic acid. Enzyme that requires NAD ⁺ and NADP ⁺ as coenzyme (EC1.4.1.3)
Is present in animals, bacteria and molds, but is most famous for those derived from bovine liver. An enzyme (EC1.4.1.4) that requires NADP ⁺ as a coenzyme is present in bacteria, yeasts and molds, and is involved in the biosynthesis of glutamic acid.

[0003] Glutamate dehydrogenase can be used for the measurement of glutamate and ammonia by utilizing oxidative deamination and reductive amination. It can be used in conjunction with various enzymes to measure urea nitrogen, creatinine, leucine aminopeptidase, and glutamate oxaloacetate transaminase.

[0004]

The glutamate dehydrogenase widely used includes those derived from bovine liver. The enzyme is an enzyme that can use NAD ⁺ and NADP ⁺ (EC 1.4.1.3), but requires ADP as an activator. It is also known that the stability is poor. Enzymes of the genus Proteus and enzymes derived from yeast are also commercially available. These are NADP ⁺ dependent glutamate dehydrogenases (EC 1.4.1.4), NAD
When P ⁺ is used, there is an advantage that contamination reaction is small when measuring ammonia or the like in a biological sample. However, on the other hand, NADPH (NADP ⁺ ) becomes NADH (NAD ⁺ )
Has the disadvantage of being more expensive than In recent years, liquid reagents that require no labor for preparation have been circulating.
It is known that PH is much less stable in liquid form than NADH.

[0005] In view of the above background, it has been desired to develop glutamate dehydrogenase which can use NAD ⁺ (NADH) as a coenzyme, is thermostable, has high stability in a reagent solution, and does not require ADP for activation. I was

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the problems of conventional glutamate dehydrogenase, and as a result, have found a novel glutamate dehydrogenase from a bacterium belonging to Pseudomonas. It was completed.

That is, the present invention is a glutamate dehydrogenase having the following physicochemical properties. (1) catalyze the next reaction

Embedded image (2) Substrate specificity: specific for L-glutamic acid. Requires NAD ⁺ and NADH as coenzymes,
It has no effect on NADP ⁺ and NADPH. (3) Thermal stability: stable up to about 60 ° C. (pH 8.3, 10
(4) Not activated by ADP.

One embodiment of the enzyme of the present invention is the glutamate dehydrogenase according to claim 1, which has the following physicochemical properties. (1) catalyze the next reaction

Embedded image (2) Substrate specificity: specific for L-glutamic acid. Requires NAD ⁺ and NADH as coenzymes,
It has no effect on NADP ⁺ and NADPH. (3) Thermal stability: stable up to about 60 ° C. (pH 8.3, 10
(4) Not activated by ADP. (5) Specific activity: 300 U / mg or more (6) Surfactant Not inhibited by Brij.

Another embodiment is the glutamate dehydrogenase according to claim 1, which has the following physicochemical properties. (1) catalyze the next reaction

Embedded image (2) Substrate specificity: specific for L-glutamic acid. Requires NAD ⁺ and NADH as coenzymes,
It has no effect on NADP ⁺ and NADPH. (3) Optimum action pH: 10.5 to 11.5 (oxidative deamination reaction) (4) pH stability: pH 5 to 10 (25 ° C, 20 hours) (5) Optimum action temperature: about 60 ° C (Oxidative deamination reaction) (6) Thermal stability: stable up to about 60 ° C (pH 8.3, 10
(7) Molecular weight: about 280,000 (gel filtration method) About 41,000 (SDS-PAGE) (8) Not activated by ADP.

Another embodiment is the glutamate dehydrogenase according to claim 1, which has the following physicochemical properties. (1) catalyze the next reaction

Embedded image (2) Substrate specificity: Specific for L-glutamic acid. Requires NAD ⁺ and NADH as coenzymes,
It has no effect on ADP ⁺ and NADPH. (3) Optimum action pH: 10.5 to 11.5 (oxidative deamination reaction) (4) pH stability: pH 5 to 10 (25 ° C, 20 hours) (5) Optimum action temperature: about 60 ° C (Oxidative deamination reaction) (6) Thermal stability: stable up to about 60 ° C (pH 8.3, 10
(7) Molecular weight: about 280,000 (gel filtration method) About 41,000 (SDS-PAGE) (8) Not activated by ADP. (9) Specific activity: 300 U / mg or more (10) Surfactant Not inhibited by Brij.

The present invention also relates to Pseudomonas (Pseudomona
s) A method for producing glutamate dehydrogenase, which comprises culturing a strain belonging to the genus and having the ability to produce glutamate dehydrogenase having the above physicochemical properties in a nutrient medium, and collecting glutamate dehydrogenase from the culture.

The enzyme of the present invention may be of any origin, such as animals, plants, and microorganisms, as long as it can produce glutamate dehydrogenase having the above properties. Preferably, Pseudomonas (Pseudomonas) capable of producing glutamate dehydrogenase having the above properties
As), a more preferable example is Pseudomonas sp. 433-3.

The pseudomonas used in the present invention
SP (Pseudomonas sp.) 433-3 is a strain isolated from the soil of Sekigahara-cho, Fuwa-gun, Gifu Prefecture by the present inventors, and its bacteriological properties are as follows. (A) Morphology (1) Bacteria type: Bacillus subtilis (2) Cell size: 0.7-1.0 × 0.3 μm (3) Cell polymorphism: No (4) Motility: Yes, It has polar flagella. (5) Presence or absence of spores: None (b) Growth state in each medium (1) Gravy agar medium: The periphery of colonies that form yellow to orange colonies after culturing at 30 ° C. for 48 hours is in the form of fragments (Labarlobulate). The convex surface is rough and has a waxy luster and is transparent. (2) Liquid broth culture: Growth is good and there is a little turbid sediment. (3) Meat gelatin culture: Growth is good and only the upper part grows in a filamentous form. Gelatin does not liquefy. (4) Litmus milk: no change in color. Milk is broken down. (5) MacConkey agar: Does not grow. (6) Phenylethyl alcohol agar: Does not grow.

(C) Physiological properties (1) Gram stainability-(2) Reduction of nitrate edge-(3) Denitrification reaction-(4) MR test-(5) VP test-(6) Formation of indole- (7) Production of hydrogen sulfide-(8) Hydrolysis of starch + (9) Decomposition of Tween 80-(10) Utilization of citric acid Koser's medium-Christe
nsen's medium-(11) Pigment production Yellow pigment was produced in the cells. (12) urease-(13) oxidase-(14) catalase + (15) β-galactosylase + (16) arginine dihydrolase-(17) lysine carboxylase-(18) ornithine carboxylase-(19) tryptophan deaminase-( 20) β-glucosylase + (21) protease-(22) deoxyribonuclease-

[0015] (24) Use of inorganic N source (C source glucose) Glutamic acid + sodium nitrate + ammonium sulfate + (25) Attitude to oxygen Aerobic (26) OF test (Hugh Leifson method) O (oxidation)

(28) Use of organic compounds D-glucose + L-arabinose + D-mannose + D-mannitol-N-acetyl-D-glucosamine + maltose + potassium gluconate-N-caprate-adipate-DL-malate- Sodium citrate-phenyl acetate-

The above-mentioned experimental method for identifying mycological properties is mainly described in a revised edition of "Classification and Identification of Microorganisms", edited by Takeharu Hasegawa.
Performed by the Society Press Center (1985). In addition, as a standard for classification and identification, the Vergees Manual of
Reference was made to "Systematic Bacteriology" (1984) and "International Journal of Systematic Bacteriology" Vol. 27 (2) p133-146 (1977).

Based on the above literature and mycological properties, the bacterium was considered to belong to the genus Pseudomonas. Furthermore, among Pseudomonas spp., There were many points that were consistent with the properties of Pseudomonas paucimobilis, but no production of deoxyribonuclease and no degradation of Tween80 were observed, and unlike the above-mentioned bacteria,
It was designated as Pseedomonas sp. 433-3 (FERMP-14092).

In producing the enzyme of the present invention, a glutamic acid dehydrogenase-producing bacterium having the above properties is cultured in a nutrient medium, and the glutamic acid dehydrogenase is collected from the culture.

The culture medium used for culturing the glutamate dehydrogenase-producing bacterium may be any of a synthetic medium and a natural medium as long as the used strain contains carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients in appropriate amounts. Can be used. As the carbon source, for example, glucose, glycerol and the like are used. Examples of the nitrogen source include nitrogen-containing natural products such as peptones, meat extracts, and yeast extracts, and inorganic nitrogen-containing compounds such as ammonium chloride and ammonium citrate. Potassium phosphate as an inorganic substance,
Sodium phosphate, magnesium sulfate and the like are used.

It is desirable that sodium glutamate be added to the medium as a glutamate dehydrogenase production inducer. The culture is usually performed by shaking culture or aeration and stirring culture. The cultivation temperature is 20 to 30 ° C, preferably 25 to 30 ° C, and the culture pH is in the range of 6 to 9, preferably pH 7 to 8. The culture period is usually 1 to 3 days, and glutamate dehydrogenase is produced and accumulated in the cells.

The method for purifying the enzyme of the present invention may be a commonly used purification method. For example, as the extraction method, any of ultrasonic crushing, mechanical crushing using glass beads, and French press may be used. Further, the extract can be purified by a salting-out method such as ammonium sulfate, an aggregation method such as polyethyleneimine, and an ion-exchange chromatography method such as DEAE-sepharose and CM (carboxymethyl) -sepharose. The crude enzyme solution and the purified enzyme solution obtained by these methods can be powdered by, for example, spray drying or freeze drying.

Next, a method for measuring the activity of the glutamate dehydrogenase of the present invention (reductive amination reaction) will be described. Prepare the following reaction mixture. 25 ml of 0.1 M Tris-HCl buffer (pH 8.3) 2 ml of 3.3 M NH ₄ Cl solution 1 ml of 0.225 M α-ketoglutaric acid solution 1 ml of 7.5 mM NADH solution 1 ml 2.9 ml of the above reaction mixture is placed in a cuvette and placed at 30 ° C. at about 30 ° C. Preheat for 5 minutes. Then, 0.1 ml of the enzyme solution was added to start the reaction.
The change in absorbance at 40 nm is recorded for 3 to 4 minutes, and the change in absorbance per minute is determined from the linear portion. In the blind test, 0.1 M Tris-HCl buffer (pH
8.3) is added, and the operation is performed in the same manner as described above. The activity of glutamate dehydrogenase is expressed as 1 unit (U) of the enzyme that oxidizes 1 μmol of NADH per minute under the above conditions.

[0024]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 0.6% sodium glutamate, 1% polypeptone,
Medium containing 1% yeast extract and 1% glycerol (pH
7.0) was dispensed in 60 ml portions and sterilized (121 ° C, 15 mi).
n) Put Pseudomonas s in a 500 ml Sakaguchi flask.
p.433-3 (FERMP-14092) was inoculated and cultured at 30 ° C. for 1 day to obtain a seed culture. Next, add 6 liters of the same medium to 10
Transfer to a 1 liter jar fermenter,
After autoclaving for 5 minutes and leaving to cool, seed culture solution 60m
transfer 300 l, aeration 2 l / min, 30
C. for 24 hours. After completion of the culture, collect the culture solution,
The cells were suspended in a 50 mM Tris-HCl buffer (pH 7.5).

The suspension was treated with a French press and centrifuged to obtain a supernatant. The resulting crude enzyme solution was subjected to ammonium sulfate fractionation, DEAE-sepharose chromatography, phenyl sepharose chromatography, and gel filtration using Sephadex G-200 to give a specific activity of 300 U / mg.
Was purified.

The obtained glutamate dehydrogenase had the following properties. (1) The following reaction was catalyzed.

Embedded image

(2) Substrate specificity The enzyme activity was measured when various amino acids were used as substrates.
The activity value for L-glutamic acid is shown in Table 1 as 100. The activity was measured by the following method. The following reaction solution was prepared. 13 ml of 0.2 M glycine-NaOH buffer (pH 9.0) 13 ml of 0.5 M various amino acids 6 ml 9 mM NAD ⁺ 2 ml H ₂ O 8 ml 2.9 ml of the above reaction solution was placed in a cuvette and preliminarily heated at 30 ° C. for about 5 minutes. The reaction was started by adding 0.1 ml of the enzyme solution, and 340 nm was measured with a spectrophotometer controlled at 37 ° C.
The change in absorbance was recorded for 3 to 4 minutes.
The change in absorbance per minute was determined. In the blind test, a 0.1 M Tris-HCl buffer (pH 9.0) was added instead of the enzyme solution, and the same operation as above was performed.

[0028]

[Table 1]

(3) Km value The Km value for ammonia was 8.5 mM. Also N
The Km values for ADH, L-glutamic acid, NAD ⁺ are:
They were 0.19 mM, 17 mM, and 0.10 mM, respectively.

(4) Optimum action pH FIG. 1 shows the relationship between the reaction pH and the relative activity of the enzyme of the present invention. FIG. 1 shows a 0.1 M Tris-HCl buffer solution (pH 6 to 8),
0.1 M glycine NaOH buffer (pH 8.5-12.
It is the result of measuring the enzyme activity in 5). In the figure, ○-
○ indicates an oxidative deamination reaction and ●-● indicates a reductive amination reaction. The optimal pH for the oxidative deamination reaction is 10.5-1.
1.5, and the optimum pH for the reductive amination reaction is 8.0.
８8.5.

(5) pH stability FIG. 2 shows the pH stability of the enzyme of the present invention. FIG.
M acetate buffer (pH 4-6), 0.1 M K-phosphate buffer (pH 6-8), 0.1 M Tris-HCl buffer (pH 8)
10 to 10) are the results of measuring the residual activity after storage at 25 ° C. for 20 hours. The stable pH was between pH 5 and 10.

(6) Optimal operating temperature FIG. 3 shows the relationship between the reaction temperature and the relative activity of the enzyme of the present invention. FIG. 3 shows the results of measuring the enzyme activity at each temperature. In the figure, −- ○ indicates an oxidative deamination reaction, and −- 反 応 indicates a reductive amination reaction. The optimum temperature for the oxidative deamination reaction is about 60 ° C, and the optimum temperature for the reductive amination reaction is about 4 ° C.
5 ° C.

(7) Thermostability FIG. 4 shows the thermostability of the enzyme of the present invention. FIG. 4 shows that the enzyme of the present invention was dissolved in 0.1 M Tris-HCl buffer (pH 8.3) for 1 hour.
This is the result of measuring the residual activity after keeping the temperature for 0 minute, and it was stable up to about 60 ° C.

(8) Molecular weight: about 280,000
(Gel filtration method) Subunit about 40,000 (SDS-PAGE)

(9) Isoelectric point 5.6 (Isoelectric focusing)

(10) Activation by ADP ADP was added to the reagent for measuring the activity of glutamine dehydrogenase (reductive amination reaction) of the present invention to a concentration of 2.5 mM, and the activity was compared with that of the reagent without addition.

[0037]

[Table 2]

(11) Comparison with Known Enzymes The properties of the enzyme of the present invention and a known NAD ⁺ -dependent glutamate dehydrogenase (EC. 1.4.1.2) were compared.

[0039]

[Table 3]

Reference Example 1 A test solution having the following composition was prepared. Glutamate dehydrogenase (Pseudomonas sp. 433-3) 0.3 U / ml urease (manufactured by Toyobo) 10 U / l α-ketoglutarate 10 mmol / l NADH 0.3 mmol / l Triton X-100 0.1% Tris-HCl buffer 50 mmol / l, pH 8.5 Preliminary heating of 3 ml of the above-mentioned test solution at 37 ° C for 5 minutes, and then diluting a sample obtained by diluting 100 mg / dl of urea nitrogen in 10 series into 0.0
5 ml was added, the mixture was reacted at 37 ° C. for 3 minutes, and the decrease in absorbance at 340 nm per minute was measured. The results are as shown in FIG. 5 and show that urea nitrogen is 100 mg / dl.
It had linearity up to that point.

Reference Example 2 A test solution having the composition of Reference Example 1 was stored at 25 ° C. for one week, and samples diluted in ten series were measured. The results are as shown in FIG. 6.
It had linearity up to g / dl.

Reference Example 3 A test solution having the following composition was prepared. Glutamate dehydrogenase (derived from Pseudomonas sp. 433-3) 20 U / ml creatinine deiminase (manufactured by Toyobo) 10 U / ml α-ketoglutarate 12 mmol / l NADH 0.4 mmol / l Tris-HCl buffer 50 mmol / l, pH 8.3 After preliminarily heating 3 ml at 30 ° C. for 5 minutes, a sample obtained by diluting creatine 50 mg / dl in 10 series with 0.1 m
was added and reacted at 30 ° C., and the value obtained by reducing the absorbance at 340 nm after 1 minute from the absorbance at 340 nm after 3 minutes was determined. The results are as shown in FIG. 7, and a proportional relationship was established up to 50 mg / dl creatinine.

Reference Example 4 A test solution having the following composition was prepared. Glutamate dehydrogenase (from bovine liver) 0.3 U / ml urease (manufactured by Toyobo) 10 U / l α-ketoglutarate 10 mmol / l NADH 0.3 mmol / l Triton X-100 0.1% Tris-HCl buffer 50 mmol / l, pH 8 5.5 Preliminary heating of 3 ml of the above test solution at 37 ° C. for 5 minutes, and then diluting a sample obtained by diluting 100 mg / dl of urea nitrogen into 10 series in 0.0
5 ml was added, the mixture was reacted at 37 ° C. for 3 minutes, and the decrease in absorbance at 340 nm per minute was measured. The results are as shown in FIG. 8 and show that urea nitrogen is 100 mg / dl.
It had linearity up to that point.

The test solution having the above composition was stored at 25 ° C. for one week, and samples diluted in ten series were measured. The result is shown in FIG.
The linearity after storage at 25 ° C. for 1 week is up to 60 mg / dl of urea nitrogen, and the enzyme derived from bovine liver is unstable without ADP, so that it has no quantification beyond that. Was.

Reference Example 5 A test solution having the following composition was prepared. α-Ketoglutaric acid 7.6 mol / l NADH 0.25 mmol / l EDTA 0.85 mmol / l Tris-HCl buffer pH 8.3 85 mmol / l NH ₄ Cl 0.22 M Brij 35 0.05% or Brij 58 0.1%

After preliminarily heating 2.9 ml of the above test solution at 30 ° C. for 5 minutes, the glutamate dehydrogenase (0.
(6 U / ml) was added and reacted at 30 ° C. for 3 minutes, and the decrease in absorbance per minute at 340 nm was measured. The result is as shown in FIG.
Brij 35 or 58 had no effect.

As a comparative example, 2.9 ml of the above test solution (however, ADP was added to be 10 mmol / l) was added to 30
After preliminarily heating at 5 ° C. for 5 minutes, 0.05 ml of bovine liver-derived glutamate dehydrogenase (0.6 U / ml) was added, reacted at 30 ° C. for 3 minutes, and the change in absorbance per minute at 340 nm was measured. The results are as shown in FIG. 10, and about 50% for Brij35 and Brij35.
At 58, 40% activity was inhibited.

[0048]

According to the present invention, inexpensive NAD ⁺ (NADH)
Can be used as a coenzyme, and a novel glutamate dehydrogenase having high stability and not requiring ADP for activation can be obtained.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the reaction pH and the relative activity of the enzyme of the present invention.

FIG. 2 is a graph showing the pH stability of the enzyme of the present invention.

FIG. 3 is a graph showing the relationship between the reaction temperature and the relative activity of the enzyme of the present invention.

FIG. 4 is a graph showing the thermostability of the enzyme of the present invention.

FIG. 5 is a graph showing urea nitrogen dilution linearity using a reagent composition for measuring urea nitrogen (including the enzyme of the present invention).

FIG. 6 is a graph showing the urea nitrogen dilution linearity after storing a urea nitrogen measurement reagent composition (including the enzyme of the present invention) at 25 ° C. for one week.

FIG. 7 is a graph showing creatinine dilution linearity using a creatinine measurement reagent composition (including the enzyme of the present invention).

FIG. 8 is a graph showing urea nitrogen dilution linearity using a urea nitrogen measurement reagent composition (including a comparative enzyme).

FIG. 9 is a graph illustrating the linearity of urea nitrogen dilution after storing a reagent composition for measuring urea nitrogen (including a comparative enzyme) at 25 ° C. for one week.

FIG. 10. Surfactant Brij for the enzyme of the invention.
5 is a graph showing the effect of the above.

────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 9/00-9/99 C12Q 1/00-1/70 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG) )

Claims (9)

(57) [Claims] 1. A glutamate dehydrogenase having the following physicochemical properties: (1) catalyze the next reaction (2) Substrate specificity: specific for L-glutamic acid. Requires NAD ⁺ and NADH as coenzymes,
It has no effect on NADP ⁺ and NADPH. (3) Thermal stability: stable up to about 60 ° C. (pH 8.3, 10
(4) Not activated by ADP. 2. The glutamate dehydrogenase according to claim 1, which has the following physicochemical properties. (1) catalyze the next reaction (2) Substrate specificity: specific for L-glutamic acid. Requires NAD ⁺ and NADH as coenzymes,
It has no effect on NADP ⁺ and NADPH. (3) Thermal stability: stable up to about 60 ° C. (pH 8.3, 10
(4) Not activated by ADP. (5) Specific activity: 300 U / mg or more (6) Surfactant Not inhibited by Brij. 3. The glutamate dehydrogenase according to claim 1, which has the following physicochemical properties. (1) catalyze the next reaction (2) Substrate specificity: specific for L-glutamic acid. Requires NAD ⁺ and NADH as coenzymes,
It has no effect on NADP ⁺ and NADPH. (3) Optimum action pH: 10.5 to 11.5 (oxidative deamination reaction) (4) pH stability: pH 5 to 10 (25 ° C, 20 hours) (5) Optimum action temperature: about 60 ° C (Oxidative deamination reaction) (6) Thermal stability: stable up to about 60 ° C (pH 8.3, 10
(7) Molecular weight: about 280,000 (gel filtration method) About 41,000 (SDS-PAGE) (8) Not activated by ADP. 4. The glutamate dehydrogenase according to claim 1, which has the following physicochemical properties. (1) catalyze the next reaction (2) Substrate specificity: specific for L-glutamic acid. Requires NAD ⁺ and NADH as coenzymes,
It has no effect on NADP ⁺ and NADPH. (3) Optimum action pH: 10.5 to 11.5 (oxidative deamination reaction) (4) pH stability: pH 5 to 10 (25 ° C, 20 hours) (5) Optimum action temperature: about 60 ° C (Oxidative deamination reaction) (6) Thermal stability: stable up to about 60 ° C (pH 8.3, 10
(7) Molecular weight: about 280,000 (gel filtration method) About 41,000 (SDS-PAGE) (8) Not activated by ADP. (9) Specific activity: 300 U / mg or more (10) Surfactant Not inhibited by Brij. 5. The method of claim 1, wherein the thermophilic bacterium produces the bacterium.
The glutamine-producing dehydrogenase described. 6. The glutamate dehydrogenase according to claim 1, which is produced by a bacterium belonging to the genus Psuedomonas. 7. Pseudomonas sp.
sp.) 433─3 (FERM P-14092). 8. A glutamate dehydrogenase, which is characterized by comprising culturing a strain belonging to the genus Pseudomonas and having the ability to produce glutamate dehydrogenase according to claim 1 in a nutrient medium, and collecting glutamate dehydrogenase from the culture. Manufacturing method. 9. A strain having the ability to produce glutamate dehydrogenase, wherein the strain has the ability to produce Pseudomonas sp.
sp.) 433-3 (FERMP-14092). The method for producing glutamate dehydrogenase according to claim 8, wherein