JPH0568543A - Alcohol dehydrogease and its production - Google Patents

Abstract

PURPOSE:To obtain the subject enzyme having high oxidizing ability of middle- chain alcohol or aldehyde by culturing a strain belonging to the genus Pseudomonas, capable of producing a specific alcohol dehydrogenase, in a nutrient medium and collecting the alcohol dehydrogenase. CONSTITUTION:A strain belonging to the genus Pseudomonas, being a reaction catalyst of converting an alcohol(aldehyde) + an electron acceptor into an aldehyde (carboxylic acid) + a reduction type electron acceptor, showing specificity to >=3C straight-chain aliphatic alcohol, aromatic alcohol and >=1C aldehyde, having substrate specificity of lower action on ethanol than on the above- mentioned compounds and of almost no action on methanol and polyethylene glycols, optimum pH at 8.4, 6.0-11.0 stable pH range and about 70,000 (gel filtration) molecular weight is cultured in a nutrient medium and the objective enzyme is collected from the culture mixture.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an alcohol dehydrogenase having novel properties and a method for producing the same. INDUSTRIAL APPLICABILITY The novel alcohol dehydrogenase of the present invention can be used, for example, for producing industrially useful medium-chain carboxylic acid and medium-chain aldehyde. Further, it has properties suitable for use as a coupling enzyme used in enzyme immunoassay widely used in the field of clinical examination.

[0002]

BACKGROUND ART Alcohol dehydrogenase is widely known as one having NAD and NADP as coenzymes (EC 1.1.1.1. And EC 1.1.1.
2. ). On the other hand, there are those that use artificial electron acceptors such as ferricyanide, phenazine methosulfate, and dichlorophenol indophenol, which use pyrroloquinolinoquinone (PQQ) as a coenzyme (Amey
ama and Adachi, Methods Enzymol., 89,450-457 (198
2)). This enzyme acts widely on alcohols, but not on aldehydes, and requires a surfactant treatment in the process of extracting and purifying the enzyme from bacterial cells. Recently, Pseudomonas aeruginosa and Pseudomonas testos
teroni), an alcohol dehydrogenase that acts on intermediate alcohols has been purified (B. Groen et al., Bioche
mj, 223, 921-924 (1984) and B. Groen et al., Bioche.
mJ234,611-615 (1986)), these enzymes act well on ethanol, but their substrate specificity for medium-chain alcohols was not sufficient.

[0003]

Based on the above background, the present inventors have higher specificity for medium-chain alcohols than known alcohol dehydrogenases, act also on aldehydes, and extract from bacterial cells. Various attempts were made to find alcohol dehydrogenases that do not require detergents.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that Pseudomonas sp. TE3493, a strain belonging to Pseudomonas sp. The inventors have found an alcohol dehydrogenase highly specific to chain alcohol and have completed the present invention.

That is, the present invention is a novel alcohol dehydrogenase having the following properties. (A) Action It catalyzes the following reaction. Alcohol (aldehyde) + electron acceptor → aldehyde (carboxylic acid) + reduced electron acceptor (b) Substrate specificity For linear aliphatic alcohols having 3 or more carbon atoms, aromatic alcohols, and aldehydes having 1 or more carbon atoms It shows remarkable specificity. The carbon number of the aldehyde refers to the carbon number other than the aldehyde group. It has a lower action on ethanol than the above compounds, and hardly acts on methanol and polyethylene glycol. (C) Optimum pH: 8.4 (d) Molecular weight: about 70,000 (gel filtration method)

The present invention also belongs to the genus Pseudomonas,
A method for producing alcohol dehydrogenase, which comprises culturing a strain having the ability to produce alcohol dehydrogenase in a nutrient medium and collecting the alcohol dehydrogenase from the culture.

The microorganism used in the present invention is a bacterium belonging to the genus Pseudomonas capable of producing alcohol dehydrogenase having the above-mentioned properties, and a preferred example thereof is Pseudomonas sp. TE3493. Pseudomonas sp.
TE3493 is a strain newly isolated from soil,
Its mycological properties are as follows.

(A) Morphology (1) Bacterial form: short bacillus (2) Cell size: 2.0 to 2.5 × 1.0 μm (3) Motility: Yes (4) Cell polymorphism: None. (5) Presence or absence of spores: None. (6) Gram stainability: negative. (7) Antibacterial property: negative.

(B) Growth state in each medium (1) Meat broth agar plate culture: Light brown circular colonies are formed by culture at 30 ° C. for 48 hours. The periphery of the colony is the whole edge, or is wavy and ridged. The surface is smooth, glossy and opaque. No dye formation. (2) Broth agar slope culture: Good growth, same as (1). (3) Broth liquid culture: Growth is good, with some cloudiness and sedimentation. (4) Meat broth gelatin stab culture: Good growth only in the upper part. Gelatin does not liquefy. (5) Litmus milk: No change in color. Milk solidifies.

(C) Physiological properties (1) Reduction of nitrate: negative (2) Denitrification reaction: negative. (3) MR test: negative. (4) VP test: negative. (5) Formation of indole: negative. (6) Generation of hydrogen sulfide: negative. (7) Hydrolysis of starch: negative. (8) Utilization of citric acid: Koser's medium, Christensen
The medium is positive. (9) Use of inorganic N source: ammonium salt is used, but nitrate is not used. (10) Dye formation: No dye is formed. (11) Urease: Negative. (12) Oxidase: Positive. (13) Catalase: Positive. (14) β-galactosidase: negative. (15) Arginine dihydrase: positive. (16) Lysine decarboxylase: negative. (17) Ornithine decarboxylase: negative. (18) Tryptophan deaminase: negative. (19) β-glycosidase: negative. (20) Growth range: Grow at pH 5.0 to 10.0.
It grows at a temperature of 20 to 37 ° C and does not grow at 42 ° C. (21) Attitude toward oxygen: aerobic (22) OF test: 0 (oxidation) (23) Production of acid from sugar L-arabinose + D-glucose + D-fructose-D-galactose + D-mannose + D-xylose-D-sorbit-D-mannitol-inosit-L-rhamnose-saccharose-mannose-lactose-D-melibiose-D-amidacrine-glycerin-starch-

The experimental method for identifying the above-mentioned mycological properties is mainly written by Takeharu Hasegawa, revised edition "Classification and Identification of Microorganisms".
It was conducted by the Academic Publishing Center (1985). As the criteria for classification and identification, the 8th edition of the "Bergiss Manual of Detergent Native Bacteriology" was referenced.

From the above literature and various mycological properties, this bacterium is considered to belong to the genus Pseudomonas. In addition, the bacterium has various properties, such as Pseudomona.
However, since this strain does not produce a water-soluble fluorescent dye, this strain is pseudomonas sp.
monas sp.) TE3493. This bacterium was submitted to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Microbiology Research Institute
Deposited as No. 8.

The enzyme of the present invention can be produced by culturing the alcohol / dehydrogenase-producing bacterium in a nutrient medium and collecting the alcohol / dehydrogenase from the culture. alcohol·
As the medium used for culturing the dehydrogenase, either synthetic medium or natural medium can be used as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance and other necessary nutrients that can be assimilated by the strain used. For example, glucose, glycerol, etc. are used as the carbon source. As the nitrogen source, for example, nitrogen-containing natural products such as peptones, meat extracts, yeast extracts and inorganic nitrogen-containing compounds such as ammonium chloride and ammonium phosphate are used. As the inorganic substance, potassium phosphate, sodium phosphate, magnesium sulfate or the like is used. In addition, it is desirable to add alcohol such as nonmaltanol to the medium as an alcohol / dehydrogenase production inducer.

The medium is usually shake culture or aeration-agitation culture. The culture temperature is controlled in the range of 20 to 40 ° C, preferably 25 to 37 ° C, and the culture pH is controlled in the range of 5 to 9, preferably 7 to 8. It can be carried out under conditions other than these if the strain to be used grows. The culture period is usually 1 to 10 days for growth, and alcohol dehydrogenase is produced and accumulated in the cells.

As the method for purifying the enzyme in the present invention, a generally used purification method may be used. For example, for the extraction method, any of ultrasonic crushing, mechanical crushing using glass beads, French press, surfactant and the like may be used. Further, regarding the extract, ammonium sulfate, salting-out method such as sodium sulfate, magnesium chloride, metal aggregation method such as calcium chloride, protamine, polyethyleneimine aggregation method, further, DEAE (diethylaminoethyl) -sepharose, It can be purified by an ion exchanger chromatography method such as 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. Further, it can be used as an immobilized enzyme by immobilizing it on a suitable carrier.

Next, a method for measuring the activity of the novel alcohol dehydrogenase of the present invention will be described. 500 mM Tris-HCl buffer, pH 8.4 0.6 ml 100 mM NH ₄ Cl 0.1 ml Enzyme solution 0.1 ml 100 mM Normal octanol 0.1 ml After preparing the above reaction solution, preheat at 25 ° C. for 5 minutes. In the blind test, 0.1 ml of water was added instead of the substrate (normal octanol), and the same operation as above was performed. The reaction is started by the addition of ferricyanide. After reacting for a certain period of time, 0.5 ml of Jupanol reagent was added to stop the reaction, and 3.5 ml of water was added, and after 20 minutes, 6
Colorimetric at 60 nm. By dividing the value (ΔOD) obtained by subtracting the blind value from the test value by the reaction coefficient and the extinction coefficient of Prussian blue produced, the mass of the group oxidized by the enzyme solution used can be known. Regarding the expression of the activity of alcohol dehydrogenase, 1 unit (U) is the enzyme activity that oxidizes 1 μmol of substrate per minute under the above-mentioned conditions.

The alcohol dehydrogenase of the present invention has the following characteristics. (1) Operation This enzyme oxidizes alcohol or aldehyde to produce the corresponding aldehyde or carboxylic acid as shown in the following reaction formula. Alcohol (aldehyde) + electron acceptor → aldehyde (carboxylic acid) + reduced electron acceptor

(2) Substrate specificity The enzyme activity when various alcohols and aldehydes were used was shown. Further, the activity when normal pentanol was used was set as 100 and shown as a relative activity. Table 1 Alcohol (aldehyde) Relative activity Methanol 2 Ethanol 20 Normal propanol 92 Normal butanol 90 Normal pentanol 100 Normal hexanol 90 Normal heptanol 83 Normal octanol 70 Isopropanol 8 Allyl alcohol 100 Benzyl alcohol 90 Phenethyl alcohol 95 Normal propione ide 95 95 Normal butyraldehyde 100 Normal hexyl aldehyde 85 Polyethylene glycol 0 From these results, the enzyme of the present invention acts well not only on medium chain alcohols and aromatic alcohols but also on various aldehydes. It easily acts on the following electron acceptors as an electron transfer system. Ferricyanide, phenazine methosulfate, nitrotetrazolium blue, dichlorophenol indophenol

(3) Km values Km values for ethanol, normal propanol and normal pentanol are 2.0 mM and 0.12 m, respectively.
It was M and 0.09 mM.

(4) Effect of ammonium ion The enzyme activity of the enzyme of the present invention was measured in the presence of 0 to 4 mM ammonium ion. The results are shown in FIG. 1, and the reaction rate was increased 1.7 times by 3 mM ammonium ion.

(5) Optimum pH 0.1 MK-phosphate buffer (pH 6.0-8.0),
The enzyme activity was measured in 0.1 M Tris-HCl buffer (pH 8.0 to 9.0). The result is as shown in FIG. 2, and the optimum pH was 8.4.

(6) Stable pH The enzyme of the present invention and a 0.1 M McClubaine buffer (pH 5.
0-6.0), 0.1M K-phosphate buffer (pH 6.0)
~ 8.0), 0.1 M Tris-HCl buffer (pH 8.0)
9.0), 0.1 M borate buffer (pH 9.0-12.
0) and stored at 5 ° C. for 5 days, and the residual activity was measured. The result is as shown in FIG. 3, and the stable pH was around 6.0 to 11.0.

(7) Optimum temperature The enzyme activity at each temperature was measured. The results are shown in Fig. 4, and the optimum temperature was around 40 ° C.

(8) Thermostability The enzyme of the present invention was added to a 50 mM K-phosphate buffer solution (pH 7.
After incubating in 0) for 10 minutes, the residual enzyme activity was measured. The results are shown in Fig. 5 and were stable up to 50 ° C.

(9) Absorption spectrum The purified enzyme showed specific absorption at 280, 418, 523 and 550 nm (Fig. 6).

(10) Prosthetic group The purified enzyme of the present invention was stirred in 90% methanol overnight to extract the coenzyme, and then methanol was removed at low temperature under reduced pressure to dissolve in water to obtain a sample. .. This was mixed with apoglucose dehydrogenase to convert the enzyme into a holoenzyme, and then the amount of pyrroloquinolinoquinone (PQQ) was calculated from the activity of the expressed glucose dehydrogenase. As a result, one molecule of PQQ was quantified per one molecule of enzyme. Therefore, it was shown that this enzyme is non-covalently bound to PQQS. When the heme content of the enzyme of the present invention was calculated from the pyridine hemochrome spectrum, 1 mole of heme was contained per 1 mole of the enzyme.

(11) Molecular weight As a result of gel filtration using Sephadex G-200 (Pharmacia), the molecular weight was about 70,000. As a result of SDS-PAGE, the molecular weight was about 70,000.
It was 0. Therefore, the novel alcohol dehydrogenase was considered to consist of a monomer having a molecular weight of 70,000.

[0028] Conventional Gluconobacter suboxydans, Ameyama and Adachi, M
ethods Enzymol., 89, 450-457 (1982)), Pseudomonas testosteroni, Pseudomonas aeruginosa alcohol dehydrogenase and the enzyme of the present invention with substrate specificity,
The molecular weight, binding mode of prosthetic group PQQ, heme content, and activation by ammonium ion were compared. The enzyme produced by Gluconobacter suboxidans acts on short-chain alcohols such as ethanol and normal hexanol, but does not act on medium-chain alcohols and aldehydes, and has a molecular weight of 85,000, 49,000, 14,400 subunits. Which is non-covalently bonded to PQQ, contains 2 molecules of heme per molecule of enzyme, and is not activated by ammonium ion. The enzyme of the present invention has substrate specificity, molecular weight, heme content, and activation by ammonium ion. Is different. The enzyme produced by Pseudomonas testosteroni acts on lower and medium chain alcohols such as ethanol and normal octanol, aromatic alcohols and aldehydes, and has a molecular weight of 6
It is a monomer of 7,000, has no PQQ, contains one molecule of heme, and is not activated by ammonium ion. The enzyme of the present invention has an action on ethanol, PQQ binding form, heme content, and activation by ammonium ion. Is different.
The enzymes produced by Pseudomonas aeruginosa include short-chain alcohols such as methanol and ethanol, medium-chain alcohols,
Acts on aldehydes and has a molecular weight of 101,000, 50,0
00 subunit, non-covalently bound to PQQ,
It is activated by ammonium ion without wiping heme, and differs from the enzyme of the present invention in the action on methanol and ethanol, the molecular weight, and the content of heme.

[0029]

EXAMPLES The present invention will be specifically described with reference to the following examples. Example 1 Polypeptone 1%, yeast extract 0.5%, NaCl1
500 ml of medium (pH 7.0) containing 0.5%
It was transferred to a ml Sakaguchi flask and autoclaved at 121 ° C. for 15 minutes. As an inoculum, Pseudomonas sp. TE3493 was inoculated with one white bacterium ear and cultured at 30 ° C. for 2 days to obtain a seed culture solution. Next, normal octanol 0.1%, yeast extract 0.01%, NaNO ₃ 0.02%, (NH ₄ ) ₂ SO
₄ 0.02%, K ₂ HPO 0.02%, KH ₂ PO
0.01%, the medium (pH 7.0) 61 containing MgSO ₄ · 7H ₂ O 0.02% was transferred to a 101 jar fermentor, performed 15 minutes autoclaving at 121 ° C., after cooling, the seed culture solution 100ml The mixture was transferred and cultured at 300 rpm and an aeration rate of 21 / min at 30 ° C. for 36 hours. The culture broth was collected by centrifugation and suspended in 650 ml of 5 mM phosphate buffer. Processed by French press (1,000kg / cm)
Then, centrifugation was performed to obtain 654 ml of the supernatant. The alcohol dehydrogenase activity of the obtained crude enzyme solution was 72 U.
/ Ml. The crude enzyme solution was further subjected to ultracentrifugation, and the supernatant was fractionated in the order of ion exchange chromatography, hydroxyapatite chromatography, and strontium apatite chromatography to obtain an enzyme preparation of 27 U / ml.

[0030]

INDUSTRIAL APPLICABILITY According to the present invention, an alcohol dehydrogenase having a high ability to oxidize medium chain alcohols and aldehydes can be obtained by extraction from bacterial cells without treatment with a surfactant.
The alcohol dehydrogenase having such properties is suitable for producing industrially useful medium-chain aldehydes and medium-chain carboxylic acids.

[Brief description of drawings]

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

FIG. 2 is a graph showing the relationship between reaction pH and relative activity of the enzyme of the present invention.

FIG. 3 is a graph showing the pH stability of the enzyme of the present invention.

FIG. 4 is a graph showing the relationship between reaction temperature and relative activity of the enzyme of the present invention.

FIG. 5 is a graph showing the temperature stability of the enzyme of the present invention.

FIG. 6 is a diagram showing an absorption spectrum of the enzyme of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazunobu Matsushita 2645-2 Yoshishiki, Yamaguchi City

Claims (2)

[Claims] 1. An alcohol dehydrogenase having the following properties. (A) Action It catalyzes the following reaction. Alcohol (aldehyde) + electron acceptor → aldehyde (carboxylic acid) + reduced electron acceptor (b) Substrate specificity For linear aliphatic alcohols having 3 or more carbon atoms, aromatic alcohols, and aldehydes having 1 or more carbon atoms It shows remarkable specificity. It has a lower action on ethanol than the above compounds, and hardly acts on methanol and polyethylene glycol. (C) Optimum pH: 8.4 (d) Stable pH range: 6.0 to 11.0 (e) Molecular weight: about 70,000 (gel filtration method) 2. A method for producing alcohol dehydrogenase, which comprises culturing a strain belonging to the genus Pseudomonas and having an alcohol dehydrogenase-producing ability according to claim 1 in a nutrient medium, and collecting the alcohol dehydrogenase from the culture.