JPS63230082A - Alcohol dehydrogenase - Google Patents

Abstract

NEW MATERIAL:The titled enzyme having 60,000 molecular weight, specific absorption zone at 280, 416, 522 and 553nm and the following physical and chemical properties. Action and substrate specificity: not requiring NAD<+> and NADP<+> as coenzyms, oxidizing at least ethyl alcohol, butyl alcohol, amyl alco hol, diethylene glycol, triethylene glycol and polyethylene glycol in the presence of an electron acceptor to form corresponding aldehydes. Electron acceptor specificity: capable of using ferricyanide, phenazine methosulfate, dich lorophenolindophenol and nitrotetrazolium Blue as an electron acceptor. Opti mum pH: 7-9. Stable pH: 7-10. USE:Useful as a conjugate enzyme to be integrated into a diagnostic kit by enzyme immunoassay by sandwich method. Measurement can be carried out in high sensitivity. PREPARATION:Flavobacterium sp. PE-10 (FERM P-9172) is cultivated in a medium and the aimed substance is isolate from the cell.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a conjugated enzyme that is suitable as a conjugate enzyme to be incorporated into a diagnostic kit for enzyme immunoassay using the sandwich method, which is widely used, for example, in the field of clinical testing. Concerning a novel alcohol dehydrogenase.

[Conventional technology]

Various alcohol dehydrogenases are already known, and alcohol dehydrogenases (EC, 1, 1, 1, 1° and EC, 1, 1, 1, 2) that use NAD+ and NADP as coenzymes are widely used in animals. Ameyama and Ada et al. have reported that ferricyanide, phenazine methosulfate (PMS) and dichlorophenolindophenol (DCP IF) are electron acceptors.
Chi.

Methods Enzymol; 89.450-4
57 (1982)).

This enzyme acts on fatty alcohols but not on polyethylene glycol (PEG). Recently, Kawai et al. obtained polyethylene glycol dehydrogenase using DCPIF as an electron acceptor by co-cultivating Pseudomonas bacteria and Flavobacterium in a medium containing PEG.

(Fusako Kainai, et al, App
l, Environ, Microbiol+.

Parallel, 701-705 (1980). This enzyme is PE
G4000 shows the highest activity, and PEG4000 shows only 20% activity. In addition, enzymes are isolated from bacterial cells,
During the purification process, solubilization using a surfactant is required.

[Problem that the invention seeks to solve]

In enzyme immunoassay using the sandwich method, as with other analytical methods, there is a need to further increase detection sensitivity, and there has been a desire to develop a new conjugated enzyme suitable for this purpose.

[Means for solving problems]

As a result of intensive studies to search for a new enzyme suitable for use in enzyme immunoassay, the present inventors discovered that Flavobacterium spp.
The present invention was completed by discovering that a strain of M. rtum) produces a constitutively novel alcohol dehydrogenase without co-cultivation or addition of an inducing substrate.

Since the enzyme of the present invention exists in the soluble fraction of bacterial cells, there is no need for solubilization treatment with surfactants etc. when separating and purifying it from bacterial cells, and a single preparation can be easily prepared using conventional methods. It can be refined to

This purified enzyme contains a cytochrome as a prosthetic group and oxidizes alcohol to the corresponding aldehyde only in the presence of a suitable redox dye as an electron acceptor.

Next, the physicochemical properties of the enzyme of the present invention will be shown.

(1) Action: Various aliphatic polyethylene glycols and polyethylene glycols of various degrees of polymerization are oxidized to aldehydes using ferricyanide, PMS, etc. as electron acceptors without using NAD and NAD as coenzymes.

Cl1z (C1z) zcHzOH-CHI3((:
I(z) tcHO.

1(OCH2CH2[OCH□-CHz)τ0- CH
,CH,OH↓ 0HCC)lz-(OCHI C-Hz)rOCHz
CHO (2) Substrate specificity The enzyme activity was measured according to the measurement method described below using various alcohols as substrates, and polyethylene glycol #4000
(Molecular weight 3000 to 3700) relative activity is shown below when the activity is set as 100.

Substrate Relative activity (%) Ethylene glycol O diethylene glycol
29 triethylene glycol
35 methyl alcohol O ethyl alcohol 58 butyl alcohol 68 amyl alcohol
26 formaldehyde
O-acetaldehyde O-propionylaldehyde O-hexylaldehyde O(3) Electron acceptors NAD" and ?1ADP" are not used as electron acceptors, but redox dyes such as ferricyanide, PMS, and DCPIF are used as electron acceptors. The activity against various electron acceptors was measured according to the measurement method described below, and the relative activity when the activity against ferricyanide was set as 100 is shown below.

Felicyanide 10100P-NT
B 3.5NTB”
8PMS-DCPIP
10DCPIP
10NAD""
0NADP”
0(4) Optimum pH and stable pH range The optimum p)l of this enzyme is between pH 7 and 1, as shown in Figure 1.
It's at 9. In the figure, △ indicates the results of measurement using acetate buffer, ◯ indicates phosphate buffer, and ◯ indicates measurement results using carbonate buffer.

As shown in FIG. 2, this enzyme is stable from near neutral to alkaline, particularly at pH 7 to 11.

The buffer used for the measurement was the same as in Figure 1, and each p
The residual activity was measured after being left for 24 hours at 5° C. in a H buffer solution.

(5) Range of suitable temperature for action The action temperature range of the enzyme of the present invention is 0°C to 50°C;
It has an optimum temperature around 7°C.

(6) pH 1 temperature and deactivation in 10mM phosphate buffer (pH 7,0), 10% at each temperature.
When treated for minutes, it does not deactivate up to 30℃, and at 50℃ it is about 4
0% inactivation.

(7) Inhibition, Activation, and Stabilization The effects on the activity of the enzyme of the present invention when various metal ions, chelating agents, and SH inhibitors were added to the reaction solution at varying concentrations are shown below.

Additive concentration (mM) Relative activity (%)
Additive-free 1 100MgZ◆
1 100Ba2÷
1 100Na” 1 10
0Co”・10Mn”1 13K”
1 100Ca”
1 100Cu"・10 Zn" 1 0Sn"
108g”
1 6 9
Kg'' 1 7 Ethylenediaminetetraacetic acid 10 318-Hydroxyquinoline 1 27 (8) Molecular weight The molecular weight determined by gel filtration and 5DS-polyacrylamide gel electrophoresis is 60.000.

(9) Absorption Spectrum Purified enzyme exhibits specific absorption at 280, 416, 522, and 553 nm (Figure 3). No specific absorption is seen in the invisible region. The measurement was carried out using a purified enzyme solution of 4.7 μ/rail.

Although the activity of the enzyme of the present invention can be measured using any of the electron acceptors described above, the case where potassium ferricyanide is used as the electron acceptor will be described below.

0.1M potassium ferricyanide solution 94 ml, 10
0mM) Lis-HCl buffer (pH 8,0) 0.5m
l.

Add an appropriate amount of the enzyme of the present invention and add 0.9 mj of water! shall be. Furthermore, 0.1 nl of alcohol solution is added and the mixture is reacted at 37°C for 5 to 30 minutes. Add FerricD to this reaction solution.
After adding 0.5 nl of upanul reagent to stop the reaction, water was added to 5.0 mj! shall be. 660n after 10 minutes
Measure the absorbance of m. On the other hand, using potassium ferrocyanide solutions of various concentrations instead of potassium ferrocyanide solution, the concentration of potassium ferrocyanide produced by conjugation with the oxidation of alcohol was determined based on the calibration curve obtained by the same procedure as described above, and 1 μmole of alcohol was
The amount oxidized per minute is defined as 1 unit.

From the above physical and chemical properties, the enzyme of the present invention is completely different from conventionally known alcohol dehydrogenases.
Recognized as a new enzyme. In particular, this enzyme is characterized by acting on polyethylene glycols having various degrees of polymerization from aliphatic alcohols.

Such an enzyme of the present invention can be produced and obtained by culturing the bacterial strain PE-10, which the inventor newly isolated from soil, in a nutrient medium.

The mycological properties of PE-10 strain are shown below.

1. Morphology (broth agar medium, 28°C, 24 hour culture) (1
) Cell size: 0.7-1.0 μm (2) Motility: None 3) Durham staining: Negative (4) Acid-fastness: Negative (5) Presence or absence of spores: Not formed 2, Growth status ( 11 Culture on broth agar plate; Colonies are raised, gold-rimmed, translucent, soft, and yellow-orange in color.

(2) Meat juice agar slant culture: Grows well in a linear pattern, grows well in condensed water, is translucent, and exhibits a yellow-orange color.

(3) Meat juice liquid culture: Forms a fungal film on the surface.

(4) Meat juice gelatin puncture culture; grows well in the upper layer. Does not liquefy. Does not produce sediment.

(5) Lidmus milk; not coagulated or peptonized.

3. Physiological properties (1) Nitrate reduction: Negative (2) MR test; Negative (3) VP test; Negative (4) Indole formation; Negative (5) Hydrogen sulfide formation; Negative (6) Starch hydration Degradation; negative (7) casein hydrolysis; positive (8) citric acid utilization; positive (9) urease; positive αφ oxidase; positive αυ catalase; positive (2) Growth pH; pH 5-10. Optimum pH 6-8α turbidity
Growth temperature i10-35℃, optimum 25-30℃α4)
OF test; Oxidative 0 Clause Production of acids and gases from sugars L-arabinose, D-xylose, D-glucose,
D-mannose, D-fructose, D-galactose 1. Maltose, Shi=II! , lactose, D-sorbitol, inosift, glycerin and starch do not produce acid or gas.

The above properties are summarized in Bergey's Manual of Systematic Bacteriology.
Manual of Systematic Bact
When compared with the classification of eriologV)l@ (1984), the country was determined to belong to the genus Flavobacterium, and Flavobacterium sp.
lavobacterium sp PE-10)
It was named.

It has been deposited in the National Institute of Microbiology, Agency of Industrial Science and Technology as FERM P-9172.

In order to produce the enzyme of the present invention, the enzyme may be cultured according to conventional enzyme production methods.

Since the enzyme of the present invention is produced constitutively, there is no need to specifically add alcohol as a substrate for enzyme induction, and carbon sources include glucose, fructose, and sucrose. , glycerol, etc. are used as nitrogen sources, various ammonium salts, urea, etc. are used as nitrogen sources, common salts, phosphates, magnesium salts, etc. are used as inorganic salts, and organic nutrients such as yeast extract, meat extract, polypeptone, etc. are used as appropriate. It will be done.

The culture is usually preferably liquid culture, and preferably carried out aerobically, such as by shaking culture, stirring culture, or aerated culture.

The culture temperature is 20 to 35°C, preferably around 30°C. The pH during culturing is in the range of pH 5 to pH 8, and the culturing time is preferably 20 to 48 hours.

General enzyme extraction and purification methods can be used to extract and purify the enzyme of the present invention from the culture thus obtained. Since this enzyme is produced and accumulated within the bacterial cells, the bacterial cells can be recovered by a normal method and used as an enzyme source. Methods for extracting enzymes from bacterial cells include grinding the bacterial cells;
A cell-free extract can be obtained by methods commonly used for enzymes, such as a method using a lytic enzyme such as lysozyme, a method using pressure shock, and a method using autolysis. Furthermore, to obtain a purified enzyme preparation, a purified enzyme can be obtained by combining a salting-out method, a gel filtration method, and an adsorption/elution method using an ion exchanger, hydroxyapatite, hydrophobic, affinity chromatography, or the like.

〔Example〕

Glycerol 0.5%, yeast extract 0.3%, meat extract 0.2%, peptone 0.2%, dipotassium phosphate 0.1
5%, salt 0.05%, magnesium sulfate 0.01%,
Medium consisting of 0.2% ammonium nitrate (pH 7.0)
Put 601 into a 901 capacity jar fermenter and heat to 120°C.
sterilized for 20 minutes. After cooling, add 1.0% glucose and 1.0% peptone to a 500 mj 2 volume Erlenmeyer flask.
, meat extract 0.5%, salt 0.3%, yeast extract 0.2
5%, dipotassium phosphate 0.03%, and magnesium sulfate 0.05% (pH 7.0) 200ni! Seed culture solution 11, which had been inoculated with Flavobacterium sp. PE-10 and cultured with shaking at 28° C. for 20 hours, was inoculated. 28℃, 300rpm, 0.5v
The cells were cultured for 24 hours under constant vm conditions to obtain 1 kg of bacterial cells (wet bacterial weight).

The bacterial cells obtained in this way were added to 10mM phosphate buffer (pH
After suspending in 7,0) 41, the suspension was crushed using a Dyno Mill, and a cell-free extract 3N was obtained by centrifugation. This cell-free extract was fractionated by ammonium sulfate salting out.
The 70% saturated ammonium sulfate fraction was collected and dialyzed against 10mM phosphate buffer. Ammonium sulfate was added to the dialysate so that it became IM, and the mixture was adsorbed onto phenyl sepharose equilibrated with 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. ) the enzyme was eluted. Add 7 more to the elution fraction
Add ammonium sulfate to 0% saturation, centrifuge to collect the precipitate, and add to 10mM phosphate buffer (pH 7,0
>dialysis. The dialysate is then adsorbed onto hydroxyapatite. After washing with IMU acid buffer (pH 7,0)
．． The enzyme was eluted with 2MIJ acid buffer (pH 7,0).

Ammonium sulfate was added to the active fraction to achieve 70% saturation, the resulting precipitate was collected by centrifugation, the precipitate was dissolved in a small amount of buffer, and the column of Sephacryl S-200 equilibrated with 10 mM phosphate buffer was applied. The gel was filtered using

Through the above operations, 200 units of a purified sample with a specific activity of 5 units/■ were obtained.

〔Effect of the invention〕

The novel enzyme of the present invention can be easily produced by culturing Flavobacterium sp. EP-10 in a medium, and utilizes the unique properties of the enzyme of the present invention not found in known enzymes. By incorporating the enzyme of the present invention as a conjugate enzyme in enzyme-linked immunosorbent assay, measurement with high sensitivity, which could not be achieved conventionally, becomes possible.

[Brief explanation of drawings]

Figure 1 shows a curve showing the optimum pH of the enzyme of the present invention, Figure 2 shows a curve showing pH stability, and Figure 3 shows an absorption spectrum of the purified enzyme. . Patent applicant: Fujirebio Co., Ltd. Figure 1 H Relative activity (@/.) Absorbance

Claims (1)

[Claims] An alcohol dehydrogenase having the following physical and chemical properties. (1) Action and substrate specificity: NAD^+ and NAD
Without using P^+ as a coenzyme, in the presence of an electron acceptor, at least ethyl alcohol, butyl alcohol, amyl alcohol, diethylene glycol, triethylene glycol, and polyethylene glycol are oxidized to produce the corresponding aldehyde. (2) Electron acceptor specificity: Ferricyanide, phenazine methosulfate, dichlorophenolindophenol, and nitrotetrazolium blue can be used as electron acceptors. (3) Optimum pH: 7-9 (4) Stable pH range: 7-10 (5) Molecular weight: 60,000 (SDS-polyacrylamide gel electrophoresis and gel filtration method) (6) Enzyme absorption spectrum: 280, 416, 522
, has a specific absorption band at 553 nm.