JPS63230081A - Aldehyde dehydrogenase - Google Patents

Abstract

NEW MATERIAL:The titled enzyme having 35,000 molecular weight, specific absorption zone at 280nm and the following physical and chemical properties. Action and substrate specificity: not requiring NAD<+> and NADP<+> as coenzyms, oxidizing at least an aliphatic aldehyde and polyethylene glycol-alpha,omega-dialdehyde in the presence of an electron acceptor to form corresponding carboxylic acids. Electron acceptor specificity: capable of using ferricyanide, phenazine me thosulfate and dichlorophenolindophenol as an electron acceptor. Optimum 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 nutrient medium, the prepared cell is ground to give a solution, from which the aimed substance is isolated.

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 aldehyde dehydrogenase.

[Conventional technology]

Various aldehyde dehydrogenases are already known, and aldehyde dehydrogenases that use NAD" and NADP" as coenzymes are widely found in animals and microorganisms. Ferricyanide, phenazine methosulfate (PMS), dichlorophenolindophenol (DCP IP), etc. are reported by Adacht et al.
ric, R4a1. Cheaa,, 44.503
(1980)), in which cytochrome-containing enzymes have been obtained from the membrane fraction of Acetobacter spp. This enzyme requires surfactants for stabilization, which often poses an obstacle to industrial use of the enzyme.

[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 Problem C] 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. rium constitutively produces a novel aldehyde dehydrogenase without the addition of an inducing substrate.

Since the enzyme of the present invention is present 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 it can be easily made into a single standard using conventional methods. It can be refined into products.

This purified enzyme differs from the enzyme reported by Sadatsu et al. in that it does not contain cytochrome C as a prosthetic group, and is an enzyme that oxidizes aldehydes to the corresponding carboxylic acids only in the presence of an appropriate redox dye as an electron acceptor. It is.

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

(11 actions) Various aliphatic aldehydes, polyethylene glycol-α, etc. are produced using ferricyanide, PMS, DCPIP, etc. as electron acceptors, without using NAD and NADP as coenzymes.
, oxidizes the ω-dialdehyde to the corresponding carboxylic acid.

C1h(C1l□)sclIQ→CH3(CH2) 5
cOOII.

0) ICCHz-(0-CHz CL:h-OC)l
zctlO↓ H00CCI□-(0-CH2-CH2 Agency0-CIl□
C00H (2) Substrate specificity The enzyme activity was measured using various aldehydes as substrates according to the measurement method described below, and the activity against heptylaldehyde was 1
The relative activity when set to 00 is shown below.

Substrate Relative activity (%) Formaldehyde 9 Acetaldehyde
37 Propionyl aldehyde
55n-butyraldehyde 59n
-Valeraldehyde 61n-hexylaldehyde 98n-heptylaldehyde 100 ethyl alcohol
O Butyl alcohol 0 (
3) Electron acceptors NAD″″ and NADP″″ are not used as electron acceptors, but redox dyes such as ferricyanide, PMS, and DCPIP 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 100PMS100P
47NTB
OPMS-DCPIP
100DCPIP
100NAD” 0
NADP”
0(4) Optimum pH and stable p
H range The optimum pH of this enzyme is pH 7 to 9 as shown in Figure 1.
It is in. In the figure, △ indicates the results of measurement using an acetate buffer, . indicates a phosphate buffer, and O indicates a result of measurement using a carbonate buffer.

As shown in Figure 2, this enzyme is stable from near neutral to alkaline, particularly at pH 6 to 10.5. The buffer solution used for the measurement was the same as that shown in FIG. 1, and the residual activity was measured after standing at 5° C. for 24 hours in the buffer solution at each pH.

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

(6) pH temperature and inactivation In 10mM phosphate buffer (pH 7,0), 10% at each temperature.
If treated for 30 minutes, it will not be inactivated up to 30℃, and at 50℃ it will not be inactivated.
0%, 70% deactivated at 60°C.

(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.

FIG” 1 7
8Ba" 1
ONa' 1
62Co2・10
Mn”・+ to 10
1 120Ca”
1 3 1 5Cu
2° 10Zn”
10Sn”・
10Ag2÷
1 9311g! +1
0 ethylenediaminetetraacetic acid 10 0 δ-hydroxyquinoline 10 0-phenanthroline 10 (8) Molecular weight The molecular weight determined by gel electrophoresis and 5DS-polyacrylamide gel electrophoresis is 35.000.

(9) Absorption Spectrum Purification The enzyme exhibits specific absorption at 280 nm (Figure 3). The measurement was carried out using a purified enzyme solution of 4.7 μ/ml.

α0) Method for Measuring Enzyme Activity 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 0.1mJ, 1n
+g/mJ pyrroloquinoquinone solution 0.02n+1.1
1001II Tris-HCl buffer (pH 8,0) 0.5
cal.

Add an appropriate amount of the enzyme of the present invention, and make up to 0.9 ml with water. Furthermore, add 0.1 mL of aldehyde solution and heat at 37℃ for 5 minutes.
Allow to react for ~30 minutes. Perr″1cD was added to this reaction solution.
After adding 0.5 mβ of Upanul reagent to stop the reaction, add water to make 5.0 ml. 660nm after 10 minutes
Measure the absorbance of On the other hand, using potassium ferrocyanide solutions of various concentrations instead of potassium ferricyanide solution, the concentration of potassium ferrocyanide produced by conjugation with the oxidation of aldehyde was determined based on the calibration curve obtained by the same procedure as described above, and 1 μmole of aldehyde was added to 1 μmole of aldehyde. The amount oxidized per minute is defined as 1 unit.

From the above physicochemical properties, the enzyme of the present invention is completely different from conventionally known alcohol dehydrogenases.
Recognized as a new enzyme.

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℃, 24 hours culture) (υ
Cell size: 0.7-1.0 μm (2) Motility: None 3) Gram staining: Negative (4) Acid-fastness: Negative (5) Presence or absence of spores: Not formed 2, Growth status (1 ) Broth agar plate culture; 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 Decomposition; negative (7) Hydrolysis of casein; positive (8) Utilization of citric acid; positive (9) Urease; positive 0ω Oxidase; positive 0υ Catalase; positive Q2 Growth pH, pH 5-10. Optimum pH 6-80■
Growth temperature: 1o~35℃, optimal 25~30''cQ4
) OF test: Oxidative Cl5) Production of acids and gases from yB L-arabinose, D-xylose, D-glucose, D-mannose, D-fructose, D-galactose, maltose,
No acid or gas formation is observed from sucrose, lactose, D-sorbitol, inosit, glycerin and starch.

The above properties are summarized in Bergey's Manual of Systematic Bacteriology (Bergey's Manual of Systematic Bacteriology).
annual of Systematic Ilact
When compared with the classification in Volume 1 (1984) of Flavobacterium sp.
avobacterium sp, PE-10
) 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 aldehydes, which are substrates, for enzyme induction, and glucose, fructose, and sucrose are used as carbon sources. , 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, in order to obtain a purified enzyme preparation, a purified enzyme can be obtained by combining a salting-out method, a gel filtration method, an adsorption/elution method using an ion exchanger, hydroxyapatite, hydrophobic, affinity chromatography, etc.

〔Example〕

Glycerol 0.5%, yeast extract 0.3%, meat extract 0.2%, peptone 0.2%, dipotassium phosphate 0.1
5%, salt O, OS%, magnesium sulfate 0.01%,
Medium consisting of 0.2% ammonium nitrate (pH 7.0)
601 to 901! Put it in a jar fermenter for 120 minutes.
Sterilized at ℃ for 20 minutes. After cooling, add 1.0% glucose and 1.0% peptone to a 500m6 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) was added, and Flavobacterium sp. PE-10 was inoculated and cultured with shaking at 28°C for 20 hours. The prepared seed culture solution 11 was inoculated. 28℃, 300rpm, 0.5vv
The cells were cultured for 24 hours under constant conditions of m to obtain 1 kg of bacterial cells (wet bacterial weight).

The bacterial cells obtained in this way were added to 10mM phosphate buffer (pH
7,0) After suspending in 4J, it was crushed with Guinomil and centrifuged to obtain cell-free extract 31. This cell-free extract was fractionated by ammonium sulfate salting out.
The precipitate of the 70% saturated ammonium sulfate fraction was collected and dialyzed against 10 mM phosphate buffer (pH 7.0). The dialysate was passed through a DEAR-cellulose column equilibrated with the same buffer, and impure proteins were adsorbed onto the column and removed. The flow-through fraction was adsorbed onto hydroxyapatite, thoroughly washed with 0.1M phosphate buffer (pH 7.0), and then washed with 0.1M phosphate buffer (pH 7.0).
The enzyme was eluted at pH 7.0). An additional 70% in the active fraction
Add ammonium sulfate to % saturation, collect the resulting precipitate by centrifugation, dissolve the precipitate in a small amount of buffer,
Sephacryl S-2 equilibrated with 10mM phosphate buffer
The gel was filtered using a 00 column.

Through the above operations, 400 units of a purified sample with a specific activity of 10 units/■ protein 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 immunoassay, it becomes possible to perform measurements at a high degree of anger, which was previously impossible to achieve.

[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. . Figure 1H Relative activity (-) Absorbance

Claims (1)

[Claims] An aldehyde dehydrogenase having the following physical and chemical properties. Action and substrate specificity: NAD^+ and NADP^+
In the presence of an electron acceptor, at least an aliphatic aldehyde and polyethylene glycol-α,ω-dialdehyde are oxidized to produce the corresponding carboxylic acid without using it as a coenzyme. Electron acceptor specificity: Ferricyanide, phenazine methosulfate, dichlorophenolindophenol can be used as electron acceptors. Optimal pH: 7-9 Stable pH range: 7-10 Molecular weight: 35,000 (SDS-polyacrylamide gel electrophoresis and gel filtration) Enzyme absorption spectrum: Has a specific absorption band at 280 nm.