JPH01108979A - Aldehyde dehydrogenase - Google Patents

Abstract

NEW MATERIAL:To titled enzyme with a molecular weight of 20,000 having the following physicochemical characteristics: (1) action and substrate specificities, (2) capable of producing the corresponding carboxylic acids by oxidation of dialdehydes, aldehyde acids, glyceraldehyde, alpha-substituted acetaldehydes in the presence of an electron acceptor without NAD<+> and NADp<+> as the coenzymes, (3) electron acceptor specificity, (4) ferricyanide, phenazine methsulfate and dichlorophenolindophenol can be used as electron acceptors, (5) optimum pH: 6-7, (6) stable pH range: 5-7, (7) absorption spectrum; having a specific absorption band at 243nm. USE:Suitable as a conjugate enzyme to be incorporated into diagnostic drug kits for enzyme immunoassay by the sandwich method. PREPARATION:Flavobacterium Sp-Et-7 (FERM P9584) is subjected to culture on a medium and the objective enzyme can be isolated and purified from the resulting cell fragments.

Description

[Detailed Description of the Invention] [Industrial Application Field] The aA of the present invention is suitable as a conjugate enzyme for incorporating 1) K in a diagnostic agent for enzyme immunoassay using the Sandermansch method, which is widely used in the field of clinical testing, for example. This invention relates to 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. 7
ericyanide, 7enazinmethtk 7a)
(PMS), -/Chlorophenol indophenol (DCPIF) etc. as electron acceptors include:
A report by Setari et al. (0, Mudachi et al., Agr.
ic, Biol.

Chem, 44, 503 (1980))
Cytochrome Kanakura enzyme has been obtained from the membrane fraction of acetic acid bacteria. This enzyme requires surfactants for stabilization, which often poses an obstacle to its industrial use.

[Problem that the invention seeks to solve]

In the enzyme immunoassay using the Sand-Deutsch method, as with other analytical methods, there is a need to increase the sensitivity for detecting the outer layer, 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 new enzymes suitable for use in enzyme immunoassay, the present inventors isolated 7 Flavobacterium from soil.
The present invention was completed based on the discovery that a strain of M. r fun produces a constitutively new aldehyde dehydration compound 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 PK to be isolated and purified from bacterial cells, solubilization treatment with surfactants, etc., and it can be easily prepared as a single standard using conventional methods. Product K
It can be split up to t#.

This purified enzyme does not contain cytochrome C as a prosthetic group, which is different from the enzyme reported by Seiri et al., and it converts the aldehyde into the corresponding carboxylic acid only in the presence of a suitable redox dye as an electron acceptor. It is an oxidizing enzyme.

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

(1) Dialdehyde, aldehydic acid, glyceraldehyde with ferricyanide, PMS, DCPIP, etc. as electron acceptors without acting as NAD and NADP 'i coenzymes.

The alpha-substituted acetaldehyde is oxidized to the corresponding carboxylic acid.

HCOCOOHHHOCOCOOH R-CH2CHO+ R-CH2C00H (2) Substrate specificity Measure the enzyme activity according to the measurement method described below using various aldehyde t-substrates, and determine the activity t- for glyoxylic acid.
The relative activity when set to 100 is shown below.

Glyoxal 3 Glyoxaldehyde 100 Glopionaldehyde 0 n-Butyraldehyde 0 Isobutyraldehyde O n-Valeraldehyde O n-Hexylaldehyde O n-Hegetaldehyde O n-Octaldehyde 0 DL-Glyceraldehyde 124 Glutaraldehyde 0 Enyl acetaldehyde 120 Methoxydiacetaldehyde 79 Methoxy vinegar fRO Ethyl vinegar #10 Diglycolic acid 0 Oxygen MO Glyceric acid 0 Methanol 0 Ethanol 0 Polyethylene glycol (average molecular weight 400)
0 (♀ro 8 children 4000) 0 (Gakushirei & 120 (ro)) 0 (3) Electron acceptor NAD + Oyohi NADP + t-m receptor Toses, 7 Elysianide, PMS, DCPIP, etc. redox pigments as electron acceptors shall be. 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.

Electron acceptor Relative activity (%) 7 Elicyanide 101 00P -NTB 32NTB
OPMS-DCPIP
15DCPIP
15NADP” 0NADP
0(4) Optimum and stable voice range The optimal range of this enzyme is at pH 6 to 7 as shown in FIG. 1.

As shown in FIG. 2, this enzyme is stable from around neutral to acidic, especially at pH 5 to 7. Jukai relatives
The cells were left in each pH 6i buffer at 5°C for 24 hours, and the residual activity was then measured. In the figure, Δ indicates the results measured using an acetate buffer, 0 a phosphate buffer, and ○ a carbonate buffer.

(5) iI of action! Range of straining The action temperature range of the enzyme of the present invention is 0°C to 50°C, and 2
It has an optimum temperature between 5 and 37°C.

(6) pH temperature and inactivation When treated in 10mM phosphate buffer (47,0) at each temperature for 30 minutes, there was no inactivation up to 25°C, and 45% at 30°C.
, 90% inactivation at 37°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.

Additive Concentration (mM) Relative activity (%
) Additive-free 1100 Na” 190K”
1 132Mg”
1 81Ba”
10Co”
1 0Mn” 1
0Ca" 1
128Cu”l
OZn” 1 0
Sn" 1 0ig"
1 70Hg”
l' 0 Ethylenediaminetetravinegar @ 10 23 Monoiodo Vinegar #R180 8-Hydroxy Nakano-Phosphorus 10o-7 Enanthroline 10 (8) Molecular weight filtration and 5DS-polyacrylamide gel -
The molecular weight determined by electrophoresis is 20,000.
-(9) Absorption spectrum purified enzyme exhibits specific absorption at 243 nm
(Figure 3).

Method for Measuring α0 Enzyme Activity Although it is possible to measure the activity of the enzyme of the present invention using any of the electron acceptors described above, the case where the potassium ferricyanide all-Kyoko acceptor is used will be described below.

0.1 M 7 potassium elycyanide solution 0.1 d
.

lv/d biloloquinolone thousandone solution 0.02117,10
Add 0mM Tris-HCl buffer (pH 7, Q), 5iu, and an appropriate amount of the enzyme of the present invention, and adjust to 0.7117 with water. After adding another 0.5 d to stop the reaction, water is added to give 5.0 icg. After 10 minutes, measure the absorbance at 660 nm by t-meter ◎Meanwhile, instead of the potassium spherocyanide solution, use a potassium 7-merocyanide solution of each degree, and use the same procedure as above to obtain the aldehyde concentration based on the calibration curve. Determine the concentration of potassium fecocyanide produced by the oxidation of 1/Jmol·acidehyde'k.
Let i oxidize in 1 minute be 1 unit.

From the above physical and chemical properties, the enzyme of the present invention is different from conventional enzymes! It is completely different from the known aldehyde dehydrogenases and is recognized as a novel element.

The enzyme of the present invention was obtained by the inventor from soil using 9'rf
It can be produced and obtained by culturing the Hosori Et-7 strain isolated in c in a gold nutrient medium.

The mycological properties of It-7 strain are shown below.

1. Morphology (broth agar medium, 28°C, 24 hour culture) (
1) Cell size; 0.4-0.5 μm (2) Motility;
None 3) Gram staining: Negative (4) Acid-fast: Negative (5) Presence of spores: Not formed 2. Growth status (1) Broth agar plate culture: Colonies are raised, soft, and milky white.

(2) Meat juice agar slant culture: Grows well in a linear pattern, grows well in condensed water, and exhibits a milky white color.

(3) Meat juice liquid culture: --like suspension, and no bacterial film is formed 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; does not coagulate or (pigment).

3. Physiological properties (υ Nitrate reduction; negative (23 MR test; negative (3 JVP test; negative (4) Indole formation; negative (5) Hydrogen sulfide formation; negative (6) Starch hydrolysis; negative (7) ) Hydrolysis of casein; positive (8) Utilization of citric acid; positive (9) Flaase; negative αQ Medium sidase; positive αη Catalase; positive (6) Growth -; p) 15-10. )
Growth temperature: 10-37°C, 620-30°C α4 0F test: Oxidative - (9) Production of acid and gas from sugar L-arabinose, D-xylose, D-glucose,
D-mannose, D-7 lactose, D-galactose, maltose, sica sugar, lactose, D-sorbitol, inodite, glycerin, and starch.No formation of sialic acid or gas was observed.

The above characteristics *'t Bergy's Manual Otsu Systematic Bacteriology (Berg@y*@M
annual of Systematlc Bact
When compared with the classification in Volume 1 (1984), this bacterium was determined to belong to the genus Flavobacterium and was classified as Flavobacterium sp.
avobact@r1um sp, Et-7).

This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Fiber Technology Research Institute No. 9584 (FERM P-9584).

In order to produce the enzyme of the present invention naked, the present bacterium may be cultured according to the conventional #accumulative production method.

Since the enzyme of the present invention is produced constitutively, there is no need to specifically add aldehydes, which are substrates for enzyme induction, to the medium used, and methoxyacetic acid, ethyl alcohol, etc. are used as carbon sources. Nitrogen sources include various ammonium salts, urea, etc., inorganic salts include table salt, phosphates, magnesium salts, etc., and organic nutrients such as yeast extract, meat extract, and polybegtone are used. Used as appropriate.

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 30°C, preferably around 28°C. The pH during culturing should be in the range of pH 6 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 a method commonly used for enzymes, such as a method using a lytic enzyme such as lysozyme, a method using pressurization, a method using pressure, and a method using autolysis. Furthermore, in order to obtain a purified enzyme preparation, a purified enzyme can be obtained by combining salting-out method, r-filtration method, and adsorption/elution method using ion exchanger, hydroxyano9tite, hydrophobic, affinity chromatography, etc. I can do it.

〔Example〕

Methoxyacetic acid 0.5%, yeast extract 0.3%, meat extract 0.3%, polybegtone 0.2%, ammonium chloride 0.2%, dipotassium phosphate 0.1%, sodium chloride 0.05%, A medium (p) 17.0) 6 (l) consisting of 9 μm of sulfuric acid 0.05% was placed in a 901 volume jar fermentor and sterilized at 120° C. for 20 minutes.

After cooling, add 1.0% glucose, 1.0% polybegtone, and 0.5% meat extract to a 500-ml Erlenmeyer flask.
, Sodium chloride 0.3%, Yeast extract 0.25
%, dipotassium phosphate 0°031. Magne sulfate column 0
．． A culture medium (pH 7.0) consisting of 0.05 ml was inoculated with the Furanobacterium sp. Seed culture solution 1j
was inoculated. The cells were cultured for 24 hours under constant conditions of 28° C., 300 rPm, and 0.5 vvm to obtain 1 kg of bacterial cells (heavy pack of wet bacteria).

The bacterial cells obtained in this way were added to 10mM phosphate buffer (pH
7,0) After suspending in 3 J, the suspension was crushed using a Dyno Mill and centrifuged to obtain a cell-free extract 41. This cell-free extract was saturated to 45% with ammonium sulfate.
The enzyme was adsorbed onto an FP-OT13 column equilibrated with 100 ml of phosphoric acid, and after thorough washing, the enzyme was eluted with a 10 mM 17-phosphoric acid solution (PH7.0). Ammonium sulfate was added to the active fraction to saturation with 7096, and the resulting precipitate was collected by centrifugation and dialyzed against phosphate buffer (pH 47, O). Same M for dialysate! DEA equilibrated with liquid]i:
- It was passed through a Sepharose column, and impure proteins were adsorbed onto the column and removed. Sotsu vii 111 minutes is after shrinking,
Glu filtration was performed using a column of Sepharose CL-68 equilibrated with 10 mM phosphorus #2 buffer (-7,0). The obtained active fraction was adsorbed onto a DEAE-Sepharose column equilibrated with the same IFt solution, and after thorough washing,
The enzyme was bathed in the same buffer containing 0.2M sodium chloride. After condensing the elution fraction, 10mM phosphoric acid grade solution (P'1
7. Glue filtration was performed using a Sephacryl S-200 column equilibrated with O).

With the above operations, the specific activity is 9 units/10 Da protein shrine products.
Obtained 0 credits.

〔Effect of the invention〕

The novel enzyme of the present invention can be easily produced by culturing Furanobacterium sp.
ArI! By utilizing the unique properties of kites and incorporating the enzyme of the present invention as a conjugate enzyme in #prime immunoassay, it becomes possible to perform measurements with high sensitivity that could not be achieved conventionally.

[Brief explanation of the drawing]

Figure 1 shows a curve showing the optimum level of the #element of the present invention, Figure 2 shows a curve showing monostability, and Figure 3 shows the absorption spectrum of the purified enzyme. be. Patents Fujirebio Co., Ltd. Figure 3 Wavelength (nm) Figure 1 pH Figure 2 H

Claims (1)

[Claims] An aldehyde dehydrogenase having the following physical and chemical properties. Action and substrate specificity: NAD^+ and NADP^+
It oxidizes dialdehyde, aldehyde acid, glyceraldehyde, and α-substituted acetaldehyde to produce the corresponding carboxylic acid in the presence of an electron acceptor without using as a coenzyme. Electron acceptor specificity: Ferricyanide, phenazine methosulfate, dichlorophenolindophenol can be used as electron acceptors. Optimum pH: 6-7 Stable pH range: 5-7 Molecular weight: 20,000 (SDS-polyacrylamide gel electrophoresis and gel filtration method) Enzyme absorption spectrum: Has a specific absorption band at 243 nm.