JPS61268178A - Fructosylamino acid oxidase and production thereof - Google Patents

Abstract

PURPOSE:To obtain the titled enzyme useful as a reagent for determination of an Amadori compound, by culturing a microbial strain belonging to Corynebacterium genus and capable of producing fructosylamino acid oxidase in a medium and separating the enzyme from the culture product. CONSTITUTION:A microbial strain belonging to Corynebacterium genus and capable of producing fructosylamino acid oxidase (e.g. Corynebacterium sp NO.2-3-1) is cultured in a medium, and the objective fructosylamino acid oxidase is collected from the culture product. The enzyme catalyzes the enzymatic reaction to oxidize iminodiacetic acid (derivative) to form glyoxylic acid or alpha-ketoaldehyde, alpha-amino acid and hydrogen peroxide. The optimum pH is 8-8.5 in the case of using fructosylglycine as a substrate. The stable pH range is 8-10 and the optimum temperature range is 35-45 deg.C. It loses >=90% of the activity by heating at 45 deg.C for 10min. The molecular weight of the enzyme is about 65,000.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel enzyme fructosyl amino acid oxidase and a method for producing the same.

Fructosyl amino acid oxidase is an enzyme that catalyzes the reaction of oxidizing iminodiacetic acid and its derivatives to produce glyoxylic acid or α-ketoaldehyde, α-amino acid, and hydrogen peroxide. Reducing sugars in food and in living organisms,
In particular, when a sugar having an aldehyde group called aldose coexists with a substance having an amine group such as a protein, peptide, or amino acid, the two bond irreversibly to form a ketoamine compound.

This compound is called an amatoli compound because it is produced as a result of amatri rearrangement of a bond between an aldehyde group and an amine group. For example, fructosylalanine of the following formula A is produced from glucose and alanine. Furthermore, hydroxyacetonylglycine of the following formula B is produced from glyceraldehyde and glycine.

〒H3 CR2-C-CH2-NE(-CH,-C0OH(B)
OHO Compounds in which an aldose and an α-amino acid combine to cause an amatoli rearrangement in this way commonly contain the basic skeleton of iminodiacetic acid in their molecules, and are oxidatively decomposed by fructosyl amino acid oxidase to form α- Produces ketoaldehyde, alpha-amino acid and hydrogen peroxide. On the other hand, amatri compounds are chemically and irreversibly produced and accumulated from the moment a substance having an aldehyde group and a substance having an amino group come into contact. It is represented by a function.Therefore,
By measuring the accumulated amount, past concentrations of sugar and amine compounds, contact time, holding temperature, etc. can be estimated. However, its quantification is relatively difficult, and the accuracy is inevitably reduced due to decomposition during processing.

For example, the following method is known as a conventional quantitative method. Method using an amino acid analyzer (Journal Agricultural Food Chemistry, Vol. 24, No. 1 (19
76) see page 70), a method in which the amatoli compound is reduced with sodium borohydride, then decomposed with hydrochloric acid and separated by column chromatography (Achieves Op Biochemistry and Biophysics (1977))
181, pp. 542-549), a method for colorimetric determination of 5-hydroxymethyl-2-furfuraldehyde produced by heating an amatri compound with a weak acid using thiobarbic acid (FEBS Letter (1976) 7f, 356-
(See page 360) etc. However, these methods were not satisfactory in terms of ease of operation and accuracy.

The present inventors conducted a wide search in nature for microorganisms that degrade fructosylclycine, which is a compound of Amateurs, and found that a culture of bacteria belonging to the genus Corynebacterium, which was newly isolated from soil, oxidized fructosylclycine. The present invention was completed by discovering an enzyme that decomposes glucosone to produce glucosone, glycine, and hydrogen peroxide.

The present invention is a fructosyl amino acid oxidase having the following physicochemical properties. The present invention also provides a method for producing fructosyl amino acid oxidase, which comprises culturing a microorganism belonging to the genus Corynebacterium that produces this fructosyl amino acid oxidase in a medium, and collecting fructosyl amino acid oxidase from the culture. .

The physicochemical properties of the enzyme (fructosyl amino acid oxidase) of the present invention are as follows.

(1) Action and substrate specificity: Catalyzes the following enzymatic reaction that oxidizes iminodiacetic acid or its derivatives in the presence of oxygen to produce glyoxylic acid or α-ketoaldehyde, α-amino acid and hydrogen peroxide. It is an enzyme that

R2 j In this formula, R1 is a group -OH, -[cH(oH))Il-
cH2oa Furthermore, this enzyme can be used for β-amino acids such as β-alanine, imino acids such as proline, methylamine,
It does not act on amatri compounds such as ethanolamine. It also does not act on reduced ketones such as gluditolylglycine.

(2) Optimal pH: The optimal pH of this enzyme is <pH 8.0 to 8.5 as shown in FIG. 1 when fructosylclycine is used as a substrate. The measurement was performed by measuring the oxygen absorption rate with an oxygen monitor.

The buffer solutions used in the figure are as follows.

0-Q: 0.1M potassium phosphate buffer x-x: 0.1
M veronal-hydrochloric acid buffer Δ-Δ: 0.1 M glycine-NaOH buffer (3) pH stability: 0.2 ml of various buffer solutions containing 0.1 unit of this enzyme was heated at 40°C for 10 minutes. , the remaining enzyme activity was investigated.

The results are shown in FIG. The buffer solutions used in the figure are as follows.

○-0: 0.1M potassium phosphate buffer Δ-△: 0.
1M sodium phosphate-0.1M sodium carbonate buffer .05% 4-aminoantipyrine and 0.015%2
．． 0 containing 4-dichlorophenol sulfonate,
Transfer 2.8 ml of 1M phosphate buffer (pH 8,0) to a test tube and add 4[]OU/ml peroxidase solution 10
Add μ2. After reaching temperature equilibrium at 37°C, 1rnl of enzyme solution Q with appropriate activity was added, and 0.1ml of 0.5M fructosylclycine was added and reacted for 10 minutes. 5 using a colorimeter
Measure the absorbance at 10 nm. Separately, prepare a graph in advance using a standard solution of hydrogen peroxide to examine its relationship with the amount of dye produced. Using this graph, 6
Calculate the micromoles of hydrogen peroxide produced per minute at 7°C, and use this number as the unit of activity in the enzyme solution used.

Method 2: Method for measuring the amount of oxygen absorbed during enzymatic reaction 2.9ml of 0.1M phosphate buffer (pH 8,0)
0°5 in a certain container of Oxycene Monitor made by SI
Add 0.1 rnl of M fructosylclycine and heat at 67°C.
Stir for 10 minutes to allow dissolved oxygen and temperature to reach equilibrium. After inserting an oxygen electrode into the tube and sealing it, 50 μl of the enzyme solution is injected, and the resulting oxygen absorption is continuously measured with a recorder connected to a monitor, and its initial rate is measured. In the same way, a standard curve is created between the oxygen concentration in the container and the recorded values, and this is used to determine the oxygen concentration from the measured values. One unit is the activity of an enzyme that absorbs 1 micromole of oxygen per minute at 67°C.

(5) Range of suitable temperature for action: Glycine produced by enzymatic reaction in 0.1M phosphate buffer (pH 8,0) using fructosylclycine as a substrate was measured by separating and quantifying it by liquid chromatography. The results are shown in Figure 2, and the optimal temperature range for this enzyme's action is 35 to 45°C.

(6) Thermostability: Enzyme solution Q containing 0.1 unit of purified enzyme, 5 ml (
0.1 M phosphate buffer, pH 8.0) was left at each temperature for 10 minutes, and the remaining enzyme activity was examined.

The results are shown in Figure 6, which shows that it is stable at temperatures below 35°C, but 90% is inactivated at 45°C.

(Ha) Inhibition, activation and stabilization: This was investigated by measuring oxygen absorption in 0.1M Tris-HCl buffer (pH 8,0).The effects of each substance at a concentration of 2mM on this enzyme are as follows: That's right.)
(g, Pb, SDS strongly inhibited, N1,
Zn moderately inhibits. Various chelators and SH
The reagent gave only weak inhibition. Furthermore, the activator and stabilizer for this enzyme are unknown.

(8) Purification method: This enzyme can be purified by the purification method described below. (9) Molecular weight: The molecular weight of this enzyme was measured by column gel filtration using Sephadex G-200. Contains 0.1M salt 0
．． 05 M phosphate buffer was 65,000.

(10) Isoelectric point: As a result of measurement by disk focus electrophoresis, PI =
It was 4.6.

(11) Disk electrophoresis: Electrophoresis was performed at 5° C. for 80 minutes at 5 mA/gel using Davis pH 9.4 gel, and the enzyme protein was stained with Coomassie brilliant blue G-250.

As a result, when the acrylamide concentration of the gel was 15%, it was 4.1 crrt on the anode side (4.1 crrt for bromophenol blue).
5 cm), and 1°7α on the anode side at 15%.
A single band with enzyme activity was observed at .

As mentioned above, this enzyme is a novel enzyme that has not been previously known in terms of its action and substrate specificity.

Next, a method for producing fructosyl amino acid oxidase according to the present invention will be explained. The microorganism used in the present invention belongs to the genus Corynebacterium and any microorganism may be used as long as it has the ability to produce fructosyl amino acid oxidase, but a specific example is Corynebacterium sp.
2-5-1, and variants or mutant strains of this bacterium can also be used.

Corynebacterium sp. A2-3-1 is a strain newly isolated from soil by the present inventors, and its mycological properties are as follows.

(a) Morphology: Microscopic observation (broth agar medium 30°C, 1~
Observation for 3 days) (1) Cell size: 0.3X O, 9-0.3X
1.0 micron bacilli (2) Cell pleomorphism: Slightly curved morphology;
No mycelial growth or branching is observed.

(3) Motility: Not observed.

(4) Presence or absence of spores: Not observed.

(5) Durham vermilion: Positive (6) Acid-fast: Negative (b) Growth status in each medium (1) Broth agar plate culture = 30°C, cultured for 48 hours in a circle with a diameter of 1.5 mm) Produces colonies with a smooth surface and a glossy surface, translucent and pale yellowish in color. It does not produce a diffusible pigment that becomes opaque over time in culture.

(2) Broth agar slant culture - Growth was good and same as (1).

(3) Meat juice liquid medium: In static culture, growth is poor and only slight turbidity and bacterial precipitation are observed, but when shaken, it grows uniformly and well.

(4) Meat juice gelatin puncture culture: Slight bacterial growth is observed at 25°C for 3 days, but no dissolution is observed.

On the sixth day, only the area around the bacteria becomes slightly liquefied.

(5) Lidomus milk: It turns purple and becomes peptonized without coagulating when cultured for a long time.

(C) Physiological properties (1) Nitrate reduction: negative (2) Denitrification reaction: negative (3) MR test: negative (4) VP test: negative (5) Indole production: negative (6) Hydrogen sulfide Formation: Two weak positives (7) Hydrolysis of starch: Negative (8) Utilization of citric acid: Positive for both Koser and Kristen Seven (9) Inorganic nitrogen, source: Utilizes both NH and NO3-.

(10) Production of pigment: Create a pale yellow pigment.

(11) Urease: Positive (12) Oxidase: Negative (e) Catalase: Positive (14) Range of growth Sericulture temperature: 10-69℃tt
pH: 4.2-10.0 (15) Attitude towards oxygen: Aerobic (16) O-F test: Very weak oxidative (17) Generation of acid and gas from sugar Acid Gas (1) L-arabinose - -(2) D-xylose -- (6) D-glucose -- (4) D-mannose -- (5) D-fructose -- (6) D-galactose -- (7
-- (8) Lactose -- (9) Lactose -- (10) ) Levalose -- (11) D-Nruvit -- (f2) D-Mannit -- (16) Inosit -- (14) Glycerin -- (Former starch powder -- No other cellulose decomposition ability is recognized.) The taxonomic position of Corynebacterium sp. A2-3-1, which has the above-mentioned mycological properties, As a result of comparison with the classification in the 8th edition of ``Manual Optical Determinative Bacteriology'' (1974), this strain was found to be a Gram-positive aerobic non-spore bacillus.
Corynebacterium is catalase positive, has no motility, slightly decomposes gelatin and casein, does not generate acid from sugar, does not exhibit extreme cell pleomorphism during its life cycle, and does not decompose cellulose. It is determined that it belongs to the genus Umbrella. Furthermore, the isolation source of this strain is not derived from animal matter, it dissolves gelatin, it produces urease, and it grows at 37°C.
Although it is recognized as a strain closely related to erium fascians), this strain was isolated from soil, and is different in that it is not a plant pathogen and does not require a growth factor and grows well in a normal medium. This strain was determined to be a new bacterial species belonging to the genus Corynebacterium, and was named Corynebacterium sp. A4 S 1. In addition, Corynebacterium sp. A2-31 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, as FERM P-a 245.

Next, the culture medium used in the present invention is a carbon source.

Either a synthetic medium or a natural medium can be used as long as it contains appropriate nitrogen sources, inorganic salts, and other nutrients. Carbon sources can include, for example, glucose, fructose, xylose, and glycerin. As the nitrogen source, protein or its digestate such as peptone, casein digest, soybean flour, or nitrogenous organic matter such as yeast extract can be suitably used. As the inorganic substance, salts of sodium, potassium, calcium, manganese, magnesium, iron, cobalt, etc. can be used.

In the present invention, fructosyl amino acid oxidase can be obtained in the highest yield when cultured in a medium containing fructosyl amino acids.

A suitable example of the medium includes, for example, glucose 0.6%
, fructosyl glycine 5%, yeast extract 0.2%,
Polypeptone 0.2%, monopotassium hydrogen phosphate 0.2%,
Magnesium sulfate 0.05%, calcium chloride 0.01
%, 0.01% ferrous sulfate (pH 6.5). Cultivation is usually carried out at a temperature in the range of 25 to 67°C, preferably around '50°C. The pH at the start of culture is in the range of 6-8.

It is preferably around 6°5. Under these conditions, 16
If the culture is performed with shaking or deep stirring for ~24 hours, fructosyl amino acid oxidase is efficiently produced and accumulated in the culture.

If the culture time is prolonged, the bacteria will dissolve and this enzyme will exist outside the bacterial cells, but since it is normally present inside the bacterial cells, the bacteria can be collected by centrifuging or filtering the culture. It is necessary to solubilize the enzyme by disrupting the bacterial cells by suspending them in an appropriate amount of buffer. Fructosyl amino acid oxidase can be obtained by removing nucleic acids, cell wall fragments, etc. from the enzyme-containing solution thus obtained. Furthermore, this enzyme can be purified according to conventional enzyme isolation and purification methods as necessary, such as (1) DEAE-cellulose column chromatography, (2) ammonium sulfate fractionation, (3) phenyl-Sepharose column chromatography, (4) Sephadex G- Purified enzymes can be obtained by using methods such as 200 column chromatography or other methods in combination as necessary. A specific example of purification of this enzyme is as follows.

After collecting the bacterial cells from the culture, they were suspended in 0.02 M phosphate buffer pE (7.5, and diluted with 10% glycerin and 1
After adding and dissolving % of Triton The supernatant was collected by centrifugation, and the DEAE-cellulose column (0.02M phosphorus rl
The enzyme is adsorbed over a buffer W (equilibrated to pH 15). 0.02M containing 0.25M salt
After washing with phosphate buffer pH 7.5, the enzyme is eluted at a concentration of 0.5M sodium chloride. Collect active fractions, 16%
Add ammonium sulfate powder so that This is passed through a phenyl Sepharose column equilibrated with 0.1 M phosphate buffer pH 7.5 containing 16% ammonium sulfate to adsorb the enzyme. The enzyme was bathed in ammonium sulfate and 0.1M phosphate buffer, and its active portion was subjected to Sephadex G-2000 column chromatography equilibrated with phosphate buffer pH 7.5 containing 0.1M sodium chloride. Purified enzyme can be obtained.

According to the present invention, a new, highly specific and simple quantitative method called an enzyme method becomes possible for quantifying Amateri compounds, which have been difficult to quantify in the past.

Example Corynebacterium sp. AA2-5-1 (FIR
P-8245), fructosylclycine 0.5%, yeast extract 0.2%, polypeptone [1.2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%
, a medium containing 0.01% calcium chloride, 0.01% ferrous sulfate, and 0.3% glucose (pH 6.5)
The cells were inoculated into a Sakaro flask (5 [10 ml volume) containing IDOml and cultured with shaking at 30°C for 18 hours. This seed culture was grown in the same medium in 301 jar fermenters.
1, and cultured at 60° C. for 20 hours at an aeration rate of 201 liters and a stirring speed of 350 rpm. Cultures at 1200 O
The cells were collected by centrifugation at rpm. A part of the cultured bacterial cells (1009) was added with 0.02 M) Lis-HCl buffer (1
Contains 0% glycerin and 1% Triton X-100)
80ml of pH 7, 5, 80ml was added to disperse the bacteria well, and the mixture was cooled to 4°C with ice. Regarding this liquid, the bacterial cells are crushed using Dyno Mill! (3000 rpm for 7 minutes).

The crushing container was sufficiently cooled with ice-cold water. 12 pieces of crushing liquid
Centrifuge at 00 rpm for 15 minutes, collect the supernatant, and
30 g of liquid was obtained. This solution was applied to a column (diameter 2.5 crn x length 30 m) packed with DEAE cellulose equilibrated with a 0.01 M hydrochloric acid buffer solution pH 7.5 to adsorb the enzyme. The active portion was collected by elution using a 0→0.5M salt concentration gradient. Next, after dissolving ammonium sulfate in this enzyme solution at a ratio of 16+9/100ml,
The enzyme was adsorbed by passing through a column (diameter 1−× length 9 crn) filled with phenyl Sepharose equilibrated in advance with 0.01 M Tris-HCl buffer (pH 7.5) containing 16% ammonium sulfate.

This is combined with the reverse concentration gradient of ammonium sulfate concentration of 16% to 0% and the concentration gradient of ethylene glycol concentration of 0% to 25% (0, 01 M)! Elution was performed with J Soot-acid buffer. The active part was filtered using an ultrafiltration device manufactured by Amicon (Fraction Membrane 1000).
0) and then a column packed with Sephadex G-200 (1, 2X 100 an ) (0.05 M containing 0.1 M salt in advance)! Jssss
Equilibrated with an acid buffer pH 7.5) and subjected to gel filtration. As a result of collecting the active parts, 2.28 units/unit of enzyme was obtained.

The yield was 23%.

[Brief explanation of drawings]

Figure 1 is a graph showing the optimum pH of this enzyme, Figure 2 is a graph showing the optimal temperature range for this enzyme to function, Figure 3 is a graph showing the thermostability of this enzyme, and Figure 4 is a graph showing pH stability. This is a graph showing.

Claims (1)

[Claims] 1. Fructosyl amino acid oxidase having the following physicochemical properties. (a) Action and substrate specificity: In the presence of oxygen, imino2
Glyoxylic acid or α is obtained by oxidizing acetic acid or its derivatives.
- Catalyzes enzymatic reactions that produce ketoaldehydes, α-amino acids and hydrogen peroxide. (b) Optimal pH and stable pH range: The optimal pH is pH 8.0 to 8.5 when fructosylclycine is used as a substrate.
(phosphate buffer), stable pH range is 8.0-10.00 (c) Suitable temperature range for action: 35-45°C. (d) Thermal stability: Stable when heated at 35°C for 10 minutes, but 90% or more inactivated by heating at 45°C for 10 minutes. (e) Molecular weight: The value measured by column gel filtration method is approximately 65
It is 000. 2. A method for producing fructosyl amino acid oxidase, which comprises culturing a microorganism belonging to the genus Corynebacterium that produces fructosyl amino acid oxidase in a medium, and collecting fructosyl amino acid oxidase from the culture.