JPH0665300B2 - Fructosyl amino acid oxidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel enzyme fructosyl amino acid oxidase.

Fructosyl amino acid oxidase is an enzyme that catalyzes a reaction of oxidizing iminodiacetic acid and its derivative to produce glyoxy acid or α-ketoaldehyde, α-amino acid and hydrogen peroxide. In foods and living organisms, reducing sugars, especially sugars having an aldehyde group called aldose and a substance having an amino group such as protein, peptide, amino acid, etc. coexist, and the two are irreversibly bound to form a ketoamino compound. Will be generated. This compound is called an Amadori compound because it is formed as a result of the Amadori transition of the combined product of the aldehyde group and the amino group. For example, fructosyl alanine of the following formula A is produced from glucose and alanine. Further, hydroxyacetonylglycine of the following formula B is produced from glyceraldehyde and glycine.

The compound in which aldose and α-amino acid are bound to each other to cause Amadori transfer contains a basic skeleton of iminodiacetic acid in the molecule in common, and is oxidatively decomposed by fructosyl amino acid oxidase to give α-amino acid. Produces ketoaldehydes, α-amino acids and hydrogen peroxide. On the other hand, the Amadori compound is chemically and irreversibly produced and accumulated from the moment when the substance having an aldehyde group is brought into contact with the substance having an amino group. The generation rate is represented by a function of the concentration of the raw material, contact time, temperature and the like. Therefore,
By measuring the accumulated amount, past concentrations of sugar and amino compounds, contact time, holding temperature, etc. can be estimated. However, its quantification was relatively difficult, and it was unavoidable that the accuracy declined due to decomposition during processing.

The following methods are known as conventional quantification methods. Method using amino acid analyzer (Journal Agricultural Food Chemistry, Vol. 24, No. 1 (1
976) p. 70), a method of reducing an Amadori compound with sodium borohydride, decomposing with hydrochloric acid, and separating by column chromatography (Achievements of Biochemistry and Biophysics (197).
7) 181, 542-549), 5-hydroxymethyl-2 produced by heating an Amadori compound with a weak acid.
-A method for colorimetrically determining furfuraldehyde with thiobarbituric acid (FEBS Letter (1976) 71 , 356-
See page 360) etc. However, these methods have not been satisfactory in terms of operability and accuracy.

Among the Amadori compounds, the present inventors extensively searched for microorganisms degrading fructosylglycine from the natural world, and as a result, newly oxidatively degrading fructosylglycine into cultures of bacteria belonging to the genus Corynebacterium separated from soil. The present invention has been completed by finding an enzyme that produces glycosone, glycine and hydrogen peroxide.

The present invention is a fructosyl amino acid oxidase having the following physicochemical properties.

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

(1) Action and substrate specificity: The iminodiacetic acid represented by the following general formula (X) or a derivative thereof, which is a basic skeleton of an Amadori compound produced by Amadori rearrangement of α-amino acid in the presence of oxygen, It is an enzyme that catalyzes the following reaction that oxidizes to produce glyoxylic acid or α-ketoaldehyde, α-amino acid and hydrogen peroxide.

In this formula, R ₁ is a group —OH, — [CH (OH)] _n —CH ₂ OH or — (CH ₂ ) _n —CH ₃ , n is an integer from 0 to 4, and R ₂ is the α-amino acid side. Chain residues are shown.

The α-amino acid side-chain residue is a structure of α-amino acid represented by the general formula When represented as R ₂ which is a residue peculiar to each α-amino acid
Means For example, when the α-amino acid is glycine, alanine, serine, aspartic acid, glutamic acid, isoleucine, leucine, phenylalanine, tyrosine, or valine, R ₂ is -H, -CH ₃ , -CH ₂ OH, respectively.
_{-CH 2 COOH, -CH 2 CH 2} COOH, -CH (CH 3) CH 2 CH 3, -CH 2 CH
(CH ₃ ) ₂ , -CH (CH _{3) 2} and the like.

Next, an example of the results obtained by examining the action of the enzyme of the present invention on iminodiacetic acid and each iminodiacetic acid derivative is shown in the table below.

The enzyme does not act on β-amino acids such as β-alanine and the like, imino acids such as proline and the like, and Amadori compounds such as methylamine and ethanolamine. Further, it does not act on a substance obtained by reducing a ketone, such as glycitolylglycine.

(2) Optimum pH: The optimum pH of this enzyme is pH 8.0 to 8.5 as shown in Fig. 1 when fructosylglycine 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.

○-○: 0.1M potassium phosphate buffer ×-×: 0.1M veronal-hydrochloric acid buffer △-△: 0.1M glycine-NaOH buffer (3) pH stability: Various buffers containing 0.1 unit of this enzyme 0.2 ml at 40 ° C, 1
After heating for 0 minutes, the remaining enzyme activity was examined. As a result, as shown in FIG. 4, the stable pH range is 8.0 to 10.0. The buffer solutions used in the figure are as follows.

○-○: 0.1M potassium phosphate buffer △-△: 0.1M sodium phosphate-0.1M sodium carbonate buffer ×-×: 0.1M glycine-NaOH buffer (4) Method for measuring titer: Method 1 : Method for colorimetric determination of hydrogen peroxide produced 0.05% 4-aminoantipyrine and 0.015% 2,4-
2.8 ml of 0.1 M phosphate buffer (pH 8.0) containing dichlorophenol sulphonate was placed in a test tube and 400 u /
Add 10 μl of ml peroxidase solution. After reaching the temperature equilibrium of 37 ° C., 0.1 ml of an enzyme solution having an appropriate activity was added, and 0.5 M fructosylglycine- was added.
0.1 ml is added and reacted for 10 minutes, and the resulting dye is measured for absorbance at 510 nm using a photoelectric colorimeter. Separately, using a standard solution of hydrogen peroxide, prepare a graph in which the relationship with the amount of the produced dye is examined. Using this graph, the micromoles of hydrogen peroxide produced per minute at 37 ° C. are calculated, and this number is taken as the activity unit in the enzyme solution used.

Method 2: Method for measuring the amount of enzyme absorbed during the enzymatic reaction 0.1 M phosphate buffer (pH 8.0) 2.9 ml was placed in a measurement container of an Oxygen monitor manufactured by YSI and 0.5 M fructosyl was added. Add 0.1 ml of glycine and stir at 37 ° C. for 10 minutes to bring dissolved oxygen and temperature to equilibrium. After inserting an enzyme electrode into this and sealing it, 50 μl of the enzyme solution is injected, and the resulting oxygen absorption is continuously measured by a recorder connected to a monitor, and the initial speed thereof is measured. Similarly, a standard curve is prepared in advance between the oxygen concentration in the container and the recorded value, and this is used to determine the oxygen concentration from the measured value. One unit is the activity of an enzyme that absorbs 1 micromol of oxygen per minute at 37 ° C.

(5) Optimum temperature range of action: By using fructosylglycine as a substrate and separating and quantifying glycine produced by the enzymatic reaction in 0.1M phosphate buffer (pH8.0) by liquid chromatograph. It was measured. The results are shown in FIG. 2, and the optimum temperature range for the action of the enzyme is 35 to 45 ° C.

(6) Thermal stability: 0.5 ml of enzyme solution containing 0.1 unit of purified enzyme (0.1 M phosphate buffer, pH 8.0) was allowed to stand for 10 minutes at each temperature.
The remaining enzyme activity was investigated. The results are shown in Fig. 3. It is stable below 35 ° C, but 90% at 45 ° C.
Will be deactivated.

(7) Inhibitory activation and stabilization: It was investigated by measuring oxygen absorption in 0.1 M Tris-HCl buffer (pH 8.0). The effects of each substance at a concentration of 2 mM on this enzyme are as follows. Hg ⁺⁺ , Pb ⁺⁺ ,
SDS strongly inhibits, Ni ⁺⁺ and Zn ⁺⁺ moderately inhibit. Various chelators and SH reagents gave only weak inhibition. The activator and stabilizer for this enzyme are unknown.

(8) Purification method: The present enzyme can be purified by the purification method described below.

(9) Molecular weight: The molecular weight of this enzyme was measured by a column gel filtration method using Sephadex G-200, and it was found to contain 0.1 M sodium chloride at 0.0.
It was about 65,000 in 5M phosphate buffer.

(10) Isoelectric point: PI = 4.6 as measured by disk focus electrophoresis
It was.

(11) Disk electrophoresis: 3 mA / gel using Davis pH 9.4 gel, 5 ° C, 8
After 0 minute migration, the enzyme protein was stained with Coomassie Brilliant Bull-G-250. As a result, when the acrylamide concentration of the gel was 7.5%, the anode side was 4.1 cm (bromphenol blue was 4.5 cm), and when it was 15%, the anode side was also 1.
A single band having enzyme activity was observed at 7 cm.

As described above, the present enzyme is a novel enzyme that has never been known in its action and substrate specificity.

Next, a method for producing fructosyl amino acid oxidase according to the present invention will be described. The microorganism used in the present invention may be any one as long as it belongs to the genus Corynebacterium and has the ability to produce fructosyl amino acid oxidase, and as a specific example, Corynebacterium sp. 3-1 is mentioned,
Variants or mutants of the bacterium can also be used. Corynebacterium sp. NO. 2-3-1 is a strain newly isolated by the present inventors from the soil, and its mycological properties are as follows.

(a) Morphology: microscopic observation (meat agar medium 30 ° C, 1-3
(1) Cell size: 0.3 × 0.9 to 0.3 × 1.0 micron rods (2) Cell polymorphism: Slightly curved morphology, no hyphal growth or branching .

(3) Motility: Not recognized.

(4) Presence of spores: Not observed.

(5) Gram stainability: positive (6) anti-acidity: negative (b) growth state in each medium (1) broth agar plate culture: diameter after culture at 30 ° C for 48 hours
It produces round, smooth, shiny colonies with a diameter of 1.5 mm, and is semitransparent and light yellowish. It does not make diffusible pigments that become opaque over the time of culture.

(2) Meat broth agar slope culture: Good growth, same as (1).

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

(4) Meat broth gelatin stab culture: Growth at about 3 days was observed at 25 ° C, but no lysis was observed. 6
At around the day, only the area around the bacteria liquefies slightly.

(5) Litmus milk: It turns purple and becomes peptone without coagulation after long-term culture.

(c) Physiological properties (1) Reduction of nitrate: negative (2) Denitrification reaction: negative (3) MR test: negative (4) VP test: negative (5) Indole formation: negative (6) Hydrogen sulfide Production: Weakly positive (7) Hydrolysis of starch: Negative (8) Utilization of citric acid: Positive in both Coeser and Christensen (9) Inorganic nitrogen source: Utilizes both NH ₄ ⁺ and NO ₃ ⁻ .

(10) Pigment formation: producing a pale yellow pigment (11) Urease: Positive (12) Oxidase: Negative (13) Catalase: Positive (14) Growth range Temperature: 10-39 ° C pH: 4.2-10 .0 (15) Attitude toward enzyme: aerobic (16) OF test: extremely weak oxidative (17) acid and gas production from sugar acid gas (1) L-arabinose-(2) D-xylose -(3) D-glucose-(4) D-mannose-(5) D-fructose-(6) D-galactose-(7) Maltose-(8) Sucrose ---- (9 ) Lactose- (10) Trehalose- (11) D-sorbit- (12) D-mannitol- (13) Inosit- (14) Glycerin- (15) Starch --- Others unacceptable.

Regarding the taxonomic position of Corynebacterium sp. NO. 2-3-1 having the above-mentioned mycological properties, refer to "Virgin AIDS Manual of Determinating Bacteriology", 8th Edition (1974). As a result of comparison with classification, this strain is a gram-positive, aerobic aspore-less bacillus, positive for catalase, non-motile, slightly degrading gelatin and casein, not producing acid from sugar, It is judged to belong to the genus Corynebacterium because it does not decompose cellulose and does not exhibit extreme cellular polymorphism along the ring. In addition, the source of isolation of this strain is not derived from animal substances, dissolves gelatin,
Since it produces urease and grows at 37 ° C, Corynebacterium fascias (Corynebacter)
It is recognized as a strain closely related to ium fascians), but this strain is isolated from soil, is not a phytopathogenic fungus, does not require a gross factor, and is well grown in a normal medium. This strain was determined to be a new strain belonging to the genus Bacterium, and this strain was identified as Corynebacterium sp.
It was named NO.2-3-1. In addition, Corynebacterium sp. NO. 2-3-1 was sent to the Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Microbial Science and Technology, Microorganisms Research Institute No. 8245 (FERM
Deposited as P-8245).

Next, as the medium used in the present invention, a carbon source, a nitrogen source,
Synthetic medium, if it contains inorganic salts and other nutrients,
Any natural medium can be used. As a carbon source,
For example, glucose, fructose, xylose, glycerin and the like can be used. As the nitrogen source, peptone, casein digest, protein such as soybean flour or its digest, or nitrogenous organic matter such as yeast extract can be preferably used. As the inorganic substance, salts such as sodium, potassium, calcium, manganese, magnesium, iron and cobalt can be used. In the present invention, fructosyl amino acid oxidase is the best known yield when cultured in a medium containing fructosyl amino acid. Preferable examples of the medium include glucose 0.3%, fructosylglycine 0.5%, yeast extract 0.2%, polypeptone 0.2%, potassium hydrogen phosphate 0.2%, magnesium sulfate 0.2%. 05%, calcium chloride 0.01%, ferrous sulfate 0.0
A 1% (pH 6.5) medium is used. Culture is usually 25 ~
It is carried out in the range of 37 ° C, preferably around 30 ° C. The pH at the start of culturing is in the range of 6 to 8, but is preferably around 6.5. Under such conditions, if it is subjected to shaking or deep-agitation culture for 16 to 24 hours, fructosyl amino acid oxidase is efficiently produced and accumulated in the culture.

This enzyme, when the culture time is prolonged, the bacteria will be dissolved and will be present outside the cells, but since it is usually present in the cells, the culture is centrifuged or passed to collect the cells, It is necessary to solubilize the enzyme by suspending it in an appropriate amount of buffer and destroying the cells. A fructosyl amino acid oxidase can be obtained by removing nucleic acids, cell wall fragments, etc. from the thus obtained enzyme-containing liquid.
Further, the enzyme is optionally subjected to a conventional method for isolation and purification of the enzyme, for example, (1) DEAE-cellulose column chromatography, (2) ammonium sulfate fractionation, (3) phenyl sepharose column chromatography, ( 4) A purified enzyme can be obtained by using a method such as Sephadex G-200 column chromatography or a combination of other methods as necessary. A specific example of purification of this enzyme is as follows.

After collecting the cells from the culture, 0.02M phosphate buffer
The suspension is suspended in pH 7.5, 10% glycerin and 1% Triton X-100 are added and dissolved, and then the cells are crushed using Dynomill (manufactured by Shinmaru Enterprise (Sweden)). The supernatant is collected by centrifugation and applied to a DEAE-cellulose column (equilibrated in 0.02 M phosphate buffer pH 7.5) to adsorb the enzyme. After washing with 0.02 M phosphate buffer (pH 7.5) containing 0.25 M sodium chloride,
The enzyme is eluted with a 5 M sodium chloride concentration. The active fractions are collected, and ammonium sulfate powder is added to make it 16%. This 16
The enzyme is adsorbed by passage through a phenyl sepharose column equilibrated in 0.1 M phosphate buffer pH 7.5 containing% ammonium sulfate. This enzyme was eluted with 0.1M phosphate buffer, which has both a reverse concentration gradient of 16% → 0% in ammonium sulfate concentration and a concentration gradient of 0 → 25% in ethylene glycol concentration. The purified enzyme can be obtained by column chromatography of Sephadex G-200 equilibrated with phosphate buffer solution pH 7.5 containing sodium chloride.

According to the present invention, in quantifying an Amadori compound, which has been difficult to quantify in the past, a new highly specific and simple quantification method called an enzyme method becomes possible.

Example Corynebacterium sp. NO. 2-3-1 (FERM P-
8245) to 0.5% fructosyl glycine, yeast extract
0.2%, polypeptone 0.2%, potassium dihydrogen phosphate 0.2.
2%, magnesium sulfate 0.05%, calcium chloride 0.0
Sakaguchi flask (5) containing 100 ml of medium (pH 6.5) containing 1%, ferrous sulfate 0.01% and glucose 0.3%
(Capacity: 00 ml) and inoculated with shaking at 30 ° C. for 18 hours.
This seed culture was inoculated on the same medium 20 in 30 jar fermenters, the aeration rate was 20 and the stirring speed was 350 rpm.
The cells were cultured under the conditions of 30 ° C. for 20 hours. 1200 culture
The cells were collected by centrifugation at 0 ram. 0.02 M Tris-hydrochloric acid buffer (containing 10% glycerin and 1% Triton X-100) pH 7.5 in a part (100 g) of the cultured cells.
80 ml was added to disperse the bacteria well, and the mixture was cooled to 4 ° C with ice. This solution was subjected to a crushing treatment of bacterial cells with Dynomill (3000 rpm, 7 minutes). The crushing container was sufficiently cooled with ice cold water. The disrupted solution was centrifuged at 12000 ram for 15 minutes, and the supernatant was collected to obtain 130 ml of solution. This solution was equilibrated with 0.01 M Tris-HCl buffer pH 7.5.
Column packed with DEAE cellulose (diameter 2.5 cm x length 3
0 cm) to adsorb the enzyme. The active part was collected by elution with a 0 → 0.5 M sodium chloride gradient. Next, ammonium sulfate was dissolved in this enzyme solution at a rate of 16 g / 100 ml, and then a column (diameter was packed with phenylsepharose equilibrated with 0.01 M Tris-hydrochloric acid buffer (pH 7.5) containing 16% ammonium sulfate in advance). The enzyme was adsorbed by passing 1 cm × 9 cm in length). This was eluted with 0.01 M Tris-hydrochloric acid buffer having a reverse concentration gradient of 16% → 0% of ammonium sulfate concentration and a concentration gradient of 0% → 25% of ethylene glycol concentration. The active part is made up of an Amicon ultrafiltration device (fractionation membrane 100
00) and then packed with Sephadex G-200 packed column (1.2 × 100 cm) (previously 0.1M).
It was then equilibrated with a 0.05 M Tris-hydrochloric acid buffer (pH 7.5) containing sodium chloride) and passed through a gel. As a result of collecting active parts,
2.28 units / mg protein of enzyme were obtained. The yield was 23%.

[Brief description of drawings]

Fig. 1 is a graph showing the optimum pH of this enzyme, Fig. 2 is a graph showing the temperature range of action of this enzyme, Fig. 3 is a graph showing the thermostability of this enzyme, and Fig. 4 is pH stability. It is a graph which shows.

Claims (1)

[Claims] 1. A fructosyl amino acid oxidase having the following physicochemical properties. (A) Action and substrate specificity: imino 2 in the presence of oxygen
It oxidizes acetic acid or its derivatives to catalyze the following reactions that produce glyoxylic acid or α-ketoaldehydes, α-amino acids and hydrogen peroxide. {Wherein R ₁ is a group —OH, — [CH (OH)] _n —CH ₂ OH or — (CH
_{2) n} -CH _{3, n} is an integer of 0 to 4, _{R 2} represents a side chain residue of α- amino acids. } (B) Optimum pH and stable pH range: The optimum pH is pH 8.0 to 8.5 (phosphate buffer solution) when fructosylglycine is used as a substrate, and the stable pH range is 8.0 to 10.0. (C) Optimum temperature range of operation: 35 to 45 ° C. (d) Thermal stability: 35 ° C., stable against heating for 10 minutes, but deactivated by 90% or more by heating at 45 ° C. for 10 minutes. (E) Molecular weight: The value measured by the column gel filtration method is about 65.
It is 000.