JPH07265074A - New creatine amidinohydrolase and its use - Google Patents

Abstract

PURPOSE:To obtain a new creatine amidinohydrolase, useful as reagents for determining creatine and creatinine, excellent in thermal stability, having a low Km value for the creatine and good in reactivity. CONSTITUTION:This creatine amidinohydrolase is obtained by culturing Alcaligenes faecalis TE3581 (FERM P-14237), etc., and has the following properties: (1) reacting with creatine and producing sarcosine and urea; (2) optimum temperature: about 40-45 deg.C; (3) optimum pH: about 8.0-9.0; (4) stable at <= about 50 deg.C when kept warm at pH7.5 for 30min; (5) stable at pH about 5-8 when preserved at 40 deg.C for 18hr; (6) about 15.2mM value of Km for creatine; (7) molecular weight: about 67000 (measured by the gel filtration method) and about 43000 [measured by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] and (8) isoelectric point: about 3.5.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel creatine amidinohydrolase, a process for producing the same, and a creatine quantifying reagent and a creatinine quantifying reagent using the enzyme.

Creatinine and creatine are found in blood or urine, and the rapid and accurate detection and measurement of the amount of creatinine and creatine are associated with diseases such as uremia, chronic nephritis, acute nephritis, giant dystrophy, and ankylosing dystrophy. Is very important in diagnosing. To make such a diagnosis, it is common to quantify creatinine and creatine in blood or urine.

As a conventional method for quantifying creatine, creatine in a sample is added to creatine amidinohydrolase,
The sarcosine oxidase is allowed to act, and the produced hydrogen peroxide is measured by a hydrogen peroxide measuring means to quantify creatine in the sample. In addition, as a method for quantifying creatinine, after eliminating creatine in the sample, creatinine in the sample is reacted with creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine oxidase, and hydrogen peroxide produced is hydrogen peroxide. There is a method of quantifying creatinine in a sample by measuring with a measuring means.

On the other hand, creatinine amide hydrolase,
Creatine amidinohydrolase and sarcosine oxidase have been widely found in the microbial world, and have already been industrially produced and used as clinical test agents.

[0005]

However, creatine amidinohydrolase produced from various known bacterial cells has low thermostability and a large Km value for creatine. For example, an enzyme derived from the genus Bacillus (Japanese Patent Publication No. 61-1)
No. 7465) has a low thermal stability of 40 ° C. or lower. Furthermore, Pseudomonas putida-derived enzyme has a low Km value of 1.33 mM for creatine. Enzymes derived from Corynebacterium, Micrococcus, Actinobacillus, Acinetobacter, and Bacillus (Japanese Patent Publication No. 3-76915)
Has a thermal stability of 50 ° C or less and K to creatine
The m value is relatively small at 20 mM. However, there has been a demand for a novel creatine amidinohydrolase which is further excellent in heat stability and has a Km value for creatine of 20 mM or less.

[0006]

Means for Solving the Invention As a result of intensive studies to solve the above problems, the present inventors have found that bacteria belonging to the genus Alcaligenes have excellent thermal stability and a higher Km value for creatine. We found a small creatine amidinohydrolase and completed the present invention.

That is, the present invention is a novel creatine amidinohydrolase having the following physicochemical properties.
It catalyzes the next reaction.

[0008]

[Chemical 2] Optimum temperature: about 40 to 45 ° C Optimum pH: about 8.0 to 9.0 Thermal stability: About 50 ° C or less (pH 7.5, 30 minutes) pH stability: pH about 5 to 8 (40 ° C, Km value for creatine: about 15.2 mM Molecular weight: about 67,000 (gel filtration method) about 43,000 (SDS-PAGE) Isoelectric point: about 3.5

The present invention also belongs to the genus Alcaligenes,
In the method for producing creatine amidinohydrolase, which comprises culturing a bacterium having a novel creatine amidinohydrolase-producing ability having the above physicochemical properties in a nutrient medium, and collecting the creatine amidinohydrolase from the culture. is there.

Furthermore, the present invention is characterized in that a novel creatine amidinohydrolase having the above-mentioned physicochemical properties is allowed to act on a sample, sarcosine oxidase is allowed to act on sarcosine produced, and hydrogen peroxide produced is measured. This is a method for quantifying creatine in a sample.

The present invention also allows creatinine amide hydrolase to act on a sample, the resulting creatine to act on a novel creatine amidinohydrolase having the above-mentioned physicochemical properties, and sarcosine oxidase to act on sarcosine produced. It is a method for quantifying creatinine in a sample, which comprises measuring the amount of hydrogen peroxide produced.

The present invention is a reagent for quantifying creatine in a sample, which contains creatine amidinohydrolase, sarcosine oxidase and a reagent for measuring hydrogen peroxide having the above-mentioned physicochemical properties.

Further, the present invention is a reagent for quantifying creatine in a sample, which contains creatinine amide hydrolase, creatine amidinohydrolase having the above-mentioned physicochemical properties, sarcosine oxidase and a reagent for measuring hydrogen peroxide. .

The creatine amidinohydrolase of the present invention may be of any origin such as animals, plants and microorganisms as long as it produces the creatine amidinohydrolase having the above-mentioned physicochemical properties. Preferably, it is a bacterium of the genus Alcaligenes capable of producing creatine amidinohydrolase having the above-mentioned properties, and more preferable examples thereof include Alcaligenes faecalis TE3581. In addition, Alcaligenes faecalis TE3581 is a strain isolated from soil in Miyama-cho, Kita-Kuwata-gun, Kyoto Prefecture, and its mycological properties are as follows.

(A) Morphology (1) Bacterial form: Short bacillus (2) Cell size: 0.5 × 0.8 to 1.5 μm (3) Cell polymorphism: None (4) Motility : Yes, with flagella. (5) Presence or absence of spores: None (b) Growth state in each medium (1) Meat broth agar plate medium: Gray to gray-yellow colonies are formed by culturing at 30 ° C. for 48 hours. The peripheral edge of the colony is the entire edge (Entire) and is convex (Convex). The surface is smooth, glossy and translucent. (2) Broth liquid culture: Growth is not good, but it becomes cloudy uniformly. (3) Meat broth gelatin stab culture: Growth is normal, and only the upper part grows in a filament (Filiform). Gelatin does not liquefy. (4) Litmus milk: No change in color. Milk does not solidify. (5) MacConkey agar: No growth. (6) Phenylethyl alcohol agar medium: Does not grow. (C) Physiological properties (1) Gram stainability :-( negative) (2) Reduction of nitrate :-( 3) Denitrification reaction :-( 4) MR test :-( 5) VP test :-( 6) Production of indole :-( 7) Production of hydrogen sulfide :-( 8) Hydrolysis of starch :-( 9) Degradation of Tween80 :-( 10) Utilization of citric acid: Koser's medium-Christensen's medium + (11) Use of inorganic nitrogen source: sodium nitrate + ammonium sulfate + sodium glutamate + (12) Dye formation :-( 13) urease :-( 14) oxidase: + (15) catalase: + (16) β-galactosidase :-( 17) ) Arginine dihydrase :-( 18) Lysine carboxylase :-( 19) Ornithine carboxylase :-( 20) Tryptophan deaminase: (21) beta-glucosidase: - (22) Protease: - (23) DNase: - (23) Attitude toward oxygen: aerobic (24) OF test (Hugh Leifson method) :-( without decomposing sugar)

(25) Production of Acid and Gas from Sugar Acid gas L-arabinose-D-xylose-D-glucose-D-mannose-D-fructose-D-galactose-maltose-sucrose -Lactose-trehalose-D-sorbitol-D-mannitol-inositol-glycerin-starch-rhamnose-D-melibiose-D-amidacrine- (26) Utilization of organic compound D-glucose -L-arabinose-D-mannose-D-mannitol-N-acetyl-D-glucosamine-maltose-potassium gluconate-n-caprate + adipic acid + dl-malic acid + phenyl acetate +

The experimental method for identifying the above-mentioned mycological properties is mainly described in Takeji Hasegawa, revised edition "Classification and Identification of Microorganisms".
It was conducted by the Academic Publishing Center (1985). In addition, "Burger's Manual of Systematic Bacteriology" (1984) is used as a criterion for classification and identification.
Was referred to.

With reference to the above literatures and mycological properties, glucose non-fermentative Gram-negative bacteria do not produce pigments, oxidase and catalase are positive, β-glucosidase and protease are negative, and further D-glucose. This bacterium is considered to belong to the genus Alcaligenes because it does not utilize sugars such as L-arabinose. Considering that nitrite is not produced from nitric acid in Alcaligenes, that D-glucose cannot be used as a C source, and that it is isolated from soil, it is considered to belong to Alcaligenes faecalis (Alcaligenes faecalis). Alcaligenes fa
ecalis) TE3581. The bacterium has been deposited under the accession number, FERM P-14237.

The enzyme of the present invention can be produced by culturing the creatine amidinohydrolase-producing bacterium in a nutrient medium and collecting creatine amidinohydrolase from the culture. As the nutrient medium used for culturing the creatine amidinohydrolase-producing bacterium, as long as it contains an appropriate amount of a carbon source, a nitrogen source, an inorganic substance, and other necessary nutrients that can be assimilated by the strain used, both synthetic medium and natural medium can be used. Can be used. As the carbon source, for example, malic acid, succinic acid, etc. are used. As the nitrogen source, for example, nitrogen-containing natural products such as peptones, meat extracts, yeast extracts, and inorganic nitrogen-containing compounds such as ammonium chloride and ammonium citrate are used. As the inorganic substance, potassium phosphate, sodium phosphate, magnesium sulfate or the like is used. In addition, it is desirable to add creatine to the medium as a creatine amidinohydrolase production inducer.

In the present invention, shaking culture or aeration-agitation culture is usually carried out. The culture temperature is about 20-40 ° C, preferably about 25-37 ° C, and the culture pH is about 5-9, preferably about 6-8. It can be carried out under conditions other than these as long as the strain to be used grows. The culture period is usually about 1 to 7 days for growth, and creatine amidinohydrolase is produced and accumulated in the cells.

As the method for purifying the enzyme of the present invention, a generally used purification method may be used. For example, as the extraction method, any of ultrasonic crushing, mechanical crushing using glass beads, French press, surfactant and the like may be used. Furthermore, regarding the extract, salting-out method such as ammonium sulfate and sodium sulfate, metal coagulation method such as magnesium chloride and calcium chloride, coagulation method such as protamine and polyethyleneimine, and further DEA
It can be purified by ion exchange chromatography such as E (diethylaminoethyl) -sepharose, CM (carboxymethyl) -sepharose and the like. The crude enzyme solution and the purified enzyme solution obtained by these methods can be powdered by, for example, spray drying or freeze drying. Furthermore, it can be used as an immobilized enzyme by immobilizing it on a suitable carrier.

The creatine amidinohydrolase of the present invention has the following physicochemical properties. It catalyzes the next reaction.

[0023]

[Chemical 3]

Optimum temperature: about 40 to 45 ° C. Optimum pH: about 8.0 to 9.0 Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes) pH stability: pH about 5 to 8 ( Km value for creatine: about 15.2 mM Molecular weight: about 67,000 (gel filtration method) about 43,000 (SDS-PAGE) Isoelectric point: about 3.5

The creatine amidinohydrolase of the present invention can be used in combination with sarcosine oxidase and a composition for detecting hydrogen peroxide to measure creatine. In addition, creatinine can be quantified in the presence of creatinine amide hydrolase.

The method for quantifying creatine in the sample of the present invention is as follows:
The novel creatine amidinohydrolase is allowed to act on a sample, the sarcosine produced is allowed to act on sarcosine oxidase, and the produced hydrogen peroxide is measured.

The method for quantifying creatine in the sample of the present invention is as follows:
Let the creatinine amide hydrolase act on the sample,
The novel creatine amidinohydrolase is allowed to act on the obtained creatine, and sarcosine oxidase is allowed to act on the produced sarcosine, and the produced hydrogen peroxide is measured. It is preferable that creatine in the sample is erased before the action of creatinine amide hydrolase.

The reagent for quantifying creatine in the sample of the present invention contains the novel creatine amidinohydrolase, sarcosine oxidase and hydrogen peroxide detection composition.

The reagent for quantifying creatinine in the sample of the present invention contains creatinine amide hydrolase, the novel creatine amidinohydrolase, sarcosine oxidase and a composition for detecting hydrogen peroxide.

The sarcosine oxidase used for the quantification of creatine or creatinine of the present invention includes Arthrobacter, Corynebacterium, Alcaligenes,
It can be obtained from microorganisms of the genus Pseudomonas, Micrococcus, Bacillus and the like, some of which are already commercially available. The creatinine amide hydrolase can be obtained from microorganisms of the genus Pseudomonas, Flavobacterium, Alcaligenes, Penicillium and the like, some of which are already commercially available.

The composition for detecting hydrogen peroxide comprises an enzyme having a peroxidase activity and a chromogen. Examples of the enzyme having peroxidase activity include peroxidase, haloperoxidase, bromoperoxidase, lactoperoxidase, myeloperoxidase and the like. The chromogen further comprises a hydrogen acceptor and a coupler. The hydrogen acceptor may be any compound that accepts hydrogen when reacting with hydrogen peroxide, peroxidase or a coupler, such as 4-aminoantipyrine and 3-methyl-2-.
Examples thereof include benzothiazoline-hydrazine derivatives.
As couplers, aniline, N, N-dimethylaniline, N, N-diethylaniline, N, N-diethyl-
m-toluidine, N, N-dimethyl-m-anthidine,
N-ethyl- (3-methylphenyl) -N'-acetylethylenediamine, N-ethyl-N- (β-hydroxyethyl) -m-toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl)- m-toluidine, N
-Ethyl-N-sulfopropyl-m-toluidine, N-
Ethyl-sulfopropyl-3,5-methoxyaniline,
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N-
(2-hydroxy-3-sulfopropyl) -m-anisidine, phenol, ρ-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, 2,
There are 4,6-trichlorophenol and the like.

In the creatine assay reagent of the present invention, the preferred amount of each component is about 1 creatine amidinohydrolase.
0-300 U / ml, sarcosine oxidase about 1-1
00U / ml, peroxidase about 0.01-50U /
ml, hydrogen acceptor about 0.1-10 mM, coupler about 0.
1 to 50 mM. In the creatinine quantification reagent of the present invention, preferred amounts of each component are creatinine addohydrolase, 10 to 300 U / ml, creatine amidinohydrolase about 10 to 300 U / ml, sarcosine oxidase about 1 to 100 U / ml, peroxidase about 0. ．
01-50 U / ml, hydrogen acceptor about 0.1-10 mM,
Coupler is about 0.1-50 mM.

The creatine assay reagent or creatinine assay reagent of the present invention is usually used together with a buffer solution having a pH of about 6-8. For example, phosphate buffer, Good's buffer,
Examples include Tris buffer and the like. If necessary, ascorbate oxidase or catalase may be added to the reagent of the present invention. Further, other compounds may be added to the reagent of the present invention in order to smoothly carry out an enzymatic reaction or a color reaction. Examples of such compounds include stabilizers, surfactants, excipients and the like.

The present invention utilizes the following measurement reaction.

[0035]

[Chemical 4]

[0036]

[Chemical 5]

[0037]

[Chemical 6]

The quinone dye produced is usually from 500 to 65
Perform by absorbance measurement at a wavelength of 0 nm. Creatine is quantified by the endo method and rate method, but the endo method is generally used.

Next, a method for measuring the activity of creatine amidinohydrolase of the present invention will be described. First, take 0.9 ml of a substrate solution (creatine dissolved in 50 mM phosphate buffer, pH 7.5 so as to be 0.1 M) in a test tube, and 3
Preheat at 7 ° C for about 5 minutes. Next, 0.1 ml of enzyme solution was added to start the reaction, and the reaction was allowed to proceed exactly at 37 ° C. for 10 minutes. Then, DAB solution (2.0 g of dimethylaminobenzaldehyde was dissolved in 100 ml of dimethyl sulfoxide, and concentrated hydrochloric acid was added thereto). Add 15 ml.) Stop the reaction by adding 2.0 ml. After standing at 25 ° C. for 20 minutes, the yellow dye (Ehrich reaction product) produced by condensation of the produced urea with dimethylaminobenzaldehyde is measured by the absorbance at 425 nm. Blind test 3 times 0.9 ml of substrate solution
After standing at 7 ° C for 10 minutes, 2.0 ml of DAB solution is added and mixed, and then 0.1 ml of enzyme solution is added to prepare the mixture, and thereafter, the mixture is allowed to stand at 25 ° C for 20 minutes, and then the absorbance is measured.
One unit (U) is defined as the amount of enzyme that produces 1 μmol of yellow pigment per minute under the above conditions.

[0040]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 1% creatine, 1% polypeptone, 0.5 yeast extract
%, Medium containing 0.5% NaCl (pH 7.0) 100
Transfer the ml to a 500 ml Sakaguchi flask, 121 ℃, 1
It was autoclaved for 5 minutes. As an inoculum, Alcaligenes faecalis TE35
81 (FERM P-14237) was inoculated with one platinum loop,
Culturing was carried out at 30 ° C. for hours to obtain a seed culture solution. Next, transfer 6 liters of the same medium to a 10 liter jar fermenter,
After autoclaving at 121 ° C. for 15 minutes and allowing to cool, 100 ml of the seed culture was transferred and cultured at 300 rpm, aeration rate of 2 liter / min for 30 hours. The culture broth was collected by centrifugation and suspended in 50 mM phosphate buffer (pH 7.0).

The above cell suspension was treated with a French press and centrifuged to obtain a supernatant. The crude enzyme solution thus obtained was subjected to ammonium sulfate fractionation, DEAE-Sepharose chromatography, phenyl sepharose chromatography, and gel filtration with Sephadex G-200 to give a specific activity of 19 U.
/ Mg. The obtained creatine amidinohydrolase had the following characteristics. 1. Enzymatic reaction: Catalyzes the following reaction.

[0042]

[Chemical 7]

2. Km value: The Km value for creatine was 15.2 mM. As a comparative example, the Km value of the enzyme derived from Actinobacillus (manufactured by Toyobo) for creatine was 20.5 mM. 3. Optimum pH: 50 mM K-phosphate buffer (pH 6.
0-8.0), 50 mM Tris-HCl buffer (pH 7.5)
~ 9.0), glycine NaOH buffer (pH 9.0-1)
The enzyme activity in 0.5) was measured. The result is as shown in FIG. 1, and the optimum pH was about 8.0 to 9.0. 4. Stable pH: Glycine hydrochloride buffer (pH 2-3), acetate buffer (pH 3-6), K-phosphate buffer (pH 6-)
8), Tris-HCl buffer (pH 8-9), glycine Na
It was stored in an OH buffer (pH 9 to 10) at 40 ° C. for about 18 hours, and its residual activity was measured. The results are shown in FIG. 2, and the stable pH was about pH 5-8. 5. Optimum temperature: The enzyme activity at each temperature was measured. The result is as shown in FIG. 3, and the optimum temperature is about 40-
It was 45 ° C. 6. Thermostability: After the enzyme of the present invention was kept warm in 50 mM K-phosphate buffer (pH 7.5) for 30 minutes, the residual enzyme activity was measured. The results are shown in Fig. 4, and were stable up to about 50 ° C. 7. Molecular weight: about 67,000 (gel filtration method) about 43,000 (SDS-PAGE) 8. Isoelectric point: about 3.5 (isoelectric focusing) 9. Comparison with known enzymes

[0044]

[Table 1]

Example 2 A test solution having the following composition was prepared. Creatine amidinohydrolase of Example 1 50U / ml sarcosine oxidase (Toyobo) 20U / ml peroxidase (Toyobo) 2.9U / ml PIPES buffer 0.1M, pH 7.0 4-aminoantipyrine 0.74mM DAOS (Dojin) (Manufactured by Kagaku) 3 ml of the above test solution was added to 100 μl of a sample containing 1 mg creatine 6 mg / dl, and the absorbance change was 37 ° C., wavelength 60.
It was measured at 0 nm. The measurement results are shown in Table 2.

[0046]

[Table 2]

Comparative Example 2 A test solution having the following composition was prepared. Creatine amidinohydrolase derived from Actinobacillus (Toyobo) 50 U / ml sarcosine oxidase (Toyobo) 20 U / ml Peroxidase (Toyobo) 2.9 U / ml PIPES buffer 0.1 M, pH 7.0 4-aminoantipyrine 0 0.74 mM DAOS (manufactured by Dojindo) 3 ml of the above test solution was added to 100 μl of a sample containing 1 mM creatine 6 mg / dl, and the absorbance change was 37 ° C., wavelength 60.
It was measured at 0 nm. The measurement results are shown in Table 3.

[0048]

[Table 3]

As is clear from Table 3, the creatine amidinohydrolase derived from the genus Actinobacillus was clearly inferior in reaction rate to the creatine amidinohydrolase of the present invention.

[0050]

EFFECT OF THE INVENTION The creatine amidinohydrase of the present invention is excellent in thermostability and has a low Km value with respect to creatine. Therefore, as a reagent for quantifying creatine and creatinine, it is possible to provide a reagent that is more stable and more reactive than conventional enzymes.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between reaction pH and relative activity of the enzyme of the present invention.

FIG. 2 is a graph showing pH stability of the enzyme of the present invention.

FIG. 3 is a graph showing the relationship between reaction temperature and relative activity of the enzyme of the present invention.

FIG. 4 is a graph showing the thermostability of the enzyme of the present invention.

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Claims (6)

[Claims] 1. A novel creatine amidinohydrolase having the following physicochemical properties. It catalyzes the next reaction. [Chemical 1] Optimum temperature: about 40 to 45 ° C Optimum pH: about 8.0 to 9.0 Thermal stability: About 50 ° C or less (pH 7.5, 30 minutes) pH stability: pH about 5 to 8 (40 ° C, Km value for creatine: about 15.2 mM Molecular weight: about 67,000 (gel filtration method) about 43,000 (SDS-PAGE) Isoelectric point: about 3.5 2. A bacterium belonging to the genus Alcaligenes and capable of producing the novel creatine amidinohydrolase according to claim 1, is cultured in a nutrient medium, and the creatine amidinohydrolase is collected from the culture. A method for producing creatine amidinohydrolase. 3. A creatine in a sample, which comprises reacting the sample with the novel creatine amidinohydrolase according to claim 1, allowing sarcosine oxidase to act with sarcosine produced, and measuring the amount of hydrogen peroxide produced. Determination method. 4. A creatinine amide hydrolase is allowed to act on a sample, the resulting creatine is allowed to act with the novel creatine amidinohydrolase according to claim 1, and sarcosine produced is allowed to act with sarcosine oxidase to generate peroxidation. A method for quantifying creatine in a sample, which comprises measuring hydrogen. 5. A reagent for quantifying creatine in a sample, which comprises the novel creatine amidinohydrolase according to claim 1, a sarcosine oxidase and a composition for detecting hydrogen peroxide. 6. A reagent for quantifying creatinine in a sample, which comprises creatinine amide hydrolase, the novel creatine amidinohydrolase according to claim 1, sarcosine oxidase and a composition for detecting hydrogen peroxide.