JPH07274961A - Production of creatine amidinohydrolase - Google Patents

Abstract

PURPOSE:To provide a creatine amidlnohydrolase useful as a clinical examination agent for the determination of creatinine and creatine by culturing a creatine amidinohydrolase-producing microorganlsm belonging to the genus Paracoccus or Sphingobacterium. CONSTITUTION:This creatine amidlnohydrolase is produced by culturing a creatine amidinohydrolase-producing microorganism belonging to the genus Paracoccus or Sphingobacterium, preferably Paracoccus denitrificans UNGS AND vamabushfungsParacoccus alkalophilus JCM7364, Sphingobacterium multivorum TE3580, etc., in a nutrient medium and separating the produced enzyme from the cultured product.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing creatine amidinohydrolase.

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,
It is very important for diagnosing giant disease, ankylosing muscular dystrophy, etc. To make such a diagnosis, it is common to quantify creatinine and creatine in blood or urine.

[0003]

2. Description of the Related Art Conventionally, as a method for quantifying creatine,
There is a method for quantifying creatine in a sample by allowing creatine amidinohydrolase and sarcosine oxidase to act on creatine in the sample and measuring the produced hydrogen peroxide by a hydrogen peroxide detection means. There is a method for quantifying creatinine in a sample by causing creatinine amide hydrolase to act on creatinine in the sample and allowing creatine to be produced to act on creatine amidinohydrolase and sarcosine oxidase.

On the other hand, creatinine amide hydrolase, creatine amidinohydrolase and sarcosine oxidase used in these quantification methods are widely found in the microbial world, and in particular, creatine amidinohydrolase is genus Pseudomonas, genus Arthrobacter, It is reported that Flavobacterium, Micrococcus, Alcaligenes, Penicillium, Acinetobacter, Corynebacterium, Actinobacillus, Bacillus, and some of them are already industrially available. It is manufactured and used as a clinical test drug.

[0005]

The present inventors have attempted to produce a new creatine amidinohydrolase from other strains of the above bacteria.

[0006]

As a result of various investigations, the present inventors have found that a strain newly belonging to the genus Paracoccus or the genus Sphingobacteria produces creatine amidinohydrolase and arrived at the present invention.

That is, the present invention cultivates a microorganism belonging to the genus Paracoccus or the genus Sphingobacteria and having the ability to produce creatine amidinohydrolase in a nutrient medium, and causes the creatine amidinohydrolase to be produced and accumulated in the culture, and the culture is performed. A method for producing creatine amidinohydrolase, which comprises collecting the enzyme from a product.

As the microorganism used in the present invention, any strain may be used as long as it belongs to the genus Paracoccus or the genus Sphingobacterium and has the ability to produce creatine amidinohydrolase. The strain belonging to the genus Paracoccus is preferably Paracoccus denitrifican.
s), Paracoccus al
kalophilus) and the like. Examples of strains belonging to the genus Sphingobacterium include Sphingobacterium multivorum.

Even more preferably, Paracoccus denitrificans IFO1
4907, Paracoccus alcalophilus
us alkalophilus) JCM7364, Sphingobacterium multivorum T
E3580 etc. are mentioned. In addition, Sphingobacterium multivorum (Sphingobacterium multivorau
m) TE3580 is a strain isolated from the soil of Fukamuike, Sakyo-ku, Kyoto, and its mycological properties are as follows.

(A) Morphology (1) Bacterial form: Bacillus (2) Cell size: 0.5 × 1.5 to 2.5 μm (3) Cell polymorphism: None (4) Motility: None (5) Presence or absence of spores: None (b) Growth state in each medium (1) Meat broth agar plate medium: Row color colonies are formed by culturing at 30 ° C. for 48 hours. The perimeter of the colony is
re) and cushion-like (Pulvinate). The surface is smooth, shiny and opaque. (2) Broth liquid culture: Growth is normal and cloudy. (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) Pigment formation: Ocher pigment is formed. (3) Reduction of nitrate :-( 4) Denitrification reaction :-( 5) MR test :-( 6) VP test :-( 7) Indole production :-( 8) Hydrogen sulfide production :-( 9) Hydrolysis of starch :-( 10) Degradation of Tween80 :-( 11) Utilization of citric acid: Koser's medium-, Christen
Sen's medium- (12) Utilization of inorganic nitrogen source: sodium nitrate + ammonium sulfate + sodium glutamate + (13) urease: + (14) oxidase: + (15) catalase: + (16) β-galactosidase: + (17) Arginine dihydrase :-( 18) Lysine carboxylase :-( 19) Ornithine carboxylase :-( 20) Tryptophan deaminase: + (21) β-glucosidase: + (22) Protease :-( 23) DNase: + (25) Attitude toward oxygen: aerobic (26) OF test (Hugh Leifson method): O (oxidation) (28) Utilization of organic compound D-glucose + L-arabinose + D-mannose + D-mannitol + N-acetyl-D-glucosamine + maltose + potassium gluconate-n-capric acid salt-adipic acid-dl-malic acid − Sodium citrate − 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, as a standard for classification and identification, the “Bergez Manual
Of Systematic Bacteriology "(198
4) and the International Journal of Systematic Bacteriology Vol. 33,580
(1983) was referred to. Generating an ocher pigment in glucose non-fermenting Gram-negative bacilli with reference to the above literature and mycological properties, oxidase, catalase, β-glucosidase, β-galactosidase, urease positive, acid from various sugars By generating
This bacterium is considered to belong to the genus Sphingobacterium. Considering that starch is not decomposed in Sphingobacterium and acid is not generated from mannitol, it is considered to belong to Sphingobacterium multivorum, and Sphingobacterium multiborum (Sp
hingobacterium multivorum) TE3580.
This bacterium has been deposited under the accession number, FERM P-14209.

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 medium used for culturing the creatine amidinohydrolase-producing bacterium, either synthetic medium or natural medium can be used as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance, and other necessary nutrients that can be assimilated by the strain used. it can. As the carbon source, glucose, glycerol acid or the like is used. Examples of the nitrogen source include peptones, meat extract,
Nitrogen-containing natural products such as yeast extract, ammonium chloride,
Inorganic nitrogen-containing compounds such as 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 to 45 ° C., preferably about 25 to 37 ° C., and the culture pH is about 5 to 9, preferably about 6 to 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 a 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 obtained by the production method of the present invention acts on creatine to produce sarcosine and urea. This creatine amidinohydrolase can be used in combination with sarcosine oxidase and a hydrogen peroxide detection composition to measure creatine. In addition, creatinine can be quantified in the presence of creatinamide hydrolase.

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.

[0018]

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, Paracoccus denitrifica
ns) IFO14907 was inoculated with one platinum loop and cultured at 30 ° C. for an hour to obtain a seed culture solution. Then, 6 liters of the same medium was added
Transfer to a liter jar fermenter, 1 at 121 ° C
Autoclave for 5 minutes, allow to cool, then seed culture solution 100
ml was transferred, and the cells were cultured at 300 rpm, aeration rate of 2 l / min, and 30 ° C. for an hour. The culture solution was collected by centrifugation to obtain 50 mM.
It was suspended in a 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 obtain a specific activity of about 1
Purified to 8 U / mg. The obtained creatine amidinohydrolase had the following characteristics. 1. The following reaction was catalyzed.

[0020]

[Chemical 1]

2. Km value: The Km value for creatine was about 22.8 mM. 3. Optimum pH: 50 mM K-phosphate buffer (pH 6.
0-8.0), 50 mM Tris-HCl buffer (pH 8.0)
~ 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 9.5. 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 25 ° 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 4-10. 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 35-35.
It was 40 ° 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 40 ° C. 7. Molecular weight: about 68,000 (gel filtration method) about 50,000 (SDS-PAGE) 8. Isoelectric point: about 4.1 (isoelectric focusing)

Example 2 Creatine 1%, Polypeptone 1%, Yeast Extract 0.5
%, 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, Paracoccus alkalophilus J
CM7364 was inoculated with one platinum loop and incubated at 30 ° C for
It was used as a seed culture. Next, 6 liters of the same medium was transferred to a 10 liter jar fermenter, autoclaved at 121 ° C. for 15 minutes, allowed to cool, 100 ml of the seed culture was transferred, and cultured at 300 rpm, aeration rate of 2 l / min for 30 hours. . The culture broth was collected by centrifugation, suspended in 50 mM phosphate buffer (pH 7.0), treated with a French press and centrifuged to obtain a supernatant. The resulting crude enzyme solution contained about 0.3 U / ml of creatine amidinohydrolase.

Example 3 Creatine 1%, Polypeptone 1%, Yeast Extract 0.5
%, 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, Sphingobacterium multivoru
m) TE3580 (FERM P-14209) was inoculated with one platinum loop and cultured at 30 ° C. for an hour to obtain a seed culture solution. Next, 6 liters of the same medium was transferred to a 10 liter jar fermenter and autoclaved at 121 ° C for 15 minutes,
After allowing to cool, 100 ml of the seed culture was transferred and cultured at 300 rpm, aeration rate of 2 l / 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 resulting crude enzyme solution was subjected to ammonium sulfate fractionation, DEAE-Sepharose chromatography, phenyl sepharose chromatography, and gel filtration with Sephadex G-200 to obtain a specific activity of about 21.
Purified to U / mg. The obtained creatine amidinohydrolase had the following characteristics. 1. The following reaction was catalyzed.

[0025]

[Chemical 2] 2. Km value: Km value for creatine is about 18.5 m
It was M. 3. Optimum pH: 50 mM K-phosphate buffer (pH 5.
5-8.0), 50 mM Tris-HCl buffer (pH 7.5)
~ 8.5), glycine NaOH buffer (pH 9.0-1)
The enzyme activity in 0.5) was measured. The result is as shown in FIG. 5, and the optimum pH was about 7.5 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 25 ° C. for 18 hours, and its residual activity was measured. As a result, the stable pH was about 4 to 10 (Fig. 6). 5. Optimum temperature: The enzyme activity at each temperature was measured. The result is shown in Fig. 7, and the optimum temperature is about 40 ° C.
Met. 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. 8 and were stable up to 45 ° C. 7. Molecular weight: about 73,000 (gel filtration method) about 44,000 (SDS-PAGE)

[0026]

EFFECT OF THE INVENTION By the production method of the present invention, creatine amidinohydrolase can be obtained from strains belonging to the genus Paracoccus or Sphingobacterium which have not been reported so far.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between reaction pH and relative activity of an enzyme of the genus Paracoccus.

FIG. 2 is a graph showing the pH stability of enzymes of the genus Paracoccus.

FIG. 3 is a graph showing the relationship between reaction temperature and relative activity of an enzyme of the genus Paracoccus.

FIG. 4 is a graph showing the thermostability of enzymes of the genus Paracoccus.

FIG. 5 is a graph showing the relationship between reaction pH and relative activity of enzymes of the genus Sphingobacteria.

FIG. 6 is a graph showing pH stability of enzymes of the genus Sphingobacteria.

FIG. 7 is a graph showing the relationship between the reaction temperature and the relative activity of enzymes of the genus Sphingobacterium.

FIG. 8 is a graph showing thermostability of enzymes of the genus Sphingobacteria.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshihisa Kawamura 10-24 Toyomachi, Tsuruga City, Fukui Prefecture Toyobo Co., Ltd.

Claims (5)

[Claims] 1. A microorganism belonging to the genus Paracoccus or the genus Sphingobacteria and having the ability to produce creatine amidinohydrolase is cultured in a nutrient medium, and creatine amidinohydrolase is produced and accumulated in the culture, and the culturing is performed from the culture. A method for producing creatine amidinohydrolase, which comprises collecting the enzyme. 2. A microorganism belonging to the genus Paracoccus and capable of producing creatine amidinohydrolase,
Paracoccus den
itrificans) or Paracoccus alcalophilus (P
aracoccus alkalophilus), The method for producing creatine amidinohydrolase according to claim 1. 3. A microorganism belonging to the genus Paracoccus and capable of producing creatine amidinohydrolase,
Paracoccus den
itrificans) IFO14907 or Paracoccus alkalophilus JCM73
64. The method for producing creatine amidinohydrolase according to claim 1, which is 64. 4. The creatine amidinohydro according to claim 1, wherein the microorganism belonging to the genus Sphingobacteria and capable of producing creatine amidinohydrolase is Sphingobacterium multivorum. Method for producing lacse. 5. A microorganism belonging to the genus Sphingobacteria and capable of producing creatine amidinohydrolase is Sphingobacterium multivorum TE3580 (FER).
MP-14209).
A method for producing the described creatine amidinohydrolase.