JPH0218068B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing creatine amidinohydrolase, and more particularly, the present invention relates to a method for producing creatine amidinohydrolase using a strain belonging to the genus Acinetobacter and having the ability to produce creatine amidinohydrolase. PRIOR ART AND ITS PROBLEMS Creatine and creatinine are found in human blood or urine, and rapid and accurate detection and measurement of their amounts is essential for human diseases such as uremia, chronic nephritis, acute nephritis, gigantism, It is very important for diagnosing tonic myodystrophy. Known methods for quantifying creatine and creatinine include (1) a chemical assay based on the Jacques reaction using picric acid, and (2) an enzymatic assay using creatinine deiminase. Among these, method (1) above has drawbacks such as requiring a boiling operation and low specificity for creatine and creatinine. In addition, method (2) above has problems such as lack of accuracy because it is affected by endogenous ammonia, and cannot be said to be a quick and accurate method. As an alternative to (1) and (2) above, there is an enzymatic quantitative method using creatinine amide hydrolase and creatine amidino hydrolase. Among these, creatine amidinohydrolase is an enzyme that hydrolyzes creatine to produce sarcosine and urea. Therefore, by measuring the produced sarcosine by a method using sarcosine dehydrogenase or sarcosine oxidase, the amount of creatine in human blood or urine can be determined, and this can be used to diagnose the various diseases mentioned above. Creatine amidinohydrolase is widely found in the microbial world, and has already been produced industrially and used as a clinical test reagent. The following strains are known to date as strains that produce creatine amidinohydrolase. Pseudomonas aeruginosa, (Kopper, PH;
Robin, L.; Arch.Biochem. vol. 26, p. 458,
1950). Pseudomonas obalis
ovalis), (Appleyard, G.; Wood, DD; J.
Gen. Microbiol. vol. 14, p. 351, 1956). Pseudomonas putida
(Yoshimoto, T.; Oka, I.; Tsuru,
D.; Arch.Biochem.Biophys. Volume 177, Page 508,
(1976). Arthrobacter ureaqaciens, (Kaplan, A.;
Naugler, D.; Mol. Cell Biochem. Volume 3, No. 9
Page, 1974). Genus Flavobacterium,
Corynebacterium genus, Micrococcus genus (Japanese Patent Application Laid-open No. 1973-
No. 118884). Genus Alcaligenes, Genus Penicillium (Japanese Patent Application Laid-Open No. 47-43281). However, creatine amidinohydrolase produced from the various known strains mentioned above is optimal.
Very narrow PH range of 7.5 to 8.5 and thermal stability of 40℃
It has the drawbacks that it is unstable to heat, has a large Km value of, for example, 2.9×10 ⁻² M, and has a narrow PH stability of 4.5 to 8.5. Therefore, there is a need for a creatine amidinohydrolase that has a wide optimum pH range, excellent thermostability, and a low Km value. OBJECTS OF THE INVENTION The object of the present invention is therefore to obtain a creatine amidinohydrolase that has a wide optimum pH range, excellent thermostability, and a small Km value. Structure of the Invention The present invention relates to Acinetobacter.
The present invention provides a method for producing creatine amidinohydrolase, which comprises culturing a strain belonging to the genus genus creatine amidinohydrolase and having the ability to produce creatine amidinohydrolase in a nutrient medium, producing and accumulating creatine amidinohydrolase in the culture, and collecting the same. The strain used in the present invention may be any strain as long as it belongs to the genus Acinetobacter, but in particular the strain of Acinetobacter belonging to the genus Acinetobacter collected by the present inventors from a soil concession in Tsuruga City, Fukui Prefecture. ) CRH-1040 strain is preferred. Acinetobacter CRH mentioned above
The mycological properties of strain -1040 are shown below. (a) Morphology When cultured on broth agar medium at 30°C for 20 hours, it is a bacillus with a size of (1.0-1.5μ) x (1.5-2.0μ), Gram staining is negative, and it is not motile. (b) Growth status in each medium (1) Broth agar plate culture Form circular colonies with a diameter of 3 to 5 mm at 30°C for 48 hours. The surface is smooth and has light selection.
The ridges are convex and circular, and the colonies are homogeneous, opaque, pale cream in color, and do not form soluble pigments. (2) Meat juice agar slant culture shows good filamentous growth at 30°C for 20 hours. Colonies are pale cream in color. (3) Meat juice liquid culture Grow in shaking culture at 30℃ for 16 hours and become cloudy. (4) Meat juice gelatin puncture culture Grows in culture at 30℃ for 16 hours and has no ability to liquefy gelatin. (5) Litmus milk No change. (c) Physiological properties (1) Reduction of nitrate; negative (2) Breaking room reaction; negative (3) MR test; negative (4) VP test; positive (5) Formation of indole; negative (6) Hydrogen sulfide Formation; negative (7) Hydrolysis of starch; negative (8) Use of citric acid; negative (9) Use of inorganic nitrogen source; negative (10) Formation of pigment; none (11) Urease; positive (12) Oxidase; Negative (13) Catalase; Positive (14) Growth range; Growth temperature 15-30℃, optimum temperature 20-25℃, growth PH5.0-8.0, optimum PH6.0-
7.5 (15) Attitude towards enzymes; aerobic (16) O-F test; oxidation (17) Production of acids from sugars; D-fructose,
Acid is produced from D-galactose, maltose, lactose, D-sorbitol, D-mannite, inositol, etc. (18) Others Degradation of arginine; negative. Lysine decarboxylation reaction; positive. Ornithine decarboxylation reaction; negative. The experimental methods for identifying the above-mentioned mycological properties were mainly carried out in "Classification and Identification of Microorganisms" edited by Takeji Hasegawa, Society Publishing Center (1975). In addition, Burgess Manual of Determinative Bacteriology No. 8 is used as a standard for taxonomic identification.
(1974) as reference. From the above literature and the above mycological properties, CRH-
Strain 1040 is considered to belong to the genus Acinetobacter. However, the above-mentioned literature does not describe much of the mycological properties of the above-mentioned bacterial species. It closely matches Acinetobacter calcoaceticus, but there are differences in the lysine decarboxylation reaction. Therefore, this bacterial strain was named Acinetobacter genus CRH-1040 strain.
This bacterium has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology under microbial accession number 7718. In carrying out the method of the present invention, a conventional nutrient medium can be used, but it is preferable to culture using creatine, creatinine, or a derivative thereof. Carbon sources for the medium include creatinine, creatine, glucose, sucrose,
Fructose, starch, molasses, alcohols, and organic acids can be used; peptone, meat extract, yeast extract, cornstarch liquor, etc. can be used as natural nutritional sources; creatinine, creatine, ammonia, ammonium sulfate, etc. can be used as nitrogen sources. Ammonium nitrate, ammonium chloride, urea, etc. can be used, and as inorganic salts, potassium phosphate, potassium chloride, sodium chloride, magnesium sulfate, etc. can be used. Each of these nutritional sources can be used alone or in combination. When culturing the bacterial strain, it can usually be carried out by shaking culture or aerated agitation culture. Generally, the culture temperature is preferably 25 to 35°C and the medium pH is 6.5 to 7.5. Creatine amidinohydrolase is produced and accumulated in the bacterial cells when the culture is generally carried out for 1 to 2 days. It goes without saying that the culture conditions should be set so as to maximize the production of creatine amidinohydrolase depending on the bacterial strain used, medium composition, etc. In collecting the creatine amidinohydrolase produced and accumulated by the method of the present invention, the bacterial cells are collected from the culture solution by centrifugation, sieving, etc., and the collected bacterial cells are crushed with beads or ultrasonic. Creatine amidinohydrolase is extracted from the bacterial cells by the following operations. In isolating creatine amidinohydrolase from the crude enzyme solution thus obtained, a method commonly used for enzyme purification can be used. For example, salting out, organic solvent precipitation, dialysis, isoelectric precipitation, ion exchange, gel filtration, and other methods can be used in combination. For example, a crude enzyme solution is centrifuged to obtain a supernatant. Furthermore, an ammonium sulfate salting out fraction (0.35 to 0.55 saturation) of the supernatant is obtained. After overnight dialysis, it was adsorbed and eluted onto DEAE-Sepharose CL4B ion exchanger. After concentrating the active fraction, highly purified creatine amidinohydrolase can be isolated by gel filtration using Sephaacrylic S200. The enzymatic and physicochemical properties of creatine amidinohydrolase obtained by the method of the present invention are as follows. (1) Action: The enzyme of the present invention hydrolyzes 1 mol of creatine to produce 1 mol of sarcosine and 1 mol of urea. (2) Substrate specificity: Acts specifically on creatine. (3) Optimal PH: The enzyme of the present invention has high activity at a pH of 7.0 to 9.0, as shown by the curve in FIG. (4) Optimal temperature: The optimal temperature for the enzyme of the present invention is between 35°C and 45°C, as shown by the curve in FIG. (5) PH stability: Figure 3 shows the PH stability when the enzyme of the present invention was treated at 25°C for 17 hours at each PH. Third
As is clear from the figure, the enzyme of the present invention has a pH of 4.5 to
It is stable between 8.5 and 8.5. (6) Thermostability: The enzyme of the present invention was incubated at pH 7.5 for 30 min at each temperature.
The thermal stability when treated for minutes is shown in FIG.
As is clear from FIG. 4, the enzyme of the present invention is heated at 50°C.
It is stable until (7) Inhibitors: As shown in Table 1 below, it was inhibited by silver nitrate, mercury chloride, and copper sulfate.

[Table] (8) Km value: The Km value of the enzyme of the present invention is approximately 2.4×10 ^-2 M. (9) Molecular weight: The enzyme of the present invention has a molecular weight of about 100,000 as determined by gel filtration using Sephaacryl S-200. (10) Enzyme activity measurement method: In the measurement of the enzyme activity of the present invention, one unit is the enzyme activity that produces 1 micromole of yellow pigment per minute under the following conditions. Reagents: (A) 0.1M creatine solution (dissolve 1.49g creatine in 50mM phosphate buffer PH7.5,
ml). (B) DAB solution (dissolve 2.0 g of p-dimethylaminobenzaldehyde in 100 ml of dimethyl sulfoxide, then add 15 ml of concentrated hydrochloric acid). (C) Enzyme solution (50mM enzyme preparation pre-cooled on ice)
Dilute to 1.0-4.0 U/ml with phosphate buffer PH7.5. Procedure: 1 Put 0.9 ml of the above substrate solution (A) into a test tube and add 37
Preheat at ℃ for about 5 minutes. 2 Add 0.1 ml of the above enzyme solution (C) to start the reaction. 3 After reacting at 37℃ for exactly 10 minutes,
Add 2.0 ml of DAB solution (B) to stop the reaction. 4 After standing at 25°C for 20 minutes, measure the absorbance at 435 nm (OD _test ). 5. A blind test is prepared by leaving 0.9 ml of the above substrate solution (A) at 37°C for 10 minutes, then adding and mixing 2.0 ml of the above DAB solution (B), and then adding 0.1 ml of the enzyme solution (C). Similarly, after leaving at 25℃ for 20 minutes, 435nm
Measure the absorbance at (OD _blaok ). Calculation formula: U/ml = ΔOD (OD _test − OD _blaok ) × 3.0 (ml) × dilution factor / 0.321 × 1.0 × 10 (min) × 0.1 (ml) = ΔOD × 9.35
x dilution factor 0.321 = millimolar molecular extinction coefficient of yellow dye (cm ² /μM) 1.0 = optical path length (cm) Description of Examples The method for producing creatine amidinohydrolase according to the present invention will be described below with reference to Examples. %
is (w/v)% unless otherwise specified. Example 1 Medium composition 0.2% creatine, 0.5% polypeptone, 0.5%
Yeast extract, 1.4% _K2HPO4 , _0.3 % _{KH2PO4 ,}
0.01% _MgSO4・_7H2O , PH7.0 Put 50ml of the above medium into a 500ml Sakaro flask.
Autoclave for 10 minutes at 121 °C. One platinum loop of Acinetobacter CRH-1040 (Feikoken Bacteria No. 7718) was inoculated into the above medium.
Culture with shaking at 30°C for 20 hours and use as a seed culture. Separately, 50 ml of the above seed culture solution is inoculated into a 10-volume jar fermentor containing medium 6 sterilized under the same conditions.
Culture at 300 rpm, aeration rate 3/min, and 30°C for 16 hours. The creatine amidinohydrolase activity of the obtained culture solution was 0.5 U/ml. Culture solution 6
Centrifuge the cells, collect the cells, suspend them in 50mM phosphate buffer, and use a bead crusher (Dynomil) as step 1.
Crush with KDL). Centrifuge the cell suspension to obtain a supernatant. Add ammonium sulfate to the supernatant to a saturation of 0.35 and centrifuge to obtain a supernatant. Add ammonium sulfate to the supernatant to a saturation of 0.55 and centrifuge to obtain a precipitate. 50mM phosphate buffer PH7.5,
Re-dissolve in 250ml. Desalt the redissolved solution using a Sephadex G-25 column (2) equilibrated with 50 mM phosphate buffer PH7.5. Adsorb the desalted solution onto a 50ml DEAE-Sepharose CL-4B column, and add 0.4M
Elute with NaCl. The eluate was concentrated by ultrafiltration and subjected to molecular sieving using a Sephaacrylic S-200 column. The specific activity of the active fraction was 15.3 U/mg protein. Effects of the Invention The creatine amidinohydrolase obtained by the method of the present invention is thermostable up to 50°C, has a wide optimum pH range of 7.0 to 9.0, and has a Km value of 2.4×10 ^-2 M.
It is small and excellent, and the PH stability is also 4.5 to 8.5.
It is wider than the conventional one.

[Brief explanation of drawings]

FIG. 1 shows the relationship between PH and activity of creatine amidinohydrolase obtained by the present invention,
Figure 2 shows the relationship between temperature and activity, and Figure 3 shows the relationship between temperature and activity.
Figure 4 shows the relationship between PH and activity when treated at PH7.5 for 17 hours at each temperature, and Figure 4 shows the relationship between temperature and activity when treated at PH7.5 and each temperature for 30 minutes.

Claims (1)

[Claims] 1. A method of culturing a strain belonging to the genus Acinetobacter and having the ability to produce creatine amidinohydrolase in a nutrient medium, producing and accumulating creatine amidinohydrolase in the culture, and collecting the same. Characteristic method for producing creatine amidinohydrolase. 2 The strain is Acinetobacter
The manufacturing method according to claim 1, which is CRH-1040.