JPS6167486A - Novel creatine amidinohydrolase - Google Patents

Abstract

PURPOSE:A novel creatine amidinohydrolase prepared by microbial engineering technique, and having broad optimum pH range, excellent thermal stability and small Km value. CONSTITUTION:A microbial strain belonging to Acinetobacter genus, Micrococ cus genus, Actinobacillus genus, etc. is cultured, and the creatine amidino hydrolase having the following characteristics is separated from the microbial cell. (i) Acts specifically to creatine, and hydrolyzes 1mol of creatine to produce 1mol of sarcosine and 1mol of urea. (ii) Optimum pH is 7-9 and optimum temperature is 35-45 deg.C. (iii) Stable at 4.5-8.5pH at 25 deg.C for 17hr. (iv) Stable at <=50 deg.C at 7.5pH for 30min. (v) The activity is inhibited with AgNO3, HgCl2, CuSO4, etc., (vi) The Km value is (2+ or -0.5)X10<-2>M. (vii) The molecular weight is 100,000+ or -5,000.

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Application The present invention relates to a novel enzyme creatinamidinohydrolase produced by microorganisms. Especially Acinetobacter (Ac)
1u@tobact@r) Jl,? Icrococcus (Miaroaoaus) genus, Corynebacterium (0oryn@baat@rium) genus, Bacillus (
BIL (lillus) genus, Actinobacillus (Ac
This invention relates to a novel enzyme creatine amidinohydrolase produced by a strain belonging to 3% of the bacteria.

PRIOR ART AND ITS PROBLEMS Creatine deficiency Creatinine is found in human blood or urine, and rapid and accurate detection and measurement of its amount is difficult for human diseases such as uremia, chronic nephritis, acute nephritis, and gigantism. This is extremely important for diagnosing diseases such as tonic muscle dystonia, nutritional disorders, etc.

Creatinine and how to quantify creatine (1)
A chemical assay based on the Jaffe reaction using picric acid and (2) an enzymatic assay using creatinine diminase are known. Among these, method (1) above has drawbacks such as requiring a boiling operation and low specificity for creatine and creatinine. In addition, the 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 amidobitolase and creatine amidinohydrolase. Among these, creatine amidinohydrolase hydrolyzes creatine to produce sarcosine and urea. Therefore, by measuring the sarcosine produced 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. can do.

Creatine amidinohydrolase is widely found in the microbial world, and the following strains are already known to produce creatine amidinohydrolase industrially.

Pseudomonas fist aeruginosa
agasruginosa) (Kopp phrase r, P,
H,HRobin, L,;Arch.

Biohsm, vol. 26, p. 458, 1950).

Pseudomonas obalis (Pseudomon&5o)
valis) (Applsyard, G, HWo
od, D, D, ;J, Gem.

MicrobloIL, 14th Spring, page 351, 195
6 years).

Pseudomonas putida (Pseudomonas p.
utida) (Yoshimoto, T,; Oka,
Ni I Tsuru, D.; Arch.

Bioch@m, Bi, ophys, vol. 177, p. 508, 1976).

Arthrobafter ureafaciens (ICapla)
ny people, i Naugler, D, I Mo1
．． 0ell Bioahem.

Volume 3, page 9, 1974).

Flavobacterium
) genus, Micrococcus (Miaroooaau+s
) genus (JP-A-5.1-118884).

Genus Alcaligenes (AICal1g@nes), Genus Peniaillium (Japanese Unexamined Patent Application Publication No. 1973-1989)
-43281).

However, those produced from the various known strains mentioned above,
The optimal pg range for creatine amidinohydrolase is 7.
．． It has a very narrow range of 5 to 8.5, has a thermal stability of 40°C or less, is unstable to heat, and has an m value of, for example, 2.9.
Large (X 10-”M) and pH stability of 4.5-8
．． 5 and had developed the drawback of being narrow. For this reason, at
A creatine amidinohydrolase is described that has a wide pH range, excellent thermal stability, and a small Km value.

Purpose of the Invention The present invention therefore has a wide optimum pH range, excellent thermal stability,
The object of the present invention is to provide a creatine amidinohydrolase with a low value.

Structure of the Invention The present invention resides in a parent creatine amidinohydrolase having the following physical and chemical properties.

(a) Action: Hydrolyzes 1 mol of creatine to produce 1 mol of sarcosine and 1 mol of urea. (b) Substrate specificity: Acts specifically on creatine.

(a) Optimal pH: 7. O~9. O.

(d) Optimum temperature: 35°c ~ 45°c0 (e) pH stability: 25"0, pH 4.5 ~ 8.5 after 17 hours treatment
Stable.

(r) Thermal stability: pH 7, 5, 5 after 30 minutes treatment
Stable up to 0'c.

(g) Vm harmful agent: AgN0. , Hg01. ．． 0u3
04 etc.

(hJ Km value: (2,0f-0,5) x 10-"
M.

(1) Molecule jl: 100000±5000 (by Kell filtration method).

The novel creatine amidinohydrolase having the above-mentioned physicochemical properties according to the present invention can be obtained by growing a microbial strain capable of producing the enzyme in a nutrient medium, and producing and storing the enzyme mainly within the bacterial cells of the culture. The vine microorganisms used in the present invention that can be obtained by collecting this include Acinetobacter
There are microbial strains belonging to the genus Aainetobaotar, @sActinobacillus, Microaoccus, Oorynebacterium, and Bacillus jQ. In particular, the present inventors collected Acinetobaote from an earthen wall in Tsuruga City, Fukui Prefecture.
r) ORH-1040 strain, Actinobacillus (Aoti
nobacillus) ORH-1271 strain, Micrococcus (IiLiarococcus) ORH-
1093 strain, Corynebacterium (Oorynebao
terium ) ORH-1268 strain and Bacillus (
Bacillus) ORH-1281 strain can be mentioned.

The mycological properties and identification standards for each of the above strains are as follows. Experimental methods for identification of mycological properties were mainly carried out by Takeharu Hase (ed.), "Classification and Identification of Microorganisms", Society Publishing Center (1975). In addition, as a standard for classification identification, reference was made to the Bersey's Manual of Determinative Bacteriology, 8th edition (1974).

The mycological properties of each of the above strains are shown below.

[Ac1netobacter]
ORH-(a) Form: Cultured on broth agar medium at 30"O for 20 hours to a size of 1.
．． It is a bacillus of 0 to 1.5 x (1.5 x 2.0)), negative in Durham staining, and non-motile.

(b) Growth status in each medium (1) Broth agar plate culture 30° C. for 48 hours to form circular colonies with a diameter of 3 to 5 gII. The surface is smooth and shiny, and the ridges are convex and circular.
Colonies are homogeneously opaque, pale cream in color, and do not form soluble pigments.

(2) Juicy 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°C for 116 hours and become turbid.

(4) Meat juice gelatin puncture culture Grows in culture at 30°C for 16 hours and has no ability to liquefy gelatin.

(5) Litmus milk no change.

(c) Physiological properties (1) Reduction of nitrate; Negative (2) Denitrification reaction; Negative (31MR test; Negative (4) VP test; Positive (5) Formation of indole; Negative (6) Formation of hydrogen sulfide; Negative (7) Hydrolysis of starch; Negative (8) Utilization of citric acid; Negative (9) Utilization of inorganic nitrogen source; Negative αO) Production of pigment; None 11) Urease; Positive α2) Oxidase; Negative α3) Catalase Positive (14) Growth range; Growth temperature 15-30°C 1 Optimum temperature 20-25°C, growth pH 5, 0-8. O1 optimal pMg, Q~7.5 α5) Attitude towards oxygen; aerobic α6) O-F test; oxidation α7) Production of acid from sugar; D-fructose, p- is lactose, maltose, lactose, -1) Generate age from Shinrubit, D-mannit, Inojit, etc.

Decomposition of α9 and other arginines; negative lysine decarboxylation; positive ornithine decarboxylation side 1 negative From the above literature and the above mycological properties, strain ORH-1040 is considered to belong to the genus Acinetobacter. However, the literature does not describe many orchidological properties of the above bacterial species. Acinetobacter calcoaceticus (A
Cin■obacterλ1aoaaeticus)
Although there is good agreement with that, there is a difference in the lysine decarboxylation reaction. Therefore, this strain is Acinetobacter (Aa
inetobacter ) OR1 (-1040 strain. Holo has been deposited with the Institute of Microbial Industry, Agency of Industrial Science and Technology under the Microbial Accession No. 7718.

[Actinobacillus
) 0RH-1271'l: 1 (a) Form Cultured on broth agar medium at 30°C for 20 hours until size 0
,3~o,s,11>x(o,s~1.0f
It is a bacillus i), is negative in Durham staining, and is not motile.

(b) Growth status in each culture medium (1) Culture on broth agar plate After 30'0148 hours, circular colonies with a diameter of 3 to 5 mm are formed. The surface is smooth and shiny, and the ridges are convex and circular.
Colonies are homogeneously opaque, cream colored, and do not form soluble pigments.

(21 Juicy agar slant culture at 30°C for 20 hours produces good filamentous growth. Colonies are cream colored and buttery.

(3) Meat juice liquid culture Grow in shaking culture at 30°C for 16 hours and become turbid.

(4) Meat juice gelatin puncture culture 30' (!, grows after 16 hours of culture and has no ability to liquefy gelatin.

(5) Lithomas fist milling It becomes acidic and causes a coagulation reaction.

((+) Physiological properties (1) Reduction of nitrate; positive (2J denitrification reaction; negative (3) MR test; negative (4) vp test; negative (5) production of indole; negative (6) hydrogen hydride Production of negative (7) Hydrolysis of starch; Negative (8) Utilization of citric acid; Negative (9) Utilization of inorganic nitrogen source; Positive α0) Production of pigment; None α1) Urease; Positive 02) Oxidase; Positive ( 13) Catalase; positive α4) Growth range; growth temperature 15-45°C, optimal temperature 2
5-35℃, growth pH 5.0-8.0, to ii! i pH6.0-7.5 α5)! ! Attitude towards substances: Aerobic α6) O-y test ↓ Fermentation α7) Production of acids from sugars: L-arabinose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbit, D-mannirat, Inozid, etc. Produces acid from.

α8) Other decomposition of arginine; positive, decarboxylation of lysine; positive decarboxylation of ornithine; negative From the above literature and the above mycological properties (!RH-1271
The strain is Actinobacillus
) is considered to belong to the genus. However, the literature does not describe much of the mycological properties of the above bacterial species.

Actinobacillus ecrii
lus aquuli), but there are differences in the liquefying ability of gelatin.

Therefore, this strain is an Actinobacillus (Aatinobaa).
illus) strain ORH-1271. Holo has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology under the microbial accession number 7721.

[Microkotsucus ('uiaroaooous)
C! F, H-1093] (a) Morphology Cultured on broth agar medium at 30'C for 20 hours, 'C1 size is 0.3-1° Ofi, Durham staining is positive, and there is no motility. .

(b) Growth status in each culture medium (1) Broth agar plate culture 30" In 48 hours, circular colonies with a diameter of 3 to 5 Wl111 are formed. The surface is smooth and glossy, the ridges are convex circular, and the colonies are homogeneous and opaque. It is pale cream in color and does not form soluble pigments.

(2) When cultured on a gravy agar slant at 30°C for 30 hours, it shows good filamentous growth. Colonies are pale cream in color.

(3) Meat juice liquid culture Grow in shaking culture at 30'C for 16 hours and become turbid.

(4) Meat juice gelatin puncture culture Grows in culture at 30°C for 16 hours and has no ability to liquefy gelatin.

(5) Lidomus milk No change c) Physiological properties (1) Nitrate reduction; negative (2) Denitrification reaction; negative (3) MR test; negative (41VP test; negative (5) Indole formation; negative (6) Production of hydrogen sulfide; negative (7) hydrolysis of starch; negative (8) use of citric acid; negative (9) use of inorganic nitrogen source; negative αO) production of pigment; none αυ urease; positive α Oxidase: Negative αΦ Catalase: Positive (1 growth range - growth temperature 15-30"C, optimum temperature 25-30"C, growth p) 15, 0-8,01 optimal pH 6, OA-7,5α9 oxygen Attitude towards; Aerobic (16) O-IF test; D-sorbitol, D-manchit,
Acid is produced from inosit etc.

Decomposition of α0 and other arginines; negative lysine decarboxylation; positive ornithine decarboxylation; negative Based on the above literature and the above mycological properties, ORH-1093
The strain is considered to belong to the genus Micrococcus. However, the literature does not describe much of the mycological properties of the above bacterial species. Microkotsukasll Lunaus (Mic
rococou81uteus). Therefore, this strain is Micrococcus luteus (Miaro).
ooccus 1uteus).

Therefore, this bacterial strain is a member of the genus Micrococcus.
cus) ORB-1093 strain. Holo has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology; Microbiology Accession No. 7719.

[Corynebacterium (Oorynebacterium)
m) ORH-xz68) (a) Form Cultured on broth agar medium at 30°C for 20 hours, and the size was 0.3 to 0.5 voices) x (1.0 to 2.0 voices). 7h Grum secretion is positive and there is no motility.

(b) Growth conditions that can be grown on each medium (1) Culture on broth agar plate at 30'C for 48 hours to form circular colonies with a diameter of 3 to 5 ws. The surface is smooth and shiny, and the ridges are convex and circular.
Colonies are homogeneously opaque, pale cream in color, and do not form soluble pigments.

(2) It shows good filamentous growth in broth agar slant culture at 30°0 for 20 hours. Colonies are pale cream in color.

(3) Meat juice liquid culture Grow in shaking culture at 30°C for 16 hours and become turbid.

(4) Meat juice gelatin puncture culture It grows in culture at 30°C for 16 hours and has no ability to liquefy gelatin.

(5) Lidomus e-milk unchanged (c) Physiological properties (1) Nitrate reduction; negative (2) Denitrification reaction; negative (3) MR test: negative (4) VP test; negative (5) Indole Production; Negative (6) Production of hydrogen sulfide; Negative (7) Hydrolysis of starch; Positive (8) Utilization of citric acid; Negative (9) Utilization of inorganic nitrogen source; Negative (Production of 101 pigment; None αυ urease; Oxidase with a negative α3) Catalase with a positive α Growth range: Growth temperature 15-45℃, optimum temperature 25
~35°C, growth pH 5, 0-10.0. Optimum pH 6,8-8.5 α5) Attitude towards oxygen; Aerobic α6) O-IF test; Fermentation α Production of acid from D-sugar; L-arabinose, D-galactose, D-xylose, maltose, sucrose, Acid is produced from lactose, mannose, fructose, trehalose, rhoneruvit, D-sorbitol, D-mannilato, inont, etc.

Decomposition of αe and other arginines; positive lysine decarboxylation; negative ornithine decarboxylation; negative Based on the above literature and the above mycological properties, the ORH-1268 strain was identified as Corynebacterium
m) considered to belong to the genus. Therefore, this strain is Corynebacterium (0aryn@b&atherium).
It was named strain ORH-1268. The heavy encyclopedia has been deposited at the Institute of Microbiology, Agency of Industrial Science and Technology under the microbial deposit number 7720.

[Bachi/L/ x (Baaillus) (JRH
-1281) (IL) Form Cultured on broth agar medium at 30'C for 20 hours, size: 0.3 x O,5fi) x (0,5-1.0
71), Durham staining is positive, and it is not motile.

(b) Growth status in each medium (1) Broth agar plate culture 30° C. for 48 hours to form circular colonies with 3 to 5 diameters. The surface is smooth and shiny, the ridges are convex and circular, and the colonies are homogeneous, opaque, pale cream in color, and do not form soluble pigments.

(2) Juicy agar slant culture at 30'C for 20 hours shows good filamentous growth. Colonies are pale cream in color and buttery.

(3) Meat juice liquid culture Grow in shaking culture at 30°C for 16 hours and become turbid.

(4) Meat juice gelatin puncture culture Grows in culture at 30°C for 16 hours and has no ability to liquefy gelatin.

(5) Lidomus milk No change 0) Physiological properties (1) Nitrate reduction; Negative (2J denitrification reaction; Negative (3) MR test; Negative (4) VP test; Negative G) Indole production; Negative (6) Production of hydrogen sulfide; negative (7) Hydrolysis of starch, negative (8) Use of citric acid; positive (9) Use of inorganic nitrogen source; positive α0) Production of pigment; none αυ urease; negative α2) Oxidase: Negative α Catalase: Positive α Growth range FMi: Growth temperature 15-35°C, optimum temperature 20-30°C, growth pH 5.0-10.0, optimum pi (6,0-7 .5 (15) Attitude towards oxygen; Aerobic (1f5) O-F test: Oxidation (formation of acid from 171%; L-arabinose, D-xylose, D-mannose, D-flagtose, D-sorbint, D - Produces acids from mannirato, inosit, etc.

(1a) Other decomposition of arginine; positive lysine decarboxylation reaction; positive ornithine decarboxylation reaction; negative Based on the above literature and the above mycological properties, strain ORH-1281 is considered to belong to the genus Bacillus. Therefore, this strain is a Neutilus (Bacillus).
s) It was named ORH-1281. Holo is assigned microbial accession number 77 to the Institute of Microbiology, Agency of Industrial Science and Technology.
It has been deposited as No. 22.

In order to obtain the novel creatine amidinohydrolase of the present invention, each of the above-mentioned bacterial strains can be cultured in a conventional nutrient medium, produced and accumulated in the culture, particularly in the bacterial cells, and then collected from there. The nutrient medium that can be used is preferably a medium containing creatinine, creatine, or a derivative thereof. Creatinine, creatine, glycose, sucrose, fructose, starch, blackstrap molasses, alcohols, and organic acids can be used as carbon sources for the nutrient medium, and natural nutritional sources include peptone, meat extract, yeast extract, cornstarch liquor, etc. is available,
Creatinine, creatine, and ammo as nitrogen sources: 7
．． Sulfuric acid, ammonium nitrate, ammonium chloride, urea, etc. can be used, and inorganic salts include potassium phosphate, potassium chloride, sodium chloride,
Magnesium sulfate etc. can be used. Each of these nutritional sources can be used alone or in combination.

In step 6, culturing the bacterial strain can be carried out by shaking culture or aerated agitation culture. Generally, the culture temperature is 25~
The pH of the 35"C1 medium is preferably 6.5-7.5, and creatine amidinohydrolase is produced and accumulated in the bacterial cells when the culture is normally carried out for 1 to 2 days.Culture conditions depend on the strain used and the medium. It goes without saying that the composition should be set so that the production amount of creatine amidinohydrolase is maximized.

To collect the creatine amidinohydrolase produced and accumulated by the above method (ζ), collect bacterial cells from the culture solution by centrifugation, filtration, etc. The creatine amidinohydrolase is extracted from the bacterial cells by a manipulation. Creatine amidinohydrolase can be isolated from the thus obtained crude fermentation X'W by a method commonly used for enzyme purification. For example, salting out, paid solvent precipitation,
Methods such as dialysis, isoelectric precipitation, ion exchange, and gel filtration can be used in combination. For example, a crude enzyme solution is centrifuged to obtain a supernatant. Furthermore, the ammonium sulfate salting out fraction of the supernatant (0.35~
0.55 saturation). - DIAI-Sepharose OI after night dialysis.

Adsorb and elute on 4B ion exchanger. After concentrating the active fraction, highly purified creatine amidinohydrolase can be isolated by gel filtration using Sephacryl 8200.

The novel creatine amidinohydrolase according to the present invention has the following enzymatic and physicochemical properties.

(a) Action: Hydrolyzes 1 mol of creatine to produce 1 mol of sarcosine and 1 mol of urea.

(b) Substrate specificity; Acts specifically on creatine.

(C) Optimal pH: The enzyme of the present invention has a high activity at an optimal pH of 7.0 to 9.0, as shown by the solid line in FIG.

(dl Optimum temperature: The optimal temperature of the enzyme of the present invention is between 35°C and 45°C, as shown by the solid line in FIG.

(e) pH stability: The pH stability of the enzyme of the present invention when treated at 25°C for 17 hours at each pH is shown by the solid line in FIG. As is clear from FIG. 3, the enzyme of the present invention is stable between pH 4.5 and 8.5.

(f) Thermostability: The thermostability of the enzyme of the present invention when treated at pH 7 and 5 for 30 minutes at each temperature is shown in FIG. As is clear from FIG. 4, the enzyme of the present invention is stable up to 50'C.

In addition, what is shown by a broken line in FIGS. 1 to 4 is a known Pseudomonas putida (Pseudomonas p.
This is a curve showing the optimum pH 1, optimum temperature, pH stability, and thermostability of creatine amidinohydrolase obtained from P. utida.

(Gl inhibitor: The enzyme of the present invention was inhibited by silver nitrate and mercury chloride as shown in Table 1 below.

(h) h value: The h value of the enzyme of the present invention is (2,0±O,S)X10M
It is.

(1) Molecular weight: The enzyme of the present invention has a molecular weight of approximately 100,000±5,000 as measured by gel filtration using Sephacryl S-200.

(j) Enzyme activity measurement method: In the measurement of 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) 0.1 M creatine solution (dissolve 1,49F creatine in 50mM phosphate buffer pH 7,5,
00-).

(B) DAB solution (2, Or p-dimethylaminobenzaldenide is dissolved in 100 d dimethyl sulfoxide, then 15 ml concentrated hydrochloric acid is added).

(0) Enzyme solution (50 mM enzyme preparation pre-cooled on ice)
1.0-4. OU
/ml).

Procedure: 1. Pour 0.9 wLl of the above substrate solution (A) into a test tube and preheat at 37"C for about 5 minutes.

2. Add 0.1 mJ of the above enzyme solution (0) to start the reaction.

3. After reacting at 37°C for exactly 10 minutes, the above DA
Solution B (B) 2.0- is added to stop the reaction.

4. After standing at 25°C for 20 minutes, measure the absorbance at 435 nm (ODtegt).

5. For blind testing, leave the above substrate solution (A) 0.9- at 37°C for 10 minutes, then add the above DAB solution (B). Oa(
Add and mix, then add the above enzyme solution (0) 0.1
Prepare by adding m. Thereafter, the absorbance is similarly measured after being left at 25°C for 20 minutes (oD'blank).

Calculation formula: % formula % 0.321 = millimolar molecular extinction coefficient of yellow pigment (al/
μM) 1.0 = Optical path length - Description of Examples The method for producing creatinamide dinohydrolase according to the present invention will be described below with reference to Examples. % is Cv/'v)% unless otherwise specified.

Example 1 Medium composition 0.2% creatine, 0.5% polypeptone, 0.5% yeast extract, 1.4% K, 1 (PO, 0,3% Kll, mushroom POa, 0.
01%MgEJO* @ 7 HsO, pH 7,
0Put 501L of the above medium into a 5001117 Sakaro flask and sterilize it by autoclaving at 121°C for 10 minutes. Aalnetobacter (Aalnetobacter)
Inoculate 1 platinum loop of ORH-1040 (Feikoken Bacteria No. 7718) into the above medium and culture with shaking for 30" (:!, 20 hours to use as a seed culture solution. Separately, 6 L of medium was sterilized under the same conditions. Transfer 5Qd of the above seed culture solution to a 10L jar fermenter containing
inoculate. 300rpm.

Culture at 30°C for 16 hours at an aeration rate of 34/min.

The creatine amidinohydrolase activity of the obtained culture solution was 0.5 U/d. Centrifuge 6t of culture solution,
The bacterial cells were collected, diluted with 50 mM'J acid buffer, and disrupted as IL using a bead crusher (Dynomil KDL). Centrifuge the cell suspension to obtain a supernatant. 0 in supernatant
．． Add ammonium sulfate to a saturation of 0.55 and centrifuge to obtain a supernatant.Add ammonium sulfate to the supernatant to a saturation of 0.55, centrifuge to obtain a precipitate. Redissolve in 250 rnl of 5QmM phosphate buffer pH 7,5. The redissolved solution is desalted using a Sephadex G-25 column (2 L) equilibrated with 50 mM J acid buffer pH 7.5. The desalted solution is adsorbed onto a DKAK-Sepharose 0L-4B column 50d and eluted with 0.4 M Mail. The eluate was concentrated by ultrapolar filtration and subjected to molecular sieving using a Sephaacrylic S-200 column. The specific activity of the active fraction is 15.3U
/Tn9 protein.

Example 2 Medium composition 0.2% creatine, 0.5% polypeptone, 0.5% yeast extract, 1.4% KjliPO*, 0
．． 3% KHzl'Os, 0.1% MgSO4.7
H80, pH 7.9 Put 50rnl of the above medium into a 50011L Sakaro flask and autoclave at 121°C for 10 minutes at 30°C.
, cultured for 20 hours and used as seed #! nutrient solution.

Separately, 50 aJ of the seed culture solution was inoculated into a 10 Ja fermenter containing 6 t of a medium sterilized under the same conditions. 300
Incubate for 16 hours at 30°C, rpm, aeration 13 L/min. The creatine amidinohydrolase activity of the obtained culture solution was 0.8 tT/-. Culture solution 6L
Centrifuge and collect the bacterial cells.50! Suspend in IIMIJ acid buffer and make 1t using a bead crusher (Dynomil KDL).
).

Centrifuge the cell suspension to obtain a supernatant. 0.3 to the supernatant
5 Add ammonium sulfate to saturation and centrifuge to obtain a supernatant.

Add ammonium sulfate to the supernatant to a saturation of 0.55 and centrifuge to obtain a precipitate.

Redissolve in 250 ml of 5QmM phosphate buffer pH 7.5. Re-dissolve the solution in 50mM') acid buffer pi-17,
Sephadex G-25 column (2t) equilibrated with 5
Desalt with. Desalinated solution]) KAK-Sepharose 0L-
Adsorb to 4B force ram 50ml, Q, 4M Mail
Elute at . The eluate was concentrated by ultrafiltration and subjected to molecular sieving using a Sephacryl S-200 column. The specific activity of the active fraction is 18. It was IU/η protein.

Example 3 Medium composition 0.2% Ri1/Atin, 0.5% Polypeptone, 0.5% Yeast Extract, 1.4% KIHP04.0.
3%KH Feces P04.0.01%Mg5O4e 7H
l O, p) 17.0 50011 5Qml of the above medium
Place in a No. 11 Sakaro flask and sterilize in an autoclave at 121°C for 10 minutes. Mic5oc
oacus) 0RH-1093 (Feikoken Bacteria No. 77
Inoculate one loopful of No. 19) into the above medium and inoculate at 30°0,20
Culture with shaking for hours and use as a seed culture. Separately, 50 m of the above seed culture solution was inoculated into a jar fermenter containing 6 t of medium sterilized under the same conditions. 300. Incubate for 16 hours at 30°C, rpm, aeration 13 L/min.

The creatinamidinohydrolase activity of the obtained culture solution was: IO, 65tr/mj! Met. Centrifuge 6 tons of culture solution, collect bacterial cells, and collect 50mM! The suspension is suspended in J acid buffer and crushed using a bead crusher (Gylenomyl KDL) as IL. 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.

Ammonium sulfate was further added to the supernatant to achieve a saturation of 0.55, and the mixture was centrifuged to obtain a precipitate. 501! LM! J-N buffer solution p
Redissolve in H7.5, 250ml. 50m of re-dissolved solution
Desalt with a Sephadex G-25 column (2 L) equilibrated with MIJ acid buffer pH 7.5.

The desalted solution was passed through a DEAEt-Sepharose 0L-4B column,
Adsorb to 50rIL1 and elute with 0.4 M Na01.

The eluate was concentrated by ultrafiltration, and Sephaacryl S-2
Perform molecular sieving using a 00 column. The specific activity of the active fraction is 1
It was 7.8U/119 protein.

Example 4 Medium composition 0.2% creatine, 0.5% polypeptone, 0.5% yeast extract, 1.4% to 21% (PO, 0.3% KH)
Mg5O4・7H20, pH 7.9 50IOm7 of the above medium 5Qrnl! Place in a Sakaro flask and autoclave at 121'C for 10 minutes to sterilize. Corynebacterium
m) Inoculate one platinum loop of ORH-1268 (Feikoken Bacteria No. 7720) into the above medium and culture with shaking for 30 hours for 0.20 hours to obtain a seed culture. A medium sterilized separately under the same conditions. 6
Transfer the above seed culture solution 5 to a 104-volume jar fermenter containing
Inoculate with 0 rul. 300rpm % ventilation Jil 3
Incubate at t/min for 16 h at 30 °C. The taleatinamidinohydrolase activity of the obtained culture solution was Q, g 5 U/1 nt.

￥Centrifuge 6 tons of nutrient solution, collect bacterial cells and make 50mM! Suspend in J acid buffer, make 1t, add ammonium sulfate using bead crusher (add ammonium sulfate), and centrifuge to obtain a supernatant. To the supernatant, add ammonium sulfate to 0.55 saturation and centrifuge to obtain a precipitate. 50
Redissolve in 52501 Ll of mMIJ acid buffer pH 7. The redissolved solution is desalted using a Sephadex G-25 column (2t) equilibrated with 50 mM IJ acid buffer pH 7.5. DKA] 1Th-Sepharose 0L-4
Adsorb to 50 ml of B force ram and elute with Q, 4M Na0L. The eluate was concentrated by ultrafiltration and subjected to molecular sieving using a Sephacryl S-200 column. The specific activity of the active fraction was 17.6υ/Misaki protein.

Example 5 Medium composition: 0.2% creatine, 0.5% polypeptone, 0.5% yeast extract, 1.4% HPOa, 0.3% KJI! P Oζ 0.01
%MgSO4.7 HsO1 pH7.0 Above medium 50
Put m1 into a 5004 Sakalo flask and heat it to 121°C for 1
Autoclave for 0 minutes to sterilize. Bacillus
lua) One platinum loop of ORH-1281 (Feikoken Bacteria No. 7722) was inoculated into the above medium and shaken for 30'0 and 20 hours. .

Then, 50 mj of the above seed culture solution was inoculated into a jar fermenter containing 6 t of medium sterilized under the same conditions. 300
Culture at 30°C for 16 hours at rpm, air flow rate 31/min. The creatine amidinohydrolase activity of the obtained culture solution was 0.78 TJ/ml. 6L of the culture solution is centrifuged, and the bacterial cells are collected, suspended in 50mM IJ brewing buffer, and crushed using a bead crusher (Dynomil KDL).

Centrifuge the cell suspension to obtain a supernatant. 0.3 to supernatant
5 Add ammonium sulfate to saturation and centrifuge to obtain a supernatant.

Ammonium sulfate is further added to the supernatant to a saturation of 0.55, and the mixture is centrifuged to obtain a precipitate. 5QmM phosphate buffer,
Redissolve in pH 7,5,250WLt. 5 redissolve solution
Desalt with a Sephadex G-25 column (2t) equilibrated with 0 mM IJ acid buffer pH 7.5. Transfer the desalting solution to a DKAK-Sepharose 0L-4B column, 50rr.
Adsorb to Ll and elute with 0.4M Na0. Ultrafiltrate the eluate! IwJ Shi, Sefa Acrylic S-200
Perform molecular sieving in a column. The specific activity of the active fraction is 17.
It was 2U/η protein.

Effects of the Invention The novel 1 noatinamidinohalolase according to the present invention is thermostable up to 50°C, and has an optimum pH of 7.0 to 9.
．． 0 and m value is also (2, Ofo, 5)X10-1M
It has a small and excellent pH stability of 4.5 to 8.5, which is wider than conventional products.

[Brief explanation of drawings]

The solid line in Figure 1 represents the relationship between pH and activity of the novel creatine amidinohydrolase of the present invention, the solid line in Figure 2 represents the relationship between temperature and activity, and Figure 3 shows the relationship between pH and activity at 25°C. - represents the relationship between pH and activity when treated for 17 hours, $4m is p! (This shows the relationship between concentration and activity when treated for 30 minutes at each temperature for 7 and 5. The broken lines in Figures 1 to 4 indicate the activity of creatine amidinohydrolase obtained from the known Pseudomonas putida. shows the relationship between

Claims (1)

[Claims] 1. A novel creatine amidinohydrolase having the following physicochemical properties. (a) Action: Hydrolyzes 1 mol of creatine to produce 1 mol of sarcosine and 1 mol of urea. (b) Substrate specificity: Acts specifically on creatine. (c) Optimum pH: 7.0 to 9.0. (d) Optimum temperature: 35°C to 45°C. (e) pH stability: pH 4.5 after treatment at 25°C for 17 hours
Stable at ~8.5. (f) Thermal stability: pH 7.5, 50°C for 30 minutes treatment
Stable until. (g) Inhibitors: AgNO_3, HgCl_2, CuSO
＿4th grade. (h) Km value: (2.0±0.5)×10^-^2M. (i) Molecular weight: 100000±5000 (by gel filtration method).