JPH0657148B2 - Method for producing creatine amidino-lolase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing creatine amidinohydrolase by culturing a strain belonging to the genus Alcaligenes and capable of producing extracellular creatine amidinohydrolase. is there.

[Conventional technology]

Serum creatine concentration increases in primary muscle disease, myositis, muscle atrophy, hyperthyroidism, etc., and causes creatine urine.
Therefore, quantifying creatine is extremely important for clinical medical diagnosis. Therefore, creatine amidinohydrolase is used for the enzyme quantification method of creatine by utilizing the property of specifically degrading creatine.

Creatine amidinohydrolase (EC3 / 5/3.
3) is a known enzyme, which is an enzyme that acts on creatine to hydrolyze it into urea and sarcosine, and its reaction formula is as follows.

Conventionally, creatine amidinohydrolase is disclosed in JP-A-47-4
It is known to exist in bacteria belonging to the genus Alcaligenes as suggested in Japanese Patent No. 3281 and Japanese Patent Laid-Open No. 55-34029.

[Problems to be solved by the invention]

However, since creatine amidinohydrolase obtained from these genus Alcaligenes is an intracellular enzyme, in order to obtain this enzyme, the cultured bacterial cells are collected,
Since it requires an operation of separating and extracting the enzyme by grinding, sonicating or lysing it, there is a problem that the enzyme purification operation is remarkably complicated and the enzyme cannot be efficiently obtained.

[Means for solving problems]

Therefore, the present invention has taken the following means for the purpose of efficiently obtaining creatine amidinohydrolase in a single-step operation without performing an extraction operation process from cultured cells.

That is, the present invention is for culturing a strain belonging to the genus Alcaligenes and having the ability to produce extracellular creatine amidinohydrolase, and separating and recovering creatine amidinohydrolase from the culture solution.

Examples of the strain used in the present invention include Microtechnical Research Institute No. 5071 strain belonging to the genus Alcaligenes and having extracellular creatine amidinohydrolase-producing ability.

Hereinafter, the bacteriological properties of this Microtechnology Research Institute strain No. 5071 will be described.

a) Morphology: Width 0.5-1.0μ, length 1.5-3.5μ, observed as monococcus or diploid.

Motility: Has one flagella and is motile.

Gram stainability: negative.

Acidic: Negative.

Capsular: None.

b) Growth state in each medium After culturing at broth agar plate culture at 28 ° C for 48 hours, a raised circular colony with a diameter of 3 to 5 mm and a smooth, glossy peripheral circular colony are formed.

Meat broth agar slant culture Grows in a widened pattern by culturing at 28 ° C for 48 hours. The color of the colonies is translucent opalescent, the surface is glossy and does not produce diffusible pigments.

Meat juice liquid culture Almost no growth is observed in static culture.

Meat broth agar stab culture It grows on the surface and puncture hole by culturing at 28 ℃ for 48 hours.

Meat broth gelatin stab culture It grows on the surface or upper layer by culturing at 20 ° C for 1 to 14 days, but no liquefaction is observed.

Litmus milk Slowly becomes alkaline with culture, reducing litmus slightly, but does not liquefy or coagulate.

Potato agar medium It grows well in a widened state by culturing at 28 ° C for 48 hours. The color of the colony is translucent milky white or gray white, and the surface is smooth and glossy.

c) Physiological properties Reduction of nitrate: None.

Denitrification reaction (Komagata et al. Method): Negative.

MR test: negative.

VP test: negative.

Generation of indole: No generation.

Generation of hydrogen sulfide: No generation.

Hydrolysis of starch: No decomposition.

Use of citric acid: Use.

(Koser Citrate medium and Christensen medium) Use of inorganic nitrogen source (Iizuka / Komagata method) Nitrate: Use.

Ammonium salt: Used.

Dye formation: No pyocyanin dye is formed.

Urease: Generate.

Oxidase: produces.

Catalase: Generate.

Growth range: pH 6.5 to 9.5, optimum pH around 8.0, temperature 20 to 3
0 ℃, optimum temperature 28 ℃ (medium agar medium used).

Attitude toward oxygen: aerobic.

OF test (Hugh-Leifson test): Does not decompose sugars oxidatively or fermentatively.

Generation of acid and gas from sugar: L-arabinose, D-
Xylose, D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbit, D-mannitol,
No acid or gas generation was observed from any sugars such as inosit, glycerin and starch.

d) Other properties Peptone formation of casein: Not produced.

Digestion of eggs: Not digested.

Hydrolysis of arginine: No decomposition.

Phenylpyruvate test: negative.

Indophenyl acid test: negative.

Acetoin production: Not produced.

Oxidation of glucuronic acid: Not oxidized.

Reaction of decarboxylation of lysine: negative (Simmons citrate medium and LIM medium).

β-galactosidase: produces.

Utilization of carbon compounds: L-arabinose, D-xylose, D-glucose, D-mannose, D-fructose, D-galactose, lactose, maltose. Sucrose, trehalose, raffinose, D-sorbit, inosit,
Glycerin, salicin, α-methyl glucosid, inulin, dextrin, starch, cellulose, L-sorbose, cellobiose, glycogen, erythriol,
Pyruvic acid, lactic acid, malonic acid, oxalic acid, d-tartaric acid,
Does not utilize butyric acid and propionic acid.

Rhamnose, succinic acid, citric acid, acetic acid and phenylalanine can be assimilated respectively.

The properties of the strain used in this invention are described in Bergey's Manual.
In contrast to the classification of Volume 1 of Systematic Bacteriology (1983), this strain is a gram-negative, aerobic rod with one flagella, motility, catalase-positive, oxidase-positive, and sugar to acid. Also, it is determined that the substance belongs to the genus Alcaligenes, because the production of gas is not recognized.

A method for producing creatine amidinohydrolase using the above-mentioned strain will be described below.

The Alcaligenes strain used in the present invention can be cultured using a solid medium, but using a liquid medium,
It is desirable to carry out shaking culture or aeration-agitation deep culture.

As the nutrient source of the medium, those usually used for culturing microorganisms are widely used. The nitrogen source may be any available nitrogen source, such as soybean flour, corn steep liquor,
Various meat extracts, peptone, yeast extract and the like are used. As the carbon source, any available carbon compound may be used, and sugars, soluble starch, glycerin and the like are used. As other inorganic salts, sodium nitrate, sodium chloride, monopotassium phosphate, dipotassium phosphate, potassium chloride, ferrous sulfate, copper sulfate, manganese chloride, zinc sulfate and the like are used as necessary. Further, addition of creatine to the medium increases the accumulation of creatine amidinohydrolase in the culture solution. The added concentration is 0.5-1.0%
A degree is preferable.

The culture temperature may be any temperature within the temperature range in which the bacterium grows and produces creatine amidinohydrolase outside the cells, and preferably about 28 ° C. The culturing time is usually 2 to 5 days, though it varies depending on the conditions. And
The culturing may be finished at an appropriate time in consideration of the time when the production amount of creatine amidinohydrolase in the culture solution reaches the maximum.

In order to separate creatine / amidinohydrolase from the culture solution, first, bacterial cells and other solids are removed by filtration, centrifugation, etc., and the resulting crude creatine / amidinohydrolase-containing solution is further subjected to known proteins, enzymes, etc. By using isolation and purification means, purified creatine amidinohydrolase can be obtained. For example, crude creatine amidinohydrolase is added with streptomycin sulfate aqueous solution and protamine sulfate aqueous solution as needed to remove nucleic acids and the like, and ammonium sulfate or the like is added thereto for salting out, or with an organic solvent such as acetone / ethanol. In addition, the precipitate is separated and collected. To further purify this precipitate, for example, the precipitate is dissolved in a solvent such as Tris-hydrochloric acid buffer solution, and diethylaminoethyl-cellulose ion exchanger, diethylaminoethyl-dextran ion exchanger, diethylaminoethyl-agarose ion exchanger, etc. Adsorption and elution method using anion exchanger, chromatographic method using gel permeation agent such as dextran gel, polyacrylamide gel,
The adsorption elution method with hydroxyapatite and the electrophoresis method using polyacrylamide gel may be performed.

These means are appropriately selected and combined, and then dried by means such as vacuum freeze-drying to obtain purified creatine amidinohydrolase powder.

Next, the physicochemical properties of the creatine amidinohydrolase obtained by the present invention will be described.

Action This enzyme has the action of hydrolyzing creatine to form urea and sarcosine, and the Km (Michaelis constant) value for creatine is 4.83 × 10 ⁻³ M (37 ° C. pH 7.
8).

Titration method 50 mM phosphate buffer containing 0.1 M creatine (pH 7.8)
1.0 ml, 0.1 ml of appropriately diluted enzyme solution was added and reacted at 37 ° C. for 10 minutes, and then P-dimethylaminobenzaldebide solution (2.0 g of P-dimethylaminobenzaldebide was dissolved in 100 ml of dimethylsulfoxide). After letting it
Add 2.0 ml of concentrated hydrochloric acid (added 15 ml) and leave at 25 ° C for 20 minutes. The absorbance (ΔOD) is measured at 435 nm with a spectrophotometer using the sample at the time of 0 minutes of the enzyme reaction as a control, and the calculation is performed as follows.

Enzyme activity unit (U / ml) in 1 ml of enzyme solution = ΔOD × 9.66
× Enzyme dilution ratio Here, for the creatine amidinohydrolase enzyme activity, the amount of enzyme that produces 1 μmol urea per minute under the above reaction conditions is 1 unit (1 U).

Optimum pH The optimum pH is around 7.0 to 8.0.

The buffer used is pH 6.0 to 8.0: phosphate buffer, pH 7.0 to
9.0: Tris-hydrochloric acid buffer solution, pH 8.5 to 9.5: carbonate buffer solution.

pH stability An enzyme was added to a buffer solution of each pH and the mixture was allowed to stand at 5 ° C. for 48 hours, and then its residual activity was measured. The buffer used is the same as that described above. As a result, it is recognized that creatine amidinohydrolase is stable around pH 7.0-8.5.

After heat-treating 1.0 ml of 50 mM phosphate buffer solution (pH 7.5) of creatine amidinohydrolase for 30 minutes at each temperature,
The residual activity of the enzyme was measured. As a result, it is confirmed that creatine amidinohydrolase is stable at around 40 ° C or lower.

Optimum temperature The optimum temperature for the reaction of creatine amidinohydrolase is
It is recognized that the temperature is around 40 ° C.

Molecular weight: about 50,000 (measured by gel permeation method) [Action] Since the bacterium used in the present invention has the ability to produce extracellular enzyme, the enzyme accumulates in the culture solution.

Therefore, it is not necessary to separate the bacterial cells from the culture solution and separate and extract the enzymes by grinding, sonication or autolysis as in the case of intracellular enzyme-producing bacteria.

〔Example〕

Hereinafter, the present invention will be described more specifically based on examples.

Example 1 Soluble starch 1.0% (w / v), 1.0% glucose (w / v), 0.75% meat extract (w / v), polypeptone 0.75% (w / v), MgSO 4 · 7H
₂ O 0.1% (w / v), trace metal solution (CuSO ₄・ 5H ₂ O 10.0 g, Mn
Dissolve 10.0 g of Cl ₂ · 4H ₂ O and 100.0 g of ZnSO ₄ · 7H ₂ O in 900 ml of distilled water, add hydrochloric acid until the liquid becomes transparent, and make 1 with distilled water) 0.1% (w / v) Nari medium 110 ml (pH 7.4)
Was added to a 500 ml-volume Sakaguchi flask and sterilized under high pressure at 120 ° C. for 20 minutes, inoculated with the Microtechnology Research Institute strain No. 5071, and cultured at 28 ° C. for 2 days with shaking to obtain seeds. Then inoculate this inoculum with glucose
20% (w / v), soybean flour 2.0% (w / v), polypeptone 0.2% (w / v),
Creatine 0.5% (w / v), NaNO ₃ 0.2% (w / v), KH ₂ PO ₄ 0.1% (w / v
_{v), MgSO 4 · 7H 2} O 0.05% (w / v), KCl 0.05% (w / v), FeSO 4
・ Transplanted into a sterilized 10 volume jar-famentor containing 7H ₂ O 0.0001% (w / v) medium (pH 8.0) 5
The culture was carried out with aeration and stirring at a rotation speed of 400 epm and an aeration rate of 0.8 l / min for 2 days.

Then, the obtained culture solution was cooled and centrifuged at 3,500 rpm for 20 minutes while cooling and centrifuging to remove bacterial cells and other solid contents, to obtain 4050 ml of a crude creatine / amidinohydrolase-containing solution. (Creatin / amidinohydrolase enzyme activity 3888 U) 2090 g of ammonium sulfate was added to this crude enzyme solution to recover a precipitate. The precipitate was dissolved in 500 ml of 20 mM Tris-HCl buffer (pH 7.8) and dialyzed overnight against the same buffer, and 20 ml of this solution was added to 20 mM Tris-HCl buffer (pH 7.
DE-AE-cellulose equilibrated in 8) (manufactured by Whatman)
Charge a column (diameter 3 cm x 21 cm) filled with 0.15
After washing with the same buffer containing M NaCl, NaCl 0.15M to 0.7M
After elution with a concentration gradient of, the active fraction was collected, and then this active fraction was concentrated using an ultrafilter (manufactured by Toyo Kagaku Sangyo Co., Ltd.), and then 20 mM Tris-HCl buffer (pH 7.8) After overnight dialysis against this, the solution was freeze-dried and creatine amidinohydrolase powder (total activity 1230 U, protein mass 350 m
g, specific activity 3.5 U / mg recovery rate 32.2%).

〔The invention's effect〕

As described above, according to the present invention, it is possible to efficiently obtain creatine amidinohydrolase from a cultured bacterial cell in a single-step operation without performing an extraction operation process.

Claims (1)

[Claims] 1. Microorganism Research Institute host belonging to the genus Alcaligenes and capable of producing extracellular creatine amidinohydrolase.
A method for producing creatine amidinohydrolase, which comprises culturing 5071 strain and separating and recovering creatine amidinohydrolase from the culture solution.