JPS6098982A - Preparation of enzyme for measuring polyamine - Google Patents

Abstract

PURPOSE:To obtain acetylpolyamine deactylase and polyamine oxidase as high- purity enzymes, efficiently, by cultivating a bacterium belonging to the genus Micrococcus, capable of producing both the enzymes, in a nutritive medium, accumulating both the enzymes in the culture, collecting them. CONSTITUTION:A bacterium capable of producing both the enzymes is cultivated in a liquid medium containing polyamine or acetylpolyamine as an induction substance for enzyme preparation. Consequently, acetylpolyamine deactylate and polyamine oxidase are simultaneously accumulated in the mold. After the cultivation is over, the mold is collected, the mold is ground, the enzymes are extracted, to give a crude enzymatic solution, which may be further purified, dried, and made into enzyme powder. The cultivation is carried out in the medium at 6-9pH by aerobic sulture such as aerated spinner culture, etc. at 20-37 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing acetylbolyamine deacetylase and polyamine oxidase by fermentation. More specifically, microorganisms belonging to the genus Micrococcus are cultured in a polyamine- or acetylpolyamine-containing medium, and both enzymes acetylfoliamine deacetylase and polyamine oxidase are cultured and stored at the same time. The present invention relates to a method for producing both enzymes such as polyamine oxidase and polyamine oxidase.

Acetylbolyamine deacetylase is an enzyme that hydrolyzes the acetyl bonds of acetyl polyamines such as acetyl butrescin, acetylspermine, and acetylspermidine to generate free polyamines.

Polyamine oxidase is an enzyme that oxidizes and decomposes various polyamines to produce various aldehydes, ammonia, and hydrogen peroxide.

In recent years, in the medical field, there has been much research into the relationship between the content of polyamines in body fluids, especially blood and cancer, and cancer diseases. Accordingly, methods for measuring polyamines include electrophoresis, thin-layer chromatography, gas chromatography, Amino acid analyzer methods, high performance liquid chromatography methods, enzyme methods, etc. have been developed. However, these measurement methods have the disadvantage that the sample is subjected to each measurement method after extensive and time-consuming pretreatments such as detensioning, hydrolysis, and extraction. The reason for this is that most of the polyamines in body fluids, blood γn, and urine exist as acetyl conjugates, that is, acetyl polyamines, and in each of the above measurement methods, it is necessary to use the acetyl polyamines in a free state for measurement. Because there is.

There is a demand for a more appropriate, faster, and easier measurement method from a clinical testing perspective, and therefore, the application of enzymes to the hydrolysis of the acetyl bond of acety)repolyamine and the measurement of free polyamines is being actively researched. There is.

Acetylbolyamine deacetylase is widely present in nature, especially in the organs of higher animals, and has traditionally been studied using cow and rat liver as a source. Furthermore, polyamine oxidases are widely present in nature, both in animals and plants, and in recent years, various persimmon polyamine oxidases have been developed by culturing microorganisms.

'Iff' due to hydrolysis of acetylpolyamine by acetylbolyamine deacetylase, depolyamine formation, and exit by polyamine oxidase. do. Quantification of hydrogen peroxide produced by the action of polyamine oxidase is extremely easy by using a well-known colorimetric method, and combining both enzymes acetylbolyamine deacetylase and polyamine oxidase allows for the determination of hydrogen peroxide. It offers great benefits for the determination of polyamines.

With the aim of industrial production of enzymes used in the measurement of acetyl polyamines using microorganisms, the present inventors searched widely for bacteria that produce enzymes that oxidize and decompose acetyl polyamines from the natural world, and selected among the strains in Fukuchiyama City, Kyoto Prefecture. Microfuccus bacteria that produce large amounts of enzymes that oxidize and decompose acetylpolyamines were discovered in the soil of Japan, and by cultivating the bacteria in their home country and purifying their cells and if''i fluid, they were able to efficiently and efficiently produce highly pure microfuccus bacteria. He devised a method for producing acetylpolyamine acetylase.As a result of detailed research on this enzyme, he discovered that it was a mixture of two types of enzymes.

One is an enzyme called acetylbolyamine deacetylase, which hydrolyzes acetyl polyamines such as acetylputrescine to produce free putrescine and acetic acid, and the other is an enzyme that oxidizes polyamines such as butledge/゛. It was discovered that it is a polyamine oxidase that produces aldehydes, ammonia, and hydrogen peroxide. Until now, it has never been known that two types of enzymes with such actions can be produced by one type of microorganism.

To measure polyamines such as acetylputrescine in body fluids, acetylbolyamine deacetylase and polyamine oxidase such as putrescine oxidase sourced from microorganisms are used together to quantify acetylputrescine. Although this is known, the present inventors are the first to reveal the existence of a microorganism that simultaneously produces extremely advantageous acetylbolyamine deacetylase and polyamine oxidase.

The present invention makes it possible for the first time to simultaneously produce the two types of enzymes mentioned above by culturing a unique type of microorganism, and this shows that the enzyme according to the present invention can be provided to society at low cost. The value is great.

The strain used in the present invention accumulates acetylbolyamine deacetylase and polyamine oxidase within the bacterial body by culturing in a liquid medium containing polyamine or acetylpolyamine as an enzyme production inducer.
After the culture is completed, the bacterial cells are collected and crushed to extract the enzyme to obtain a crude enzyme solution, which can also be further purified and dried to obtain an enzyme powder.

That is, this bacterium is cultured in a suitable medium, for example, a medium containing a suitable charcoal* source, nitrogen source, inorganic salts, polyamines or acetyl polyamines necessary for inducing enzyme production, and an organic promoter; As such, glucose, soluble starch, etc. are used. As a nitrogen source, organic nitrogen sources such as defatted soybean extract, 100% alcohol extract, polypeptone, and meat extract are effective. As the inorganic salts, dipotassium phosphate, magnesium sulfate, ferrous sulfate, common salt, etc. are used. Enzyme Raw Snake 14> As the reed substance, polyamines such as putrescine, spermine, and spermidine, or acetyl polyamines such as acetylputrescine and acetylspermine are used. Added dragon is 0.02-0.5% by weight
, preferably 0.05 to 0.2 weight ltt%. As organic promoters, yeast extract, meat extract, and corn stew brewer are recommended. The pH of the medium is 6.
-9, preferably 7-8, and aerobic cultivation such as aeration and stirring is carried out at 20-37°C, preferably 27-33°C. Cultivation time (varies depending on the grain) When reaching the maximum temperature, select 113, usually 16 to 36
It's time.

The enzymes acetylbolyamine deacetylase and BoIJ amine oxidase are extracted and purified from the culture obtained by the above method as follows.

After culturing, collect the bacteria from the culture by a method such as r-filtration or centrifugation, and disperse the wet bacteria in an appropriate amount of water as a solvent, preferably a buffer such as phosphate buffer. Suspension, preferably at a temperature below 30 degrees Celsius, can be easily lysed by lysis with lysozyme, crushed with Guinomi 11, crushed with ultra-H waves, etc., alone or in combination, to remove acetylbolyaminide from the bacterial body. Since acetylase and polyamine oxidase are simultaneously extracted into the solvent,
Collect this as a crude enzyme solution.

Furthermore, a crude enzyme can be obtained by subjecting this crude enzyme solution to ammonium sulfate fractionation and acetone/alcohol fractionation.

To obtain a more highly purified enzyme preparation, apply known protein and enzyme purification methods such as ion-exchange chromatography using DEiAK cellulose, gel filtration, etc. 1. In this way, a purified enzyme containing both of the above enzymes is obtained. I can do it.

Further, each of the obtained enzyme solutions can be made into a dry powder using a conventional drying method, or can be made into an immobilized powder by immobilizing it on a suitable carrier by a known method.

In the present invention, it is also possible to separately separate and separate acetyl ΔflJ amine deacetylase and lamine oxidase by changing the conditions such as min-1 in the above purification step. It also includes a method for producing VJ as a single enzyme agent. In order to use the enzyme containing acetyl polyamine deacetylase and bo-1 noamine oxidase according to the present invention for measuring the bo-1 noamine content in body fluids, the above-mentioned crude enzyme solution, m, t
N! ”v.

Purified enzymes, enzyme dry powders, and immobilized enzymes can be used, and the respective enzymes that have been single-treated as described above can be mixed again and used (T3-T3), and none of them are practical. No problem l/1゜Micrococcus 131 which has the ability to produce both acetylbolyamine deacetylase and polyamine oxidase of the present invention. Describe the mycological stinging quality of ;

a) Sugi Jin (1) Cocci (1.0-1.5μm) (2) Soren or Yotsutsuji (3) No interlocking, no flagella 4) No spores 5) Durham staining: positive (6) Anti-acidity: negative b) Growth status (2) Meat juice agar slant culture: Good growth. Stringy and shiny.

C) Physiological properties (1) Nitrate superconductivity: Negative (2) Vaccination reaction: Negative (3) M, R test: Negative (4) V, P test: Negative (5) Indole formation: Negative (6) Generation of hydrogen sulfide: Positive (7) Hydrolysis of starch: Negative (8) Utilization of citric acid: Positive (9) @Nitrogen source) H: Both nitrate and ammonium salt are positive (10 Pigment production: negative (11) Urease: positive (12) Oxidase double positive (13) Catalase: #, sex (14) Growth pH: 6.Q ~ 9.0 (15) Growth temperature = 20 ~ 376C (16) Attitude towards M element: Aerobic (17) o -F test: Does not decompose sugars (18) Generation of gas from sugars: Negative (19) Generation of acids from sugars: (G) is positive , ←) is negative L-albinose (-)
D-xylose ←) D-glucose ←) D-mannose ←) D-fructose (→ maltose (
→Sucrose ←) Lactose ←) Trehalose ←) D-Sorbitol (→D-Mannitol←)
Inositol ←) Glycerin ←) Starch ←) D-galactose ←) The properties described above were summarized in Bergey's Manual of Determinative Bacteriology, 8th edition (1977).
According to the classification of 4th year), this strain is Micrococcoccus.
It was recognized that the microorganism should be identified as Micrococcus luteus, and the present strain was named Micrococcus luteus-11. This strain has been submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Microbiological Research Center No. 73 (FIRM P-730).
0).

Below, various properties of acetylbolyamine deacetylase obtained by the present invention will be described.

(1) Action and substrate specificity This enzyme acts on acetylputrescine and hydrolyzes the acetyl bond to produce putrescine and acetic acid. It has little effect on acetylspermine and acederspermidine. The substrate specificity for each acetylspermine was shown by relative activity.

Acetyl spermidine 3.5 Acetyl spermine 261 (2) Titer measurement method The titer of this enzyme is measured as follows.

(Reaction solution composition) 0.5M Tris buffer (pH 8,0) 1.0rr
r14-aminoantipyrine (4q/y) solution 0.1
ml phenol (IOW/me) solution 01ml peroxidase (120 units/-) solution 0.05-acetylputrescine (2v/m/) solution 2.0ml Reaction solution 3.3- with the above composition was heated to 37℃ for 5 minutes. After incubating for 0.5 minutes, add 0.5 minutes of acetylbolyamine deacetylase enzyme solution of the present invention.
-Additionally, react at 37°C and measure the increase in absorbance at 505 nm per unit time. The titer is displayed once every 60 minutes.
The amount of enzyme that produces micromoles of putrescine was defined as one unit.

(3) Optimal pH As shown in FIG. 1, the optimum pH of this enzyme is between 8 and 9.

(4) Optimal temperature As shown in FIG. 2, the optimal temperature of this enzyme is 30 to 40°C.

(5) Stability The pH stability of this enzyme is stable at a pH of 7 to 9, as shown in FIG. Temperature stability is +! As shown in Figure 84, it is stable up to 40°C.

(6) Km value The Km value of book 1+if+ for acetylputrescine was 0.5mM.

(7) Effects of inhibitors and metal ions Table 1 shows the results of investigating the effects of 1S14 inhibitors and metal ions on this enzyme. The fermentation activity is 1 without additives.
The relative activity was expressed as 00%.

Table 1 describes the properties of the polyamine oxidase obtained by the present invention.

(1) Substrate specificity of action This enzyme acts on putrescine to produce ammonia and hydrogen peroxide. It has little effect on spermidine, spermine, and cadaverine. Furthermore, it does not act on acetyl polyamines such as acetylputrescine, acetylspermidine, and acetylspermine. The substrate specificity for each polyamine was expressed as relative activity.

Putrescine 100 Spermidine 4.1 Spermine 2.3 Cadaverine 0 Lysine 0 (2) Titer measurement method The titer of this enzyme is measured as follows.

(Reaction liquid composition) 0.5M) Risbara yr -(11)H8,0) 1.
0-4-aminoantipyrine (4~/+d) solution 0.
1-Phenol (10 units/-) solution 0.1-Peroxidase (60 units/-) solution 0.1-Putrescine (2 sv/mg) solution 2.0-Reaction solution with the above composition3.
After incubating 3- at 37°C for 5 minutes, 0.5- of the polyamine oxidase enzyme solution of the present invention is added and reacted at 17°C, and the increase in absorbance at 505 nm per unit time is measured.

The titer was expressed as 1 unit, which was the amount of enzyme that produced 1 micromole of hydrogen peroxide in 60 minutes.

(3) Optimum pH As shown in Figure 5, the optimum pH of this enzyme is between 8 and 9.

(4) Optimal temperature As shown in Figure 6, the optimal temperature for this enzyme is 30 to 50°C.

(5) Stability The pH stability of this enzyme is stable between 6 and 9 as shown in FIG. As for temperature stability, as shown in FIG. 8, it is stable up to 50°C.

(6) Km value The Km value of this enzyme for putrescine was O62mM.

(7) Effects of inhibitors and metal ions Table 2 shows the results of a W comparison of the effects of inhibitors and metal ions on this enzyme. The kr41 elementary activity is expressed as a relative activity, with the case of no addition being taken as 100%.

Acetylputrescine can be quantified using the acetylbolyamine deacetylase and dClyamine oxidase of the IWJ of the present invention having the properties as described above. The specific method will be explained below using reference examples.

Reference example (enzyme test solution composition) 0.5M Tris/'777 (pH 8,0) 1.0 shoulder/4-aminoantipyrine (4ki/-) solution 01Fg
Peroxidase (60 units/-) solution o, i y
Phenol (io-y, "*) solution 0.1 is the polyamine oxidase of the present invention C20M'-mlne) 0.
25rd Add acetylputrescine solutions of various concentrations to the enzyme reagent solution containing the acetylbolyamine deacetylase and polyamine oxidase of the present invention having the above composition. 〇－Additionally,
After reacting at 37° C. for 60 minutes, absorbance at 505 nm was measured. This result shows that there is a linear relationship between the concentration of acetylputrescine (μm) and the absorbance at 505 nm, as shown in Figure 9. It is recognized that putrescine can be quantified.

The present invention will be explained in detail with reference to the following examples, but the present invention is not limited thereby.

Percentages are by weight.

Example 1 Polypeptone 0.5%, yeast extract 0.05%, dietary mO
, 1%, acetylputrescine 0.05%, pH 7.0
Micrococcus of the present invention was placed in a jar fermenter (20 Or, p, m) containing 201 medium consisting of
Luteus was inoculated with -11, kept at 30°C for 24 hours with aeration and agitation, and the culture solution was centrifuged using a Sharpless type centrifuge for 1iflt.
#I41ζ was harvested 7, and the obtained wet bacterial cells 160F were used as an enzyme source. Disperse these bacterial cells in 1/100M phosphate buffer (1)H7,0)1600-! M and was ground in a dyno mill while cooling to 25° C. or less. The homogenated solution was clarified by centrifugation to obtain 50,600 units of acetylboriaminthiacetylase and 11 units of polyamine oxidase.
0.300 units of crude enzyme solution was obtained. Then 1/100 M
DEA in phosphorus exchange buffer (pH 7,0)
I-cellulose (manufactured by Pharmacia) column (packed @
2000 ml) was subjected to IR filtration to adsorb both enzymes, and after washing with the same buffer as above, both enzymes were eluted with the same buffer containing 0.4M NaCl. The eluate was desalted and concentrated using the ultraviolet 1 filtration method, and freeze-drying was not performed. 2.17
A dry powder of was obtained. This powder was an MLIC containing 35,400 units of acetylboriamin thiacetylase and 76,200 liters of polyamine oxidase.

Example 2 The same procedure as in Example was carried out except that putrescine was used instead of acetylputrescine in the medium shown in Example 1, and enzymes containing 26,000 units of acetylbolyamine deacetylase and 89,400 units of polyamine oxidase were used. Obtained.

Example 3 DI of the extract obtained by the same culture method shown in Example 1! tA11! - In the operation of a cellulose column, instead of eluting with the same buffer containing 0.4M NaCl, if the adsorbed protein is eluted by a saline concentration gradient elution method from 0 to IM, 0.2~0.25M NaCl 171
4! The elution class is divided into the elution class of 0.3-0.4M NaCl and the elution class of 0.3-0.4M NaCl.
0 units), the latter is acetylbolyamine deacetylase (
39,100 units). Each was desalted and concentrated by ultrafiltration and freeze-dried to obtain 1.2z polyamine oxidase powder (70,900 units) and 1.41 acetylboramine deacetylase powder (29,700 units).

[Brief explanation of drawings]

FIG. 1 shows the pH activity curve, FIG. 2 shows the temperature activity curve, FIG. 3 shows the pH stability curve, and FIG. 4 shows the temperature stability curve of the acetylborium deacetylase according to the present invention. Fifth
The figure shows the pH activity curve, FIG. 6 shows the temperature activity curve, FIG. 7 shows the pH stability curve, and FIG. 8 shows the temperature stability curve of the polyamine oxidase according to the present invention. FIG. 9 shows the linear relationship between the acetylputrescine p degree and the absorbance of the reaction solution at 505 nm using the enzyme agent of the present invention. Special feature person Nagase Seikagaku Kogyo Co., Ltd. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

Claims (1)

[Claims] 1. A microorganism belonging to the genus Micrococcus and having the ability to produce both acetylbolyamine deacetylase and polyamine oxidase is cultured in a nutrient medium, and the culture contains acetylbolyamine deacetylase and polyamine oxidase. A method for producing acetyl polyamine deacetylase and polyamine oxidase, which comprises accumulating both polyamine oxidase enzymes at the same time and collecting them. 2. An acetylbolyamine, which is characterized by culturing a microorganism belonging to the genus Micrococcus and having the ability to produce acetylbolyamine deacetylase in a nutrient medium, accumulating acetylbolyamine deacetylase in the culture, and collecting the acetylbolyamine deacetylase. Method for producing amine deacetylase. 3. A method for producing polyamine oxidase, which comprises culturing Micrococcus luteus -11 in a nutrient medium, accumulating polyamine oxidase in the culture, and collecting the polyamine oxidase. 4. The production method according to claim 1, 2 or 3, wherein the nutrient medium is a medium to which 0.02 to 0.5 weight percent of polyamine or acetyl polyamine is added. 5. The 1# manufacturing method according to claim 1, science, 2 or 4, which uses Micrococcus luteus -11 as the microorganism.