JPS6050438B2 - Method for liberating polyamine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for liberating polyamines by hydrolysis of acyl polyamines.

More specifically, microbial cells having the ability to decompose acyl polyamines, crude enzyme extracts or purified enzymes obtained from the cells, or crude enzyme extracts or purified enzymes having the ability to decompose acyl polyamines obtained from animal organs are used to act on acyl polyamines. This is a method for liberating polyamines, which is characterized by: Polyamines such as putrescine, spermidine, and spermine are widely present in living organisms ranging from bacteria to higher animals and humans, and are involved in cell proliferation.

Since the study by Ratsell et al. in 1971 [Cancer Research, Vol. 31, 1555-1558 (1971)], the correlation between cancer and polyamine concentrations in body fluids has attracted attention, and many studies have been conducted.

According to these studies, polyamines are abundantly contained in tissues in which protein synthesis and nucleic acid synthesis are active in living organisms, and are said to be deeply involved in nucleic acid metabolism and cell proliferation. It is becoming clear in many cases that in cancer patients who have cancer cells that are actively proliferating, the amount of polyamines in body fluids such as blood and urine is increased compared to healthy people. Therefore, measurement of polyamines in body fluids such as urine and blood has the potential to become a promising clinical test method for diagnosing cancer.

Conventional cancer diagnosis methods include X-ray examinations, histological examinations, endoscopy, and gastrocamera examinations, but all of these methods are inadequate for early diagnosis, and a new and simple method is needed. The development of early diagnosis methods for cancer is desired.

In this sense, there are great expectations for the development of a new cancer diagnosis system that focuses on polyamine levels in body fluids.
Conventionally, many methods such as high performance liquid chromatography and enzymatic methods have been proposed as methods for quantifying polyamines in living organisms.

A problem when analyzing polyamines in biological samples is that polyamines do not often exist in free form in living organisms, but as polyamine complexes.
Although the actual nature of polyamine complexes is not necessarily clear,
There are one or two reports regarding this polyamine composite. Journal of Marmode et al.
67, Volume 1, 1671-3 (197
According to 8), polyamines in human urine exist as acetylpolyamines such as acetylputrescine, acetylspermidine, and acetylspermine, and Chiang et al.
a) Acta), Volume 91, 233-241 (1979
) reported that polyamines in human amniotic fluid exist as polyamine complexes covalently bonded to peptides. Therefore, it can be said that the polyamine complex is an amide bond between a polyamine and an acyl group such as acetyl or a peptide, that is, it has an acyl polyamine as a main component.

1 However, when quantifying polyamines in urine, whether the quantitative method is liquid chromatography or enzyme method, pretreatment is required to decompose the acyl polyamines and convert the polyamines into free forms prior to analysis. It is.

The conventional pretreatment method is hydrochloric acid hydrolysis.

One example of this is to add concentrated hydrochloric acid in the same amount as urine, seal it in a glass ampoule, and heat it at 120°C for 3 hours. However, such pretreatment is complicated and dangerous, and no matter how good the subsequent quantitative method itself is, this pretreatment step remains a bottleneck in the analysis of polyamines in biological samples. I can say that. The present inventors have conducted intensive research to find a simpler pretreatment method to replace the conventional hydrochloric acid decomposition method in order to establish a method for analyzing polyamines in biological samples that is easier and more convenient to operate.
As a result, cells of microorganisms belonging to a specific genus that have the ability to decompose acyl polyamines, crude enzyme extracts or purified enzymes obtained from the cells, or crude enzyme extracts or purified enzymes that have the ability to decompose acyl polyamines obtained from animal organs are obtained. We discovered that polyamines can be liberated by acting on amyl polyamines, and that this can be applied to liberate polyamines from polyamine complexes in urine.
The present invention has now been completed. The microorganisms having the ability to decompose acyl polyamines used in the present invention include Escherichia sp., Enteropacter sp., Serratia sp., Bacillus sp., Agrobacterium sp., Micrococcus sp., Staphylococcus sp., Arthrobacter sp., Pseudomonas sp. , Xanthomonas, Proteus, Plevibacterium, Asbergillus, Penicillium, Rhizopus, Mucor, Pericyularia, Trichophyton,
Fusarium, Neurospora, Monascus, Pyricillaria, Cladosporium, Satucharomyces,
Satucharomycopsis, Schizosatucharomyces, Pichia, Hansenula, Kuriyiberomyces, Debaryomyces, Cryptococcus, Cretschella, Bitskelhamia, Quiandeida, Rhodotorula, Trichosporon, Cytelomyces , Endomyces, Rhodosporidium, Streptomyces, Actinomyces,
Examples include microorganisms belonging to the genus Nocardia, Mycobacterium, Streptoverteicilum, Micromonospora, Actinoplanes, Micropolyspora, Spirillospora, or Streptosporangium, and among these, especially Pseudomonas, Microorganisms belonging to the genus Proteus, Rhizopus, Fusarium, Rhodotorula, Trichosporon, Streptomyces, Nocardia, or Streptoverteicilum are preferably used.

Strains of these microorganisms include, for example, Escherichia
Cori BIF'013168, Enteropacter Cloake IAMl22l, Serratia, Marcescens IFOO
3O54, Bacillus subtilis IFO3O26, Agrobacterium rhizogenes IFO13257, Micrococcus roseus IFO376tun Micrococcus ●
Roseus IFO3768, Staphylococcus ●Aureus IF'012732, Arthrobacter ●Simplecus IF'012069, Pseudomonas iodium IFO355F3sPseudomonas ●Ovaris IF'
0373 Pseudomonas Aeruginosa IFO344
＼Pseudomonas●Aureofaciens IF'035
21. Pseudomonas chlorolahuis IFO39O4
, Pseudomonas denitrificiens IF'013
302, Xanthomonas campestris IFOl35
5l, Proteus lettuge IFOl35Ol, Previbacterium ● Ammoniagenes IFOl2O72,
Aspergillus niger 1F06661, Aspergillus Tereus IFO6l2λ Penicillium Chrysogenum IF'04626, Rhizopus Deremer 1F0540
Sakamukor ●Jiabanicus IFO457 Hepericularia filamentosa IFO8985, Trichophyton
Mentagrophytes IFO58Oλ Fusarium oxysporum IFO7l9Ol Pyricularia oryzae 1
F'05994, Neurospora◆Curasa IFO3O2l
5. Monascus ● Anka IFO654Ol Cladosporium ● Herbalum IAMF5l7, Saccharomyces ● Saccharomyces cerevisiae IFOO25λ Saccharomyces cerevisiae I
F'00216, Schizocalomycopsis Capsularis ■FOO672, Schizosaccharomyces bombe IFOO
346, Pihia, Membrane Huaciens IFOOl2
& Pihia, Bitskelhamii IFOl27F3s Hansenula saturnus IFOOll7, Hansenula, Anomala IFOO569s Kuryuiberomyces lactis IFO
lO9 Hedevariomyces globozaus [F'00794
, Cryptococcus neofuormans IFOO 4l Hekletsukura ● Magna IF'00868, Bitzkelhamia fluorescens IF'00868, Candida lipolytica IFOO7l7, Candida albikiyans IFOO 57gs Rhodotorula ● Glutinis Var.
Direnensis IFOO4l\Rhodotorula●Glutinis Var. Lusitanica IFOllf! K Trichosporon cutaneum IFOll9 & Citelomyces pineuritensis IFOO954, Endomyces obetensis IF
Ol2Ol, Rhodosporidium toruloides IFOO
55λ Streptomyces Avelanius IFOl345
l, Streptomyces Avelanius R-20 (Feikoken Bacteria No. 5443), Streptomyces gricius I
FOl2875, Streptomyces 1st Kellicolor 1F
01285tun Streptomyces hygroscopicus IF
Ol3472, Streptomyces ◆ Frazier IFOl2
77 Streptomyces●Alps IFOl3Oltun Streptomyces●Lavenjiure IFOl278λ Streptomyces viridoclo1 Mogenes■FOl3347,
Streptomyces aureofaciens IFOl28
4 Nuactinomyces fluorescens IFOl286l
, Nocardia●Erythropolis! FOl2682, Nocardia corallina IFO3338, Streptoverteicirum, Hiroshi 2 Mense IFOl2785, Mycobacterium prey IFOl3l6Ol Mycobacterium smegmatis IFO3l54, Micromonospora chiarse Var. ismensis IFOl 298 & Actinoplanes armeniacus IFOl 255＼ Micropolyspora●Cesia IFOl 299O, Spirillospora albida IFOl 2248, Streptosporangium●Roseum IFOl 377 Sow.

Among these strains, Pseudomonas obalis IFO3
738, Pseudomonas denitrificiens IFO
l33O2, Proteus retsutoge JanuaryFOl35Ol
, Rhizopus delemer 1F05406, Fusarium *Oxysporum IFO7l9O, Rhodotorula *Glutinis Var. Lusitanica IFOO688, Trichosporon
Cutaneum IFOll98, Streptomyces abellanius R-201 Nocardia Erythropolis IFO
l2682, Nocardia corallina IFO3338s
Streptoverteicilum hiroshimense IFOl27
85 etc. are preferably used. The mycological properties of Streptomyces averanius R-20, which is a strain newly isolated by the present inventors among the above-mentioned strains, will be explained in detail below.

(1) Morphology Under a microscope, this strain extends aerial hyphae from branched basal hyphae, and the tips of these hyphae exhibit a straight or wavy shape.

It does not show whorled branching, and no formation of special organs such as sclerotia is observed. The mature spore chain has a spore chain of 1C@ or more, the size of the spore is cylindrical, 0.6 to 1.1 x 1 to 1.9 microns, and the surface of the spore is smooth. Growth status and physiological properties in various media Unless otherwise specified, cultivation was carried out at 30°C.

In addition, no production of soluble pigment was observed in any of the following media. 1 No growth on sucrose/nitrate agar is observed.

2 Glucose●Asparagine agar The growth is olive gray, and the aerial mycelium is light brownish gray.

3 Glycerin/asparagine agar The growth is light olive color, and the aerial mycelium is light gray.

4. Growth on starch/inorganic salt agar is gray-olive color, and aerial mycelium is light olive-gray color.

Water decomposition of starch was observed from around the 5th day after culturing. 5
The tyrosine agar growth is gray-olive, and the aerial mycelium is gray-yellow-brown.

6 Nutrient agar The color grows to a light olive gray color, and no aerial mycelium is observed.

7 Yeast/malt agar Growth is grayish-yellow-brown, with brownish-gray aerial mycelia attached.

8 Oatmilk agar The growth is gray-olive, and the aerial mycelium is light gray.

9 Glucose ● Peptone ● Gelatin puncture culture Aerial adhesion of mycelium is not observed.

Liquefaction of gelatin was observed from around 9 days after culturing.

10 skimmed milk powder No aerial mycelium was observed.

From around the 9th day after cultivation, the color changes to a light cream color, and from around the 16th day after the cultivation, it changes to a light yellowish brown color, indicating coagulation of skim milk powder.

11 Production of melanin-like pigments No melanin-like pigments were produced in either tyrosine agar or peptone yeast iron agar media.

12 Utilization of carbon sources in Pridham Goshita Sorb agar D
- Glucose ● Sucrose is often used, and D-fructose is also used, but D-arabinose, D-xylose, inositol, L-rhamnose, raffinose, D-
-Do not use mannitol.

13 Optimum growth temperature 6 using maltose yeast agar (PH7.O)
℃, 12PC119℃, 26℃, 35℃, 40℃, 43
As a result of tests at various temperatures of 177°C to 35°C, growth is possible at 177°C to 35°C, but around 26°C seems to be the optimum temperature.

To summarize the above results, this strain belongs to the genus Streptomyces, the tips of the hyphae are linear or wavy, and the surface of the spores is smooth.

Bright olive-gray to brown-gray aerial mycelium grows on bright olive-gray to gray-olive growth on various media, and soluble pigments and melanin-like pigments are not observed. It has proteolytic ability and starch water decomposition ability. D as a carbon source
- Utilizes glucose, sucrose, and D-fructose. Based on these properties, similar known bacterial species were identified by ISP (
InternationalOnalStreptOmyces
A search using Streptomyces averaneus (PrObject) revealed that Streptomyces averaneus (StreptOmy
cesaVellanellS).

This strain meets all the characteristics of Streptomyces averaneus described by ISP. Ru. Also, Bergey's ManualOfDetermi
nativeBacteriOIOgy. ．． 8th Ed
．． ), but the morphology of the spore chain is 0pen100p. ．． c0i1
Although there are some differences in that it belongs to Streptomyces averaneus or HOOks, in other respects this strain satisfies all the characteristics of Streptomyces averaneus. Therefore, this bacterial strain was identified as a strain belonging to Streptomyces avellaneus, and was identified as Streptomyces avellaneus ◆R-20 (
StreptOmycesavellaneusR-2
0). This bacterial cell is StreptOmyc.
It has been deposited at the Microbial Technology Research Institute as es.Avellaneus R-20. (Fiber Engineering Research Institute No. 544
No. 3) Animal organs used in the present invention include cow, pig,
Examples include liver, kidney, pancreas, heart, etc. of animals such as chicken, and chicken and pig liver are particularly preferably used. The medium used for culturing each of the above-mentioned microorganisms may be either a synthetic medium containing a carbon source, a nitrogen source, an inorganic salt, or other nutrient source, or a natural medium.

As the carbon source, commonly used carbon sources such as glucose, sucrose, fructose, glycerol, starch, etc. may be used. As the nitrogen source, inorganic nitrogen compounds such as ammonium sulfate and ammonium chloride, or organic nitrogen compounds such as peptone, meat scratches, corn steep liquor, yeast scratches, and asparagine amino acids are used. Monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, etc. are used as the inorganic salt, and a surfactant such as Adekanol LG-805 is added as an antifoaming agent, if necessary. It is also important to add acylpolyamines such as atylputrescine and benzoylputrescine to the medium as enzyme production inducers.Acylpolyamines may be added to the medium from the beginning or , may be added during the cultivation. Also, trace components necessary for growth promotion such as yeast scratches, vitamins, nucleotides, etc. may be added as necessary. The medium may be in either liquid or solid form. Also, the cultivation method may be cultured statically, with shaking, or with aeration, but liquid culture with aeration and agitation is suitable for mass culture.The culture temperature is 1.
The temperature is 2-35°C, preferably 26-30°C. When carrying out the method of the present invention, the method can be roughly divided into (1) a method using bacterial cells, (2) a method using a crude enzyme extract obtained by crushing bacterial cells or an enzyme purified from this, and (3) a method using an enzyme purified from animal organs. There is a method using the obtained crude enzyme extract or an enzyme purified from it.

Among these, (1) the method of using bacterial cells includes modes such as using the culture as it is, using bacterial cells separated by centrifugation from the culture, and using freeze-thawed bacterial cells. Furthermore, in the case of a method using bacterial cells, adding a small amount of an organic solvent such as toluene, a surfactant, etc. has the effect of making the reaction proceed smoothly. Among the above methods, the bacterial cells obtained by culturing in the presence of acyl polyamines are isolated,
It is preferable to use an enzyme preparation prepared by purifying a crude enzyme extract obtained by disrupting cells by ultrasonication or the like by ammonium sulfate fractionation, dialysis, column chromatography, gel filtration, or the like.

An example of such a preparation is an enzyme preparation obtained by purifying acylpolyamine amidohydrolase produced by Streptomyces averanius R-20. Streptomyces Avelanius R-20 was inoculated into a medium containing 0.1% acetylputrescine and cultured for 1 turbidity at 30°C in a 20' jar fermenter.The cells were separated from the culture solution and cultured in a Dynomil. The physicochemical properties of the acyl polyamine amide hydrolase obtained by crushing the bacterial cells and purifying the obtained crude enzyme extract by ammonium sulfate fractionation, DEAE-cellulose, hydroxyapatite column chromatography, Sephadex G-200 gel filtration, etc. are as follows. That's right. To measure the activity of this enzyme, mix 2.0ml of 100rnM phosphate buffer (PH8.O) and 0.05ml of 2507nM acetylputrescine 0.2mt1 enzyme solution.
A two-part reaction is carried out at 25°C, the reaction is immediately immersed in boiling water for 1 minute to stop the reaction, and the putrescine produced is quantified by a putrescine oxidase assay method.

That is, the reaction mixture and mixture were mixed with 1007N, M phosphate buffer (P
H8. 0.2 ml of 0.1% 0-aminobenzaldehyde solution and 0.05 mt of putrescine oxidase (20 uImt) were added to 2.0 ml of the diluted solution, mixed and reacted at 30°C for 1 hour at 435 nm. Absorbance of (A
435). Putrescine oxidase ●The concentration of putrescine in the assay solution is determined by the following formula. Putrescine (μMOle ml) = A435/2.1 Enzyme activity is defined as 1 unit (1 u.) of enzyme activity that produces 1 μMole of putrescine in one powder.

In addition, the enzyme specific activity is 1.0 mgI of protein when the absorbance (A28O) at 280r1TrL is 1.0.
The activity per unit protein amount (U
．． lmg). 1 substrate specificity 25771. The relative activity of this enzyme for each substrate at the concentration of M is 100% for the activity for acetylputrescine.
When displayed as , it becomes as shown in Table 1.

Optimal pH The optimum pH of this enzyme is between 7.2 and 7.8 (Figure 1).

Optimal temperature for action The optimal temperature for action of this enzyme is 30-40°C (Figure 2).

Stability This enzyme is stable at pH 6.5 to 8.5, but the most stable pH is around 7.5 ft (Figure 3).

As for temperature, it is stable up to 35°C, but its activity decreases at temperatures higher than that, and at 60°C, it is almost completely inactivated after one drop (Figure 4). In addition, 20mM phosphate buffer (
It is stable for more than two weeks at 5°C in pH 7.2), and is stable for more than one month if stored frozen at -20°C. )
Effects of inhibitors, etc. As shown in Table 2, this enzyme is susceptible to copper, zinc, manganese,
Although it is inhibited by metal ions such as cobalt and nickel, parachloromercribenzoate (hereinafter abbreviated as PCMB), iodoacetamide, and O-phenanthroline, ethylenediaminetetraacetate (hereinafter abbreviated as EDTA), sodium azide, It is not inhibited by β-mercaptoethanol or the like.

It is also not inhibited by spermidine, spermine, acetylspermine, etc. 5.5″-dithiobis(2-
Nitrobenzoate) (hereinafter abbreviated as DTNB) and dipyridyl promote the enzymatic reaction.

7Km value The Km value of this enzyme for acetylputrescine is 2.0×
It is 10-3M.

In addition, for acetylspermidine and benzoylputrescine, 1.1 x 10-3 and 6.7 x 10-3, respectively.
It is 5M. In the present invention, there are no particular limitations on the method of allowing the bacterial cells, crude enzyme extract, or purified enzyme to act on the acylpolyamine, and a method of adding these to a solution containing the acylpolyamine is generally employed. Furthermore, it is also possible to use cells, crude enzyme extracts, or purified enzymes that have been immobilized by a commonly used method for immobilizing cells and enzymes.
The reaction conditions for the reaction that decomposes the acyl polyamine to generate free polyamine are as follows: pH of the reaction solution is 6.5 to 9.
．． A value of 7 to 8 is preferable.

The reaction temperature is usually selected appropriately below 10°C, but preferably from 25 to 35°C. The acylpolyamine to be used in the method of the present invention includes putrescine on the polyamine side,
Acylpolyamines such as spermine and spermine, or organic acid residues such as acetyl and benzoyl groups, and amino acid residues such as glycyl and alanyl groups,
Examples include acylpolyamines, which are peptide residues. The method of the present invention can also be carried out in cases where these acyl polyamines are present in human body fluids such as urine and blood. Therefore, the method of liberating polyamines of the present invention provides an extremely simple method to replace the conventional hydrochloric acid decomposition method as a pretreatment method when analyzing polyamines in body fluids.

For analysis of polyamines in body fluids after the polyamines have been liberated, known methods for quantifying polyamines such as high performance liquid chromatography and enzymatic methods can be used. Next, the present invention will be explained with reference to Examples, but the present invention is not limited to these Examples.

Examples 1 to 59 Glucose 0.5%, Meat processing scratches 0.2%
, peptone 0.4%, yeast scratches 0.2%, salt 0.2
% and acetylputrescine 0.1% (PH 7.O) into large test tubes, sterilized at 121°C, and inoculated with 1 platinum loop of various microorganisms from slant culture at 30°C. C124~4? It was cultured.

After culturing, the bacterial cells were collected by centrifugation, and 0.85%
After washing with saline, add 5 m of 100 Tn, M phosphate buffer.
1 and ultrasonic treatment (20kC1) under cooling at -5℃.
5 minutes). This sonicated solution was centrifuged to obtain an enzyme extract. To 0.2 mt of enzyme extract, add 1.8 ml of 100TrLM phosphate buffer (PH8.O), 0.2 ml of 0.1% ortho-aminobenzaldehyde, 0.05 ml of putrescine oxidase (25 U.), and 0.2 mt of 107 nM acetylputrescine. The reaction was carried out at 30° C. for 30 minutes, and the absorbance of 435 nrr1 was measured. That is, a method was adopted in which a reaction for releasing putrescine by allowing an enzyme extract to act on acetylputrescine and a reaction for enzymatically quantifying the produced putrescine using putrescine oxidase were carried out simultaneously. The results are shown in Table 3. The produced putrescine (μMOle) in the table is the amount of putrescine produced by the action of 0.2 mt of crude enzyme extract in the reaction of 30° C13 powder, and the activity per ml of medium is the amount of putrescine produced by the reaction of 30° C13 powder. The enzyme activity is calculated per 1 mt of the original culture solution. Examples 60-73 Acetylputrescine 0.5%, ammonium chloride 0.3
%, magnesium sulfate 0.1%, monopotassium phosphate 0.
1%, dipotassium phosphate 0.1%, sodium chloride 0.
2%, yeast scratch 0.02% medium (PH7.O) 10
Tests were conducted in the same manner as in Examples 1 to 59 except that TILL was used.

The results were as shown in Table 4. Example 74~
80 chicken liver, cow liver, pancreas, heart, pig liver, pancreas, heart, frozen fresh organs (30y each) were sliced with a knife and added to 50mM phosphate buffer (PH7.
O) After suspending in 150ml and crushing with a mixer, the supernatant was collected by centrifugation, and this was used as a crude enzyme solution.

For the hydrolysis reaction of acetylputrescine, crude enzyme solution 0.1
Using mt, 17TL. M putrescine solution 2.35ml
I did it inside. Free putrescine was produced by an enzymatic method using putrescine oxidase, and the reaction was carried out in a reaction system in which the hydrolysis reaction of acetylputrescine and the enzyme assay were carried out simultaneously. The enzyme assay used in this case was to colorimetrically quantify hydrogen peroxide produced by the oxidation of putrescine using a peroxidase/4-aminothipyrine/dichlorophenol system. PH of reaction solution
The reaction was carried out at 8.0130°C for 2 hours. 5th result
Shown in the table. Example 81 Glucose 0.5%, Polypeptone 0.8%, Yeast Scratch 0.2%, Salt 0.1%, Acetylputrescine 0.1%, Adekanol LG-8050
．． A medium containing 0.01% (PH 7.0) 10' was placed in a 20e jar fermenter and sterilized for one batch at 121°C.

To this, Streptomyces averanius R-20 (Feikoken Bacteria No. 5443) was added to the medium with the same composition as above for 500 g.
Inoculate the cultured material in a mL Sakaro flask as a seed,
Culture was carried out at 30° C. for 2 hours at a stirring speed of 600 r'Pml and an aeration amount of 1 vvm to obtain 180 q of wet bacterial cells. Wet bacterial cells 15
0y was suspended in 400ml of ion-exchanged water, the cells were disrupted by ultrasonication, and the supernatant was obtained by centrifugation to obtain 420ml of crude enzyme extract.
m1 was obtained. Using 500μ' of this crude enzyme solution, 250r
It was added to a reaction solution consisting of 0.2 ml of rLM acetylputrescine and 2.0 mL of 100 wLM phosphate buffer (PH8.O), and reacted at 25°C for 20 minutes. The reaction solution was immersed in a boiling water bath to stop the reaction, and then the free putrescine produced was quantified by an enzymatic method using putrescine oxidase. That is, the reaction solution was diluted, and 0.1% o-aminobenzaldehyde solution 0.2TI111 putrescine oxy,dase (
After adding 0.05 mL of 25UIm1) and incubating at 30°C for 11 hours, the absorbance of 435n7n was measured. In addition, for quantitative determination, the absorbance was subtracted using a control sample that was subjected to the same procedure except that 0.2 mL of the substrate solution was not added. As a result, the reaction of 2 Gin was 3.
6pm01e of free putrescine was produced. (Conversion rate 7
2%) This is 36U1mL (as the enzyme activity of the crude enzyme solution)
36 μMOLelminITnt). Example 82 In Example 81, ammonium sulfate was added to the crude enzyme extract obtained from the cells cultured with Streptomyces Avelanius R-20 to make it 70% saturated, and the resulting precipitate was collected by centrifugation (10n1.M It was suspended in phosphate buffer and dialyzed against the same buffer for 2 hours.

The dialysis fluid was preliminarily mixed with 10mM phosphate buffer (PH7.2).
A DEAE-cellulose column (4.5 x 4
5cm) and salt (a). Concentration gradient elution was performed with the above buffer containing 1 to 0.5 M). The active fractions were collected, precipitated again with ammonium sulfate (70% saturation), and dialyzed in the same manner. M phosphate buffer (PH7.2)
A hydroxyapatite column (1.2X20
CTf1). After concentrating the active fraction by ammonium sulfate precipitation and dialysis, it was added to a 20mM phosphate buffer (PH7).
．． Sephadex GT-200 column prepared in 2) (
Purified by gel filtration (1.7 x 55 cm). The purification pattern is shown in FIG. In this way, protein 1
50ml of purified enzyme solution containing 2.5m9 was obtained. The hydrolysis reaction of acetylputrescine was carried out in exactly the same manner as in Example 81, except that 500 μe of this purified enzyme solution was used. The free putrescine produced in the reaction between the two particles was 29 μMole.
(Conversion rate 56%).

This corresponds to an enzyme activity of 28 UImt in the purified enzyme solution, and a specific activity of 112 U1m9-protein. Example 83 Purified enzyme solution obtained in Example 82 500
Using Pe, 100w1,M solution of various acyl polyamines 0.5nt10. IM phosphate buffer (PH7.8) 1
．． Add to the reaction solution consisting of 35mt and mix at 30℃
6 Conflict reaction was carried out.

After terminating the reaction by adding 0.1 Tnt of 50% Torikuyamareacetic acid, the free polyamine produced was quantified by high performance liquid chromatography.

The results are shown in Table 6. In addition, the purified enzyme was added to 0.2 ml of 1.25 mM acetylputrescine at various concentrations (1, 2,
3u. /2.5mt) and the total volume was made up with 100TrLM phosphate buffer (PH8.O). After reacting 15, 30, 6 powders of the mixed solution in a volume of 2.5 ml at 30°C, immediately immerse it in boiling water for 1 minute to stop the reaction,
The putrescine produced was quantified by high performance liquid chromatography. The results are shown in FIG. Example 84 Using the purified enzyme solution 500Pe obtained in Example 82,
007nM acetylputrescine solution 0.5mt1100
771. It was added to a reaction solution consisting of 1.35 ml of M phosphate buffer (pH 7.8), and three-powder reactions were carried out at each temperature.

The generated free putrescine was quantified by the same enzymatic method as in Example 81. The results are shown in Table 7. Example 85 20 ml of urine was collected and the pH was adjusted to 7.0 with 1N caustic soda.
After adjusting to 87, 1 ml of the purified enzyme solution obtained in Example 82
1 (enzyme activity 28 UImt) was added and incubated at 30°C for 1 hour.

After filtering the reaction solution, the filtrate was passed through a 700.5 ml column of cation exchange resin Biorex, and then the column was washed with about 20 ml of 0.2N sodium citrate solution (pH 7.0), followed by 0.5N hydrochloric acid solution. Elute with eluent 4.
m1 was obtained. When putrescine in this eluate was analyzed by high performance liquid chromatography, the concentration of putrescine was 9.
It was 8.0 μM. This corresponds to a putrescine concentration of 19.6 μM in the urine sample. In addition, except that 20 ml of 10 μM acetylputrescine was used instead of 20 ml of the urine sample, the same procedure as above was performed to obtain 4 ml of 0.5N hydrochloric acid eluate, and the putrescine concentration was analyzed by high performance liquid chromatography. It was 49.0 μM. This value corresponds to the concentration of acetylputrescine in the original sample of 9.8
Corresponds to μM. This shows the validity of the intermediate operations in this embodiment. Comparative Example 1 20 ml of the same urine sample used in Example 85 was subjected to the same procedure as in Example 85, except that no enzyme solution was added, to obtain 4 ml of a 0.5N hydrochloric acid eluate.

The concentration of putrescine in this solution was analyzed by high performance liquid chromatography and was found to be 8.2 μM. This corresponds to a concentration of putrescine in urine of 1.6 μM. This comparative example shows that the concentration of free putrescine present in the urine sample used in Example 85 was as low as 1.6 μM, and most of it existed as a complex, which was liberated by the action of the enzyme. Ru. Comparative Example 2 10 ml of concentrated hydrochloric acid was added to 10 ml of the same urine sample used in Example 85, sealed in a glass ampoule, and heat-treated at 1200C for 3 hours.

After opening the ampoule, it was concentrated to dryness under reduced pressure, dissolved again in 10 mt of water, and after filtration, the filtrate was adjusted to pH 7. Example 8
In the same manner as in 5, 0.5 mL of Biorex was treated with the column and the following operations were performed. The putrescine concentration of 4 TnL of the 0.5N hydrochloric acid eluate was measured by high performance liquid chromatography and found to be 51.0 μM.

This corresponds to a concentration of 20.4 μM in the original urine sample. The hydrochloric acid decomposition treatment of the urine sample in this comparative example is a conventional method that has been used to analyze polyamines in urine. Comparing this comparative example with Example 85, it is clear that the treatment of urine according to the method of the present invention is an extremely effective method for decomposing polyamine complexes in urine and liberating polyamines. Example 8
6 Collect 20ml of urine sample and adjust the pH to 7 with 1N caustic soda.
．． 8, and then 1 ml of the purified enzyme solution obtained in Example 82.
L was added and incubated at 30'C for 1 hour.

Putrescine and spermidine in the reaction solution 5 were analyzed by high performance liquid chromatography after the same treatment as in Example 85. The concentrations of putrescine and spermidine in urine were 15 μM and 4.2 μM. Comparative Example 3 When the same urine sample as in Example 86 was subjected to the same procedure as in Example 86 except that the enzyme solution was not used, neither putrescine nor spermidine was detected.

Example 87 20 ml of urine sample (different from Example 85) was collected, 3.0 ml of 1M Tris buffer (PH8.O) was added thereto, 1 ml of the purified enzyme solution obtained in Example 82 was added, and the mixture was heated at 30°C.
and incubated for 1 hour.

After adjusting the pH to 6 by adding 1N hydrochloric acid to the reaction solution, 700.5 ml of cation exchange resin Biolex was added, and 2 ml of water was added.
After washing with 0ml, elute with 4.0mL of 0.5N hydrochloric acid,
After adding 1.2 ml of ΔHillis solution to the eluate and adjusting the pH to 8.0, polyamines were analyzed by an enzymatic method.
That is, an enzymatic method using putrescine oxidase was used, and hydrogen peroxide produced was determined by a colorimetric method using a peroxidase/4-aminoantipyrine/dichlorophenol system. The polyamine concentration in urine measured by this method was 23 μM. Example 88 (Hydrolysis of acetylputrescine by living microorganisms) 30 microorganisms were added to 10 ml of the culture medium used in Examples 1 to 59.
Culture medium 1 with the same composition was placed in a 500 m1 saka flask using 1 ml of the culture solution obtained by culturing at ℃ for 24 to 4 hours as a seed.
00mt and cultured at 30°C for 24 hours.

The bacterial cells were collected by centrifugation, washed with 200 ml of water, and then washed with 1007 n. M phosphate buffer (PH7.8) 50ml
to prepare a bacterial cell suspension. 250 for that 5m1
mM acetylputrescine 0.2mt (50 μMole)
was added and reacted at 30°C for 1 hour, and then the bacterial cells were removed by centrifugation. Putrescine in the reaction solution thus obtained was quantified by a putrescine oxidase assay method. In addition, 250 Tr1. 100 Tr1. instead of 0.2 ml M acetylputrescine. M
A control was prepared by adding 0.2 ml of phosphate buffer (pH 7.8) and performing the same operation, and the difference was taken as the amount of putrescine produced. The results are shown in Table 8. Example 89 (
Hydrolysis of acetylputrescine by freeze-treated bacterial cells) 40 ml of the bacterial cell suspension obtained in Example 8 was sufficiently frozen at -20°C for 3 days, thawed at room temperature, and collected by centrifugation. The bacterial cells were grown for 100 m. M phosphate buffer (PH
7.8) The cells were resuspended in 40ml to obtain a frozen cell suspension.

Using the 10 mt, the same procedure as that of the Example feathers was carried out, and the putrescine produced was quantified. The results are shown in Table 9. Example 90 (Effect of addition of organic solvent and surfactant) Streptomyces spp. prepared in the same manner as in Example 88
Prepare three sets of 2 mt of Avelanius R-20 bacterial suspension, add the additives shown in the first cough, and add 2 mt of bacterial suspension of Avelanius R-20.
0.2 mt (50 μMole) of M acetylputrescine was added, and the mixture was reacted at 30° C. for 1 hour.

After the completion of the reaction, the reaction was stopped by immersing one particle in boiling water, and the bacterial cells were removed by centrifugation. Putrescine in the obtained reaction solution was quantified by the same procedure as in Example 88.
The results are shown in the first cough. *Nonionic surfactant,
1%BL-9EX (product name NikkOl manufactured by Nikkoku Chemical Co., Ltd.)

[Brief explanation of the drawing]

FIG. 1 shows the relative activity when an enzyme solution was subjected to an enzyme reaction at 25° C. for 2 minutes at each pH.

Claims (1)

[Scope of Claims] 1 Escherichia, Enterobacter, Serratia, Bacillus, Agrobacterium, Micrococcus, Staphylococcus, Arthrobacter, Pseudomonas, Xanthomonas, Proteus, Brevi Bacterium, Aspergillus, Penicillium, Rhizobus, Mucor, Pericyularia, Trichophyton, Fusarium, Neurospora, Monascus, Pyricyularia, Cladosporium, Satucharomyces, Satucharomycopsis, Schizophrenia Satucharomyces, Pihia, Hansenula, Cryuyveromyces, Debaryomyces, Cryptococcus, Cretschella, Bitschelhamia, Chiandeida, Rhodotorula, Trichosporon, Cytelomyces, Endomyces, Rhodosporidei The group consisting of the genera P. genus, Streptomyces, Actinomyces, Nocardia, Mycobacterium, Streptoverteicilum, Micromonosvora, Actinoplanes, Micropolyspora, Spirillospora and Streptosporangium. Cells of microorganisms that belong to one genus selected from the following and have the ability to decompose acyl polyamines, crude enzyme extracts or purified enzymes obtained from the cells, or crude enzyme extracts or purified enzymes that have the ability to decompose acyl polyamines obtained from animal organs. A method for liberating polyamines, which comprises causing a purified enzyme to act on an acyl polyamine. 2 The microorganism is Escherichia coli BIFO13168,
Enterobacter cloacae IAM1221, Serratia marcescens IFO03054, Bacillus subtilis IFO03026, Agrobacterium rhizogenes IFO13257, Micrococcus roseus IFO
3764, Micrococcus roseus IFO3768,
Staphylococcus aureus IFO 12732, Arthrobacter simplex IFO 12069, Pseudomonas iodium IFO 3558, Pseudomonas obalis IFO 3738, Pseudomonas aeruginosa IFO 3445, Pseudomonas aureofaciens IFO 3521, Pseudomonas spp. Lahuis IFO3904, Pseudomonas denitrificans IFO13302, Xanthomonas gampestris IFO13551, Proteus lettugelii IF
O13501, Brevibacterium ammoniagenes IFO12072, Aspergillus niger IFO66
61, Aspergillus terreus IFO6123, Penicillium chrysogenum IFO4626, Rhizopus deremer IFO5406, Mucor jiavanicus IFO
4570, Pericularia filamentosa IFO89
85, Trichophyton mentaglophytes IFO58
09, Fusarium oxysporum IFO7190, Pyricularia oryzae IFO5994, Neurospora crassa IFO30216, Monascus anchora IFO
6540, Cladosporium herbalum IAMF51
7. Satucharomyces cerevisiae IFO0259, Satucharomyces cerevisiae IFO0216, Satucharomycopsis capsularis IFO0672, Schizosaccharomyces pombe IFO0346, Pihia memplanefascens IFO0128, Pihia bitskelhamii I
FO1278, Hanzenula saturnus IFO0117
, Hansenula anomara IFO0569, Kryuberomyces lactis IFO1090, Debaryomyces globozas IFO0794, Cryptococcus neoformans IFO0410, Kretzchella magna IFO0
868, Bitskelhamia fluorescens IFO111
6, Candeida lipolytica IFO0717, Candeida albicans IFO0579, Rhodotorula glutinis var. direnensis IFO0415,
Rhodotorula glutinis var. Lusitanica IFO06
88, Trichosporon cutaneum IFO1198, Cytelomyces maturitensis IFO0954, Endomyces obetensis IFO1201, Rhodosporidium toruloides IFO0559, Streptomyces avelanius IFO13451, Streptomyces avelanius R-20, Streptomyces gricius IF
O12875, Streptomyces coericolor IFO1
2854, Streptomyces hygroscopicus IFO
13472, Streptomyces fraziae IFO127
73, Streptomyces alpus IFO13014,
Streptomyces labenziuree IFO12789, Streptomyces viridoccamogenes IFO13347
, Streptomyces aureofaciens IFO12
843, Actinomyces fluorescens IFO128
61, Nocardia erythropolis IFO12682
, Nocardia corallina IFO3338, Streptoverteicilum hiroshimaense IFO12785, Mycobacterium freyi IFO13160, Mycobacterium smegmatis IFO3154, Micromonospora chiarce var. ismensis IFO12988,
Actinoplanes armeniacus IFO12555,
The method according to claim 1, which is Micropolyspora caesia IFO 12990, Spirillospora albida IFO 12248, and Streptosporangium roseum IFO 3776. 3. The method according to claim 1, wherein the animal organ is liver, kidney, pancreas, or heart of a cow, pig, or chicken. 4. The method according to claim 1, wherein the acyl polyamine is an acyl polyamine found in urine.