JPS597435B2 - Acyl polyamine amide hydrolase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel enzyme acyl polyamine amide hydrolase.

More specifically, the present invention provides an enzyme acyl polyamine amide hydrolase which has the action of cleaving and hydrolyzing the amide bond of an acyl polyamine to generate a free polyamine.

Also provided is a method for producing the enzyme acylamine amide hydrolase.

Polyamines such as putrescine, spermidine, and spermine are present in a wide range of living organisms, from bacteria to higher animals to humans, and are involved in cell proliferation.

Since the study by Russell 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 found in many tissues in living organisms where protein synthesis and nucleic acid synthesis are active, and are 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 these methods are insufficient 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 have been proposed for quantifying polyamines in living organisms, such as high performance liquid chromatography and enzymatic methods.

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.

Marmoud et al., Journal of Pharmaceutical Science (J.Pharmaceutical
&ience), 6 7, No. 12, 1671-3 (
According to (1978), polyamines in human urine exist as acetyl polyamines such as acetyl ftrescine, acetylspermidine, and acetylspermine, and W.Y. Chan et al.
a) Vol. 91, 233-241 (1979) reports 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 of polyamine with an acyl group such as acetyl or peptide, that is, it has an acyl polyamine as a main component.

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

The conventional pretreatment method is chlorine hydrolysis.

One example of this is to add the same amount of concentrated hydrochloric acid as urine, seal it in a glass ampoule, and heat it at 120°C for 3 hours.

However, such pretreatment is complicated and dangerous for soil, and no matter how good the subsequent quantitative method itself is, this pretreatment step becomes a bottleneck in the analysis of polyamines in biological samples. I can say that there is.

The present inventors have conducted intensive research to find a simple 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, we discovered a new enzyme, acyl polyamine amide hydrolase, which has the ability to cleave and hydrolyze the amide bonds of acyl polyamines to generate free polyamines.

The present inventors are the first to isolate and purify this enzyme and clarify its properties.

That is, the present invention is an enzyme acyl polyamine amide hydrolase which has basic physicochemical properties and has the action of cleaving and hydrolyzing the amide bond of an acyl polyamine to generate a free polyamine.

■ Substrate specificity: Shows action on acetylputrescine, acetylspermine, and penzoylputrescine.

■ Normal PH; PH7.2-7.8 ■ Stable PH range; PH6.5-8.5 ■ Suitable temperature range for action; 30-40°C ■ Temperature stable range; Stable up to 35°C ■ 1. , Metal Ion Shadow Chi; PCMB. Light damage is caused by 0-phenanthroline, Zn ions, and Ni ions.

The physicochemical properties of oxygen acyl polyamine amide hydrolase are as described below.

The activity of this enzyme was measured using 100 ffl MIJ acid buffer (
pH8.0) 2. Ornl, 250mM a7cetylputrescine 0.2ml, enzyme solution 0. 0 5
ml is mixed, reacted at 250°C for 20 minutes, immediately immersed in boiling water for 10 minutes to stop the reaction, and the putrescine produced is quantified by putrescine oxidase assay.

That is, the reaction mixture was diluted with 100 mM' acid buffer (pH 8.0), and 2. 0.1 to 0 ml
%0-Aminebenzaldehyde koji solution 0.2rul and putrescine oxidase (20u/rILl) 0.05rI
1 liter, mix and react at 30°C for 1 hour.
Measure the absorbance at 35 nm (A4 35 ).

The putrescine concentration in the putrescine oxidase assay solution is determined by the following formula.

Putrescine (μmol eAl) = A 4 3
5/2. One unit (lu.) of enzyme activity is the enzyme activity that produces 1 μmole of putrescine in 10 minutes.

In addition, the enzyme specific activity is determined by the absorbance at 280 nm (
A280) is 1.0, the protein is 1.0'IIt
Activity per unit protein amount (calculated assuming that ≠l)
u. /III9).

1. Substrate specificity: The relative activity of this enzyme for each substrate at a concentration of 25 mM is shown in Table 1, when the activity for acetylputrescine is expressed as 100.

2, Optimum pH The optimum pH of this enzyme is between 7.2 and 7.8.

(Figure 1) 3. Optimal working temperature The optimum temperature for action of this enzyme is 30-40°C.

(Figure 2) 4. Stability This enzyme has a pH of 6. It is stable at a pH of 5 to 8.5, but the most stable pH is around 7.5.

(Figure 3) It is stable up to 35℃ with respect to temperature, but
At higher temperatures, the activity decreases, and at 60°C, most of the activity is lost in 10 minutes.

(Figure 4) Also, 20 mM phosphate buffer (pH
7.2) is stable for 2 weeks or more at 5°C, and is stable for 1 month or more if stored frozen at -20°C.

5. Effects of inhibitors, etc. As shown in Table 2, this enzyme is inhibited by metal ions such as copper, zinc, manganese, cobalt, and nickel, parachloromercribenzoate (hereinafter abbreviated as PCMB), iodoacetamide, and O-phenanthroline. Although it is inhibited by ethylenediaminetetraacetate (hereinafter referred to as EDTA)
), sodium azide, β-mercaptoethanol, etc.

It is also not inhibited by spermidine, spermine, acetylspermine, etc.

5,5'-dithiobis(2-nitropenzoate) (hereinafter abbreviated as DTNB) and dipyridyl activate the enzyme reaction.

6. Km value The Km value of this enzyme for acetylputrescine is 2. O
X 1 0-3M.

and 1.1 x 10-3. for acetylspermidine and penzoylputrescine, respectively. 6.7
x 1 0-”M.

7. Molecular weight The molecular weight of the enzyme acyl polyamine amide hydrolase was determined to be approximately 52 by gel filtration using Sephacryl S-300.
,0 0 0.

It was also estimated to be about 51,000 by SOS-polyacrylamide gel electrophoresis.

From these facts, it is presumed that the enzyme acyl polyamine amide hydrolase is a monomer enzyme without a subunit structure.

8. The isoelectric point of the enzyme acyl polyamine amide hydrolase was electrophoresed in the presence of ampholine, and the isoelectric point was measured.4
．． It was estimated to be around 5.

The enzyme acyl polyamine amide hydrolase in the present invention has the above-mentioned properties, but in particular, its substrate specificity is that it hardly decomposes acylamino acids and acts specifically on acyl polyamines such as acylputrescine and acylspermidine. This can be cited as a characteristic.

Further, this enzyme is characterized by having an optimum pH range of 7.5 to 8.0 and a stable pH range of 6.5 to 8.5.

The enzyme acyl polyamine amide hydrolase in the present invention can be sourced from many microorganisms and animal organs.

Examples of acyl polyamine amide hydrolase-producing bacteria include the many strains listed below.

Escherichia coli BIFO1316'8, Enterobacter cloacae IAM1221, Serratia marcescens IFOO3054, Bacillus subtilis IFO
3026, Agrobacterium rhizogenes IFO13
257, Micrococcus roseus IFO3764, Micrococcus roseus IFO3768, Staphylococcus aureus IFO12732, Arthrobacter simplex IFO12069, Pseudomonas iodium IF03558, Pseudomonas obalis IF03738, Pseudomonas aeruginosa IF
O3445, Pseudomonas aureofaciens I
FO3521, Pseudomonas chlorolahuis IFO
3904, Pseudomonas denitrificans IF
O13302, Xanthomonas campestris IFO
13551, Proteus letgeri IFO13501
, Previbacterium ammoniagenes IFO120
72, Aspergillus niger IFO666 1, Aspergillus terreus IFO6123, Penicillium
Chrysogenum IFO4626, Rhizopus delemer IF
O5406, Mucor Javanicus IFO4570,
Pelicularia filamentosa FO8985, Trichophyton mentagrophytes IFO5809, Fusarium oxysporum IFO7190, Neurospora crassa IF030216, Monascus anchora IFO
6 5 4 0, Satsurikuchi Myces cerevisiae IFO
0259, Satucharomyces cerevisiae IFO216,
Satucharomycopsis cabsularis IFOO672,
Schizosanki Romyces pombe IFOO346, Pihia
Memplanefaciens IF'00128, Pihia pickelhami IFO1278, Hansenula saturnus I
FOO117, Hansenula anomala IFOO569,
Kluyveromyces lactis IFO1090, Debaryomyces globozas IFOO794, Cryptococcus neoformans IFOO410, Kretschella magna IFOO868, Pickelhamia fluorescens IFOO868, Candida lipolyteica IFOO
717, Candeida albicans IFOO579
, Rhodotorula glutinis var-dyrenensis IF
OO415, Rhodotorula glutinis var. Lusitanica ■FO0688, Trichosporon cutaneum IF
O1198, Cytelomyces pineuritensis IFO09
54, Endomyces opertensise FO1201, Rhodosporidium toruloides IFO0559, Strephtomyces averanius IFO13451, Streptomyces averanius R-20, Streptomyces
gricius IFO12875, Streptomyces chericolor IFO12854, Streptomyces hygroscopicus IFO13472, Streptomyces fragii-r-IFO12773, Streptomyces alpus IFO13014, Streptomyces labengere I
FO12789, Streptomyces viridocumogenes IFO13347, Streptomyces aureofaciens IF012843, Actinomyces fluorescens IF012861, Nocardia erythropolis IFO12682, Nocardia corallina IF03
338, Streptocarticilum hiroshimense I
F 0 12785, Mycobacterium freyi IFO
Examples of animal organs that can be used as a source of the enzyme acyl polyamine amide hydrolase include liver, kidney, pancreas, and heart of cows, pigs, and chickens.

Although the enzyme acylboriamine amide hydrolase can be produced from many of the sources exemplified above, it is preferred to use a strain belonging to the genus Streptomyces.

In particular, Streptomyces aveela-ne isolated by the present inventors
us) R-20 bacteria [Feikoken Bacteria No. 5443 (
F E R M P A . 5 4 4 3) is preferably used.

The mycological properties of this Strephtomyces averanius R-20 bacterium are as shown 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.

No whorled branches are shown, and no formation of special organs such as sclerotia is observed.

A mature spore chain consists of 10 or more spores, and the size of the spores is 0.6 to 1. The spores have a cylindrical shape with a diameter of 1 to 1.9 microns and a smooth surface.

(2) Growth status and physiological properties in various media Unless otherwise specified, culturing was carried out at 30°C.

Furthermore, no production of soluble pigment was observed in the following media.

■ Sucrose/nitrate agar No growth was observed.

■ Glucose/asparagine agar The growth is olive gray, and the aerial mycelium is light brownish gray.

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

■ Starch/mineral salt agar growth is gray-olive color, and aerial mycelium is light olive-gray color.

From around the 5th day after culturing, scooch ice thaw is observed.

■ Tyrosine agar The growth is grayish-olive, and the aerial mycelium is grayish-yellow-brown.

■ Growth on nutrient agar bright olive gray, with no aerial mycelium observed.

■ Yeast/malt agar The growth is grayish-yellow-brown, and brown-gray aerial mycelia are attached.

■ Oatmeal agar The growth is gray-olive, and the aerial mycelium is light gray.

■ Glucose/peptone/gelatin puncture culture Aerial hyphae growth is not observed.

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

[Phase] Skimmed milk powder No epiphyte of aerial mycelia was observed.

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

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

@ Utilization of carbon sources in Pridham-Godelive agar D - Glucose, sucrose, < Use, D
-Fructose is also used, but D-arabinose, D-
Xylose, inositol, L-rhamnose, raffinose, and D-mannitol are not used.

[Phase] Optimal growth temperature: 6℃ using maltose yeast agar (pH 7.0)
, 12°C, 19°C, 26°0135°C, 40°C, 43°C
As a result of tests at various temperatures, it is possible to grow at 12°C to 35°C, but it seems that around 26°C is 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 surfaces of the spores are smooth.

Bright olive-gray to brown-gray aerial mycelia grow on bright olive-gray to gray-olive growth on various media, and soluble pigments and melanin-like pigments are not observed.

It has proteolytic power and starch deicing ability.

D-glucose, sucrose, and D-fructose are used as carbon sources.

Based on these properties, similar known bacterial species are classified as ISP (Int
ernational Streptomyces P
When searching for Streptomyces avellanius, the most closely related bacterium was found.
s avel laneus).

This strain meets all the characteristics of Streptomyces avelanius described by ISP.

Also, Bergey's description (Bergeys Manua)
l of Determinative Bacteri
ology, 8th Ed. ), the spore chain morphology is open loop coil (openloop + e
Although there are some differences in that it belongs to the genus Streptomyces avelanius or Streptomyces hooks, in other respects this strain satisfies all the characteristics of Streptomyces avelanius.

Therefore, this bacterial strain was fixed with a strain belonging to Streptomyces averanius, and Streptomyces averanius R.
-20 (Streptomyces avel lan)
eus R-20).

The medium used for culturing the acyl polyamine amide hydrolase-producing bacteria may be either a synthetic medium or a natural medium to which a carbon source, nitrogen source, inorganic salt, or other nutrient source is added.

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 extract, constipation liquor, yeast extract, and asparagine are used.

Inorganic salts include monopotassium phosphate, dipotassium phosphate,
Magnesium sulfate, sodium chloride, etc. are used, and if necessary, a surfactant such as Adekanol LG-805 is added as an antifoaming agent.

It is also desirable to add an acyl polyamine such as acetylputrescine or penzoylputrescine to the medium as an enzyme production inducer.

Acetylputrescine may be added to the medium from the beginning or during the growth of the bacterial cells.

The amount of acetylputrescine added is 0.05% or more to induce enzyme production, but the amount is preferably 0.1 to 0.3%.

Cultivation can be carried out at a pH range of 5 to 8, but preferably at a pH of 7 to 8.

When culturing in a jar fermenter, the initial pH may be set to 7.0 to 8.0, preferably 7.0, but it is necessary to ensure that the pH does not fall below 5 during culturing.

The culture temperature can range from 12 to 35°C, but is preferably from 26 to 30°C.

Culture requires oxygen supply and aeration and agitation.

The culture time in a jar fermenter is usually 16 to 24 hours, but in order to obtain highly active bacterial cells, it is desirable that bacterial cell growth has reached the stationary phase.

When recovering the enzyme after culturing the acyl polyamine amide hydrolase producing bacterium, in the case of Streptomyces averanius R-20, the enzyme is mainly located within the bacterium, so the bacterium is first separated from the culture solution.

This bacterial cell or a product treated with a surfactant, lysozyme, etc. can be used as an enzyme agent, but the bacterial cell can also be crushed to obtain a crude enzyme solution, and it can also be used for ammonium sulfate fractionation, dialysis, column chromatography, etc. It can also be an enzyme preparation purified by gel filtration or the like.

The enzyme acyl polyamine amide hydrolase of the present invention can be used not only as a purified enzyme, but also as a culture of microorganisms, live bacterial cells, frozen and thawed bacterial cells, crushed tooth bodies, crude enzyme extract, and during enzyme purification. Any graded standard product may be used.

It is also possible to carry the enzyme or bacterial cells on a water-insoluble carrier and use them in the form of an immobilized enzyme or an immobilized cup.

The enzyme acyl polyamine amide hydrolase of the present invention is used to act on acyl polyamines in an aqueous medium and hydrolyze the acyl polyamines to produce free polyamines. It is used to liberate polyamines by decomposing them.

The reaction conditions for the action of the enzyme acyl polyamine amide hydrolase are as follows: pH of the reaction solution: 6. 5-8.
5, particularly 7 to 8 are preferred.

The reaction temperature is usually appropriately selected at 50°C or lower, preferably from 25 to 35°C.

This reaction is shown in the case of acetylputrescine as follows.

Regarding the acylpolyamine as a substrate on which this enzyme acts, as described in the section regarding the substrate specificity of this enzyme, examples of the polyamine side of the acylpolyamine include putrescine, spermidine, and spermine.

The acyl group can be selected from a wide range and includes, for example, organic acid residues such as acetyl group and benzoyl group, and amino acid residues such as glycyl group and alanyl group.

The method of the present invention in which the enzyme acyl polyamine amide hydrolase is applied to human body fluids, such as urine, to liberate polyamines is an extremely simple and convenient alternative to the conventional hydrochloric acid decomposition method as a pretreatment method when analyzing polyamines in body fluids. This is an excellent method.

For analysis of polyamines in body fluids after the enzyme acyl polyamine amide hydrolase is activated to liberate polyamines, 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.

Example 1 (Culture of bacteria, purification of enzymes) Streptomyces averanius R-20 (microbial storage entrustment application acceptance number 5443) was grown in a mixture of 0.1% acetylputrescine, 0.5% glucose, and 0.4% peptone. %
, 10.4 medium containing 0.2% yeast extract and 0.2% salt (pH 7.0) was placed in a 20 t jar fermenter, sterilized at 121°C for 15 minutes, inoculated, and incubated at 30°C for 18
Cultured for hours.

A culture solution cultured in a 500rrLl volume Sakaro flask containing a medium of the same composition was used as a seed, and the inoculation amount was 5 parts.

For jar fermenter culture, stirring speed is 600 r.
pm, aeration volume of 1vvm, and the initial pH was 7.
The pH during culture was adjusted so that the lower limit did not fall below 5.0.

After completion of the culture, 144 g (wet weight) of bacterial cells collected by centrifugation was collected at 0.00 g. The cells were suspended in OIM phosphate buffer (pH 7.2), disrupted with a Dynomill, and centrifuged to obtain 1,625 rrL of a crude enzyme extract as a soil solution.

This crude enzyme extract contains 50g of protein and has an enzyme activity of 3
It was 0,000 units.

767 g of ammonium sulfate (70% saturation) was added to the crude enzyme extract, and the resulting precipitate was collected by centrifugation and diluted with 10 mM
It was suspended in phosphate buffer (pH 7.2) and dialyzed against the same buffer for 24 hours.

The dialysate solution was 300 wLl, contained 24 g of protein, and had an enzyme activity of 25,000 units.

This dialyzed fluid was adsorbed onto a DEAE-cellulose column (4.5x45c*) equilibrated in advance with 10mM' acid buffer (pH 7.2), and added with sodium chloride (0.1
~0. Concentration gradient elution was performed with the above buffer containing 5 M).

The active fractions were collected and after ammonium sulfate precipitation (70% saturation), 10
70 TLl of enzyme solution was obtained by dialysis against mM') acid buffer.

This enzyme solution contained 2,500 my of protein and had an oxygen activity of 24,000 units.

This enzyme solution was applied to a DEAE-Sephadex A-50 column (1 column) equilibrated with 20 mM' acid buffer.
．． 7
Elution was performed with the above buffer solution having a salt concentration gradient of .

The active fractions were collected and concentrated by ammonium sulfate precipitation (70% saturation) to obtain an enzyme preparation of 27.5 rrLl.

This enzyme preparation contains 457 proteins, and the enzyme activity is 11,
000 units.

This enzyme preparation was added to 2mM 'J acid buffer (pH 7.2).
) prepared with hydroxyapatite powder (1.2X2
0 crrL).

Most of the active fraction passed through the column.

Most of the impure proteins were adsorbed and eluted with a 5-50 mM phosphate buffer gradient.

The enzyme preparation (64Ull) obtained here is 16.67I
It contained the IJ1 protein, and its enzyme activity was 2,800 units.

This specimen was dialyzed against saturated ammonium sulfate solution, precipitated and concentrated, and then columned on a Sephadex G-200 column (1.7X55crr) prepared with 20mM phosphate buffer (pH 7.2).
Purified by gel filtration with L).

The purification pattern is shown in FIG. The thus obtained purified enzyme preparation (40 ml) contained 9.2 m9 of protein, and its enzyme activity was 1,016 units.

(Effect of the present enzyme) The effect of the present enzyme on acyl polyamines was investigated using the acyl polyamine amide hydrolase preparation obtained above.

0 using 10mM acylpolyamine shown in Table 3 as a substrate.
．． Take 5rrLl and add 100m phosphate buffer (.pH7
．． 8) After adding 1.35TLl and further adding 0.05ml of the above enzyme preparation and reacting at 30°C,
The reaction was stopped by adding 0.1 ml of 50% trichloroacetic acid (TCA).

As a result of analyzing the reaction mixture by high-performance liquid chromatography, a unique peak of free polyamines corresponding to each acyl polyamine newly appeared.

The quantitative results of the free polyamine are shown in Table 3.

Furthermore, the same reaction mixture was spotted on a thin layer plate of silica gel, and chromatography (TLC) was performed using a developing solvent of methyl cellosolve: propionic acid: water (salt saturated) = 14:3:3 to develop ninhydrin color. When this was done, only new spots of free polyamine corresponding to each acyl polyamine appeared.

The Rf values are also listed in Table 3.

Note that the Rf values of acetylputrescine and spermidine were 0.50 and 0.24, respectively.

Also, 1. 2 5 mM acetylputrescine 0. 2
rfLlK, the enzyme preparation was prepared at various concentrations (1, 2
, 5 u. /2.5rILl) and 100
Total volume is 2.5 rIL with mM phosphate buffer (pH 8.0)
15.30.60 l of the mixture at 30°C.
After reacting for a minute, the reaction was immediately immersed in boiling water for 10 minutes to stop the reaction, and the putrescine produced was quantified by high performance liquid chromatography. The results are shown in FIG.

These results confirmed that acyl polyamines were hydrolyzed by this enzyme to liberate polyamines corresponding to the acyl polyamines.

(Substrate specificity of this enzyme) Substrate specificity was investigated using the enzyme preparation obtained above.

0.001 containing 25 μmole each of the substrates listed in Table 4.
Add 1 part of the enzyme preparation to 1 M IJ acid buffer (pH 7.8).
．． 3 9-1 3.9 u. Add 0. 9 5 r
The ul solution was reacted at 30° C. for 20 to 150 minutes.

The reaction was stopped by adding 0.05 d of 50% (w/v)}lichloroacetic acid.

The precipitated protein was heated at 4,000 rpm for 10 min.
After removal by centrifugation for a minute, the compound released by deacylation was analyzed and quantified by high performance liquid chromatography.

The relative activity was also determined.

These results are also listed in Table 4.

In addition, relative activity was expressed based on the rate at which putrescine was released by hydrolyzing acetylputrescine as 100%.

(Optimum action pH of this enzyme) Using the enzyme preparation obtained above, determine the optimum action pH of this enzyme.
I looked into it.

50mM acetylputrescine 0.2rul and 100mM
Phosphate buffer (pH 5.3, 5.6, 5.9,
6.4, 6.9, 7.5, 8.0) or 100771M Tris-HCl buffer (pH 7.2,
7.6, 8.1, 9.0)2. O rnl
0.05 wLl of the above enzyme preparations were added to the mixed solution, and the mixture was allowed to react at 25° C. for 20 minutes, and then immediately immersed in boiling water for 10 minutes to stop the reaction.

The putrescine produced in the reaction mixture was quantified by a putrescine oxidase assay to determine the enzyme activity, and the relative activity, with the highest enzyme activity as a factor of 100, was as shown in FIG.

As is clear from Fig. 1, the optimum working pH of this enzyme is 7.
It can be seen that it is in the range of 2 to 7.8.

(Optimal temperature for action) The optimal temperature for action of this enzyme was investigated using the enzyme preparation obtained above.

10mM acetylputrescine 0.2ml and 100
mM phosphate buffer (pH 8.0) 2. Add 0.05rrLl of the above enzyme preparation to the mixture of OrrLl,
15, 20, 25, 30, 35, 40, 50, 60.
5.10, 20. at each temperature of 70°C. After reacting for 40 minutes, it was immediately immersed in boiling water for 10 minutes to stop the reaction.

When the putrescine produced in the reaction mixture was quantified by the putrescine oxidase assay method, 1 and 2
The results shown in the figure were obtained.

From Figure 2, it is clear that the optimal temperature for the action of this enzyme is 3.
The temperature was 0-40°C.

(pH stability of the present enzyme) Using the enzyme preparation obtained above, the stability of the present enzyme against pH was investigated.

100mM! J acid buffer (pH 5.3, 5.6
, 5.9, 6.4, 6.

9, 7.5, 8.0) or 100 mM
}Lys-HCl buffer (pH 7.2, 7.6, 8
．． 1, 9.0) 0.8ml and the above enzyme preparation 0
．． After mixing 2r/Ll and leaving it at 25°C for 1 hour, the enzyme activity of each enzyme solution was measured, and the relative activity was examined with the highest enzyme activity as 100%. The results were as shown in Figure 3. there were.

As is clear from Figure 3, this enzyme is ph17. It was most stable around 5.0 (Thermal stability of the present enzyme) Using the enzyme preparation obtained above, the stability of the present enzyme against heat was investigated.

100 mM 'J acid buffer (pH 7.8)
2. Mix OrI'Ll and 0.05rrLl of the above enzyme preparation, and prepare 10,20,25,30,35,40,45
,50,60. After maintaining each temperature at 70°C for 10 minutes, each enzyme activity was measured at 25°C.

The activity of enzyme preparations that were not subjected to this temperature treatment was reduced to 100
The relative activity expressed as % was examined and the results were as shown in FIG.

As is clear from FIG. 4, this enzyme is stable up to 35°C at pH 7.8.

Example 2 Acyl polyamines shown in Table 5 were added to a medium (p}i7.0) containing 0.5% glucose, 0.4% Peftone, 0.2% Ei-C Kiss, and 0.2% salt. , 1OrfLl
Dispense each into large test tubes, sterilize them at 121°C for 15 minutes, inoculate them with Streptomyces averanius R-120 (Microorganism Storage Consignment Application Acceptance No. 544:l.), and heat them at 30°C for 40 hours. Cultured.

After the completion of the culture, the bacterial cells were collected by centrifugation, washed with 0.85% saline, suspended in 5rrL of 100mM IJ acid buffer, and sonicated (2
0kc, 5 minutes).

This sonicated solution was centrifuged to obtain an enzyme extract.

Measure the acyl polyamine amide hydrolase activity of each enzyme extract, determine the enzyme specific activity, and use the results as
It was as shown in the table.

Application example 1 Collect 20rd urine and adjust the pH to 7.8 with IN caustic soda.
After preparing 1rrLl of the enzyme preparation obtained in Leprosy Example 1 (enzyme activity 2 5.4 u/rul!), 30
Incubate for 1 hour at ℃.

After filtering the reaction solution, the filtrate was passed through a column of cation exchange resin Biorex 700.5rILl, and then 0.2
N sodium citrate solution (pH 7.0) approximately 20 rIL
After washing the column with 0.5 N hydrochloric acid solution, 4 ml of eluate was obtained.

When putrescine in this eluate was analyzed by high performance liquid chromatography, the concentration of putrescine was 980 μM.

This corresponds to a putrescine concentration of 19.6 μM in the urine sample.

Application example 22 IM Tris-HCl buffer (1) H8. O) After adjusting the pH (pH 7.8) by adding 3.0 ml, the enzyme preparation 1r obtained in Leprosy Example 1
Ill was added and incubated at 30°C for 1 hour.

After adding IN hydrochloric acid to the reaction solution and adjusting the pH to 6.0, cation exchange resin Biolex 70, 0.5rI was added.
After washing with 20 ml of water, 4.0 ml of 0.5N hydrochloric acid.
Elute at O rnl.

Add 1.2 ml of 2M TolJ solution to the eluate and adjust the pH to 8.
After adjusting to 0, polyamines were analyzed by enzymatic method.

That is, 1. O ml eluate after pH adjustment, 0.5 m
lO. IM} Lys-HCl buffer (20 p9 peroxidase), 120 μg 4-amine antihyrine,
Contains 16μg 2,4-dichlorophenol), 0,0
After incubating the reaction mixture consisting of 5rI'Ll putrescine oxidase (1 unit) at 30°C for 1 hour,
Measure the absorbance at 14 Hm.

Absorbance at 5 1 4 nm: OD514 is 0.5
It was 37.

The amount of polyamine by this method is calculated according to the following formula.

Amount of polyamine (μM) = 4 2.9 X O D, 14
The measured amount of polyamine in the urine sample was 23 μM.

[Brief explanation of drawings]

FIG. 1 shows the relative activity when an enzyme solution was subjected to an enzyme reaction at each pH for 20 minutes at 25°C. Buffer is 100mM phosphate buffer for pH 5.3-80,
pH 7.2-9.0 is 100mM To'J-HCl buffer. Figure 2 shows the amount of putrescine produced when the enzyme solution was reacted at each temperature for 5.10 and 20.40 minutes. FIG. 3 shows the relative activity of the enzyme solution after it was left at 25° C. for 1 hour at each pH. The buffer solution has a pH of 5. 3 to 8.0 is 100771M phosphate buffer, and pH 7.2 to 9.0 is 100mM Tris-HCl buffer. FIG. 4 shows the residual activity after maintaining the enzyme solution at each temperature for 10 minutes as relative activity. FIG. 5 shows the purification pattern of this enzyme by treatment with Sephadex G-200. FIG. 6 shows the change over time in the concentration of putrescine produced by the hydrolysis of 100 μM acetylputrescine by this enzyme. The amount of enzyme used is 1, 2, 5u. /2.5ml.

Claims (1)

[Scope of Claims] 1. Acyl polyamine amide hydrolase, an enzyme which has the following physical and chemical properties and has the action of cleaving and hydrolyzing the amide bonds of acyl polyamines to generate free polyamines. ■Substrate specificity: shows action on acetylputrescine, acetylspermidine and penzoylputrescine. ■ Optimum PH; PH7.2-7.8 ■ Stable PH range; PH6.5-8.5 ■ Suitable temperature range for action; 30-40°C ■ Temperature stability range; stable up to 35°C ■ Light-impairing agent ，
Effect of metal ions; PCMB, 0-phenanthroline+
Light damage is caused by Zn ions and Ni ions.