JPH0420287A - Acylpolyamine oxidase, production thereof and use thereof - Google Patents

Abstract

NEW MATERIAL:Acylpolyamine oxidase, an enzyme acting on an acylpolyamine and catalyzing production of hydrogen peroxide and an acylaminoalkyl aldehyde. Active for acetylputrescine, acetylcadaverine and benzoylputrescine and inactive for acetylspermidine and acetylspermine. USE:A catalyst for a reaction producing hydrogen peroxide and an acylaminoalkyl aldehyde and useful for direct analysis of an acylpolyamine in the body liquid. Excellent in thermal stability and pH stability. PREPARATION:A strain (e.g. Thermomonospora.fusca IFO 14071, Metschnikowia.pulcherima IFO 0561 or Issatchenkia.orientales IFO 1279) belonging to Thermomonospora genus, Metschnikowia genus or Issatchenkia genus is aerobically cultured in a nutrient medium at 30-45 deg.C and pH 6.5-8.5 for 1-10 days.

Description

Detailed Description of the Invention (Field of Application of Industry-1-) The present invention relates to an acyl polyamine oxidase, which is a novel enzyme. The acyl polyamine oxidase of the present invention is an enzyme that catalyzes the reaction of acetylputrescine, acetylcadaverine, and benzoylputrescine to produce hydrogen peroxide and acylaminoalkyl aldehyde, and has excellent thermal stability and pH Polyamines such as pretocin, cadaverine, spermidine, and spermine, which are characterized by a wide stability range, are widely present in living organisms. 1971
In 2007, Russell et al. reported that polyamine levels in the urine of cancer patients were significantly higher than those of normal subjects [Cancer Research 31, 1555-1558 (+97
1) Since then, quantifying polyamines in living bodies has been useful for cancer diagnosis and as an indicator of the effectiveness of anticancer drugs.

(Prior Art) Many methods have been proposed for quantifying polyamines, such as electrophoresis, high performance liquid chromatography, gas chromatography, and amino acid analysis. However, these methods lack speed.

In addition, methods for quantifying free polyamines using enzymes (Japanese Patent Publication No. 56-36918 and Japanese Patent Publication No. 50-949
2s;-Refer to the Publication Q(f) has been proposed and has been improved in terms of speed, but all of these methods are aimed at constant electricity as a free polyamine.

Therefore, when acetyl polyamine is present, free polyamine is generated by hydrolysis with hydrochloric acid or deacetylation using an enzyme (see Publication No. 60-50438), and the method described above is used. The amount of free polyamine must be quantified using a method, which makes the operation complicated, and it is not possible to directly conduct constant voltage measurement of acetyl polyamine.

However, it has become clear that the majority of polyamines in human urine exist as acetyl compounds, and that they are acetylputrescine.Therefore, there has been a need for a quick and simple method for producing acetylputrescine, and recently acetylputrescine oxidase has been developed. Quantitative method of polyamine used (Special Publication Publication 1983-
037839 and JP-A-61-88897) have been reported. Although this method enables direct electrostatic measurement of acetylputrescine quickly and easily, the known acetylputrescine oxidase used has only a moderate thermostability and pH stability. In other words, the acetylputrescine oxidase derived from Aspergillus oryzae IAM2BB2 described in Japanese Patent Publication No. 83-037639 and Japanese Patent Application Laid-Open No. 61-88897 has a 90%
residual activity at 37°C at pH 6.0 to 8.5.
The residual activity was 85% or more after treatment for 30 minutes. Therefore, the stability of the reagent in the polyamine quantitative method was not satisfactory.

(Problems to be Solved by the Invention) Based on the background described above, it has been desired to find a new enzyme that oxidizes acetyl polyamines, which is more stable than the known acetylputrescine oxidase.

(Means for Solving the Problems) In order to solve the above problems, the present inventors have conducted extensive research and found that Thermomonospora (Thermomonospora)
pora), Metchnikow
genus la) and Issatchenchia (1ssatchen)
The present invention has been achieved by discovering a novel acetylputrescine oxidase from a microorganism related to the genus Kia).

That is, the first aspect of the present invention is oxygen that acts on polyamines and catalyzes the reaction to produce hydrogen peroxide and acylaminoalkyl aldevide, and acts on acetylputrescine, acetylcadaverine, and benzoylputrescine, and acts on acetylspermidine and acetylputrescine. It is an acyl polyamine oxidase characterized by not acting on spermine.

The second aspect of the present invention is that a strain belonging to the genus Thermomonospora, the genus Metinichoia, or the genus Isatchenchia and having the ability to produce the above-mentioned acyl polyamine oxidase is cultured in a nutrient medium, and the acyl polyamine oxidase is collected from the culture. This is a method for producing acyl polyamine oxidase.

The third aspect of the present invention is a polyamine quantitative reagent containing the above-mentioned acyl polyamine oxidase and a reagent for measuring hydrogen peroxide or a reagent for measuring acylaminoalkyl aldebide.

The acyl polyamine oxidase of the present invention acts on acyl polyamines, catalyzes the reaction to produce hydrogen peroxide and acylaminoaldebide, and further has the above-mentioned physicochemical properties.

■Substrate specificity: Acts on acetylputrescine, acetylcadaverine and benzoylputrescine, but not on acetylspermidine and acetylspermine.

■ Thermal stability: pH 7, 0, 1 after 30 minutes treatment at 55℃
00% residual activity.

■ pH stability: pH 5.0 even after 1 hour treatment at 37℃
It has a residual activity of more than 90% at ~9°6.

■ Optimum pH: around 8 ■ Optimum temperature: 55°C ■ 2.0% for acetylputrescine under conditions of km(a;I)H7.5. OX 10-'M, 7.9X 10-'M for acetylcadaverine, 2.5X 10-'M for benzoylputrescine, approximately 140.
000 The method for measuring enzyme activity according to the present invention is described below.

0.2M phosphate buffer pH7, 511mG'0.2%
4-aminoantipyrine solution 2IlIQ 0.4%
E HS P T (*) solution 2 m (!9
0U/m (! Peroxidase solution 2 m
Q30mM N-acetylputrescine solution 4 mQ Distilled water 7 mQ (*): N-ethyl-N-(2hydroxy-3-sulfopropyl)-m-toluidine After preparing the above reaction mixture, 2.8 ml! Aliquot it into a test tube and prewarm it at 37°C for 5 minutes. Then enzyme solution 0
．． Add 2 m (!) and mix gently, then add 3 m
1 at 565 nm in a spectrophotometer controlled at 7°C.
Calculate the change in absorbance per minute (△OD test)
. Blind test: 20mM phosphate buffer pH instead of enzyme solution
Add 0.2+J of 7,5 and perform the same operation as above to find the change in absorbance per minute (△0I) blank)
. Acyl polyamine oxidase activity is determined under conditions 1-1)
The enzyme activity that produces 1M mol of hydrogen peroxide in 1 minute at °C is defined as IU (medium), and is determined by the following formula.

=(△Q1)test-△01)blank)Xo,
718X dilution factor 34.12: Quinone imine dye 5
Millimolar extinction coefficient % at 65 nm: Coefficient due to the fact that the quinone imine dye formed from one molecule of hydrogen peroxide produced in an enzymatic reaction is % molecule.

1.0: Optical path length The novel acyl polyamine oxidase of the present invention belongs to the genus Thermomonospora, the genus Metinicoia, or the genus Isatchenchia, and a strain having the ability to produce the acyl polyamine oxidase of the present invention is cultured in a nutrient medium, and the culture is It is produced by collecting acyl polyamine oxidase from. Examples of Thermomonospora strains include Thermomonospora fusca (Thermomonospora fusca).
fusca) I F 014071, and examples of strains belonging to the genus Metchnikoia include Metchnlkowla pulch.
errlma) I F OO561, etc., and strains belonging to the genus Isatchenkia include, for example, Isatchenkia orientalis (Isatchenkia orientalis).
orlentars) IFO1279, etc.

As the medium used in the present invention, either a synthetic medium or a natural medium can be used as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients that can be assimilated by the bacterial strain used. Examples of carbon sources used include evaculucose, glycerol, maltose, and the like. As the nitrogen source, for example, nitrogen-containing natural products such as peptone, meat extract, and yeast extract, and inorganic nitrogen compounds such as ammonium chloride and ammonium citrate are used. Potassium phosphate, sodium phosphate, sodium chloride, magnesium sulfate, etc. are used as the inorganic substance. Furthermore, it is desirable to add acetylpolyamines such as acetylputrescine and diacetylputrescine to the medium as an acylpolyamine oxidase production inducer.

Culture is usually carried out by shaking culture or aerated agitation culture. The culture temperature is in the range of 20 to 55°C, preferably 30 to 45°C.
, the culture pH is in the range of 5 to 9, preferably 6.5 to 8.5.
It is better to control the Conditions other than these can also be carried out if the strain used grows. The culture period is usually 1 to 1 O
B., and acyl polyamine oxidase is accumulated within the bacterial cells.

The purification method used in the present invention may be any commonly used purification method. For example, as an extraction method, any method such as ultrasonic crushing, mechanical crushing using glass beads, French press, surfactant, etc. may be used.

Furthermore, for extracts, there are salting-out methods such as ammonium sulfate and Glauber's salt, metal flocculation methods such as magnesium chloride and calcium chloride, flocculation methods such as protamine and polyethyleneimine, and even more.
DEAE (diethylaminoethyl) Sepharose, CM
It can be prepared in advance by ion exchange chromatography using (carboxymethyl) Sepharose or hydrophobic chromatography using phenyl Sepharose.

Further, the crude enzyme solution or purified enzyme solution obtained by these methods can be powdered by, for example, spray drying or freeze drying. Furthermore, it can be immobilized on a suitable carrier to obtain an immobilized enzyme.

The novel acyl polyamine oxidase of the present invention can be used as a reagent for polyamine constant M by using it in combination with a reagent for measuring hydrogen peroxide or a reagent for measuring acylaminoalkyl aldehyde.

Reagents for measuring hydrogen peroxide include, for example, peroxidase, 4-ami/antipyrine, and phenol derivatives or aniline derivatives. Reagents for measuring acylaminoalkyl aldehyde include, for example, aminoalkyl aldehyde dehydrogenase (NAI), and may further include reagents for measuring generated N A I) H, such as diaphorase and formazan dyes. Aminoalkyl aldehyde dehydrogenase is, for example, aminobutyraldehyde dehydrogenase (E
, C, 1, 2, 1, 1, 9), etc., and may be of any origin, including those produced by microorganisms such as the genus Shutomonas.

Since the novel acyl polyamine oxidase of the present invention has excellent thermal stability and pH stability, the polyamine quantitative reagent of the present invention has excellent retention stability and good color development sensitivity when polyamine is determined. be.

(Example) Hereinafter, the present invention will be specifically described with reference to specific examples.

Example 1 Diacetylfurycin 0.5%, Yeast Nitrogen Base (manufactured by Dlfco) 0.1%, Thymine 0.01
%, NaC10.5%, MgSO4・7H200.02
%, medium containing 30mM phosphate buffer (pH 7,5) (
pH 7, 2) 100 m (! 500 mQ Yosaka] -
The mixture was transferred to a flask and autoclaved at 121°C for 15 minutes. Thermomonospora fusca (T.
hermomonospora fusca) I F
One platinum loop of 014071 was inoculated and cultured with shaking at 37°C for 2 days to prepare a seed culture. Next, add 500 mQ of the same medium to 2
The flask was transferred to a Qonzaka 1-1 flask and autogelated at 121°C for 15 minutes. Add to this the seed culture solution IO+, IQ
were inoculated and cultured with shaking at 37°C for 311 days. Culture solution 2Q was collected by centrifugation, and 200mM phosphate buffer (pH 7,5) was added. , (suspended in J. followed by super 7
17. The mixture was treated with a wave crusher for 30 minutes, and the bacterial cells were removed by centrifugation to obtain 200 mQ of a crude enzyme solution.

This crude enzyme solution was fractionated using DEAE-Sepharose,
After desalting and concentrating, HPLC using an anion exchanger was performed, and the acyl polyamine oxidase fractions were pooled, desalted and concentrated to obtain a purified enzyme preparation of IOU/m (!).

The physicochemical properties of the obtained enzyme were as follows.

(2) Substrate specificity The relative activity of this enzyme against each substrate at a concentration of 4mM is as shown in Table 1, assuming that the activity against N-acetylputrescine is 100.

Table 1 ■ Z Thermostability As for the stability of this enzyme against heat, it showed 100% activity up to 55°C when treated in a phosphate buffer solution of pH 7.0 for 30 minutes at each temperature (Figure 1). reference).

3. pH stability Regarding the I)H stability of this enzyme, it showed more than 90% residual activity at pH 5.0 to 9.6 even after being treated at 37°C for 1 hour at each pH (Figure 2). Reference +1 (().

4. Optimal 1) H The optimal pH for this enzyme is around 8.0 (see Figure 3)
.

5. Optimal temperature The optimal temperature for this enzyme is based on the activity measurement conditions shown in this patent.
The temperature was 5°C (see Figure 4).

6. km value pH 7.5 condition F, k of this enzyme for acetylputrescine, acetylcadaverine, benzoylputrescine
The m value is 2. OX 10-”M17.9X l
O-'M, 2.5X I 0-4M.

7. Gel filtration method using molecular fil HPLC (column: TSKG300
0SW (manufactured by Toyo Soda), the molecular weight of this enzyme is approximately 14
It was estimated to be 0,000.

(Example 2) Using the enzyme preparation obtained in Example 1, a reaction mixture consisting of the following reaction composition was added with O, 1 m (!) of acetylputrescine solution of a known concentration, and the mixture was heated at 37°C for 20 A calibration curve for acetylputrescine at 565 nm was prepared by reacting for a minute (see Figure 5).

0.2M phosphate buffer pH7.51.5ml! 0, 2
% 4-aminoantipyrine 0.2m
l! 0.4% EH8PT (*)
o, 2mQ90U/m (' Peroxidase 0.2mQ10U/m'c'
Acyl polyamine oxidase of the present invention 0.3 mQ
(*): The linearity of the N-ethyl-N-(2hydroxy-3-sulfopropyl)-m-toluidine test line decreased up to 110 μM (final concentration) of N-acetylputrescine at the absorbance at 565 nm.

(Example 3) Using the enzyme preparation obtained in Example 1, 0.1 mQ of acetylbutrenone solution with a known concentration was added to a reaction mixture consisting of the following reaction composition, and the mixture was reacted at 37°C for 20 minutes. let me,
The amount of NAD H produced was measured by absorbance at 3401 m, and a test line for acetylputrescine was created (see Figure 6).

0.2M phosphate buffer I) H7,51,5mf! 7
．． 5mM NAI) dissolved? &0.1m('lOU/IT
IQ aminoalkyl aldehyde dehydrogenase
0.1mR10U/□Q Acyl polyamine oxidase of the present invention 0.3m (! Distilled water
0.4mQ inspection] Company line is 340n
N-acetylputrescine 110/z at absorbance of m
Linearity was shown up to M (a variation).

(Example 4) Medium 10011. same as Example 1. Q was transferred to a 500 mQ Saka 11 flask and autoclaved at 121°C for 115 minutes. As an inoculum, Metchnlkowlapulcherrlma (Metchnlkowlapulcherrlma)
) One platinum loop of I F 00561 was inoculated and cultured with shaking at 30°C for 3 hours.The culture titer of acyl polyamine oxidase was 20 mU/mQ. and obtained similar results t'J.

(Example 5) Diacetylputrescine 0.5%, yeast 1-1 extract 0.3
%, Chimi 70.01%, NaCl O, 5%, MgS
O4・7H200.02%, phosphate buffer (1) H7,
5) 2m, medium containing M (pH 7,2) room! Transfer 500 mQ Yosaka "1 flask" and heat at 121℃ for 15 minutes.
Autoclave for 1 minute.

As a seed fungus, Isachenchia orientalis (l5
satchenkla orlentars) I
One platinum loopful of F 01279 was inoculated and cultured with shaking at 30°C for 30 minutes. The culture titer of acyl polyamine oxidase was 2 mU/mQ. Thereafter, the same procedure as in Example 1 was carried out, and similar results were obtained.

(Effects of the Invention) The acyl polyamine oxidase of the present invention has the properties as shown in 1-1, and is excellent in thermostable f/l: and pH stability. The acyl polyamine oxidase of the present invention acts on acyl polyamines to produce hydrogen peroxide and acylaminoaldehyde, and is furthermore very useful for direct determination of acyl polyamines in body fluids.

[Brief explanation of the drawing]

FIG. 1 shows the thermostability of the acyl polyamine oxidase of the present invention. FIG. 2 shows the pH stable region of the acyl polyamine oxidase of the present invention. In the figure, △-△ is 20mM dimethylglutarate-NaOH buffer, ... is 20mM'J acid buffer, and O-○ is 20mM dimethylglutarate-NaOH buffer.
mM) Lis-HCl buffer, Muum 20mM boric acid-
KCI-NaOH buffer is shown. FIG. 3 shows p of the acyl polyamine oxidase of the present invention.
Shows reactivity towards H. In the figure, -- indicates 73mM phosphate buffer, O-O indicates 73mM Tris-HCI buffer, and Moum indicates 73mM boric acid-KCl-NaOH buffer. FIG. 4 shows the reactivity of the acyl polyamine oxidase of the present invention to black 1 degree. Figure 5 shows 565 nm.
The calibration curve for acetylputrescine is shown in FIG. Figure 6 shows the calibration curve for acetylputrescine at 340 nm. FIG. 6 shows a calibration curve for acetylputrescine at 340 nm.

Claims (5)

[Claims] (1) An enzyme that acts on acyl polyamines and catalyzes the reaction that produces hydrogen peroxide and acylaminoalkyl aldehyde, acts on acetylputrescine, acetylcadaverine, and benzoylputrescine, but does not act on acetylspermidine and acetylspermine. An acyl polyamine oxidase characterized by: (2) The acyl polyamine oxidase according to claim 1, which has 100% residual activity after treatment at pH 7.0, 55° C., and 30 minutes. (3) The acyl polyamine oxidase according to claim 1, which has a residual activity of 90% or more at pH 5.0 to 9.6 after treatment at 37°C for 1 hour. (4) A strain belonging to the genus Thermomonospora, the genus Metinicoia, or the genus Isachenchia and having the ability to produce the acyl polyamine oxidase according to claim 1 is cultured in a nutrient medium, and the acyl polyamine oxidase is collected from the culture. A method for producing acyl polyamine oxidase. (5) A polyamine quantitative reagent comprising the acyl polyamine oxidase according to claim 1 and a reagent for measuring hydrogen peroxide or a reagent for measuring acylaminoalkyl aldehyde.