JPS5871886A - Monomethylamine oxidase and its preparation - Google Patents

Abstract

PURPOSE:To prepare a novel monomethylamine oxidase having extremely high substrate specificity to monomethylamine, by culturing bacteria belonging to Bacillus genus. CONSTITUTION:Bacteria belonging to Bacillus genus and capable of producing monomethylamine oxidase, e.g. Bacilus sp. N-101 (FERM BP-59) are cultured usually at 20-35 deg.C and 6-8pH under aerobic conditions for 10hr-1 week, and the objective monomethylamine oxidase is separated mainly from the cultured bacterial cells.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel monomethyl-amino acid converting enzyme and a method for producing the same.

Conventional amine oxidases include Aspergillus niger, Penicillium chrysogenum, Monascus Φ
It has been reported that a strain belonging to Fusarium pulvigenum produces amine oxidase [Agricultural Biological Chemistry (Agr.
Biol, Chem, ) Voi, 29.

Bian 2, P, //7~/23. /9 Otori.

However, none of these known amine oxidases exhibits enzymatic activity on monomethylamine.

Therefore, the present inventors investigated the species and found that by culturing a strain belonging to the genus Bacillus in a medium, monomethylamine oxidase with extremely high substrate specificity for monomethylamine could be obtained from the culture. The invention has been completed.

That is, the present invention is a monomethylamine oxidase having the following physical and chemical properties.

a) Action: O has the action of oxidatively deaminating the amino group of monomethylamine in the presence of water and oxygen to generate formaldehyde, ammonia and hydrogen peroxide b) Substrate specificity: Substrate specificity for monomethylamine ethylamine, n-propylamine, benzylamine, dimethylamine, trimethylamine,
It has no effect on ethylenediamine and tyramine.

C) The h value for monomethylamine is t, 2×1'0-
' M (pH 9,0).

d) Optimal pH and stable pH range: Optimal pH+r~9 Stable pH range 2~9 e) Range of suitable temperature for action: ≠0-SO℃ when pH 7, 30~≠θ℃ when pH 9
The range of is the optimum temperature for action.

f) Conditions for inactivation by temperature: pH 7, treatment at a temperature of 70°C or higher for 70 minutes, or pHq
, tWhen treated at a temperature of 3°C or higher for 70 minutes, each is inactivated.

(3) Titer determination method Substrate monomethylamine hydrochloride was added to 0.7M KHaPOg
- Dissolve in NaxBλ0 buffer (pH 70) to adjust substrate concentration/mM. This substrate solution i, Omg
Mix the enzyme solution θ,/− diluted with the same buffer solution, and
After reacting at ℃/θ0 minutes, add J-N-KOH/ml to stop the enzyme reaction. Monomethylamine is oxidatively decomposed by such an enzymatic reaction, and formaldehyde, ammonia, and hydrogen peroxide are produced in the enzymatic reaction termination solution. Next, add O0j-amino-3-hydrazino-j-mercapto-/, 2. ≠-triazole (hereinafter abbreviated as AHMT) O,,5-NH
After adding /d of QI solution and reacting for lj minutes at 30°C, 0.7j-% KOy (7) 0.2 N
7m7 of KOH solution? The formaldehyde is quantitated by adding the coloring solution to a transparent cell and measuring the absorption with zsorxm.

Under such measurement conditions, the activity is expressed as the amount of enzyme that oxidizes monomethylane and produces /micromol (μmol) of formaldehyde in 7 minutes.

(4) Optimal pH As a buffer solution, 0. / mo/l/ -KHaPOp
-Na: lHPOg diluted solution (pHjj-4,0), 0.
t##% LeKHaPOp-NaaBp07H buffer solution (p
H4,0-4,3), 0.04M Velonal (Ver
onal) buffer (pH 7, 3-g!), 0.
/-E /l/ @ HaBO*-KCI-NaxCO
J buffer (pHr, OP-//, 0) and 0.
/MoA/ NaJCOJ-NaHC, Oi buffer (pH
As a result of measuring the enzymatic activity of monomethylamine oxidase against monomethylamine at various pH values using the following method, the optimum pH was found to be around r~9 as shown in Figure 1. It was recognized that there is.

(5) 0.1M acetate buffer (pHJJ, Hiro θ) as a pH stability and thermostability buffer
,Q,/M@KHaPO4t-NaaBaO2 buffer (pHJ: θ, t, o.

7.011019.0), Q, / M * Vero
nal buffer (put, olr, s, 5!θ) Q,
/M NaaCOr-NaHCO3 buffer (pH
/ 0.01/ 1.0), pH 3,3-//
After holding each sample at 30° C. for 30 minutes, the enzyme activity was measured, as shown in FIG. 2 (pH was found to be around 6 to 9).

After treating this enzyme at pH 7.0 for 70 minutes at each temperature, 7mM
When the residual activity of this enzyme is measured using a monomethylamine solution as a substrate, as shown in FIG. 3, it is completely inactivated at 70°C or higher. Also, pH! ; and θ, it is completely inactivated at 3°C or higher.

(6) Range of suitable temperature for action The pH of the reaction system was set to 70 with 0 and 1 mol KH Coder, PO, NaJB, and 02 buffers, and the enzyme activity at each temperature was examined. The pH of the reaction system was set at θ and the enzyme activity at each temperature was investigated. As shown in the figure, the optimum temperature for action was at pH 7;
It was recognized that the temperature was around -≠θ℃.

This enzyme having the above-mentioned physicochemical properties can be suitably used for the quantitative determination of monomethylamine.

Note that quantifying monomethylamine is carried out by the leather industry,
This is not just a stupid thing that is important from the standpoint of manufacturing control analysis in the organic compound synthesis industry, but it is extremely important for understanding food safety for the first time and for diagnosing abnormalities in living organisms.

The present enzyme can also be suitably used in a method for quantifying methylguanidine.

That is, since this enzyme has extremely high substrate specificity for monomethylamine, a methylguanidine-degrading enzyme is applied to a sample in which methylguanidine is to be determined, and this enzyme is allowed to act on the monomethylamine produced by this action.
Methylguanidine can be specifically quantified by quantifying the amount of formaldehyde generated or by quantifying hydrogen peroxide.

Next, a method for producing monomethylamine oxidase will be specifically described.

The microorganism used in the method for producing the enzyme of the present invention may be any fungus that belongs to the genus Bacillus and has the ability to produce monomethylamine oxidase, including newly isolated strains from nature and existing cultured strains. , and these strains were subjected to conventional microbial mutagenesis methods, such as ultraviolet light,
Any mutant strain with high production of monomethylamine oxidase obtained by physical treatment such as X-ray or γ-ray irradiation, or chemical treatment with a drug such as nitrosoguanidine may be used.

Bacillus sp, @N, which the present inventors isolated from the soil of a field in Noda City, Chiba Prefecture.
-10/ is especially capable of producing monomethylamine oxidase. ~2 x 3-1 micron short rods.

■Presence or absence of cell pleomorphism.

■Motility: Has periflagella and is motile.

■Presence or absence of spores: Yes.

sex ■Gram staining '4#: Positive.

b) Growth status in each medium ■ Juicy agar plate culture at 30°C, ≠? Incubation for hours, diameter 0. Round colonies of j~/mm. The surface is smooth, yellow and shiny.

■Juice agar slant culture When cultured at 30°C for ≠r hours, it is filamentous and the growth is weak. The surface is smooth, yellow, and shiny.

■ Meat juice liquid culture Slight turbidity occurs when statically cultured at 30°C for ≠r hours.

■ Meat juice gelatin puncture culture, 20℃, static culture for ≠ 2 days to liquefy the meat juice and gelatin.

■ Lidomus milk culture No change after static culture at 30°C for ≠ days.

C) Physiological properties ■Reduction of nitrate: Although weak, reduction occurs.

■Denitrification reaction: Positive, no gas produced.

■MR test: Negative.

■VP test: Negative.

■Indole generation: Not generated.

■Generation of hydrogen sulfide: Not generated.

Hydrolysis of C9 Tenbun - Decomposes weakly.

■Use of citric acid: Use it.

■Inorganic nitrogen source: Not used.

(9) Formation of pigment: Not formed.

■Urease: Positive.

■ Oxidase di negative.

0 catalase: positive.

0 growth range: temperature:/! ~≠≠℃.

pH: J'~L? ,evening.

[Phase] Attitude towards oxygen: Aerobic.

@l O-F test (Hugh'Leifson method)
:negative.

Utilization of O carbon source: Production of acid from fructose.

[Phase] Has the ability to assimilate methanol.

Classifying the above physiochemical properties according to the edition of Burgess Manual of Determinative Bacteriology, this bacterium forms spores, is a bacillus, and is an aerobic bacterium. It was determined that this bacterium belongs to the genus Bacillus because it has the ability to produce catalase and is a Durham-positive bacterium.Furthermore, when this bacterium is compared with known bacteria belonging to the genus Bacillus, the cell size is extremely large. It is similar to Carabacillus cereus, Carabacillus anthrans, Carabacillus thuringiensis, and Carabacillus megaterium, but compared to these bacteria, this bacterium produces acid from glucose. It was recognized as a new species belonging to the genus Bacillus because of the differences in that it does not produce methanol and has the ability to assimilate methanol.

Next, the method and conditions for culturing the present microorganism will be described. The method and conditions for culturing the microorganism for producing monomethylamine oxidase are not restricted as long as they do not particularly impede the purpose of the present invention. That is, a culture method and culture conditions that provide an environment in which monomethylamine oxidase-producing bacteria belonging to the genus Bacillus can grow and produce monomethylamine oxidase can be adopted.

It has been found that when a wild strain of Bacillus sp. N-/θ/ is used, production of monomethylamine oxidase is more advantageous in a liquid culture method than in a solid culture method. However, if strain improvement techniques are applied to this bacterium, it is possible to create a strain suitable for solid culture.

However, when monomethylamine hydrochloride is added at O0θ/~2 times, the production amount of monomethylamine oxidase increases dramatically. The above carbon sources include glucose, glycerin,
Examples of nitrogen sources include various amino acids, peptone, soybean flour, various protein hydrolysates, cornstarch liquor, meat extract, yeast extract, nitrate, etc. In addition, trace components include various sodium salts, potassium salts, manganese salts, magnesium salts, calcium salts, zinc salts, iron salts,
Examples include phosphates and sulfates.

Specific examples of the medium include glucose/1, yeast extract 2 t, potassium phosphate 0.7'16, magnesium sulfate 0. /%, ferrous sulfate O0θ/%, manganese chloride O1θ/% (pH 7,2) (hereinafter referred to as A
(abbreviated as medium), monomethylamine hydrochloride 0. /C, Cucrose/C, Yeast extract/%, Phosphoric acid-potassium 0.
/*, medium containing magnesium sulfate O1θtS, ferrous sulfate O0θ/%, manganese chloride 0.0/% (hereinafter abbreviated as B medium), meat juice substitute test sample (meat extract/%, polypeptone border, sodium chloride 0.0/%). j), wort medium (wort 2 t, glucose 2 t, peptone 0./%)
, Safro medium (maltose < z, peptone/%),
Examples include YpSs medium (soluble starch/, 5-thi, yeast extract 02t%, dipotassium phosphate 0./9b, magnesium sulfate heptahydrate o, osts), but aerobic Cymonomethylamine oxidase can be produced by culturing under these conditions. Regardless of which medium is used, culturing is preferably carried out under sufficient oxygen supply, and shaking culture methods and aerated agitation culture methods are applicable for this purpose.

Culture conditions are usually temperature 20-33℃, pH 6~? The culture period is 70 hours to 1 week.

In the solid culture method, the weight ratio of commercially available wheat bran is 0 to 0.
A so-called bran medium prepared by sprinkling 20% water, or a medium to which a carbon source, a nitrogen source, and trace components that can be assimilated by the bacteria were appropriately added, and these were granulated into an appropriate composition, size, and shape. A medium or the like is used as a solid medium.

The culture conditions are usually a temperature of θ to 315°C, a pH of 4 to ♂, and a culture period of 1 to 10 days.

As described above, monomethylamine oxidase can be produced by culturing monomethylamine oxidase-producing bacteria.

To collect monomethylamine oxidase from the culture, monomethylamine oxidase may be collected directly from the liquid culture filtrate or the aqueous extract of the solid culture, but this enzyme is not secreted outside the bacterial cells. The cells are collected from the culture, suspended in an appropriate buffer, and then subjected to ultrasonic treatment and Triton sterilization.
Monomethylamine oxidase within the bacterial cells can be efficiently collected by disrupting the bacterial cells by treatment with a surfactant such as IOθ, mechanical grinding, or a lytic enzyme such as lysozyme.

The obtained crude enzyme solution is extracted using known methods such as salting out using salts such as ammonium sulfate, sodium sulfate, and common salt, organic solvent precipitation using acetones and ethanol, and Toyobar TSK-j (Toyo Soda). ), Sephadex, G, 200, Sepharose B (manufactured by Pharmacia, Sweden), Biogel P30θ (
Gel filtration method using omega-aminohexyl agarose (manufactured by Miles Laboratory), adsorption chromatography method using hydroxyabatide (Bio-Rad Ring, USA), diethyl fist Aminoethyl Sephadex (manufactured by Pharmacia, Sweden), diethylamine ethylcellulose, triethylaminocellulose (
Column chromatography using an ion exchanger such as Bio-Rad Ring (USA), isoelectric focusing using Ampholine (manufactured by 1000K, Sweden), electrophoresis using azetate membrane, starch, acrylamide gel, etc. The enzyme is purified by using individual methods such as dialysis, dialysis, fractionation using various membranes, isoelectric focusing precipitation, or an appropriate combination of these methods, and is electrophoretically purified by single monomethylamine oxidation. A purified sample of the enzyme can be obtained.

Example 1 Bacillus sp. N-10/
Add sterile physiological saline to the storage slant of No., FERM BP 3-9), suspend it, and prepare the seed culture solution (/θ' cells/
me) was prepared.

Place the above A medium/θ punishment in a large test tube and incubate at 720°C for 3
After autoclaving for 0 minutes, 0.3 ml of the above seed culture solution! was aseptically inoculated and cultured with shaking at 30°C for 2≠ hours. The culture solution thus obtained was centrifuged to collect wet bacterial cells, and then the bacterial cells were collected in 0.1 molar phosphate buffer ('
After suspending at pH 70)2-, the cells were disrupted for j minutes using an ultrasonic disruptor while cooling on ice.

Next, this was centrifuged, the supernatant was collected, and the crude enzyme solution/,
-c5' ml (enzyme activity 0.29 units/me)
I got it.

Example 2 The above-mentioned B medium (adjusted to pH 72) 21 was placed in the culture tank of an agitated small culture device (If) (manufactured by Ja Co., Ltd.), and /, 2
Inoculum solution 2 prepared in Example 2 by autoclaving at 0°C for 30 minutes
θrnl was inoculated aseptically, and submerged culture with aeration and stirring was performed at 30°C for ≠r hours.

The culture thus obtained; 11 minutes, =i heart separation to collect wet bacterial cells, and then this 61.g4:291r
')! J)NX-100 (Wako Tsumugi Pharmaceutical Co., Ltd.) solution”
00 rrre, leave to stand at 30°C for 1 hour for bacteriolysis (7), centrifuge and collect supernatant to obtain crude enzyme solution.

Add ammonium sulfate to the crude enzyme solution with O1! When added to saturation concentration, a precipitate separates out. Collect this precipitate section o, o
The mixture was dissolved in iM phosphate buffer (p'H7, θ) and desalted by dialysis with the same buffer. Next, the dialyzed fluid was concentrated 70 times using an ultrafiltration system (Mikasei Mini Module, M-3), and this was equilibrated with a 0.01M phosphate buffer containing 0.05M KCI. TSK-3! ;
The mixture was passed through a column packed with B (manufactured by Toyo Soda) and subjected to high-speed gel filtration chromatography. The active fraction from the previous operation was then collected and added to a 0.0
Dialyzed against 1 molar phosphate buffer, adsorbed on an omega-aminohexyl agarose (Miles Laboratories, IJ's) column equilibrated with the same buffer, and impure proteins were sufficiently eluted and removed with the same buffer. Then, the KC1 concentration was reduced to 0.
．． Affinity chromatography was performed with 0.01 molar phosphate buffer raised to 2M.

The active fraction thus obtained was dialyzed against distilled water and freeze-dried to obtain purified enzyme preparation 7 of monomethylamine oxidase (enzyme activity/?year unit: 1 ng).

[Brief explanation of drawings]

FIG. 1 shows the optimal p of monomethylamine oxidase of the present invention.
Figure 2 shows the pH stability, Figure 3 shows the thermal stability, and Figure ≠ shows the range of suitable temperature for action. Patent Applicant: Kikkoman Corporation No.) Figure B H Section Figure QO, Ir/--Grobu-no L"a#yt@
Q, Ir'l・NaxCOs-NaHCO25l) y mourning' OpH7,0 fist--rHq sword o-oP) 17. . Reference-Φ IH'1.0

Claims (3)

[Claims] (1) Monomethylamine oxidase having the following physical and chemical properties. a) Action: It has the action of oxidatively deaminating the amino group of monomethylamine in the presence of water and oxygen to produce formaldehyde, ammonia and hydrogen peroxide. b) Substrate specificity: The substrate specificity is highest for monomethylamine, followed by ethylamine and n-propylamine, and does not affect benzylamine, dimethylamine, trimethylamine, ethylenediamine and tyramine. C) h value for monomethylamine is z, 2×lθ−′
M (pH 9,0). d) Optimal pH and stable pH range Optimum pH: / ~ 9 Stable pH range: O ~ 9 e) Range of suitable temperature for action: When pH 7 ≠ θ ~! θ℃, pH 9: 30~≠θ℃
The range of is the optimum temperature for action. f) Conditions for inactivation by temperature: pH 7, treatment at a temperature of 70°C or higher for 70 minutes, or pH 9
, When treated for 70 minutes at a temperature of 63°C or higher, each of the following is inactivated. (2) The monomethylamine oxidase according to claim 1, wherein the monomethylamine oxidase is obtained from a bacterium belonging to the genus Bacillus. (3) A method for producing monomethylamine oxidase, which comprises culturing a strain belonging to the genus Bacillus and having the ability to produce monomethylamine oxidase in a medium, and collecting monomethylamine oxidase from the culture.