JPH0440988B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel enzyme, amine dehydrogenase M. In general, amines are toxic to living organisms, and there have been problems with their biodegradability and impact on nature. Therefore, it has been desired to make amines non-toxic and to analyze amines remaining in nature. However, amines are also toxic to microorganisms, and there have been few examples of bacteria that can assimilate amines. Amine oxidase and amine dehydrogenase are known enzymes contained in bacteria that assimilate amines, and these enzymes oxidize amines to generate aldehydes. Among them, there are few reports of amine dehydrogenase, which does not involve oxygen in the enzymatic reaction. Conventionally, enzymes produced from Pseudomonas AMI have been reported as amine dehydrogenases (RREady et al, Biochem.J., 106 ,
245 (1968); 123 , 757 (1971), these oxidized only low-molecular amines and had narrow substrate specificity. Furthermore, no amine dehydrogenase has been found that oxidizes amines with large molecular weights such as long-chain alkyl amines. In view of this current situation, the present inventors have been searching for amine dehydrogenases that can also oxidize large molecular weight amines such as long-chain alkyl amines and have broad substrate specificity. It was discovered that amine dehydrogenase M contained in the producing bacteria satisfies the above conditions, and the present invention was completed. That is, an object of the present invention is to provide a novel amine dehydrogenase. The present invention will be explained in detail below. Amine dehydrogenase M used in the present invention
is Pseudomonas sp. K95
(Pseudomonas sp.K95). This strain was isolated from soil by the present inventors and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 7041, and has the following mycological properties. There is. (a) Morphology 1 Cell shape and size: Bacillus, 0.4-0.6×1.5
×2.0μ 2 Pleomorphism: None 3 Motility: Motile and has polar flagella 4 Spores: Not formed 5 Gram staining: Negative 6 Acid-fastness: Negative (b) Growth status in each medium 1 Broth agar plate Culture: Growth is abundant, colonies are greenish-brown in color and have a dull luster. 2. Broth agar slant culture: Growth is abundant and produces a greenish-brown pigment. 3 Meat juice liquid culture: Forms a thin film and causes overall turbidity. 4 Meat juice gelatin puncture culture: Liquefied from the surface layer. 5 Litmus milk: coagulation, peptonization. Both are positive. (c) Physiological properties 1 Nitrate reduction: No reduction 2 Denitrification reaction: Negative 3 MR test: Negative 4 VP test: Negative 5 Indole formation: Negative 6 Hydrogen sulfide formation (TSI agar): Negative 7 Starch hydration Degradation: Negative 8 Use of citric acid Koser's medium: Used Christensen's medium: Used 9 Use of inorganic nitrogen sources Nitrate: Used Ammonium salt: Used 10 Pigment production: Produces fluorescent dyes. 11 Urease: positive 12 Oxidase: positive 13 Catalase: positive 14 Growth range: 22-41℃ (optimum 28-39℃) PH5.0-8.5 (optimum 5.0-7.0) 15 Attitude towards oxygen: aerobic 16 OF test: O type (oxidized type) 17 Generation of acid and gas from sugars ^** :

[Table] **10% peptone water, 30℃, 14 days culture Indicator: BCP (Bromcresol Purple) For bacteria with the above mycological properties, please refer to Bergey's Manual of
Determinative Bacteriology) 8th edition (1975)
As a result of a search based on this, it was determined that this bacterial strain belongs to the genus Pseudomonas. In the present invention, as the nutrient source for the medium used for culturing the amine dehydrogenase-producing microorganism, a wide variety of nutrients commonly used for culturing microorganisms can be used. That is, carbon sources include carbohydrates (e.g. glucose, glycerol, fructose, etc.);
Organic acids (e.g. citric acid, succinic acid, etc.), amino acids (e.g. glutamic acid, asparagine, etc.),
Hydrocarbons (eg, n-dodecane, etc.), or aliphatic amines and/or aliphatic diamines (eg, hexylamine, dodecylamine, dodecamethylene diamine, etc.) can be used. Nitrogen sources include ammonia, inorganic and organic ammonium salts (e.g. ammonium chloride, ammonium phosphate,
ammonium sulfate, ammonium nitrate, ammonium succinate, etc.), nitrogen-containing organic substances (such as urea,
Peptone, NZ amine, meat extract, yeast extract,
corn steep liquor, casein hydrolyzate, etc.)
Alternatively, amino acids (eg, glutamic acid, asparagine, tyrosine, etc.) can be used. As the inorganic salt, various phosphates, magnesium sulfate, etc. can be used. In addition, trace amounts of heavy metal salts are used, but their addition is not necessarily required in media containing natural products. Furthermore, when using a mutant strain that exhibits nutritional requirements, a substance that satisfies the nutritional requirements must be added to the medium. Cultivation is usually preferably carried out under aerobic conditions such as shaking or aerated agitation culture. When using a carbon source that is poorly soluble in water, it is also possible to add various surfactants such as polyoxyethylene sorbitan or organic solvents such as acetone and ethanol to the medium. The pH of the medium is 6.0 to 10.0, the culture temperature is 20 to 40°C, and the culture period is usually 18 to 96 hours, and the culture may be terminated at the time when the accumulated amount of enzyme production reaches its maximum. As for the extraction method of amine dehydrogenase from cultured bacterial cells, a general enzyme extraction method from bacterial cells can be applied. For example, live bacterial cells are collected from the obtained culture solution by a method such as centrifugation, and the live bacterial cells or their lyophilized bacterial cells are destroyed by grinding, autolysis, or sonication, and the enzyme is released. . Furthermore,
The supernatant containing the crude enzyme is separated from the bacterial fragments by centrifugation or the like from this bacterial cell treatment product. The above crude enzyme can be further processed by ion exchange resin method, DEAE
Cellulose, CM cellulose, DEAE Cephadex A-50, QAE Cephadex A-50 or SE
Ion exchange Cephadex such as Cephadex, Cephadex G-100, Cephadex G-
200, etc. and Toyo Pearl HW-
Gel filtration method using 55SF etc., isoelectric point fractionation method,
Purified enzymes can be obtained by using individual methods such as acetate membrane starch, electrophoresis using acrylamide gel, isoelectric precipitation, or an appropriate combination thereof. Amine dehydrogenase M used in the present invention does not necessarily have to be pure. That is, it is also possible to use a treated bacterial cell product in which the bacterial cells have been destroyed or a crude enzyme in which bacterial fragments have been removed from the treated bacterial cell material. Of course, these immobilized enzymes may also be used. The activity of this enzyme is determined by the amount produced in this enzyme reaction.
The amount of 2.6-dichlorophenol indophenol (DCPIP) reacting with PMSH ₂ (PMS; N-methylphenazonium mesosulfate) was 600 mμ.
The absorbance was measured at . For measurements, 40mM
Tris-HCl buffer (PH7.3), 1mM KCN,
Using a solution of 5mM PMS, 0.05mM DCPIP,
Add 1mM substrate and enzyme solution and incubate for 30-120 min at 30°C.
The reaction was carried out for minutes. 1μM per minute of reaction time
The amount of enzyme that produces aldehyde is defined as 1 unit. Next, the physicochemical properties of amine dehydrogenase M, a novel enzyme produced by Pseudomonas sp. K95 used in the present invention, will be described. Action and substrate specificity It shows broad substrate specificity and acts on secondary amines and tertiary amines in addition to monoamines and diamines.
It acts on the amino group of amines to release ammonia and produce the corresponding aldehyde. Stable PH and optimal PH Stable PH between PH6 and 9, optimal PH between PH7 and 7.5
has. Operating temperature and optimum temperature It has an operating temperature in the range of 20 to 45°C, and an optimum temperature in the vicinity of 30 to 40°C. Deactivation conditions based on pH, temperature, etc. Completely deactivated by heat treatment at 70℃ for 5 minutes. PH
is inactivated below 5.5 or above 9.5. Inhibition, activation and stabilization Inhibited by 1mM carbonyl reagent (isoniazid, semicarbazide), no activator or stabilizer. Molecular Weight It has a molecular weight of approximately 42,000. (SDS-polyacrylamide electrophoresis method) Isoelectric point It has an isoelectric point at PH8.4. In the present invention, amine dehydrogenase M is allowed to act on amines to oxidize them to produce the corresponding aldehydes. The generated aldehyde can be confirmed by gas chromatography. The enzyme of the present invention has broad substrate specificity and can be applied to various amines. Examples of amines used as substrates include methylamine, ethylamine, n-propylamine, n-
Butylamine, i-butylamine, n-pentylamine, i-pentylamine, n-hexylamine, n-heptylamine, n-octylamine,
Examples include n-nonylamine, n-decylamine, n-undecylamine, n-dodecylamine, and n-tetradecylamine. Examples of the primary diamine include ethylene diamine, propylene diamine, butylene diamine, pentamethylene diamine, hexamethylene diamine, heptamethylene diamine, octamethylene diamine, nonamethylene diamine, decamethylene diamine, and dodecamethylene diamine. Examples of the ω-amino acid include 6-aminohexanoic acid, 8-aminooctanoic acid, 11-aminoundecanoic acid, and 12-aminododecanoic acid. Examples of substituted primary amines include benzylamine, phenethylamine, tyramine, 1-noradrenaline, histamine, and the like. As the secondary amine, di-n-butylamine,
Examples include di-n-hexylamine. Examples of the tertiary amine include triethylamine, tri-n-butylamine, and tri-n-hexylamine. Examples of polyamines include spermine and spermidine. The amount of amines as a substrate in the reaction system is not particularly limited, but it is usually used in a concentration range of 0.01 to 10% by weight in the solution. During the reaction, it is necessary to add a small amount of an electron acceptor such as PMS, which is a coenzyme, in addition to the substrate. The amount of enzyme in the reaction system varies depending on the separation and purification or processing method of the enzyme, but is not particularly limited and depends on the amount ratio of each substrate, the activity of the enzyme,
It may be changed as appropriate depending on other conditions. The reaction temperature in this reaction is usually in the range of 20 to 45 °C,
Further, the pH during the reaction is in the range of 6 to 9. The aldehyde produced in the reaction solution can be easily isolated using commonly used separation means such as column chromatography using silica gel or ion exchange resin. The enzyme of the present invention can be applied to a wide range of fields, including decomposition of harmful amines, clinical analysis, and a method for producing useful aldehydes. Next, the present invention will be specifically explained using Examples, but the present invention is not limited thereto. Example 1 Pseudomonas sp. K-95 strain was cultured for 60 hours at 30° C., 600 rpm, and 1 VVM (aeration volume) in a jar fermentor containing a liquid medium 20 having the following composition.

[Table] After culturing, bacteria were collected using a centrifuge, and the cells were crushed using a French press (20,000 PSI), and ultracentrifuged to remove cell debris and the supernatant was used as a crude enzyme fraction. Streptomycin sulfate dissolved in 40 mM Tris-HCl buffer (PH7.2) is added dropwise to the enzyme solution containing amine dehydrogenase M under ice cooling, and after standing for 30 minutes, centrifugation is performed to obtain a supernatant. Add purified ammonium sulfate to the obtained supernatant and adjust to 40% at pH 7.2.
After standing for 30 minutes at % saturation, centrifuge to obtain the supernatant. Furthermore, add purified ammonium sulfate to make it 70% saturated, and make 2N
Adjust the pH to 7.6 with ammonia water. After standing for 30 minutes, centrifuge to obtain a precipitate, collect the precipitate with 40mM Tris-HCl buffer (PH7.2) - 70% ammonium sulfate saturated solution,
This is dissolved in 10mM Tris-HCl buffer (PH8.3) and dialyzed overnight. DEAE-cellulose (Watmann DE-
52), washed with the same buffer, eluted with 0-150mM saline using a linear concentration gradient, and absorbed the active fraction of amine dehydrogenase M with 10mM sodium phosphate buffer (PH6.8). ) averaged with CM-cellulose (CM manufactured by Watmann)
-52) column, washed with the same buffer, and
The active fraction of amine dehydrogenase M was collected by elution with a linear gradient of ~70 mM saline. Furthermore, this active fraction was added to Toyopearl HW-55SF.
(manufactured by Toyo Soda Co., Ltd.) to perform gel filtration and elution with 40 mM Tris-HCl buffer (PH7.2) to obtain a single enzymatic specimen of amine dehydrogenase M. This enzyme was analyzed by disk electrophoresis and SDS.
It was electrophoretically homogeneous. The results are shown in Table 1 and Figures 1, 2, and 3. By the above operations, disk electrophoresis and SDS electrophoresis became uniform. Example 2 n-hexylamine was used as a substrate.
A solution containing 10 mg of n-hexylamine and 20 units of amine dehydrogenase M in 100 ml of 51 mM phosphate buffer solution (PH 7.0) containing 5 mM PMS was placed in a 300 ml Erlenmeyer flask, and the reaction was performed at 30°C for 120 minutes. Summer. After the reaction was completed, extraction was performed with diethyl ether, and 5.7 mg of n-hexylaldehyde was obtained by gas chromatography. This product matched the standard product by gas chromatography and was identified as n-hexylaldehyde. Example 3 Various amines as shown in Table 2 were used as substrates.
Use 1mM, 40mM Tris-HCl (PH7.3),
1mM KCN, 5mM PMS, 0.05mM DCPIP
and 10ml containing amine dehydrogenase M1 units
was placed in a large test tube and reacted at 30°C for 120 minutes. Enzyme activity was measured by absorbance at 600 mμ of the amount of reacting DCPIP. Table 2 shows the specific activity as a result, with the activity when dodecamethylene diamine being used as a substrate being 100.

[Brief explanation of drawings]

FIG. 1 is a column chromatogram of the present enzyme in Example 1 using DEAE-cellulose, and FIG. 2 is a column chromatogram of the same enzyme using CM-cellulose. Furthermore, FIG. 3 is a column chromatogram obtained by gel filtration.

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Claims (1)

[Claims] Amine dehydrogenase M having the following physical and chemical properties. Action and substrate specificity It shows broad substrate specificity and acts on secondary amines and tertiary amines in addition to monoamines and diamines.
It acts on the amino group of amines to release ammonia and produce the corresponding aldehyde. Stable PH and optimal PH Stable PH between PH6 and 9, optimal PH between PH7 and 7.5
has. Operating temperature and optimum temperature It has an operating temperature in the range of 20 to 45°C, and an optimum temperature in the vicinity of 30 to 40°C. Deactivation conditions based on pH, temperature, etc. Completely deactivated by heat treatment at 70℃ for 5 minutes. It is inactivated when the pH is below 5.5 or above 9.5. Inhibition, activation and stabilization Inhibited by 1mM carbonyl reagent (isoniazid, semicarbazide), no activator or stabilizer. Molecular Weight It has a molecular weight of approximately 42,000. (SDS-polyacrylamide electrophoresis method) Isoelectric point It has an isoelectric point at PH8.4.