JPS6058068A - Novel amine dehydrogenase and oxidation of amine using it - Google Patents

Abstract

NEW MATERIAL:Amine dehydrogenase M having the following physical and chemical properties. (A) Action and substrate specificity: showing wide substrate specificity, acting on monoamine, diamine, seconary-tertiary amine. Acting on amino group of amine to release ammonia, forming a corresponding aldehyde: (B) the fittest pH is 7-7.5, stable pH is 8-9; (C) action temperature is 20-45 deg.C; (D) being deactivated by heat treatment at 70 deg.C for 5min, being deactivated at <=5.5pH and >=8.5pH; (E) molecular weight; about 42,000; and (F) alkali protein having an isoelectric point of 8.4pH, etc. USE:An enzyme for oxidizing an amine such as long-chain alkylamine, etc. having high molecular weight. PREPARATION:Pseudomonas sp. K95 (FERM-P 7041) is cultivated in a liquid medium preferably under aerobic conditions at 6-10pH at 20-40 deg.C for 18-96hr.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel enzyme, amine dehydrogenase M, and a method for oxidizing amines using the same.

Generally, amines are toxic to living things, and there are problems with their biodegradability and their impact on nature. Therefore, it has been desired to detoxify amines and analyze amines remaining in nature.

However, amines are also toxic to microorganisms;
Until now, 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 (R, R
, Eady et al., Biochem, J.

Moon ■ Yu 245 (19θ8); same day 757 (1871)
, these oxidized only low-molecular-weight amines and had narrow substrate specificity. Furthermore, no amine dehydrogenase has been found that oxidizes large molecular weight amines such as long-chain alkyl amines.

In view of the current situation, the present inventors have been searching for amine dehydrogenases that can also oxidize large molecular weight amines such as long-chain alkylamines 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.

Furthermore, another object is to provide a method for oxidizing amines using the amine dehydrogenase.

The present invention will be explained in detail below.

Amine dehydrogenase M used in the present invention is Pseudomonas sp. K 95 (Pseudomonas sp.
It is an enzyme produced by As sp, K95).

The strain was isolated from soil by the present inventors, and
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 7041, and has the following mycological properties.

(a) Morphology l) Cell shape and size: Bacillus, 0.4-0.8 xl, 5 X2.0 #1゜2
) Pleomorphism: None 3) Motility: Motile and has polar flagella 4) Spores: Not formed 5) Durham staining: Negative 6) Acid-fastness: #1 (b) Growth status in each medium l ) Broth agar plate culture: The growth is abundant, and the colonies are greenish-brown in color and have a dull luster.

2) Meat juice 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) Lidmus milk: Both coagulation and peptonization were positive.

(C) Physiological properties 1] Nitrate reduction: No reduction 2) Denitrification reaction: Negative 3) MR test: Negative 4) VP test: Negative 5) Indole production: Negative 6) Hydrogen sulfide production (TSI agar) : Negative 7] Hydrolysis of starch: #R property 8) Use of citric acid Kose r's medium: Use of Ghristensen's medium J1! l: Use 8)
Utilizing inorganic nitrogen sources Nitrate: Utilizing ammonium salt: Utilizing 10) Pigment production: Producing fluorescent pigment.

II) Urease: positive 12) Oxidase: positive 13) Catalase: positive 14) Growth range: 22-41"O (optimal 28-38°C) p) 15.0-8.5 (optimal 5.0-7 , 0) 15) Attitude towards oxygen: Aerobic 1B) OF test: O type (oxidized type) ** 17) Production of acids and gases from sugars Second, 1 oz peptone water, 30°C, 14 days Culture indicator:
For bacteria with mycological properties of 5cp (Bromcresol Purple) or higher, Bergey's Manual of De-te
rminative Bacteriology) No. 8
(1975), it was found that this 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 (e.g., n-dodecane, etc.), or fats. Group 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.), nitrogenous organic substances (e.g. urea, peptone, NZ amines, meat extract, yeast extract, corn steep liquor). , casein hydrolyzate, etc.), or amino acids (eg, glutamic acid, asparagine, tyrosine, etc.).

As the inorganic salt, various phosphates, magnesium sulfate, etc. can be used. In addition, trace amounts of heavy metal salts are used, but they are not necessarily needed in media containing natural products, and when using mutant strains that exhibit nutritional requirements, it is natural to add substances that satisfy the nutritional requirements to the media. There must be.

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 energize the medium with various surfactants such as polyoxyethylene sorbitan, or organic solvents such as acetone and ethanol. 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 amount of enzyme production and accumulation reaches its maximum.

Regarding 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 bacteria or their lyophilized bacterial cells are ground and autolyzed.

The bacterial cells are destroyed by sonication and the enzyme is released. Furthermore, the supernatant containing the crude enzyme is separated from the bacterial cell fragments by centrifugation or the like.

The above crude enzyme can be further processed by ion exchange resin method, DEAE cellulose, 0M cellulose, DEAE Sephadex A.
-50, QAE Cef 7 Days XA-5g or 5E-
Gel filtration method using ion exchange Sephadex such as 1!77 Dex, Sephadex such as Sephadex G-100, Sephadex G-200, Toyopearl HW-55SF, etc., isoelectric point fractionation method, acetate membrane bracken Purified enzymes can be obtained by using individual methods such as powder, electrophoresis using acrylamide gel, and other methods such as electrophoretic precipitation, or an appropriate combination thereof.

The amine dehydrogenase M used in the present invention does not necessarily have to be pure; in other words, it is also possible to use a treated bacterial cell product in which the bacterial cells have been destroyed or a crude enzyme obtained by removing bacterial cell fragments from the treated bacterial cell material. Of course, these immobilized enzymes may also be used.

Next, the physicochemical properties of amine dehydrogenase M, a novel enzyme produced by Pseudomonas sp. 95 used in the present invention, will be described.

■Action and substrate specificity: This enzyme exhibits broad substrate specificity, acting on secondary amines and tertiary amines in addition to monoamines and diamines. It acts on the amine group of amines to release ammonia and produce the corresponding aldehyde.

RCH, NH, +PMS+H, 0 → RCHO + PMSH, 10 NH3 PMSH, + Oma → PMS + HtO* (non-enzymatic) *PMS: N-methylphenazonium mesosulfate (coenzyme) ■Optimal pH and stable pH range: Optimal The pH is 7-7.5, but varies depending on the substrate. Stable pH range is 6-9.

■Measurement method of titer The activity of the two enzymes is determined by the PMSH produced in the enzyme reaction,
The amount of DCP IF reacted with was measured at an absorbance of 800 ts g.

CDCPIP: 2.8-dichlorophenolindophenol, ε-21.5X 103) For measurement, 4
0 cod Tris-HCl buffer (pH 7,3), 1 d KCN, 5+sN PMS, 0.05+al) DCP
Using IP c7) solution, 1 mM substrate and enzyme solution were added, and reaction was carried out at 30° C. for 30 to 120 minutes. The amount of enzyme that produces 1M aldehyde per minute of reaction time is defined as 1 unit.

■ Range of suitable temperature for action: The suitable temperature for action is within the range of 20 to 45℃, especially 30 to 45℃.
The optimum temperature is around 0°C.

(Deactivation conditions such as 51 pH 1 temperature, etc.) It is completely inactivated by heat treatment at 0°C for 5 minutes.

It is inactivated when pi is less than 5.5 or more than 9.5.

■Inhibition, activation, and stabilization: Inhibited by 1 riM carbonyl reagent (inniacit, semicarbazide), and no activator or stabilizer is used.

■Molecular weight: As a result of measurement by 5O5-polyacrylamide electrophoresis, it is approximately 42,000.

■Isoelectric point: It is an alkaline protein with an isoelectric point of pH 8.4.

■Purification method: Add 40m to the crude enzyme solution containing amine dehydrogenase M.
M) Streptomycin sulfate dissolved in lithium-hydrochloric acid buffer (pH 7.2) is cooled with water, allowed to stand for 30 minutes, and then centrifuged 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, centrifugation is performed to obtain a supernatant. Furthermore, purified ammonium sulfate was added to make the mixture saturated with 701, and the pH was adjusted to 7.8 with 2N aqueous ammonia. After standing for 30 minutes, centrifuge to obtain a precipitate, 40d Tris-salt #CPH7,2)
-70! Collect the precipitate with a saturated ammonium sulfate solution, and add it for 10 d.
Dissolve in Tris-HCl buffer (pH 8.3) and dialyze overnight.

DEAE-cellulose (Whatman DE-52) equilibrated with 10mM Tris-HCl buffer (pH 8.3)
) was adsorbed with the above crude enzyme, precipitated with the same buffer solution, and
Elute with a linear concentration gradient with 50 d of saline, collect the active fraction of amine dehydrogenase M, and then collect this amine dehydrogenase fraction with C-tocellulose (equilibrated with 10 d of sodium phosphate buffer (pH 8,8)). The solution was passed through a Whatman CM-52) column, washed with the same buffer, eluted with a linear concentration gradient of 0 to 70 mM saline, and the active fraction of amine dehydrogenase M was collected.

Furthermore, this active fraction was passed through a column of Toyo Pearl HW-55SF (manufactured by Toyo Soda Co., Ltd.) to perform gel filtration.
A single enzymatic preparation of amine dehydrogenase M was obtained by elution with Lis-HCl buffer (pH 7.2). The enzyme was homogeneous by disk electrophoresis and SO8 electrophoresis.

In the present invention, amine dehydrogenase M is allowed to act on amines to oxidize them and generate 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, and primary monoamines.
n-propylamine, n-butylamine, i-butylamine, n-pentylamine, i-pentylamine, n-
Examples include hexylamine, n-hebutylamine, n-octylamine, n-nonylamine, n-decylamine, n-undecylamine, n-dodecylamine, and n-tetradecylamine.

As primary diamines, ethylene diamine, propylene diamine, butylene diamine, pentamethylene diamine,
Examples include hexamethylene diamine, heptamethylene diamine, octamethylene diamine, nonamethylene diamine, decamethylene diamine, and dodecamethylene diamine.

Examples of the ω-amino acids include 6-7 minohexanoic 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, di-n-
Examples include 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 1O2 by weight in the solution.

During the reaction, in addition to the substrate, PMS, which is a coenzyme, is
It is necessary to add a small amount of electron acceptor such as

The amount of enzyme in the reaction system is not limited to the amount of the enzyme mentioned above, and can be changed as appropriate depending on the amount ratio of each substrate, enzyme activity, and other conditions.

The reaction temperature in this reaction is usually in the range of 20 to 45°C, and the pH during the reaction is in the range of 6 to 8.

In order to isolate the aldehyde produced in the reaction solution,
This can be easily carried out using commonly used separation means such as column chromatography using silica gel or ion exchange resin.

The oxidation method of the present invention can be applied to a wide range of fields, such as decomposition of harmful amines, clinical analysis, and the production of useful aldehydes.

Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Example 1 Pseudomonas sp. K-95 strain was placed in a jar fermenter containing 20 volumes of liquid medium with the following composition for 30 minutes.
°C, θ00 rpm, I VVM (ventilation volume) Te80
Time culture was performed.

Liquid medium Composition Na3 HPO4” 12 Hl 0 1.5 gNa
ffiso4o, e g MgSO4 fist 7Ht0 0.1 g FeSO4@ 7H105tsg MnSO4@ 4H105mg ZnSO4m 7 Hl OQ, 8 mgCuSO4-
5Hl 0 0.061gNa2Mo04 *4)1
t0 0.03 mgNap B 407 e 10
Hto 0.03 tsgCall @ * 2) 1BO
f (Omg yeast extract 5 rag ion-exchanged water 1 sentence) After culturing, collect the bacteria using a centrifuge, then crush the bacteria using a French press (20,000 PSI), perform ultracentrifugation to remove cell debris, and remove the cells. The supernatant was used as a crude enzyme fraction.

Next, the crude enzyme fraction was treated with streptomycin.

Ammonium sulfate precipitation (40-70% fraction), 11EAE-cellulose column chromatography, C cellulose column chromatography, gel filtration (Toyobar HW-
55SF) to purify amine dehydrogenase M.

The results are shown in Table 1 and Figure 1.2.3. Through the above operations, disk electrophoresis and SDS electroelectrodynamics were obtained.

Example 2 n-hexylaldehyde was used as a substrate. 5 mM P
51mM phosphate buffer solution (pH”0) containing MS
) A solution containing 20 units of IOB n-hexylamine and amine dehydrogenase M in 100+Jl.
The mixture was placed in a 0+Jj Erlenmeyer flask and reacted for 120 minutes at 30"O.

After the reaction was completed, extraction was performed with diethyl ether, and n-hexylaldehyde was quantitatively determined by gas chromatography, and 5.7+wg was obtained.

Motoichi confirmed that it matched the standard product by gas chromatography, and n
-Identified as hexylaldehyde.

Example 3 Various amines shown in Table 2 were used as substrates for lsN, and 4011% (7) )! J Soo-hydrochloric acid (pH 7,3
), 1 mH KCN, 5mM PMS, 0.0
10 ff1n containing 5mM DCPIP and amine dehydrogenase M1 units were placed in a large test tube and reacted at 30°C for 120 minutes.

Enzyme activity was measured by absorbance at 800 mg of reacting DCPIP amount. Table 2 shows the specific activity as a result, setting the activity when dodecamethylene diamine as a substrate as 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. Patent applicant: Kao Soap Co., Ltd. Agent: Ronono Mochizuki Keibe

Claims (1)

[Claims] 1. Amine dehydrogenase M having the following physical and chemical properties. 0 action and substrate specificity: This enzyme exhibits broad substrate specificity and acts not only on monoamines and diamines but also on 21I & amines and tertiary amines. It acts on the amino group of amines to release ammonia and generate the corresponding aldehyde. RCHt N Ht + P M S + Ht O→
RCHO+ PMS H2+ N HsPMSH,
+O, +PMS+HtO. (Non-enzymatic) *PMS: N-methylphenazonium mesosa ■Optimal pH
and stable pH range: The optimum pH is 7 to 7.5, but varies depending on the substrate. Stable pH range is 6-8. ■Method for measuring titer: The activity of this enzyme is determined by the FMSH produced in this enzyme reaction,
The amount of DCPIF reacting with was measured at an absorbance of 800 ysμ. CDCPIP: 2.8-dichlorophenolindophenol, 9g21.5XIO') When measuring, 4
0d Tris-salt #buffer (pH 7,3), 1 ta
M KGN, 5 mH PNS, 0.05 mN ncp
Using rp(7) solution, 11N (71 substrate and enzyme solution were added, and the reaction was carried out at 30°C for 30 to 120 minutes.
The amount of enzyme that produces 1 N aldehyde per minute during reaction is defined as 1 unit. ■ Range H of suitable temperature for action: The suitable temperature for action is within the range of 20 to 45°C, especially! ? n~
in"t'rm:FF-1s<a:a-taMsu-■p
Deactivation conditions such as H1 temperature are completely deactivated by heat treatment at near 0°C for 5 minutes. It is inactivated when the pH is below 5.5 or above 8.5. ■Inhibition, activation and stabilization: Inhibited by a carbonyl reagent (isoniacyto, semicarbazide) at 1 mH, and no activator or stabilizer is used. ■Molecular weight: It is approximately 42,000 as measured by S[11S-polyacrylamide electrophoresis method. ■Isoelectric point: It is an alkaline protein with an isoelectric point of PH8.4. ■Purification method: Add 40m to the crude enzyme solution containing amine dehydrogenase M.
Streptomycin sulfate dissolved in M1 lithium-hydrochloric acid buffer (pH 7.2) is added dropwise under water cooling, left to stand for 30 minutes, and then centrifuged to obtain upper #. Purified ammonium sulfate was added to the obtained supernatant and 40X was added at pH 7.2.
After standing for 30 minutes to achieve saturation, centrifugation is performed to obtain a supernatant. Furthermore, purified ammonium sulfate was added to make the mixture 70% saturated, and the pH was adjusted to 7, f3 with 2N aqueous ammonia. After standing for 30 minutes, centrifugation was performed to obtain a precipitate, and 4 hM Tris-HCl CPH7,2) -
The precipitate was collected with a 70X ammonium sulfate saturated solution, dissolved in 10 d Tris-HCl buffer (pH 8,3), and dialyzed overnight. DEAE-cellulose (Whatman DE-
52) was adsorbed with the above crude enzyme, washed with the same buffer, and
The active fraction of amine dehydrogenase, Naase M, was collected by eluting with a linear concentration gradient with ~150 mM saline, and this amine dehydrogenase fraction was then equilibrated with 10 mM sodium phosphate buffer (pH 8.8). The liquid was passed through a column of cellulose C (ON-52 manufactured by Whatman),
It was washed with the same buffer and eluted with a linear concentration gradient of 0 to 70 mM saline to collect the active fraction of amine dehydrogenase M. Furthermore, this active fraction was passed through a column of Toyo Pearl HW-55SF (manufactured by Toyo Soda Co., Ltd.) to perform gel filtration.
A single enzymatic preparation of amine dehydrogenase M was obtained by elution with Tris-HCl buffer (pH 7.2).The enzyme was subjected to disk electrophoresis and SDS electrodynamic analysis. 2. A method for oxidizing amines, which comprises causing amine dehydrogenase M to act on amines.