JP2584907B2 - Method for producing diacetylpolyamine amide hydrolase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing diacetylpolyamine amide hydrolase.

[0002] The enzyme, diacetylpolyamine amide hydrolase, according to the present invention is used for the synthesis of monoacetylpolyamine useful as an intermediate for the synthesis of pharmaceuticals and agricultural chemicals, and for the quantification of diacetylpolyamine in biological samples.

[0003]

2. Description of the Related Art Diacetylpolyamine amide hydrolase is an enzyme that hydrolyzes diacetylpolyamine to produce monoacetylpolyamine, and catalyzes, for example, the following reaction.

Diacetyl putrescine + H ₂ O → monoacetyl putrescine + CH ₃ COOH Heretofore, there has been little knowledge about amidohydrolases acting on diacetylpolyamines, and thus there has been no report on a method for producing diacetylpolyamine amidohydrolases. There is no established technology that can be used.

[0005]

From the above background, an enzyme which acts on diacetylpolyamine to generate monoacetylpolyamine, diacetylpolyamine amide hydrolase, has been established, and a technique for industrially producing diacetyl polyamine amide hydrolase has been established. Was desired.

[0006]

Means for Solving the Problems The present inventors have made intensive studies in order to solve the conventional drawbacks. As a result, microorganisms belonging to the genus Alcaligenes, Pseudomonas, Fusarium, Candida, or Trichosporon were found to be diacetylpolyamine. They have found that they produce a large amount of amide hydrolase, and have completed the present invention.

That is, the present invention provides a method for culturing a microorganism belonging to the genus Alcaligenes, Pseudomonas, Fusarium, Candida, or Trichosporon and having the ability to produce diacetylpolyamine amide hydrolase, and collecting diacetyl polyamine amide hydrolase from the culture. A method for producing diacetylpolyamine amide hydrolase.

As the diacetyl polyamine used in the present invention, known diacetyl polyamines can be used without particular limitation. Particularly preferred examples include diacetyl-1,2-diaminoethane, diacetyl-1,3-diaminopropane,
Diacetylputrescine, diacetylcadaverine, diacetyl-1,6-diaminohexane, diacetyl-1,8-diaminooctane, diacetylspermidine, diacetylspermine and the like.

The microorganism used in the present invention belongs to the genus Alcaligenes, Pseudomonas, Fusarium, Candida or Trichosporon, and may be any of existing strains and nature as long as it has the ability to produce diacetylpolyamine amide hydrolase. Newly isolated strains and any of these mutants can be used.

Further, a gene encoding diacetylpolyamine amide hydrolase is isolated from a microorganism belonging to the genus Alcaligenes, Pseudomonas, Fusarium, Candida, or Trichosporon and capable of producing diacetylpolyamine amide hydrolase. A diacetylpolyamine amide hydrolase-producing bacterium obtained by introducing a gene so as to be expressed in a microorganism belonging to the same genus or another genus can also be used.

[0011] The method for producing diacetylpolyamine amide hydrolase of the present invention is a method for producing diacetylpolyamine amide hydrolase from a culture of a microorganism belonging to the genus Alcaligenes, Pseudomonas, Fusarium, Candida, or Trichosporone capable of producing diacetylpolyamine amide hydrolase. It is a method of collecting hydrolase,
Genus Alcaligenes, Pseudomonas, Fusarium,
As strains of microorganisms belonging to the genus Candida or the genus Trichosporon, for example, Alcaligenes faecalisIF
O 1311) Alcaligenes faecalisIF
O 14479) Alcaligenes Sp. IFO
14130) Pseudomonas aureofaciens
aureofacieuns IFO 3521) Pseudomonas synzantha
IFO 3912) Pseudomonas synzantha
IFO 3913) Pseudomonas aerug
inosa IFO 3080) Pseudomonas chloraffis
oraphis IFO 3904) Fusarium oxysporum I
FO 7190) Fusarium solani IFO 9955 Candida tropicalisIFO
0007) Candida guillermondii
IFO 0454) Trichosporon cutaneum
IFO 1198) Trichosporon cutaneum
IFO 0173) and the like.

A known medium is used for culturing each of the above microorganisms. For example, glucose, molasses, carbon sources such as soluble starch, meat extract,
It is not particularly limited as long as it contains a nitrogen source such as yeast extract and polypeptone, and inorganic salts such as potassium phosphate monobasic, potassium phosphate dibasic, sodium chloride, and magnesium sulfate. Diacetylpolyamines such as putrescine and diacetylcadaverine; monoacetylputrescine; monoacetyl-1,6-
Inclusion of a monoacetylpolyamine such as diaminohexane is advantageous in increasing the productivity of the enzyme.

The culturing conditions for culturing the producing bacterium which produces the diacetylpolyamine amide hydrolase of the present invention include culturing at a temperature of 15 to 40 ° C., preferably 20 to 35 ° C. under aeration and stirring conditions. Is preferred.
The pH conditions during the culture are in the range of pH 5.0 to 9.0, preferably pH
A range of 6.0 to 8.0 is appropriate. The culturing time is not particularly limited, but is suitably in the range of from the time reaching the latter stage of the growth to 10 to 30 hours after entering the dormant state in consideration of economy such as productivity of the enzyme.

As a method for separating a culture solution and cells from a culture obtained by culturing, a conventional method such as centrifugation or filtration can be used, but centrifugation is preferred. .

The separation and purification of the diacetylpolyamine amide hydrolase of the present invention can be performed as follows.

As a method for extracting the enzyme accumulated in the cells from the cells, there are conventionally used cell disruption by ultrasonic waves, cell disruption by a Dynomill cell disrupter rotating together with glass beads, Alternatively, a method of destroying a cell membrane with an enzyme such as lysozyme or an organic solvent such as toluene may be used. The enzyme can be collected by selecting an appropriate method from these and extracting the enzyme from the cells.

When it is necessary to further purify diacetylpolyamine amide hydrolase from the crude enzyme solution extracted by these methods, ammonium sulfate precipitation, ion exchange column, which is a commonly used means for purifying enzymes, is generally used. Purification can be carried out by appropriately combining or repeating methods such as chromatography, gel filtration, hydroxyapatite column chromatography, affinity column chromatography, and preparative electrophoresis.

The diacetylpolyamine amide hydrolase obtained according to the present invention has the following physicochemical properties.

Action Diacetylpolyamine is hydrolyzed to produce monoacetylpolyamine. For example, it catalyzes the following reaction.

[0020] Temperature stability Under a treatment condition of pH 7.5 and 30 minutes, it has a residual activity of 80% or more at 4-35 ° C.

PH stability When stored at 30 ° C. for 30 minutes, it has a residual activity of 80% or more at pH 5.6 to 9.4.

Further, the physicochemical properties of the diacetylpolyamine amide hydrolase obtained according to the present invention will be described in more detail in Alcaligensfaecalis.
IFO14479), and Candida Giramondi (Candi)
da guillermondii IFO 0454) is illustrated below for diacetylpolyamine amide hydrolase obtained by culturing.

[Diacetylpolyamine amide hydrolase derived from the genus Alcaligenes] 1. Action Diacetylpolyamine is hydrolyzed to produce monoacetylpolyamine. That is, it catalyzes the following reaction.

Diacetyl polyamine + H ₂ O → monoacetyl polyamine + CH ₃ COOH 2. Substrate specificity The relative activity of this enzyme for each substrate at a concentration of 10 mM is shown in Table 1 when the activity for diacetyl putrescine is expressed as 100. Become like

[0025]

[Table 1] * N ¹ -acetyl spermidine formation 3.Optimal pH: pH8.5-9.5 (Fig.1) 4.pH stability When stored at 30 ° C for 30 minutes, it has more than 80% residual activity at pH5.6-9.4. . (Figure 2) 5. Optimum temperature: 37-42 ° C in a reaction at pH 9.0 for 15 minutes.

(FIG. 3) 6. Temperature stability Under a treatment condition of pH 7.5 for 30 minutes, it has a residual activity of 80% or more at 4-55 ° C., and is completely treated at 67 ° C. for 30 minutes. Deactivate. (Figure 4) [Diacetylpolyamine amide hydrolase derived from Candida sp.] 1. Action Diacetylpolyamine is hydrolyzed to produce monoacetylpolyamine. That is, it catalyzes the following reaction.

Diacetyl polyamine + H ₂ O → monoacetyl polyamine + CH ₃ COOH 2. Substrate specificity The relative activity of this enzyme for each substrate at a concentration of 10 mM is shown in Table 2 when the activity for diacetyl putrescine is expressed as 100. Become like

[0028]

[Table 2] * N 1 ^- acetyl spermidine product 3. Optimum pH: pH 7.0-8.0 (Figure 5) 4.pH when stored for 30 minutes at stability 30 ° C., with 80% or more residual activity in pH5.2~11.4 . (Fig. 6) 5. Optimum temperature: 37-42 ° C in a reaction at pH 7.5 for 15 minutes.

(FIG. 7) 6. Temperature stability Under the conditions of pH 7.5 for 30 minutes, at 4 to 35 ° C.
It has a residual activity of 80% or more and is completely inactivated by treatment at 55 ° C for 30 minutes. (FIG. 8) The method for measuring the enzyme activity of diacetylpolyamine amide hydrolase and the method for displaying the enzyme activity in the present invention are as follows.

1.0 ml of 20 mM diacetylputrescine,
Acylpolyamine amide hydrolase (JP-B-59-743)
5) 6.8 units, putrescine oxidase 0.28 units, peroxidase 0.9 units, 2,4-dichlorophenol 0.7
A mixture of 1.0 ml of 0.1 M Tris-HCl buffer (pH 8.0) containing 5 mg and 0.06 mg of 4-aminoantipyrine, and 50 μl of a test sample containing diacetylpolyamine amide hydrolase were mixed.
After reacting at a temperature of 1 ° C. for 1 hour, the reaction is performed by measuring the absorbance at 510 nm.

The enzyme activity value was determined by measuring the amount of an enzyme that produces 1 μmole of monoacetyl putrescine per minute per unit (μmole).
mole / min).

[0032]

According to the present invention, a microorganism belonging to the genus Alcaligenes, Pseudomonas, Fusarium, Candida or Trichosporon and having the ability to produce diacetylpolyamine amide hydrolase is cultured, and diacetyl polyamine amide hydrolase is produced from the culture. A method for producing diacetylpolyamine amide hydrolase, which comprises collecting.

The diacetylpolyamine amide hydrolase according to the present invention is very advantageous when monoacetylpolyamine useful as an intermediate for medical and agricultural chemicals is synthesized purely and simply.

The present invention also provides a technique for industrially producing an amide hydrolase acting on diacetylpolyamine.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[0036]

EXAMPLES Examples 1-5 0.5% polypeptone, 0.01% yeast extract, 0.1% salt, 0.1%
Medium consisting of 2% diacetyl putrescine, 0.05% potassium phosphate monobasic, 0.15% potassium phosphate monobasic (pH 7.0)
Each 10 ml was dispensed into a large test tube and autoclaved at 120 ° C. for 15 minutes. One platinum loop was inoculated from the slant culture with various microorganisms shown in Table 3. After culturing with shaking at 28 ° C for 48 to 120 hours, add 400 ml (100 ml x 4) of a medium having the same composition as above to a previously sterilized Sakaguchi flask and perform the main culture. Was. After shaking culture at 28 ° C. for 48 to 120 hours, the culture was centrifuged to collect cells. The obtained cells were lysed in 10 mM phosphate buffer (pH
7.5) The cells were suspended in 50 ml, and the suspension was sonicated to disrupt cells. The crushed liquid was centrifuged by a centrifuge to obtain a crude enzyme extract. Each crude enzyme extract 0.05
In ml, 0.95 ml of 100 mM phosphate buffer (pH 7.5) and 20 mM
1.0 ml of diacetylputrescine was added and reacted at 30 ° C. for 1 hour. After stopping the reaction by adding 0.2 ml of 50% trichloroacetic acid, the resulting monoacetylputrescine was quantified by high performance liquid chromatography. Table 3 shows the results. The produced monoacetyl putrescine in the table is at 30 ° C,
1 hour reaction, produced by the action of 0.05ml crude enzyme extract
The amount of monoacetyl putrescine formed, and the activity per 1 ml of the medium is the enzyme activity of the crude enzyme extract,
It is converted per ml.

[0037]

[Table 3] Example 6 A medium consisting of 0.5% polypeptone, 0.01% yeast extract, 0.2% diacetylputrescine, 0.1% salt, 0.05% potassium monophosphate, 0.15% potassium phosphate, 0.02% antifoam (pH 7.0) ) Put 0.5L into a 2L Erlenmeyer flask and add 12L
After autoclaving at 0 ° C for 20 minutes, Alcaligenes faecal
is IFO 14479). After shaking culture at 28 ° C. for 48 hours, this culture solution was added to a jar fermenter which had been previously sterilized with 20 L of a medium having the same composition as above, and main culture was performed. After culturing for 48 hours at 28 ° C. under agitation speed of 100 rpm and aeration of 20 L / min, the culture was centrifuged to collect cells. Of the obtained cells
200 g (wet cell weight) was suspended in 1 L of 10 mM phosphate buffer (pH 7.5), and the suspension was disrupted by an ultrasonic disrupter. The crushed liquid is centrifuged using a centrifuge,
A supernatant was obtained. The total activity of diacetylpolyamine amide hydrolase in this supernatant was 1,080 units, and the specific activity was 0.
It was 014 units / mg-tank.

The supernatant was passed through a 600 ml DEAE-cellulose column previously equilibrated with 20 mM phosphate buffer (pH 7.5) to adsorb the enzyme. After washing the column with the same phosphate buffer, diacetylpolyamine amide hydrolase was eluted with a linear concentration gradient of sodium chloride. [Total enzyme activity = 950 units, specific activity = 0.074 units / mg-tank]
After desalting the eluate by ultrafiltration, ammonium sulfate was added to a concentration of 20%, and then 100 ml of butyl preliminarily equilibrated with a 20 mM phosphate buffer (pH 7.5) containing 20% ammonium sulfate. The enzyme was adsorbed through a column of Toyopearl 650M (manufactured by Tosoh Corporation). After the column was washed with the same phosphate buffer, diacetylpolyamine amide hydrolase was eluted with a reverse linear gradient of ammonium sulfate. [Total enzyme activity = 845 units,
Specific activity = 1.6 units / mg-protein] After concentrating the obtained enzyme solution by ultrafiltration, 1.8 L of Sephacryl S-300
(Pharmacia) was passed through a column filled with the mixture, and the mixture was subjected to gel filtration to collect an active fraction. [Total enzyme activity = 762 units, specific activity = 2.2 units / mg-protein]
40m equilibrated with 20mM phosphate buffer (pH 7.5)
1 EAH-Sepharose (Pharmacia) column for adsorption. After washing the column with the same buffer,
Diacetylpolyamine amide hydrolase was eluted with a linear salt concentration gradient. [Total enzyme activity = 670 units, specific activity = 5.1 units / mg-tank] This eluate was desalted by ultrafiltration, and then preliminarily 20 mM phosphate buffer (pH 7.
70 ml of DEAE-Toyopearl 650S equilibrated in 5)
(Tosoh Corporation) column to adsorb the enzyme.
After washing the column with the same phosphate buffer, diacetylpolyamine amide hydrolase was eluted with a linear concentration gradient of sodium chloride. [Total enzyme activity = 570 units, specific activity
= 8.5 units / mg-tank] To this eluate, add ammonium sulfate to a concentration of 15%, and then equilibrate with 100 ml of a 20 mM phosphate buffer (pH 7.5) containing 15% ammonium sulfate in advance. The enzyme was adsorbed through a column of phenyl-Sepharose 4B (manufactured by Pharmacia).
After washing the column with the same phosphate buffer, diacetylpolyamine amide hydrolase was eluted with a reverse linear gradient of ammonium sulfate. [Total enzyme activity = 480
Unit, specific activity = 13 units / mg-protein] The purity of the enzyme thus obtained was examined by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Was observed and confirmed to be pure diacetylpolyamine amide hydrolase.

Next, the purified enzyme thus obtained was added to 10 mM
The optimal pH, substrate specificity, pH stability and temperature stability of the present enzyme were examined using an enzyme sample prepared by appropriately diluting with a phosphate buffer (pH 7.5).

[Optimal pH] 0.1 M Britton-Robinson broad buffer containing 10 mM diacetylputrescine (pH 4.
4, 5.7, 7.1, 7.6, 8.7, 9.4, 10.3, 11.3, 11.8, 12.
5) 0.05 ml of the enzyme preparation (0.04 unit) was added to 0.95 ml, mixed, and reacted at 37 ° C. for 15 minutes. This reaction solution
Add 0.1ml of 50% aqueous solution of trichloroacetic acid to 1.0ml,
Leave at 0 ° C for 20 minutes, then add 1M phosphate buffer (pH
7.5) 0.9 ml was added. 0.2 ml of this solution, 0.8 ml of 10 mM phosphate buffer (pH 7.5), 6.8 units of acylpolyamine amide hydrolase, putrescine oxidase 0.
Mix 1 unit of 0.1 M Tris-HCl buffer (pH 8.0) containing 28 units, 0.9 units of peroxidase, 0.75 mg of 2,4-dichlorophenol, and 0.06 mg of 4-aminoantipyrine, and add 3
After reacting at 7 ° C. for 15 minutes, the absorbance at 510 nm was measured, and each enzyme activity value was calculated. After the above operation, the relative activity (Relativ
e activity) was calculated and graphed to obtain FIG. FIG. 1 shows that the optimum pH of the present enzyme is in the range of 8.5 to 9.5.

[Substrate Specificity] 0.05 ml (1.3 units) of the enzyme preparation was added to 0.1 ml of a 100 mM solution of various diacetylpolyamines,
It was added to a reaction solution consisting of 0.85 ml of 0.1 M carbonate buffer (pH 9.0), and reacted at 37 ° C for 15 minutes. 50% trichloroacetic acid 0.1
After the reaction was stopped by adding ml, the produced monoacetylpolyamine was quantified by high performance liquid chromatography. The strength of the action on diacetylpolyamine and the action on diacetylputrescine, respectively.
Table 1 shows the relative activity value as 100%.

[PH stability] 0.1 M Britton-Robinson wide area buffer (pH 3.7, 4.4, 5.7, 7.1, 7.6, 8.7, 9.
4, 10.3, 11.3, 11.8, 12.5) 0.95 ml and enzyme standard
After mixing 0.05 ml (0.4 units) and standing at 30 ° C for 30 minutes, 0.1 ml of each solution and 0.9 ml of 1 M phosphate buffer (pH 7.5)
Was mixed. 0.05 ml of this enzyme solution and 1.0 ml of 20 mM diacetylputrescine, 6.8 units of acylpolyamine amide hydrolase, 0.28 units of putrescine oxidase, 0.9 units of peroxidase, 0.75 mg of 2,4-dichlorophenol, and 0.06 mg of 4-aminoantipyrine including
Mix 1 ml of 0.1 M Tris-HCl buffer (pH 8.0), 37 ℃
After reacting for 15 minutes at, the absorbance at 510 nm was immediately measured to calculate each enzyme activity value. After the above operations, the relative activity was calculated with the highest enzyme activity value taken as 100%, and graphed to obtain FIG. As is clear from FIG. 2, this enzyme has a residual activity of 80% or more in the pH range of 5.6 to 9.4.

[Temperature stability] 10 mM phosphate buffer (pH 7.
Prepare the enzyme preparation (0.8 units) diluted in 5) in 4, 23, 30, 37, 4
It was left at each temperature of 5, 53, 60, 67 and 75 ° C for 30 minutes. Diluent obtained by diluting this enzyme solution 10-fold with 10 mM phosphate buffer
0.05 ml and 1 ml of 20 mM diacetylputrescine, 6.8 units of acylpolyamine amide hydrolase, 0.28 units of putrescine oxidase, 0.9 units of peroxidase,
0.1 M Tris-HCl buffer containing 0.75 mg of 2,4-dichlorophenol and 0.06 mg of 4-aminoantipyrine (pH 8.
0) Mix 1 ml and react at 37 ° C for 15 minutes.
Was measured, and each enzyme activity value was calculated. After the above operation, the relative activity was calculated with the highest enzyme activity value taken as 100%, and graphed to obtain FIG. As is evident from FIG. 4, the enzyme is 80% at temperatures up to 55 ° C.
% Or more of the remaining activity.

Example 7 Medium consisting of 0.5% polypeptone, 0.01% yeast extract, 0.2% diacetylputrescine, 0.1% salt, 0.05% potassium phosphate, 0.15% potassium phosphate, 0.02% antifoam (PH 7.0) 0.5L into a 2L Erlenmeyer flask, 120
After autoclaving at 20 ° C for 20 minutes, Candidaguilliermondii was added to this medium at 28 ° C or lower.
IFO 0454). After performing shaking culture at 28 ° C. for 24 hours, the culture solution is previously cultured in a medium having the same composition as above.
The main culture was performed by adding 20 L to the jar fermenter that had been sterilized. Culture conditions: 28 ° C, stirring speed: 10
After culturing for 24 hours at 0 rpm and aeration of 20 L / min, the culture was centrifuged to collect the cells. 460 g of the obtained cells
(Wet cell weight) was suspended in 1 L of 10 mM phosphate buffer (pH 7.5), and the suspension was crushed with a Dynomill crusher. The crushed liquid is centrifuged using a centrifuge,
A supernatant was obtained. The total activity of diacetylpolyamine amide hydrolase in this supernatant was 220 units, and the specific activity was 0.003.
Unit / mg-tank.

The supernatant was passed through a 1.5 L DEAE-cellulose column previously equilibrated with 20 mM phosphate buffer (pH 7.5) to adsorb the enzyme. After washing the column with the same phosphate buffer, diacetylpolyamine amide hydrolase was eluted with a linear concentration gradient of sodium chloride. [Total enzyme activity = 170 units, specific activity = 0.087 units / mg-protein] After desalting this eluate by ultrafiltration, ammonium sulfate is added to a concentration of 25%, and then contains 25% ammonium sulfate in advance. The enzyme was adsorbed by passing through a 100 ml column of butyl toyopearl 650M (manufactured by Tosoh Corporation) equilibrated with a 20 mM phosphate buffer (pH 7.5). After the column was washed with the same phosphate buffer, diacetylpolyamine amide hydrolase was eluted with a reverse linear gradient of ammonium sulfate. [Total enzyme activity = 160 units, specific activity = 0.87 units / mg protein] The obtained enzyme solution is concentrated by ultrafiltration, and then passed through a column filled with 1.8 L of Sephacryl S-300 (Pharmacia). The active fraction was collected by gel filtration. [Total enzyme activity = 130 units, specific activity = 2.0 units / mg-protein]
40 ml of E equilibrated with phosphate buffer (pH 7.5)
It was adsorbed through a column of AH-Sepharose (Pharmacia). After washing the column with the same buffer, diacetylpolyamine amide hydrolase was eluted with a linear concentration gradient of sodium chloride. [Total enzyme activity = 77 units, specific activity = 2.7 units / mg protein] The eluate was desalted by ultrafiltration, and then 70 ml of DEAE which had been equilibrated with 20 mM phosphate buffer (pH 7.5) in advance. -The enzyme was adsorbed through a column of Toyopearl 650S (manufactured by Tosoh Corporation). After washing the column with the same phosphate buffer, diacetylpolyamine amide hydrolase was eluted with a linear concentration gradient of sodium chloride. [Total enzyme activity = 54 units, specific activity = 5.2 units / mg-tank] The eluate was desalted by ultrafiltration, and then 20 ml pre-equilibrated with 20 mM phosphate buffer (pH 7.5). Was passed through a column of DEAE-cellulose to adsorb the enzyme. After washing the column with the same phosphate buffer, diacetylpolyamine amide hydrolase was eluted with an inverse linear gradient of sodium chloride. [Total enzyme activity =
33 units, specific activity = 15 units / mg-protein] The purity of the enzyme thus obtained was examined by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Is observed,
It was confirmed to be pure diacetylpolyamine amide hydrolase.

Next, the purified enzyme thus obtained was added to 10 mM
The optimal pH, substrate specificity, pH stability and temperature stability of the present enzyme were examined using an enzyme sample prepared by appropriately diluting with a phosphate buffer (pH 7.5).

[Optimal pH] 0.1 M Britton-Robinson broad buffer containing 10 mM diacetylputrescine (pH 4.
5, 5.0, 5.6, 6.5, 7.0, 7.5, 8.2, 8.7, 9.0, 9.5, 1
0.1, 11.0, 11.3, 11.5, 11.9), 0.95 ml, 0.05 ml of enzyme preparation (0.03 unit) was added and mixed, and reacted at 37 ° C. for 15 minutes. 0.1 ml of a 50% aqueous solution of trichloroacetic acid was added to 1.0 ml of the reaction solution, left at 0 ° C. for 20 minutes, and then
0.9 ml of 1 M phosphate buffer (pH 7.5) was added. This solution
0.4 ml, 0.6 ml of 10 mM phosphate buffer (pH 7.5), 6.8 units of acylpolyamine amide hydrolase, 0.28 units of putrescine oxidase, 0.9 units of peroxidase, 0.75 mg of 2,4-dichlorophenol, and 0.06 of 4-aminoantipyrine 0.1M Tris-HCl buffer (mg
H 8.0) 1 ml was mixed and reacted at 37 ° C. for 15 minutes.
The absorbance at m was measured, and each enzyme activity value was calculated. After the above operation, relative activity (Relativeactivity) was calculated with the highest enzyme activity value taken as 100%, and graphed to obtain FIG. From Fig. 5, the optimal pH of this enzyme is 7.0-8.0
It can be seen that it is within the range.

[Substrate specificity] 0.05 ml (0.05 unit) of the enzyme preparation was added to 0.1 mM of a 100 mM solution of various diacetylpolyamines.
1, 0.85 ml of 0.1 M phosphate buffer (pH 7.5), and reacted at 37 ° C. for 15 minutes. After the reaction was stopped by adding 0.1 ml of 50% trichloroacetic acid, the produced monoacetylpolyamine was quantified by high performance liquid chromatography. Table 2 shows the strength of the action on diacetylpolyamine as a relative activity value when the action on diacetylputrescine is 100%.

[PH stability] 0.1 M Britton-Robinson broad buffer (pH 4.5, 5.0, 5.6, 6.5, 7.0, 7.5, 8.
2, 8.7, 9.0, 9.5, 10.1, 11.0, 11.3, 11.5, 11.9)
Mix 0.95 ml and enzyme standard 0.05 ml (0.4 units),
After 30 minutes, 0.1 ml of each solution and 0.9 ml of 1M phosphate buffer (pH 7.5) were mixed. 0.05 ml of this enzyme solution
0.1 M Tris-HCl containing 1.0 ml of 20 mM diacetylputrescine and 6.8 units of acylpolyamine amide hydrolase, 0.28 units of putrescine oxidase, 0.9 units of peroxidase, 0.75 mg of 2,4-dichlorophenol and 0.06 mg of 4-aminoantipyrine Buffer solution (pH 8.0)
Mix 1 ml and react at 37 ° C for 15 minutes.
The absorbance at 0 nm was measured and each enzyme activity value was calculated. After the above operations, the highest enzyme activity value is 100%
The relative activity was calculated and graphed to obtain FIG. Figure 6
As is clear from this, this enzyme has a residual activity of 80% or more in the pH range of 5.2 to 11.4.

[Temperature stability] 10 mM phosphate buffer (pH 7.
Enzyme sample (0.03 units) diluted in 5) was added to 4, 25, 30, 3
It was left at each temperature of 7, 40, 43, 45, 55 and 65 ° C for 30 minutes. 0.05 ml of a diluent obtained by diluting the enzyme solution 10-fold with 10 mM phosphate buffer and 1 ml of 20 mM diacetylputrescine,
Acylpolyamine amide hydrolase 6.8 units, putrescine oxidase 0.28 units, peroxidase 0.9
After mixing the unit, 0.75 mg of 2,4-dichlorophenol and 1 ml of 0.1 M Tris-HCl buffer (pH 8.0) containing 0.06 mg of 4-aminoantipyrine, and reacting at 37 ° C. for 15 minutes,
The absorbance at 510 nm was measured and each enzyme activity value was calculated. After the above procedure, the highest enzyme activity
The relative activity was calculated as 0%, and graphed to obtain FIG.
As is clear from FIG. 8, the enzyme has a residual activity of 80% or more at temperatures up to 35 ° C.

Examples 8 to 10 Each crude enzyme extract obtained by culturing in Examples 2, 3 and 5 was mixed with DEAE-cellulose (100 ml), butyl toyopearl (10
0 ml) and Sephacryl S-300 (500 ml) by column chromatography. As a result of examining the purity of the obtained enzyme by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate,
In both cases, only one band was observed, confirming that it was pure diacetylpolyamine amide hydrolase. The purified enzyme thus obtained was added to a 10 mM phosphate buffer (pH
PH stability, temperature stability and optimal pH were examined using enzyme preparations prepared by appropriate dilution according to 7.5).

[PH Stability] The pH of each of the 0.1 M Britton-Robinson wide area buffers was 4.6, 5.4, 6.2, 7.5, 8.5, 9.2,
Except being 10.0, 10.8, p is exactly the same as in Example 6.
H stability was investigated. Graph in the same manner as in Example 6,
9, 10, 11 were obtained. As is clear from FIGS. 9, 10 and 11, diacetylpolyamine amide hydrolases derived from Pseudomonas sp., Fusarium sp. And Trichosporon sp., Respectively, have pH 5.2 to 9.6, pH 5.4 to 10.2, and pH 5.6 to 1.
It has a residual activity of 80% or more in the range of 0.4. [Temperature stability] The temperature left for 30 minutes is 4, 3
Except that the temperature was 0, 37, 43, 50 and 56 ° C., the temperature stability was examined in exactly the same manner as in Example 6. Graphing was performed in the same manner as in Example 6, and FIGS. 12, 13, and 14 were obtained. As is clear from FIGS. 12, 13, and 14, the Pseudomonas-derived, Fusarium-derived and Trichosporone-derived diacetylpolyamine amide hydrolases have a residual activity of 80% or more at temperatures up to 45 ° C., 40 ° C., and 38 ° C., respectively. Have. [Optimal pH] 0.1 M Britton-Robinson Broad Buffer
Except that the pH was 5.6, 6.3, 7.2, 8.6, 9.4, 11.0, the optimum pH was examined in exactly the same manner as in Example 6. Graphing was performed in the same manner as in Example 6, and FIGS. 15, 16, and 17 were obtained. Figures 15, 16, 1
As is clear from FIG. 7, the optimum pH of the diacetyl polyamine amide hydrolase derived from Pseudomonas sp., Fusarium sp. And Trichosporon sp.
1 to 9.1, pH 8.9 to 9.9, pH 5.8 to 6.8.

[Brief description of the drawings]

FIG. 1 is a view showing a pH activity curve of a diacetyl polyamine amide hydrolase derived from the genus Alcaligenes.

FIG. 2 shows the pH stability of the enzyme.

FIG. 3 is a view showing a temperature activity curve of the enzyme.

FIG. 4 shows the temperature stability of the enzyme.

FIG. 5 is a view showing a pH activity curve of diacetylpolyamine amide hydrolase derived from Candida.

FIG. 6 shows the pH stability of the enzyme.

FIG. 7 is a diagram showing a temperature activity curve of the enzyme.

FIG. 8 shows the temperature stability of the enzyme.

FIG. 9 shows the pH stability of Pseudomonas-derived diacetylpolyamine amide hydrolase.

FIG. 10 is a graph showing pH stability of diacetylpolyamine amide hydrolase derived from Fusarium.

FIG. 11 shows the pH stability of Triacetylsporone-derived diacetylpolyamine amide hydrolase.

FIG. 12 shows the temperature stability of Pseudomonas-derived diacetylpolyamine amide hydrolase.

FIG. 13 shows the temperature stability of diacetylpolyamine amide hydrolase derived from Fusarium.

FIG. 14 is a view showing the temperature stability of Triacetylsporone-derived diacetylpolyamine amide hydrolase.

FIG. 15 is a view showing a pH activity curve of Pseudomonas-derived diacetylpolyamine amide hydrolase.

FIG. 16 is a view showing a pH activity curve of a diacetylpolyamine amide hydrolase derived from Fusarium.

FIG. 17 is a view showing a pH activity curve of Triacetylsporone-derived diacetylpolyamine amide hydrolase.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12N 9/80 C12R 1:72)

Claims (1)

(57) [Claims] 1. The genus Alcaligenes, the genus Pseudomonas, and the fusarium
m) A diacetylpolyamine characterized by culturing a microorganism belonging to the genus, Candida or Trichosporon and having the ability to produce diacetylpolyamine amide hydrolase, and collecting diacetylpolyamine amide hydrolase from the culture. A method for producing an amide hydrolase.