JP2680665B2 - Putrescine: Pyruvate transaminase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to putrescine: pyruvate transaminase.

The enzyme according to the present invention, putrescine: pyruvate transaminase, synthesizes L-alanine, and also traces of putrescine, pyruvate, 4-aminobutanal and L.
-Used for quantification of alanine. Further, it is also used for the purpose of analytically quantifying polyamine. For example, polyamine in a biological sample can be analyzed, and its increase or decrease can be used for diagnosis such as tumor development.

[Prior Art] Putrescine: pyruvate transaminase is an enzyme that catalyzes the following reaction.

Putrescine + Pyruvate 4-Aminobutanal + L-alanine Conventional transaminase acting on putrescine is described in Journal of Biological Chemistry (J. Biol. Chem), Vol. 239, page 783 (1964). Diamine produced by Escherichia coli B: α-ketoglutarate transaminase, Agricultural Biological Chemistry, 43, 1043 (1979) Pseudomonas (1979). Ω-amino acid produced by Pseudomonas bacterium F-126: pyruvate transaminase,
Furthermore, Journal of Bacteriology (J. Bacter
iol), 128, 722 (1976), γ derived from Pseudomonas aeruginosa.
-Aminobutyric acid: α-ketoglutarate transaminase is known.

[Problems to be Solved by the Invention] Putrescine: pyruvate transaminase is L
-It is expected to be used for the synthesis of alanine, the quantification of trace amounts of putrescine, pyruvic acid, 4-aminobutanal and L-alanine. Further, it is also used for the purpose of analytically quantifying polyamine. However, in order to be used for those purposes, conventionally known transaminase has the following drawbacks. That is, Journal of Biological Chemistry, 239.
Escherichia Collie B, Vol. 783 (1964)
The derived diamine: α-ketoglutarate transaminase does not act on polyamines such as spermidine and spermine and is therefore unsuitable for the purpose of polyamine quantification. Further, in terms of storage stability, the enzyme is not industrially sufficient. Also, Agricultural Biological Chemistry, 43, 1043 (19
1979) ω from Pseudomonas bacterium F-126
-Amino acid: pyruvate transaminase also has no effect on spermidine or spermine.

Γ-Aminobutyric acid: Pseudomonas aeruginosa-derived γ-aminobutyric acid: α-ketoglutarate transaminase described in Journal of Bacteriology, Vol. 128, p. 722 (1976) is known to act on putrescine, spermidine and spermine. , L-lysine and L-ornithine also act, so that the amount of polyamine in the sample cannot be measured accurately.

Furthermore, these enzymes had the drawback that the Km value for putrescine and pyruvic acid was large, and the reaction rate was slow for a small amount of putrescine and pyruvic acid.

[Means for Solving Problems] As a result of earnest studies to solve the above-mentioned conventional drawbacks, the present inventors acted on putrescine, spermidine, and spermine without acting on L-ornithine and L-lysine. In addition, a Km value for putrescine is about 1/10 of the Km value of a conventional enzyme, and a putrescine: pyruvate transaminase having higher thermostability was found in a microorganism and the present invention was completed.

That is, the present invention has a molecular weight of 19 having the following physicochemical properties.
Provides 2,000 ± 5,000 putrescine: pyruvate transaminase.

Action As shown in the following formula, it catalyzes a transamination reaction that produces 4-aminobutanal and L-alanine by acting on an amino group donor putrescine and an amino group acceptor pyruvic acid, and its reverse reaction. To do.

NH ₂ (CH ₂ ) ₄ NH ₂ + CH ₃ COCOOH NH ₂ (CH ₂ ) ₃ CHO + CH ₃ CH (NH ₂ ) COOH Substrate specificity (1) Amino group donor Acts on putrescine, cadaverine, spermidine, spermine, It has no effect on L-lysine and L-ornithine.

(2) Amino group receptor It acts on pyruvic acid and glyoxylic acid, and does not act on α-ketoglutarate.

Table 1 shows the strength of the action on the amino group donor and the amino group acceptor as relative activity values with the action on putrescine and pyruvic acid as 100, respectively.

Optimum pH: 9.5 to 10.5 (shown in FIG. 1) pH stability: When stored at 30 ° C. for 1 hour, it has 90% or more residual activity at pH 4.5 to 13.0. (Shown in FIG. 2) Further, the physicochemical properties of the putrescine: pyruvate transaminase of the present invention other than the above-mentioned to are as follows.

1. Optimum temperature: PH10.5, 30-60 minutes, 55-60 ℃
It is. (Shown in Fig. 3) 2. Temperature stability: PH7.5, under treatment condition of 1 hour, 70
It has 90% or more residual activity at temperatures up to ° C. Also, 80
Complete inactivation occurs at 1 ° C for 1 hour. (Shown in Figure 4) 3. Molecular weight: 192,000 ± 5,000 (Biosil TSK-250; measured by gel filtration method by Bio-Rad) 4. Molecular weight of subunit: 48,000 ± 5,000 (by SDS-polyacrylamide gel electrophoresis method) (Measurement) 5. Number of subunits: 4 6. Km value: PH1 with putrescine and pyruvate as substrates
The Km value for putrescine determined under the conditions of 0.5 and 30 ° C. is 0.2 mM, and the Km value for pyruvic acid is 1.0 mM.

7. Coenzyme: pyridoxal phosphate 8. Absorption spectrum: Absorption maximum at 280 nm and 416 nm.
(Shown in FIG. 5) 9. Inhibitors: Table 2 shows the effects of various reagents and the metal ion on the enzyme of the present invention at a concentration of 1 mM. It is strongly inhibited by pyridoxal phosphate inhibitors such as hydroxylamine, phenylhydrazine and D-cycloserine. It is strongly inhibited by parachloromercuribenzoic acid, silver ions and mercury ions.

As a result of comparison between putrescine having the above-mentioned physicochemical properties: pyruvate transaminase and a conventionally known transaminase capable of acting on putrescine, the enzyme of the present invention is an enzyme having properties different from those of conventional transaminase. Became clear.

Table 3 shows a comparison of the physicochemical properties of putrescine: pyruvate transaminase according to the present invention and conventionally known transaminase.

The conventional transaminase in Table 3 is defined in Journal of Biological Chemistry (J. Bio
l.Chem), 239, 783 (1964), a diamine derived from Escherichia coli B:
α-Ketoglutarate transaminase, Agricultural Biological Chemistry (Agric.Biol.C
hem), 43, 1043 (1979), ω-amino acid from Pseudomonas bacterium F-126:
Pyruvate transaminase, and Pseudomonas aeruginosa (Ps) described in Journal of Bacteriology, 128, 722 (1976).
γ-aminobutyric acid: α-ketoglutarate transaminase from Eudomonas aeruginosa).

As can be seen from Table 3, the putrescine: pyruvate transaminase of the present invention is an enzyme having a different substrate specificity from the conventionally known transaminase.

This putrescine: pyruvate transaminase has Km values of 0 for putrescine and pyruvate, respectively.
It is as low as 2 mM and 1.0 mM, and the reaction proceeds rapidly even with a small amount of putrescine and pyruvic acid.

In addition, even though it was obtained from Streptomyces avellaneus which grows at room temperature, it has a high heat resistance of 90% or more residual activity even after heat treatment at 70 ° C for 1 hour. . It was also found to have the excellent property of being stable in a wide pH range of pH 4.5 to 13.0. The fact that the putrescine: pyruvate transaminase of the present invention is excellent in affinity for substrates, heat resistance, and pH stability as described above is a very useful property when industrially or clinically using this enzyme. .

The method for displaying the enzyme activity of putrescine: pyruvate transaminase and the method for displaying the enzyme activity in the present invention are as follows.

0.1 M phosphate buffer containing 2 mM putrescine, 2 mM pyruvate and 0.02% O-aminobenzaldehyde (pH 7.
5) 2.0 ml and 0.2 ml of a sample containing putrescine: pyruvate transaminase are mixed, and the mixture is mixed at 30 ° C. for 30 minutes to 1 minute.
Immediately after the reaction, the absorbance at 435 nm is measured. The enzyme activity value of putrescine: pyruvate transaminase enzyme transaminase per 1.0 ml of the test substance is calculated from the increase amount (A) of the absorbance per minute using the following conversion formula (1). The enzyme activity value is expressed as 1 unit (μmole / min) of the amount of enzyme that produces 1 μmole of 1-pyrroline per minute.

The putrescine: pyruvate transaminase of the present invention can be collected from a culture of a microorganism capable of producing the putrescine: pyruvate transaminase. The microorganism is, for example, a strain belonging to the genus Streptomyces, and preferably Streptomyces aberanius R-20 [Ministry of Industrial Research, No. 5443].

As the medium used for culturing the production bacteria, glucose, molasses, carbon sources such as soluble starch, meat extract, yeast extract, nitrogen sources such as polypeptone, and potassium monophosphate, dipotassium phosphate, sodium chloride, etc. It is not particularly limited as long as it contains inorganic salts, but in addition to these components, an acylpolyamine such as acetylputrescine, benzoylputrencine or putrescine, spermidine, diaminopropane, or polyamine such as calzine may be contained. It is advantageous in increasing the productivity of the enzyme.

The culture conditions for cultivating the putrescine: pyruvate transaminase-producing bacterium of the present invention include:
A method of culturing under aeration and stirring conditions at a culturing temperature of 15 to 40 ° C, preferably 20 to 35 ° C is suitable. The pH condition during the culture is in the range of 5.0 to 9.0, preferably pH 6.0 to 8.0.
Is appropriate. The culturing time is not particularly limited, but in consideration of the economic efficiency such as the productivity of the enzyme, it is appropriate that the culturing time is within the range from the time when the latter stage of growth is reached to 10 hours after entering the resting state.

As a method for separating the culture solution and the bacterial cells from the culture obtained by the culturing, conventionally used methods such as centrifugation and filtration can be used, but the method of centrifugation is preferred.

Separation and purification of putrescine: pyruvate transaminase of the present invention can be performed as follows.

As a method for extracting the enzyme accumulated in the microbial cells from the microbial cells, microbial cell disruption by ultrasonic waves that has been conventionally performed,
Alternatively, methods such as cell disruption by a Dynomill cell disruptor rotated with glass beads or cell membrane disruption by an enzyme such as lysozyme or an organic solvent such as toluene may be mentioned. The enzyme can be collected by selecting an appropriate method from these and extracting the enzyme from the bacterial cells.

When it is necessary to further purify putrescine: pyruvate transaminase from the crude enzyme solution extracted by these methods, ammonium sulfate precipitation method and ion-exchange column chromatography, which are commonly used means for purifying enzymes, are usually carried out. The purification can be carried out by appropriately combining or repeating methods such as the method, gel filtration method, hydroxyapatite column chromatography method, affinity chromatography method, preparative electrophoresis method and the like.

Further, a method of denatured contaminating proteins by heat treatment and then separating and removing them by utilizing the high heat resistance of putrescine: pyruvate transaminase according to the present invention can also be an effective means of purification.

The specific activity value of the fully purified enzyme of putrescine: pyruvate transaminase according to the present invention is about 4.5 units / mg-protein. In addition, a single protein band is observed in both the presence and absence of sodium dodecyl sulfate in polyacrylamide gel electrophoresis.

[Effect] The putrescine: pyruvate transaminase of the present invention has a low Km value for putrescine and pyruvate, acts also on spermidine and spermine, is stable in a wider pH range, and has temperature stability. It is characterized by being excellent. These properties are very advantageous in using this enzyme industrially and as a clinical test agent. Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited to these examples.

[Example] 0.5% glucose, 0.4% polypeptone, 0.5% fish meat extract, 0.3% acetylputrescine, 0.2% sodium chloride, 0.02%
A medium (pH 7.5) 1 composed of an antifoaming agent was placed in an Erlenmeyer flask of 5 and autoclaved at 120 ° C for 20 minutes.
Streptomyces aberanius R-20 in this medium
[Microtechnology Research Institute, No. 5443] was inoculated. After shaking culture at 28 ° C. for 24 hours, this culture solution was added to a jar fermenter in which a medium 150 having the same composition as the above was charged and sterilized in advance for main culture. Culture conditions are 28
After culturing at 18 ° C. for 18 hours at a stirring speed of 300 rpm and aeration of 100 / min, the culture was centrifuged to collect the cells.

About 2.7 kg (wet cell weight) of the obtained cells was suspended in 10 mM phosphate buffer (pH 7.5) 12 containing 40% ethanol,
The suspension was continuously passed through a Dynomill cell disrupter to disrupt cells. The disrupted liquid was centrifuged using continuous centrifugation to obtain a supernatant liquid. The total activity of putrescine: pyruvate transaminase in this supernatant was 22,0.
The specific activity was 00 units and 0.072 units / mg-protein.

This supernatant was added to 4 DEAE-cellulose (trade name: Whatman), which had been equilibrated with 10 mM phosphate buffer (pH 7.5) in advance, and the mixture was stirred for 1 hour and then 10 mM containing 40 mM ammonium sulfate. It was washed with phosphate buffer (pH 7.5) 15. Then, a 10 mM phosphate buffer solution (pH 7.
5) The enzyme component was eluted in step 5. [Total enzyme activity = 15,40
0 unit, specific activity = 0.56 unit / mg-protein] This enzyme solution was desalted by ultrafiltration, treated at 65 ° C. for 30 minutes, and the resulting precipitate was removed by centrifugation. [Total enzyme activity = 12,200 units, specific activity = 2.1 units / mg-protein] The enzyme solution thus obtained was loaded onto 1 DEAE-cellulose column which had been equilibrated with 10 mM phosphate buffer (pH 7.5) in advance. It was adsorbed through. After washing the column with the same buffer 2, putrescine:
Pyruvate transaminase was eluted. [Total enzyme activity = 8,820 units, specific activity = 3.5 units / mg-protein] This eluate was desalted by ultrafiltration, and then ammonium sulfate was added to 20%, and then 20% ammonium sulfate was previously contained. The enzyme was adsorbed by passing it through a column of 0.1 butyl Toyopearl 650M (trade name: manufactured by Tosoh Corporation) equilibrated with 10 mM phosphate buffer (pH 7.5).
After washing the column with the same phosphate buffer, putrescine: pyruvate transaminase was eluted with an inverse linear concentration gradient of ammonium sulfate. [Total enzyme activity = 7,
430 units, specific activity = 4.4 units / mg-protein] The obtained enzyme solution was concentrated by ultrafiltration, and then passed through a column packed with 1.7 Sephacryl S-400 (trade name: Pharmacia) for gel filtration. The active fractions were collected. [Total enzyme activity = 7,360 units, specific activity = 4.5 units / mg-protein] The purity of the thus obtained enzyme was examined by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In each case, only one band was observed, and it was confirmed that the band was pure putrescine: pyruvate transaminase.

In addition, the molecular weight of this enzyme was measured by a gel filtration method using Biosil TSK-250 (trade name: manufactured by Bio-Rad), and was estimated to be about 192,000. Furthermore, the molecular weight of the subunit was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
It was estimated. From the molecular weight and the molecular weight of the subunit, it can be seen that the enzyme of the present invention is an oligomeric enzyme composed of four subunits.

Next, using the enzyme preparation prepared by diluting the purified enzyme thus obtained to an appropriate concentration with 20 mM phosphate buffer (pH 7.5), the optimum pH, pH stability, , Temperature stability, and absorption spectrum.

[Optimal pH] 0.1 acetate buffer (pH 3.2, 4.4, 5.7) or 0.1 M phosphate buffer (pH 5.0, containing 2 mM putrescine and 2 mM pyruvate)
6.0,7.0,7.5) or 0.1M Tris (hydroxymethyl)
Aminomethane-hydrochloric acid buffer (pH 7.0, 8.2, 9.2) or 0.1
Add 0.2 ml of enzyme preparation (0.002 units) to 1.8 ml of M carbonate buffer (pH 9.0, 9.4, 10.6, 11.7) or 0.1 M tetraborate buffer (pH 10.5, 11.4, 12.1, 13.0) and mix Then, the reaction was carried out at 30 ° C. for 30 minutes. 0.2 ml of a 20% aqueous solution of trichloroacetic acid was added to 1.0 ml of the reaction solution, and the mixture was allowed to stand at 0 ° C. for 20 minutes. Then, 1.0 ml of 0.5 M phosphate buffer (pH 7.5) containing 0.02% O-aminobenzaldehyde was added and allowed to stand at room temperature for 30 minutes, and then the absorbance at 435 nm was measured to calculate the enzyme activity value of each. . After the above operation, the highest enzyme activity value
Relative activity was calculated as 100%,
A graph was obtained to obtain FIG. From Fig. 1, the optimum of this enzyme
It can be seen that the pH is in the range of 9.5 to 10.5.

[PH stability] 50 mM acetate buffer (pH 3.5, 4.5, 5.5, 5.9) or 50 mM phosphate buffer (pH 5.6, 6.2, 6.7, 7.5, 8.3) or 50 mM tris (hydroxymethyl) aminomethane-hydrochloric acid Buffer solution (pH
7.6, 8.1, 8.9) or 50 mM borate buffer (pH 8.2, 9.3, 9.
9) or 50mM carbonate buffer (pH9.4,10.6,11.7) or 50
0.95 ml of mM tetraborate buffer (pH 10.8, 12.7, 13.2, 13.7) and 0.05 ml of enzyme preparation (50 units) were mixed and allowed to stand at 30 ° C for 1 hour, then 0.02 ml of each solution was added to 20 mM phosphoric acid. Buffer solution (pH 7.
5) 1.98 ml were mixed. 0.2 ml of this enzyme solution was added to 2.0 ml of 0.1 M phosphate buffer (pH 7.5) containing 2 mM putrescine, 2 mM pyruvic acid and 0.02% O-aminobenzaldehyde, and immediately reacted at 30 ° C. for 1 hour, then immediately at 435 nm. The absorbance at was measured and the enzyme activity value of each was calculated.

After the above operation, calculate the relative activity with the highest enzyme activity value as 100%,
A graph was obtained to obtain FIG. As is clear from FIG. 2, this enzyme has a residual activity of 90% or more in the pH range of 4.5 to 13.0.

[Optimum temperature] 1.8 ml of 0.1 M carbonate buffer (pH 105) containing 25 mM putrescine and 25 mM pyruvate and 0.2 ml of enzyme preparation (0.013 units) were mixed, and the mixture was added to 30,39,44,50,56,60, The reaction was carried out for 30 minutes at each temperature of 68 and 79 ° C. 0.2 ml of a 20% trichloroacetic acid solution was added to 1 ml of each reaction solution, and left at 0 ° C. for 20 minutes. Next, 1.0 ml of 0.5 M phosphate buffer (pH 7.5) containing 0.02% O-aminobenzaldehyde was added, and the mixture was allowed to stand at room temperature for 30 minutes, and then the absorbance at 435 nm was measured to calculate each enzyme activity value. . After the above operation, the relative activity was calculated with the highest enzyme activity value taken as 100%, and graphed to obtain FIG. From Fig. 3, the optimal temperature of this enzyme is 55-
It turns out that it is 60 degreeC.

[Temperature stability] An enzyme preparation (0.0%) diluted with 25 mM phosphate buffer (pH 7.5)
34 units / ml) was allowed to stand for 1 hour at each temperature of 4,30, 40, 50, 60, 70, 75, 80 ° C. 0.2 ml of this enzyme solution was added to 2.0 ml of 0.1 M phosphate buffer (pH 7.5) containing 2 mM putrescine, 2 mM pyruvic acid and 0.02% O-aminobenzaldehyde, and immediately reacted at 30 ° C. for 1 hour, then immediately at 435 nm. The absorbance at was measured and the enzyme activity value of each was calculated. 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. 4, the enzyme has a residual activity of 90% or more at temperatures up to 70 ° C.

[Absorption Spectrum] Using an enzyme preparation- (a) (180 μg / ml) prepared by diluting the purified enzyme with 20 mM phosphate buffer (pH 7.5), the absorption spectrum in the ultraviolet region was measured. FIG. 5- (a) was obtained. An enzyme preparation prepared in the same manner-(b) (910 μg / m
Using l), the absorption spectrum in the visible region was measured to obtain FIG. 5- (b). FIG. 5 shows that the enzyme was 280n
It can be seen that there is an absorption maximum at m, 416 nm.

[Brief description of the drawings]

FIG. 1 is a pH activity curve of putrescine: pyruvate transaminase of the present invention (◯: acetate buffer, Δ: phosphate buffer, □: tris-hydrochloric acid buffer, ●: carbonate buffer, ▲: tetraborate FIG. 2 also shows pH stability (◯: acetate buffer, Δ: phosphate buffer, □: Tris-hydrochloric acid buffer, ●: borate buffer, ▲: carbonate buffer, ■ : Tetraborate buffer), FIG. 3 shows the temperature activity curve, FIG. 4 shows the temperature stability, and FIG. 5 shows (a) in the ultraviolet region and (b) in the visible region. 3A and 3B respectively show the absorption spectra of.

Claims (1)

(57) [Claims] 1. A molecular weight of 192,000 having the following physical and chemical properties.
± 5,000 putrescine: pyruvate transaminase. Action: By acting on putrescine and pyruvate, 4-
It catalyzes a transamination reaction that produces L-alanine with aminobutanal. Substrate specificity: (1) Acts on putrescine, cadaverine, spermidine and spermine as an amino group donor. (2) Acts on pyruvic acid and glyoxylic acid as amino group receptors. Optimum pH: 9.5 to 10.5 pH stability: When stored at 30 ° C for 1 hour, it has 90% or more residual activity at pH 4.5 to 13.0.