JPH0141307B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing polyamine oxidase by fermentation. In recent years, the relationship between increased polyamine concentrations in blood, urine, etc. and cancer diseases has attracted attention, and the use of polyamine oxidase for diagnosis of cancer diseases has been considered. Although polyamine oxidase obtained from animal tissues is used as a source of polyamine oxidase, it is impossible to obtain it industrially at low cost.
Furthermore, polyamine oxidases originating from microorganisms such as those of the genus Aspergillus and Mucor have been reported [Agric.Biol.Chem., 44 2469 (1980)]. These mainly contain polyamines and
In order to produce oxidase, the enzyme must be extracted from the bacterial cells using an ultrasonic crusher or the like. The present inventors aimed to obtain polyamine oxidase without using an ultrasonic crusher or the like, and mainly searched for microorganisms that produce polyamine oxidase outside the bacterial body. As a result, it was discovered that a strain of the genus Streptomyces mainly produces polyamine oxidase outside the bacterial body, and the present invention was completed by successfully extracting and purifying polyamine oxidase. The microorganism used in the present invention belongs to the genus Streptomyces and has the ability to produce polyamine oxidase. Any polyamine oxidase producing strain obtained by irradiation with ultraviolet rays or treatment with nitrosoguanidine or the like can be used. Furthermore, the present invention utilizes the function of a gene involved in polyamine oxidase synthesis of a microorganism belonging to the genus Streptomyces, and uses methods such as introducing this gene into other microorganisms, such as transduction using a plasmid. It also includes the use of polyamine oxidase-producing microorganisms obtained by cell fusion using protoplasts.
The polyamine oxidase-producing bacterium belonging to the genus Streptomyces isolated by the present inventors was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on October 7, 1981, and the accession number is Fiber Science and Technology Research Institute No. 6172. , its taxonomic properties are as follows. (1) Morphology: The basal hyphae of this strain elongate and branch without dividing. The aerial hyphae are well elongated, and the sporophytes on the aerial hyphae are straight or gently wavy. The shape of the spores is circular or cylindrical (0.3 to 0.5 x 0.7
~1.0 μm) and the surface is smooth. The number of spores per sporophyte ranges from 10 to 50, and larger numbers can be found. (2) Growth status on various media 1. Seuucrose nitrate agar medium (cultured at 27°C): Gray-white aerial mycelium grows loosely on the gray-white growth, and no soluble pigment is observed. 2 Glucose-asparagine agar medium (27℃
Culture): Gray-white aerial mycelium grows loosely on the gray-white growth, and no soluble pigment is observed. 3 Yeast malt agar medium (ISP medium 2, 27℃
Culture): Gray-white aerial mycelium grows on the pale yellow-brown growth, and no soluble pigment is observed. 4 Oatmeal agar medium (ISP medium 3, 27℃
Culture): Gray-white aerial mycelium grows on the white growth, and no soluble pigment is observed. 5. Starch inorganic salt agar medium (ISP4, cultured at 27°C): Grayish-white aerial mycelium grows on the white growth, and no soluble pigment is observed. 6 Glycerin-asparagine agar medium (ISP
Medium 5, culture at 27°C): Grayish-white aerial mycelium grows on the grayish-white growth, and no soluble pigment is observed. 7. Peptone yeast iron agar medium (ISP medium 6, cultured at 27°C): Gray aerial mycelium grows on the gray-brown growth, and the soluble pigment is brown. 8. Tyrosine agar medium (ISP medium 7, cultured at 27°C): Gray aerial mycelium grows on the gray-brown growth, and the soluble pigment is only slightly yellowish. 9 Nutrient agar medium (cultured at 27°C): White to gray aerial mycelium grows on gray-brown growth, and no soluble pigment is observed. 10 Bennet's agar medium (cultured at 27°C): White aerial mycelium grows on the yellow-brown growth, and the soluble pigment is only slightly yellowish. (3) Physiological properties 1 Growth temperature range: 12-37℃ 2 Growth PH range (Tryptone Yeast Pross medium ISP medium 1, 27℃ culture): PH5-9 3 Production of melanin-like pigment (Tryptone Yeast Pross medium ISP medium 1, 27℃ culture) Culture medium ISP medium 1, peptone yeast iron agar medium ISP medium 6, tyrosine agar medium ISP medium 7, cultured at 27°C): Negative on tyrosine agar medium, but positive on the other two media. 4 Hydrolysis of starch (starch agar medium)
ISP medium 4, 27℃ culture): Positive 5 Liquefaction of gelatin (glucose, peptone,
Gelatin medium, cultured at 27℃): Negative 6 Coagulation and peptonization of skim milk (skimmed milk,
27℃ culture): Negative 7 Hydrogen sulfide production (hydrogen sulfide generation medium, 27℃
Culture): Positive 8 Nitrate reduction reaction (1% nitrate-containing peptone water, cultured at 27°C): Positive 9 Salt tolerance (yeast malt agar medium containing sodium chloride, cultured at 27°C): No growth at 4 g/dl 10 Carbon source Usability (Pridham, Gotlieb agar medium ISP medium 9, 27°C culture): D-glucose, sucrose, D-fructose,
D-galactose is often used for growth, and L
- Growth in arabinose, D-xylose, D-mannitol, and raffinose is weak, and i-inositol and L-rhamnose are not utilized. To summarize the above characteristics, this strain belongs to the genus Streptomyces, the color of the aerial hyphae is grayish, the shape of the sporophyte is straight or wavy, the spore shape is circular or cylindrical, and the surface is smooth. ,
Produces melanin-like pigment. When searching for known bacterial species based on these properties and the availability of carbon sources, Streptomyces tanashiensis, International
Journal of Systematic Bacteriology18 380,
(1968)] are listed as closely related species. Comparing the properties of this strain with Streptomyces thanasiensis described in ISP, the two agree well as shown in Table 1. Therefore, this strain was considered to be a species closely related to Streptomyces thanasiensis and was identified as Streptomyces thanasiensis 97SY-4.

[Table] The medium used in the present invention may be either a synthetic medium or a natural medium as long as a carbon source, a nitrogen source, inorganic substances, and other nutrients are present in suitable ratios. Glucose, soluble starch, maltose, and the like are used as carbon sources as a medium suitable for producing polyamine oxidase. Yeast extract, polypeptone, soybean extract (Ssan Meat), etc. are used as nitrogen sources. Further, as the inorganic substance, potassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, etc. are used. As a culture method, a liquid culture method (shaking culture method or aerated agitation culture method) is preferred, and an aerated agitation culture method is also suitable for industrial purposes. The culture temperature is preferably around 27°C, and the optimal pH is around neutrality. The culture period varies depending on the conditions, but is usually about 3 to 7 days. This enzyme mainly accumulates outside the bacterial cells. Separation and purification of polyamine oxidase is carried out as follows. After the cultivation is completed, the bacterial cells are removed from the culture by filtration or centrifugation to obtain a culture solution. A purified enzyme can be obtained by treating this culture solution with methods commonly used for enzyme purification, such as salting out, dialysis, ion exchange chromatography, and gel filtration. In order to obtain polyamine oxidase from bacterial cells, the bacterial cells collected by filtration or centrifugation are disrupted by an appropriate means, and then centrifuged to obtain a cell-free extract. The subsequent operations can be performed in the same manner as for the culture solution. The enzymatic properties of the polyamine oxidase obtained according to the present invention are as follows. (1) Action This enzyme acts on spermine and spermidine to produce putrescine, 3-aminopropionaldehyde and hydrogen peroxide. (2) Enzyme activity measurement method 1 Using spermidine as a substrate, hydrogen peroxide produced during the reaction, 4-aminoantipyrine, and phenol are reacted in the presence of horseradish peroxidase, and the resulting dye is measured at 500 nm.
Measure the enzyme titer. That is, 4-
Dissolve 0.82 m mole of aminoantipyrine, 14 m mole of phenol, and 1900 units of horseradish peroxidase in 1 part of 0.1 M phosphate buffer (PH 6.8). 2ml of this coloring liquid
0.5ml of 20mM spermidine and 0.1ml of this enzyme solution
Add and react at 37℃ for 30 minutes, then measure at 500nm. For the enzyme titer, one unit was defined as the amount of enzyme that produced 1 μmole of hydrogen peroxide per minute. 2. Method 1 is quick and simple, but because it uses horseradish peroxidase, it is not suitable for examining the effects of inhibitors. Therefore, the method of R. Pad-manabhan et al., which measures the absorption of thiosemicarbazone of 3-aminopropionaldehyde at 256 nm [Biochem.Biophys.Res.
Commun. 19, 1 (1965)]. (3) Substrate specificity Table 2 shows a comparison of the effects on various amines. The activity was expressed as a relative activity when the value for spermidine was set as 100.

[Table] (4) Optimal PH Figure 1 shows the PH activity curve of this enzyme. As is clear from FIG. 1, the optimum pH is between 9 and 10. (5) PH stability Figure 2 shows the PH stability curve of this enzyme. As is clear from the figure, this enzyme is stable at pH 6.5 to 10. (6) Temperature stability Figure 3 shows the temperature stability of this enzyme. As is clear from Figure 3, this enzyme is stable up to 50°C. (7) Effects of inhibitors Table 3 shows the effects of various drugs on this enzyme. This enzyme was strongly inhibited by copper ions, mercury ions, and silver ions.

【table】

[Table] Next, the present invention will be specifically explained with reference to Examples. Example 1 In a 500ml Sakaguchi flask, 1% glucose, 1% soybean extract (Ssan Meat),
K ₂ HPO ₄ 0.1%, KCl 0.05%, MgSO ₄・
Add 100ml of medium (PH7.0) containing _7H200.05 % to 120
After autoclaving for 20 min at °C, Streptomyces
thanaciensis 97SY-4 was inoculated with 1ase and 27
The cells were cultured with shaking at ℃ for 5 days. The potency of polyamine oxidase contained in the culture solution and bacterial cells is
They were 0.27 units/ml and 0.01 units/1g. Example 2 One portion of glucose in a 25 volume jar fermenter
%, soybean extract (essan meat) 1%,
K ₂ HPO ₄ 0.1%, KCl 0.05%, MgSO ₄・7H ₂ O 0.05
A medium containing 12% (PH7.0) was prepared and the medium was sterilized by a conventional method. 1% soluble starch, yeast extract
500 ml of Streptomyces thanasiensis 97-SY4 seed culture cultured with shaking in a medium containing 0.2% (PH7.0) for 3 days was transplanted, and the aeration rate was 10 per minute and the number of stirring was
Aerated agitation culture was performed at 250 rpm and 27°C for 4 days. The titers of polyamine oxidase contained in the culture solution and bacterial cells are 0.76 units/ml and 0.01 units/g, respectively.
It was hot. Example 3 Culture solution obtained by the same operation as Example 2
3.04 kg of ammonium sulfate was added to No. 10, left overnight, and centrifuged to obtain a precipitate. 0.1M of this precipitate
After dissolving in phosphate buffer (PH7.0), dialysis was performed against the same buffer. DEAE equilibrated with the same buffer
- The dialyzed enzyme was adsorbed onto a Sephadex column (5 x 30 cm). 0.1M phosphate buffer (PH
After washing the column with 7.0) sodium chloride 0~
Concentration gradient elution was performed using the same buffer containing 1M. The active fractions were collected, dialyzed against distilled water, and then lyophilized to obtain 1.54 g of dry powder.

[Brief explanation of drawings]

Figure 1 shows the optimal PH curve of polyamine oxidase, Figure 2 shows the PH stability curve of the same enzyme, and Figure 3 shows the temperature stability curve.

Claims (1)

[Claims] 1. A method for producing polyamine oxidase, which comprises culturing a microorganism belonging to the genus Streptomyces and having the ability to produce polyamine oxidase, and collecting polyamine oxidase from the resulting culture.