JPS63219373A - Phospholipase d and production thereof - Google Patents

Abstract

PURPOSE:To obtain phospholipase D having phosphatidyl group converting ability and high thermostability, by cultivating Streptomyces prunicolor SK-02-92 strain and collecting phospholipase D from the culture solution. CONSTITUTION:Streptomyces prunicolor SK-02-82 strain (FERM P-9225) is cultivated by the use of a medium generally adopted in culture of ordinary ray fungus and the culture is stopped when production of phospholipase D of the aimed substance reaches optimum. The phospholipase D has the following physical and chemical properties. Optimum temperature: 50-55 deg.C. Optimum pH: about 6.0. Stable at pH 5-8 and not deactivating at all under heating at pH 5 or 6 at 50 deg.C for 3 months or under heating at 55 deg.C for 100min.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing phospholipase D, which has transphosphatidyl group activity in water and various organic solvents and has excellent thermal stability. be.

Phospholipase D (particularly the one that has the ability to catalyze the transfer of phosphatidyl groups described below) is a phosphatase D that is contained in soybeans and egg yolks.
7 It is used to produce phospholipid derivatives useful as emulsifiers, dispersants, agricultural chemicals, pharmaceuticals, industrial reagents, etc. from phospholipids such as tinorcholine and phosphatidylethanolamine and various alcohols.

PRIOR ART Phospholipase D (EC3,1,4,4) is an enzyme that hydrolyzes the ester bond between the phosphatidyl group of phospholipids and a base to liberate phosphatidic acid and base, but depending on its origin, , hydrolyzing the ester bond between the phosphatidyl group of the glycerophospholipid and the base in the coexistence of a compound having an alcoholic hydroxyl group such as glycerol or ethanol, and simultaneously forming a new ester with the phosphatidyl group and the above-mentioned compound having an alcoholic hydroxyl group. Generating reaction (phosphatidyl group transfer reaction)
cause to occur.

It has been known for a long time that this enzyme exists widely in plants such as cabbage and carrots, and other plant kingdoms, and it has been common to produce it by extracting it from plants containing this enzyme. In the genus Streptomyces,
A method of producing by fermentation using microorganisms of the genus Micromonospora, genus Norkadiopsis, genus Actinomadura, and genus Mecardia has been proposed and partially implemented (Japanese Patent Publication No. 52-39918).

JP 58-52633, JP 58-63388, JP 58-67183, JP 60-16448
No. 3).

However, as far as phospholipase D obtained by these conventional production methods is concerned, it has poor thermal stability under optimal conditions, and therefore the transfer reaction cannot be carried out efficiently at high temperatures. There was a problem.

In addition, the conventional production method using microorganisms not only does not provide enzymes with good thermostability as described above, but also has the problem of poor enzyme productivity per culture solution, resulting in expensive products. Ta. Furthermore, it has been pointed out that the microorganisms used in some manufacturing methods are pathogenic, and there are safety issues when using them in the manufacturing of food emulsifiers.

Problems to be Solved by the Invention In view of the fact that the conventional phospholipase D having phosphatidyl group transfer activity has the above-mentioned drawbacks, and that its production method also leaves room for improvement, the present invention has developed a highly advanced phospholipase D. The present invention aims to provide phospholipase D exhibiting thermostability and phosphatidyl group transfer activity and an efficient method for producing the same.

The present inventors investigated the ability of a large number of microorganisms to produce phospholipase D, which exhibits transphosphatidyl group activity, and further developed selected highly efficient phospholipase D producing bacteria. We repeated experiments to search for strains that produce particularly thermostable enzymes. As a result, a new bacterial strain, Streptomyces spp., was isolated from soil collected in the Tama area of Tokyo.
Purnicolor (S trepto-+ayces pr
It was discovered that SK-02-81 (unicolor) has excellent performance, and the present invention was completed.

That is, the present invention provides a method for producing phospholipase D using the Streptomyces pur-color 3K-02-81 strain, and highly thermostable phospholipase D obtained thereby.

The Streptomyces purnicara-3K-02-81 strain used in the production method of the present invention has the following mycological properties.

■ Morphological characteristics Aerial hyphae: Mainly straight, slightly curved. Sometimes nested or sclerotium-like forms are seen.

Width 0.4-0.6um.

Spore: Size 0.4-0.611mX1.1-1.3X
Im's short cylindrical shape 1 surface is smooth.

Substratum hyphae: somewhat wavy, with short axes branching. There was no hyphal fragmentation or spore settlement.

Flagellated spores and sporangia: not formed.

The growth status on various media is as shown in the secondary table (however, at 28℃
The display of 0 colors after 2 weeks is in accordance with the color classification according to the JIS Z8721 standard color chart. ).

■ Amino acid composition and sugar composition of cell wall meso-noaminopimelic acid - and L-noaminopimelic acid
Deca-glycine + Glutamic acid Deca-alanine + Arabinose - Galactose - Fructomin - Glucosamine + From the above, the cell wall of the 5K-02-81 strain is type 1.

■ Physiological properties a, Growth temperature range: 20-40℃ b, Optimal growth temperature: 28-35℃ C0 optimum p) 1:6-7 d, Liquefaction of gelatin: None e, Hydrolysis of starch: Yes f, Coagulation of skimmed milk, pepnization: None g, Melanoid pigment production: Eggplant, Carbon source availability (30″C916 days @n>: L・
Arabinose 10 〇−Xylose 10−
Glucose 10.7 Lactose 10-
Mannitol + sucrose + wild boar F-ru + rhamnose + raffinose + i, GC content near 0.4% ([Experimental method for identifying actinomycetes] Japan Actinomycetes Research Society, 151-
The fact that this strain is Streptomyces purnicolor (according to the method on page 160) is based on the above-mentioned mycological properties, according to Bergey's Manu.
al 8th edition, pp. 807-808, and Cooper
Active Description of Type
Culture of SL reptomyees
W 5pecies*Descripti
onsfrom the 5econcLThird
and Fourth 5tudies by Elw
ood.

B, Shirling and Gottliebt, pages 468-469.

Streptomyces purnicara-9K-02-81 is
It has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology, and its deposit number is FEIKEN Bibori No. 9225.

The cultivation of the above-mentioned Treb H Seth Brunicolor 5K-02-81 for producing phospholipase D of the present invention is as follows:
This can be carried out according to methods commonly employed for culturing actinomycetes in general.

That is, glucose, fructose, sucrose, lactose, molasses, starch, dextrin, glycerin, etc. can be used as a carbon source in the medium alone or in combination, as appropriate.
Fatty acids, fats and oils/lecithin, alcohols, etc. can also be used. In addition, as a nitrogen source, ammonium sulfate,
Ammonium nitrate, ammonium chloride, urea, sodium nitrate, peptone, meat extract, yeast extract, corn steep liquor, casamino acids, defatted soybean flour, soybean protein, destiller soluple, and the like can be used. The medium also contains salt, potassium chloride, phosphate, magnesium salt, calcium salt, potassium salt, iron salt, manganese salt,
Various vitamins and other substances effective for promoting bacterial growth and phospholipase D production can be added as appropriate. Preferred medium pH is 5-9, particularly preferably 6-7. As the culture method, a deep culture method is preferable, but a solid culture method can also be adopted. Cultivation can be carried out at about 20-40°C, but the preferred culture temperature is 25-35°C. The preferred culture period varies depending on the temperature and pH1 medium, but is usually about 1 to 6 days, and the culture is stopped when the production of the target product, phospholipase D, has not reached its maximum.

After the cultivation is completed, the bacterial cells are filtered from the culture solution, and phospholipase D is collected from the filtrate. There is no particular difficulty in collecting phospholipase D, and it can be carried out by appropriately combining methods commonly employed for the separation and purification of various enzymes. For example, methods such as ultrafiltration, vacuum concentration, salting out, organic solvent precipitation, dialysis, Delfiltration, adsorption chromatography, ion exchange chromatography, isoelectric focusing, and lyophilization can be applied to the phospholipase of the present invention as described below. It may be adopted under conditions that take into account the physical and chemical properties of D.

The phospholipase D of the present invention produced by Streptomyces purnicara-3K-02-81 has the following physicochemical properties.

(a) Action ■ Hydrolyzes the ester bond between the phosphatidyl group of the phospholipid and the base, liberating phosphatidic acid and the base.

■ In the coexistence of a compound having an alcoholic hydroxyl group such as glycerol or ethanol, the ester bond between the phosphatidyl group of the glycerophospholipid and the base is hydrolyzed, and at the same time, a new ester is formed with the phosphatidyl group and the above-mentioned compound having an alcoholic hydroxyl group. A phosphatidyl transfer reaction occurs to produce the phosphatidyl group.

(b) Substrate specificity ■ Hydrolytic activity for phosphatidylfurin is 100
In this case, the relative activity is 1.5 for lysophosphatidylfurin and 0.1 for sphingomyelin.

■Km value for phosphatidylcholine is 0.9nM
It is.

(c) Optimal pH: about 6.0 (common to hydrolysis reaction and phosphatidyl group rearrangement reaction) (d) Optimal temperature: 50 to 55°C (common to hydrolysis reaction and phosphatidyl group rearrangement reaction) (e) pH Stability: Stable at pH 5-8 Heat stability: Heated at pH 5 or 6 at 50°C for 3 months or 5
It is not inactivated at all by heating at 5°C for 100 minutes.

(g) Effects of metal ions When metal ions are present at a concentration of 10 mM, the hydrolytic activity is inhibited by 30% outside the throat of Mn2+, but Ca2'', Ni2+, Mg+, Ba2+, Z
, 24, is not inhibited when co2+.

(h) Effects of various inhibitors Although inhibited by 46% by monoiodoacetic acid, diisopropylfluorophos-7ate, N-ethylmaleimide, potassium cyanide, p-chloromercury-benzony)
, o-71 nanthroline, 2-mercaptoethanol, and disodium ethylenedi7minetetraacetate.The molecular weight was 52,000 (according to SDS-PAGE method).

The enzymatic activity of phospholipase D is determined by quantifying the amount of base produced when it acts on the phospholipid substrate to decompose the ester bond between phosphoric acid and base.

The enzyme activity described in this specification was measured by the following method using phosphatidylcholine as a substrate, and one unit is the enzyme activity that releases 1 μmol of choline per minute.

Phosphatidylcholine degrading activity measurement method 2 Mix 900 μm of isopropanol solution 50IJ1 containing 0.8% phosphatidylcholine and 0.6% Triton xioo, and 0.1M sodium citrate buffer (pH 6) containing 0.1% Triton xioo Add 50% enzyme solution to this.
1 and react at 50°C for 10 minutes. Thereafter, the mixture is immediately boiled at 100° C. for 2 minutes to completely stop the reaction.

Next, reagents for measuring choline (325 units of choline oxidase, 500 units of peroxidase, 123 mg of calcium chloride, 14 g of Lydone X100, 250 g)
-7 Minoantipyrine 50 mg and phenol 78 mg dissolved in 250 ml of Tris-HCl buffer at pH 8.0) Add 50 μl of the reaction solution of the above enzyme reaction to 1.
After reacting at 37° C. for 10 minutes, the absorbance at 500 nm is measured using a control solution prepared by performing a similar reaction using an enzyme solution that has been heat-inactivated in advance.

Example Hereinafter, the present invention will be explained with reference to Examples.

Example 1 As a medium, 4% soluble starch, 5% soybean flour, 0.1% potassium phosphate, 0.2% dipotassium phosphate, 0.3% sodium chloride, 0.05% magnesium sulfate, and 7 pH 7.0 containing 0.2% decanol LG294
Prepare 100 ml of it, put 100 ml of it into a 500 ml volume with 7 grains, and after steam sterilization, inoculate 5 rings of Streptomyces purnicolor-3K"02-81 preculture solution,
Culture was carried out at 28° C. for 3 days with shaking at 120 spm.

After the culture was completed, the bacterial cells were filtered to obtain 100+ sl of the culture filtrate with an enzyme activity of 75.5 units/ml.

Next, 96 g of ammonium sulfate was gradually added to the above filtrate with stirring, and the resulting precipitate was collected by centrifugation and dried under reduced pressure to give a pale brown phospholipase D (880
1 g of μm, 5300 units were obtained. The recovery rate of phospholipase D activity from the culture filtrate was 70%.

Example 2 The same medium 20e as the medium used in Example 1 was placed in a 30 g Jar 7 Amengu, and after steam sterilization at 120°C for 15 minutes, Streptomyces Brunicolor 5K-02-
81 preculture solution 1e was inoculated and cultured at 28°C for 40 hours.

After culturing, solid matter such as bacterial cells is removed by centrifugation.
18 g of supernatant having an enzyme activity of 80.0 units/1 was obtained.

Next, the supernatant liquid was cooled to 4°C and filtered through an ultrafiltration membrane/AIL.
Ultrafiltration was performed using -1010 (manufactured by Asahi Kasei Corporation) to obtain a concentrated solution of 1.88. 40-6 for concentrate
Perform ammonium sulfate salting out at 0% saturation, and reduce the resulting precipitate to 50mM)
It was dissolved in JS hydrochloric acid buffer (pH 7, 0) and dialyzed against the same buffer. Then DE equilibrated with the same buffer
It was passed through an AE-Sepharose CL-6B column to adsorb impurities such as protein and dye.

The eluted phospholipase D fraction was dialyzed against the same buffer, and then dialyzed with 4.8mM citrate buffer (pH 5,4
) to adsorb the activity through a column of CM-Sepharose CL-6B equilibrated with
The active fraction was eluted and separated using M) and lyophilized to obtain white phospholipase D1301ag.

The activity recovery rate of phospholipase D in the above purification process is approximately 30%, and the specific activity of the obtained purified product is 3300 units)
/mg protein.

Next, the following physical and chemical property tests were conducted on the purified product.

■Optimal pH Phosphatidylcholine degrading activity: The EIH dependence of the phosphatidylcholine degrading activity of this enzyme was investigated by replacing the buffer in the enzyme activity measurement method described above with other buffers with various pH values and measuring the enzyme activity. . The result is the first
As shown in the figure, the optimum pi-t is around 6.0.

Phosphatidyltransferase activity: In the presence of phospholipase D, the initial rate of the reaction to produce phosphatidylglycerol from phosphatidylrefrin and glycerin was determined at various pH values.
It was measured with

The results are shown in FIG. 2, and the optimum pH is 6 to 7 in both organic solvent and aqueous systems. [Initial reaction rate measurement method: In the case of Ariiso solvent (ethyl acetate) system, 1
Inpropateur solution 25IJ+ containing 0% phosphatidylfurin, 50 μl of glycerin, 50 μl of buffer
1, ethyl acetate 4001+1, and enzyme solution number p1,
After the reaction is completed at 50°C for 30 minutes, the p of the reaction solution is
H is adjusted to 2 or less with dilute hydrochloric acid, all phospholipids are transferred to an ethyl acetate layer, and phosphatidylglycerol in the same layer is analyzed using IatroScan to determine the reaction phase rate. In the case of an aqueous system, 25 μm of Impropatol solution containing 10% phosphatidylcholine, 50 μm of glycerin, 50 μm of buffer solution 1.0.1M aqueous calcium chloride solution.
, mix several micrometers of the enzyme solution and react at 50°C for 30 minutes.After the reaction, adjust the pH of the reaction solution to 2 or less with diluted hydrochloric acid,
Hexane/ether/isopropanol (1/110.
5) After adding a mixed solvent to extract all the phospholipids, analyze the phos-7-acylglycerol in the organic solvent layer using an IATROScan to determine the initial reaction rate. ]■ Optimum Temperature The temperature dependence of the activity of this enzyme was investigated by measuring the enzyme activity while varying the enzyme reaction temperature in the above-mentioned method for measuring phosph-7tinlucholine decomposition activity. The results are shown in FIG. 3, and the optimum temperature is around 50-55°C.

■ Stable pH Dissolve the sample in buffer solutions of various pH containing 0.1% aliquots at a concentration of 1 unit/ml, and incubate at 50'C for 1 hour or at 4°C for 24 hours, respectively. The enzyme activity was then measured and compared with the enzyme activity before the test.The results are shown in Figure 4 (stored at 50°C) and Figure 5 (4
The pH at which the phospholipase D according to the present invention is stable is about 5 to 8, and particularly stable at a pH of about 5 to 7.
It can be seen that it is.

(2) Thermostability A sample was dissolved in a 0.1 M sodium citrate buffer (pH 6.0), kept at 20-70°C for 30 minutes, and the remaining enzyme activity was measured. The results are shown in Figure 6, with no deactivation at 55°C, and a decrease in activity of about 5% at 60°C.

Separately, changes in activity were investigated when the enzyme was kept at pH 5 or 6 for a long period of time at 50°C, which is the optimum temperature for this enzyme, and showed 100% residual activity even after three cycles.

■ Effect of metal ions In the enzyme activity measurement method described above, aqueous solutions of various metal salts are used, and the metal ion concentration in the enzyme reaction system is 1mM or 10mM.
Enzyme activity was measured in mM. Relative activities are shown in Table 1, with the activity when no metal ions are added as 100.

Table 1 Metal ion concentration types of metal ions '10nM 1mMCa'
92 99Ni” 98
99Mg2+105 101 Ba” 1 0 2
1 0 1Zn” 1 0
0 9 9Mn2+70 1
04 Co” 1 0 4
1 00■ Influence of inhibitors In the enzyme activity measurement method described above, aqueous solutions of various inhibitors (substances known to have inhibitory effects on various enzymes) are used, and the inhibitor concentration is 1mM in the enzyme reaction system. Enzyme activity was measured as follows. Relative activities are shown in Table 2, with the activity measured when no inhibitor is added as 100.

Table 2 Inhibitor Relative activity diisopropylfluorophos7ate 104 mo/iodoacetic acid
54N-ethylmaleimide
111 potassium cyanide
105p-90 Romarqui+) -Henso s-
-) 101o-7 enanthrolin
1112-Mercaptoethanol
96 Ethylenediaminetetraacetic acid disodium
1120 Molecular Weight 5 Molecular weight was measured by DS-PAGE method. The molecular weight of the enzyme estimated from the results was 52,000.

Effects of the Invention As mentioned above, the phospholipase D of the present invention exhibits 100% residual activity even when used continuously for three months or more at its optimum reaction temperature of 50°C, and virtually no activity decrease occurs at this temperature. It exhibits extremely high thermal stability. It is also less susceptible to the effects of metal ions and inhibitors. Therefore, the phospholipase D of the present invention has the feature that it can be stored for a long period of time at a temperature of about 50°C or higher without the risk of the growth of microorganisms such as mold, and it also enables the production of phospholipid derivatives with much higher efficiency than conventional methods. This is an excellent feature that allows for easy implementation with low enzyme costs.

Further, according to the production method of the present invention, phospholipase D having excellent properties as described above can be produced with extremely high productivity. That is, the enzyme production amount per ml of culture medium of the phospholipase D-producing strain that has been reported so far is 0.5 for Streptomyces taromofuscus.
Unit (J, Biochem, .

Vol. 85, p. 79, 1979), Micromonomata Bora Charcea 0.13 units (Special Publication No. 58-5263)
3), mecardiopsis sp is 0.54 units
(Unexamined Japanese Patent Publication No. 58-63388), Actinomadura s.
p is 1.7 units (Japanese Patent Application Laid-open No. 58-67183), 7
Caldei 7sp is 0.1 munits
4483), the production method of the present invention using bacteria producing a high titer of 80 units)/ml makes it possible to dramatically improve the production efficiency of phospholipase D.

[Brief explanation of the drawing]

FIG. 1: A graph showing the pH dependence of the phosphatidylcholine degrading activity of the enzyme of the present invention. FIG. 2 p of the phosphatidyl group transfer activity of the enzyme of the present invention
Graph showing H dependence. Part 3: Graph showing the temperature dependence of the phosphatidylcholine degrading activity of the enzyme of the present invention. Part 4: Graph showing the influence of pH on the stability of the enzyme of the present invention (storage temperature 50°C). Part 5: Graph showing the influence of pH on the stability of the enzyme of the present invention (storage temperature 4°C). No. 6l: Graph showing the thermostability of the enzyme of the present invention.

Claims (1)

[Claims] (1) Phospholipase D having the following physicochemical properties:
(B) Action [1] Hydrolyzes the ester bond between the phosphatidyl group of the phospholipid and the base to liberate phosphatidic acid and the base; [2] In the coexistence of a compound having an alcoholic hydroxyl group such as glycerol or ethanol , hydrolyzes the ester bond between the phosphatidyl group of the glycerophospholipid and the base, and at the same time causes a phosphatidyl group transfer reaction to produce a new ester with the phosphatidyl group and the above-mentioned alcoholic hydroxyl group-containing compound; (b) substrate specificity; [1] The hydrolysis activity for phosphatidylcholine is 1
00, the relative activity is 1.5 for lysophosphatidylcholine and 0.1 for sphingomyelin.
[2] Km value for phosphatidylcholine is 0.9
(c) Optimal pH: about 6.0 (d) Optimum temperature: 50-55°C (e) pH stability: Stable at pH 5-8 (f) Thermal stability at 50°C at pH 5-6 Heating for 3 months or 55
No deactivation at all after heating for 100 hours at °C; (g) Influence by metal ions When metal ions are present at a concentration of 10 mM, hydrolytic activity is inhibited by 30% when the metal ions are Mn^2^+; Ca^2^+, Ni^2^+, Mg^2^+, B
It is not inhibited when it is a^2^+, Zn^2^+, or Co^2^+; (h) Effects of various inhibitors It is inhibited by 46% by monoiodoacetic acid, but diisopropylfluorophosphate, N-ethylmaleimide, Potassium cyanide, p-chloromercury benzoate, o-phenanthroline, 2-mercaptoethanol,
Not affected by disodium ethylenediaminetetraacetate; (li) Molecular weight of 52,000 (according to SDS-PAGE method). (2) Streptomyces purnicolor SK-02-8
A method for producing phospholipase D, which comprises culturing one strain and collecting phospholipase D from the culture solution.