JPH088866B2 - Phospholipase D and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to phospholipase D having phosphatidyl group transfer activity in water and various organic solvents and having excellent thermal stability, and a method for producing the same.

Phospholipase D (particularly having the phosphatidyl group transfer catalytic activity described later) is a phospholipid such as phosphatidylcholine or phosphatidylethanolamine contained in soybean or egg yolk and various alcohols as an emulsifier, dispersant, agricultural chemical, pharmaceutical, industrial reagent, etc. Used to produce a phospholipid derivative useful in

BACKGROUND ART Phospholipase D (EC3.1.4.4) is an enzyme that hydrolyzes an ester bond between a phosphatidyl group of a phospholipid and a base to release phosphatidic acid and a base. , Hydrolyzes the ester bond between the phosphatidyl group of glycerophospholipid and the base in the coexistence of a compound having an alcoholic hydroxyl group such as ethanol, and simultaneously forms a new ester with a compound having a phosphatidyl group and the above alcoholic hydroxyl group. A reaction (phosphatidyl group transfer reaction) occurs.

It has been known for a long time that this enzyme is widely present in plants such as cabbage and carrot, and other plant kingdoms, and it was usual to produce it by a method of extracting from plants containing this enzyme. In, a method of producing by fermentation using a microorganism such as Streptomyces, Micromonospora, Norcadiopsis, Actinomadura, Nocardia, etc. has been proposed and partially implemented (Japanese Patent Publication No. -39918, Japanese Patent Publication No. 58-52633, Japanese Patent Laid-Open No.
58-63388, JP-A-58-67183, JP-A-60-164483
issue).

However, the phospholipase D obtained by these conventional production methods has poor thermal stability under optimum conditions as long as it has phosphatidyl group transfer activity, and therefore the transfer reaction cannot be efficiently performed at high temperature. was there.

Further, the above-mentioned conventional method utilizing microorganisms not only does not give an enzyme having good heat stability as described above, but also has a problem that the productivity of the enzyme per culture solution is poor and thus the product becomes expensive. It was Further, it has been pointed out that the microorganisms used in some of the production methods are pathogenic, and there is a problem in terms of safety when used in the production of food-grade emulsifiers.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention In view of the fact that the conventional phospholipase D having a phosphatidyl group transfer activity has the above-mentioned drawbacks and the production method thereof also has room for improvement, An object of the present invention is to provide phospholipase D exhibiting thermostability and phosphatidyl group transfer activity and an efficient production method thereof.

Means for Solving the Problems The present inventors searched for the ability of producing phospholipase D exhibiting phosphatidyl group transfer activity in a large number of microorganisms, and further, for selected phospholipase D high-efficiency producing bacteria, particularly thermostable. The experiment for searching for a strain producing an enzyme with good properties was repeated. As a result, a new strain, Streptomyces prunicolor SK-02-8, was isolated from soil collected in the Tama area of Tokyo.
Knowing that 1 has excellent performance, the present invention has been completed.

That is, the present invention provides a method for producing phospholipase D using the above Streptomyces purnicolor SK-02-81 strain,
And a highly thermostable phospholipase D obtained thereby.

The Streptomyces purnicolor SK-02-81 strain used in the production method of the present invention has the following mycological properties.

Morphological characteristics Aerial mycelium: Mainly linear, slightly bent. Occasionally nest or sclerothium-like stuff is seen. width
0.4-6 μm.

Spores: A short cylinder with a size of 0.4 to 0.6 μm x 1.1 to 1.3 μm.
The surface is smooth.

Basal hyphae: somewhat wavy, short-axis branching. There is no hyphal fragmentation and spore settlement.

Flagella spores and sporangia: do not form.

Growth conditions on various media: As shown in the table below (however, 28 ℃,
Results after 2 weeks. The colors are displayed according to the JIS Z8721 standard color chart. ).

Amino Acid Composition and Sugar Composition of Cell Wall meso-diaminopimelic acid-LL-diaminopimelic acid + glycine + glutamic acid + alanine + arabinose-galactose-galactosamine-glucosamine + From the above, the cell wall of the SK-02-81 strain is type I.

Physiological properties a. Growth temperature range: 20-40 ℃ b. Optimum growth temperature: 28-35 ℃ c. Optimum pH: 6-7 d. Gelatin liquefaction: None e. Starch hydrolysis: Yes f. Coagulation of skim milk, peptone formation: None g. Production of melanoid pigment: None h. Utilization of carbon source (30 ° C, 16-day culture): L-arabinose + D-xylose + D-glucose + D-fructose + D -Manitol + sucrose + inositol + L-rhamnose + raffinose + i.GC content: 70.4% ("Experimental method for identifying actinomycetes", Japanese Society for Actinomyces, 151-160
According to the method of page) This strain is Streptomyces purnicolor, the above-mentioned mycological properties are explained by Bergey's Manual 8th edition,
Pages 807-808, and Cooperative Description of Typ
e Culture of Streptomyces IV Species, Descriptions
from the Second, Third and Fourth Studies by Elwoo
d, B. Shirling and Gottlieb, pp. 468-469.

Streptomyces purnicolor SK-02-81 has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, and the deposit number is Microbiology Research Institute No. 9225.

Cultivation of the above-mentioned Streptomyces purnicolor SK-02-81 for producing the phospholipase D of the present invention can be carried out according to a method usually adopted in general culturing of actinomycetes. That is, glucose as a carbon source in the medium,
Fructose, sucrose, lactose, molasses, starch, dextrin,
Glycerin and the like can be appropriately used alone or in combination, and fatty acids, oils and fats, lecithin, alcohols and the like can also be used. Further, as the nitrogen source, ammonium sulfate, ammonium sulfate, ammonium chloride, urea, sodium nitrate, peptone, meat extract, yeast extract, corn steep liquor, casamino acid, defatted soybean powder,
Soy protein, Destilers solubles, etc. can be used. In addition to sodium chloride, potassium chloride, phosphate, magnesium salt, calcium salt, potassium salt, iron salt, manganese salt, various vitamins, other substances effective for the growth of bacteria and the promotion of phospholipase D production are appropriately added to the medium. It can be added. The preferred medium pH is 5-9, particularly preferably 6-7. As a culturing method, a submerged culturing method is preferable, but a solid culturing method can also be adopted. Culture is about 20 ~
It can be carried out at 40 ° C, but a preferable culture temperature is 25-35.
° C. The preferable culturing period varies depending on the temperature, pH and medium, but it is usually about 1 to 6 days, and the culturing is stopped when the production of the desired product, phospholipase D, reaches a maximum.

After the culture 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 is possible to appropriately combine the methods usually used for separating and purifying various enzymes. For example, ultrafiltration, vacuum concentration, salting out, organic solvent precipitation, dialysis, gel filtration,
A method such as adsorption chromatography, ion exchange chromatography, isoelectric focusing, lyophilization or the like may be adopted under the conditions considering the physicochemical properties of the phospholipase D of the present invention described later.

The phospholipase D of the present invention produced by Streptomyces purnicolor SK-02-81 has the following physicochemical properties.

(A) Action Hydrolyzes the ester bond between the phosphatidyl group of the phospholipid and the base to release 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 glycerophospholipid and the base is hydrolyzed, and at the same time, a new ester is formed with the compound having a phosphatidyl group and the alcoholic hydroxyl group. To cause a phosphatidyl group transfer reaction.

(B) Substrate specificity Hydrolyzing activity against phosphatidylcholine is 100
Relative activity is 1.5 for lysophosphatidylcholine and 0.1 for sphingomyelin.

The Km value for phosphatidylcholine is 0.9 mM.

(C) Optimum pH: about 6.0 (common to hydrolysis reaction and phosphatidyl group rearrangement reaction) (d) Optimum temperature: 50 to 55 ° C (common to hydrolysis reaction and phosphatidyl group rearrangement reaction) (e) pH stability : Stable at pH 5 to 8 (f) Thermal stability At pH 5 or 6, heating at 50 ° C for 3 months or 55 ° C 1
It is not deactivated at all after heating for 00 hours.

(G) Effect of metal ions When a metal ion with a concentration of 10 mM is present, the hydrolysis activity is inhibited by 30% when the metal ion is Mn ²⁺ , but Ca ²⁺ ,
It is not inhibited when Ni ²⁺ , Mg ²⁺ , Ba ²⁺ , Zn ²⁺ and Co ²⁺ .

(H) Effects of various inhibitors Although it is inhibited by 46% by monoiodoacetic acid, diisopropylfluorophosphate, N-ethylmaleimide, potassium cyanide, p-chloromercury benzoate, o-phenanthroline, 2-mercaptoethanol, ethylenediaminetetraacetic acid diacetate. Not affected by sodium.

(I) Molecular weight 52,000 (by SDS-PAGE method).

The enzyme activity of phospholipase D is determined by quantifying the base produced when the ester bond between phosphate and base is decomposed by acting on the substrate phospholipid. The enzyme activity described in this specification is measured by the following method using phosphatidylcholine as a substrate, and the enzyme activity of releasing 1 μmol of choline per minute is defined as 1 unit.

Phosphatidylcholine-degrading activity assay method: 50 μl of isopropanol solution containing 0.8% phosphatidylcholine and 0.6% Triton X100, and 900 μl of 0.1M sodium citrate buffer (pH 6) containing 0.1% Triton X100 were mixed, and the enzyme solution was 50 μl. Is added and reacted at 50 ° C for 10 minutes. Then, immediately boil at 100 ° C. for 2 minutes to completely stop the reaction. Next, 1 ml of choline measuring reagent (325 units of choline oxidase, 500 units of peroxidase, 123 mg of calcium chloride, 250 mg of Triton X100, 50 mg of 4-aminoantipyrine, 78 mg of phenol dissolved in 250 ml of pH 8.0 Tris-HCl buffer) 50 μl of the reaction solution of the above enzyme reaction was added to the above, reacted at 37 ° C. for 10 minutes, and then reacted in the same manner with the enzyme solution that had been heat inactivated in advance, and the absorbance was measured at 500 nm as a control solution. To do.

EXAMPLES Hereinafter, the present invention will be described with reference to examples.

Example 1 As a medium, pH 7.0 containing soluble starch 4%, soybean flour 5%, monopotassium phosphate 0.1%, dipotassium phosphate 0.2%, sodium chloride 0.3%, magnesium sulfate 0.05%, and adecanol LG294 0.2%. Prepare the one, 100
ml in a 500 ml Sakaguchi flask, and after steam sterilization, 5 ml of pre-cultured solution of Streptomyces purnicolor SK-02-81
Was inoculated and cultured at 28 ° C. for 3 days with shaking at 120 spm.

After culturing, the bacterial cells were filtered off and the enzyme activity was 75.5 units.
100 ml of culture filtrate of / ml was obtained.

Next, 96 g of ammonium sulfate was gradually added to the above filtrate with stirring, and the formed precipitate was collected by centrifugation and dried under reduced pressure to give a pale brown phospholipase D (880 mg, 53
I got 00 units. The recovery rate of phospholipase D activity from the culture filtrate was 70%.

Example 2 The same medium 20 as that used in Example 1 was placed in 30 jar fermenters and steam sterilized at 120 ° C. for 15 minutes, and then the preculture liquid 1 of Streptomyces purnicolor SK-02-81 was inoculated. And cultured at 28 ° C for 40 hours.

After completion of the culture, solids such as bacterial cells were removed by centrifugation to obtain a supernatant 18 having an enzyme activity of 80.0 units / ml.

Then, the above supernatant was cooled to 4 ° C, and the ultrafiltration membrane / AIL
Ultrafiltration was performed using -1010 (a product of Asahi Kasei Corporation) to obtain a concentrated liquid 1.8. Ammonium sulfate salting out at 40-60% saturation was performed on the concentrated solution, and the resulting precipitate was dissolved in 50 mM Tris-HCl buffer (pH 7.0) and dialyzed against the same buffer. Then, DEAE-Sepharose CL- equilibrated with the same buffer solution.
It was passed through a 6B column to adsorb protein and dye as impurities. The eluted phospholipase D fraction was dialyzed against the same buffer and then 4.8 mM citrate buffer (pH 5.
The activity was adsorbed by passing it through a column of CM-Sepharose CL-6B equilibrated in 4), the active fraction was eluted and separated by a salt concentration gradient method (0 to 0.2 M), and freeze-dried to give 130 mg of white phospholipase D. Got

The activity recovery of phospholipase D in the above purification step was about 30%, and the specific activity of the obtained purified product was 3300 units / mg.
It was protein.

Next, the purified product was tested for physicochemical properties as described below.

Optimum pH Phosphatidylcholine-degrading activity: The pH dependence of the phosphatidylcholine-degrading activity of this enzyme was investigated by changing the buffer solution used in the above-mentioned enzyme activity measuring method to other buffer solutions having various pH values and measuring the enzyme activity. The results are shown in Fig. 1, and the optimum pH is around 6.0.

Phosphatidyl group transfer activity: In the presence of phospholipase D, the initial rate of the reaction for producing phosphatidylglycerol from phosphatidylcholine and glycerin was measured at various pHs. The results are shown in FIG. 2, and the optimum pH is 6 to 7 in both organic solvent system and aqueous system. [Initial reaction rate measurement method: In the case of organic solvent (ethyl acetate) system, 25 μl of isopropanol solution containing 10% phosphatidylcholine, 50 μl of glycerin, 50 μl of buffer solution.
l, 400 μl of ethyl acetate and several μl of enzyme solution are mixed, and the temperature is 50 ° C.
Let react for 30 minutes. After completion of the reaction, adjust the pH of the reaction solution to 2 or less with dilute hydrochloric acid, and transfer all phospholipids to the ethyl acetate layer,
The phosphatidylglycerol in the same layer is analyzed by an iatroscan to determine the initial reaction rate. 10 for water
% Phosphatidylcholine in isopropanol 25 μl, glycerin 50 μl, buffer 400 μl, 0.1M
Mix 50 μl of calcium chloride aqueous solution and several μl of enzyme solution,
Incubate at 50 ° C for 30 minutes. After the reaction was completed, adjust the pH of the reaction solution to 2 or less with dilute hydrochloric acid, add a hexane / ether / isopropanol (1/1 / 0.5) mixed solvent to extract all phospholipids, and then remove the phosphatidylglycerol in the organic solvent layer. Analyze by Toroscan to obtain the initial reaction rate. Optimum temperature The temperature dependence of the activity of the present enzyme was examined by measuring the enzyme activity by changing the temperature of the enzyme reaction in the above-mentioned phosphatidylcholine splitting activity measuring method. The results are shown in Fig. 3, and the optimum temperature is around 50 to 55 ° C.

Stable pH Dissolve the sample in buffer solutions of various pH containing 0.1% Triton X100 to 1 unit / ml, 50 ℃ each
At room temperature for 1 hour or at 4 ° C. for 24 hours. Then, the enzyme activity was measured and compared with the enzyme activity before the test. The results are shown in FIG. 4 (stored at 50 ° C.) and FIG. 5 (stored at 4 ° C.). The phospholipase D according to the present invention has a stable pH of about 5-8, particularly a stable pH of 5-7. I understand.

The sample was dissolved in a thermostable 0.1 M sodium citrate buffer (pH 6.0) and kept at 20 to 70 ° C for 30 minutes, and the remaining enzyme activity was measured. The results are shown in FIG. 6, and at 55 ° C., inactivation was not observed at all, and at 60 ° C., a decrease in activity of about 5% was observed. Separately, the activity change of the enzyme was examined at pH 5 or 6 when it was kept at the optimum temperature of 50 ° C. for a long period of time, and 100% residual activity was shown even after 3 months.

Effect of metal ions In aqueous solution of various metal salts in the above-mentioned enzyme activity measurement method, the metal ion concentration was 1 mM or 10 mM in the enzyme reaction system.
The enzyme activity was measured as follows. Table 1 shows the relative activity when the activity when no metal ion is added is 100.

Effect of inhibitors Use an aqueous solution of various inhibitors (substances that are known to have an inhibitory effect on various enzymes) in the above-mentioned enzyme activity measurement method so that the inhibitor concentration becomes 1 mM in the enzyme reaction system. Then, the enzyme activity was measured. Table 2 shows the relative activity when the activity measured without addition of an inhibitor is 100.

Table 2 Inhibitor relative activity Diisopropyl fluorophosphate 104 Monoiodoacetic acid 54 N-Ethylmaleimide 111 Potassium cyanide 105 p-Chlormercury benzoate 101 o-Phenanthroline 111 2-Mercaptoethanol 96 Ethylenediaminetetraacetic acid disodium 112 Molecular weight SDS-PAGE method The molecular weight was measured by. The molecular weight of this enzyme estimated from the result was 52,000.

EFFECTS OF THE INVENTION As described above, the phospholipase D of the present invention can be continuously used for 3 months or more at its optimum reaction temperature of 50 ° C.
It shows a very good thermal stability, showing 100% residual activity and virtually no loss of activity at this temperature. It is also less susceptible to 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 at which there is no fear of growth of microorganisms such as mold, and the production of phospholipid derivatives is much more efficient than before. It is excellent because it can be easily performed at low enzyme cost.

Moreover, 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 1 ml of the medium of the phospholipase D-producing strain that has been reported so far is 0.5 unit for Streptomyces chromofuscus (J. Biochem., 85, 79, 1979), and Micromonospora charlesea. 0.13 unit (Japanese Patent Publication 58-52633)
No.), 0.54 units of Nocardiopsis sp (JP-A-58)
-63388), Actinomadura sp is 1.7 units (JP-A-58-67183), Nocardia sp is 0.17 units (JP-A-60-164483), so it is a low level product, so 80 units / ml. According to the production method of the present invention using a high titer producing bacterium, the production efficiency of phospholipase D can be dramatically improved.

[Brief description of drawings]

FIG. 1 is a graph showing the pH dependence of the phosphatidylcholine-degrading activity of the enzyme of the present invention. FIG. 2: pH of phosphatidyl group rearrangement activity of the enzyme of the present invention
A graph showing the dependency. FIG. 3: A graph showing the temperature dependence of the phosphatidylcholine-degrading activity of the enzyme of the present invention. FIG. 4 is a graph showing the effect of pH on the stability of the enzyme of the present invention (storage temperature 50 ° C.). FIG. 5: Graph showing the effect of pH on the stability of the enzyme of the present invention (storage temperature 4 ° C.). FIG. 6: Graph showing the thermostability of the enzyme of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Tsunekazu Watanabe 1-1-19 Higashishimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Akio Kuroda 1-1-19 Higashishimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Takamasa Oki 1-9-3 Kamatahonmachi, Ota-ku, Tokyo Inside Niigata Iron Works Co., Ltd. (56) Reference JP-A-58-152481 (JP, A)

Claims (2)

[Claims] 1. A phospholipase D having the following physicochemical properties: (a) Action Hydrolyzes an ester bond between a phosphatidyl group of a phospholipid and a base to release phosphatidic acid and a base; In the coexistence of a compound having an alcoholic hydroxyl group, a phosphatidyl group transfer reaction that hydrolyzes an ester bond between a phosphatidyl group of glycerophospholipid and a base and simultaneously forms a new ester with the compound having a phosphatidyl group and the alcoholic hydroxyl group. (B) Substrate specificity Hydrolyzing activity against phosphatidylcholine is 100
The relative activities are 1.5 for lysophosphatidylcholine and 0.1 for sphingomyelin; Km value for phosphatidylcholine is 0.9 mM; (C) Optimum pH: about 6.0 (D) Optimum temperature: 50-55 ℃ (e) pH stability: Stable at pH 5-8 (f) Thermal stability At pH 5-6, heating at 50 ℃ for 3 months or heating at 55 ℃ for 100 hours does not deactivate at all. Effect of metal ions In the presence of metal ions at a concentration of 10 mM, the hydrolysis activity is inhibited by 30% when the metal ions are Mn ²⁺ , but Ca ²⁺ , Ni
Not inhibited when ²⁺ , Mg ²⁺ , Ba ²⁺ , Zn ²⁺ , Co ²⁺ ; (h) Effects of various inhibitors 46% inhibition by monoiodoacetic acid, but diisopropylfluorophosphate, N-ethylmaleimide , Potassium cyanide, p-chloromercury benzoate,
Not affected by o-phenanthroline, 2-mercaptoethanol, disodium ethylenediaminetetraacetate; (i) molecular weight 52,000 (by SDS-PAGE method). 2. Streptomyces purnicolor SK-02-
A method for producing phospholipase D, which comprises culturing 81 strains and collecting phospholipase D from the culture solution.