JPH0117675B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing phospholipase D using a microorganism. That is, the present invention is a method for producing phospholipase D, which comprises culturing phospholipase D-producing bacteria belonging to the genus Actinomadura in a medium, and employing phospholipase D from the culture. Phospholipase D (EC3.1.4.4) is an enzyme that decomposes the ester bond between the phosphoric acid of glycerophospholipid and the nitrogen-containing base to liberate phosphatidic acid and the base. It is also known that when phospholipase D acts on glycerophospholipids in the presence of a compound having an alcohol group such as ethanol, glycerol, or ethanolamine, it transfers phosphatidic acid to an alcohol group. Phospholipase D has been known for a long time to exist widely in the plant kingdom, such as cabbage and carrots, and is mainly produced by extracting it from the tissues of cabbage. In addition, recently, as a method for producing phospholipase D using microorganisms, actinomycetes belonging to the genus Streptomyces (Japanese Patent Publication No. 52-39918) and the genus Micromonospora (Japanese Patent Publication No. 44094/1989) have been used. A method of manufacturing by fermentation is known. Phospholipase D is used as a research reagent related to phospholipid metabolism, a reagent for quantifying phospholipids contained in serum, and the like, and can also be used in the production of phosphatidic acid from various phospholipids. The present inventors isolated a wide range of microorganisms from soil in the natural world and searched for strains that produce phospholipase D. As a result, when a bacterial strain isolated from the soil of Hachioji City, Tokyo (referred to as Actinomadeura sp. No. 362) was cultured in a medium, it was found that it acts on glycerophospholipids in the medium to release phosphatidic acid and base. It was confirmed that a certain enzyme was produced, and it was discovered that this bacterium produces phospholipase D. Furthermore, when this enzyme was allowed to act on glycerophospholipids in the presence of a compound having an appropriate alcohol group such as ethanol, sorbitol, ethanolamine, or glycerol, it was also observed that phosphatidic acid was transferred to an alcohol group. The mycological properties of the above bacterial strain are as follows. (a) Morphology It grows well on starch inorganic salt agar, tyrosine agar, yeast/malt agar, oatmeal agar, etc., and grows moderately on glycerin-asparagine agar, with colonies of aerial mycelium forming epiphytes. The color of the bacterial flora that has attached spores varies slightly depending on the type of culture medium and the time of observation, but it is generally grayish-white to gray with a slight purplish tinge. On seuucrose nitrate agar, nutrient agar, and glucose asparagine agar, aerial mycelia do not attach or attach only poorly. When this strain grown on an agar medium is observed under a microscope, the airtight filaments are branched with a width of 0.8 to 1.2μ, some are looped or spiral, and extend long and mainly straight with some bends. The tip is often loosely wound into a loop. The spores settle in chains of several tens to more than 100, forming almost the entire aerial hyphae. The size of the spores is 0.8-1.2 x 1.2-1.7μ, short cylindrical or oval, and somewhat irregular in size and shape. The basal hyphae are 0.6 to 1.0 μm in width and elongate with irregular branches and bends, and zoospores, sporangia, sclerotia, etc. are not formed. In addition, septa and hyphal division are not usually observed, but hyphal division may be observed in liquid culture. (b) Properties on various media The experimental method described below is mainly based on E.
Bee Shearing (Int.J.Syst.Bacteriol.16
Vol. 313-340, 1966). The hue was determined using the "Color Standard" (Japan Color Research Institute, 1964), and along with the hue name, the hue symbol was written in parentheses in the order of hue name, saturation number, and brightness number. Cultivation is carried out at 25℃, and 2 to 3 are the most vigorously growing.
Table 1 shows the observation results on the ground for each week. However, in Table 1, the color of the surface of the basal hyphae listed in the growth items shows the observation results during the first week of culture before spores are attached, and it is difficult to judge the color of the surface of the basal hyphae in a medium where spores attach quickly. There is no mention of.

【table】

[Table] (c) Physiological properties Growth temperature: Grows around 10-37℃, 20-30℃
Grows best at ℃. Liquefaction of gelatin: Not liquefied (cultivated on glucose-peptone-gelatin medium at 25°C for 3 weeks). Starch hydrolysis: Decompose (cultivate on starch agar medium at 25°C for 3 weeks). Coagulation and peptonization of skim milk: No coagulation,
Peptonize (cultivate at 30°C for 3 to 4 weeks). Production of melamine-like pigment: Produced on peptone yeast iron agar, tyrosine agar (25℃, 2
~4 days). (d) Assimilation of carbon sources (cultured at 30°C for 10-16 days) L-arabinose + sucrose - D-xylose + inositol ± D-glucose + L-rhamnose - D-fructose - raffinose - (e) Cell chemistry Analysis Diaminopimelic acid of this strain is meso type, and the sugar composition of the cell wall does not contain arabinose, xylose, rhamnose, etc., but contains madeulose, galactose, mannose, etc. Regarding the above analysis results, Bergey's Manual of
The Determinative Bacteriology, 8th edition, pp. 657-658 (1974), and L'Essier Varier (Inter.J.
System. Bacteriol. vol. 20, pp. 435-443, 1970)
When judged according to the classification method of , etc., this bacterium is of cell wall type and cell wall sugar pattern type B. As described above, this bacterium is an actinomycete that has madeulose, whose cell wall type is B, and whose sugar composition type is C, which does not have madeulose.
It is different from the Datsuson belay type of the genus Actinomadeura (genus Nocardiopsis). Therefore, this bacterium belongs to any of the genus Microbispora, Streptosporangium, Spirillospora, Planomonospora, Dermatophilus, and Actinomadeula. However, this fungus forms a spore chain consisting of many spores,
Since no sclerotia, sporangia, or zoospores were found, the genus Actinomadeura (Genus
Taxonomically, it is most appropriate to identify it as (Acinomadura). Therefore, this bacterium is Actinomadeura sp. No.362.
(Actinomadura sp No. 362). This bacterium has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is FERM BP-511. As the bacteria used in the present invention, not only the above-mentioned Actinomadeura sp. No. 362 and mutant strains obtained by mutating this strain, but also any bacteria that belong to the Actinomadeula sp. and produce phospholipase D can be used. I can do it. In carrying out the present invention, both liquid culture and solid culture can be used as the culture mode, but it is industrially advantageous to perform deep aeration agitation culture. The culture sources used include carbon sources, nitrogen sources, inorganic salts, and other micronutrients that are generally used for microbial culture, as well as any nutrient source that can be used by microorganisms belonging to the genus Actinomadeula. It can be used. As carbon sources for the medium, for example, in addition to glucose, fructose, sucrose, lactose, starch, glycerin, dextrin, molasses, sorbitol, fatty acids, fats and oils, crude lecithin, alcohol, organic acids, etc. are used alone or in combination. As the nitrogen source, either an inorganic nitrogen source or an organic nitrogen source can be used, and examples of the inorganic nitrogen source include ammonium nitrate, ammonium sulfate, urea, sodium nitrate, monoammonium phosphate, diammonium phosphate, ammonium chloride, etc. Examples of organic nitrogen sources that can be used include flour, bran, and defatted lees of soybeans, rice, corn, cottonseed, rapeseed, and wheat, as well as corn steep liquor, peptone, yeast extract, meat extract, casein, and amino acids. Examples of inorganic salts and micronutrients include salts such as phosphoric acid, magnesium, potassium, iron, aluminum, calcium, manganese, and zinc;
Vitamins, nonionic surfactants, antifoaming agents, and other substances that promote bacterial growth and phospholipase D production can be used as needed. Cultivation is carried out under aerobic conditions. The culture temperature can be changed as appropriate within the temperature range at which the bacteria grow and produce phospholipase D, but particularly preferred is 20°C to 35°C.
It is ℃. Although the culture time varies depending on the conditions, it is sufficient to culture until the maximum production amount of phospholipase D is reached.
In the case of liquid culture, it usually takes about 1 to 3 days. Since phospholipase D produced in the culture is mainly dissolved in the culture solution in submerged culture, phospholipase D is collected from the culture solution obtained by separating the solid matter from the culture solution. In collecting phospholipase D from the culture solution, any method commonly used for enzyme purification can be used. For example, methods such as salting out with ammonium sulfate, common salt, etc., precipitation with organic solvents such as acetone, ethanol, methanol, etc., dialysis, ion exchange chromatography, adsorption chromatography, gel filtration, adsorbent, isoelectric point precipitation, etc. can be used. Furthermore, if a suitable combination of these methods improves the purification effect of phospholipase D, the methods can be used in combination. Enzymes obtained by these methods are converted into liquid or by methods such as vacuum concentration, vacuum drying, and freeze-drying, with or without adding various salts, carbohydrates, proteins, lipids, surfactants, etc. as stabilizers. Solid phospholipase D can be collected. The enzymatic activity of phospholipase D is determined by acting on the substrate glycerophospholipid to decompose the ester bond between phosphoric acid and a nitrogen-containing base, and measuring the amount of base produced. Phospholipase D activity was measured by the choline oxidase method described below unless otherwise specified. Titer determination method: 0.1 ml of 1% egg yolk purified lecithin emulsion (0.1 g lecithin, 1 ml ethyl ether, 10 ml distilled water ultrasonic emulsion), 0.1 ml of 0.2M PH7.2 Tris-HCl buffer, 0.1M CaCl _2Aqueous solution 0.05ml, distilled water
Mix 0.15ml, add 0.1ml of enzyme solution, and
Contains 50mM EDTA・2Na after incubating for 20 minutes at °C.
Add 0.2ml of 1M Tris-HCl buffer (PH8.0),
Immediately boil for 5 minutes to completely stop the reaction. Next, 4 ml of a solution of the choline coloring agent contained in the kit of cholinesterase measurement reagent [manufactured by Nippon Shoji Co., Ltd.] dissolved in the coloring solution was added, and after reacting at 37°C for 20 minutes, the absorbance at 500 nm was measured. Measure. As a control, the absorbance of a similar reaction using an enzyme solution that has been heat-inactivated in advance is measured. The enzyme activity that releases 1 μmol of choline per hour is defined as 1 unit. Next, the physicochemical properties of phospholipase D using the enzyme preparation purified by the method shown in Example 3 will be described. Action Decomposes the ester bond between phosphoric acid of glycerophospholipid and nitrogen-containing base to liberate phosphatidic acid and base. Substrate specificity: Use 0.1 ml of emulsion containing 0.5 μmol of one of lecithin, lysolecithin, and sphingomyelin as a substrate, and use 1% instead of distilled water.
Except for using an aqueous solution containing Triton X-100,
The amount of choline released by the reaction was measured in the same manner as the titer measurement method described above, and the phospholipase D activity for each substrate was measured. As a result, the relative activity was 3.6 for lysolecithin and 0.3 for sphingomyelin when the activity against lecithin was set as 100. Optimum PH instead of buffer used in titer assay
For 3.0 to 4.0, formic acid/sodium formate buffer, PH4.0 to
Acetic acid/sodium acetate buffer for pH 5.5, Tris/maleic acid/caustic soda buffer for pH 5.5 to 8.5, pH
For 7.0 to 9.0, use Tris/HCl buffer, PH9.0 to 10.0
Then, the activity of phospholipase D was measured using a glycine/caustic soda buffer to determine the optimal pH.
Also, instead of 0.15ml of distilled water used in the same measurement method,
The optimum pH when using 0.15 ml of Triton X-100 (Wako Pure Chemical Industries) aqueous solution was also determined. The results are shown in Figure 1, and the optimal pH when using distilled water is around 7.0, which is 1%
Optimal pH when using Triton X-100 aqueous solution
was found around 5.5. Optimal temperature In the titer measurement method, the reaction temperature condition is 10,
20, 25, 37, 40, 50, 55, 60, 70, 80 and 90
Enzyme activity was measured at °C. The results are shown in Figure 2, and it is recognized that the optimum temperature is in the range of 55°C to 80°C. PH stability 0.9ml of 0.1M various buffers in 0.1ml of enzyme solution,
In other words, glycine/hydrochloric acid buffer for pH 3.0 to 3.5, acetic acid/sodium acetate buffer for pH 3.5 to 7.0,
Tris/maleic acid/caustic soda buffer was added for pH 5.0 to 8.0, Tris/hydrochloric acid buffer was added for pH 7.0 to 9.0, and glycine/caustic soda buffer was added for pH 9.0 to 9.5, and the mixture was kept at 25°C for 2 hours. after that,
9.0 ml of 0.5M Tris/HCl buffer solution (PH7.2) was added to these enzyme buffer solutions to adjust the pH to 7.0 to 7.3. Using 0.1 ml of this solution, the titer was measured according to the titer measurement method and the stable PH range was investigated.As shown in Figure 3, the particularly stable PH range of this enzyme was PH4.0.
It was found to be ~8.0. In addition, the pH stability range was investigated using the same procedure as above except that 0.15 ml of a 1% Triton X-100 aqueous solution was used instead of the 0.15 ml of distilled water used in the titer measurement method. Not much had changed. Thermostability Add 9.9 ml of 0.1 M Tris/HCl buffer (PH7.2) to 0.1 ml of enzyme solution, add 20, 30, 37, 40, 50,
After standing at 60 and 65°C for 30 minutes, the remaining enzyme activity was measured. The results are shown in Figure 4, with almost no loss of activity after heat treatment at 30°C for 30 minutes, and 60% activity remaining after heat treatment at 50°C for 30 minutes. Effects of Various Substances In the titer measurement method, 0.05 ml of aqueous solutions of various substances were added in place of the CaCl ₂ aqueous solution, and the activity was measured at a concentration of 1 mM in the enzyme reaction system.
As a result, those that had an activating effect, for example,
_AlCl3 , _CaCl2 , _FeCl3 , _FeSO4 , _MgCl2 ,
These include primary, secondary, or tertiary alcohols such as SnCl ₂ , sodium deoxycholate, ethanol, isopropanol, and t-butanol, while the inhibitory agent is cetylpyridinium chloride. Method for measuring titer As described above. Purification method It is as described above, and the specific example is as described in Example 3. Isoelectric point 6.4±0.1 (measured by ampholine electrophoresis) Transfer action Cabbage phospholipase D generates phosphatidic acid from lecithin, transfers it to a linear primary alcohol with 1 to 6 carbon atoms, and converts it into an ester. known to form. As a result of similarly examining the transfer effect of this enzyme, it was found that this enzyme transfers to a wider range of alcohols to form esters. As the substrate phospholipid, diacyl ester type, monoacyl ester type, plasmalogen type, cycloalkylidene type, dialkyl ether type, monoalkyl ether type α-glycerophospholipid and β-glycerophospholipid are used, Sphingophospholipids are also good substrates. Alcohols that undergo metastasis include the following categories: A Primary alcohols (1) Aliphatic alcohols having 1 to 22 carbon atoms and substitution thereof with primary, secondary, tertiary amines, halogens, hydroxyl groups, carboxylic acids and their esters, ethers, aldehydes, ketones, etc. (2) Pentose, hexose, and those with substituents such as amino groups and acid amides (3) Sugar alcohols and polyhydric alcohols (4) Disaccharides (5) Aromatic alcohols and amino groups,
Those with substituents such as halogen and carboxylic acid (6) Alicyclic alcohols (7) Carbon polycyclic alcohols (8) Furan ring, phthalimide ring, pyrrole ring, indole ring, pyridine ring, morpholine ring, pyrimidine ring, Multi-ring alcohols such as piperazine rings and imidazopyrimidine rings B Secondary alcohols (1) Aliphatic alcohols with 3 to 10 carbon atoms and those with various substituents (2) Aromatic alcohols (3) Alicyclic alcohols Table 2 shows the results of examining whether a transposition effect occurred when these substrates were combined with alcohol. In Table 2, cases in which production of a transition product (ester) was observed are indicated by +, cases in which a small amount of production was observed are indicated by ±, and cases in which no production was observed are indicated by -. Further, the symbol in front of each alcohol indicates the classification number of the alcohol. A similar test was conducted using commercially available phospholipase D obtained from cabbage.
was used, but no transition products (esters) were produced. Next, we will describe the experimental method for the transfer effect. 0.4M, PH5.7 acetate buffer 0.1ml, 0.1M _CaCl2
0.05 ml of aqueous solution, 0.1 ml of enzyme solution containing 250 units of phospholipase D, 1% phospholipid emulsion (0.1 g phospholipid, 1 ml ether, 10 ml sonicated water emulsion) and 0.1 ml of 10% alcohol solution (water depending on solubility). , ether or acetone) were mixed and reacted at 37°C for 1 to 5 hours. After the reaction is complete, 50 mmol of
0.2 ml of 1M Tris-HCl buffer containing EDTA, pH 8.0, and 5 ml of a chloroform-methanol mixture (2:1) were added and mixed to extract the transfer product (ester). After standing still, the lower layer was separated, dried under reduced pressure, dissolved in a small amount of chloroform-methanol mixture (1:1), and the transition product (ester) was detected by thin layer chromatography. The results are shown in Table 2.

【table】

[Table] Molecular weight: Approximately 52,000 (measured by Ultrogel ACA34 gel filtration method) Approximately 58,000 (measured by SDS electrophoresis method) Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereby. It's not something you can do. Example 1 10g of defatted wheat germ, 0.1g of ammonium sulfate, 0.1g of peptone
Put g, and 8 ml of water into a 500 ml Erlenmeyer flask,
After steam sterilization at 121°C for 15 minutes, 2 ml of an aqueous spore suspension of Actinomadeula sp. No. 362 was inoculated. Then, the cells were left to stand and cultured for 30 days at a culture temperature of 25°C. After the culture was completed, 100 ml of water was added to extract phospholipase D, the solid matter was separated, and the activity of phospholipase D in the liquid was measured. The result is
It was 7.4u/ml. Example 2 Starch 1%, (NH ₄ ) as seed medium
_{H2PO4 0.25} %, peptone 0.25%, _{K2HPO4 0.2} %,
100ml of aqueous medium (PH6.8) containing MgSO4 _0.01 %
Place it in a 500ml Sakaguchi flask, steam sterilize it, and inoculate it with a loopful of Actinomadeula sp. No. 362 spores.
A seed culture solution was obtained by culturing at a culture temperature of 30° C. and shaking at 120 revolutions/min for 2 days. Next, the main medium, namely glucose 1.0%, corn steep liquor 1.0%, peptone 0.5%, powdered yeast extract 0.1%, NH ₄ NO ₃ 0.5%, K ₂ HPO ₄ 0.2
%, medium consisting of _{MgSO4.7H2O0.01} % ₍ PH6.0)
Pour 50 ml into a 500 ml Sakaguchi flask, steam sterilize at 121℃ for 10 minutes, then transplant 5 ml of the above seed culture solution.
The cells were cultured at ℃ for 2 days. After culturing, solid matter was removed by centrifugation to obtain 50 ml of culture solution (77 u/ml). To this, 22.7 g of ammonium sulfate was gradually added with stirring to precipitate phospholipase D. The precipitate was collected by centrifugation, dissolved in 0.02M Tris-HCl buffer (PH7.2), and phospholipase D activity was measured. At this time, the recovery rate of phospholipase D activity from the culture solution was 78%. Example 3 Soy flour 3.0%, corn steep liquor 1.0%, peptone 0.5%, powdered yeast extract 0.1%, glucose
1.0%, _NH4NO3 0.25 _% , _{K2HPO4 0.4} %,
_MgSO4・_7H2O0.01 %, Tween-85
A medium (PH6.0) consisting of approximately 0.1% was placed in a 30 jar fermentor, and after sterilization at 120°C for 15 minutes,
Inoculate the seed culture solution 1.5 described in Example 2,
Culture was performed at 27°C for 40 hours. After culturing, the bacterial solids are removed by centrifugation,
Centrifugation supernatant 13 (100u/ml) was obtained. After cooling the centrifuged supernatant to 5°C, acetone at -20°C was added, and a precipitate containing phospholipase D corresponding to an acetone concentration fraction of 30 to 70% was collected by centrifugation.
This precipitate was dissolved in PH6.5 Tris-maleic acid,
After dialyzing against 0.02M of the same buffer, it was passed through a column of DEAE-cellulose equilibrated with the same buffer, and the passed-through fraction was collected. Next, the method of Horiuchi et al. [J.Biochem.
81, 1639 (1977)] was packed into a column, thoroughly washed with water, and then
The DEAE-cellulose permeate was injected to adsorb the activity. This was mixed with 0.05M Tris-HCl buffer (PH
After washing with 7.2), the same buffer containing 0.2% Triton X-100 was added to elute the activity. After collecting the active fraction and concentrating it using an ultrafiltration membrane (Type G-10T) manufactured by Bio Engine Airing Co., Ltd., Toyo Pearl HW-55F (Toyo Soda Co., Ltd.) was used as a gel supercarrier.
The active fraction was collected and freeze-dried. Add this dry powder to 0.025M tris-acetic acid (PH8.3).
After dissolving in the solution, it was passed through a column packed with Poly Buffer Exchanger PBE ^TM 94 (20 ml) manufactured by Pharmacia Fine Chemicals to adsorb the activity, and then evaporated by pH gradient using the same company's Poly Buffer Exchanger (PH5.0) for elution. It eluted. The active fraction of eluted phospholipase D was collected and concentrated using an ultrafiltration membrane, and then filtered into Sephadex G-
The phospholipase D active fraction was collected and lyophilized. In this way, phospholipase D was recovered with an activity recovery rate of about 43%, and the specific activity at this time was 13100 u/mg protein.

[Brief explanation of drawings]

The drawings relate to phospholipase D obtained by the method of the present invention; FIG. 1 is a curve showing the optimum pH, FIG. 2 is a curve showing the optimum temperature, FIG. 3 is a curve showing PH stability, and FIG. Figure 4 is a curve showing thermal stability.

Claims (1)

[Claims] 1. A method for producing phospholipase D, which comprises culturing phospholipase D-producing bacteria belonging to the genus Actinomadeura in a medium, and collecting phospholipase D from the culture.