JPS5867183A - Production of phospholipase d - Google Patents

Abstract

PURPOSE:A microorganism in Actinomadura is cultured to produce phospholipase D. CONSTITUTION:A microorganism in Actinomadura, capable of producing phospholipase D, e.g., Actinomadura NO 362 (FERM-6132) is inoculated in an enriched culture medium and cultured by the deep tank aeration process at 20- 35 deg.C and phospholipase is obtained mainly from the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing phospholipase D using a microorganism. Specifically, one aspect of the present invention is a method for producing phospholipase D, which is characterized in that a phospholipase D-producing bacterium belonging to the genus Actinomadura is cultured in a medium, and phospholipase D is collected from the culture. C, 3, t, tt, tt) is an i element that decomposes the ester bond between the phosphoric acid of the -glycerophospholipid and the nitrogen-containing base to liberate monophosphatidic acid and the base. D is monoethanol, riI
When acted on glycerophospholipids in the coexistence of compounds with alcohol groups such as J-cellol and ethanolamine,
It is also known that phosphatidic acid can be transferred to an alcohol group.

Phospho-1J hase D has been known for a long time to exist widely in the plant kingdom of cabbage, carrots, etc., and is produced by extracting it from the tissues of cabbage. In addition, recently, as a method for producing phospholipase D using microorganisms, Streptomyces (Japanese Patent Publication No. 62-399/g)
-Micromonospora
A method is known in which 5g of the product can be produced by a single fermentation method using actinomycetes belonging to Publication No. 1).

Phospholipase D is used as a research reagent related to the metabolism of phospholipids, 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 invention first isolates microorganisms from a wider range of soil than in the natural world,
We 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 Actinomadura f4No 3-t2) was cultured in a medium, it was found that it acts on glycerophospholipids in the medium to release phosphatidic acid and base. Confirm that a certain enzyme is produced.

It was discovered that this bacterium produces phospholipase D. Furthermore, when this enzyme was allowed to act on monoglycerophospholipids in the presence of a compound having an appropriate alcohol group such as ethanol, sorbitol, ethanolamine-glycerol, etc., it was observed that phosphatidic acid was transferred to an alcohol group. The mycological properties of the above-mentioned strain are as follows. Form 0 (a) performed well on starch inorganic salt agar, tyrosine agar, yeast/malt agar, oatmeal agar, etc., and showed moderate performance on glycerin asparagine agar. It grows to a certain extent and forms a colony of aerial mycelia.

The color of the bacterial flora with spores attached depends on the type of medium - sucrose nitrate agar at the time of observation, nutrient agar - glucose asparagine agar, and whether aerial mycelia are attached.

Only poorly established.

When this strain grown on an agar medium is observed under a microscope, the hyphae are branched with a width of 00g-1.2μ, some are looped or spiral, and some are mainly linear (elongated) with some bends. The tip is often loosely wound into a loop.

The spores settle in a chain of 100 or more from number/Q,
Forms entire hyphae.

The size of the spores is 01g-/, 2Xj, ko~), 7μ, short cylindrical or oval, and somewhat irregular in size and shape.

The basement hyphae are 0.1-1. Qμ, it elongates while bending with irregular branches, and no single zoospore, single sporangium, or sclerotium is formed.

In addition, septa and division of hyphae are usually not observed, but division of hyphae may be observed in liquid culture.

(b) Properties on various media The experimental methods described below are mainly based on E.B., Shearing (Int. J., 5yst, Bac3er).
iol.

It was carried out according to the method of Vol. 16, 3) 3-3qO17966).

Color tone is - “Color Standard” (Japan Color Research Institute, 19
411), and one hue name and a hue symbol were written in parentheses in the order of hue name - saturation number - brightness number.

Cultivation was carried out at 23C, and Table 1 shows the observation results on each medium during the 1st to 3rd week when growth was most vigorous. However, the first
In the table, the color of the surface of the basal hyphae described in the growth items shows the observation results during the 1st week of culture before spore attachment, and the description is given for media where spore attachment is rapid and it is difficult to determine the color of the surface of the basal hyphae. I haven't.

(c) Physiological properties ■Growth temperature: Grows around lO~37C, 120~30C
It's the most (grows).

■ Liquefaction of gelatin: Not liquefied (cultivated on glucose e peptone gelatin medium, U, fC, 3 weeks) 0 ■ Hydrolysis of starch: Decomposed (cultured on starch agar medium, U, tC, 3 weeks).

■Coagulation and peptonization of skim milk: No coagulation.

Peptonize (30C, 3-Q, weekly culture).

■ Production of melanin-like pigment: Produced in peptone yeast iron agar-tyrosine agar (2 to q days).

(d) Assimilation of carbon source (30C,/Q-/6 days culture)
L-arabinose + Cylose -D-xylose + Inositol ±D-glucose +
L-rhamnose-D-fructose-raffinose-(e) Chemical analysis of cells The diaminopimelic acid of this strain is men type, and the sugar composition of the cell wall does not contain arabinose-xylose-rhamnose. Bergey's Manu for analysis results of 0 or more containing galactose, mannose, etc.
al of the Determinative Ba
cteriology 3rd edition, pages 6S7-6J ()
979) and - Lessier Vallier (Inter, J, S
system, Bacteriol, Volume 2C, '13
According to classification methods such as those published in 1970 (pp.
ltype) Type III-sugar composition type (cell w
all sugar pattern) becomes type B.

As described above, this bacterium has a cell wall type of III and a sugar composition type of B.
Therefore, the genus Microbispora - the genus Strebutosborangium, the genus Spirillospora, the genus Planomonospora,
It belongs to either the genus Dermadophilus or the genus Actinomadura. However, since a single bacterium forms a spore chain consisting of many spores, and one sclerotium, one sporangium, and zoospores were not found, it was found that the genus Actinomadura (Ge.
From a taxonomic point of view, it is most appropriate to identify it as nusActinomadura).

Therefore, this bacterium is of the genus Actinomadura N03p;
tinomadura sp NO,? I decided to call it 12). This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is ``FEBM P-Otsu 13.''

The bacteria to be used in the present invention include not only the above-mentioned Actinomadura genus NOJ Otsuko and mutant strains obtained by mutating this strain, but also all bacteria that belong to the Actinomadura genus and produce phospholipase D. I can do it.

In carrying out the present invention, both liquid culture and solid culture can be used as the culture mode, but from an industrial perspective, deep aeration agitation culture is advantageous.

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 Actinomadura. It can be used.

As a carbon source for the medium - for example glucose-fructose.

In addition to sucrose, lactose-starch, glycerin, dextrin-molasses, nrubitol, etc., fatty acids, fats and oils, crude lecithin-alcohol, organic acids, etc. are used alone or in combination.

As the nitrogen source, any one of an inorganic nitrogen source and an organic nitrogen source can be used. Examples of the inorganic nitrogen source include ammonium nitrate, ammonium sulfate, and urea.

Sodium nitrate, monoammonium phosphate, diammonium phosphate - ammonium chloride, etc. are listed.Organic nitrogen sources include soybean, rice, corn, cottonseed, rapeseed, wheat flour, etc., bran, oil cake, and 1% Uchipo liquor. 1.
7. Yeast eyayu, meat extract-casein-amino acids, etc. are used.

Inorganic salts and micronutrients that promote the production of phosphoric acid, magnesium-potassium, iron-aluminum, capase D, for example, can be used as required.

Cultivation is carried out under aerobic conditions. Although the culture temperature can be changed as appropriate within the temperature range at which the bacteria grow and produce phospholipase D, a temperature of 100 to JjC is particularly preferred.

The culture time varies depending on the conditions, but 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.

Phospholipase D produced in the culture is mainly dissolved in the culture solution in single-liquid culture, so phospholipase D is extracted from the culture solution obtained by separating the solid matter from the solution after one culture.
Collect.

In collecting phospholipase D from culture fluid F, any method commonly used for enzyme purification can be used. For example, methods such as salting out with ammonium sulfate, precipitation with an organic solvent such as acetone or ethanol-methanol, dialysis, ion exchange chromatography, adsorption chromatography, gel filtration, and isoelectric focusing precipitation with an adsorbent can be used.

"Furthermore, if the effectiveness of purifying phospholipase D can be improved by appropriately combining these methods, a combination of these methods can be used.

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, and surfactants 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: /'Iz egg yolk purified lecithin emulsion (Q, 1ji
l-Lecithin, 1mt, ethyl ether, 10ml distilled water ultrasonic emulsion)
, u Tris-HCl buffer 0, /me, o,)M
CaCl2 aqueous solution o, oom6 - distilled water 0. Mix ljm O, add 1 ml of enzyme solution θ, and mix at -370.
After 0 minute reaction--containing t OmM EDTA・2Na/
-M) Add lis-hydrochloric acid buffer (pHg, o) and yumt - Immediately boil for 5 minutes to completely stop the reaction. Next, reagent for measuring cholinesterase [manufactured by Nippon Shoji Co., Ltd.]
Add 1 ml of a solution prepared by dissolving the choline coloring agent included in the kit into the coloring solution, react at 37C for 12θ minutes, and then measure the absorbance of -J-00fim.

As a control, the absorbance of a similar reaction using an enzyme solution that has been previously heat-inactivated is measured.

The enzyme activity that releases 78 moles of choline per hour is defined as 7 units.

Next, the physicochemical properties of phospholipase D using the enzyme preparation purified by the method shown in Example 3 will be described.

0 action Decomposes the ester bond between phosphoric acid of glycerophospholipid and nitrogen-containing base to liberate phosphatidic acid and base.

(2) Substrate specificity An emulsion containing 0.18 mol of either lecithin-lysolecithin or sphingomyelin as a substrate.
Use 1ml/4Triton X instead of distilled water
The amount of choline released by the reaction was measured in the same manner as the titer measurement method described above, except that an aqueous solution containing -/o was used, and the phospholipase D activity for each substrate was measured. As a result, the relative activity of lecithin when expressed as active ioo is -lysolecithin 3. Otsu, sphinco minirin 0
Met.

■Optimal pH pH 30-4 instead of buffer used in titer measurement method
．． At 0, formic acid/sodium formate buffer - pHtt, or,
In s, acetic acid/sodium acetate buffer, pH! ,5~g,! So, Tris-maleic acid-caustic soda buffer, pH 7.0
~9. For θ, Tris-Fist hydrochloric acid buffer, pH 9,0~/Q;
In Q, the activity of phospholipase D was measured using a glycine/caustic soda buffer to determine the optimum pH. Also, instead of 0.11ml of distilled water used in the same measurement method, / % Trit
on X-to.

(Wako Tsumugi Pharmaceutical) Optimal p when using aqueous solutions o, t, rmb
H was also determined.

The results are shown in Figure 7, and the optimum pH when using distilled water is around 7.0.
What is the optimal pH when using an OO aqueous solution? ,! Found nearby.

■Enzyme activity was measured at the optimum temperature C. The results are shown in FIG. 2, and it is recognized that the optimum temperature is in the range of jjC to goc.

■pH stable enzyme solution o, 1 ml; qml of 011M various buffers, i.e. pH 3-0 to 3. In j, glycine, salt -
Buffer, pH 3, j ~ 7.0: acetic acid/sodium acetate buffer - PH 3, 0~g, 0: Tris/maleic acid/caustic soda buffer - pH 7, 0 - 9.0: Tris/hydrochloric acid buffer, pH 9 , 0-9. At f, glycine and caustic soda buffers were added, respectively, and maintained at 2j'7 for 7 hours. After that-
Add these enzyme buffer solutions to 0.6M Tris-HCl buffer solution (PH
7,2) Add q and ome and adjust the pH to 7.0-7.
It was set as 3. Using this solution 0,'/ml, the titer was measured according to the titer measurement method, and the stable pH range was investigated - Part 3
As shown in the figure, the particularly stable pH range of this enzyme is PHII.
, 0-, za, O.

Distilled water used in the titer measurement method also has 0. /! Instead of ML/
4Triton X-'00 aqueous solution 0. /jm
The stable range of pI (pI) was investigated using the same procedure as above except for using 1; but the results were almost the same as in Figure 3.

■ Add 0.1 to 1 g of thermostable enzyme solution to 0.1 M Tris/HCl buffer (p
) (7, 2) 9, add 9~g -YuQ, 30. ．．
After being left in 77゜Io, ro, OtsuO, and tSC for 30 minutes, the remaining enzyme activity was measured.The results are as shown in Figure 9. No inactivation occurred - 60 ml of activity remained after 30 minutes of heat treatment at RO'6.

■In the influence potency measurement method using various substances, o, osml of aqueous solutions of various substances are added instead of CaC12 aqueous solution in an enzyme reaction system.
Activity was measured as f. As a result, 1, for example, AlCl3-CaCl2.
FeCl3. Fe50. - MgCl2-5nC12
, sodium deoxycholate, etc., and the one that had an inhibitory effect was cetylpyridinium chloride.

■Method for measuring titer As mentioned above.

(2) Purification method The method is as described above; a specific example thereof is as described in Example 3.

[Phase] Isoelectric point 6, q±0.7 (the present invention is not limited by the ampholine electrophoresis method.

Example l Ammonium sulfate Q, lf, peptone Q, /f to defatted wheat germ IQf
- and water g me so o ml volume triangle 7-ra
After steam sterilization for lj minutes with no IC, Actinomadura spp. 4 u of spore water suspension λm O was inoculated. Then, the cells were left to stand and cultured for 30 days at a culture temperature of -IC.

After culturing, add 100ml of water and add phospholipase D.
After extraction, the solid matter was separated from P and the activity of phospholipase D in the P solution was measured. The result is 7, l/-11/
It was ml.

Example 2 Starch/4 as seed medium. (N11()II, l
'0. θ, -7 4, Het yo, -1r%, K
2) Aqueous medium containing LPO+0.24-Mg50°0.014 (pH, gj')/. Oml room
l After steam sterilization of Sakaro flamelo flask.

One platinum loop of spores of Actinomadura sp. NQ3t2 was inoculated and cultured with shaking at a culture temperature of 307:0/0 rpm for one day to obtain a seed culture solution.

Next, the main medium - glucose 1. θ#I.

Cone steeple force - 1.04. Peptone O0j-powdered yeast extract Q, l, NH4No3o, r*.

K2HPO40, λ, Mg5O, 7H200,
Cultivation consisting of O no staff! (pHt, o) SOME was placed in a 300ml volume Sakaro flask, steam sterilized at 121C for 10 minutes, and then the above seed culture solution RME was transplanted and
The cells were cultured between columns.

After culturing, centrifuge to remove solids, culture f liquid zOm
b (77u/me) was obtained. To this, 2.lambda. and 2.mu. of ammonium sulfate were gradually added with stirring to precipitate phospholipase D. The Gisha menmai precipitate was collected by centrifugation, dissolved in 0.02M Tris-HCl buffer (pH 2.2), and phospholipase D activity was measured.

At this time, the activity recovery rate of phospholipase D from the culture liquid was 7 g%.

Example 3 @ Nako 3.04-Corn Stew Rica-1.0 Part, Peptone O, s%, Powdered Yeast Extract O, i4°Glucose 1. θ Wound, NH4N030, Month 1. K2HPO4
θ, 44. A medium consisting of MgSO4・7H20θ, θl-Tween-gso, t4 (
pH 4,0) approx. lj! 30Jl Jar Fur Mentor
After sterilizing the seed culture solution 1. in Example 2 for 1,5 minutes at 100% OC. ! ! was inoculated and cultured in core C for a□ hours.

After culturing - remove solid bacteria by centrifugation and centrifuge supernatant/? A(/θθu/m6) was obtained. This centrifuged supernatant was
After cooling to -200 °C, acetone was added and a precipitate containing phospholipase D corresponding to an acetone concentration fraction of 30-70% was collected by centrifugation. This precipitate is p[d
, j) DEAE dissolved in lis-maleic acid, dialyzed against 0.02M of the same buffer, and equilibrated with the same buffer.
- Cellulose was passed through the column and the 1-pass fraction was collected. Next, the method of Horiuchi et al. (J, Biochemlg/, tt3q(
A column was filled with valmitoyl gauze prepared in Example 1/977), washed thoroughly with water, and the DEAE-cellulose permeate was injected to adsorb the activity. This is o, orM
After washing with Tris-HCl buffer (PH7, -2) -0,1
Same buffer i containing % Triton X −/o
7JO, t The 0 activity fraction from which the activity was eluted was collected and filtered using an ultrafiltration membrane (Type G-
10T), and then injected into a packed column of Toyovar HW-jj F (manufactured by Toyo Soda Co., Ltd.) as a gel f overcarrier, passed through the column using mono-distilled water, and collected the active fraction and frozen. It was dried.

This dry powder was mixed with 0.02-tM trisulfuric acid (pHg
, ,? ) After K dissolution, the polybuffer exchanger PBE”9 a created by Pharmacia Fine Chemicals Co., Ltd.
After passing through a column packed with 2 omε) to adsorb the activity, it was eluted with a pH gradient using the same company's elution polybuffer CPHj, 0). The active fraction of eluted phospholipase D was collected and concentrated using an ultrafiltration membrane, followed by Sephatex G-2.
The active fraction of phospholipase D was collected and lyophilized using 3 packed columns, and phospholipase D was recovered with an activity recovery rate of about 34 μl, and the specific activity at this time was /J/.

It was Qu/In9 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. 1 is a curve showing the optimum temperature, and FIG. 3 is a curve showing PU stability.
Figure q is a curve showing thermal stability. Figure 11-i44 pl<hzm 0 20 40 60 80 100'Cf
Rokokuya 7r15iI Procedural Amendment Document July 1999 Showa SG Special Review Agency Director-General Kazuo Wakasugi 1, Indication of the case Showa 2015 Special Request No. i63ri 2S No. 3, Person making the amendment' 4, Representative Personal address Postal code /71 Voluntary amendment 6, Detailed description of the invention column 7 of the specification subject to amendment, Contents of amendment (1) Specification letter ig page 9th line to 10th line ``With sodium deoxycholate etc. Yes," should be corrected as follows: ``M1 grade, secondary grade, or tertiary grade such as sodium deoxycholate, ethanol, impropatol, t-butanol,
(2) Ming #l, page 7, page 1
Insert the following sentence between the 9th line and the 1st θ line. ``0-rearrangement cabbage phospholipase D generates phosphatidic acid from lecithin and converts it into linear 7
It is known that esters are rearranged into alcohols to form esters. As a result of similarly examining the rearrangement effect of this enzyme, it was found that this enzyme rearranges a wider range of alcohols to form esters. As the substrate phospholipid, diacyl ester type, monoacyl ester type, flamelogen type, cycloalkylidene type, dialkyl ether type, monoalkyl ether type α-glycerophospholipid, and Uβ-glycerophospholipid are used as substrate phospholipids. and sphingophospholipids are also good substrates. Alcohols that undergo rearrangement include the following categories: A. /Class alcohol (1) Aliphatic alcohol with carbon number from 1 to 22, primary, secondary, tertiary amine, halogen, hydroxyl group, carboxylic acid and its ester, nityl, aldehyde,
Those with substituents such as ketones (2) Pentoses, hexoses, and those with substituents such as amine groups and acid amides (3) Sugar alcohols and polyhydric alcohols (4) Disaccharides (5) Aromatic alcohols and Those with substituents such as amine groups, halogens, and carboxylic acids (6) Alicyclic alcohols (7) Carbon polycyclic alcohols (8) Furan rings, phthalimide rings, virol rings, indole rings, pyridine rings, morpholine rings , heterocyclic alcohols such as pyrimidine rings, piperazine rings, imidazopyrimidine rings B, secondary alcohols (1) aliphatic alcohols having 7 to io carbon atoms and those having various substituents (2) aromatic alcohols (3) ) Alicyclic alcohol Table 2 shows the results of examining whether rearrangement effects occurred when these substrates and alcohols were combined. In Table 2,
Those in which production of rearranged product (ester) was observed were indicated by +, those in which a small amount of production was observed were indicated by -, and those in which no production was observed were indicated by -. Further, the symbol in front of each alcohol indicates the classification number of the alcohol. A similar test was conducted using a commercially available cabbage-derived homeolipase D, but none of the products produced rearranged products (esters).Next, we will describe the experimental method for rearrangement effects. , II M, pH 3,7 acetate buffer o, imb,
0-07M CaCl2 aqueous solution o, osmt, enzyme solution containing phospholipase DJso units o, imp, , l easy lipid emulsion co, tyniphospholipid fr, /
ml; :r-te/l/, iome ultrasonic emulsion of distilled water) 0. imb and 10% alcohol solution (use water, ether or acetone depending on solubility) 0. I
jrnb was mixed and reacted at j7C for /-3 hours. After the reaction is complete, 1 mol containing SO mmol EDTA, pH
Then, 0.2 mb of Tris-HCl buffer and chloroform-
3 ml of a methanol mixture (λ:l) was added and mixed to extract the rearrangement product (ester). After standing still, separate the lower layer, dry under reduced pressure, and add a small amount of chloroform-methanol mixture 11
ffl(/:/), and the rearrangement product (ester) was detected by thin layer chromatography. The results are shown in Table 2.

Claims (1)

[Claims] A method for producing phospholipase DF, which comprises culturing a phospholipase D-producing bacterium belonging to the genus Actinomadura in a medium, and collecting phosphorine (-zeD) from the culture.