JPS5863388A - Preparation of phospholipase d - Google Patents

Abstract

PURPOSE:To obtain phospholipase D useful as a reagent for research, etc. efficiently, by cultivating a microorganism belonging to the genus Nocardiopsis capable of producing phospholipase D in a medium, collecting phospholipase D from the culture. CONSTITUTION:A novel strain belonging to the genus Nocardiopsis NO 779 (FERM-P 6133) separated from soil is cultivated in a nutrient medium under aerobic conditions about 20-35 deg.C for about 1-3 days. A solid substance is filtered off from the culture solution, the culture solution is treated by a proper combination of salting-out, dialysis, ion chromatography, absorption chromatography, gel filtration, precipitation at isoelectric point, etc., so that phospholipase D contained in the filtrate is separated and purified. Phospholipase D is an enzyme to decompose an ester bond of phosphoric acid of glycerophospholipid and a nitrogen-containing base and to liberate phosphatidic acid and a base.

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 comprises culturing phospholipase D-producing bacteria belonging to the genus Nocardiopsis in a medium and collecting phospholipase D from the culture.

Phospholipase D (E, C, 3,), da, da) 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 phospholipase D transfers phosphatidic acid to an alcohol group when it acts on a monoglycerophospholipid in the presence of a compound having an alcohol group such as ethanol, chrycerol, or ethanolamine.

Phospholipase D has been known for a long time to be widely present in plants 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 a single microorganism, actinomycetes belonging to the genus Streptomyces (Japanese Patent Publication No. 12-3991g) and the genus Micromonospora (Japanese Patent Publication No. 12-3991) have been used. 1. A method of manufacturing by fermentation method is known.

Phospho IJ Hase D is used as a research reagent related to phospholipid metabolism, a reagent for quantifying phospholipid contained in serum, etc., and can also be used for 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. Results: When a strain isolated from the soil of Hachioji City, Tokyo (referred to as Nocardiopsis No. 77 q) is cultured in a medium, it acts on glycerophospholipids in the medium to liberate phosphatidic acid and base. Confirm that an enzyme is produced.

It was discovered that this bacterium produces phospholipase D.

This enzyme can also be used in combination with ethanol and nrubitol.

When acting on glycerophospholipids in the presence of a compound having an appropriate alcohol group such as ethanolamine or glycerol, it was also observed that phosphatidic acid was transferred to an alcohol group.

The dental properties of the above bacterial strain are as follows.

(a) Form It grows well on glucose asparagine agar, glycerin asparagine agar, yeast malt agar medium, etc., and grows moderately on starch inorganic salt medium, and forms colonies of aerial mycelia.

The color of the bacterial flora on which the spores have settled varies slightly depending on the type of culture medium and the time of observation, but it is generally white or grayish-white to light gray.

Indications: On nitrate agar, nutrient agar, and oatmeal agar media, aerial mycelia do not or only poorly attach.

When this strain grown on agar medium is observed under a microscope,
Aerial hyphae are straight and loosely wavy or curved or branched and elongate long with θ, j to θ1gμ, and the entire aerial hyphae is formed by a chain consisting of several 10 to 100 or more spores.

The size of the spore is 0, j-0, g X O, ? ×l,
0μ, short cylindrical shape and slightly irregular size.

Substrate hyphae elongate in a branched manner with widths o, J-10, and tμ, and although they do not necessarily divide on an agar medium, in most cases, the fine hyphae divide by liquid culture.

However, zoospores, spore paste, sclerotia, etc. are not formed.

(b) Properties on various media The experimental methods described below are mainly based on E.P. Shearing (Int. J. 5yst, Baeteri)
ol.

16, 313-34tO, 7966).

Color tones are "color standards" (Japan Color Research Institute).

/944') and entered the hue symbol in the order of hue name, saturation number, and lightness number in parentheses together with one hue name.

Cultivation was carried out in CojC, and Table 1 shows the observation results on each medium during the 2nd to 3rd week when growth was most vigorous. However, the first
In the table, the color of the surface of the basal hyphae listed in the growth item indicates the observation result during the 1st week of culture before spore settlement. Not listed.

(c) Physiological properties ■Growth temperature: Grows around JC to 30C, YuO to 30C
It grows best in

■ Liquefaction of gelatin: No liquefaction (cultured on glucose-peptone-gelatin medium, YujC, 3 weeks).

■Hydrolysis of starch: Decompose (culture on starch agar medium for 2!t'l::, 3 weeks).

■ Coagulation-peptonization of skim milk: Neither coagulation nor peptonization was performed (30C, 3-q week culture).

■ Production of melanin-like pigment: Produced in peptone yeast iron agar, tyrosine agar (CojC, -~1 day) 0 (d) Assimilation of carbon source (307:, 10~) 6 days culture)
L-arabinose-sucrose-D-
Xylose - Inositol - D-Glucose + L-Rhamulase - D-Fructose - Raffinose - (e) Chemical analysis of cells The diaminopimelic acid of this strain is meso type and does not contain hydroxydiaminopimelic acid.

The sugar composition of the cell wall is monoarabinose and xylose.

Does not contain madurosurumnose, galactose,
Contains mannose etc. Moreover, this strain does not have nocardomicolic acid.

Regarding the above analysis results - Bergey's Manu
al of the Determinative Be
cteriology g edition, pages 117-tjg (i
q7a) and Recimopalier (Inter, J, Sy
stem, Bacteriol, vol. 20, a, ys
Page~#<ZJ page,)97Q)-Mayer (Int,
J.

5yst, Bacteriol, Volume 6, 4'ff7
When judged according to the classification method of p.-4I93 (1976) et al.

Desire is cell wall type
Type III, sugar composition type (cell wall sug
ar pattern) 0 or more, which is type C. Since the cell wall type is III and the sugar composition type is C, according to the classification method of Lessier-Varié, it is a Dannvillei type Actinomadura genus.

It belongs to the genus Thermoactinomyces, Actinobiifidus, or Geodermatophyllus.

However, the main hope is that all of the aerial hyphae consist of long chains of spores, dividing monobasic hyphae into small pieces, but since no endospores, zoospores, or sporangia are found, Dasonbireikippu. Actinomadura genus (Gen
usActinomaduradassonville
It is taxonomically appropriate to identify it as ei type), and it is generally integrated into the genus Iopsis and handled under the name of the genus Inocardiopsis.

Therefore, I wanted to use Ichinocardiopsis sp. N0779 (Ge).
nus Nocardiopsis 8p No 77
9). The desired bacteria have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the accession number is ``Feikokenkanyori No. 6-33.'' The bacteria used in the present invention include Nocardiopsis genus N0779 and Not only the mutant strain obtained by mutating the present strain, but also any strain that produces phospholipase D belonging to the genus Nocardiopsis (formerly the genus Actinomadura dasonbirequipe) can be used.

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 use deep aeration agitation culture.

Culture sources to be used include carbon sources, nitrogen sources, inorganic salts, and other micronutrients commonly used in microbial culture, as well as nutrient sources that can be used by microorganisms belonging to the genus Nocardiopsis. All can be used.

Examples of carbon sources for the medium include glucose and fructose.

Sucrose-lactose, starch, glycerin, dextrin.

Used together.

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

Examples include sodium nitrate, monoammonium phosphate, diammonium phosphate, ammonium chloride, etc. Organic nitrogen sources include soybean, rice, corn, cottonseed, rapeseed, flour such as wheat, rice bran, fat lees, and other organic nitrogen sources. Stee liquor, peptone, yeast extract, meat extract, casein, amino acids, etc. are used.

Inorganic salts and micronutrients include, for example, magnesium phosphate, potassium, iron, aluminum, calcium, -
In addition to salts such as zinc 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 a temperature of 10 to 3 IC is particularly preferred.

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.The phospholipase D produced in the culture is mainly dissolved in the culture solution in submerged culture, so the culture obtained by separating the solid matter from the culture solution. Phospholipase D is collected from P solution.

In collecting phospholipase D from the culture solution, any method commonly used for enzyme production can be used. For example, salting out with ammonium sulfate, etc.

Methods such as dialysis-ion exchange chromatography, adsorption chromatography, gel filtration, and isoelectric precipitation using an adsorbent such as acetone, ethanol, and methanol can be used. Furthermore, if the purification effect of phospholipase D is improved by appropriately combining these methods, a combination of these methods can be carried out.

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, protein-lipid-surfactants, etc. as stabilizers. It can be made into solid phospholipase D.

Phospholipase Dnl! The elementary activity measurement method is determined by measuring the amount of base produced by acting on the substrate glycerophospholipid to decompose the ester bond between phosphoric acid and a nitrogen-containing base. The activity of phospholipase D is particularly relative to that of zolecithin q, when the descriptive property is lOO. Sphingomyelin 0.
It was 3.

■Optimal pH pH 3.0~ instead of buffer used in titer measurement method
11. For Q, formic acid/sodium formate buffer, pHa, o
-z, t for acetic acid/sodium acetate buffer, pHj, 5~
Tris Φ maleic acid/caustic soda buffer for g and j, p
For H7,0~q,o, Tris-competent hydrochloric acid buffer, pH 9,0
-10.0, the activity of phospholipase D was measured using a glycine-based caustic soda buffer to determine the optimum pH. In addition, the distilled water used in the same measurement method is 0. '4 instead of ljmt
Triton X-Zo.

(Wako Tsumugi) Optimal p when using aqueous solution o, 1 srul
H was also determined.

The results are shown in Figure 1, and the optimum pH when using distilled water is 4. j~7.0, /4Trito
When an aqueous solution of nX-/o was used, the optimum pH was found to be around s and o.

■Optimal temperature In titration method, 1 reactivity condition is IQ, -〇.

+23.37,40. ! 0. j, t, 60, 70
Enzyme activity was measured with ゜go and qoC. The results are shown in Figure 2, and the optimum temperature ranges from 60C to 0.
It is recognized that it is within the range of C.

(2) pH stability Enzyme solution 0.1-g, Ol-mA Q, 1M various buffer solutions, pH 3, 0-3. For j, glycine/hydrochloric acid buffer, pHJ, for j ~ 7.0, acetic acid/sodium acetate buffer, for pH 1,0~g, for O, Tris Φ maleic acid/caustic soda buffer, for pH 7, 0~9.0, Tris. Hydrochloric acid buffer, pH 9.0-9. ! Then M Tris-HCl buffer (
pH 7, add -mb, PR 7.0 to 2.3
And so. Using this solution O07mA, the titer was measured according to the titration method, and the stable pH range was investigated. As shown in Figure 3, the particularly stable pH range of this enzyme is a, O to 7.
It was found to be 0.

Distilled water used in the titer measurement method also has 0. instead of ljme'
4Triton X-too aqueous solution 0. ir
The pH stability range was investigated using the same procedure as above except that ml was used, but the results were almost the same as in Figure 3.

■Thermostable enzyme solution 0. Add IJ sous acid buffer (pH 7,2) to imtr, add 0,3
0.3? .

After being left in ao, to, to, and tIC for 30 minutes, the remaining enzyme activity was measured. The results are shown in Figure 4. Heat treatment at -3OC for 30 minutes hardly deactivated the enzyme, and lOO g after 30 minutes of heat treatment.

In the influence titer measurement method using various substances (:: Add 0.0jml of aqueous solutions of various substances instead of aC1t aqueous solution,
The activity was determined by adjusting the M concentration. The results are based on the activity at the time of water addition as 100, and the relative activity of substances with an activating effect and powder 1, such as AlCl5-CuSO4,
ZnSO4, Coal, CaCl2. FeC1,
, FeSO4°MgClt -5nC1z-sodium deoxycholate, Triton

■Method for measuring titer As mentioned above.

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

[Phase] Isoelectric point aogjthO,'C Measured by ampholine electrophoresis) Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited by these Examples. 1 Ammonium sulfate 0.0% to defatted wheat germ IQf. ll-, peptone 0.1
? , and aqueous mb were placed in a 100 mIV Erlenmeyer flask, and after steam sterilization with 1 Juno C for 13 minutes, an aqueous spore suspension of Nocardiopsis N0779-mb was inoculated. After the culture was completed, t□oml of water was added and phospholipase D was incubated at the culture temperature for 30 days.
After extraction, the solid matter was separated and the activity of phospholipase D in the P solution was measured. The result was 3.1y/ml.

Example 2 Starch (NH*)HzPO4o as seed medium
, 2s arc, peptone θ, cor %, K2HPO4θ,
λ4, an aqueous medium containing MgSO40,014 (pH
4.g) NoθOm6 was placed in a 100mb slope flask, and after steam sterilization, spores of NQ779 strain of Nocardiopsis sp. A seed culture solution was obtained.

Next, the main medium: glucose 1.0%, corn steep liquor 7. θ Yu-peptone θ, jll.

Powdered yeast extract O0) 1% NO30, Width-K2
Put rOml of a medium (pH 6,0) consisting of HPO40, Ko-MgSO, 7H200,0 into a zoomA Yosaka flask, steam sterilize it with /JIC for 70 minutes, transfer the seed culture medium jm6, and incubate at 2JC for 2 hours. After culturing for 1 day - centrifuge to remove solid matter, culture liquid jOmA (
j, cou/at) were obtained. Add ammonium sulfate to this, 7
Phospholipase D was precipitated by gradually adding f while stirring. The precipitate was collected by centrifugation, dissolved in 0.0 M Tris-HCl buffer (pH 7), and phospholipase D activity was measured.

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

Example 3 Soy flour 3.0%, corn steep liquor/, QO, 4'
4- MgSO4117H2Q O, 0/4.
Approximately 1 Jl of a medium (PH 6,0) consisting of Tween-g, tO,/% Kawasaki was placed in a 3θ! jar fermenter.

After sterilization at 120C for 7f, seed culture 1. as described in Example 2. jlj, and cultured at 2C for eo hours. 1 After culturing, the solid bacterial cells were removed by centrifugation to obtain a centrifuged supernatant 13I/(3 u/iJ'). After cooling the supernatant obtained by centrifugation, 207: of acetone was added, and a precipitate containing phospholipase D corresponding to an acetone concentration of 30 to 704 fractions was collected by centrifugation. This precipitate was dissolved in pH 4, O) lis-maleic acid, dialyzed against 0.02M of the same buffer, and then equilibrated with the same buffer.
The EAE-cellulose was passed through the column and the 1-pass fraction was collected. Next, the method of Hauchi et al. (J, Biochem, g/,
The column was filled with valmitoyl gauze prepared with tt3q (/977)).

After thorough washing with water, the above DEAE-cellulose permeate was injected to adsorb one activity. After washing this with 0.01M Tris-HCl buffer (pH 7.2), 0.24'l'ri
The same buffer containing ton X-'00 was added to elute the activity. The active fractions were collected and transferred to an ultrafiltration membrane (Type G −/o T ) (=
The dried powder was injected into 0.021 M imidazole-hydrochloric acid (pH
2. After dissolving in u) - Polybuffer exchanger PBE manufactured by Pharmacia Fine Chemicals"?<1(-〇at
) After passing through a packed column to adsorb the activity.

Elution was carried out using a pH gradient using a polybuffer for elution (pH-t, 0) manufactured by the same company. The eluted active fraction of phospholipase D was collected and concentrated using an ultrafon membrane, and then separated into Sephadex G-7! The mixture was passed through a packed column, and the phospholipase D active fraction was collected and freeze-dried.

In this way, phospholipase D was recovered with an activity recovery rate of about yo4 - the specific activity at this time was 700u7J11 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; FIG. 3 is a curve showing pH stability;
FIG. 4 is a curve showing thermal stability. rit 1'fA -? G44A Person making the amendment 4, Agent address: Postal code 171 5, Date of amendment order Voluntary amendment 6, Detailed explanation of the invention in the specification subject to amendment 7, Contents of the amendment (1) Specification, page 1g, Correct "Sodium deoxycholate, TritonJ" in line 79 as follows: "Sodium deoxycholate, primary, primary, or tertiary alcohol such as ethanol, Impropatol, t-butanol , Triton J (2) Specification page 19, insert the following sentence between line 1O and line 1/. ``◎Transposition action Cabbage phospholipase D produces phosphatidic acid from lecithin, and converts it into carbon number Straight chain 1 from No to O
It is known that esters are formed by rearrangement to alcohols. 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. Other than lecithin, diacyl ester type, monoacyl ester type, flasmalogen type, cycloalkylidene type, dialkyl ether type, monoalkyl ether type α-glycerophospholipid, and β-glycerophospholipid are used as substrate phospholipids. and sphingophospholipids are also good substrates. Alcohols that undergo rearrangement include the following categories: A. Primary alcohols (1) Aliphatic alcohols with carbon numbers from 1 to 1, and primary, secondary, and tertiary amines, halogens, hydroxyl groups, carboxylic acids and their esters, ethers, aldehydes,
Those with substituents such as ketones (2) Pentoses, hexoses, and those with substituents such as amino groups and acid amides (3) fiflucol and poly(i11i alcohols) (4) Trisaccharides (5) Aromatic alcohols and those having a substituent such as a minino group, norogen, or carboxylic acid (6) alicyclic alcohol (7) carbon polycyclic alcohol (8) furan ring, phthalimide ring, pyrrole ring, indole ring, pyridine ring, Morpho-11 ring, pyrimidine ring,
Heterocyclic alcohols B such as piperazine rings and imidazopyrimidine rings, secondary alcohols (1) Aliphatic alcohols with carbon numbers from 1 to 10 and those having various substituents [2] Aromatic alcohols (3) Alicyclic alcohols Alcohol When these substrates are combined with alcohol, a small amount of rearrangement is produced, and those in which no production is observed.
It was shown in 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, but the rearrangement

[Ester] was not produced at all. Next, the experimental method of rearrangement effect will be described. 0.17M, pH 3,7 acetate buffer Q, / 1M
CaC1, aqueous solution o, osrrte, phospholipase D, enzyme solution containing SO units a, /111, /11J lipid emulsion CO, IP+) lipid, /mA ether, ioms ultrasonic emulsion of distilled water] o, tab and I
O alcohol solution (use water, ether or acetone depending on solubility) 0. /! ; Mix mA and 3C
The reaction was carried out for 1-3 hours. After the reaction is complete, add 1 mol of SO containing mmol of EDTA, 1 g of p'f, 0 Tris-HCl buffer O, Coll 6 and chloroform-methanol mixture C.
Add koni/) jlRl and mix to form the rearrangement product (
ester) was extracted. After standing still, the lower layer was separated, dried under reduced pressure, dissolved in a small amount of chloroform-methanol mixture (/:/), 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 D, which comprises culturing phospholipase D-producing bacteria belonging to the genus Nocardiopsis in a medium, and collecting phospholipase D from the culture.