JPH0488981A - Production of phospholipase d-k - Google Patents

Abstract

NEW MATERIAL:A DNA coding a phospholipase D-K originated from microorganism belonging to genus Streptomyces. USE:Production of phospholipase D-K useful for the determination of phospholipid, the improvement of the physical properties of foods, etc. PREPARATION:The objective phospholipase D-K is produced e.g. by culturing a microbial strain belonging to genus Streptomyces (e.g. Streptomyces acidomyceticus IFO 3125) in a medium, collecting the bacterial cells from the cultured product by centrifugal separation, extracting chromosome DNA from the cell by conventional process, partially decomposing the DNA by restriction enzyme treatment, collecting a fraction containing DNA fragments of 3-6kb by electrophoresis, linking to a vector, inserting into a cell of microorganism of genus Streptomyces to effect the transformation of the cell, culturing the transformant, selecting a colony having phospholipase D activity to obtain a microbial strain containing recombinant DNA consisting of a DNA coding phospholipase D-K and integrated into a vector DNA and culturing the obtained microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel method for producing phospholipase D-, which is used for quantifying phospholipids and improving physical properties of foods.

Prior Art Phospholipase D (EC3, 1, 4, 4) is an enzyme that hydrolyzes phosphatidylcholine, particularly glycerophospholipids, and catalyzes the reaction that produces phosphatidic acid and choline, and the transfer reaction of phosphatidyl groups.

Phospholipase D is found in plants such as carrots and cabbage, and in the genus Streptomyces [Journal of Biochemistry].
y ; Tokyo), vol. 78, p. 363, 1975
Year: Vol. 185, p. 79.

1979; Acrylicultural and Biological Chemistry
d Biolo-gical Chemistry),
Volume 37, page 1667, 1973;

53, p. 3083, 1989] and the genera Actinomadura and Nocardia [Agricult
Ural and Biological Chemistry
try, Vol. 48, p. 2181 (1984)].

Phospholipase D- produced by Streptomyces andmyceticus strain has a molecular weight of 54,000 and uses phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatisilyl-glycerol, lysophosphatidylcholine, etc. as a substrate and hydrates its phosphate ester. It is an enzyme that decomposes [Patent Application Hei 1-
No. 44394].

The enzyme has an optimum temperature of 50-75°C and a pH of 4.0-75°C.
It is a thermostable enzyme whose activity does not decrease even when heated for 1 hour at a temperature of 50°C in the range of 8.5, and is not activated by Ca ions or surfactants.
It is a novel phospholipase D with different properties from [
Patent Application No. 1-44394]. Furthermore, phospholipase D-, which has such properties, is useful for emulsifying foods, and eggs have been modified using this enzyme [Patent Application No. 2-124]
No. 637].

Livestock disease endotoki! Regarding phospholipase D derived from a microorganism of the genus Corynebacterium, the DNA of the enzyme
was extracted and cloned into Escherichia coli [Infection and Immiunity].
and Immunity), vol. 58, p. 131.

1990], but the production amount of this enzyme is 0.005% in the culture solution when sphingomyelin is used as a substrate.
It is extremely low at U/mg. The base sequence of the DNA of phospholipase D derived from a microorganism of the genus Corynebacterium obtained from Escherichia coli from which the DNA of the enzyme was cloned has also been reported [Journal of Bacteriology
l of Bacteriology).

Volume 172, page 1256, 1990 (hereinafter referred to as Document 1)
)].

According to Document 1, phospholipase D derived from a microorganism of the genus Corynebacterium is phospholipase D-K [Patent Application No. 1-4]
No. 4394], it has a molecular weight of 31.7 KJal and requires calcium ions to exhibit activity. Table 1 shows the base sequence (upper row) and the corresponding amino acid sequence (lower row) of phospholipase D derived from a microorganism of the genus Corynebacterium shown in Document 1.

The problem to be solved by the invention is phospholipase D derived from a microorganism of the genus Streptomyces.
The enzyme is useful in food processing, etc., but the production amount of the enzyme in Streptomyces microorganisms is small and supply is limited. In order to produce the enzyme in large quantities, it is desired to develop a production method using microorganisms that can produce larger quantities, especially microorganisms that can easily separate the enzyme into the culture solution.

Means for Solving the Problems The present invention incorporates DNA encoding phospholipase D-K derived from a microorganism of the genus Streptomyces into a vector DNA. recombinant DNA,
A microorganism containing a recombinant DNA in which DNA encoding phospholipase D-K derived from a microorganism of the genus Streptomyces is incorporated into a vector DNA, and a DNA encoding phospholipase D-K derived from a microorganism of the genus Streptomyces is incorporated into a vector DNA. A microorganism belonging to the genus Streptomyces containing the recombinant DNA incorporated into the culture medium is cultured in a nutrient medium, phospholipase D-K is produced and accumulated in the culture, and phospholipase D-K is collected from the culture. The present invention relates to a method for producing phospholipase D-.

The present invention will be explained in detail below.

1. Extraction of DNA encoding phospholipase D-K derived from Streptomyces microorganism and the DNA
Production of recombinant DNA incorporating into vector DNA: Chromosomal DNA containing the gene for phospholipase D from a microorganism belonging to the genus Streptomyces is isolated by a conventional method. (Current
Topics in Microbiology and
Immunology), Vol. 95, 1982 (hereinafter referred to as Document 2), p. 72.

Any microorganism capable of producing phospholipase D- can be used as the microorganism that donates the chromosomal DNA, and microorganisms of the genus Strebtomyces are preferred.

Next, the chromosomal DNA is integrated according to a conventional method, for example, by cutting the chromosomal DNA and the vector DNA with restriction enzymes to prepare fragments of both DNAs, and then dividing the mixture of the two into DNA fragments.
This can be done by treating with NA ligase [Molecular cloning].
Cloning).

Edited by T. Maniatis et al., Cold Spring Harbor Laboratory (Co.
lcl Spring )Iarbor Labora
Tory), 1982 (hereinafter referred to as Document 3)]
.

As the vector DNA used here, any vector can be used as long as it can express the incorporated DNA in a microorganism belonging to the genus Streptomyces. Preferably, a plasmid capable of using Streptomyces lividans as a host is used, particularly pENlol (Japanese Patent Application Laid-open No. 1-304885).

It is preferable to use plJ'702 (Reference 1, page 72).

In addition, as restriction enzymes used for cutting DNA, for example, commercially available (7) Bgβ■, BamHI, PstlSau3A
Examples include I. The cleavage site by 5au3AI is Bg
Since a protruding end having the same structure as the cleavage site with β■ and BamHI is generated, the DNA fragment obtained by limited digestion of chromosomal DNA with 5au3AI and Bgβ■ or Ba
Vector DNA fragments cut with mHI can be easily ligated. Commercially available T4 is used as DNA ligase.
T4 DNA ligase derived from phage-infected E. coli is used.

An example of the recombinant DNA thus obtained is plasmid pPLD1.

2. Introduction of recombinant DNA into a Streptomyces microorganism and production of phospholino(-zeD-) by the microorganism: The recombinant DNA is transformed into Streptomyces by a conventional method, for example, the transformation method described on page 2.78 of the literature. introduced into microorganisms of the genus Myces [JP-A-2-131577].

The microorganism containing the recombinant DNA is selected from colonies of transformed microorganisms of the genus Streptomyces, for example, by the following screening method.

Screening method: The transformed strain of the genus Streptomyces regenerated on the regeneration medium is inoculated onto a suitable nutrient agar medium containing 10 jtg/- of thiopeptone, and cultured at 30°C for 2 days. When colonies have grown, an egg yolk agar medium (10% egg yolk, 0.8% agar) kept at 42° C. is layered. Colonies of strains containing recombinant DNA are selected because the activity of phospholipase D splits the yolk and forms a clear zone around it. The fact that the activity of the phospholipase D- obtained in this screening is due to the genetic information derived from the chromosome of the producing strain in the phospholipase D- belonging to the genus Streptomyces linked to the plasmid vector is that the plasmid DNA extracted from the strain is When Streptomyces lividans was transformed again using the transfectant, all of the transformants obtained were confirmed to be productive of phospholipase D-.

Examples of microorganisms containing recombinant DNA obtained in this manner include, for example, Streptomyces lividans P.
LDI stock <Feikoken Joyori No. 2936 May 25, 1990
day) can be given.

By liquid culturing the thus obtained microorganisms such as PLDI in a nutrient medium, a significant amount of phospholipase D-K is accumulated in the culture supernatant.

As the culture medium, either a synthetic medium containing a carbon source, a nitrogen source, and an inorganic substance, or a natural medium can be used.

As the carbon source, various carbohydrates such as glucose, starch, starch hydrolyzate, glycerol, and molasses are used, and the amount used is preferably about 5 to TOg/l. In addition, as a nitrogen source, for example, ammonium sulfate,
Nitrogen-containing organic substances such as ammonium phosphate, ammonium carbonate, ammonium acetate, peptone, yeast extract, corn steep liquor, casein hydrolyzate, meat extract, etc. are used, and the amount used is preferably about 5 to 20 g/f. . Further, as inorganic substances, for example, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, magnesium chloride, etc. are used, and the amount used is 0.0
About 5 g/β to 5 g/A is preferable.

Phospholipase D- can be obtained by purification from culture supernatant.

Purification of phospholipase D- is carried out by removing the culture supernatant from the culture solution by a method such as centrifugation, using ammonium sulfate salting out method, organic solvent precipitation method, chromatography using an ion exchange resin, gel filtration, This is carried out using conventional methods such as affinity chromatography.

3. Determination of the base sequence of DNA encoding phospholipase D-K: Determination of the base sequence of DNA encoding phospholipase D-K was performed using Streptomyces spp. containing recombinant DNA.
After cloning a 2 to 3 kb DNA fragment excised from the insertion plasmid present in the lividans using BamHI into the BamHI cleavage site of an E. coli plasmid, various deletions from both ends of the DNA fragment to be determined were determined. Plasmids are created and transformants carrying these plasmids are infected with Hertz (-phage) to convert double-stranded DNA into single-stranded phage to prepare single-stranded DNA (hereinafter referred to as 5sDNA). , the 5sDNA
This is done by determining the nucleotide sequence of according to a modification of the guideeoxy method.

The present invention will be specifically described below with reference to Examples.

Example t, Cloning of the gene in phospholipase D- (1) Chromosome D containing the gene in phospholipase D-
Preparation of NA Streptomyces acidomyceticus IF031
25 strains were placed in SK#2 medium [Stabilose K 20g/j
! , peptone 5g/β, meat extract 3g/A, KH
2P0.0.2g/I, MgCL 0.6g/l (
[pH 7,6)] and cultured with shaking at 30°C for 3 days. The obtained culture was transferred to a refrigerated centrifuge (SCR18B
; manufactured by Hitachi Koki) for 10 minutes at 4° C. and 11,000 rpm to collect bacteria. After removing the supernatant, 10
．． After washing with a 3% sucrose solution, cells were collected by centrifugation again. Chromosomal DNA was extracted from the bacterial cells according to the method described in Reference 2, page 72, and about 1 inch of chromosomal DNA was extracted.
I got it.

(2) Introduction of chromosomal DNA fragment into vector DNA (1
) Chromosomal DNA10■ obtained in
t 10mM, NaC 150mM, dithiothreitol (hereinafter referred to as DTT > 1mM) 5[10mM] dissolved in 10mM. Add restriction endonuclease L/7-5 to this DNA solution.
10 units of au3AI (manufactured by Zenshuzo Co., Ltd.) were added, and 37
Partial decomposition was carried out for 30 minutes at °C.

The partially degraded DNA was subjected to 10-40% sugar density gradient centrifugation according to the method described in Reference 3. Centrifugation (SCR18B; manufactured by Hitachi Koki) at 20°C, 2
The centrifugation was performed at 600 rpm for 16 hours, and fractionation was performed after centrifugation.

The size of DNA fragments contained in each fraction was measured by agarose gel electrophoresis according to the method described in Reference 3. 3~5k
Only the fractions containing the DNA fragment of b were collected and subjected to ethanol precipitation, and then the ethanol was removed to obtain 4J1 g of the remaining precipitate (DNA fragment). The obtained precipitate was added to a ligation buffer [Tris-HCl buffer (pH 7,6) 66mM.

MgClx 5mM 5DTT 5mM, AT'P
1mM) and stored at 60ρ.

pENlol was used as the vector, and 5 g of it was added to 1 ml of M buffer.
It was dissolved in 50ρ. Restriction endonuclease B
LM
) IJ soot-acid buffer (pH 8,6) 20I11 and 2 units of calf small intestine alkaline phosphatase (manufactured by Boehringer Mannheim) were added, and a dephosphorylation reaction was carried out at 37°C for 1 hour. After the reaction solution was heated at 65° C. for 10 minutes to completely inactivate the enzyme, the precipitate collected by ethanol precipitation was dissolved in ligation buffer 40°C.

Solution 16I in which chromosomal DNA was partially degraded with 5au3Ar
Bgj jJt and pENlol! Mix 5 ml of the partially decomposed solution with If, and make up the total volume to 75 ml with ligation buffer.
After that, 2 units of T4 DNA ligase (manufactured by Boehringer Mannheim) were added, and ligation reaction was carried out at 16° C. for 16 hours to obtain recombinant DNA.

(3) Streptomyces lividans T using recombinant DNA containing DNA encoding phospholipase D-K
Transformation of the K23 strain The recombinant DNA containing the DNA encoding phospholipase D-K was used to transform the Streptomyces lividans TK23 strain.

Protoplasts of Streptomyces lividans TK23 strain were prepared according to the method described on page 2.78 of the literature.

10 g of the recombinant DNA preparation obtained in (2), P buffer [sucrose 10.3%, N-tris(hydroquinemethyl)methyl-2-aminoethanesulfonic acid (hereinafter referred to as TMS)]
pH 7,2) (side lid) 25mM.

MgC1210mM, CaC12 (side lid) 25+++
M.

K H2P O4 (side lid) 0.005%, ZTIC
1280j'g/l, Fe C13・6 R2
0200g/l.

CuCl2・2H201hg/I, MnCl2-4H2
0Log/l, Na2B<Oi'1OH2010■/
], (NH4) 6M07024・4H2010
100 m (approximately 10"/-) of Streptomyces lividans strain TK23 protoplasts suspended in 440 g of T buffer (25% polyethylene glycol 1000 and A solution dissolved in P buffer) was added and mixed. P buffer was added to the protoplast solution and the total volume was diluted to 10 μm, and then R5 regenerated agar medium [sucrose 103 g/l, glucose 10 g/l, East Extract (
Difco) 5g/I, casamino acid (Difco)
co) 0.1g/l, 2S○40.25g/l,
MgC1z・6H2010, 1g/l.

CaC12・2Ha○ 2.94g/j! (lateral lid).

KH2PO40.05g/l (side lid), Na
NO30, 3g/l (side lid), sodium glutamate 0.03g/l (side lid), NaOH 3g/l.

TES 5.73g/l, ZnC1, 80g/].

FeCl3” 6H20200/l1g/l, CuC1
a'2H2010■/I, MnC1,・4H201
0ttg/l, Na2B40t・10H2010ttg
/l.

(NH4)sMOtOa4' 4H2010,q/1° Bacto Agar (manufactured by Difco) 22 g/I Cp
H1,2)) was coated at 100n/plate, and the medium was cultured at 30°C for 16 hours. The medium coated with the mixed solution of protoplasts and recombinant DNA was coated with a thiopeptin-containing soft agar overlay medium kept at 42°C [regular bouillon medium (manufactured by Difco) 8 g/j! .

Bact agar (manufactured by Difco) 5g/I1. Thiopebutin 200μ/1] per plate, 2.5rnI
! After layering, culture was performed at 30°C for 4 days.

When the recombinant DNA was introduced into protoplasts, they became resistant to thiopebutin, and about 30,000 such transformed strains were obtained.

(4) Selection of transformants containing a recombinant DNA containing DNA encoding phospholipase D-K A metal sterilized transformant of Streptomyces lividans TK23 obtained by the method shown in (3) GPL medium containing thiovabutin 10 g/l [glycerol 20 g/l, peptone 20 g/j! , crude lecithin 10 g/j', KNO30.5
g/j! , KH2P0゜0.5 g/1. M
g 304.7H200, 5g/f.

Fe50<7H202Xl0-5g/1, Mn
C1a ・4H202xlO-"g/l, Zn5O,-
78,02xlO-5g/J, agar 20g/L (p
H7,2):] and cultured at 30°C for 2 days. When colonies have grown, for screening, layer 10 ml of egg yolk agar medium (10% egg yolk, 0.8% agar) kept at 42°C per plate, and
It was kept warm at ℃ for about 15 hours. Five colonies in which the egg yolk was decomposed and a clear zone was formed around them were selected, and the transformed strains were treated with thiopeptin at 10 Jtg/mi! SK containing
The cells were inoculated into a large test tube containing 10 - of #2 medium, and cultured with shaking at 30°C for 2 days. This culture solution was transferred to a refrigerated centrifuge (S
CR18B (manufactured by Hitachi Koki) at 4°C 10,000
Centrifugation was performed at rpm for 10 minutes to obtain a culture supernatant. The phospholipase D activity in the culture supernatant was determined using Japanese Patent Application No. 1-44394.
A transformed strain (PLDI strain, Kaikoken Joyori No. 2936, May 25, 1990) that strongly expressed phospholipase D activity was obtained.

From the transformed strain PLDI strain obtained here, reference 2.78
Plasmid pPLD1 according to the method described on page
was extracted. Using plasmid pPLDl, Streptomyces was transformed again according to the transformation method shown in (3).
Lividans TK'23 strain was transformed. When the phospholipase productivity of the transformed strain that appeared was measured according to the above-mentioned screening method, the measured transformed strain showed productivity for phospholipase D-.

Plasmid pPLD contained in this transformed strain PLDI strain
The restriction enzyme cleavage map of No. 1 is shown in FIG.

(5) Determination of DNA base sequence for phospholipase D- DNA10 of plasmid (pPLDl) in PLDI strain
x M buffer (NaCl! 50mM.

Tris-HCl buffer (pH 7,5) 10mM.

MgCJ! 210mM, DTT 1rnM]301I
! 1 and cut with 30 units of BamHI (manufactured by Boehringer Mannheim), and 3 2.3 kb DNA fragments were recovered according to the method described in Reference 3, pp. 150-163.

Escherichia coli plasmid pUc119 [purchased from Takara Shuzo Co., Ltd.] 2■ was dissolved in M buffer 20%, BamHI 10%
After cutting into units, phenol treatment and ethanol precipitation were performed. The obtained precipitate 2■ was added to Tris-HCl buffer (pH
8,6) After dissolving in 200mM and dephosphorylating the 5' end using 1 unit of calf thymus alkaline phosphatase (manufactured by Boehringer Mannheim),
The mixture was heated at 65° C. for 15 minutes, and further subjected to phenol treatment and ethanol precipitation. A 2 to 3 kb fragment excised from pPLDl with BamHI and pUc1191 which had been cut with BamHI and dephosphorylated were dissolved in 20 ρ of ligation buffer and mixed, and T 4 DNA was added.
'Ligation was performed using 1 unit of J gauze.

When E. coli strain MV1184 [purchased from Takara Shuzo Co., Ltd.] was transformed with this DNA preparation according to the method described in Reference 3, pp. 249-251, a 2 to 3 kb BamHI gene containing the gene for phospholipase D- in both directions was transformed. The cut piece is p
Plasmid clones pPL Dseq l and pPL inserted into the BamHI cleavage site of Ucl, 19
Dseq 7 was obtained.

From these plasmids [Method in Enzymology]
A deletion plasmid was constructed according to the method described in [Vol. 155, p. 156, 1987].

Using the deletion plasmids, E. coli MV1184 strain was transformed according to the method described in Reference 3, p. 249, to produce transformants having deletion plasmids of various sizes.

From these transformed strains, [Method in Enz
ymology vol. 153, p. 3, 1987 and the same,
101, p. 20, 1978], 5sDNA 2■ was prepared.

The base sequence of the 5sDNA fragment was determined using 5equanas.
Modification of the dioxin method using e■ (Toyobo-Ll, S, manufactured by Biochemica 1) [Procedure of the National Academy of Sciences,
Nee Guess Nee (Proc, Natl, ^ca
d5ciII, S, A,), vol. 84, 4767
Page, 1987].

The obtained Streptomyces-derived phospholipase D-
The nucleotide sequence (upper row) and the corresponding amino acid sequence (lower row) are shown in Table 2.

According to Table 2, among the determined DNA base sequences,
Phospholipase D- protein was converted to phospholipase D using the Endoman method
, 5cand, ), vol. 227, p. 283.

1 from the N-terminal side when analyzed by [1950]
0 amino acid sequences (Asp Ser Pro Thr
Pr.

There was a base sequence showing His Leu Asp Ala Ala), and the results of partial protein analysis and base partial analysis matched.

Furthermore, when the molecular weight is calculated from the amino acid sequence of phospholipase D- deduced from the base sequence, it is 53,208, and a purified single specimen of the enzyme is subjected to electrophoresis [sodium dodecyl sulfate polyacrylamide gel (hereinafter referred to as 5DS- Molecular weight measured by PAGE)
4 Kdal, the molecular weight of phospholipase D derived from Corynebacterium microorganisms is 31.7 Kda.
It was very different from l. Furthermore, the amino acid sequence of phospholipase D- and the amino acid sequence of phospholipase D derived from a microorganism of the genus Corynebacterium [Journal of
Bacteriology, vol. 172, p. 1256, 1990].

Example 28 Fermentative production of phospholipase D- by a microorganism containing recombinant DNA (1) Cultivation of Streptomyces lividans transformed strain PLD1 strain containing a recombinant plasmid containing the gene for phospholipase D- Example 1 The Streptomyces lividans transformed strain PLDI strain obtained was inoculated into a large test tube containing 10 - of SK#2 medium containing 10/- of thiobeptin, and cultured with shaking at 30°C for 2 days. . Add this to thiopeptin 10u/rnl! SK#2 medium containing 50
Inoculate a 21-type Erlenmeyer flask with a double-sided tube containing 0-,
Shaking culture was performed at 30°C for 16 hours. The culture solution thus obtained was centrifuged at 4°C and 8000 rpm for 20 minutes using a refrigerated centrifuge (SCR18B; manufactured by Hitachi Koki).
Culture supernatant 400ml excluding bacterial cells! I got it.

When the activity of phospholipase D in the obtained cultured Tsuchiura was measured according to the method described in Japanese Patent Application No. 144394, it showed a maximum titer of 15 U/mf. This titer was determined from Streptomyces acidomyceticus IF, which is the gene donor strain for phospholipase D- used for transformation.
It was 15 times higher than the maximum titer of 10/- for the 03125 strain.

(2) Purification of recombinant phospholipase D-120 ammonium sulfate was added to 400-40% of the culture supernatant obtained in this example.
g was gradually added, stirred, and left for 1 hour. The resulting precipitate was centrifuged at 4°C at 8°C using a refrigerated centrifuge (SCR18B; manufactured by Hitachi Koki).
It was removed by centrifugation at 000 rpm for 20 minutes.

120 g of ammonium sulfate was added to the obtained supernatant, stirred, allowed to stand for 1 hour, and then centrifuged under the same conditions as above to obtain a precipitate. The obtained precipitate was dissolved in 20 ml of 50 mM) lis-maleic acid buffer (pH 8,0), and desalted by dialysis. The yield of the ammonium sulfate fraction was about 90%.

Add the elution fraction to 50mM) IJS hydrochloric acid buffer (pH 7.5)
Equilibrated DEAE-3epharose
It was passed through a CL6B column (approximately 100 me). Add another 5QmM) to the column! When washed with JS hydrochloric acid buffer, both components containing phospholipase D-K were eluted and collected.

The yield of phospholipase D- at this stage was 90%.

When a part of the elution fraction was subjected to 5DS-PAGE electrophoresis, a single band was observed at the position corresponding to phospholipase D-K.

Effects of the Invention According to the present invention, a recombinant DNA in which a DNA encoding phospholipase D-K derived from the genus Streptomyces is incorporated into a vector DNA and a microorganism containing the recombinant DNA are obtained, and these It can be used for mass production of phospholipase D- derived from Myces.

[Brief explanation of drawings]

FIG. 1 shows a restriction enzyme cleavage map of plasmid pPLD1 containing the gene for phospholipase D-. Figure 1

Claims (4)

[Claims] (1) DNA encoding phospholipase D-K derived from a microorganism of the genus Streptomyces. (2) A recombinant DNA in which a DNA encoding phospholipase D-K derived from a microorganism of the genus Streptomyces is incorporated into a vector DNA. (3) A microorganism containing a recombinant DNA in which DNA encoding phospholipase D-K derived from a Streptomyces microorganism is incorporated into a vector DNA. (4) A microorganism belonging to the genus Streptomyces containing a recombinant DNA in which DNA encoding phospholipase D-K derived from a microorganism of the genus Streptomyces has been incorporated into a vector DNA is cultured in a nutrient medium, and a A method for producing phospholipase D-K, which comprises producing and accumulating phospholipase D-K and collecting phospholipase D-K from the culture.