JP3006856B2 - Method for producing phospholipase DK - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel method for producing phospholipase DK used for quantification of phospholipids, improvement of food properties, and the like.

2. Prior Art Phospholipase D (EC 3.1.4.4) is an enzyme that hydrolyzes phosphatidylcholine, particularly glycerophospholipid, and catalyzes a reaction to generate phosphatidic acid and choline and a transfer reaction of a phosphatidyl group.

Phospholipase D is used for plants such as carrots and cabbage;
Streptomyces [Journal of Biochemistry; Tokyo, 78, 363]
85, 79, 1979; Agricultural and Biological Chemistry (Agricultur)
al and Biological Chemistry), 37, 1667, 1973;
Ibid., 53, p. 3083, 1989) and the genera Actinomadura and Nocardia (Agricultural and Biological
Chemistry, 48, 2181 (1984)].

Phospholipase DK produced by Streptomyces acidumyticus strain has a molecular weight of 54,000 and hydrolyzes its phosphate ester using phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidyl-glycerol, lysophosphatidylcholine or the like as a substrate. Enzyme [Japanese Patent Application No. 1-44
394].

The enzyme is a thermostable enzyme whose optimal temperature is as high as 50 to 75 ° C and its activity does not decrease even when heated at a temperature of 50 ° C for 1 hour in a pH range of 4.0 to 8.5, and it is activated by Ca ions or surfactants. This is a novel phospholipase D having properties different from the above-mentioned phospholipase D, for example, it is not converted to a phospholipase D (Japanese Patent Application No. 1-4439).
No. 4]. In addition, phospholipase DK having such properties
Is useful for emulsifying food, and eggs are modified using the enzyme [Japanese Patent Application No. 2-124637].

Regarding phospholipase D derived from a microorganism of the genus Corynebacterium, which is an endotoxin for livestock diseases, the DNA of the enzyme
Was extracted and cloned into E. coli [Infection and Immunity
ty), vol. 58, p. 131, 1990], but the production of the enzyme is as low as 0.005 U / ml in the culture broth when sphingomyelin is used as a substrate. The nucleotide sequence of the DNA of phospholipase D derived from Corynebacterium microorganism obtained from Escherichia coli in which the DNA of the enzyme has been cloned has also been reported [Journal of Bacteriology (Journal of B.
acteriology), vol. 172, p. 1256, 1990 (hereinafter referred to as reference 1)].

According to Document 1, phospholipase D derived from a microorganism of the genus Corynebacterium is phospholipase DK [Japanese Patent Application No.
No. 44394], and requires a calcium ion for expression of activity at a molecular weight of 31.7 Kdal. Base sequence of phospholipase D derived from Corynebacterium genus microorganism shown in Reference 1 (upper row) and its corresponding amino acid sequence (lower row)
Are shown in Table 1.

PROBLEM TO BE SOLVED BY THE INVENTION Phospholipase D derived from Streptomyces microorganism
Although -K is useful for food processing and the like, the production amount of the enzyme in a microorganism of the genus Streptomyces is small and the supply is limited. In order to produce the enzyme in large quantities, it is desired to develop a production method using a microorganism having a higher production amount, particularly a microorganism capable of easily secreting the enzyme into a culture solution.

Means for Solving the Problems The present invention relates to a DAN encoding phospholipase DK derived from a Streptomyces genus microorganism, and a set in which a DNA encoding a phospholipase DK derived from a Streptomyces genus microorganism is incorporated into a vector DNA. A recombinant DNA, a microorganism containing a recombinant DNA obtained by incorporating a DNA encoding a phospholipase DK derived from a microorganism of the genus Streptomyces into a vector DNA, and a DNA encoding a phospholipase DK derived from a microorganism belonging to the genus Streptomyces. DNA
A microorganism belonging to the genus Streptomyces containing the recombinant DNA incorporated into the medium is cultured in a nutrient medium, phospholipase DK is produced and accumulated in the culture, and phospholipase DK is collected from the culture. And a method for producing phospholipase DK.

 Hereinafter, the present invention will be described in detail.

1. Extraction of DNA encoding phospholipase DK derived from a microorganism belonging to the genus Streptomyces and structure of recombinant DNA obtained by incorporating the DNA into vector DNA: Phospholipase DK gene from a microorganism belonging to the genus Streptomyces Isolation of the chromosome DAN containing the chromosome DAN is carried out by a conventional method, for example, by using Current Topics in Microbiology and Immunology (Current Topics
in Microbiology and Immunology), vol. 96, 1982 (hereinafter referred to as reference 2), p. 72.

As the microorganism that provides chromosomal DNA, any microorganism capable of producing phospholipase DK can be used, and a Streptomyces microorganism is preferred.

Then, the chromosomal DNA can be integrated by a conventional method, for example, by cutting a chromosomal DNA and a vector DNA with a restriction enzyme to prepare a fragment of both DNAs, and then treating the mixture of the two with DNA ligase [ Molecular Cloning, edited by T. Maniatis et al., Cold Spring Harbor Laboratories
atory), 1982 (hereinafter referred to as Reference 3)].

As the vector DNA used here, any vector DNA can be used as long as it can express the DNA incorporated in a microorganism belonging to the genus Streptomyces. Preferably, a plasmid capable of using Streptomyces lividans as a host is used. In particular, pEN101 (Japanese Patent Application No. 1-304885) and pIJ702 (References 1, 7)
2) is preferably used.

Examples of the restriction enzyme used for cutting DNA include commercially available Bgl II, Bam H I, Pst I, Sau3A I, and the like. S
The au3A I cleavage site produces a protruding end with the same structure as the Bgl II and Bam HI cleavage sites,
A DNA fragment obtained by limited digestion with au3A I and a vector DNA fragment cut with Bgl II or Bam HI can be easily ligated. As the DNA ligase, a commercially available T4 ligase derived from Escherichia coli infected with T4 phage is used.

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

2. Introduction of recombinant DNA into a microorganism of the genus Streptomyces and structure of phospholipase DK by the microorganism: Recombinant DNA can be prepared by a conventional method, for example, by a transformation method described in Reference 2, p. It is introduced into microorganisms (JP-A-2-131577).

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

Screening method: The transformed strain of Streptomyces sp. Regenerated on the regeneration medium is inoculated on a suitable nutrient agar medium containing 10 μg / ml of thiopeptone, and cultured at 30 ° C. for 2 days. When the colony has grown, overlay the egg yolk agar medium (yolk 10%, agar 0.8%) kept at 42 ° C. In a colony of a strain containing the recombinant DNA, such a colony is selected because the yolk is divided by the phospholipase DK activity and a transparent zone is formed around the yolk. The fact that the phospholipase DK activity obtained by the screening depends on the chromosome-derived genetic information of the phospholipase DK producing strain belonging to the genus Streptomyces linked to the plasmid vector is based on the plasmid DNA extracted from the strain. When Streptomyces lividans is again transformed, all of the resulting transformants are phospholipase D
It is confirmed by showing -K productivity.

Examples of microorganisms containing the recombinant DNA thus obtained include, for example, Streptomyces lividans PL
The D1 strain (Jikoken No. 2936, May 25, 1990) can be mentioned.

By subjecting a microorganism such as PLD1 thus obtained to liquid culture in a nutrient medium, a considerable amount of phospholipase DK in the culture supernatant is accumulated.

As a medium used for the culture, any of 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, molasses, and the like are used, and the amount of use is preferably about 5 to 70 g / about. As the 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 and the like are used, and the use amount thereof is preferably about 5 to 20 g / g. Further, as the inorganic substance, for example, potassium hydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, magnesium chloride and the like are used, the amount of which is 0.05 g / ~
About 5 g / preferably.

Phospholipase DK can be obtained by purifying from the culture supernatant.

Phospholipase DK can be purified by removing the cells from the culture solution by centrifugation or the like, and then removing the culture supernatant by ammonium sulfate precipitation, organic solvent precipitation, chromatography using an ion exchange resin, gel permeation, affinity. This is performed using a conventional method such as chromatography.

3. Determination of nucleotide sequence of DNA encoding phospholipase DK: The nucleotide sequence of DNA encoding phospholipase DK was determined by inserting the insertion plasmid present in Streptomyces lividans containing recombinant DNA into BamH. The 2.3 kb DNA fragment excised by I was ligated with BamH
DNA to be determined after cloning into the I fragment site
Create various deletion plasmids from both ends of the fragment,
Transformants carrying these plasmids should have double-stranded DN
A single-stranded DNA (hereinafter referred to as ssDNA) was prepared by infecting a helper phage for converting A into a single-stranded phage.
It is performed by determining the nucleotide sequence of the DNA according to a modification of the dideoxy method.

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

Example 1. Cloning of phospholipase DK gene (1) Chromosomal DNA containing phospholipase DK gene
Preparation of Streptomyces acidomycesticus IFO312
5 strains were used in SK # 2 medium [Stabilose K 20g /, peptone
5g /, meat extract 3g /, KH ₂ PO ₄ 0.2g /, MgCl ₂ 0.6g
/(PH7.6)] in 30 ml with shaking at 30 ° C for 3 days. The obtained culture is cooled and centrifuged (SCR18B; manufactured by Hitachi Koki)
The cells were centrifuged at 4 ° C. and 1000 rpm for 10 minutes to collect the cells. After removing the supernatant, the cells were washed with a 10.3% sucrose solution and centrifuged again to collect the cells. Chromosomal DNA was extracted from the cells according to the method described on page 2, 72
mg of chromosomal DNA was obtained.

(2) Introduction of chromosomal DNA fragment into vector DNA 10 μg of the chromosomal DNA obtained in (1) was added to an M buffer [Tris-HCl buffer (pH 7.5) 10 mM, MgCl ₂ 10 mM, NaCl 50 mM, dithiothreitol (hereinafter referred to as “dithiothreitol”). DTT) (1 mM). To this DNA solution, 10 units of restriction endonuclease Sau3A I (Takara Shuzo) was added and partially digested at 37 ° C. for 30 minutes.

The partially digested DNA was subjected to 10-40% sucrose density gradient centrifugation according to the method described in Reference 3. Centrifugation (SCR18B; manufactured by Hitachi Koki) was performed at 20 ° C. and 26000 rpm for 16 hours, and fractionation was performed after completion of centrifugation.

The size of the DNA fragment contained in each fraction was measured by agarose gel electrophoresis according to the method of Reference 3. 3-6kb
After collecting only the fractions containing the DNA fragment and performing ethanol precipitation, 4 μl of the remaining precipitate (DNA fragment) except ethanol was removed.
g was obtained. The obtained precipitate was subjected to ligation buffer [Tris-HCl buffer (pH 7.6) 66 mM, MgCl ₂ 5 mM, DTT 5 mM, ATP
1 mM] and stored.

The vector used was pEN101, and 5 μg thereof was added to M buffer 150.
dissolved in μ. This is the restriction endonuclease Bgl
Add 20 units of II (Boehringer Mannheim) and add 37 units
After 3 hours of reaction at 37 ° C. for partial decomposition, 20 μl of 1M Tris-HCl buffer (pH 8.6) and 2 units of calf intestinal alkaline phosphatase (Boehringer Mannheim) were added, followed by dephosphorylation at 37 ° C. for 1 hour. The following reaction was performed. Reaction solution
After heating at 65 ° C. for 10 minutes to completely inactivate the enzyme, the precipitate collected by ethanol precipitation was added to the ligation buffer solution 40.
dissolved in μ.

Chromosomal DNA partially digested with Sau3A I 16μ and pEN1
5 was mixed with 5 μl of a solution obtained by partially decomposing 01 with Bgl II, and the mixture was adjusted to a total volume of 75 μl with a ligation buffer.
A ligation reaction was performed at 16 ° C. for 16 hours to obtain a recombinant DNA.

(3) Streptomyces lividans TK23 by recombinant DNA containing DNA encoding phospholipase DK
Transformation of strain Recombinant D containing DNA encoding phospholipase DK
NA was used for transformation of 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 μm of the recombinant DNA sample obtained in (2) and a P buffer [sucrose 10.3%, N-tris (hydroxymethyl) methyl-2
-Aminoethanesulfonic acid (hereinafter referred to as TMS) (pH 7.2)
(Separate steam) 25 mM, MgCl ₂ 10 mM, CaCl ₂ (Separate steam) 25 mM, KH ₂ PO
₄ (another _{steam) 0.005%, ZnCl 2 80μg /} l, FeCl 3 · 6H 2 O 200μ
_{g / l, CuCl 2 · 2H} 2 O 10μg / l, MnCl 2 · 4H 2 O 10μg / l, Na 2 B
₄ O ₇・ 10H ₂ O 10μg / l, (NH ₄ ) ₆ MO ₇ O ₂₄・ 4H ₂ O 10μg / l]
100 μl (approximately 10 ¹⁰ cells / ml) of Streptomyces lividans TK23 suspended in a suspension, and left at room temperature for 1 minute. (A solution dissolved in a buffer). After adding a P buffer to the protoplast solution to dilute the total volume to 10 ml, R5 regenerated agar medium [103 g / l sucrose, 10 g / l glucose, 5 g / l yeast extract (Difco), 5 g / l casamino acid (Difco) 0.1g / l,
K ₂ SO ₄ 0.25 g / l, MgCl ₂・ 6H ₂ O 10.1 g / l, CaCl ₂・ 2H ₂ O 2.94 g / l
(Another _{steam), KH 2 PO 4 0.05g /} l ( separate steam), NaNO ₃ 0.3 g / l (separate steam), sodium glutamate 0.03 g / l (separate steam), NaOH3g
_{/l,TES5.73g/l,ZnCl 2 80μg / l, FeCl 3} · 6H 2 O200μg / l, CuC
_{l 2 · 2H 2 O10μg / l} , MnCl 2 · 4H 2 O10μg / l, Na 2 B 4 O 7 · 10H 2 O1
0 μg / l, (NH ₄ ) ₆ MO ₇ O ₂₄・ 4H ₂ O ₁₀ μg / l, Bacto agar (D
(ifco) 22 g / (pH 7.2)] at 100 μ / plate, and the medium was cultured at 30 ° C. for 16 hours. On a medium coated with the mixed solution of the protoplast and the recombinant DNA, thiopeptin-containing soft agar overlay medium (normal broth medium (Difco) 8 g /, Bacto agar (Difco) 5 g /, thiopeptide 200 mg) kept at 42 ° C. /] Was overlaid with 2.5 ml per plate and cultured at 30 ° C. for 4 days. When recombinant DNA is introduced into protoplasts, it becomes resistant to thiopeptides.
000 shares were obtained.

(4) Selection of Transformant Containing Recombinant DNA Containing Phospholipase DK-Encoding DNA The transformant of Streptomyces lividans TK23 strain obtained by the method shown in (3) was sterilized with a metal. GPL medium containing thiopeptide 10 μg / ml [glycerol 20 g /, peptone 20 g /, crude lecithin 10
g /, KNO ₃ 0.5g /, KH ₂ PO ₄ 0.5g /, MgSO ₄・ 7H ₂ O0.5g /,
_{FeSO 4 · 7H 2 O2 × 10} -5 g /, MnCl 2 · 4H 2 O2 × 10 -5 g /, ZnSO
^{_{4 · 7H 2 O2 × 10 -5}} g /, agar 20 g /, was inoculated into (pH 7.2)] were cultured for 2 days at 30 ° C.. When the colony has grown, add 1 egg / agar medium (10% egg yolk, 0.8% agar) incubated at 24 ° C for screening.
The mixture was overlaid with 0 ml and kept at 37 ° C. for about 15 hours. Five colonies in which the yolk was decomposed to form a transparent zone around them were selected, and the transformed strain was SK containing 10 μg / ml thiopeptin.
The cells were inoculated into a large test tube containing 10 ml of # 2 medium and cultured at 30 ° C. for 2 days with shaking. This culture solution is cooled and centrifuged (SCR18
B; manufactured by Hitachi Koki Co., Ltd.), and centrifuged at 10,000 rpm at 4 ° C. for 10 minutes to obtain a culture supernatant. Phospholipase D activity in the culture supernatant was measured according to the method described in Japanese Patent Application No. 1-444394, and a transformant that strongly expresses phospholipase D activity (PLD1 strain, Microkoken Kenjiro No. 2936 Heisei May 25, 2)
I got

The plasmid pPLD1 was extracted from the thus obtained transformant strain PLD1 according to the method described on page 2, 78 of the literature. Using the plasmid pPLD1 according to the transformation method described in (3), Streptomyces lividans TK
Transformation of 23 strains was performed. The phospholipase productivity of the emerged transformants was measured according to the above-mentioned screening method, and all the measured transformants showed phospholipase DK productivity.

FIG. 1 shows a restriction map of the plasmid pPLD1 contained in the transformed strain PLD1.

(5) Determination of base sequence of phospholipase DK DNA 10 μg of plasmid (pPLD1) DNA in PLD1 strain was added to M buffer [NaCl 50 mM, Tris-HCl buffer (pH 7.5) 10 mM, MgCl ₂
[10 mM, DTT1 mM] 30 µm, and cut with 30 units of BamHI (manufactured by Boehringer Mannheim), and 3 µg of a 2.3 kb DNA fragment was recovered according to the method described on pages 3,150 to 163 of the literature.

E. coli plasmid pUC119 (purchased from Takara Shuzo) 2μ
g was dissolved in 20 μl of M buffer and cut with 10 units of BamHI, followed by phenol treatment and ethanol precipitation. 2 μg of the resulting precipitate was tris-hydrochloric acid buffer (pH 8.6) 200 mM 20 μm.
After dephosphorylation of the 5'-terminal using 1 unit of calf thymus-derived 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. pPLD1 to B
A 2.3 kb fragment excised with amHI and 1 μg of pUC119 cleaved and dephosphorylated after digestion with BamHI were each ligated to ligation buffer.
After dissolving in 20μ, they were mixed and ligated using 1 unit of T4 DNA ligase.

The DNA preparation was transformed with Escherichia coli MV1184 strain (purchased from Takara Shuzo Co., Ltd.) according to the method described on pages 3,249 to 251 of the literature, and it contained the phospholipase DK gene in both directions.
Plasmid clones pPLDseq1 and pPLDseq7 in which a 2.3 kb BamHI cleavage fragment was inserted into the BamHI cleavage site of pUC119 were obtained.

From these plasmids [Method in Enzymology, 155, 156, 1987]
Year], a deletion plasmid was prepared.

Escherichia coli MV1184 strain was transformed using the deletion plasmid according to the method described on page 3,249 of the literature to prepare transformants having deletion plasmids of various sizes.

From these transformants, [Method in Enzymology, 153
Volume 3, page 1987, and 101, page 20, 1978], 2 μg of ssDNA was prepared.

 Determination of the base sequence of the ssDNA fragment was performed using Sequanase (Toyobo
-U.S.Biochemical Co., Ltd.)
Law [Proceedings of National Academy
ー of Science, USA (Proc. Natl.
Acad.Sci.U.S.A.), 84, 4767, 1978].
Was.

Phospholipase D derived from Streptomyces obtained
Table 2 shows the base sequence of -K (upper row) and the corresponding amino acid sequence (lower row).

According to Table 2, among the determined DNA base sequences,
The phospholipase DK protein was analyzed by the Endman method [Acta. Chem. Scand., 2
27, p. 283, 1950], the 10 amino acid sequence from the N-terminal side (Asp Ser Pro Thr Pro His
Leu Asp Ala Ala), and the results of partial analysis of proteins and the results of partial analysis of bases agreed.

Further, phospholipase DK deduced from the nucleotide sequence
Calculated molecular weight from the amino acid sequence of 53208,
A purified single sample of the enzyme was subjected to electrophoresis [Sodium dodecyl sulfate polyacrylamide gel (hereinafter SD)
S-PAGE)] and a molecular weight of 54 Kdal, which is significantly different from the molecular weight of phospholipase D derived from Corynebacterium sp. 31.7 Kdal. Furthermore, there was no complementarity between the amino acid sequence of phospholipase DK 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 2 Fermentative Production of Phospholipase DK by a Microorganism Containing Recombinant DNA (1) Culture of Streptomyces lividans Transformant PLD1 Containing a Recombinant Plasmid Containing Phospholipase DK Gene The Streptomyces lividans transformant PLD1 obtained in Step 1 was inoculated into a large test tube containing 10 ml of SK # 2 medium containing 10 μg / ml of thiopeptin, and cultured with shaking at 30 ° C. for 2 days. . This is thiopeptin 10μg /
The cells were inoculated into a 21-ml baffled Erlenmeyer flask containing 500 ml of SK # 2 medium containing ml, and cultured at 30 ° C. for 16 hours with shaking. The culture solution thus obtained is cooled and centrifuged (SCR1
8B; manufactured by Hitachi Koki Co., Ltd.) and centrifuged at 8,000 rpm for 20 minutes at 4 ° C. to remove the cells and obtain 400 ml of culture supernatant.

When the phospholipase DK activity in the obtained culture supernatant was measured according to the method described in Japanese Patent Application No. 1-444394, the highest titer was 15 U / ml. This titer was 15 times higher than the highest titer of 1 U / ml in the case of the strain of Streptomyces acidomyceticas IFO3125 which is the donor strain of the phospholipase DK gene used for the transformation.

(2) Purification of recombinant phospholipase DK Ammonium sulfate 1 was added to 400 ml of the culture supernatant obtained in this example.
20 g was gradually added and stirred and left for 1 hour. The resulting precipitate was cooled at 8000 rpm at 4 ° C. using a cooling centrifuge (SCR18B; manufactured by Hitachi Koki).
And centrifuged for 20 minutes.

120 g of ammonium sulfate was added to the obtained supernatant and stirred,
After standing for 1 hour, the mixture was centrifuged under the same conditions as described above to obtain a precipitate. The resulting precipitate is washed with 50 mM Tris-maleate buffer (pH
8.0) It was dissolved in 20 ml and desalted by dialysis. The yield in the same ammonium sulfate fraction was about 90%.

The eluted fraction was passed through a DEAE-Sepharose CL6B column (about 100 ml) equilibrated with 50 mM Tris-HCl buffer (pH 7.5). When the column was further washed with 50 mM Tris-HCl buffer, a fraction containing phospholipase DK was eluted and collected.

The yield of phospholipase DK at this stage was 90%.

When a part of the eluted fraction was subjected to SDS-PAGE electrophoresis, a single band was shown at a position corresponding to phospholipase DK.

Effects of the Invention According to the present invention, a recombinant DNA obtained by incorporating DNA encoding a phospholipase DK derived from Streptomyces into a vector DNA and a microorganism containing the recombinant DNA can be obtained. It can be used for mass production of phospholipase DK derived from the genus Seth.

[Brief description of the drawings]

FIG. 1 shows a restriction map of the plasmid pPLD1 containing the phospholipase DK gene.

Claims (4)

(57) [Claims] (1) A phospholipase DK comprising an amino acid sequence represented by
DNA that encodes (2) A phospholipase DK comprising an amino acid sequence represented by
DNA that incorporates DNA encoding DNA into vector DNA
A. (3) A phospholipase DK comprising an amino acid sequence represented by
DNA that incorporates DNA encoding DNA into vector DNA
Microorganisms containing. (4) A phospholipase DK comprising an amino acid sequence represented by
DNA that incorporates DNA encoding DNA into vector DNA
A method for producing phospholipase DK, comprising: culturing a microorganism containing, in a nutrient medium, producing and accumulating phospholipase DK in the culture, and collecting phospholipase DK from the culture.