JP3121890B2 - DNA having phospholipase DP gene information and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a phospholipase DP
The present invention relates to a gene and a method for producing a recombinant enzyme phospholipase DP using the gene.

[0002]

2. Description of the Related Art Phospholipase D (Phosphatidylcholin)
e Phosphatidohydrolase is known per se. For example, it hydrolyzes an ester bond between phosphoric acid and a choline residue of lecithin (phosphatidylcholine), which is a kind of phospholipid, to release phosphatidic acid and choline. Plant tissues such as cabbage, spinach, turnip, potato, lettuce leaves, carrots, sugar beet, beet roots, barley malt, cottonseed, mung bean sprouts and peas, etc. (Enzyme Handbook No. 451- Microorganisms such as Streptomyces (Japanese Patent Publication No. 52-39918) and Nocardinapsis genus (Japanese Patent Publication No. 63-62195) are known to exist. Also, there has been a report on the purification of phospholipase D extracted from a culture solution of Streptomyces chromofuscus (Journal Biochemistry. 85 , 79-95 (197)
9)). On the other hand, Streptomyces sp. (Streptom
yces sp.) AA586 strain (FERM BP-3578)
A novel phospholipase D, phospholipase D-
It is known that P is contained (Japanese Unexamined Patent Publication No.
No. 152481). This phospholipase DP
Is an enzyme characterized by a weak effect on lysolecithin and little effect on sphingomyelin. Phospholipase D is known to be an enzyme that is used not only as a reagent for measuring phospholipids in blood, but also for the production of derivatives by base exchange reaction utilizing hydrolysis of phospholipids and reversible reaction of phospholipases ( JP-A-63-36790 and JP-A-61-236
No. 793).

[0003]

As described above, various phospholipase D-producing bacteria have been reported. In addition, the present inventors have previously described Streptomyces kuromovsukasu ( Streptom
yces chromofuscus ), and the nucleotide sequence and amino acid sequence of the gene were determined (JP-A-3-187382). As described later, the amino acid sequence of the phospholipase D-P of the present invention and the amino acid sequence of the same were determined. It did not show any homology with the nucleotide sequence of the DNA encoding the amino acid sequence of the enzyme (hereinafter sometimes referred to as phospholipase DP gene). As described above, the specificities of the phospholipase DP for the substrate by the various producing bacteria are different from each other, and it was not possible to predict at all what the amino acid sequence of the phospholipase DP by the producing bacteria was. Under these circumstances, the production efficiency of phospholipase DP, which is obtained by extracting and purifying from a culture solution of a microorganism belonging to the genus Streptomyces, is low, so that efficient and high-purity and high-quality phospholipase D are obtained.
A -P production method was desired. In addition, phospholipase D-
The primary structure of the polypeptide constituting P and the nucleotide sequence of DNA encoding the amino acid sequence of the enzyme have not been published yet. Accordingly, it has been desired to determine the primary structure of the polypeptide constituting phospholipase DP, determine the base sequence of DNA encoding the amino acid sequence of the enzyme, and produce the enzyme by genetic engineering.

[0004]

Means and Action for Solving the Problems The present inventors have made intensive studies on the above problems, and have conducted many trial and errors. As a result, by using oligonucleotide 3 described below, fortunately, phospholipase D -P gene was successfully caught, its base sequence was determined, and Streptomyces lividans could be transformed with the recombinant plasmid incorporating this DNA to produce phospholipase DP. The present invention has been completed by obtaining a novel microorganism. That is, the present invention is characterized by being a microorganism belonging to the genus Streptomyces transformed by a recombinant plasmid having a DNA that expresses phospholipase DP having a specific amino acid sequence. It is a substantially pure microorganism that produces phospholipase DP. Further, the present invention provides a method for culturing a substantially pure microorganism producing phospholipase DP, which is a transformed microorganism belonging to the genus Streptomyces, in a medium, and then collecting the phospholipase DP from the microorganism. A method for producing phospholipase DP.

[0005] The phospholipase DP-expressing DNA used for producing the transformed microorganism belonging to the genus Streptomyces according to the present invention can be obtained, for example, from chromosomal DNA of various microorganisms producing phospholipase DP. , 5'-CGTANCCCGAAGTCTG
The oligonucleotide 3 represented by NA-5 'can be appropriately collected by screening using the oligonucleotide 3 as a probe. More specifically, for example, first, chromosomal DNA of a microorganism that produces phospholipase DP is separated and purified, and then purified and purified from BamHI, E.
Cleavage is performed using an appropriate restriction enzyme such as coRI and KpnI, and the resultant is ligated to a cloning vector to prepare a gene library. On the other hand, an oligonucleotide 3 probe prepared by labeling with an isotope or a non-radioactive compound is prepared. Next, by using this oligonucleotide 3 probe, a gene DNA expressing phospholipase DP is picked up from the gene library to screen a gene DNA of interest.
In the present invention, the DN of the phospholipase DP is
As a donor microorganism for A, Streptomyces sp. AA586 strain (FERM BP-
3578) is preferably used. When the present strain is used, the bacteriological properties of the strain, culture means and purification means are described in Japanese Patent Publication No. 1-124774 (page 2, column 3, line 30 to page 3, column 6, column 32). Line) should be referred to.

[0006] This Streptomyces sp. AA
A specific example of a method for screening a gene DNA expressing the enzyme from the 586 gene library will be described. First, 100 to 20 chromosomal DNAs of the microorganism
About 00 μg is extracted by a commonly used method. Thereafter, about 1 to 10 μg of the restriction enzyme KpnI was used.
And ligated, for example, to the KpnI cleavage site of the cloning vector plasmid pUC118. Next, this recombinant DNA is introduced into a host microorganism, and the chromosomal DNA is cloned to prepare a chromosomal DNA library consisting of 10 ⁴ to 10 ⁵ clones. The host microorganism used at this time may be any microorganism as long as the recombinant DNA is stable and can grow autonomously. Usually, Escherichia coli and the like are preferable, and Escherichia coli DH1, Escherichia
E. coli HB101, Escherichia coli W3110, Escherichia coli C600 and the like can be used. Examples of plasmid vectors other than the plasmid pUC118 include plasmids pBR322 and pBR
325, pACYC184, pUC12, pUC18,
pUC19 and pIN I can be used.

[0007] As a method of transferring the recombinant DNA to the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, the recombinant DNA may be transferred in the presence of calcium ions. The choice of whether or not the target recombinant DNA has been introduced into the host microorganism is determined by selecting the recombinant DNA.
May be selected by a selective culture method using a selective medium based on the drug resistance marker of the vector constituting Next, a partial amino acid sequence is determined, and based on this, various oligonucleotides having a base length of about 15 to 30, for example, oligonucleotide 3 are synthesized, and then, for example, labeled with an isotope to prepare a radioactive oligonucleotide probe. By using this oligonucleotide probe and selecting by the colony hybridization method, it can be confirmed whether or not the target gene DNA is contained.

Next, from the transformed host microorganism containing the gene DNA of interest, for example, the method of T. Maniatis et al. (Maniitiatis, T. et.
al. Molecular Cloning, AL
laboratory Manual, cold Spr
ing Harbor Laboratory 86-
94, 1982), and phospholipase DP.
Can be prepared, for example, a plasmid DP135a can be obtained. FIG. 10 is a schematic diagram showing the structure of this plasmid. Next, as described above, an expression vector incorporating DNA expressing phospholipase DP is constructed from the gene library. As this expression vector, a vector constructed for gene recombination from a plasmid capable of autonomous propagation in a host microorganism is suitable. As a plasmid vector, for example, when Escherichia coli is used as a host microorganism, the plasmid vector is used. When Streptomyces lividans is used as a host microorganism, plasmid pSEV1 [Molacu is used.
1ar & General Genetics (19
87) 210: 468-475 (FERM BP-26
48), pIJ680, pIJ702 and the like can be used, respectively. Furthermore, a shuttle vector capable of autonomous propagation in two or more types of host microorganisms such as Escherichia coli and Streptomyces lividans can also be used.

Preferably, the phospholipase D-
Prior to incorporating the P gene DNA into the plasmid, an expression promoter was incorporated upstream of the gene,
The promoter and the phospholipase DP gene DNA may be simultaneously incorporated into a plasmid. As an expression promoter, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, a promoter of the β-galactosidase gene of Escherichia coli can be used, and when the host microorganism is a microorganism belonging to the genus Streptomyces, The promoter derived from Streptomyces griseus incorporated in the plasmid pSEV1 can be used. The method for incorporating the phospholipase DP gene DNA and promoter into a vector is not particularly limited, and a conventionally used method can be used. For example, using an appropriate restriction enzyme, the recombinant plasmid containing the phospholipase DP gene DNA or the expression vector is treated to obtain a phospholipase DP gene DNA fragment and a vector, respectively.
After annealing each adhesive end, the appropriate DN
An expression vector is obtained by ligation using A ligase.

[0010] The thus constructed expression vector is inserted into a microorganism belonging to Escherichia coli or Streptomyces lividans and, when the host microorganism is transformed, a substantially pure protein producing phospholipase DP is obtained. Microorganisms can be obtained. There is no particular limitation on the method for introducing the expression vector.For example, when the host microorganism is a microorganism belonging to the genus Escherichia, the expression may be carried out in the presence of calcium ions, or by using the competent cell method. Good. When the host microorganism is a microorganism belonging to the genus Streptomyces, a competent cell method or an electroporation method can be employed. Selection of the presence or absence of the introduction of the expression vector into the host microorganism may be performed by selecting a microorganism that grows by culturing the microorganism in a selective medium based on a drug resistance marker of the expression vector. Specific examples of the transformants include the actinomycetes promoter derived from the plasmid pSEV1 and FIG .
The DNA linked to the phospholipase DP gene DNA shown continuously up to 7 is incorporated into plasmid pIJ680, and the host microorganism Streptomyces lividans T
K (FERM BP-2685) is transformed and Streptomyces lividans TK • DP obtained by selecting a microorganism that produces phospholipase DP.
108 (FERM BP-3577). FIG. 12 is a schematic diagram showing the structure of this plasmid. The chromosomal DNA of Streptomyces sp. AA586 is shown in FIG.
FIG. 8 shows a restriction enzyme map of the origin site. A schematic of the above construction method is illustrated in Figure 11.

[0011] DNA expressing phospholipase D-P in the present invention, specifically, Ru DNA der having a nucleotide sequence encoding the amino acid sequence represented by continuously from the N-terminal side in FIGS. 1 and 2 . The N-terminal side and the C-terminal side of the amino acid sequence continuously shown in FIG. 1 and FIG. 2 may include an amino acid residue or a polypeptide residue. It may have one or more amino acid residues, and examples of the amino acid residues or polypeptide residues include amino acid residues such as Met corresponding to the initiation codon, and signal-like peptides. Ala on the C-terminal side
Downstream may further have one or more amino acid residues. The DNA having a base sequence encoding the amino acid sequence continuously shown in FIGS. 1 and 2 of the present invention is:
A D consisting of any one of a series of codons for each amino acid in the amino acid sequence including the N-terminal and C-terminal amino acid residues or polypeptide residues
NA is sufficient .

A representative example of the DNA is a DNA having the base sequence shown in FIG. 3 from the 5 'end. The DNA may have one or more codons encoding amino acids upstream of the 5 ′ end,
Any codon other than A, TAG and TGA may be used. Further, preferably, ATG, GTG, and those having a start codon other than these or a codon corresponding to a signal peptide can be mentioned. Downstream of the 3′-terminal GTG, there may be one or more amino acid-encoding codons or a translation termination codon, and further, a 3′-terminal codon encoding an amino acid. When it has one or more, it is preferable to have a translation termination codon at the 3 'end of the codon encoding this amino acid. More preferably, the upstream side of the 5 'terminus of the nucleotide sequence represented by Figure 3, the amino acid sequence And a DNA having a base sequence encoding a signal-like peptide represented by the following formula. The DNA can efficiently secrete phospholipase outside the cells.

[0013] The DNA encoding the amino acid sequence of the signal-like peptide is The base sequence represented by is particularly preferable. In addition, as a preferable base sequence including the base sequence portion encoding the signal-like peptide and the ribosome binding site, a base sequence represented continuously from the 5 ′ end to FIGS. 4 to 7 can be used. Can be mentioned. The nucleotide sequence of the DNA encoding the amino acid sequence of the polypeptide constituting phospholipase DP obtained by the above method was determined by the dideoxy method (Science 214 1205-
1210 (1981)), and the entire amino acid sequence of the polypeptide constituting phospholipase DP is:
Predicted from the nucleotide sequence. The N-terminal amino acid sequence of one of the amino acid sequences whose N-terminal amino acid sequence was predicted and determined by a liquid-phase protein sequencer (BECKMAN System 890 ME) using the phospholipase DP polypeptide cultured and purified by the following method. Part. We have:
First, Streptomyces kuromovsukasu ( Strep
Tomyces chromofuscus ), the gene for phospholipase D was isolated, and the amino acid sequence constituting the enzyme was determined. Phospholipase DP of the present invention
When the amino acid sequence of phospholipase D was compared with the nucleotide sequence of the gene, no homology was recognized in both the amino acid sequence and the nucleotide sequence. Therefore, the phospholipase D gene was isolated from the phospholipase DP gene of the present invention. Could not be used as a probe for

According to the transformant, the phospholipase DP
When producing the transformant, the transformant is cultured in a nutrient medium, and the phospholipase D-P
After completion of the culture, the obtained culture is collected by filtration or centrifugation to collect cells, and then the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme. EDTA and / or depending on
Alternatively, an aqueous solution of the phospholipase DP may be concentrated by adding an appropriate surfactant or the like, or, without concentration, ammonium sulfate fractionation, gel filtration, adsorption chromatography such as affinity chromatography, and ion exchange chromatography. To obtain the phospholipase DP with high purity. The culturing conditions of the transformant microorganism may be selected in consideration of the nutritional and physiological properties thereof.In most cases, the culturing is carried out by liquid cultivation. Is advantageous. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used.

The carbon source may be any carbon compound that can be assimilated by the transformant microorganism, and examples thereof include glucose, saccharose, lactose, maltose, fructose, and molasses. Any nitrogen source may be used as long as it is a nitrogen compound that can be used by a transformant microorganism, such as peptone, meat extract, yeast extract, and casein hydrolyzate. In addition, phosphates, carbonates, sulfates, metal salts such as magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids and specific vitamins are used as necessary. The culture temperature may be within a temperature range in which the microorganism can grow and proliferate and produce phospholipase DP, and may be appropriately changed depending on the microorganism.
28-30 for Streptomyces lividans
It is about ° C. The culture period varies somewhat depending on the culture conditions, but the culture may be terminated at an appropriate time in anticipation of the time when the phospholipase DP reaches the maximum yield. For example, in the case of Streptomyces lividans, 48 to 50% is used.
It is about 72 hours. The culture medium pH allows microorganisms to grow and proliferate,
It is sufficient that phospholipase DP can be produced,
The pH can be appropriately changed depending on the microorganism. For example, in the case of Streptomyces lividans, the pH is about 7 to 7.5.

[0016] Phospholipase DP in the culture can be used by directly collecting and using a culture solution containing bacterial cells. However, in general, when phospholipase DP is present in a culture solution, it can be used in a usual manner. Is used after separating a phospholipase DP-containing solution from microbial cells by filtration or centrifugation. On the other hand, when phospholipase DP is present in the cells, the obtained culture is collected by filtration or centrifugation, and then the cells are collected. The phospholipase DP is solubilized by adding a chelating agent such as EDTA and / or a surfactant as necessary, and separated and collected as an aqueous solution. The thus obtained phospholipase DP-containing solution is concentrated, for example, under reduced pressure, membrane concentration, salting out treatment with ammonium sulfate, sodium sulfate or the like, or a hydrophilic organic solvent such as methanol, ethanol and acetone. May be precipitated by the fractional precipitation method described above. The precipitate can then be dissolved in water and dialyzed through a semi-permeable membrane to remove lower molecular weight impurities. Further, purification by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography such as affinity chromatography, ion exchange chromatography, etc., and concentration under reduced pressure from the phospholipase DP-containing solution obtained using these means are performed. Purified phospholipase DP is obtained by a treatment such as drying.

Examples of the phospholipase DP obtained by the above-mentioned production method include phospholipase DP having the following physical properties. (1) Action The compound acts on an ester bond between phosphoric acid and a nitrogen-containing base of a phospholipid to release a phosphorus compound and a corresponding nitrogen-containing base. Action for lecithin is illustrated by Figure 13. In addition, lecithin is hydrolyzed with phospholipase DP, and a nucleic acid-based nucleoside phospholipid complex can be produced by a base exchange reaction using a nucleic acid-based nucleoside and a phospholipid such as lecithin as a substrate in the reversible reaction. .

(2) Method for measuring activity 0.4 ml of a substrate solution [1.1 mM CaCl ₂ , 0.33% Triton X-100, 44 mM 3,3-dimethylglutaric acid-NaOH buffer (pH 5.5), 2.2 mM lecithin], add 0.05 ml of an activity measurement sample, heat at 37 ° C. for 10 minutes, and then add 0.5 ml of a reaction stop solution [1 M Tris-HCl
H8.0), 10 mM EDTA, 1.0% acetyltrimethylammonium chloride].
Color developing solution [choline oxidase (5 u./ml), peroxidase (5 u./ml), 0.02% phenol, 0.0
3% 4-aminoantipyrine, 0.1 M Tris-HCl (pH
8.0)] and heated at 37 ° C. for 20 minutes.
5 ml of 1.0% Triton X-100 was added, and the absorbance at 500 nm was determined. One unit of phospholipase DP (Uni
t. or u.) is defined as an enzyme activity that produces 1 μmole of choline per minute. The enzyme activity is calculated by the following equation. u./ml=ΔA ₅₀₀ × 0.55 (in the formula, ΔA ₅₀₀ indicates absorbance at ₅₀₀ nm)

(3) Substrate specificity In the reaction solution for the above-mentioned activity measurement, using the respective emulsions (10 ml, 0.1 ml) of lecithin, lysolecithin and sphingomyelin as substrates, the same operation was carried out to determine choline. Then, the relative activity is determined with the enzymatic action on lecithin as 100. Table 1 shows the relative activities.

[0020]

[Table 1]

(4) pH stability pH 4 to 7.5: dimethyl glutaric acid-NaOH buffer pH 8 to 8.5: Tris-HCl buffer pH 9.5: glycine-NaOH buffer pH 6.5 to 7: phosphoric acid After incubating the enzyme solution (48 u./ml) at 37 ° C. for 60 minutes at each pH of the buffer, the residual activity of phospholipase DP is determined according to the above-mentioned activity measurement method. This enzyme has a pH
It is relatively stable at 4.2 to 8.5.

(5) Optimum pH pH 4 to 7.5: Dimethyl glutaric acid-NaOH buffer pH 6.5 to 8: Phosphate buffer pH 8 to 9: Tris-HCl buffer Using each of the above buffers, The activity of phospholipase DP is determined according to the above activity measurement method. Its optimum pH is around 5.5.

(6) Thermostability An enzyme solution (25 u./ml) dissolved in 10 mM dimethylglutaric acid-NaOH buffer (pH 6.0) containing 0.05% bovine serum albumin was heated at 42-75 ° C. For 10 minutes, and then cooled on ice, and the residual activity of phospholipase DP on lecithin was measured according to the enzyme activity method described above. This enzyme is stable up to 60 ° C.

(7) Molecular weight and isoelectric point Biogel P-200 has a molecular weight of about 46,
000, and the isoelectric point (pI) by isoelectric focusing is pH 4.2.

[0025]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Extraction of DNA from Actinomycetes Tryptic Soy Medium [30 g / l Tryptic Soy Brot
h (manufactured by Difco)] in 50 ml of Streptomyces sp., a Streptomyces sp.
yces sp.) AA586 (FERM BP-3578) was inoculated with 1 ase and cultured with shaking at 30 ° C for 3 days. The cells were collected from the culture by centrifugation (3000 × G, 10 minutes, 4 ° C.), and after removing the supernatant, 10 ml of TEL [10 [10] was used to cut the peptidoglycan on the cell wall and facilitate lysis.
mM Tris-HCl pH 8.0, 1 mM EDTA disodium salt, 1 mg / ml egg white lysozyme (Sigma)] and heated at 37 ° C. for 30 minutes. 2 ml 10%
SDS (Sodium Dodecyl Sulfate) was added and mixed gently to prepare a lysate. Subsequently, 10 ml was added to the obtained lysate.
TE (10 mM Tris-HCl pH 8.0, 1 mM EDT
(A2 sodium salt) Saturated phenol was added and the whole was suspended. The same centrifugation was repeated, and the upper layer was transferred to another container again (in this way, an aqueous solution containing DNA having a volume equivalent to that of the aqueous solution containing DNA). The operation of adding TE-saturated phenol, suspending the whole, and separating and separating the upper layer containing DNA from the lower layer (corresponding to the phenol layer) by centrifugation at around room temperature is hereinafter referred to as phenol treatment). This operation is D
Denatures and inactivates proteins attached to NA, restriction enzymes and other nucleases that coexist in the solution, and collects the denatured protein at the bottom of the centrifuge tube or at the interface between the upper and lower layers by centrifugation. To handle by sorting
It increases the purity of A and enables removal of enzymatic activity acting on coexisting DNA.

Subsequently, 10 ml of chloroform was added to remove the phenol in the obtained upper layer, and the whole was suspended and centrifuged (25,000 × G, 20 minutes, 25 ° C.).
The upper layer containing the chromosomal DNA was transferred to another container (glass beaker for 100 ml, autoclaved). This D
In order to precipitate chromosomal DNA from the NA solution, gently overlay 30 ml of ethanol on the upper layer, gently stir the vicinity of the interface between the two layers with the tip of the glass rod, and entangle the chromosomal DNA that precipitates in white with the glass rod. I took it. D obtained
Transfer NA to another container, add 1 ml of 70% ethanol,
The supernatant was discarded by centrifugation (15,000 × G, 1 minute, 4 ° C.), and the DNA precipitate was dried under reduced pressure. This DNA was
It was dissolved in 4 ml of TE, and a portion thereof was quantified by absorbance measurement and gel electrophoresis. As a result, about 0.5 mg of chromosomal DNA of Streptomyces sp.
Was obtained.

Example 2 Preparation of Gene Library 2 μg of the chromosomal DNA of Streptomyces sp. AA586 strain obtained by the procedure of Example 1 and 2 μg of the cloning vector pUC118 (Takara Shuzo Co., Ltd.) were separately restricted in different containers. Enzymes (BamHI, Eco
RI, KpnI, PstI, SacI, SalI, Xb
Cleavage by aI). The reaction conditions for cleavage were in accordance with the reaction conditions indicated for each enzyme. pUC118
Is added to the reaction solution for further dephosphorylation of the 5 'end with a quarter volume (the same applies to the following volume) of 250 mM Tris-HCl (pH 8.8) and 1 to 3 units of BAP (bacteria). -Alkaline phosphatase: manufactured by Toyobo Co., Ltd. was added, and the mixture was heated at 65 ° C for 0.5 to 1 hour (this operation is hereinafter referred to as BAP treatment). In this way, a solution for cutting chromosomal DNA of Streptomyces sp. AA586 strain and a solution for cutting cloning vector pUC118-BAP treatment- were prepared. Subsequently, phenol treatment was performed twice for the purpose of removing the enzyme activities in these two kinds of solutions. This treatment was performed after 100 μl of TE was added to each solution to make the volume manageable.

Each D obtained after phenol treatment
In order to remove phenol from the upper layer containing NA,
After adding 0.4 ml of diethyl ether to each and vigorously stirring with a vortex mixer, the mixture was allowed to stand still for about 10 seconds to remove the separated diethyl ether layer containing phenol in the upper layer (this operation is hereinafter referred to as ether treatment). I will). After the ether treatment, the lower layer containing the DNA was mixed with a 1/10 volume of a 5 M potassium acetate solution, and then 2.5 to 3 times the volume of the lower layer with ethanol.
A cooling treatment was performed at a temperature of 110 ° C to -80 ° C for 10 minutes or more. Subsequently, the mixture was centrifuged (15,000 × G, 10 minutes, 4 ° C.), and the DNA was precipitated at the bottom of the centrifuge tube to remove the supernatant. To remove salts in the precipitate, 0.4 ml of ethanol was added and centrifuged again (15,000 × G, 30 seconds, 4 ° C.). The supernatant was removed, and the obtained DNA precipitate was dried under reduced pressure to remove ethanol (this series of operations is hereinafter referred to as ethanol precipitation).

The dried chromosomal DNA of the cleaved Streptomyces sp. AA586 strain obtained by ethanol precipitation and the cloning vector pUC118 that had been cleaved-BAP-treated were dissolved in 20 μl of distilled water. Was obtained. Two μl of each of the two DNA solutions thus obtained was added to one container, and 100 units of T4 DNA ligase (manufactured by Takara Shuzo) was added to 66 mM Tris-HCl (pH 7.6), 6.6 mM M
gCl ₂ , 10 mM Dithiothreitol, 660 μM ATP
(Boehringer Mannheim), final volume 1
Ligation was performed at 0 ° C. for 16 hours at 16 ° C. Using this, Shigesada method (Cell Engineering 2, 616-626,
1983) and made Escherichia coli DH1 (ATCC 338) a competent cell.
^{49) (F -, recA1,} endA1, gyrA96, thi-1, hsdR17 (rk -, m
^{k +), supE44, relA1,} λ -. (T.Maniatis et al Molecular
Cloning: Cold Spring Harbor (1982), 504-506)) was transformed, and an L-plate agar medium containing 50 μg / ml ampicillin [Bactotryptone (manufactured by Difco) 10
g / l, yeast extract (Difco) 5 g / l, NaCl 10 g / l,
Bacto agar (manufactured by Difco) 15 g / l] overnight, and a total of 100,000 ampicillin-resistant colonies were obtained from each restriction enzyme digested sample to obtain a gene library.

Example 3 Preparation of Radioactive DNA Probe The enzyme phospholipase DP produced from Streptomyces sp. AA586 strain was purified, fragmented with trypsin or V8 protease, and these fragments were purified. Sequence was determined by Edman degradation. D corresponding to each amino acid in this sequence
The DNA sequence is predicted from the NA codon, and the predicted DN
Designing an oligonucleotide probe to be used for screening the phospholipase DP gene based on the A sequence,
The method of R.L. Lessinger and others (RL Le.
stinger. , W.S. B. Lursford Jou
rnal Am. Chem. Society 98,3
655), using a DNA synthesizer (manufactured by Beckman: System l plus).
The determined amino acid sequence, the DNA sequence deduced therefrom, and the base sequences of three types of oligonucleotides prepared based on the determined amino acid sequence are shown in FIG. 9 (also, oligonucleotide 1, oligonucleotide 2 and oligo Nucleotides 3 are shown). In FIG. 9 , a portion indicated by two or more bases in the base sequence means any one of them. However, all of the oligonucleotides are prepared and mixed. “N” indicates all four types of bases, A, G, C and T. Oligonucleotide 3 is designed based on a complementary strand. The aforementioned synthetic oligonucleotide 10
0 ng of a buffer (50 mM Tris-H) in the presence of 8.5 μCi of γ-ATP (Amersham Japan) and 8.5 units of T4 polynucleotide kinase (Toyobo Co., Ltd.)
Cl (pH 8.0), 10 mM MgCl ₂ , 10 mM
5 'phosphorylation in 2-mercaptoethanol) to give a radioactive probe. The reaction was performed in a total reaction volume of 20 μl.
At 37 ° C for 30 minutes, frozen at -80 ° C at the end and thawed at the time of use.

Example 4 Screening of Phospholipase DP Gene-Containing Clones The gene library obtained in Example 2 was replicated on a nylon membrane filter (Biodyne A, manufactured by PALL), and this filter was separated by another 50 μg / ml. The cells were overlaid on an L-plate agar medium containing ampicillin so that the colony surface was facing upward, and cultured at 37 ° C. for 16 hours. After cultivation, filter this filter with alkaline denaturing solution (0.5N NaOH, 1.5M NaCl)
The filter paper was moistened with the filter paper and placed on top of the filter paper so that the colony face was facing upward, and allowed to stand for 5 minutes, after which the filter was air-dried. The filter was further heated under reduced pressure at 80 ° C. for 2 hours to fix the DNA to the filter. This filter is immersed in a pre-hybridization solution (Membrane 1).
(About 4 ml per 00 cm ² ) and heated at 42 ° C. for 16 hours. Prehybridization solution: 6 × SSC, 5 × De
nhardt solution, 0.1% SDS, 100 μg / ml salmon sperm DNA (manufactured by Pharmacia, a 10 mg / ml stock solution was heated at 100 ° C. for 10 minutes immediately before use, and immediately quenched with ice for denaturation) and 0. 05% sodium pyrophosphate 6 × SSC: 0.9 M NaCl and 0.09 M sodium citrate (pH 7.0) 5 × Denhardt solution: 0.1% (w / v) ficoll (molecular weight 400,000, manufactured by Pharmacia) , 0.1% (w /
v) Polyvinylpyrrolidone (molecular weight 360,000) and 0.1% (w / v) bovine serum albumin (Boehringer Mannheim)

After heating, the pre-hybridization solution was discarded, the hybridization solution was added (about 2 ml per 100 cm ^{2 of the} membrane), and a quarter of the radioactive DNA probe prepared in Example 3 was added. Hybridization was performed for hours. Hybridization solution: 6 x SSC, 5 x Denhar
dt solution, 20 μg / ml E. coli tRNA, 100 μg / ml
Salmon sperm DNA (Pharmacia, 10m immediately before use)
The g / ml stock solution was warmed at 100 ° C. for 10 minutes, immediately quenched on ice for denaturation) and washed with 0.05% sodium pyrophosphate (6 × SSC, 0.
0.05% sodium pyrophosphate) 3 times wash the filter with (room temperature, filter 100 cm ² per about 50ml / dose) followed in 50 ° C. of the cleaning liquid (filter 100 cm ² per about 5
0ml) After washing for 10 minutes, again about 100ml of room temperature washing solution
After washing with water, the filter was air-dried. This drying filter is applied to an X-ray film (Fuji Photo Film New
RXO-H) and subjected to autoradiography at -70 ° C for 24 hours under light shielding. After completion of the autoradiography, the film was developed, and from each library and combination of oligonucleotide probes, colonies showing a positive signal were obtained from about 10 strains obtained by oligonucleotide 1, about 35 strains obtained by oligonucleotide 2, and A total of about 50 strains of 5 strains obtained with oligonucleotide 3 were obtained.

Example 5 Extraction of Recombinant Plasmid and Confirmation of Phospholipase DP Gene Colonies showing a positive signal selected in Example 4 were isolated from 50 μg / ml ampicillin-containing LB liquid medium [Bactotripton (Difco)]. 10g / l, yeast extract (Difco
5 g / l, NaCl 10 g / l] 1 ml and inoculated at 37 ° C
After shaking culture for 16 hours, the method of Tea Maniatis et al. (Maniatis T. et al. Molecular Cloning, A Labora
tory Manual. Cold Spring Harbor Laboratory 86-94 1
982), the plasmid was extracted. Streptomyces sp. AA586 carried by these plasmids
The nucleotide sequence of the site hybridizing with the oligonucleotide probe in the derived DNA fragment was analyzed by the dideoxy method (Science 214 1205-1210 1981).
As a result, Streptomyces sp.
Of the library (20,000 colonies) prepared by cutting the chromosome of AA586 and pUC118, and one of five positive clones obtained by colony hybridization using oligonucleotide 3 as a probe, one was an oligonucleotide design The plasmid was confirmed to have a base sequence encoding a known amino acid sequence, and this plasmid was confirmed to be a phospholipase DP gene-carrying clone and named plasmid DP135a. Four other positive clones and all the positive clones obtained with oligonucleotides 1 and 2 did not have the phospholipase DP sequence.

Example 6 Restriction Map of Plasmid DP135a and Determination of Phospholipase DP Structural Gene Sequence The structure of plasmid DP135a obtained in Example 5 is shown in FIG.
It is shown in FIG. FIG. 3 shows the results of base sequence determination of the portion encoding phospholipase DP from this plasmid by the dideoxy method. Further, connecting the amino acid sequence deduced based on the nucleotide sequence in Figures 1 and 2
Shown next .

Example 7 Construction of Actinomycete Expression Plasmid DP108 Plasmid DP135a was reacted with a restriction enzyme BamHI (manufactured by Toyobo) at 37 ° C. for 20 minutes to perform partial digestion. After the digested plasmid was subjected to phenol treatment, ether treatment and ethanol precipitation, it was further digested with a restriction enzyme SacI (manufactured by Toyobo Co., Ltd.) at 37 ° C. for 2 hours under the specified reaction conditions to complete digestion. This was mixed with 1% low melting point agarose gel (Bethesda Research Labora)
tories LMP Agarose, 40 mM Tris-acetate buffer pH
After electrophoresis at 150 V for 1.5 hours at 7.4 V and 2 mM EDTA, about 2.0 Kbp (Kbp: 1,000 base pairs) of BamH containing the phospholipase DP structural gene was obtained.
A gel containing the I-SacI DNA fragment was cut out. The cut gel was melted by heating at 65 ° C. for 10 minutes, and 100 μl of TE-saturated phenol was added and stirred,
The agarose was denatured by standing for 5 minutes. This was centrifuged (14,000 × G, 5 minutes) to separate the upper aqueous layer to remove agarose in the solution. The separated aqueous layer was treated with phenol, treated with ether and precipitated with ethanol to obtain about 2.0 Kbp containing the phospholipase DP structural gene.
A dried product of the BamHI-SacI DNA fragment was obtained (a series of operations for separating this DNA fragment having a certain length is hereinafter referred to as agarose electrophoresis isolation). This was dissolved in 2 μl of distilled water. In addition, Escherichia coli plasmid vector pUC118 (manufactured by Takara Shuzo Co., Ltd.) was reacted at 37 ° C. for 2 hours under conditions specified by restriction enzymes BamHI and SacI to completely digest, and further subjected to BAP treatment, phenol treatment, ether treatment and ethanol precipitation. 5 μl of the resulting BamHI and SacI digested dephosphorylated plasmid vector pUC118
In distilled water.

Approximately 2.0 Kbp of BamHI containing the phospholipase DP structural gene obtained by the above operation.
-SacI DNA fragment and BamHI, S
acI digested dephosphorylated plasmid vector pUC118
Are mixed in one container, and T4 DN
The reaction was carried out at 16 ° C. for 16 hours with 100 units of A ligase (manufactured by Takara Shuzo) under the designated reaction conditions, and the ligation was carried out. Using this, Escherichia coli DHI transformed into competent cells by the method of Shigesada was transformed and cultured overnight on an L-plate agar medium containing 50 μg / ml ampicillin.
/ Ml Ampicillin-containing LB liquid medium (1 ml) at 37 ° C
After shaking culture for 16 hours, the plasmid was extracted by the method of T. Maniatis et al.
No. 4. Furthermore, the vector pSEV for expression of actinomycetes
1 (Horinouchi.S., Nishiyama.M., Nakamura.A., Beppu.
T., Molecular & General Genetics 210 168-475 1987)
(FERM BP-2684) was reacted with a restriction enzyme KpnI (manufactured by Toyobo Co., Ltd.) at 37 ° C. for 2 hours under the specified reaction conditions, and completely digested. An about 500 bp KpnI DNA fragment containing the actinomycetes promoter was isolated from the digested plasmid by agarose electrophoresis isolation. The fragment was reacted with T4 DNA polymerase (manufactured by Toyobo Co., Ltd.) at room temperature for 1 hour at room temperature to blunt both ends of the fragment. This was subjected to agarose gel electrophoresis isolation, and the KpnI DNA fragment having blunt-ended ends was separated again, and 2 μl
Dissolved in 1 l of distilled water.

Further, plasmid DP104 was replaced with restriction enzyme X.
The reaction was carried out at 37 ° C. for 2 hours under the conditions specified by baI (manufactured by Toyobo) to complete the digestion, and the reaction was carried out at room temperature for 1 hour under the conditions specified by T4 DNA polymerase (manufactured by Toyobo). Was subjected to agarose gel electrophoresis isolation to separate a blunt-ended linear plasmid DP104. This was further treated with BAP, phenol, ether and precipitated with ethanol to obtain an XbaI-digested blunt-ended dephosphorylated plasmid D.
P104 was dissolved in 5 μl of distilled water. Approximately 500 bp blunt-ended KpnI DNA fragment containing the actinomycetes promoter obtained by the above operation and XbaI
2 μl of each of the digested end blunt-ended dephosphorylated plasmid DP104 was mixed in one container, and 100 units of T4 DNA ligase (Takara Shuzo) was added under the specified conditions.
Ligation was performed at 16 ° C. for 16 hours. Using this
Escherichia coli DHI transformed into competent cells by the method of Shigesada was transformed,
After culturing overnight on an L-plate agar medium containing 50 μg / ml ampicillin, the grown cells were inoculated into 1 ml of an LB liquid medium containing 50 μg / ml ampicillin, and cultured at 37 ° C. for 16 hours with shaking, followed by tea maniatis et al. The plasmid was extracted by the method described above. This plasmid was named DP107.

Further, plasmid DP107 was digested by reacting at 37 ° C. for 2 hours under the reaction conditions specified by restriction enzymes XbaI and SacI. The digested plasmid was subjected to agarose electrophoresis isolation to obtain a 1.2K plasmid containing an actinomyces promoter and a phospholipase DP gene.
The bp Xbal-SacI DNA fragment was isolated. The fragment was reacted with T4 DNA polymerase (manufactured by Toyobo Co., Ltd.) at room temperature for 1 hour at room temperature to blunt both ends of the fragment. This was again subjected to agarose gel electrophoresis isolation to separate the blunt-ended Xbal-SacI DNA fragment, which was dissolved in 2 μl of distilled water. In addition, actinomycete plasmid vector pIJ680 (Kieser, T. Genetic Manupila)
tion of Streptomyces. A Laboratory manual. The Joh
n Innes Foundation John Innes Institute (298-29
9)) (Hopwood (DAHopwood, John Innes Instit)
ute) from plasmid DP107 with restriction enzyme Ec
Under the reaction conditions specified by oRV (manufactured by Toyobo Co., Ltd.), 37
The mixture was reacted at 2 ° C. for 2 hours for complete digestion. This is BA
After P treatment, phenol treatment, ether treatment and ethanol precipitation, the obtained EcoRV digested and dephosphorylated plasmid pIJ680 was dissolved in 5 μl of distilled water.

Approximately 1.2 containing the actinomyces promoter and phospholipase DP gene obtained by the above operation.
Kbp end-blunted Xbal-Sacl DNA fragment and EcoRV digested dephosphorylated plasmid pI
Mix 2 μl of each of J680 in one container and add T
4 Ligation was performed with 100 units of DNA ligase (manufactured by Takara Shuzo) at 16 ° C. for 16 hours under the designated reaction conditions. Using this, the method of Hopwood et al. (DA Ho
ppwood et al. , GeneticMani
lotion of Streptomyces.
A Laboratory Manual. The
John Innes Foundation, John
nInstitute Institute. 103-12
2) Streptomyces lividans TK-24 (FERM BP-2685) which was made competent cells
And a tryptic soy plate agar medium containing 10 μg / ml neomycin (30 μg / l Tryp).
tic soy broth (manufactured by DIFCO)) for 3 to 4 days, and the grown cells were inoculated into 1 ml of a tryptic soy liquid medium containing 10 μg / ml of neomycin, and cultured with shaking at 28 ° C. for 2 days. The method of Hopwood et al. (DA Hoppwood et al., G.
enetic Manipulation of St
reptomyces. A Laboratory M
anual. The John Innes Foun
date, John Innes Institute
te81-84) to extract the plasmid. This plasmid was designated as DP-108, and the strain carrying this plasmid was named Streptomyces lividans TK2.
4 ・ DP108 ( Streptomyces live
Dans TK24 / DP108) (FERM BP-
3577). Figure 11 shows the above procedure. The structure of plasmid DP108 is shown in FIG.

Further, the nucleotide sequence of the about 2.0 Kbp fragment held by the plasmid DP108 and the amino acid sequence encoding the open reading frame region thereof are represented by the following:
In addition it is shown in succession in FIGS. 4 through 7. Further, a plasmid equivalent to the plasmid vector pIJ680 can also be obtained by the following method. That is, plasmid DP10
Lividans TK24 showing 8
Extraction from DP108 by Hopwood et al., Restriction enzymes BanIII and PvuII (both manufactured by Toyobo Co., Ltd.)
, And blunt at both ends by reaction with T4 DNA polymerase (manufactured by Toyobo Co., Ltd.) at room temperature for 1 hour, and agarose gel electrophoresis is carried out. Separate the blunted linear plasmid. This was further treated with BAP, phenol, ether and precipitated with ethanol, and then T4DN in the presence of a suitable phosphorylation restriction enzyme linker (preferably a restriction enzyme having no site in pIJ680).
An A ligase reaction (same as in Example 2) is performed. This is introduced into Streptomyces lividans TK24, which was made into competent cells by the method of Popwood et al. Plasmids extracted again from the transformants having acquired neomycin resistance by the method of Hopwood et al. Were prepared by using the restriction enzyme site of the linker used as a cloning site and the neomycin resistance as a drug resistance marker using the plasmid pI.
It can be used equivalent to J680.

Example 8 Expression of Phospholipase DP in Actinomyces Streptomyces lividans TK24.DP108 (FERM BP-357), which is a strain carrying the plasmid DP108 for expressing phospholipase DP obtained in Example 7
7) was inoculated into 10 ml of a tryptic soy medium (30 μg / l Tryptic soy broth (manufactured by DIFCO)) containing 10 μg / ml neomycin, and cultured at 28 ° C. for 2 days with shaking. Streptomyces lividans TK for comparison
The same culture was carried out for Streptomyces lividans TK24.pIJ680 in which plasmid pIJ680 was introduced into No.24. After the culture, centrifugation (15,000)
0 × G, 1 minute, 4 ° C.), and the supernatant was transferred to another container to obtain a sample.

Example 9 Confirmation of phospholipase DP enzyme activity in the cell culture broth The phospholipase DP enzyme activity in the cell culture supernatant prepared in Example 8 was measured according to the method for measuring the activity described above. . Table 2 shows the results. Thus, the expression of phospholipase DP activity in Streptomyces lividans TK24 / DP108 was confirmed.

[0043]

[Table 2]

The molecular weight of the expressed enzyme protein is about 46.0.
The isoelectric point was identical to that of phospholipase DP enzyme protein produced by Streptomyces sp. AA586 strain at pH 4.2, and it was confirmed that the expressed protein was the same as phospholipase DP.

[0045]

The present invention uses an oligonucleotide synthesized based on the partial amino acid sequence of phospholipase DP,
Chromosomal DNA derived from phospholipase DP producing strain
Total DN of phospholipase DP gene from library
A novel phospholipase DP gene having a defined A sequence is isolated.
It made it possible to produce and culture phospholipase DP in a vector system. Furthermore, phospholipase DP
By isolating the gene, various protein engineering such as improvement of heat resistance of phospholipase DP and conversion of substrate specificity became possible.

[Brief description of the drawings]

FIG. 1 shows a part of the amino acid sequence of a polypeptide constituting phospholipase DP.

FIG. 2 shows the remainder of the amino acid sequence of the polypeptide constituting phospholipase DP.

FIG. 3 shows DNA encoding the amino acid sequence of the polypeptide constituting phospholipase DP.

FIG. 4 shows the amino acid sequence of the polypeptide constituting phospholipase DP, the DNA encoding the amino acid sequence, and the upstream of the DNA suitable for its expression, in conjunction with FIG. 5, FIG. 6 and FIG. The part and the downstream part are shown together.

FIG. 5 is an amino acid sequence of a polypeptide constituting phospholipase DP, a DNA encoding the amino acid sequence, and an upstream of a DNA suitable for the expression thereof, in conjunction with FIG. 4, FIG. 6 and FIG. The part and the downstream part are shown together.

FIG. 6 shows the amino acid sequence of a polypeptide constituting phospholipase DP, DNA encoding the amino acid sequence, and upstream of a DNA suitable for its expression, in conjunction with FIG. 4, FIG. 5 and FIG. The part and the downstream part are shown together.

FIG. 7 is an amino acid sequence of a polypeptide constituting phospholipase DP, a DNA encoding the amino acid sequence, and an upstream of a DNA suitable for the expression thereof, in conjunction with FIG. 4, FIG. 5 and FIG. The part and the downstream part are shown together.

FIG. 8 shows a restriction map of a phospholipase DP gene.

FIG. 9 shows the nucleotide sequence of an oligonucleotide probe (N in the figure indicates A, G, C or T).

FIG. 10 shows the structure of plasmid DP135a.

FIG. 11 shows an outline of the procedure for preparing plasmid DP108.

FIG. 12 shows the structure of plasmid DP108.

[Figure 13] is indicative of the effect of the lecithin enzyme of the present invention shows the reaction course of lecithin (e scan choline) is hydrolyzed.

──────────────────────────────────────────────────続 き Continuation of front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 9/20 C12R 1: 465) (56) References JP-A-58-152481 (JP, A) JP-A-4-88981 (JP, A) JP-A-3-187382 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1/21 C12N 9/20 C12N 15/00 BIOSIS (DIALOG) GenSeq / DDBJ / pir WPI (DIALOG)

Claims (19)

(57) [Claims] 1. The amino acid sequence described in SEQ ID NO: 2 in the sequence listing
Wherein the microorganism is a microorganism belonging to the genus Streptomyces transformed by a recombinant plasmid having a DNA that expresses phospholipase DP having a sequence from 1 to 514. Pure microorganisms. 2. The base sequence of the DNA is SEQ ID NO: 2 in the Sequence Listing.
It expresses in from 205 1746 in the nucleotide sequence described
請 Motomeko 1 microorganism according. [3] the amino acid sequence of SEQ ID NO: 2
In which the phospholipase has a sequence from 1 to 514
In the amino acid sequence described in SEQ ID NO: 2 with DP
Signal-like peptide having a sequence from -68 to -1
Recombinant plasmid having DNA expressing adduct with
Belongs to the genus Streptomyces transformed by
A phospholipase DP characterized by being a microorganism
A substantially pure microorganism that produces . 4. The nucleotide sequence of the DNA is SEQ ID NO: 2 in the Sequence Listing.
The microorganism according to claim 2, which is represented by 1 to 1746 in the base sequence described above. 5. A microorganism belonging to the genus Streptomyces which is transformed microorganisms according to claim 1, wherein the microorganism transformed with plasmid DP108. 6. The microorganism according to claim 1, wherein the transformed microorganism belonging to the genus Streptomyces is a microorganism belonging to Streptomyces lividans. 7. The transformed microorganism belonging to Streptomyces lividans is Streptomyces lividans TK24.DP108 (FERMBP-357).
The microorganism according to claim 6, which is 7). 8. The amino acid sequence described in SEQ ID NO: 2 in the sequence listing
Characterized in that the phospholipase DP has a base sequence encoding an amino acid represented by 1 to 514.
A substantially pure DNA that produces 9. The DNA according to claim 8 , wherein the nucleotide sequence is derived from DNA isolated from Streptomyces sp. AA586. 10. A salt whose base sequence is represented by SEQ ID NO: 2 in the Sequence Listing.
請 Motomeko In group sequence represented by 2 05 1746 8
The described DNA. 11. An amino acid sequence according to SEQ ID NO: 2 in the Sequence Listing.
A phospholipper having a sequence from 1 to 514
In the amino acid sequence described in SEQ ID NO: 2
Characterized by having a base sequence encoding an adduct with a signal-like peptide having a sequence from -68 to -1
Substantially pure DNA producing phospholipase DP . 12. The nucleotide sequence as set forth in SEQ ID NO: 2 in the Sequence Listing.
Claim 1 in the nucleotide sequence you express 1746 10
The described DNA. 13. An amino acid sequence according to SEQ ID NO: 2 in the sequence listing.
Substantially producing phospholipase DP, a microorganism belonging to the genus Streptomyces, transformed by a recombinant plasmid having DNA expressing phospholipase DP having a sequence from 1 to 514 A method for producing phospholipase DP, comprising culturing a highly pure microorganism in a medium, and then collecting phospholipase DP from the culture. 14. The nucleotide sequence of DNA is as shown in SEQ ID NO:
The production method according to claim 13, which is represented by 205 to 1746 in the base sequence according to (2 ). An amino acid sequence according to SEQ ID NO: 2 in the sequence listing
A phospholipper having a sequence from 1 to 514
In the amino acid sequence described in SEQ ID NO: 2
Recombinant plasmid having a DNA expressing an adduct with a signal-like peptide having a sequence from -68 to -1
Belongs to the genus Streptomyces transformed by
Substantially producing phospholipase DP, a microorganism
A pure microorganism is cultured in a culture medium, and then the culture is
Phospholipase DP is collected.
A method for producing holipase DP . 16. The nucleotide sequence of the DNA is as shown in SEQ ID NO:
2 process according to claim 14, wherein is Ru represented by 1 to 1746 in the nucleotide sequence described. 17. The method according to claim 13 , wherein the transformed microorganism belonging to the genus Streptomyces is a microorganism transformed by plasmid DP108. 18. microorganism belonging to transformed Streptomyces <br/> The production method of claim 13, wherein the microorganism belonging to Streptomyces lividans. 19. The transformed Streptomyces strain.
Microorganisms belonging to Streptomyces lividans TK24 DP-108 (FERM BP-
3577) The process according to claim 18, wherein a. [Sequence list] SEQ ID NO: 2 Sequence length: 1749 Sequence type: Number of nucleic acid strands: Double-stranded topology: Type of linear sequence: Genomic DNA origin Organism: Streptomyces sp.
sp.) Strain name: Characteristic of AA586 sequence Characteristic code: CDS location: 1..1746 Method for determining characteristic : E Characteristic code: sig peptide Location: 1..204 Method for characteristic determination: Symbol for E characteristic: mat peptide Location: 205..1746 Method for determining characteristic: E sequence ATG CCT CTC TCC CAG GCT GTC GCA CGC TCC CGT CGT CGT CCG GTG CGG 48 Met Pro Leu Ser Gln Ala Val Ala Arg Ser Arg Arg Arg Pro Val Arg -65 -60 -55 ACC GTA CTG AGC CTC GCG CTC GTC CTC GCC GGA GCG TCG GCG TCG GCC 96 Thr Val Leu Ser Leu Ala Leu Val Leu Ala Gly Ala Ser Ala Ser Ala -50 -45 - 40 ACA CCC GCG CTC GCC GCC CCG CCG AGC GGC GCG TCC GCG GCC ACG GCC 144 Thr Pro Ala Leu Ala Ala Pro Pro Ser Gly Ala Ser Ala Ala Thr Ala -35 -30 -25 GCA GTC CCG GCC GCA GCC TCG GTC GCG GCC CCT ACC GGG AGT CCT GCC 192 Ala Val Pro Ala Ala Ala Ser Val Ala Ala Pro Thr Gly Ser Pro Ala -20 -15 -10 -5 GGG AGC CCT GGT GGG AGC CCT GGT GGG AGT CCG ACC CCG CAC CTG GAC 240 Gly Ser Pro Gly Gly Ser Pro Gly Gly Ser Pro Thr Pro His L eu Asp 1 5 10 GCC GTC GAG CAG GTC CTG CGC CAG GTC TCG CCC GGC CTG GAG GGC ACG 288 Ala Val Glu Gln Val Leu Arg Gln Val Ser Pro Gly Leu Glu Gly Thr 15 20 25 GTC TGG CAG CGC ACC GAG GGC AAC GCG CTG GAC GCG CCG GCC GGT GAC 336 Val Trp Gln Arg Thr Glu Gly Asn Ala Leu Asp Ala Pro Ala Gly Asp 30 35 40 CCG GGC GGC TGG CTG CTG CAG ACG CCG GGC TGC TGG GGC GAC CCG TCC 384 Pro Gly Gly Trp Leu Leu Gln Thr Pro Gly Cys Trp Gly Asp Pro Ser 45 50 55 60 TGC GCC ACC CGT CCG GGC AGC CAG GCG CTG CTG GCG AAG ATG ACC GCG 432 Cys Ala Thr Arg Pro Gly Ser Gln Ala Leu Leu Ala Lys Met Thr Ala 65 70 75 AAC ATC GCC GCG GCC ACC CGC ACG GTC GAC ATC TCC TCG CTC GCC CCG 480 Asn Ile Ala Ala Ala Thr Arg Thr Val Asp Ile Ser Ser Leu Ala Pro 80 85 90 CTG CCG AAC GGC GCC TTC GAG GAC GCC ATC GTG GCC GGG CTC AAG TCG 528 Leu Pro Asn Gly Ala Phe Glu Asp Ala Ile Val Ala Gly Leu Lys Ser 95 100 105 GCG GTG GCG TCC GGA CAC CGG CTC CAG GTG CGG ATC CTG GTC GGC GCC 576 Ala Val Ala Ser Gly His Arg Leu Gln Val Arg Ile Leu Val Gly Ala 110 115 120 GCC CCG CTG TAC AAC ATC ACC ACG CTG CCC TCC TCC TAC CGG GAC GAA 624 Ala Pro Leu Tyr Asn Ile Thr Thr Leu Pro Ser Ser Tyr Arg Asp Glu 125 130 135 140 CTG GTC GGC AAG CTG GGC GAC GCG GCC GGC TCC GTC ACG CTC AAC GTG 672 Leu Val Gly Lys Leu Gly Asp Ala Ala Gly Ser Val Thr Leu Asn Val 145 150 155 GCC TCG ATG ACC ACC GCC AAG ACC TCC TTC TCG TGG AAC CAC GCC AAG 720 Ala Ser Met Thr Thr Ala Lys Thr Ser Phe Ser Trp Asn His Ala Lys 160 165 170 CTG CTG GTG GTC GAC GGG CAG AGC GTG ATC ACC GGT GGC ATC AAC GAC 768 Leu Leu Val Val Asp Gly Gln Ser Val Ile Thr Gly Gly Ile Asn Asp 175 180 185 TGG AAG GCC GAC TAC CTG GAG ACC AGC CAC CCC GTC ACC GAC GCC GAC 816 Trp Lys Ala Asp Tyr Leu Glu Thr Ser His Pro Val Thr Asp Ala Asp 190 195 200 CTC GCG CTG ACC GGG CCG GCC GCC GCC ACG GCG GGA CGC TAC CTG GAC 864 Leu Ala Leu Thr Gly Pro Ala Ala Ala Thr Ala Gly Arg Tyr Leu Asp 205 210 215 220 ACC CTG TGG AGC TGG ACC TGC CGC AAC AGC GGC CCG TTC TCG GCT GCC 912 Thr Leu Trp Ser Trp Thr Cys Arg Asn Ser Gly Pro Phe Ser Ala Ala 225 230 235 TGG TTC GCC TCC TCG AAC GGC GCC GGC TGC CTG GCC ACC CTC GAA CAG 960 Trp Phe Ala Ser Ser Asn Gly Ala Gly Cys Leu Ala Thr Leu Glu Gln 240 245 250 GAC AGC AAC CCG GCC TCC CCG GCC GCC ACC GGC AGC CTG CCG GTG ATC 1008 Asp Ser Asn Pro Ala Ser Pro Ala Ala Thr Gly Ser Leu Pro Val Ile 255 260 265 GCG GTC GGC GGC CTG GGC GTC GGC ATC CAG AGC GTC GAC CCG GCC TCG 1056 Ala Val Gly Gly Leu Gly Val Gly Ile Gln Ser Val Asp Pro Ala Ser 270 275 280 ACC TTC CAG CCG ACC CCG GTC AAC CCG GCC GGC ACC CCG GCC ACC TCC 1104 Thr Phe Gln Pro Thr Pro Val Asn Pro Ala Gly Thr Pro Ala Thr Ser 285 290 295 300 TGC GGC CCG ATC AAG GTG CCG GAC CAC ACC AAC GCC GAC CGC GAC TAC 1152 Cys Gly Pro Ile Lys Val Pro Asp His Thr Asn Ala Asp Arg Asp Tyr 305 310 315 GCC ACG GTG AAC CCC GAG GAG AGC GCC CTG CGC GCG CTG GTC GCC AGC 1200 Ala Thr Val Asn Pro Glu Glu Ser Ala Leu Arg Ala Leu Val Ala Ser 320 325 330 GCG ACC AGC CAC ATC GAG ATA TCG CAG CAG GAC CTC AAC GGC ACC TGC 1248 Ala Thr Ser His Ile Glu Ile Ser G ln Gln Asp Leu Asn Gly Thr Cys 335 340 345 CCG CCG CTG CCC CGC TAC GAT GCC CGG CTC TAC GAC ACC CTG GCG GCC 1296 Pro Pro Leu Pro Arg Tyr Asp Ala Arg Leu Tyr Asp Thr Leu Ala Ala 350 355 360 AAG CTG GCG GCC GGT GTG AAG GTG CGG ATC GTG GTC AGT GAC CCG GCC 1344 Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val Val Ser Asp Pro Ala 365 370 375 380 AAC CGG GGC GCG GTC GGC AGC GGC GGC TAC TCG CAG ATG AAG TCG CTC 1392 Asn Arg Gly Ala Val Gly Ser Gly Gly Tyr Ser Gln Met Lys Ser Leu 385 390 395 TCC GAG ATC AGT GAC GTG CTG CTG GAC CGG ATC GGC GCG GCC ACC GGC 1440 Ser Glu Ile Ser Asp Val Leu Leu Asp Arg Ile Gly Ala Ala Thr Gly 400 405 410 CAG GAC CGG GCC GGC GCC AAG GCC ACC ATG TGC CAG AAC CTC CAG CTC 1488 Gln Asp Arg Ala Gly Ala Lys Ala Thr Met Cys Gln Asn Leu Gln Leu 415 420 425 GCC GCC TTC CGC GCG GCC CCC GGC GAC ACC TGG GCC GAC GGC CAC CCG 1536 Ala Ala Phe Arg Ala Ala Pro Gly Asp Thr Trp Ala Asp Gly His Pro 430 435 440 TAC GCG CTG CAC CAC AAG CTG GTG TCC GTG GAC GGC GCC GCG TTC TAC 1584 Tyr Ala Leu Hi Hi s His Lys Leu Val Ser Val Asp Gly Ala Ala Phe Tyr 445 450 455 460 CTC GGC TCC AAG AAC CTC TAC CCG GCC TGG CTG CAG GAC TTC GGC TAT 1632 Leu Gly Ser Lys Asn Leu Tyr Pro Ala Trp Leu Gln Asp Phe Gly Tyr 465 470 475 GTG ACG GAG GAC CAG ACG GCC GCC GCC CAG CTG GAC GCC CAG TTG CTG 1680 Val Thr Glu Asp Gln Thr Ala Ala Ala Gln Leu Asp Ala Gln Leu Leu 480 485 490 490 GCC CCC GAG TGG CAG TAC TCG CAG GCC GCC GCG ACC GTC GAC TAC ACC 1728 Ala Pro Glu Trp Gln Tyr Ser Gln Ala Ala Ala Thr Val Asp Tyr Thr 495 500 505 CGC GGG CTC TGC TCG GCC TGA 1749 Arg Gly Leu Cys Ser Ala *** 510