JPH10155493A - Gene coding for phospholipase a1 derived from aspergillus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene consisting of a DNA coding for a phospholipase-active polypeptide derived from the genus Aspergillus having a specific amino acid sequence, capable of giving an enzyme for producing lysophospholipid useful as e.g. a surfactant for foods. SOLUTION: This new gene consists of a polypeptide containing an amino acid sequence indicated by amino acid numbers 1 to 269 and shown by the formula and having phospholipase activity, or consists of such an amino acid sequence that one or more amino acids are replaced, deleted or inserted at one or more sites on the above-mentioned amino acid sequence of the formula, coding for polypeptide having phospholipase activity, and being useful for e.g. producing phospholipase A1 to be used for producing lysophospholipid useful as a surfactant for foods. This gene is obtained by screening the chromosome DNA of Aspergillus oryzae SANK 11870 (FERM BP-3887) strain by use of a probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene encoding phospholipase A1 of Aspergillus oryzae, a recombinant DNA vector comprising the gene, a host cell transformed with the vector and a host cell transformed with the vector. The present invention relates to a method for producing recombinant phospholipase A1 using host cells.

[0002]

2. Description of the Related Art Lecithin is a phospholipid having a surfactant action, an antioxidant action, a physiologically active action and the like.
Widely used for feed and pharmaceuticals. Since the surfactant activity of lecithin is inferior to emulsification stability as compared with other food surfactants, studies have been made to improve the emulsification stability using enzymatic decomposition. Lysophospholipids are partially degraded phospholipids obtained by hydrolyzing a part of the fatty acid ester-linked to the glycerin residue of the phospholipid. The temperature range where the emulsifying power is exhibited is expanded, and the emulsifying power is exhibited even when added to any of oil and water systems, and the emulsifying power does not decrease even if metal ions such as calcium and magnesium coexist at a high concentration. And good properties such as good emulsification stability under acidic conditions [Yamano, Y. and G
ohtani, S. (1996) Abstract of 4th World Surfactants
Congress P-84].

[0003] At present, lysophospholipids obtained by decomposing phospholipids using enzymes are commercially available, which use the phospholipase A2 activity contained in the enzyme agent pancreatin prepared from pig liver. Enzymes have odor peculiar to organs, and there are problems such as limited supply amount and high price. Therefore, a source of phospholipase A instead of pancreatin is required. Also, phospholipase A already produced by some microorganisms [Japanese Patent Laid-Open No. 58-2]
12783] and lipases [JP-A-63-4269]
No. 1] has been shown, and Takaji Astase (registered trademark) derived from koji mold and Aspergillus oryzae, which is referred to as “a treasure trove of enzymes”, The existence of lipases containing phospholipid degrading activity was known [Contardi, A. a
nd Ercoli, A. (1933) Biochem. Z.261, 275-302 and Blain, JA, et al. (1978) FEMS Microbiology L
etters 3, 85-87], but the methods using these enzymes have lower enzymatic activity than pancreatin, and when enzymes with poor substrate specificity are used, the yield of lysophospholipids may deteriorate. However, there is a drawback that the quality of lysophospholipid is deteriorated due to coexistence of by-products. Therefore, in order to overcome the above problems and drawbacks, there has been a demand for the development of an enzyme which has excellent activity and selectivity and can be supplied constantly at low cost. Hattori et al. Have isolated and purified a phospholipase produced by a filamentous fungus of the genus Aspergillus, and have found highly purified phospholipase A1 having excellent activity and selectivity [JP-A-6-62850].
reference].

[0004]

[Problems to be Solved by the Invention] Phospholipase A1
If it becomes possible to clone the gene coding for and to express it in large amounts in other host cells, it is expected that the production efficiency of the enzyme will be greatly improved.

That is, an object of the present invention is to provide a phospholipase A of Aspergillus oryzae.
, A recombinant DNA vector comprising the gene, a host cell transformed with the vector, and a recombinant phospholipase A using the host cell
It is another object of the present invention to provide a manufacturing method.

[0006]

Means for Solving the Problems The present invention comprises (1) a polypeptide having an amino acid sequence represented by amino acid numbers 1 to 269 of SEQ ID NO: 2 in the sequence list, and a polypeptide having phospholipase activity, or An amino acid sequence having one or more amino acids substituted, deleted or inserted at one or more sites in the amino acid sequence represented by amino acid numbers 1 to 269 of SEQ ID NO: 2, and having a phospholipase activity (2) a DNA encoding a polypeptide having an amino acid sequence represented by amino acid numbers 1-269 of SEQ ID NO: 2 in the sequence listing, and encoding a polypeptide having a phospholipase activity; D comprising the nucleotide sequence represented by nucleotide numbers 1 to 885 of SEQ ID NO: 1 in the column list. NA, (4) a DNA that hybridizes with the DNA according to (3) and encodes a polypeptide having phospholipase activity, and (5) hybridizes with the DNA according to (3) under stringent conditions to obtain a phospholipase. DNA encoding an active polypeptide, (6) a DNA encoding (6)
SSC, which hybridizes under conditions of 60 to 70 ° C. and encodes a polypeptide having phospholipase activity
A, (7) D according to any one of (1) to (6)
(8) a recombinant DNA vector comprising NA
(7) the recombinant DNA vector according to (7), which is plasmid pCY339; (9) the recombinant DNA vector according to (7), which is plasmid pAAP;
(7) The recombinant DNA according to any one of (9) to (9).
A host cell transformed with the vector, (11) Saccharomyces cerevisiae KS58-2D / pCY339
(12) Aspergillus oryzae M-2-3 / pAAP, (1)
0) the host cell described in (1), a polypeptide obtained by genetic manipulation, comprising the amino acid sequence represented by amino acid numbers 1-269 of SEQ ID NO: 2 in the sequence listing, and having a phospholipase activity, or An amino acid sequence having one or more amino acids substituted, deleted or inserted at one or more sites in the amino acid sequence represented by amino acid numbers 1 to 269 of SEQ ID NO: 2, and having a phospholipase activity A polypeptide having the amino acid sequence represented by amino acid numbers 1-269 of SEQ ID NO: 2 obtained by genetic manipulation and having phospholipase activity, (15) (10) Or (1
2) culturing the host cell according to any one of the above, under conditions capable of producing phospholipase, and then recovering the polypeptide having phospholipase activity from the culture. A manufacturing method.

The present inventors cloned a gene encoding phospholipase A1 (hereinafter referred to as "PLA1") from Aspergillus oryzae, and expressed the gene by introducing it into cultured cells, yeast and koji mold. As a result, the present invention was completed.

In the present invention, "phospholipase activity"
The term “enzyme handbook (1982 edition; published by Asakura Shoten) p427” refers to the activity of releasing free fatty acids from lecithin or lysolecithin.

In the present invention, the term “recombinant PLA”
"1" refers to an amino acid sequence obtained by genetic manipulation and represented by amino acid numbers 1-269 of SEQ ID NO: 2 in the sequence listing, or amino acid numbers 1-26 of SEQ ID NO: 2 in the sequence listing
A polypeptide having a phospholipase activity, comprising an amino acid sequence in which one or more amino acids have been substituted, deleted or inserted at one or more sites in the amino acid sequence represented by No. 9 Say. Particularly preferred as the recombinant PLA1 of the present invention are amino acids 1 to 269 of SEQ ID NO: 2 in the sequence listing.
And a polypeptide having the amino acid sequence represented by

Further, as the DNA of the present invention, a DNA encoding a polypeptide having an amino acid sequence represented by amino acid numbers 1-269 of SEQ ID NO: 2 in the sequence listing is preferable. More preferably, a DNA having a nucleotide sequence represented by nucleotide numbers 1 to 888 of SEQ ID NO: 1 in the sequence listing can be mentioned, but is not limited thereto.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The DNA of the present invention
It can be obtained by preparing mRNA encoding PLA1 polypeptide from a microorganism producing LA1 polypeptide, and then converting the mRNA to double-stranded DNA by a known method. In the present invention, Aspergillus oryzae (Aspergillus oryzae) is preferable as the microorganism that can be a source of the mRNA, and furthermore, Aspergillus oryzae (Ahlburg) Cohn SANK 11870.
(FERM BP-3887) strain is preferred.

For the extraction of mRNA, a guanidine thiocyanate hot phenol method, a guanidine thiocyanate-guanidine hydrochloride method or the like can be employed, but a guanidine thiocyanate cesium chloride method is preferred.

The mRNA obtained as described above is expressed in PL
To confirm that it encodes the A1 polypeptide, it is necessary to translate the mRNA and examine the physiological activity,
A method such as identifying the protein using an antibody specific to the protein can be used. For example, mRN is added to Xenopus laevis oocytes.
A can be injected and translated [Gurdon, JB, et al. (1
972) Nature 233 , 177-182], or translation reactions using rabbit reticulocytes or wheat germ lines have been performed [Schleif, RF and Wensink, PC (1981) "Prac".
tical Methods in Molecular Biology "Springer Verla
g, NY].

After synthesizing a single-stranded DNA using reverse transcriptase with the mRNA obtained by the above-described method as a template, a double-stranded DNA is synthesized from the single-stranded DNA. Method [Efstratiadis, A., et.
al. (1976) Cell 7 , 279-288], the Rand method [Lan
d, H., et al. (1981) Nucleic Acids Res. 9 , 2251-22
66], O. Joon Yoo method [Yoo, OJ, et al. (198
3) Proc. Natl. Acad. Sci. USA 79 , 1049-1053], but for the purpose of the present invention, the Okayama-Berg method [Okayama, H. and Berg, P. (1982) Mol. Cell. Biol.
2 , 161-170].

Next, the obtained recombinant plasmid is introduced into a microorganism such as Escherichia coli and transformed, and a recombinant can be selected using tetracycline resistance or ampicillin resistance as an index. Transformation of host cells was performed by the method of Hanahan [Hanahan, D. (1983) J. Mol. Biol. 166 ,
557-580], that is, by adding the recombinant DNA to competent cells prepared in the presence of calcium chloride, magnesium chloride or rubidium chloride. In addition, a phage vector such as a lambda system can be used in addition to the plasmid.

As a method for selecting a strain having a DNA encoding the desired PLA1 polypeptide from the transformants obtained as described above, for example, the following various methods can be used.

[1] Synthetic oligonucleotide probe
When the whole or a part of the amino acid sequence of the target protein is clarified (the sequence may be that of any region of the target protein as long as the sequence is a plurality of specific sequences), An oligonucleotide corresponding to the amino acid sequence is synthesized (in this case, either a nucleotide sequence derived using codon usage or a plurality of nucleotide sequences obtained by combining possible nucleotide sequences, and in the latter case, inosine is included. Can be used as a probe (labeled with a radioisotope such as ³² P, biotin, digoxigenin, etc.) and hybridized with the DNA of a transformant immobilized on a nitrocellulose filter or nylon filter. Let it soy, and use the method appropriate for the probe labeling method. Search for and select Tive strains.

[2] Prepared by polymerase chain reaction
Screening method using the probe, when all or a part of the amino acid sequence of the target protein has been elucidated, oligonucleotides of sense strand and antisense strand corresponding to a part of the amino acid sequence are synthesized, and these are combined. Polymerase chain reaction [Saiki, RK, et al. (1988) Science
239 , 487-491] to amplify a DNA fragment encoding the desired PLA1 polypeptide. The template DNA used here is cDN synthesized by reverse transcription from mRNA of a cell producing PLA1 polypeptide.
A, or genomic DNA can be used. The DNA fragment thus prepared is labeled as in the above [1], and colony hybridization or plaque hybridization is performed using this as a probe to select a desired clone.

[3] Antibodies to PLA1 polypeptide
In advance, cDNA is incorporated into an expression vector, a protein is produced in a transformant, and a desired PLA1 polypeptide-producing strain is detected using an antibody against the PLA1 polypeptide and a secondary antibody against the antibody. And select the target strain.

[4] Selective hybridisation
After the cDNA resulting from the process transformants using a system ® emission Translation, blotted to nitrocellulose filters, and the like, and hybridized with mRNA from PLA1 polypeptide-producing cells, cDNA
The mRNA bound to A is dissociated and recovered. Injecting the recovered mRNA into a protein translation system such as Xenopus oocytes, or translating the protein into a cell-free system such as rabbit reticulocyte lysate or wheat germ, and examining the PLA1 activity of the protein or PLA
A target strain is selected by detection using an antibody against one polypeptide.

Using the mRNA described above as a material, cDN
In addition to the method for preparing A, a DNA fragment encoding a certain enzyme can be generally prepared by removing DNA containing a nucleotide sequence corresponding to the enzyme from a donor cell and treating it with a restriction enzyme or the like. .

That is, the Aspergillus oryzae of the present invention
A method for collecting DNA encoding the PLA1 polypeptide of Orize can be obtained by a known method [Maniatis, T., et al. (19)
82) "Molecular Cloning: A Laboratory Manual" Cold S
pring Harbor Laboratory, NY].
For example, it can be carried out by separating a fraction corresponding to plasmid DNA from cells and cutting out a DNA region encoding the polypeptide from the plasmid DNA.

In general, eukaryotic genes are known as polymorphisms (poly-
morphism) [eg Nishi, T., et.
al. (1985) J. Biochem. 97 , 153-159], one or more amino acids may be substituted by this polymorphism, or the amino acid may be changed completely even though the nucleotide sequence changes. Not always. PLA1 consisting of amino acid numbers 1-269 represented by SEQ ID NO: 2 in the sequence listing
A protein in which one or more amino acid residues have been deleted, inserted or substituted at one or more sites in the amino acid sequence of a polypeptide may also have PLA1 activity. In the present invention, these proteins are referred to as PLA1 synonyms.

For example, it has already been known that a protein obtained by converting a nucleotide sequence corresponding to cysteine of the interleukin 2 (IL-2) gene to a nucleotide sequence corresponding to serine retains IL-2 activity. [Wang, A., et al. (1984) Science 224 , 1431-14
33]. Therefore, as long as these naturally occurring or artificially synthesized proteins have PLA1 activity, all DNAs consisting of the same nucleotide sequence encoding those proteins are also included in the present invention.

Such various DNAs of the present invention can be prepared, for example, by the phosphite triester method [Hunkapiller, M., et al. (1)
984) See Nature 310 , 105-111] and the like, and can be produced by chemical synthesis of nucleic acid.

The codon for the desired amino acid sequence is known per se, and its selection may be arbitrarily determined. For example, it can be determined according to a conventional method in consideration of the codon usage of the host to be used [Grantham, R., et al. . (1981) Nucleic Acid
s Res. 9 , r43-r74]. Further, partial modification of the codons of these nucleotide sequences can be performed by a conventional method using site-specific mutagenesis (Mark, DF, et a) using a primer composed of a synthetic oligonucleotide encoding the desired modification.
l. (1984) Proc. Natl. Acad. Sci. USA 81 , 5662-5666.
Reference].

Further, a certain DNA is the DNA of (3) of the present invention.
Whether to hybridize with A can be examined as follows. That is, first, the DNA of the test sample is subjected to agarose gel electrophoresis as necessary, and then blotted on a membrane such as nitrocellulose or nylon, and the adsorbed DNA is fixed to the membrane by heat treatment, ultraviolet irradiation, or the like. The DNA of (3) of the present invention was prepared by the random primer method [Fein
berg, AP, et al. (1983) Anal.Biochem., 132 , 6-1
3], nick translation method [Maniatis, T.,
et al. (1982) "Molecular Cloning: A Laboratory Ma
radiopharmaceuticals such as ³² P, biotin,
A probe labeled with digoxigenin or an enzyme is prepared. After immersing the membrane in a hybridization solution containing the probe and incubating at a predetermined temperature, the membrane is washed and the probe is detected by a method suitable for each label.

The concentration of SSC (salined-sodium citrate: “sodium citrate-saline”; 1 × SSC contains 0.15 M sodium chloride and 15 mM sodium citrate) contained in the hybridization solution is preferable. Is 4 to 8 × SSC. The incubation temperature is preferably 30 to 70 ° C. In the present invention, a combination of the SSC concentration and the incubation temperature in the above range is referred to as “stringent conditions”. Such conditions include, in particular, 6 × SS
C, the condition of 60 ° C. is most preferable.

By utilizing the above method, a DNA that hybridizes with the DNA of (3) of the present invention can be cloned from various cDNA libraries or genomic libraries.

The nucleotide sequence of the thus obtained DNA of the present invention can be determined, for example, by the chemical modification method of Maxam-Gilbert [Maxam, AM and Gilbert, W. (1980).
Methods in Enzymology 65 , 499-559], M13
Dideoxynucleotide chain termination using phage [Me
ssing, J. and Vieira J. (1982) Gene 19 , 269-276].

A fragment containing the gene encoding the cloned PLA1 polypeptide is ligated to an appropriate vector DNA.
Can be transformed into other prokaryotic or eukaryotic host cells. further,
Genes can be expressed in each host cell by introducing an appropriate promoter and a sequence related to expression into these vectors.

As prokaryotic host cells, for example, Streptomyces lividans, Escherichia coli and Bacillus subtili
s) and the like. In order to express the gene of interest in these host cells, the host cells may be transformed with a replicon derived from a species compatible with the host, that is, a plasmid vector containing an origin of replication and regulatory sequences. In addition, the vector provides the phenotype (phenotype)
Those having a sequence capable of imparting selectivity to are preferred.

As a vector, pBR322 or p
UC-based plasmids are often used, but are not limited thereto, and any of various known strains and vectors can be used. As a promoter, tryptophan (trf) promoter, lactose (la
c) promoter, tryptophan lactose (ta)
c) promoter, lipoprotein (lpp) promoter, the lambda-derived bacteriophage (lambda) P _L promoter, polypeptide chain elongation factor Tu (tufB) promoter.

As eukaryotic microorganisms, yeast and Aspergillus oryzae are generally used, and among them, yeast of the genus Saccharomyces, for example, Saccharomyces cerevisiae or Aspergillus oryzae is preferable. In preparing an expression vector for a eukaryotic microorganism such as the yeast, for example, an alcohol dehydrogenase gene promoter [Bennetzen, JL and Hall, B. et al.
D. (1982) J. Biol. Chem. 257 , 3018-3025] and the promoter of the acid phosphatase gene [Miyanohara,
A., et al. (1983) Proc. Natl. Acad. Sci. USA 80 , 1
-5] can be preferably used. As an expression vector for Aspergillus oryzae, pTAex3 [Fu
jii, T., et al. (1995) Biosci. Biotech. Biochem. 5
9 , 1869-1874].

The transformed cells of the present invention thus obtained include Saccharomyces cerevisiae KS58
The -2D / pCY339 strain or the Aspergillus oryzae M-2-3 / pAAP strain are preferred.

The desired transformed cells obtained above can be cultured according to a conventional method, and the recombinant PLA1 polypeptide is produced intracellularly or extracellularly by the culture.
As the medium used for the culture, various types commonly used depending on the employed host cells can be appropriately selected.

For example, when E. coli is used as a host,
As the medium, any of those well known to those skilled in the art such as LB medium, 2 × YT medium, L medium, broth medium, M9 minimum medium and the like can be used. The culture temperature is preferably 15
To 40 ° C, more preferably 25 to 37 ° C. The time required for the culture is usually within 10 days under anaerobic or aerobic conditions, preferably 8 hours to 3 days.

When yeast is used as a host, any medium known to those skilled in the art such as YPD medium, MY medium, malt extract medium, and synthetic minimum medium can be used. The culture temperature is preferably 15 to 40.
° C, more preferably 20 to 30 ° C. The time required for the culture is usually within 20 days under anaerobic or aerobic conditions, preferably 3 to 7 days.

Further, when COS-1 cells are used as a host, a minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), Ham's F12 medium, RPMI1640 medium and the like can be used as the medium. The culture temperature is preferably between 30 and 37 ° C, most preferably 37-37 ° C.
° C. Further, it is desirable that the concentration of carbon dioxide in the air in the incubator is adjusted to 5 to 10%, most preferably 5%.

In addition, depending on the host cell to be used, a medium and culture conditions capable of growing the host cell and producing a protein can be used.

The PLA1 polypeptide produced intracellularly or extracellularly by the above-mentioned method is the PLA1 polypeptide.
Various known separation procedures utilizing the physical and chemical properties of the A1 polypeptide can be used to separate and separate from them.
It can be purified. Specific examples of the method include treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography (HPLC). ), Various liquid chromatography methods, dialysis methods, combinations thereof, and the like.

Whether or not the recombinant PLA1 polypeptide obtained by the present invention has phospholipase activity can be confirmed, for example, by the method described below.

[0043] [1] lecithin as a substrate, a method for quantifying the free fatty acids [see JP-A 6-62850]; [2] ^1-[1-14 C] oleoyl glycerol or ^1-[1-14 C] palmitoyl A method for measuring the radioactivity of free fatty acids using -2-acyl-sn-glycero-3-phosphoethanolamine as a substrate [Waite, M., et al. (1)
974) J. Biol. Chem. 249 , 6401-6405].

According to the above method, the desired recombinant PLA1 polypeptide can be easily produced on an industrial scale with high yield and high purity. The recombinant PLA of the present invention thus obtained
The properties of 1 polypeptide are common to those of natural PLA1 (see Japanese Patent Application Laid-Open No. 6-62850) already described in the literature.
It can be widely used in the pharmaceutical industry, mainly for lysophospholipid production.

The recombinant PLA1 of the present invention is a very useful phospholipase which has excellent activity and selectivity, and can be constantly supplied in large quantities at low cost. The enzymatic reaction for decomposing phospholipids using the present enzyme is carried out by contacting the enzyme with a substrate in an aqueous solvent or in a wet state. The substrate used is, for example, lecithin such as flour, soybean and egg yolk, and the concentration thereof is preferably 1 to 50.
% By weight. Well water or tap water can be used as it is, but for more efficient enzyme reaction,
Add acid (eg acetic acid), alkali (eg sodium hydroxide) or buffer (eg acetate buffer)
H may be adjusted to 3 to 7 (more preferably, 3.5 to 5.5) before use. The reaction temperature is between 10 ° C and 7
0 ° C (preferably 20 ° C to 65 ° C, particularly preferably 30 ° C
To 60 ° C.), and the time required for the reaction varies depending on the substrate, reaction temperature, pH and the like, but is usually from 10 minutes to 10 days (preferably from 1 hour to 2 days).

After completion of the enzymatic reaction, the obtained lysophospholipid can be directly used for processing without separation. In addition, an insoluble substance can be filtered off by a conventional method and used directly, or a suitable lysophospholipid can be obtained by concentration. Furthermore, if necessary, water can be added, extraction can be carried out with a water-immiscible organic solvent, and the extraction solvent can be distilled off. Further, if necessary, it can be further purified by a conventional method, for example, a column chromatography, a recrystallization method or the like.

[0047]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

Embodiment 1 Partial amino acid sequence of PLA1
PLA1 was determined by Aspergillus oryzae (Ahlburg) Cohn SANK
11870 (FERM BP-3887) strain (hereinafter “SAN
K11870 strain) according to the method of Hattori et al. [See JP-A-6-62580]. The molecular weight calculated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (hereinafter referred to as “SDS”) is 37.
Of PLA1a (0.1 mg / ml) corresponding to 2,000
0 mM sodium acetate buffer (pH 4.5) 0.45 m
Then, 45 μl of trichloroacetic acid containing 0.4% (w / v) deoxycholic acid (manufactured by Sigma) was added to
Centrifugation was performed at 0 rpm and 4 ° C. for 10 minutes. 0.68 ml of cold acetone was added to the precipitate and 10,000 rpm
m, and centrifuged at 4 ° C for 10 minutes, and the precipitate was dried under reduced pressure.
This precipitate was washed with a tris-guanidine solution [0.5 M Tris-HCl buffer containing 6 M guanidine hydrochloride (pH 8.
5)] and dissolved in 90 μl, and nitrogen gas was blown for 1 minute. To this, 4.5 μl of a tris-guanidine solution containing 100 mM dithiothreitol was added, and nitrogen gas was blown again for 2 minutes. After standing at room temperature for 2 hours, 100m
M iodoacetamide solution (9 μl) and add
Let stand for 0 minutes. This solution was dialyzed against distilled water at 4 ° C. overnight and dried under reduced pressure. Add 8M urea to 30μ
The mixture was kept at 37 ° C. for 1 hour. To this, 43 μl of a digestion buffer (CTFF kit, manufactured by Shimadzu Corporation) was added, and 13 μl of 10 mg / ml lysyl endopeptidase was further added, followed by incubation at 37 ° C. overnight.

Next, 0.1% (v / v) was added to the reaction solution.
0.3 ml of trifluoroacetic acid was added, and the whole amount was fractionated by reverse phase HPLC. HPLC conditions were as follows.

Column: Cosmoseal 5C8-AR-30
0 (size 4.6 × 10 mm, manufactured by Nacalai Tesque); flow rate: 0.5 ml / min; mobile phase: 0.1% (v / v) trifluoroacetic acid;
The concentration of acetonitrile was eluted with an initial concentration of 5% (v / v) and a final concentration of 80% (v / v) with a 15 minute gradient; detection wavelength: 210 nm.

The elution time was 5.2 minutes and 6.8 under the above conditions.
Fraction, 8.0 minutes, 9.2 minutes and 9.8 minutes were fractionated, and each peak was separated using a gas phase protein sequencer (model PPSQ-10; manufactured by Shimadzu Corporation). Automatic Edman method [Edman, P., et al. (1967) E
ur. J. Biochem. 1 , 80]. The amino acid sequence of each of the above peaks was as described below: Elution time (minutes) Sequence 5.2 SEQ ID NO: 3 6.8 Sequence Listing SEQ ID NO: 4 8.0 Sequence Listing SEQ ID NO: 59 .2 SEQ ID NO: 6 in the Sequence Listing 9.8 SEQ ID NO: 7 in the Sequence Listing

Embodiment 2 FIG . Aspergillus oryzae m
Preparation of RNA The SANK 11870 strain was cultured in a medium (1% (w / v) polypeptone (Nippon Pharmaceutical Co., Ltd.), 5% (w / v) sucrose, 0.5% (w / v) yeast extract ( 0.1% (w / v) magnesium sulfate.7
Hydrate, 0.5% (w / v) calcium carbonate, 0.8%
(W / v) Tween 80 (manufactured by Daiichi Pure Chemicals Co., Ltd.), 0.5% (w / v) sodium glutamate,
% (W / v) lecithin (manufactured by Hoshinen Co., Ltd.), 0.25%
(W / v) dipotassium hydrogen phosphate-0.25% (w / v)
v) Potassium dihydrogen phosphate buffer (pH 6.6)) 10
Inoculate 500 ml Erlenmeyer flask containing 0 ml
The cells were cultured at 210 ° C. and 210 rpm. 3 days, 5 days and 7 days
After culturing for one day, the cells were collected, immediately frozen with liquid nitrogen, and stored at -80 ° C.

Next, 3 g of the cryopreserved cells were crushed in a mortar previously cooled on dry ice until they became powdery. This was mixed with a guanidine thiocyanate solution (4M guanidine thiocyanate (Fluka), 4% (w / v) sarkosyl (Sigma), 0.1% (w / v) Antiform A (Sigma), 20 mM EDTA. 2Na, 4
The mixture was transferred to a centrifuge tube filled with 15 ml of mM 2-mercaptoethanol and 25 mM trisodium citrate (pH 7.0), vigorously stirred, and then disrupted for 30 seconds using a polytron homogenizer. This is 900
Centrifuge at 0 rpm for 15 minutes at 4 ° C.
Centrifugation was performed at 000 rpm and 4 ° C. for 15 minutes. 7m of supernatant
l was placed in an ultracentrifuge tube (1) containing 3 ml of a 5.7 M cesium chloride solution containing 0.1 M EDTA · 2Na.
3PA: manufactured by Hitachi, Ltd.)
The mixture was centrifuged at 4 rpm at 4 ° C for 15 hours. After centrifugation, the precipitate was washed with 10 ml of 0.3 ml of 1 mM EDTA · 2Na.
It was dissolved in a Tris-HCl buffer (pH 8.0) (hereinafter referred to as “TE”). To this was added 30 μl of a 3M acetic acid-sodium acetate buffer (pH 5.2) and 1 ml of ethanol. This is 14500 rpm at 4 ° C.
The mixture was centrifuged for 0 minutes, 80% (v / v) ethanol was added to the precipitate, and the mixture was centrifuged again at 14,500 rpm at 4 ° C. for 5 minutes. The precipitate was dried under reduced pressure, and dissolved in 40 μl of TE to obtain a total RNA sample.

The total RNA thus obtained was subjected to oligo (dT) cellulose column chromatography to purify poly (A) ⁺ RNA. That is, the total RNA (540 μg) was added to the adsorption buffer (0.5 M
Sodium chloride, 20 mM Tris-HCl (pH 7.
5) 1 mM EDTA, 0.1% (w / v) SDS)
And heated at 65 ° C. for 5 minutes, and then an oligo (dT) cellulose column (Type) filled with an adsorption buffer.
7; Pharmacia) and an elution solution (10 mM
Tris-HCl (pH 7.5), 1 mM EDTA,
Poly (A) ⁺ RNA was eluted with 0.05% (w / v) SDS and recovered. By such an operation, 20 μg of poly (A) ⁺ RNA was obtained.

Embodiment 3 FIG . Aspergillus oryzae c
Preparation of DNA library mRNA of SANK 11870 strain obtained in Example 2
(3 μg) in 5 mM Tris-HCl buffer (pH 7.
5) Dissolved in 15 μl. This was heated at 65 ° C. for 5 minutes, then cooled to 37 ° C., and a 10-fold concentration of reverse transcriptase buffer (Tris-HCl buffer containing 80 mM magnesium chloride, 300 mM potassium chloride, and 3 mM dithiothreitol (pH 8.5) )) 3 μl, 20 mM dATP,
20 mM dCTP, 20 mM dGTP and 20 mM
3 μl of a mixed solution of dTTP and 3 μl of 0.5 μg / μl vector primer (pCDV1 oligo (dT) tailed plasmid; manufactured by Pharmacia), 27 units / μl reverse transcriptase (avian myeloblastosis virus (avian myeloblastosis virus (avian
myeloblastosis virus); manufactured by Seikagaku Corporation
1.7 μl and 4.3 μl of distilled water were added, and the mixture was added at 37 ° C. for 30 minutes.
Incubated for a minute.

To the reaction mixture, 1/2 volume of TE-saturated phenol and 1/2 volume of chloroform were added, vigorously shaken, and then centrifuged at 10,000 rpm for 5 minutes. The aqueous layer was collected, １ ／ volume of TE-saturated phenol and 容量 volume of chloroform were added thereto, vigorously shaken, and centrifuged at 10,000 rpm for 5 minutes. The aqueous layer was collected again, the same amount of chloroform was added thereto, and the mixture was vigorously shaken, followed by centrifugation at 10,000 rpm for 1 minute.
The aqueous layer was collected (hereinafter, this operation is referred to as "phenol / chloroform extraction").

To the collected aqueous layer, 1/10 volume of a 3M acetic acid-sodium acetate buffer (pH 6.5) and 2.5 volumes of ethanol were added, and the mixture was cooled at -80 ° C. for 15 minutes, and then cooled.
Centrifugation was performed at 0000 rpm for 5 minutes. 80% precipitation
(V / v) After washing with ethanol, it was dried under reduced pressure (hereinafter, referred to as
This operation is called "ethanol precipitation").

13 μl of distilled water and 2 μl of 1
Buffer solution for terminal deoxynucleotidyl transferase reaction at 0-fold concentration (0.3 M Tris-HCl (pH 7.6) containing 1.4 M sodium cacodylate, 10 mM cobalt chloride), 2 μl of poly (A), 1 μl
2 mM dithiothreitol, 0.6 μl 2 mM d
ATP, 2 mM dCTP, 2 mM dGTP and 2
0.5 μl of a mixed solution of mM dTTP and 21 units / μl of terminal deoxynucleotidyl transferase (Pharmacia) were added, and the mixture was kept at 37 ° C. for 5 minutes. The reaction solution was extracted with phenol / chloroform and precipitated with ethanol. The resulting precipitate was dissolved in 24 μl of TE, and 30 units of HindIII was added thereto to add 3 units of HindIII.
Incubated at 7 ° C overnight. The reaction solution was extracted with phenol / chloroform and precipitated with ethanol.
E dissolved in Aspergillus oryzae cDNA
A sample was used.

On the other hand, the plasmid vector pcDL-SR
α296 [Takebe et al., (1989) Experimental Medicine Vol. 7, 95-9
9] with the restriction enzyme PstI, and further 3 ′
DGTP was added to the terminal by terminal deoxynucleotidyl transferase (Pharmacia). Then, this was digested with the restriction enzyme HindIII, whereby oligo (dG) was added to the SRα promoter.
A linker DNA with oligo (dG) to which was added was prepared.

To 1 μl of the Aspergillus oryzae cDNA sample, 0.5 μl of the above linker DNA with oligo (dG) (equivalent to 0.08 pmol), and 2 μl of a 5-fold concentration hybridization buffer (5 mM EDTA, 5 mM) were added.
A 50 mM Tris-HCl buffer (pH 7.5) containing 00 mM sodium chloride and 6.5 μl of distilled water were added, and the mixture was heated at 65 ° C. for 5 minutes, and then kept at 43 ° C. for 30 minutes. To this, 9 μl of 10-fold concentration of ligation buffer (manufactured by Amersham), 68 μl of distilled water and 1
Add 10 μl of 10 mM β-nicotinamide adenine dinucleotide (Boehringer Mannheim), cool on ice for 10 minutes, then add 2 μl of 0.28 μg / μl DNA
Ligase (Pharmacia) was added, and the mixture was kept at 12 ° C. overnight. In this reaction solution, 2 mM dATP, 2 mM
2 μl of a mixed solution of dCTP, 2 mM dGTP and 2 mM dTTP, 0.5 μl of 10 mM β-nicotinamide adenine dinucleotide, 0.28 μg / μl of D
NA ligase (derived from E. coli; Pharmacia) 1.5
1 μl of 4 units / μl of DNA polymerase I (manufactured by Pharmacia) and 4 μl of 1 units / μl of ribonuclease H (manufactured by Pharmacia) were added, and the mixture was kept at 12 ° C. for 1 hour. The reaction solution was extracted with phenol / chloroform and precipitated with ethanol, and the precipitate was dissolved in 100 μl of distilled water.

Embodiment 4 FIG . Aspergillus orize dyeing
Preparation of Chromosome DNA Library 3 g of the SANK 11870 strain cells cultured and frozen at -80 ° C. by the method described in Example 2 were crushed in a mortar cooled with dry ice until they became powdery. The crushed cells were previously added to 20 ml of 62.5 mM E.
50 mM containing DTA · 2Na, 5% (w / v) SDS
The mixture was transferred to a centrifuge tube filled with a Tris-HCl buffer (pH 8), stirred vigorously, and allowed to stand on ice for 1.5 hours. To this, 10 ml of TE-saturated phenol was added and gently shaken at 50 ° C. for 1 hour. Thereafter, 5 ml of TE-saturated phenol was further added, and the mixture was centrifuged at 3000 rpm for 10 minutes. The aqueous layer was collected in another centrifuge tube, and 7.5 ml of TE-saturated phenol and 7.5 ml of chloroform were added.
After vigorous stirring, the mixture was centrifuged at 3000 rpm for 10 minutes. The aqueous layer was collected again in another centrifuge tube, and １ ／ volume of 8M
Add ammonium acetate solution and 25 ml of ethanol,
After cooling at -80 ° C for 15 minutes, the mixture was centrifuged at 10,000 rpm and 4 ° C for 10 minutes. Dissolve the precipitate in 5 ml TE,
100 μl of a solution containing 2 mg / ml ribonuclease A and 2500 units / ml ribonuclease T1
The mixture was added and kept at 37 ° C. for 20 minutes. 20 ml of isopropanol was added thereto, and the mixture was gently mixed. The resulting filamentous DNA was picked up with tweezers.
Dissolved in 8 ml TE.

[0062] To this DNA solution was added 1/2 volume of TE-saturated phenol and 1/2 volume of chloroform, and after vigorous stirring, centrifuged at 15000 rpm for 5 minutes to collect an aqueous layer. This operation was repeated three more times, and an equal amount of chloroform was added to the obtained aqueous layer.
After centrifugation at 00 rpm for 5 minutes, the aqueous layer was collected. 1/10 volume of 3M sodium acetate (pH 6.
After adding 5) and 2.5 volumes of ethanol, the mixture was cooled at -80 ° C for 15 minutes, and then centrifuged at 15000 rpm for 5 minutes to collect a precipitate. 1 ml of 80% (v /
v) Ethanol was added, and the mixture was centrifuged at 15000 rpm for 5 minutes to collect a precipitate. The precipitate is dried under reduced pressure,
What was dissolved in E was used as purified Aspergillus oryzae chromosomal DNA.

Next, Aspergillus oryzae chromosome D
NA (equivalent to 15 μg) was converted to 400 units of restriction enzyme Pst
After digestion with I (37 ° C., 12 hours), ethanol precipitation was performed.

On the other hand, the plasmid pUC18 equivalent to 3 μg was used.
(Manufactured by Takara Shuzo Co., Ltd.) with 100 units of restriction enzyme PstI
And precipitated with ethanol. The precipitate was washed with 200 μl of T
E, 2.5 μl / μl of alkaline phosphatase (CAP-101; manufactured by Toyobo Co., Ltd.) (1 μl) was added, the mixture was incubated at 37 ° C. for 30 minutes, extracted with phenol / chloroform, and precipitated with ethanol. .

The Aspergillus oryzae chromosomal DNA (equivalent to 500 ng) digested with the restriction enzyme PstI was ligated to pUC18 similarly digested with PstI using a DNA ligation kit (Takara Shuzo Co., Ltd.). Escherichia coli JM109 was transformed with this reaction solution [Hanahan, D. (1
983) J. Mol. Biol. 166 , 557-580] to prepare a chromosomal DNA library of Aspergillus oryzae.

Embodiment 5 FIG . PLA1 inheritance by PCR method
Partial amino acid sequence of the cloned child PLA1: Asp Leu Phe Ala Gln Tyr mixing 17mer encoding (amino acid numbers 11-16 of SEQ ID NO: 6 of the Sequence Listing) sense primer: 5'- GA (CT) (TC ) T ( ACGT) TT (CT) G CICA (AG) TA -3 ′ (SEQ ID NO: 8 in the sequence listing; the portion enclosed in parentheses in the sequence represents any one of the nucleotides in the parentheses) By the solid phase phosphoramidite method [Beaucage, et al. (1981)
See Tetrahedron Letters 22 , 1859-1862].

A 16-mer mixed antisense primer encoding another partial amino acid sequence of PLA1: Asp Glu Phe Asn Glu Ser (amino acids 20 to 25 of SEQ ID NO: 5 in the sequence listing): 5 ′-(GC) ( AT) TC (AG) TT (AG) AA (CT) TC (AG) TC-3 ′ (SEQ ID NO: 9 in Sequence Listing) was chemically synthesized. The primer was synthesized using a DNA synthesizer (Model 380A; manufactured by Applied Biosystems).

Using the Aspergillus oryzae cDNA library prepared in Example 3 as a template, 925 ng of the mixed sense primer, 925 ng of the mixed antisense primer, 0.2 mM dATP, 0.2 mM dC
TP, 0.2 mM dGTP, 0.2 mM dTTP,
1 containing 50 mM potassium chloride, 1.5 mM magnesium chloride and 2.5 units of Taq DNA polymerase
In 0.1 ml of 0 mM Tris-HCl buffer (pH 8.3), 1 cycle at 94 ° C for 1 minute, 50 ° C for 1 minute,
30 cycles of PCR were performed at 2 ° C. for 2 minutes.

From the reaction product obtained by PCR, 0.1
A 14 kbp fragment was cut out and blunt-ended using a DNA blunting kit (Takara Shuzo Co., Ltd.).
Plasmid pUC18 (manufactured by Takara Shuzo Co., Ltd.) was prepared using NA ligation kit (manufactured by Takara Shuzo Co., Ltd.).
By inserting into the aI cleavage site, the plasmid pRA
V12 was constructed.

0.14 kbp inserted into pRAV12
The nucleotide sequence of the fragment was analyzed by the dideoxy method. That is, the plasmid pRAV12 was transformed by the method of Chang et al. [Zhang, H., et al. (1988) Nucleic Aci
ds Res. 16 , 1220], and denatured with template DNA
Was prepared, and the nucleotide sequence was analyzed using 7-Deaza Seek Nace Version 2.0 kit (manufactured by Toyobo Co., Ltd.). The primer DNA for the DNA polymerase reaction of the kit may be M13 primer M4 (Takara Shuzo Co., Ltd.) or M13 primer R
V (Takara Shuzo Co., Ltd.) was used.

As a result, 0.14 kb in pRAV12
p DNA fragments are Ser Ala Ala Ala Tyr Cys Asp Glu Asn Leu and Ser Val Gly Asn Cys Pro Leu Val Glu Ala Ala Ser Th
It contained an open reading frame encoding the amino acid sequence of rGln Ser Leu. The above sequence is the P determined in Example 1.
Since the partial amino acid sequence of LA1 was identical to a part of the partial amino acid sequence (SEQ ID NO: 6 and SEQ ID NO: 5 in the sequence listing, excluding cysteine which cannot be analyzed), it was confirmed that this DNA fragment encoded the PLA1 gene. Was.

Embodiment 6 FIG . Colony hybridization
Chromosome D of Aspergillus oryzae produced in ® emissions Example 4
Escherichia coli JM109 strain containing the NA library was added to a TY medium (10 g / l Bacto tryptone (Difco), 5 g / l yeast extract (Difco), 1.5% (w / v) agar powder. And 100
(g / ml ampicillin) was inoculated so as to have about 2000 colonies per culture plate (9 cm in diameter). After overnight culture at 37 ° C., a circular nitrocellulose filter (HA, pore diameter 0.45 μm, diameter 82 m
m: Millipore Co.) was placed on the medium to transfer E. coli colonies in the plate. This filter
Placed on fresh TY medium and cultured overnight at 37 ° C.

A nitrocellulose filter on which colonies of Escherichia coli grew was filtered with a filter paper impregnated with 0.5 M sodium hydroxide (No. 2: manufactured by Advantech Co., Ltd.).
And left standing at room temperature for 5 minutes to lyse Escherichia coli. Next, the filter was placed on a filter paper impregnated with 1 M Tris-HCl buffer (pH 8), and allowed to stand at room temperature for 5 minutes. Further, the filter was placed on a filter paper impregnated with 1 M Tris-HCl buffer (pH 8) containing 1.5 M sodium chloride, and allowed to stand at room temperature for 5 minutes.
C solution (0.03M containing 0.3M sodium chloride)
(Trisodium citrate dihydrate solution) was placed on a filter paper impregnated with the solution, and allowed to stand at room temperature for 5 minutes. After air-drying the filter, it was heated at 80 ° C. for 2 hours, and then 6 × SSC (0.9%).
Washed thoroughly in 0.09 M trisodium citrate dihydrate solution containing M sodium chloride) and air dried.

The labeling of the probe was performed by the method described below. First, 20 μg of pRAV12 was digested with 200 units of EcoRI (37 ° C., 12 hours). This was subjected to 1 × using 6% (w / v) polyacrylamide gel.
TBE buffer (50m containing 1mM EDTA · 2Na)
After electrophoresis at 100 V in M Tris-borate buffer (pH 8.3), the D of the band corresponding to 0.14 kbp was measured.
The NA fragment was cut out. This 0.14 kbp DNA
The fragment was labeled using a nick translation kit (Amersham) and [α- ³² P] -dCTP.
Unreacted [α- ³² P] -dCTP was removed using a nick column (Pharmacia).

Hybridization was performed by the following method. That is, the air-dried nitrocellulose filter was passed through a hybridization solution (40% (v / v
v) formamide, 5 × SSC (0.15 M sodium chloride, 0.015 M trisodium citrate), 5 ×
Denhardt's solution (bovine serum albumin (Sigma) 1
g / liter, Ficoll 400 (Pharmacia)
1 g / liter, polyvinylpyrrolidone (Sigma)
1 g / liter), 0.5% (w / v) SDS and 1
It was immersed in 10 mM phosphate buffer (pH 7.0) containing 00 μg / ml salmon sperm DNA, and kept at 42 ° C. for 2 hours. Thereafter, the probe labeled as described above was added, and the mixture was kept at 42 ° C. overnight in the same hybridization solution. The filters were then filtered with 0.1% (w / v) S containing 40% (v / v) formamide and 5 × SSC.
It was transferred into a DS solution and washed at 42 ° C. for 30 minutes. Further, the filter was washed in 2 × SSC at 42 ° C. for 1 hour, air-dried, and subjected to autoradiography.

Approximately 52,500 colonies were screened by the above method, and three positive clones were selected. The plasmids prepared from these positive clones were each digested with the restriction enzyme PstI to select a clone that produced a band equivalent to 4.3 kbp in addition to the band equivalent to 2.7 kbp derived from pUC18, and the plasmid retained by the clone was selected. Was named pRAV43.

Embodiment 7 FIG . CDN encoding PLA1
Cloning of A Analysis of the nucleotide sequence of PLA1 genomic DNA contained in pRAV43 was carried out by using a dideoxy method and a DNA sequencer in combination. In the case of the dideoxy method, pRAV43 was alkali-denatured by the method of Chang et al. To prepare a template DNA. The sequence was determined using 7-Deaza Seek Nace version 2.0.
This was performed using the kit.

When a DNA sequencer is used, a sample is prepared using a Dye Terminator Cycle Sequencing Kit (manufactured by PerkinElmer Japan), and a nucleotide sequence is prepared using a DNA sequencer model 373A (manufactured by PerkinElmer Japan). It was determined.

From the nucleotide sequence of PLA1 genomic DNA determined as described above, 3
The presence of two introns was expected. In addition, primers were designed by estimating positions encoding the N-terminal amino acid and the C-terminal amino acid of PLA1 in the base sequence of the PLA1 genomic gene. That is, nucleotide primers having the following sequences: 5′-CGTCCTGCAC GGCATTCAAA-3 ′ (sense primer:
SEQ ID NO: 10 in the sequence listing) and 5'-TCCCTTCTCA AAGCCAGAAT-3 '(antisense primer: SEQ ID NO: 11 in the sequence listing) were synthesized.

Next, 1 μg of the Aspergillus oryzae cDNA library prepared in Example 3 was digested with 20 units of the restriction enzyme PstI (37 ° C., 2 hours). This PstI-treated cDNA library 5n
g, sense primer 200 nM, antisense primer 200 nM, 0.2 mM dATP, 0.2 mM
dCTP, 0.2 mM dGTP, 0.2 mM dTT
P, 10 mM potassium chloride, 6 mM ammonium sulfate, 2 mM magnesium chloride, 0.1% (w / v) Triton X-100, 0.01 mg / ml bovine serum albumin, 20 mM containing 5 units of Pfu DNA polymerase (manufactured by Stratagene) Dissolve in 50 μl of Tris-HCl buffer (pH 8.0) and add 9 per cycle.
30 cycles of PCR were performed at 4 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute. The reaction solution after PCR was extracted with phenol / chloroform and precipitated with ethanol, and the precipitate was dissolved in TE.

This PCR reaction product (equivalent to 20 μg)
10 mM magnesium chloride, 1 mM ATP, T
4 polynucleotide kinase (Takara Shuzo Co., Ltd.) 10
0 units, and 50 containing 5 mM dithiothreitol
400 μl of mM Tris-HCl buffer (pH 7.5) was added, and the mixture was kept at 37 ° C. for 30 minutes. The reaction solution was extracted with phenol / chloroform, and precipitated with ethanol, and the precipitate was dissolved in TE.

On the other hand, after digesting 10 μg of pUC18 with 100 units of SmaI (25 ° C., 3 hours), the reaction solution was extracted with phenol / chloroform and ethanol precipitated, and the precipitate was dissolved in 200 μl of TE. To this, 5 units of alkaline phosphatase (manufactured by Toyobo Co., Ltd.) was added, and the mixture was incubated at 37 ° C. for 30 minutes, extracted with phenol / chloroform, and ethanol-precipitated.
Was dissolved.

The SmaI digested p obtained as described above
UC18 (corresponding to 100 ng) and the phosphorylated PCR reaction product (corresponding to 50 ng) were ligated using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.) to construct plasmid pRAV885.

The nucleotide sequence of the cDNA encoding PLA1 contained in pRAV885 was determined using a dye terminator cycle sequencing kit and a DNA sequencer. By comparing this nucleotide sequence (SEQ ID NO: 1 in the sequence listing) with the nucleotide sequence of the PLA1 genomic DNA, the position of the intron on the genome was determined.

Embodiment 8 FIG . Structure of expression vector for animal cells
Restriction enzyme Sa of pRAV885 100 units of built 10μg
After digestion with cI (37 ° C., 3 hours), the mixture was extracted with phenol / chloroform and precipitated with ethanol. The cut end of this DNA is used as a DNA blunting kit (Takara Shuzo).
Then, the reaction solution was extracted with phenol / chloroform and ethanol precipitated, and the precipitate was dissolved in 0.4 ml of TE. Add 5 units of alkaline phosphatase (Toyobo Co., Ltd.) to this,
After keeping at 37 ° C. for 30 minutes, phenol / chloroform extraction was performed and ethanol precipitation was performed. To this DNA, 0.5
μg of PstI linker (Takara Shuzo Co., Ltd.) was added, and a DNA ligation kit (Takara Shuzo Co., Ltd.) was added.
By closing with Sac, the Sac of pRAV885 is
Plasmid p in which the I-cut sequence was replaced with the PstI-cut sequence
RAV820 was constructed.

Next, pRAV820 was replaced with the restriction enzyme SalI.
And PstI, digested with about 1.
The 0 kbp fragment was isolated and purified.

On the other hand, the high expression vector pME18S [Har
a, T., et al. (1992) EMBO J. 11 , 1875], digested with the restriction enzymes XhoI and PstI, and then reacted with T4 DNA ligase to obtain the SalI-PstI.
The digested DNA fragment was ligated. Escherichia coli was transformed with this DNA, and plasmid DNA was extracted from each of the resulting transformants. Of these transformants, plasmid DNA was digested with the restriction enzyme EcoRI to generate fragments of 0.3 kbp and 3.6 kbp, and digested with the restriction enzyme XbaI to obtain 0.9 kbp and 3.0 kbp.
A clone producing a kbp fragment was selected, and the plasmid carried by the clone was designated as pRAV825.

Embodiment 9 FIG . PL in COS-1 cells
Transfection of COS-1 cells expressing the A1 gene was performed by an electroporation method using a gene transfer apparatus (model GTE-1; manufactured by Shimadzu Corporation). First, COS-1 cells were grown in cell culture flasks in serum-free Dulbecco's modified Eagle's medium (hereinafter referred to as "DMEM") until they became semi-confluent.
Cells are peeled and collected by EDTA treatment, and PBS
(-) After washing twice with a buffer solution (manufactured by Takara Shuzo Co., Ltd.),
The cells were suspended in a PBS (-) buffer solution to a concentration of 6 × 10 ⁷ cells / ml.

On the other hand, pRAV825 and pRAV825 prepared using a DNA purification kit (Wizard Maxiplex DNA Purification System: manufactured by Promega)
ME18S was washed with PBS (-)
g / ml.

The above-mentioned cell suspension and the DNA solution were mixed in an amount of 20 μl each, and the mixture was placed in a chamber with an electrode spacing of 2 mm.
A pulse of 600 V-30 sec was applied twice at 1 second intervals. After cooling the chamber at 4 ° C. for 5 minutes, the cell / DNA mixture in the chamber was diluted with 10% fetal bovine serum.
The cells were added to 10 ml of MEM, transferred to a cell culture dish, and cultured at 37 ° C. under 5% carbon dioxide gas overnight. afterwards,
After removing the culture supernatant, washing the cells with serum-free DMEM, adding 10 ml of DMEM and culturing for 3 days,
The culture supernatant was collected.

Test Example 1 COS-1 cell culture supernatant prepared in Example 9 was 2.5 m each.
1 at 5000 rpm, 4 ° C. using a centrifuge tube with an ultrafiltration membrane (Centricon-10; manufactured by Grace Japan).
Centrifugation for 1.5 hours at 220
It was concentrated to μl and used as a sample for measuring phospholipase activity described below (hereinafter, referred to as “COS-1 / pRAV825” and “COS-1 / pME18S”).

2% (w / v) SLP-white (manufactured by True Lecithin Co .: 4% (v / v
v) 10 ml of Triton X-100 dissolved therein) 0.05 ml of 0.1 M calcium chloride dihydrate solution and 0.2 M acetate buffer (pH
4.0) 0.25 ml was added, and the mixture was preliminarily heated at 37 ° C. for 5 minutes. Then, 0.1 ml of a sample was added, and the mixture was kept at 37 ° C. for 10 minutes. The reaction was stopped by adding 0.1 ml of 1N hydrochloric acid thereto. Immediately after stopping the reaction, the reaction solution
The amount of free fatty acid contained in 1 was determined using an enzyme kit for determining free fatty acid (Determiner: manufactured by Kyowa Medix Co., Ltd.). The enzyme activity for producing 1 μmole of free fatty acid per minute was defined as one unit.

The phospholipase activity of each sample measured by the above method was as shown in Table 1.

[0094]

[Table 1] 検 体 Sample activity (unit / liter) ────────── ─────────────────── COS-1 / pRAV825 4.8 × 10 ² COS-1 / pME18S 0.0 ──────────── Example 10 Construction of Expression Vector for Yeast pCY303 (see Japanese Patent Application Laid-Open No. 5-17132) was used as a starting material plasmid for construction of an expression vector for yeast. In order to secrete PLA1 out of yeast cells, the pre-region, pro-region derived from yeast caloxypeptidase Y (hereinafter referred to as "CPY"), and the N-terminal two amino acids (lysine) DNA encoding [isoleucine] [Valls, LA,
et al. (1987) Cell 48 , 887-897] by polymerase chain reaction (hereinafter referred to as "PCR") [Saiki, RK,
et al. (1988) Science 239 , 487-491].
By ligating to a DNA encoding a mature PLA1 gene, a PLA1 gene expression plasmid for yeast pCY33
9 was constructed. Specifically, the method was performed as described below.

First, primers having the nucleotide sequences described below: 5′-GGGGTCGACAT GAAAGCATTC ACCAGTTT -3 ′ (S1: SEQ ID NO: 12 in the sequence listing) and 5′-AGAAGGGAAG AGCTGACATC AATCTTGTTG ACACGAAGCT-
3 ′ (A2: SEQ ID NO: 13 in Sequence Listing) was synthesized.

Next, 5 ng of pCY303 digested with the restriction enzyme HindIII and 200 gram of the sense primer S1 were used.
nM, antisense primer A2 at 200 nM, 0.
2 mM ATP, 0.2 mM dCTP, 0.2 mM
dGTP, 0.2 mM dTTP, 10 mM potassium chloride, 6 mM ammonium sulfate, 2 mM magnesium chloride, 0.1% (w / v) Triton X-100,
0.01 mg / ml bovine serum albumin, Pfu D
50 μl of 20 mM Tris-HCl buffer (pH 8.0) containing 5 units of NA polymerase (manufactured by Stratagene)
After performing 30 cycles of PCR under the conditions of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute, a further 7 cycles
Incubated at 2 ° C for 10 minutes.

In order to remove unreacted primers, the PCR reaction solution was subjected to agarose gel electrophoresis at 100 V in 1 × TBE buffer, and a gel corresponding to a band corresponding to 0.37 kbp was cut out. 12000
-14000: Gibco BRL)
Elution at 00V for 1 hour. The solution in the dialysis tube was treated with phenol / chloroform and precipitated with ethanol. After the precipitate was air-dried, it was dissolved in TE (the same operation is hereinafter referred to as "cutting out").

On the other hand, a primer having the following nucleotide sequence: 5′-AGCTTCGTGT CAACAAGATT GATGTCAGCT CTTCCCTTCT −
3 ′ (S2: SEQ ID NO: 14 in the sequence listing) and 5′-CCCAAGCTTC TATGAACATT CGCTAATAT -3 ′ (A1:
SEQ ID NO: 15) in the sequence listing was synthesized.

Then, using pRAV885 (5 ng) digested with the restriction enzyme HindIII as a template, 200 nM of sense primer S2, 200 nM of antisense primer A1, 200 mM dATP, 0.2 mM dC
TP, 0.2 mM dGTP, 0.2 mM dTTP,
10 mM potassium chloride, 6 mM ammonium sulfate,
20 mM Tris-HCl buffer containing 2 mM magnesium chloride, 0.1% (w / v) Triton X-100, 0.01 mg / ml bovine serum albumin, and 5 units of Pfu DNA polymerase (Stratagene) (pH
8.0) 30 sec at 94 ° C, 30 sec at 55 ° C in 50 μl
After performing PCR for 30 cycles at 72 ° C. for 1 minute for 1 second, the temperature was further kept at 72 ° C. for 10 minutes. The reaction solution was subjected to agarose gel electrophoresis at 100 V, and 0.84 kbp
The DNA corresponding to the band was cut out.

The 0.37 kbp DNA fragment (2.5 ng) obtained by the above PCR and 0.84 kbp DN
Using fragment A (2.5 ng) as a template, sense primer S
1 at 200 nM and antisense primer A1 at 200
nM, 0.2 mM dATP, 0.2 mM dCTP,
0.2 mM dGTP, 0.2 mM dTTP, 10 m
M Potassium chloride, 6 mM ammonium sulfate, 2 mM
Magnesium chloride, 0.1% (w / v) Triton X
-100, 0.01 mg / ml bovine serum albumin,
Pfu DNA polymerase (Stratagene) 5
20 mM Tris-HCl buffer (pH 8.
0) 30 sec at 94 ° C. per cycle in 50 μl
After performing PCR for 30 cycles under the conditions of 55 ° C. for 30 seconds and 72 ° C. for 1 minute, the temperature was further kept at 72 ° C. for 10 minutes. The reaction solution was treated with phenol / chloroform and precipitated with ethanol. The precipitate was air-dried and dissolved in TE. The DNA thus obtained was digested with restriction enzymes SalI and HindIII, and then subjected to agarose gel electrophoresis at 100 V in 1 × TBE buffer to give 1.2 k
The DNA of the band corresponding to bp was cut out.

The 1.2 kbp S obtained by the above method
The alI-HindIII fragment was ligated into pCY303 digested with restriction enzymes SalI and HindIII,
By inserting using a ligation kit (manufactured by Takara Shuzo Co., Ltd.), the expression plasmid for yeast pCY339
Was built.

Embodiment 11 FIG . Transformation of yeast Transformation of baker's yeast Saccharomyces cerevisiae KS58-2D strain (γ, ssl1, leu2, his1 or 3, ura3)
Reddy and Murray [Reddy, A. an
d Maley, F. (1993) Anal. Biochem. 208 , 211-212]. That is, one colony of KS58-2D strain
100 ml containing 1 ml of YPD medium [1% (w / v) yeast extract (manufactured by Difco) and 2% (w / v) glucose containing 2% (w / v) bactopeptone (manufactured by Difco). Inoculate Erlenmeyer flask, 28 ℃, 21
Preculture was performed at 0 rpm for 3 days. Add 1 ml of preculture to 100 ml
The culture was inoculated into a 500 ml Erlenmeyer flask containing 500 ml of YPD medium, cultured at 28 ° C. and 210 rpm, and when the turbidity measured by the wavelength of 600 nm became 1.5, the culture solution was cooled to 5000 rpm at 4 ° C. for 10 minutes. After centrifugation for minutes, the cells were collected.

[0103] 1 mM EDTA · 2Na was added to the recovered cells.
10 mM Tris-HCl buffer (pH 7.5) containing
(Hereinafter referred to as “W solution”), and the cells were sufficiently washed, and then centrifuged again at 5000 rpm at 4 ° C. for 10 minutes. 10 ml of a W solution containing 10 mM calcium chloride and 15 mM dithiothreitol was added to the precipitate, and the mixture was allowed to stand at room temperature for 20 minutes. This suspension is heated at 1100 rpm at 4 ° C.
, And 10 ml of a cooled W solution was added to the precipitate, the cells were washed well, and then again at 1100 rpm,
After centrifugation at 4 ° C. for 5 minutes, the precipitate was suspended in 2 ml of W solution. 0.2 ml of the suspension was collected and transferred to a 1.5 ml microtest tube, and the plasmid pCY was added thereto.
339 was added in an amount of 5 μg, and the mixture was shaken well. Further, 1 mM EDTA · 2Na, 0.1 M lithium acetate,
0.8 ml of a 10 mM Tris-HCl buffer (pH 8.0) containing 40% (w / v) polyethylene glycol (manufactured by Aldrich, molecular weight: 3400) was added, and the mixture was shaken well and kept at 30 ° C. for 45 minutes. . Then, 42
After keeping at 10 ° C for 10 minutes, the mixture was centrifuged at 4000 rpm and 4 ° C for 5 minutes, and the precipitate was suspended in 1 ml of distilled water. This suspension was centrifuged at 4000 rpm at 4 ° C. for 5 minutes.

After suspending the precipitate in 0.5 ml of distilled water,
SD plate (0.667% (w / v) yeast nitrogen base (manufactured by Difco), 2% (w / v)
Glucose, 2% containing 1 × amino acid mixture ^a) (w /
v) The bacterial cells were spread on an agar plate). The plate was cultivated at 30 ° C. to obtain a grown colony. This transformant was named KS58-2D / pCY339.

A) 1 × amino acid mixture: L-tryptophan 0.002% (w / v) uracil 0.02% (w / v) L-histidine monohydrochloride 0.002% (w / v) L- Lysine monohydrochloride 0.003% (w / v) L-arginine monohydrochloride 0.002% (w / v) L-threonine 0.02% (w / v) L-methionine 0.002% (w / v) v) L-tyrosine 0.003% (w / v) L-phenylalanine 0.005% (w / v) L-isoleucine 0.003% (w / v) L-valine 0.015% (w / v) 0.002% adenine sulfate (w / v) L-glutamic acid 0.01% (w / v) 0.01% L-aspartic acid (w / v) L-0.375% serine (w / v) example 12. Expression of PLA1 gene in yeast Saccharomyces cerevisiae KS58-2D / pCY3
39 strains, and KS58-2 without plasmid
The D strain was transformed to 100 m each containing 20 ml of YPD medium.
The cells were inoculated into a 1-liter Erlenmeyer flask and pre-cultured at 28 ° C. and 210 rpm for 3 days. 0.5 ml of the pre-culture solution was completely synthesized
^{b) The} cells were inoculated into a 100 ml Erlenmeyer flask containing 20 ml and then cultured at 28 ° C. and 210 rpm. However, in the case of the KS58-2D strain, leucine was added to the medium to a final concentration of 0.003% (w / v).
A part of the culture solution was collected over time, and 5 mL at 15000 rpm.
After centrifugation for minutes, the supernatant was recovered and used as a sample for measuring phospholipase activity.

B) Complete synthetic medium: thiamine hydrochloride 400 μg / l biotin 2 μg / l boric acid 500 μg / l copper sulfate 40 μg / l potassium iodide 100 μg / l ferric chloride 200 μg / l manganese sulfate 400 μg / l sodium molybdate 200 μg Per liter zinc sulfate 400 μg / liter ammonium sulfate 0.5% (w / v) dipotassium phosphate 0.5% (w / v) magnesium sulfate 0.05% (w / v) sodium chloride 0.01% (w / v) v) Calcium chloride 0.01% (w / v) Glucose 2% (w / v) Galactose 2% (w / v) Potassium phosphate buffer (pH 7.6) 0.2M Bovine serum albumin 0.5% ( in w / v) amino acid mixture 3 times concentration test example 2 example 12 The manufactured sample of phospholipase activity was determined by the method described in Test Example 1. The phospholipase activity of each sample was as described in Table 2.

[0107]

[Table 2] ──────────────────────────────── Sample culture days (days) 3 5 7 10 13 17 ── ────────────────────────────── KS58-2D (control) 0 0 0 0 0 0 KS58-2D / pCY339 3.9 6.2 7.7 10.3 12.1 13.8 ──────────────────────────────── unit: U / ml example 13. Expression in Aspergillus oryzae (1) Construction of expression plasmid vector 3 μg of plasmid pRAV885 prepared in Example 7
Was digested with the restriction enzyme SacI, the DNA was recovered by ethanol precipitation, and further digested with the restriction enzyme BamHI.
The DNA was recovered by ethanol precipitation. This DNA
Was subjected to agarose gel electrophoresis, a gel corresponding to a band corresponding to 0.94 kbp was cut out, and DNA was recovered. The ends of this 0.94 kbp fragment were blunted using a DNA blunting kit. Then, phenol / chloroform extraction was performed twice and ethanol precipitation was performed once to obtain DN.
A was recovered.

On the other hand, an expression plasmid vector pTAex3 for Aspergillus oryzae [Fujii, T., et al. (1)
995) Biosci. Biotech. Biochem. 59 , 1869-1874]
1.5 μg was digested with the restriction enzyme EcoRI, and the DNA was recovered by ethanol precipitation. The end of this DNA is
After blunting using a DNA blunting kit, phenol / chloroform extraction was performed twice and ethanol precipitation was performed once to recover DNA. Furthermore, the end of this DNA was dephosphorylated with 30 units of calf intestinal alkaline phosphatase (Takara Shuzo Co., Ltd.), followed by phenol / chloroform extraction twice and ethanol precipitation once to recover the DNA.

The EcoRI-digested pTAex3D
NA 1 μg and 0.94 kb obtained from pRAV885
0.4 μg of the BamHI-SacI fragment of p
Ligation was performed using the A ligation kit. Using this ligation reaction solution, competent E. coli E. coli was used. coli HB
LB agar medium (1% Bacto-tryptone, 0.1%) containing 100 μg / ml ampicillin.
Colonies that grew on 5% Bacto yeast extract, 0.5% sodium chloride, 1.5% Bacto agar) were selected. Among these ampicillin-resistant strains, the plasmid DNA retained was replaced with the restriction enzyme EcoR.
5.5 kbp when digested with I and BglII;
Those that produced three fragments of 3 kbp and 0.7 kbp were selected. This strain was mass-cultured to prepare an expression plasmid vector pAAP for Aspergillus oryzae of PLA1.

(2) Transformation of Aspergillus oryzae The expression vector pAAP prepared in (1) above was introduced into Aspergillus oryzae by the method of Gomi et al. [Gomi, K.,
et al. (1987) Agric. Biol. Chem. 51 , 2549-2555]. That is, first, the DP medium (2
% Dextrin, 1% polypeptone, 0.5% monopotassium phosphate, 0.05% magnesium sulfate)
In a 500 ml Erlenmeyer flask charged with 0 ml, place Aspergillus oryzae strain M-2-3 [available from Brewery Research Institute:
See above Gomi, K., et al.] And inoculate one platinum loop at 30 ° C.
For 20 to 40 hours with shaking. The obtained pelleted bacterial cells were passed through a glass filter 3G1 (manufactured by Hario Corporation).
Collect by filtration, wash with sterile water, drain
1 conical tube. A filter-sterilized protoplast solution (5 mg / ml Novozyme 234 (manufactured by Novo), 5 mg / ml cellulase R-10 (manufactured by Seikagaku Corporation), 0.8 M sodium chloride, 10 mM phosphate Buffer (pH 6.0)
After adding 0 ml, the mixture was shaken at 30 ° C. and 100 rpm for 3 hours. This reaction solution is passed through a glass filter 3G3 (Hario
The filtrate was washed twice with 0.8 M sodium chloride solution, and then washed with solution I (0.8 M sodium chloride, 10 mM calcium chloride, 10 mM Tris-hydrochloric acid (pH 7.5)). And centrifuged at 2000 rpm for 5 minutes to obtain precipitated protoplasts.

This protoplast was treated with 2.5 × 10 ⁸ / m
1/4 of the final volume of solution I so that
To this, 1/5 volume of solution II (40% polyethylene glycol 4000, 50 mM calcium chloride, 50 mM
Tris-hydrochloric acid (pH 7.5) and 1/100 amount of dimethyl sulfoxide were added. 10 μg of pAAP DNA was added to 0.2 ml of the protoplast suspension,
Ice-cooled for 30 minutes. Next, 1 ml of solution II was added, mixed well, and allowed to stand at room temperature for 20 minutes.
And centrifuged at 2000 rpm for 5 minutes. The precipitated protoplasts were suspended in an appropriate amount of solution I, and 0.8 M
Czapek-Dox agar medium containing sodium chloride (0.
2% sodium nitrate, 0.1% dipotassium phosphate,
0.05% magnesium sulfate, 0.05% potassium chloride, 0.001% ferrous sulfate, 3% sucrose, 2% agar). Since Aspergillus oryzae strain M-2-3 is arginine-requiring, it cannot normally grow on Czapek-Dox medium, which is the minimum medium, and pAA
Only the strain into which P has been introduced can grow. Czapek-Doxplate
The transformants grown above were further cultured on Czapek-Dox medium for 3 hours.
Aspergillus oryzae M-2
-3 / pAAP strain was obtained.

(3) Culture of Transformant and Confirmation of PLA1 Activity Aspergillus oryzae M-2 grown on slant
From the -3 / pAAP strain, one platinum loop was scraped off from spores and mycelia, and DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5%
Potassium phosphate, 0.05% magnesium sulfate, p
H6.5) was inoculated into a 500 ml Erlenmeyer flask charged with 80 ml. This was cultured at 26 ° C. and 210 rpm, and the PLA1 activity of the culture supernatant on day 7 of the culture was measured by the method described in Test Example 1.
U / ml activity was observed. This secretory production was determined by the plasmid-free Aspergillus oryzae M-2-
This corresponds to 300 times or more of the three strains, indicating that PLA1 gene overexpression greatly increased the secretory production of PLA1.

This Aspergillus oryzae M-2-3
10 μl of the culture supernatant of the pAAP strain
SDS-polyacrylamide gel electrophoresis using a -20% gradient gel was performed. After the electrophoresis, the proteins in the gel were transferred to a transfer device (Sart Blot II-S:
Was transferred to a nitrocellulose membrane (manufactured by Amersham) using Sartorius Corporation. The nitrocellulose membrane was subjected to Western blot analysis using an anti-PLA1 rabbit IgG antibody as a primary antibody and a horseradish peroxidase-labeled anti-rabbit IgG goat antibody (manufactured by Bio-Rad) as a secondary antibody. As a result, a large amount of PLA1 was detected. It was confirmed that PLA1 was produced in large quantities in Aspergillus oryzae M-2-3 / pAAP strain.

[0114]

As described above, the recombinant protein produced by the method of the present invention has excellent phospholipase activity. According to the present invention, it has become possible to carry out industrial-scale production of recombinant PLA1 with high yield and high purity.

[Brief description of the drawings]

FIG. 1 is a restriction map of plasmid pRAV12.

FIG. 2 is a restriction map of plasmid pRAV43.

FIG. 3 is a restriction map of plasmid pRAV885.

FIG. 4 is a diagram showing the construction of plasmid pRAV825.

FIG. 5 is a construction diagram of a plasmid pCY339.

FIG. 6 is a construction diagram of a plasmid pAAP.

[Sequence list]

SEQ ID NO: 1 Sequence length: 888 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical: No Antisense: No Origin Organism name: Aspergillus oryzae (Aspergillus oryza
e) Sequence features Symbol indicating characteristics: CDS position: 1..885 Symbol indicating characteristics: mat_peptide Position: 79..885 Symbol indicating characteristics: sig_peptide Position: 1..78 Sequence ATG TTT AGT CTC GCG CGA TTG GGG ACC GTT GCA GGT CTA TTT TTA CTG 48 Met Phe Ser Leu Ala Arg Leu Gly Thr Val Ala Gly Leu Phe Leu Leu -26 -25 -20 -15 GCT CAG GCT GCC CCG GCT TCA CTG CGC AGA GAT GTC AGC TCT TCC CTT 96 Ala Gln Ala Ala Pro Ala Ser Leu Arg Arg Asp Val Ser Ser Ser Leu -10 -5 1 5 CTC AAT AAC CTG GAT CTC TTT GCA CAG TAC AGC GCC GCC GCA TAC TGT 144 Leu Asn Asn Leu Asp Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys 10 15 20 GAT GAG AAC CTG AAC TCT ACG GGG ACC AAG TTG ACA TGC TCT GTT GGC 192 Asp Glu Asn Leu Asn Ser Thr Gly Thr Lys Leu Thr Cys Ser Val Gly 25 30 35 AAC TGT CCT TTG GTA GAA GCG GCC TCT ACC CAA TCA TTG GAT GAA TTC 240 Asn Cys Pro Leu Val Glu Ala Ala Ser Thr Gln Ser Leu Asp Glu Phe 40 45 50 AAC GAA TCG TCA TCC TAC GGC AAC CCC GCC GGG TAC CTC GCC GCT GAT 288 Asn Glu Ser Ser Ser Tyr Gly Asn Pro Ala Gly Tyr Leu Ala Ala Asp 55 60 65 70 GAG ACT AAC AAG CTC CTA GTC CTG TCC TTC CGG GGT AGC GCT GAC TTG 336 Glu Thr Asn Lys Leu Leu Val Leu Ser Phe Arg Gly Ser Ala Asp Leu 75 80 85 GCC AAT TGG GTC GCC AAC CTG AAT TTT GGT CTC GAG GAT GCC AGC GAT 384 Ala Asn Trp Val Ala Asn Leu Asn Phe Gly Leu Glu Asp Ala Ser Asp 90 95 100 CTG TGT TCT GGG TGC GAA GTG CAC AGC GGC TTC TGG AAG GCA TGG AGT 432 Leu Cys Ser Gly Cys Glu Val His Ser Gly Phe Trp Lys Ala Trp Ser 105 110 115 GAA ATC GCC GAC ACC ATC ACT TCC AAA GTG GAA TCA GCT TTG TCG GAT 480 Glu Ile Ala Asp Thrle Ile Thr Ser Lys Val Glu Ser Ala Leu Ser Asp 120 125 130 CAT TCC GAT TAT TCC TTG GTC TTG ACC GGA CAT AGT TAC GGC GCT GCG 528 His Ser Asp Tyr Ser Leu Val Leu Thr Gly His Ser Tyr Gly Ala Ala 135 140 145 150 CTG GCA GCC CTC GCA GCG ACT GCT CTG CGG AAC TCC GGC CAT AGT GTT 576 Leu Ala Ala Leu Ala Ala Thr Ala Leu Arg Asn Ser Gly His Ser Val 155 160 165 GAG CTG TAC AAC TAC GGT CAA CCT CGA CTT GGA AAC GAG GCA TTG GCA 624 Glu Leu Tyr Asn Tyr Gly Gln Pro Arg Leu Gly Asn Glu Ala Leu Ala 170 175 180 ACA TAT ATC ACG GAC CAA AAC AAG GGT GGC AAC TAT CGC GTT ACG CAC 672 Thr Tyr Ile Thr Asp Gln Asn Lys Gly Gly Asn Tyr Arg Val Thr His 185 190 195 ACT AAT GAT ATT GTG CCT AAA CTG CCA CCC ACG CTG CTC GGG TAT CAC 720 Thr Asn Asp Ile Val Pro Lys Leu Pro Pro Thr Leu Leu Gly Tyr His 200 205 210 CAC TTC AGC CCA GAG TAC TAT ATC AGC AGC GCC GAC GAG GCA ACG GTG 768 His Phe Ser Pro Glu Tyr Tyr Ile Ser Ser Ala Asp Glu Ala Thr Val 215 220 225 230 ACC ACC ACT GAT GTG ACT GAG GTT ACG GGA ATC GAT GCT ACG GGC GGT 816 Thr Thr Thr Asp Val Thr Glu Val Thr Gly Ile Asp Ala Thr Gly Gly 235 240 245 AAT GAT GGA ACC GAC GGA ACT AGC ATC GAT GCT CAT CGG TGG TAC TTT 864 Asn Asp Gly Thr Asp Gly Thr Ser Ile Asp Ala His Arg Trp Tyr Phe 250 255 260 ATT TAT ATT AGC GAA TGT TCA TAG 888 Ile Tyr Ile Ser Glu Cys Ser 265 SEQ ID NO: 2 Sequence length: 295 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Met Phe Ser Leu Ala Arg Leu Gl y Thr Val Ala Gly Leu Phe Leu Leu -26 -25 -20 -15 Ala Gln Ala Ala Pro Ala Ser Leu Arg Arg Asp Val Ser Ser Ser Leu -10 -5 1 5 Leu Asn Asn Leu Asp Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys 10 15 20 Asp Glu Asn Leu Asn Ser Thr Gly Thr Lys Leu Thr Cys Ser Val Gly 25 30 35 Asn Cys Pro Leu Val Glu Ala Ala Ser Thr Gln Ser Leu Asp Glu Phe 40 45 50 Asn Glu Ser Ser Ser Tyr Gly Asn Pro Ala Gly Tyr Leu Ala Ala Asp 55 60 65 70 Glu Thr Asn Lys Leu Leu Val Leu Ser Phe Arg Gly Ser Ala Asp Leu 75 80 85 Ala Asn Trp Val Ala Asn Leu Asn Phe Gly Leu Glu Asp Ala Ser Asp 90 95 100 Leu Cys Ser Gly Cys Glu Val His Ser Gly Phe Trp Lys Ala Trp Ser 105 110 115 Glu Ile Ala Asp Thr Ile Thr Ser Lys Val Glu Ser Ala Leu Ser Asp 120 125 130 His Ser Asp Tyr Ser Leu Val Leu Thr Gly His Ser Tyr Gly Ala Ala 135 140 145 150 Leu Ala Ala Leu Ala Ala Thr Ala Leu Arg Asn Ser Gly His Ser Val 155 160 165 Glu Leu Tyr Asn Tyr Gly Gln Pro Arg Leu Gly Asn Glu Ala Leu Ala 170 175 180 Thr Tyr Ile Thr Asp Gln Asn Lys Gly Gly Asn Tyr A rg Val Thr His 185 190 195 Thr Asn Asp Ile Val Pro Lys Leu Pro Pro Thr Leu Leu Gly Tyr His 200 205 210 His Phe Ser Pro Glu Tyr Tyr Ile Ser Ser Ala Asp Glu Ala Thr Val 215 220 225 230 Thr Thr Thr Asp Val Thr Glu Val Thr Gly Ile Asp Ala Thr Gly Gly 235 240 245 Asn Asp Gly Thr Asp Gly Thr Ser Ile Asp Ala His Arg Trp Tyr Phe 250 255 260 Ile Tyr Ile Ser Glu Cys Ser 265 SEQ ID NO: 3 Sequence length : 15 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: protein Fragment type: middle fragment Sequence Gly Gly Asn Tyr Arg Val Thr His Thr Asn Asp Ile Val Pro Lys 1 5 10 15 SEQ ID NO: 4 Sequence length: 12 Sequence type: amino acid Number of chains: single-chain Topology: linear Sequence type: protein Fragment type: middle fragment SEQ ID NO: 5 Sequence length: 39 Sequence type: amino acid Number of chains: single-chain Topology: linear Sequence type: protein Fragment type: middle fragment Sequence Leu Thr Xaa Ser Val Gly Asn Xaa Pro Leu Val Glu Ala Ala Ser Thr 1 5 10 15 Gln Ser Leu Asp Glu Phe Asn Glu Ser Ser Ser Tyr Gly Asn Pro Ala 20 25 30 Gly Tyr Leu Ala Ala Xaa Glu 35 SEQ ID NO: 6 Sequence length: 26 Sequence type: Number of amino acids Chain: single chain Topology: linear Sequence type: protein Fragment type: middle fragment sequence Asp Val Ser Ser Ser Leu Leu Asn Asn Leu Asp Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Xaa Asp Glu Asn Leu 20 25 SEQ ID NO: 7 Sequence length: 30 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: protein Fragment type: middle fragment sequence Val Glu Ser Ala Leu Ser Asp His Ser Asp Tyr Ser Leu Val L eu Thr 1 5 10 15 Gly His Ser Tyr Gly Ala Ala Leu Xaa Xaa Leu Xaa Ala Thr 20 25 30 SEQ ID NO: 8 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Type of sequence: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GAYYTNTTYG CNCARTA 17 SEQ ID NO: 9 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Type Sequence type: Other nucleic acid (synthetic DNA) Hypothetical: No antisense: Yes Sequence SWTCRTTRAA YTCRTC 16 SEQ ID NO: 10 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Straight Chain Sequence type: Other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence CGTCCTGCAC GGCATTCAAA 20 SEQ ID NO: 11 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Type of linear sequence: other nucleic acids (synthetic D NA) Hypothetical: No Antisense: Yes Sequence TCCCTTCTCA AAGCCAGAAT 20 SEQ ID NO: 12 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids ( Synthetic DNA) Hypothetical: No Antisense: No Sequence GGGTCGACAT GAAAGCATTC ACCAGTTT 28 SEQ ID NO: 13 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other Nucleic acid (synthetic DNA) Hypothetical: No Antisense: Yes Sequence AGAAGGGAAG AGCTGACATC AATCTTGTTG ACACGAAGCT 40 SEQ ID NO: 14 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type : Other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence AGCTTCGGTGT CAACAAGATT GATGTCAGCT CTT
CCCTTTCT 40 SEQ ID NO: 15 Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Hypothetical: No Antisense: Yes sequence CCCAAGCTTC TATGAACATT CGCTAATAT 29

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:69) (C12N 1/19 C12R 1: 865) (C12N 5/10 C12R 1:91) (C12N 9/16 C12R 1:91) (C12N 9/16 C12R 1:69) (C12N 9/16 C12R 1: 865) (72) Inventor Toshiaki Tsuji 2-58 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Shinki Serizawa 1-58, Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Yoichiro Shiba 389-4 Otsugi, Shimokawa, Izumicho, Iwaki-shi, Fukushima Sankyo Co., Ltd. (72) Hiroshi Yoshikawa, Inventor Osamu Shimokawa, Izumi-cho, Fukushima Prefecture

Claims (15)

[Claims] 1. The amino acid numbers 1 to 1 of SEQ ID NO: 2 in the sequence listing
269, a polypeptide having phospholipase activity, or one at one or more sites in the amino acid sequence represented by amino acid numbers 1-269 of SEQ ID NO: 2 in the sequence listing. Alternatively, a DNA comprising an amino acid sequence in which two or more amino acids have been substituted, deleted or inserted, and encoding a polypeptide having phospholipase activity. 2. The amino acid sequence of SEQ ID NO: 2 in the sequence listing
269, which comprises the amino acid sequence of SEQ ID NO: 269, and encodes a polypeptide having phospholipase activity.
NA. 3. A DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 885 of SEQ ID NO: 1 in the sequence listing. 4. A DNA that hybridizes with the DNA according to claim 3 and encodes a polypeptide having a phospholipase activity. 5. A DNA which hybridizes with the DNA according to claim 3 under stringent conditions and encodes a polypeptide having phospholipase activity. 6. The DNA of claim 3 and 6 × SSC,
A DNA that hybridizes under conditions of 60 to 70 ° C. and encodes a polypeptide having phospholipase activity. 7. A recombinant DNA vector comprising the DNA according to any one of claims 1 to 6. 8. The recombinant DNA vector according to claim 7, which is a plasmid pCY339. 9. The plasmid of claim 7, which is a plasmid pAAP.
The recombinant DNA vector according to the above. A host cell transformed with the recombinant DNA vector according to any one of claims 7 to 9. 11. Saccharomyces cerevisiae KS5
The host cell according to claim 10, which is 8-2D / pCY339. 12. Aspergillus oryzae M-2-3
11. The host cell of claim 10, which is / pAAP. 13. A polypeptide having an amino acid sequence represented by amino acid numbers 1 to 269 of SEQ ID NO: 2 obtained by genetic manipulation and having a phospholipase activity, or an amino acid of SEQ ID NO: 2 in Sequence Listing. A polypeptide comprising an amino acid sequence in which one or more amino acids have been substituted, deleted or inserted at one or more sites in one or more of the amino acid sequences represented by Nos. 1-269, and having a phospholipase activity. 14. A polypeptide having the phospholipase activity, which is obtained by genetic manipulation and comprises an amino acid sequence represented by amino acid numbers 1-269 of SEQ ID NO: 2 in the sequence listing. 15. A method for culturing the host cell according to any one of claims 10 to 12 under conditions capable of producing phospholipase, and then recovering the polypeptide having phospholipase activity from the culture. A method for producing the polypeptide, which is characterized in that: