WO2004090137A1 - Novel gene - Google Patents

Novel gene Download PDF

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Publication number
WO2004090137A1
WO2004090137A1 PCT/JP2004/004789 JP2004004789W WO2004090137A1 WO 2004090137 A1 WO2004090137 A1 WO 2004090137A1 JP 2004004789 W JP2004004789 W JP 2004004789W WO 2004090137 A1 WO2004090137 A1 WO 2004090137A1
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Prior art keywords
polypeptide
dna
vacuolar
gene
filamentous fungus
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PCT/JP2004/004789
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French (fr)
Japanese (ja)
Inventor
Chikara Tokunaga
Chiaki Saitoh
Katsuhiko Kitamoto
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Kyowa Hakko Food Specialties Co., Ltd.
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Application filed by Kyowa Hakko Food Specialties Co., Ltd. filed Critical Kyowa Hakko Food Specialties Co., Ltd.
Priority to JP2005505251A priority Critical patent/JPWO2004090137A1/en
Publication of WO2004090137A1 publication Critical patent/WO2004090137A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus

Definitions

  • the present invention relates to a polypeptide having an activity of localizing a vacuolar enzyme having homology to VFS5 polypeptide of Saccharomyces cerevisiae in a cell-a DNA encoding the polypeptide, a DNA encoding the polypeptide, how the function of suppressing this and by the liquid ⁇ element producing filamentous fungi that secrete extracellularly, and t further relates filamentous fungus liquid ⁇ element made secreted extracellularly by said method, the filamentous fungus
  • the present invention relates to a method for producing a vacuolar enzyme used, and a method for producing a protein hydrolyzate using the enzyme.
  • Vacuoles are one of the intracellular organs commonly observed in yeast, plant cells, filamentous fungi, etc., and have physiological functions such as recycling of intracellular components, osmotic pressure regulation, detoxification, and storage of nutrients. Is responsible for. Yeast vacuoles are known to contain many hydrolases, including proteases, phosphatases, esterases, lyases, ribonucleases, and glycosidases
  • vacuolar enzyme is industrially useful, and in particular, the vacuolar enzyme of Aspergillus oryzae, which is GRAS dale, is highly useful in food production. Since it is necessary to autolyze or physically or enzymatically disrupt and lyse the koji mold, it takes time and effort, so that an efficient method for obtaining vacuolar enzymes is desired.
  • Kitamoto mentions the possibility of efficient extracellular secretion of vacuolar enzymes from the point of view of sorting engineering (Biotechnical Society, 1999, 9th, 7th edition). Vol. 7, p. 1-3-3) Force There has been no example of koji mold that secreted vacuolar enzymes extracellularly.
  • VPS Vacuolar protein sorting
  • the Saccharomyces cerevisiae VPS5 gene is a vacuolar enzyme selection gene that encodes a polypeptide having homology to sorting nexin I.Saccharomyces cerevisiae, which broke the VPS5 gene, is a vacuolar enzyme cell. Missed out was seen
  • Filamentous fungi also have intracellular organs that are morphologically similar to yeast vacuoles. Like yeast vacuoles, they are thought to have various hydrolases. 'In filamentous fungi, it is expected that vacuolar enzymes are selected from secretory enzymes and transported to the vacuoles by a mechanism similar to yeast.
  • VPS45 homologous gene, vpsB also known to be involved in the selection of vacuolar enzymes involved in vesicle transport from the trans-Golgi to the pre-vacuolar compartment, was isolated from Aspergillus nidulans (Nippon Agriculture) Journal of the chemical Society, Chapter 7 3 Certificates, extra edition No., E 9 9 9 fiscal Japan Society for Bioscience, Biotechnology, and Agrochemistry, Abstracts for the Annual Meeting, 1 9 9 9 9 years, p. 2 1
  • VPS21 Asuperugi Angeles homologous gene vpsC of .. - was isolated from Doransu (for example, Japan Agricultural Chemistry Journal, 7 Volume 3, extra edition No., 1 9 9 9 fiscal Japan agrochemical Proceedings of the Annual Meeting of the Congress, 1 1989, p213).
  • Ohneda also isolated from Aspergillus nidulans. Expressed a fusion protein in which a fluorescent protein was linked to the identified carboxypeptidase Y gene cpyA in Aspergillus oryzae cells, and the gene product was expressed as Aspergillus oryzae. Oryzae was localized in the vacuole [Fungal Genetics and Biology, 37, 29 (2002)], and a mutant strain with increased fluorescence intensity in the medium was isolated and its phenotype was analyzed. Conference Abstracts, 2001, p. 231). However, the causative gene has not been identified (Second Abstract of the 2nd Conference on Molecular Biology of Filamentous Fungi, 2002, p. 49). Disclosure of the invention
  • a novel gene encoding Aspergillus oryzae which encodes a polypeptide having an activity of localizing vacuolar enzymes to cells, and the use of the gene to efficiently utilize the vacuolar enzymes of filamentous fungi There is a need for a method of producing in a medium.
  • the present invention provides the following (1) to (34).
  • a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and having an activity of localizing vacuolar enzymes to cells.
  • a DNA comprising the nucleotide sequence of SEQ ID NO: 2 or 3.
  • the transformant according to (8) is cultured in a medium, and the polypeptide according to any one of (1) to (3) is produced and accumulated in the culture.
  • a DNA having a sequence of 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 2 or 3 and a DNA having a sequence complementary to a sequence of 15 or more nucleotides of the consecutive nucleotide sequence of SEQ ID NO: 2 or 3 A method for detecting a raRNA encoding the DNA according to any one of (4) to (6) or the polypeptide according to any one of (1) to '(3) .
  • a method for producing a filamentous fungus that secretes a vacuolar enzyme extracellularly comprising suppressing the function of a polypeptide having an activity of localizing the vacuolar enzyme to a cell.
  • the polypeptide having an activity of localizing a vacuolar enzyme to a cell is the polypeptide according to any one of (1) to (3), (12)
  • the filamentous fungus is a filamentous fungus belonging to the genus Aspergillus (12) ⁇
  • a recombinant vector comprising the DNA according to (22).
  • a filamentous fungus obtained by the method according to any one of (12) to (21) is cultured in a medium, to produce and accumulate a vacuolar enzyme in the medium, and to form a vacuole from the medium.
  • a method for producing a vacuolar enzyme comprising collecting an enzyme.
  • Vacuolar enzyme is triptidyl peptidase (27) ⁇ (
  • a method for producing a protein hydrolyzate which comprises causing a culture or a culture-treated product containing a vacuolar enzyme obtained by culturing the obtained filamentous fungus to act on a protein serving as a substrate.
  • the polypeptide of the present invention includes a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 and an amino acid sequence represented by SEQ ID NO: 1.
  • Polypeptides comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added and having an activity of localizing vacuolar enzymes to cells can be given.
  • the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is a polypeptide encoded by a homologous gene of Aspergillus oryzae to the VPS5 gene of Saccharomyces cerevisiae (hereinafter also referred to as Aovps5 gene).
  • Polypeptides consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and having the activity of localizing vacuolar enzymes to cells are known as Molecular Cloning: A Laboratory Manual, 3rd Edition, Cola Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular Kroichi Jung, 3rd edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-2001) (hereinafter abbreviated as Current Molecular 'Norology and B'), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Natl. Acad. Sci.
  • a site-specific mutation was introduced into the DNA encoding the polypeptide having It can be obtained by expressing the polypeptide encoded by the DNA into which the difference has been introduced.
  • the number of amino acids to be deleted, substituted or added is not particularly limited, but is a number that can be deleted, substituted or added by a well-known method such as the above-described site-directed mutagenesis, and is 1 to several tens.
  • the number is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
  • deletion, substitution or addition of an amino acid is not limited to one position in the amino acid sequence represented by SEQ ID NO: 1, and may occur simultaneously at two or more positions. Examples of the position of the amino acid at which the amino acid can be deleted or added include the N-terminal and the C-terminal of the amino acid sequence represented by SEQ ID NO: 1.
  • One or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 An acid is deleted, substituted or added when one or more amino acids are deleted, substituted or added at any and one or more amino acid sequences in the same sequence. And deletion, substitution or addition may occur simultaneously, and the amino acid to be substituted or added may be either natural or non-natural.
  • Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, Examples include L-arginine, L-methionine, L-phenyalarayun, L-prolin, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valin, and L-cysteine. -The following are examples of mutually substitutable amino acids. Amino acids included in the same group can be substituted for each other. .
  • Group A leucine, isoleucine, nonoleucine, norin, nornoline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-ptynoreglysine, t-butinolealanine, cyclohexinoleranine
  • Group B aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosveric acid
  • the amino acid set forth in SEQ ID NO: 1 must be used. It is preferable to have at least 60% or more homology with the sequence, usually at least 80%, especially at least 95%.
  • These polypeptides have the activity to localize vacuolar enzymes to cells This can be confirmed by that the vacuolar enzyme of the filamentous fungus is secreted extracellularly when the function of the polypeptide is suppressed in the filamentous fungus by the method described later in 3. .
  • vacuolar enzymes outside the cells can be confirmed by culturing the filamentous fungus in the medium and detecting the vacuolar enzymes in the medium after the culture.
  • vacuolar enzymes include triptidyl peptidase and lipoxypeptidase Y.
  • Methods for detecting vacuolar enzymes include (a) a method for measuring the enzyme activity of the vacuolar enzyme, and (b) immunological detection by Western blotting using an antibody that recognizes the vacuolar enzyme.
  • C Expression of a marker protein such as daline fluorescent protein (GFP) or a fusion protein of an epitope peptide and a vacuolar enzyme such as a FLAG tag in a filamentous fungus, and specifically recognizing the marker protein peptide epitope
  • GFP daline fluorescent protein
  • a vacuolar enzyme such as a FLAG tag in a filamentous fungus
  • a method for detecting a fusion protein using an antibody [Fungal Genet. Biol., 37, 29 (2002)] and the like.
  • tripeptidyl peptidase can be used as a substrate for alanine as a substrate according to the method for measuring the activity of tripeptidyl peptidase I described in the literature [Biochem. Mol. Biol. Int., 47, 1079 (1999)].
  • a sample solution such as a culture solution of a filamentous fungus is added to a substrate solution containing alanine-bye-alanine-para-nitrolide, and the mixture is allowed to react at 30 ° C for 10 minutes.
  • the absorbance at 384 nm is then measured by a spectrophotometer. By measuring the activity, the activity can be measured.
  • Example 3 using the Aovps5 gene-disrupted strain of Aspergillus oryzae described later in 3. and Example 3 as a host, expressing the polypeptide by the method described in 2. or the homologous recombination method described in 3. Expressions of (1) extracellular secretion of vacuolar enzymes, (2) secretion of reddish brown pigment, (3) resistance to heavy metals, (4) inhibition of conidium formation, etc. possessed by the AovpsS gene-disrupted strain. Even when the type is restored to the original phenotype, it can be confirmed that the polypeptide has an activity of localizing vacuolar enzymes to cells.
  • the DNA of the present invention encodes the above-described polypeptide of the present invention.
  • DNA As a DNA encoding the amino acid sequence of SEQ ID NO: 1 Is a DNA containing the nucleotide sequence of SEQ ID NO: 2 or 3.
  • the nucleotide sequence of SEQ ID NO: 2 is the sequence of the Aovps5 gene on the genome of Aspergillus oryzae
  • the nucleotide sequence of SEQ ID NO: 3 is the sequence of the cDNA coding region of the gene.
  • the DNA of the present invention is a DNA that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or 3, and has a vacuolar enzyme.
  • DNA that hybridizes under stringent conditions refers to, for example, a colony hybridization method using, as a probe, a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or 3 or a partial DNA fragment thereof as a probe.
  • hybridization was performed with the probe at 65 ° C in the presence of 0.7 to 1.Omol / L sodium chloride. After that, use a SSC solution with a concentration of 0 :! to 2 times (the composition of a 1% concentration SSC solution is composed of 150 mmol / L sodium chloride and 15 mmol / L sodium tate). DNA that can be identified by washing the filter at 65 ° C can be mentioned.
  • Hybridization is performed by Molecular Yura Closing Third Edition, Current Protocol / Les' in Morekiura Pyrology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (199o, etc.) Specifically, as a hybridizable DNA, at least 60% of the nucleotide sequence represented by SEQ ID NO: 1 or 3 is used. DNA having the above homology, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more homology. Can be done.
  • the base sequence of the Saccharomyces cerevisiae VPS5 gene (GenBank accession number: U73512) was queried for the sequence (SEQ ID NO: 4) of the VPS5 gene.
  • various filaments such as Aspergillus edulans and Neurospora crassa were used.
  • a homology search program BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] was used to search the genome sequence database of the fungus for high homology with the Saccharomyces cerevisiae VPS5 gene.
  • the genome sequence of the VPS5 homologous gene such as Neurosbora classa with For example, SEQ ID NO: 5 can be mentioned as the nucleotide sequence of the VPS5 homologous gene of Neurosbora classa.
  • a degenerate primer is designed based on the amino acid sequence encoded by the Neurospora crassa VPS5 homologous gene.
  • the amino acid sequence for primer design can be selected from any two amino acid sequences from the amino acid sequence encoded by the VPS5 homologous gene of Neurospora crassa.
  • the degenerate primers have the base sequences of all the codons corresponding to the N-terminal amino acid sequence and all the codons corresponding to the C-terminal amino acid sequence. Are designed as DNAs each containing a complementary nucleotide sequence. In order to reduce the number of degeneracy, inosine was used as the third base of the codon, and the codon usage of Aspergillus dylans thiol. Gen. Genet.,
  • the length of the primer is preferably 14 to 30 bases.
  • An example of a degenerate primer is SEQ ID NO:
  • DNAs having the nucleotide sequences of 6 and 7, respectively, can be mentioned.
  • the measured degenerate primer can be prepared, for example, using a DNA synthesizer manufactured by Applied Biosystems.
  • PCR By performing PCR using the above degenerate primer and chromosomal DNA or cDNA of Aspergillus oryzae as a template, a partial fragment of the Aovps5 gene of Aspergillus oryzae can be amplified and isolated.
  • the chromosomal DNA or cDNA of Aspergillus oryzae can be prepared by the method described later in (2).
  • PCR can be performed using conditions and means known to those skilled in the art. For example, PCR reaction conditions include a reaction at 94 ° C for 5 minutes, followed by 30 cycles of a reaction cycle consisting of 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds. The conditions to be performed are given.
  • the annealing temperature during the reaction cycle should be appropriate based on the TJ estimated from the primer length and base composition.
  • a commercially available thermal cycler such as Perkin Elmer 9600, Astec program.temp.control.system PC-700, etc. can be used.
  • Commercial products such as Taq DNA polymerase (Takara Shuzo) and ExTaq DNA polymerase (Takara Shuzo) can be used as the heat-resistant DNA polymerase.
  • the composition of the reaction solution follows the instructions attached to the polymerase.
  • a DNA fragment amplified by PCR is isolated, and labeled with digoxigenin (hereinafter abbreviated as DIG), a radioisotope or the like is used as a probe. Further, by adding a nucleotide labeled with DIG, a radioisotope, or the like to the reaction solution of the above PCR and performing PCR, the labeling of the probe can be performed simultaneously with the amplification.
  • DIG digoxigenin
  • a radioisotope or the like
  • Examples of the partial fragment of the Aovps5 gene of Aspergillus oryzae obtained as described above, which can be used as a probe include a DNA having the nucleotide sequence of SEQ ID NO: 8.
  • the amplified DNA fragment is cloned into an appropriate plasmid vector and used with a DNA sequencer such as DSQ2000L DNA Sequencer (manufactured by Shimadzu Corporation) or ABI310 Genetic Analyzer (manufactured by PerkinElma). Then, it is preferable to determine the nucleotide sequence and confirm whether or not it has homology with the VPS5 syngeneic gene of Neurosbora classa.
  • a DNA sequencer such as DSQ2000L DNA Sequencer (manufactured by Shimadzu Corporation) or ABI310 Genetic Analyzer (manufactured by PerkinElma). Then, it is preferable to determine the nucleotide sequence and confirm whether or not it has homology with the VPS5 syngeneic gene of Neurosbora classa.
  • Genomic DNA extracted from Aspergillus oryzae is cleaved with an appropriate restriction enzyme, and inserted into a beta cleaved with a restriction enzyme having the same cleaved end.
  • Lambda vector vectors such as ⁇ DASHI I (Stratagene), ⁇ FIXI I (Stratagene), and pUC118 as vectors
  • plasmid vectors such as (Takara Shuzo) and PBR322 (Takara Shuzo).
  • Aspergillus oryzae genomic DNA can be extracted according to the method of Gomi et al., C J. Gen. Appl. Microcrobio l., 35, 225, (1989)].
  • insertion into the phage vector ⁇ DASHI I can be performed by ligating a vector cleaved with BamHI and chromosomal DNA degraded with BamHI using T4 DNA ligase.
  • a chromosomal DNA library can be obtained by introducing the vector into which the genomic DNA fragment thus obtained has been inserted into an appropriate Escherichia coli host.
  • a cDNA library can be prepared as follows. First, the cells of Aspergillus oryzae frozen in liquid nitrogen are finely pulverized, and then homogenized with phenol or phenolic orifice-form solution containing guanidine isothiocynate, and then centrifuged into an aqueous layer and an organic layer by high-speed centrifugation. To separate. Then, mix the aqueous layer with isopropanol, and collect by precipitating and collecting the total RNA contained in the aqueous layer, or recover by sucrose or cesium chloride density gradient centrifugation. Or a commercially available total RNA extraction kit
  • RNA RNA
  • oligo (dT) cellulose chromatography to purify mRNA (ie, poly (A) RNA).
  • cDNA is synthesized from mRNA in the presence of reverse transcriptase, and an appropriate restriction enzyme site is created so that it can be linked to a phage or a plasmid vector.
  • the cDNA is ligated to a plasmid vector, and Escherichia coli is transformed with the vector obtained in this manner to prepare a cDNA library.
  • the Superscript Plasmid System for cDNA synthesis and cloning using gateway technology SUPERSCRIPT Plasmid System
  • kits such as with GATEWAY "Technology for cDNA Synthesis and Cloning (manufactured by Invitoguchi Gen), can also be used.
  • clones containing the Aovps5 gene are selected by plaque hybridization or colony hybridization (Molecular cloning, 3rd edition, ucleic Acids Res., 9, 879 (1981)). be able to.
  • plaques are transferred to nitrocellulose or nylon membrane, and the DNA is immobilized on the membrane by irradiating with UV light at 80 ° C for 2 hours in a vacuum.
  • denaturation using an alkaline solution containing 0.5 mol / l sodium hydroxide and 1.5 mol / l sodium chloride, and 0.5 mol / l tris-hydrochloric acid (pH 7.5), 3 mol / l chloride Neutralize with a solution of sodium.
  • the probe was put in a solution of 5XSSC; 50% formamide, 0.1% N-lauroyl sarcosine, 0.2% SDS, 1% blocking reagent (Roche) at 42 ° C, 6 ° C. After incubating for 2 hours at 42 ° C, wash sequentially with 2XSSC containing 0.1% SDS for 5 minutes, then with 0.1XSSC containing 0.1% SDS for 15 minutes, or 4 XSSC, 50% formamide, 50 mmol / 1 HEPES — From sodium hydroxide, lithium (pH 7.0), lOXDenhardt's solution (0.2% polyester 400, 0.2 ° /.
  • the phage DNA or plasmid DNA is extracted and isolated from the positive clones or plasmids selected as described above, and the inserted fragment is cut out from the vector with an appropriate restriction enzyme to obtain the DNA of the present invention. be able to.
  • the obtained DNA is subcloned into an appropriate plasmid vector, and the DNA is sequenced using a DNA sequencer such as DSQ2000L DNA Sequencer (manufactured by Shimadzu Corporation) or ABI .310 Genetic 'Analyzer (manufactured by PerkinElma).
  • the nucleotide sequence of the DNA can be determined.
  • the intron region of the genomic DNA can be determined by determining the sequence of the genomic DNA and the sequence of the cDNA of the Aovps5 gene, and comparing the nucleotide sequences of both.
  • a region containing the coding region of the polypeptide is appropriately selected, and the sequence of 20 to 40 nucleotides at the 5 'end of the nucleotide sequence of the selected region is 3'.
  • Use a DNA synthesizer to synthesize the DNA at the end and the DNA at the 3 'end that is complementary to the base sequence 20 to 40 bases at the 3' end of the selected region using a DNA synthesizer c Genomic DNA or cDNA from Aspergillus cerevisiae
  • the DNA of the present invention can be amplified and isolated by PCR using two types of synthetic DNAs as primers as a template. PCR can be performed under the same conditions as described in (1).
  • the DNA of the present invention obtained as described above includes the genomic DNA of the Aovps 5 gene of Aspergillus oryzae comprising the nucleotide sequence of SEQ ID NO: 2, and the Aovps 5 of Aspergillus oryzae comprising the nucleotide sequence of SEQ ID NO: 3 Examples include the cDNA of the gene.
  • genomic DNA library or cDNA library of another filamentous fungus is prepared according to the method described in (2) and (3) according to ( By screening by hybridization under the condition of 3), genomic DNA or cDNA of Aovps5 homologous gene of other filamentous fungi can be obtained. These DNAs are also included in the DNA of the present invention.
  • Sequences of 20 or more consecutive bases of a sequence complementary to the base sequence of the DNA of the present invention A DNA fragment containing the sequence or oligo DNA labeled with a radioisotope, digoxigenin, biotin, or the like is used as a probe to perform hybridization, thereby obtaining the mRM encoding the DNA of the present invention or the polypeptide of the present invention. Can be detected.
  • a DNA fragment containing a continuous sequence of 20 or more bases complementary to the base sequence of the DNA of the present invention can be obtained by preparing a region containing any region of the DNA of the present invention according to the method described in (4) above.
  • DNA containing a sequence of 20 to 40 bases at the 5 'end of the base sequence of the selected region at the 3' end, a sequence complementary to 20 to 40 bases of the base sequence of the selected region at the 3 'end can be prepared by PCR using, as primers, DNAs containing 3 ′ at the 3 ′ end.
  • Oligo DNA can be prepared using a DNA synthesizer.
  • the length of the prope can be appropriately selected by those skilled in the art according to the detection target and the like, but is usually 15 to 3000 bases, preferably 20 to 1000 bases.
  • Hybridization can be performed on a nitrocellulose membrane or a nylon membrane to which genomic DNA or mRNA has been transcribed from an electrophoresis gel or a colony according to the method of hybridization described in (3). .
  • a DNA chip [GenomeRes., 639 (1996)] is used in which oligo DNA or DNA fragments are immobilized on a substrate, and the resulting DNA is hybridized with labeled mRNA or DNA and then detected as a dot. Can also detect mRNA encoding the DNA of the present invention or the polypeptide of the present invention.
  • a DNA having a sequence of 15 or more consecutive bases of the DNA of the present invention and a DNA having a sequence complementary to a sequence of 15 or more consecutive bases of the DNA of the present invention are used as primers.
  • PCR By using PCR as a template with genomic DNA or cDNA prepared from a colony or the like, mRNA encoding the DNA of the present invention or the polypeptide of the present invention can be detected. PCR can be performed in the same manner as described in (1).
  • the polypeptide of the present invention can be prepared, for example, by the following method, by preparing a transformant in which a recombinant DNA containing the DNA encoding the polypeptide of the present invention has been introduced into a host cell, Can be prepared by cultivating 2004/004789
  • a DNA of an appropriate length containing a portion encoding the polypeptide of the present invention is prepared. If necessary, a DNA is prepared by substituting the nucleotide sequence of the portion encoding the polypeptide of the present invention so that the nucleotide sequence becomes an optimal codon for expression in a host cell.
  • a recombinant vector is prepared by inserting the DNA downstream of the promoter of an appropriate expression vector.
  • the recombinant vector is introduced into a host cell compatible with the expression vector.
  • any bacteria, yeast, filamentous fungi, animal cells, insect cells, plant cells and the like can be used as long as they can express the gene of interest. '
  • the expression vector those which are capable of autonomous replication in the host cell or capable of integration into the chromosome and which contain a promoter which functions in the host cell are used. Further, it preferably contains a gene serving as a transformation marker for selecting a transformant.
  • the recombinant vector containing the DNA encoding the polypeptide of the present invention is capable of autonomous replication in the prokaryote, and has a promoter
  • the vector is preferably a vector containing a structure in which a ribosome binding sequence, DNA encoding the polypeptide of the present invention, and a transcription termination sequence are linked.
  • a gene that controls the promoter ' may be included.
  • prokaryotes examples include pGEMEX-II (promega), pQE-30 (Qiagen), pKYP200 [Agric. Biol. Chem., 48,
  • any promoter can be used as long as it functions in the host cell.
  • trp-flop opening motor P trp
  • lac flop port motor p L promoter
  • Pr promoter can be mentioned, such as ⁇ 7 promoter, a promoter one derived from large ⁇ Ya off Aji like.
  • artificially designed and modified promoters such as a promoter in which two P trps are connected in series (P trp X 2), a tac promoter, a lacT7 promoter, and a let I promoter can be used.
  • a plasmid in which the distance between the Shine-Dalgarno sequence, which is the ribosome-binding rooster S row, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
  • the transcription termination sequence is not always necessary, but it is preferable to arrange the transcription termination sequence immediately below the DNA encoding the polypeptide of the present invention.
  • Examples of the host cell include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevipacterium, Corynepacterium, Microbatteryrum, Pseudomonas, and the like, for example, Escherichia coli XL1-Blue, Escherichia coli XL2- Blue, Escherichia coli BL21, Escherichia coli DH1 Escherichia coli M and 1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli, W3110, Escherichia coli NY49 N Escherichia coli GI698, Escherichia coli TB1, Serratia f icaria Serratia f ontico ⁇ a, Serratia liquef aciens
  • amylol iquef acines Brevibacterium ammoniagenes Brevibacterium immariopnilum ATCC14068, Brevi bacterium saccharolvticum ATCC14066, Brevibacterium f lavum ATCC14067 Brevibacterium lactof ermentum ATCC13869, Corynebacterium glutamicum ATCC 130g, Corynebacterium glutamicum ATCC13032g , Microbacterium ammonia Dhilum ATCC15354, Pseudomonas putida, Pseudomonas sp. D-0110 and the like.
  • Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)], the protoplast method (JP-A-63-2483942), or the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979).
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)]
  • the protoplast method JP-A-63-2483942
  • the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979). Can be.
  • examples of expression vectors include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.
  • promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP Promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFal promoter, CUP 1 promoter and the like.
  • Hyakuo Itoda S-package is a microorganism belonging to the genus Saccharomycesyu, Scnizosaccharomyces, Kluyveromyces, Trichpsooron3 ⁇ 4 Schwanniomvces, Pichiafe, Candida, etc. Trichosporon oullulans, Schwanniomyces alluvius, Candi ⁇ ____ utilis, etc. are available. '
  • any method for introducing DNA into yeast can be used.
  • an electroporation method can be used.
  • pPTRI manufactured by Hakutsuru Shuzo
  • pPTRII manufactured by Hakutsuru Shuzo
  • pAUR316 manufactured by Takara Shuzo
  • Any promoter may be used as long as it functions in a filamentous strain, and examples thereof include amyB promoter, enoA promoter, gpd promoter, melO promoter, alcA promoter, and prA promoter. Can be given.
  • Host cells include the genus Aspergillus, the genus Penicillium, the genus Tricoderma, the genus Fusarium, the genus Humicola, the genus Mucor, and lysozoa. Filamentous fungi belonging to the genus Rhizopus, Monascus, etc., such as Aspergillus niger and Aspergillus niger, Aspernoluginus
  • filamentous fungi can be used as a method for introducing a recombinant vector, such as squid (Aspergillus ficcum), trichothenorema li- 1 , fr ichoderma re_e sei), and Rhizopus 'Rhizopus nibeus'. Any method for introducing DNA can be used, and examples include a protoplast method [GENETICS of ASPERGILLUS NIDULANS: EMBO Practical Course Manual, 8 (19
  • an expression vector for example, pEGFP-C2 (manufactured by Clontech), pAGE107 (JP-A-3-22979;
  • any promoter can be used as long as it functions in animal cells.
  • the promoter of the IE (immediate early) gene of cytomegalovirus (CMV) the early promoter of SV40, and the promoter of retrodinores Promoter, metallothionein promoter, human 9
  • One-shot promoter SRct promoter and the like. Further, the enhancer of the IE gene of human CMV may be used together with the promoter.
  • Examples of the host cell include Namalwa cell, a human cell, COS cell, a Sal cell, CH0 cell, a Chinese hamster cell, and HBT5637 (JP-A-63-299). it can.
  • any method for introducing a recombinant vector into animal cells any method can be used as long as DNA can be introduced into animal cells.
  • an electoral poration method [Cytotechnology, 3, 133 (1990) )]
  • Calcium phosphate method Japanese Patent Application Laid-Open No. 2-227075
  • Lipofexion method Proc. Natl. Acad. Sci. USA, 8> 7413 (1987)
  • Virology, 52, 456 (1973), etc. Can be raised.
  • the recombinant gene transfer vector and the baculovirus are co-transfected into insect cells to obtain a recombinant virus in the culture supernatant of insect cells, the recombinant virus is further infected into insect cells, and the polypeptide is Can be expressed.
  • Is a transgenic base compactors used in the method can be exemplified pVL1392, pVL1393, P BlueBac4.5 (both manufactured by Lee Nbitorojen Co.), P BacPAK9 (Clontech) and the like.
  • paculovirus for example, a virus that infects nocturnal insects such as Atographa californica-force, nucleus, polyhedrosis, ⁇ noreth (Autographa californica nuclear polyhedrosis virus) and the like are used. Can be.
  • Sf9 ⁇ an egg cell of Spodoptera f rugiperda
  • Examples of the method for co-transferring the above-mentioned recombinant gene introduction vector and the above-mentioned PacuMouth virus into insect cells for preparing a recombinant virus include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075). Acad. ScI. USA, 84, 7413 (1987)].
  • examples of the expression vector include Ti plasmid and tobacco mosaic virus vector.
  • any promoter can be used as long as it functions in plant cells, and examples thereof include the 35S promoter of potato flower mosaic virus (CaMV) and the inactin 1 promoter.
  • CaMV potato flower mosaic virus
  • Examples of the host cell include plant cells such as tobacco, potato, tomato, carrot, soybean, oilseed rape, alf alfa, rice, wheat, oats and the like.
  • any method for introducing DNA into plant cells can be used.
  • Agrobacterium Agrobacterium
  • JP-A-59-140885, JP-A-60-1985 700.80, W094 / 00977
  • an electoral-portion method JP-A-60-251887
  • a method using a partake noregan Gene gun
  • Patent Publications 2606856 and 2517813 Patent Publications 2606856 and 2517813
  • the transformant of the present invention obtained as described above is cultured in a medium, the polypeptide of the present invention is produced and accumulated in a culture, and the polypeptide of the present invention is collected from the culture. Peptides can be produced.
  • the method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
  • the transformant of the present invention is a transformant obtained using a prokaryotic organism such as Escherichia coli or a eukaryotic microorganism such as yeast or filamentous fungus as a host
  • the transformant is used as a medium for culturing the transformant.
  • Nitrogen, a carbon source that the body can utilize Any of a natural medium and a synthetic medium may be used as long as the medium contains a source, inorganic salts, and the like, and can efficiently culture the transformant.
  • Any carbon source may be used as long as the transformant can be assimilated, and glucose, fructoses, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, and the like can be used.
  • Organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; other nitrogen-containing compounds; and peptone and meat. Extract, yeast extract, corn steep liquor, casein hydrolysate, wheat protein and wheat protein hydrolysate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used. .
  • Inorganic salts include potassium potassium phosphate, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. Can be used.
  • wheat bran, rice bran, soy bran, defatted soy protein, steamed rice, and the like can be used as a carbon source, a nitrogen source, and an inorganic substance source, and those supplemented with appropriate salts can be used as a medium.
  • the culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH during the cultivation is kept between 3.0 and 9.0.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium as necessary.
  • indoleacrylic acid or the like may be added to the medium.
  • a solid medium used for culturing filamentous fungi a medium having lower water activity than a normal agar medium can be used.
  • the solid medium include a wheat bran medium in which an aqueous medium is infiltrated into wheat bran, steamed rice, and the like.
  • the culture is allowed to stand still for 3 to 10 days at a temperature of usually 15 to 50 ° C, preferably 25 to 37 ° C, and a humidity of usually 80 to 100%, preferably 90 to 100%.
  • a commonly used RPMI 1640 medium [: III. Am. Med. Assoc., 199, 519 (1967)], Awakening of Eagl e (Mimimum Es sential Medium) [Science, 122, 501 (1952)], Dalbecco modified Eagle medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol Med., 73, 1 (1950)] or a medium obtained by adding fetal bovine serum or the like to such a medium.
  • Culture is usually pH 6-8, carried out between 30 ⁇ 40 ° C, 5% C0 2 1 ⁇ under conditions such presence 7 days. If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
  • Insect cells as a medium for culturing a transformant obtained by the host commonly used TNM- FH medium (Faminjiwen Inc.), Sf -. 9 (manufactured by Lee Nbitorojiwen Co.) 00 II SFM medium , ExCel l400, ExCel l405 (all manufactured by JRH Biosciences), Grace insect medium [Nature, 195, 788 (1962)], and the like.
  • the cultivation is usually performed under conditions of pH 6 to 7 and 25 to 30 ° C for 1 to 5 days.
  • antibiotics such as gentamicin may be added to the medium during the culture. JP2004 / 004789
  • a transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ.
  • a culture medium for culturing the transformant commonly used Murashige 'and' Sturg (MS) medium, white medium, or a plant hormone such as auxin or cytokinin is added to these mediums.
  • MS Murashige 'and' Sturg
  • white medium or a plant hormone such as auxin or cytokinin is added to these mediums.
  • An added medium or the like can be used.
  • Cultivation is usually performed at pH 5-9 and 20-40 ° C for 3-60 days.
  • antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.
  • a transformant derived from a microorganism, animal cell, or plant cell having a recombinant vector into which a DNA encoding the polypeptide of the present invention has been incorporated is cultured according to a conventional culture method. By producing and accumulating the polypeptide and collecting the polypeptide from the culture, the polypeptide can be produced.
  • a sugar or a sugar chain-added polypeptide When expressed by yeast, filamentous fungi, animal cells, insect cells or plant cells, a sugar or a sugar chain-added polypeptide can be obtained.
  • the polypeptide of the present invention can be produced in a host cell, secreted out of the host cell, or produced on the outer membrane of the host cell.
  • the method can be selected by changing the structure of the polypeptide to be made.
  • the polypeptide of the present invention can be secreted out of the host cell by expressing the polypeptide of the present invention with a signal peptide added in front thereof using a gene recombination technique.
  • the production amount can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like.
  • the polypeptide of the present invention can be produced using a tro transcription / translation system. That is, DNA encoding the polypeptide of the present invention is connected downstream of promoters such as SP6, T7, and ⁇ 3, and a large amount of the polypeptide of the present invention is coded by reacting each promoter-specific RNA polymerase. After synthesizing RNA in vitro, the polypeptide of the present invention can be produced using a cell-free translation system, for example, a translation system using a heron reticulocyte lysate wheat germ extract.
  • a conventional enzyme isolation and purification method can be used.
  • the polypeptide of the present invention when expressed in a lysed state in cells, the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, The cells are disrupted using a mantongaulin homogenizer, dynomill, etc. to obtain a cell-free extract.
  • a normal enzyme isolation and purification method can be used, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent. Law, Jechilami Noechil (DEAE)-Sepharose, DIAI0N ⁇ -75
  • Anion-exchange chromatography using a resin such as (Mitsubishi Kasei), cation-exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), petitinoresepharose, Hydrophobic chromatography using resin such as pheno-recepharose, gel filtration using molecular sieve, affinity chromatography, chromatophoresis, electrophoresis such as isoelectric focusing
  • a method such as the method alone or in combination, a purified sample can be obtained.
  • Affinity purification can also be performed using a suitable carrier such as nickel resin and glutathione sepharose.
  • the cells are similarly collected, crushed, and centrifuged to obtain an insoluble form of the polypeptide as a precipitate fraction.
  • Collect. Solubilize the insoluble recovered polypeptide with a polypeptide denaturant.
  • the polypeptide is returned to a normal three-dimensional structure by diluting or diffusing the solubilized solution and reducing the concentration of the polypeptide denaturing agent in the solubilized solution.
  • a purified preparation of the polypeptide can be obtained by the same isolation and purification method as described above.
  • the polypeptide of the present invention is secreted extracellularly, the polypeptide can be recovered in the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above. Can be obtained.
  • polypeptide of the present invention examples include a polypeptide containing an amino acid sequence represented by SEQ ID NO: 1.
  • vacuolar enzymes such as triptidyl peptidase, carboxypeptidase, aminopeptidase, and serine protease are used. Since aspartyl protease, preferably, tripidyl peptidase and carboxypeptidase are efficiently secreted extracellularly, vacuolar enzymes can be produced in the medium.
  • a polypeptide having an activity of localizing a vacuolar enzyme to a cell means that when the function of the polypeptide is suppressed, all or a part of the vacuolar enzyme is normally localized in the vacuole.
  • Vacuolar enzymes secreted extracellularly may be either precursors or matures.
  • Such polypeptides include: Examples include polypeptides encoded by genes involved in the selection and transport of vacuolar enzymes, vacuolar acidification, morphogenesis, and maintenance.For example, Saccharomyces cerevisiae VPS genes, PEP genes, VAC genes, A filamentous fungus that is homologous to one of the TLG gene group, VAM gene group, APG gene group, CVT gene group, GRD gene group, or a gene group such as syntaxin family and sorting-nexin family in animal and plant cells. And a polypeptide encoded by the gene.
  • polypeptides examples include polypeptides encoded by the vpsA gene, vpsB gene and vpsC gene of Aspergillus nidulans, the VAM3 homologous gene of Aspergillus oryzae, the Aovps 5 gene, and the like.
  • the polypeptide encoded by the Aovps5 gene is also hereinafter referred to as Aovps5 polypeptide), and Aovps5 polypeptide is preferably used.
  • a filamentous fungal gene encoding such a polypeptide can be obtained in the same manner as in the method for obtaining ⁇ A of the present invention described in 1.
  • filamentous fungi examples include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, Fusarium, Humicola, Mucor, Monascus, etc., and those belonging to the genus Aspergillus. And a filamentous fungus belonging to the genus Aspergillus used in the brewing industry.
  • filamentous fungi belonging to the genus Aspergillus used in the brewing industry include, for example, Aspergillus' oryzae, Aspergillus soja, Aspernoreginoles niger, Aspernoreginoles caschi (
  • A a method for disrupting a gene, (b) a method for transcribing an antisense mRNA, and (c) a method for suppressing the function of a polypeptide having an activity of localizing a vacuolar enzyme to a cell.
  • DNA for gene disruption In the method for gene disruption, first, DNA encoding polypeptide A whose function is to be inhibited is isolated, and polypeptide A of the DNA is coded. The structure is destroyed by inserting or deleting one or more DNAs, usually several hundred bp to several kb, using the restriction enzyme site in the region to be ligated, and the function of polypeptide A (the present invention) In this case, DNA that no longer encodes a polypeptide having the activity of localizing vacuolar enzymes to cells (hereinafter referred to as DNA for gene disruption) is prepared.
  • the gene encoding polypeptide A in the genome is transformed by homologous recombination with the DNA for gene disruption.
  • the body is selected by PCR or Southern blotting.
  • Genes that serve as transformation markers are genes that are related to auxotrophy and drug resistance of the host filamentous fungi, such as sC inheritance +, argB crawling, ni aD3 ⁇ 4 fe +, pyr pyrGja na and amdS 1 child, ptrA exclusion ⁇ gene, pabaA gene, niiA gene, BenA R gene, etc.
  • the vector used for the construction of the recombinant vector may be any ordinary plasmid vector that can autonomously replicate in E. coli and has an appropriate restriction enzyme site for closing.For example, pBluescript SK II (+) (Stratagene) Manufactured).
  • a transformation marker gene is not used to prepare the DNA for gene disruption, a transformant is selected by using a vector in which the transformation marker gene has been inserted into the vector described above to prepare a recombinant vector. can do.
  • a filamentous fungus used as a host is a strain deficient in a transformation marker gene.
  • (C) In the method of introducing a mutation into a promoter region, first, DNA containing a promoter region upstream of the coding region of a genomic gene encoding polypeptide A whose function is to be suppressed is isolated. A DNA having a promoter function disrupted is produced by inserting or deleting a DNA of several lOObp to several kb by using a restriction enzyme site in a promoter region of the DNA. Next, a filamentous fungus is transformed with a recombination vector containing the DNA, and the promoter region of the gene encoding polypeptide A in the genome is transformed by homologous recombination with DNA whose promoter function has been destroyed. Transformants are selected by PCR or Southern blotting. As the vector used for the preparation of the recombinant vector, the same vector as the vector used for the gene disruption in (a) is used. .
  • a dominant negative mutant is a polypeptide prepared by adding a modification such as deletion, substitution, or addition to a functional domain of a certain polypeptide, loses the function of the original polypeptide, and furthermore, A variant of the sex stomach that interferes with the normal function of the polypeptide.
  • a DNA encoding a dominant negative mutant of polypeptide A whose function is to be suppressed is prepared.
  • an expression vector for filamentous fungi is prepared according to the method described in 2. above. In the transformant obtained by transforming the filamentous fungus with the expression vector, a dominant negative mutant is expressed to inhibit the function of normal polypeptide A in the cell.
  • the PX domain of the Saccharomyces cerevisiae VAM7 polypeptide showed The amino acids involved in the binding include arginine at position 87, lysine at position 95, arginine at position 128, and arginine at position 129.
  • the amino acid sequence from position 87 to position 89 (Arg Arg Tyr) is particularly important. Therefore, a dominant negative mutant can be prepared by introducing a mutation such as deletion or substitution into any of these amino acids or a plurality of amino acids selected from these amino acids.
  • a mutant comprising only a coiled coil region in which a portion other than the coiled coil region has been deleted is also an example of a dominant negative mutant that inhibits formation of a normal polypeptide polymer.
  • a mutant comprising only the PX domain or a mutant in which part or all of the coiled coil region is deleted may be used. .
  • the function of the polypeptide having the activity of localizing the vacuolar enzyme to the cell when the function of the polypeptide having the activity of localizing the vacuolar enzyme to the cell is suppressed, the formation of conidia tends to be inhibited.
  • the expression of the dominant negative mutant can be performed using an expression vector that can be induced by administration of a drug, etc., the function of the gene is not suppressed in the process of growth and conidiation, and the gene By suppressing the function, there is an advantage that the vacuolar enzyme can be produced extracellularly without impairing the conidial ability.
  • any method can be used as long as DNA can be introduced into filamentous fungi.
  • AOV PS 5 polypeptide mutants function is deficient in by selecting (hereinafter, also referred to as Ao V ps5 function deficient mutant), without using the Do genetic manipulation will Yo above, Aovps 5 Filamentous fungi in which the function of the polypeptide is suppressed can be obtained.
  • Aovps5 gene disrupted strain of Aspergillus oryzae secretes a reddish brown pigment into the medium when cultured on an agar medium. Therefore, Aovp S 5 polypeptides of the functions are possible scan cleaning mutant strain deficient by utilizing this property.
  • a colony whose peripheral medium turns reddish brown can be selected as a candidate strain for Aovps5 function-deficient mutants.
  • tryptidyl peptidase activity in the medium in which the above candidate strains were cultured was measured, and those with increased activity compared to the parent strain were identified as Aovps 5 function-deficient mutants. And select.
  • the SPS strain of Saccharomyces cerevisiae has improved resistance to heavy metals such as cobalt and nickel compared to its parent strain [Proc. Natl. Acad. USA, 98, 9660 (2001)]. Therefore, the Aovps 5 gene-disrupted strain of Aspergillus oryzae is also expected to have acquired resistance to heavy metals. Therefore, it is possible to screen A0 V ps5 function-deficient mutants using the resistance to heavy metals as an index.
  • heavy metals include chromium, manganese, iron, cobalt, nickel, copper, zinc, molybdenum, silver, cadmium, tin, barium, mercury, and lead. can give. '
  • a parent medium and Aov-ps5 gene were added to a medium in which these heavy metals were not contained or contained only at the concentration that is contained in a normal medium. Culture the disrupted strain and determine the concentration of heavy metals in the selection medium so that the parent strain cannot grow but the Aovps 5 gene-broken strain can grow. Conidia which ultraviolet was irradiated to induce mutations in the genome, cultured on selective media containing heavy metals determined concentration, the colonies grown in the candidate strains of Ao V ps 5 loss-of-function mutant select.
  • the activity of triptidyl peptidase in the medium in which the candidate strain was cultured was measured, and those with higher activity compared to the parent strain were identified as Aovps5 function-deficient mutants. Select as stock.
  • the Aovps 5 function-deficient mutant obtained in this manner is suitable for production of foods, beverages, and the like because it has not been subjected to genetic manipulation.
  • the filamentous fungus is cultured in a medium, the vacuolar enzyme is produced and accumulated in the medium, and the vacuolar enzyme can be collected from the medium.
  • the method for culturing the filamentous fungus includes solid culture, liquid culture, porous membrane culture, gel entrapment immobilization culture, and the like.
  • spores can also be inoculated.
  • Aovps 5 gene-disrupted strains do not form spores, they can be inoculated after, for example, dispersing hyphae in sterile physiological saline or the like.
  • a liquid medium glucose, starch, dextrin, fructose, sucrose, etc. as a carbon source, sodium nitrate, ammonium chloride, ammonium sulfate, polypeptone, yeast extract, defatted soy protein, etc. as a nitrogen source
  • a mineral component sodium phosphate, potassium phosphate, magnesium sulfate, iron sulfate and the like can be added.
  • the cultivation is carried out under aerobic conditions such as shaking culture or deep aeration stirring culture, usually at 15 to 50 ° C, preferably 25 to 37 ° C, usually for 1 to 7 days.
  • the raw material of the medium includes wheat bran, steamed rice, defatted soybean protein, etc. in which an aqueous medium is moistened, and the aqueous medium includes an aqueous solution containing water and inorganic salts.
  • the cultivation is performed at a temperature of usually 15 to 50 ° C, preferably 25 to 37 ° C, and a humidity of 80 to 100%, preferably 90 to 100%, usually for 3 to 10 days.
  • the culture can be performed according to the method described in JP-A-11-225574.
  • vacuolar enzyme in which a filamentous fungus having the ability to secrete a vacuolar enzyme out of a cell is cultured in a liquid medium.
  • the vacuolar enzyme can be isolated and purified according to the method described below.
  • tags such as pRSET-based vector (Invitrogen) and pGEX-based vector (Amersham Biosciences) are attached to vacuolar enzymes and expressed, appropriate tags such as nickel resin and gnoletathion sepharose can be used. Abity purification can also be performed using a carrier.
  • vacuolar enzymes are isolated from the supernatant.
  • the vacuolar enzyme can be isolated and purified from the enzyme extract by the same method as the purified method.
  • a vacuolar enzyme can be isolated and purified from a medium that has been brought into contact with a gel in which cells have been embedded, as in the case of culturing in a liquid medium.
  • a protein hydrolyzate can be produced by adding a protein-containing raw material to the culture.
  • the vacuolar enzyme obtained by the method described in 3. (2) is added to the protein-containing raw material and mixed, and the mixture is usually treated at 20 ° C to 60 ° C, preferably 30 ° C to 50 ° C.
  • the pH during the reaction may be any pH at which the vacuolar enzyme and secretory enzyme of the present invention can act, but is preferably adjusted to pH 3 to 8, and more preferably to pH 5 to 8.
  • vacuolar enzyme used in this production method a purified vacuolar enzyme can be used, or the vacuolar enzyme described in 3. above is secreted extracellularly without purifying the vacuolar enzyme.
  • a culture containing a vacuolar enzyme or a culture-treated product obtained by culturing a filamentous fungus in a medium can also be used.
  • the culture product include a solution obtained by removing cells from the culture by filtration, centrifugation, or the like, a concentrate of the solution, a dried product, and the like.
  • the filamentous fungus may be any as long as it is used in the brewing industry, and examples include Aspergillus oryzae, Aspergillus soja, Aspergillus.
  • the protein contained in the raw material used in the method for producing a protein hydrolyzate is not particularly limited, but a protein having a high glutamic acid content is preferable.
  • a protein having a high glutamic acid content examples include wheat dart, corn meal gluten, defatted soy, isolated soy protein, and the like. Wheat dart, corn meal darten, and the like are preferably used.
  • the raw material containing the protein is not particularly limited, but those having a high protein content are preferable.
  • the obtained protein hydrolyzate can be used as it is as a seasoning, but it is mixed with amino acids, nucleic acids, extracts, acidulants, sweeteners, etc. to adjust the taste and flavor to give a strong umami. Good seasoning can be made.
  • FIG. 1 Comparison of the amino acid sequence encoded by the VPS5 gene of Saccharomyces cerevisiae and the amino acid sequence encoded by the VPS5 homologous gene of Neurosbola classa.
  • the upper and lower sequences are Saccharomyces cerevisiae, respectively.
  • the amino acid sequence of Neurospora crassa is shown in the middle row, and the amino acids corresponding to both sequences are shown in the middle row
  • the numbers indicate the positions of amino acid residues from the N-terminus of each polypeptide.
  • the amino acid sequence corresponding to is shown.
  • FIG. 2 shows the structure of a transforming DNA used for disruption of the Aovps5 gene.
  • Fig. 3 shows PCR detection of the shattered Aovps5 gene in the genomes of CT6118 strain, CT9103 strain and CT96 strain. Lanes 1 to 3 show the genomes of Aovps5 mutant strains CT6118, CT9103 and CT96, respectively. Lane 4 shows plasmid pDV51 for Aovps5 gene disruption, and lane 5 shows wild type (RIB40 This shows the PCR product when genomic DNA of the strain was used as a template.
  • FIG. 4 shows the nitrogen solubilization rate (total nitrogen in the filtrate ⁇ total nitrogen in the raw material ⁇ 100) of the hydrolyzed protein produced using the culture solution of the Aovps5 gene-disrupted strain.
  • Fig. 5 Protein protein produced using a culture solution of Aovps 5 gene disrupted strain Shows the concentration (mmol / 1) of free amino acids in the hydrolyzate.
  • Genomic DNA was prepared from the cells of Aspergillus oryzae RIB 40 strain (ATCC No. 42144) according to the method of Gomi et al. [Gen. Appl. Microbiol., 35, 225 (1989)] as shown below.
  • Aspergillus oryzae RIB 40 strain in 150 ml of DPY medium (2% dextrin, 2% polypeptone, 0.5% yeast yeast, 0.5% potassium phosphate, 0.05% magnesium sulfate heptahydrate)
  • DPY medium 2% dextrin, 2% polypeptone, 0.5% yeast yeast, 0.5% potassium phosphate, 0.05% magnesium sulfate heptahydrate
  • the cells were collected with a Buchner funnel and ground in a mortar in the presence of liquid nitrogen. This was added to a 10 ml protease solution (50 mmol / l EDTA (pH 8.0), 0.5% SDS,
  • the cells were suspended in 0.1 mg / ml proteinase K) and incubated at 50 ° C for 4 hours. This solution was sequentially subjected to phenol treatment twice, phenol / cloth form treatment twice, and closform form treatment once. The treatment with these organic solvents was carried out by gently mixing the solution and the organic solvent and collecting the supernatant. Then 1/10 volume of a solution 3 mol / 1 sodium acetate (P H5.2
  • the nucleotide sequence of the Saccharomyces cerevisiae VPS5 gene was obtained from the public database GenBank (registration number: U73512).
  • GenBank registration number: U73512
  • Various filamentous fungi databases were searched using the obtained Saccharomyces cerevisiae VPS5 gene coding region sequence (SEQ ID NO: 4) as a query sequence.
  • SEQ ID NO: 4 the genomic database of Neurospora crassa (http: // www-genome, // www-genome, wi.mit.edu/annotat ⁇ o ⁇ ) / from f ungi / neurospora /)
  • Saccharomyces' Serepishe of VPS 5 genes highly homologous (E-value 3E-54 ) gene sequence (SEQ ID NO:.
  • FIG. 1 shows a comparison between the Saccharomyces cerevisiae VPS5 gene and the amino acid sequence encoded by the gene of Neurospora crassa having high homology to the gene.
  • the degenerated primers V5P-F and V5P-R were designed from the regions that are relatively highly conserved and are likely to be functionally conserved.
  • the nucleotide sequences of the primers are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.
  • the nucleotide sequences of SEQ ID NOS: 6 and 7 are the amino acid sequence of amino acids 181 to 189 (Val Gly Asp Pro His Lys Val Gly Asp) and the amino acids 267 to 274 of the amino acid sequence encoded by the VPS5 homologous gene of Neurosbora classa. U (Pro Pro Glu Lys Gin Al a Val Gly) respectively.
  • PCR is the above-mentioned O Li Gore-nucleotide primer, Asuperugirusu-cage zero of genomic DNA template, was carried out under the following conditions by the GeneAmp PCR Syst em 2400 (Nono 0 manufactured by one Kin. Elma one company). After heating at 94 ° C for 5 minutes to denature the template DNA, one cycle was performed at 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 cycles. A 30-second reaction at C was performed for 30 cycles. As a result of electrophoresis of the reaction solution on a 2% agarose gel, a DNA fragment of about 250 bp was detected.
  • the genomic DNA of Aspergillus oryzae obtained in (1) was cut with BamHI and inserted into the BamHI site of lambda phage vector ⁇ DASH II (Stratagene). This was packaged in vitro and used as a genomic DNA library for Aspergillus oryzae. Using this library
  • a plaque was formed using E. coli P2392 as a host according to a conventional method.
  • the plaques are blotted onto Hybond-N + (Amersham Biosciences) and denatured (1.5 mol / 1 sodium chloride, 0.5 mol / 1 sodium hydroxide) for 5 min.
  • a neutralizing solution 1.5 mol / 1 sodium chloride, 0.5 mol / 1 tris-hydrochloric acid (pH 8.0)
  • 2XSSC 0.3 mol / 1 sodium chloride, (70 mmol / 1 sodium citrate) for 5 minutes and storm-dried.
  • the membrane was added to a buffer solution (5XSSC, 50% formamide, 0.1% N-lauroyl sarcosine, 0.2% SDS, 1% blocking reagent (Roche).
  • the phage DNA is purified from the positive clone obtained in the above (5) according to a conventional method, cut with Sail, and a 3.2 kbp Sail fragment is inserted into the Sail site of pBluescript SK II (+) (Stratagene). And plasmid pVPS5S.
  • the base sequence of the Aovps5 gene of Aspergillus oryzae was determined using DSQ2000L DNA Sequencer (manufactured by Shimadzu Corporation). The results are shown in SEQ ID NO: 2.
  • Aspergillus strain RIB 40 strain was inoculated with 60 ml of DPY medium (inoculated and shake-cultured at 300 rpm for 2 days at 150 rpm in a 300 ml Erlenmeyer flask with a notch / resp.).
  • the wet cells lg were turned into a fine powder in a mortar in the presence of liquid nitrogen.
  • the recovered mRNA is used for cDNA synthesis using gateway technology and Superscript 1 for the Crowjung, 'plasmid system (SUPERSCRIPT).
  • PCR was performed by Geneamp PCR system 2400 (manufactured by Parkin Elma) using the template cDNA and primers described above. The reaction is performed by heating at 94 ° C for 5 minutes to denature the DNA of the template, and then the reaction is performed at 94 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 2 minutes in a 3: 0 cycle. Went under.
  • a homologous recombination of Aspergillus oryzae was performed by homologous recombination using an Aovps5 gene (hereinafter, also referred to as a “broken Aovps5 gene”) in which a marker gene was inserted into the coding region of the polypeptide of the present invention to break the original function of the Aovps5 gene.
  • An Aovps5 gene disrupted strain in which the Aovps5 gene on the genome has been broken has been prepared. The method is described below.
  • the BamHl / BamHI fragment of pIAD containing the sC gene was inserted into a Bglll site present in the coding region of the Aovps5 genomic gene of pVPS5S to prepare a plasmid pDV51 having the broken Aovps5 gene.
  • the DNA for transformation was obtained by cleaving pDV51 with Sail, separating on a 0.8% agarose gel, and purifying a 3.5 kbp fragment by GeneClean 'kit.
  • Fig. 2 shows the structure of the transforming DNA.
  • Aspergillus an sC gene-deficient strain.
  • Oryzae NS4 strain (National Institute of Liquor Research, RIB No. NS4) was inoculated into 150 ml of DPY medium, shake-cultured at 30 ° C for 20 hours, and the cells were collected by filtration and washed with sterile water. did. The collected cells were suspended in 10 ml of an enzyme reaction solution [0.6 mol ammonium sulphate, 50 mmol / 1 maleate buffer (pH 5.5), 1% catalase (Takara Shuzo)]. Shake gently for 3 hours at C. The reaction solution was filtered using Miracloth (manufactured by Calbiochem) to collect protoplasts.
  • buffer A 1.2 mol / 1 sorbitol, 50 mmol / 1 calcium chloride, ⁇ 35 mmol / 1 sodium chloride, 10 mmol / 1 tris-hydrochloride (pH 7.5)
  • the precipitate was gently dispersed and centrifuged at 2000 rpm for 8 minutes to collect the precipitate.
  • the above buffer was added and suspended at a concentration of 1 ⁇ 10 7 to 1 ⁇ 10 8 cells / ml to prepare a protoplast suspension.
  • the 200 At 1 pro-plast suspension was mixed with 5 g of the transforming DNA and allowed to stand on ice for 30 minutes.
  • buffer B 60% polyethylene glycol 4000, 50 mmol / 1 calcium chloride, 10 mmol / 1 bets Risu hydrochloric acid (P H7.5)] were mixed 250 1 added gently, the buffer B 250 ⁇ 1 was added and mixed gently, and 850 1 of buffer ⁇ ⁇ was further added and mixed gently. This was allowed to stand at room temperature for 20 minutes to introduce DNA into the cells.
  • a selective medium (1.2 mol sorbitol, 0.2% ammonium chloride, 0.1% ammonium sulfate, 0.05% chloride, 0.05% sodium chloride, 0.1% dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.002% iron sulfate Layered over heptahydrate, 2% glucose, 1.5% agar, pH 5.5).
  • the selective medium is used to transform a strain into which a DNA containing the disrupted Aovps5 gene into which the sC gene has been introduced has been introduced. You can choose your body. After culturing at 30 ° C for 7 days, about 100 transformants were obtained. The obtained transformant was subcultured three times on the above selective medium.
  • strain homologous recombination has occurred in the gene heritage
  • Aovps5 is obtained from each of the transformants by the method of (1) described in Example 1
  • the genomic DNA is used as a template, and PCR can be performed by using as a primer the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 9 and SEQ ID NO: 10 used for cloning of the cDNA.
  • PCR was performed using a Program Temp Control System PC-700 (manufactured by Astec).
  • the agar medium around the colony changed from milky white to reddish brown as a result of culturing at 30 ° C for more than 5 days. Therefore, AOV P s 5 gene disruption strain would be expected to secrete reddish brown dye in the culture conditions.
  • a parenter strain of Aspergillus oryzae (NS4 strain) and three Aovps 5 gene-broken strains (CT6118, CT9103 and CT96 strains) were inoculated into 60 ml of DPY medium, respectively.
  • the cells were cultured with shaking at 180 rpm for 2 days.
  • the culture was filtered through a 0.45 m membrane filter to remove the cells, and 435 ⁇ l of the filtrate was added with 24151 50 ramol / l acetic acid buffer to give 2.85 ml of the enzyme solution.
  • the substrate solution 50 mmol octaacetic acid buffer ( ⁇ 4.5) containing 8 ramol / 1 alanine-araphen-phenylanilane-paranitroanilide
  • was added in 150 ml. ⁇ 1 was mixed to initiate the decomposition of the substrate.
  • the mixture was reacted at 30 ° C for 10 minutes, sampled with time, and the absorbance at 384 nm was measured with a spectrophotometer.
  • the parent strain (NS4 strain) and three Aov P s 5 gene-disrupted strains (CT6118 palm, CT9103 strain and CT96 strain) were inoculated respectively in 60 m of DPY medium and placed in a 300 ml baffled triangular flask at 30 ° C.
  • the cells were cultured with shaking at 180 rpm for 2 days. After the culture, the cells were separated into bacterial cells and supernatant by suction filtration. The weight of the cells was measured in a wet cell state.
  • the nitrogen solubilization rate is the ratio of the total nitrogen in the filtrate to the total nitrogen contained in the raw material.
  • the total nitrogen was measured using NA2100 (manufactured by Thermofinigan).
  • the measured value (%) was normalized by the wet cell weight (g), and the average of the results of three experiments was calculated.
  • the results are shown in FIG.
  • the method for measuring the amount of free amino acids was in accordance with the orthophthalaldehyde (0PA) method [Food Chem., 62, 363 (1998)].
  • the measured amount of free amino acid (nimol / 1) was standardized by the wet cell weight (g), and the average of the results of three experiments was calculated (FIG. 5).
  • the protein hydrolyzate produced using the culture solution of the Aovps5 gene disrupted strain significantly increased the nitrogen solubilization rate and the amount of free amino acids per wet cell weight (g) compared to the parent strain. I found that. Therefore, a protein hydrolyzate having a higher hydrolysis rate than before can be produced by using a culture solution of the Aovps5 gene-broken strain.
  • the present invention relates to the Aovps 5 gene, a polypeptide encoded by the gene, a filamentous fungus in which the function of the gene is suppressed, and a protein using the filamentous fungus.
  • a method for producing a protein hydrolyzate having a significantly increased hydrolysis rate is provided.

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Abstract

Aovps5 gene, a polypeptide coded for by the gene, filamentous fungi wherein the functioning of the gene has been inhibited, and a process for producing a protein hydrolyzate at strikingly enhanced protein hydrolysis ratio with the use of the filamentous fungi.

Description

明 細 書  Specification
新規遺伝子  New gene
技術分野 Technical field
本発明は、 サッカロミセス ' セレビシェの VFS5ボリぺプチドと相同 性を有する液胞酵素を細胞内に局在させる活性を有するポリぺプチド- 該ポリぺプチドをコ一ドする DNA、該ポリぺプチドの機能を抑制するこ とにより液胞酵素を細胞外に分泌する糸状菌を作製する方法、 および 該方法により作製される液胞酵素を細胞外に分泌する糸状菌に関する t さらには、 該糸状菌を用いた液胞酵素の製造方法、 およぴ該酵素を用 いたタンパク加水分解物の製造方法に関する。 背景技術 The present invention relates to a polypeptide having an activity of localizing a vacuolar enzyme having homology to VFS5 polypeptide of Saccharomyces cerevisiae in a cell-a DNA encoding the polypeptide, a DNA encoding the polypeptide, how the function of suppressing this and by the liquid胞酵element producing filamentous fungi that secrete extracellularly, and t further relates filamentous fungus liquid胞酵element made secreted extracellularly by said method, the filamentous fungus The present invention relates to a method for producing a vacuolar enzyme used, and a method for producing a protein hydrolyzate using the enzyme. Background art
液胞は酵母、 植物細胞、 糸状菌などに共通して観察される細胞内器 官の一つであり、 細胞内成分のリサイクル、 浸透圧調節、 解毒、. 栄養 素の貯蔵などの生理的機能を担っている。 酵母の液胞はプロテアーゼ、 ホスファターゼ、 エステラーゼ、 リ / ーゼ、 リボヌク レアーゼ、 グリ コシダーゼなど多くの加水分解酵素を含むことが知られている  Vacuoles are one of the intracellular organs commonly observed in yeast, plant cells, filamentous fungi, etc., and have physiological functions such as recycling of intracellular components, osmotic pressure regulation, detoxification, and storage of nutrients. Is responsible for. Yeast vacuoles are known to contain many hydrolases, including proteases, phosphatases, esterases, lyases, ribonucleases, and glycosidases
[Mi crob io logi ca l revi ews , 54, 2り 6 、丄 990) ]。 [Mi crob io logi ca l revi ews, 54, 2-6, 990990)].
醤油醸造などにおいては、 熟成中に麹菌 〔ァスペルギルス ' ォリゼ (Aspergi l lus orvzae ま 7こ fまァスへノレギメレス · ソーャ ( Aspergi l lus soj ae) 〕 が溶菌し、 溶出した細胞内タンパク質により窒素可溶化率が 上昇し （醤油の科学と技術、 1 9 8 8年、 p . 1 2 2 ) 、 味質の向上 に寄与しているが、 その分解活性の大部分は液胞酵素に因っていると 予想される。 このよ うに液胞酵素は産業上有用であり、 特に GRASダレ ードである麹菌の液胞酵素は食品製造上、 利用価値が高い。 しかし、 液胞酵素を取得するためには、 自己消化または物理的もしくは酵素的 に麹菌を破砕、 溶菌することが必要であり時間と手間がかかる。 その ため効率的な液胞酵素の取得方法が望まれる。  In soy sauce brewing, etc., aspergillus (Aspergillus orvzae) is lysed during ripening, and nitrogen is solubilized by the eluted intracellular proteins. The rate has increased (Soy sauce science and technology, 1988, p. 122), contributing to the improvement of taste quality, but most of its decomposition activity is due to vacuolar enzymes Thus, the vacuolar enzyme is industrially useful, and in particular, the vacuolar enzyme of Aspergillus oryzae, which is GRAS dale, is highly useful in food production. Since it is necessary to autolyze or physically or enzymatically disrupt and lyse the koji mold, it takes time and effort, so that an efficient method for obtaining vacuolar enzymes is desired.
北本はソーティング工学の見地から、 液胞酵素を効率的に細胞外に 分泌できる可能性に言及している （生物工学会誌、 1 9 9 9年、 第 7 7卷、 p . 1 - 3 3 ) 力 これまで麹菌において液胞酵素を細胞外に 分泌した例はない。 Kitamoto mentions the possibility of efficient extracellular secretion of vacuolar enzymes from the point of view of sorting engineering (Biotechnical Society, 1999, 9th, 7th edition). Vol. 7, p. 1-3-3) Force There has been no example of koji mold that secreted vacuolar enzymes extracellularly.
液胞酵素を分泌酵素から選別し、 液胞まで輸送する機構についての 解析は、 酵母 （サッカロミセス . セ レピシェ） で研究が進んでいる。 サッカロミセス ' セレビシェの液胞酵素の選別と輸送には 40以上の遺 伝子が関与しており、 該遺伝子の破壊、 変異によりその機能が欠失、 低下した場合、 液胞酵素の輸送は正常に行なわれず細胞外にミス ソー トされる。 これらは液胞酵素選別 （Vacuolar protein sorting： VPS) 遺伝子として知られてレヽる [Annual Review of Cell and Developmental Biology, 11， 1 (1995)]。 サッカロミセス ' セレピシェの VPS5遺伝子 は、 ソーティング · ネキシン I と相同性を有するポリペプチドをコー ドする液胞酵素選別遺伝子であり、 VPS5遺伝子を破壌したサッカ口ミ セス · セレビシェは、 液胞酵素の細胞外へのミスソー トが見られた  Studies on the mechanism of sorting vacuolar enzymes from secretory enzymes and transporting them to the vacuole are being studied in yeast (Saccharomyces cerevisiae). More than 40 genes are involved in the selection and transport of Saccharomyces cerevisiae vacuolar enzymes.If the function is deleted or reduced due to disruption or mutation of the gene, transport of the vacuolar enzymes is normal. Miscellaneous and missorted outside the cell. These are known as Vacuolar protein sorting (VPS) genes [Annual Review of Cell and Developmental Biology, 11, 1 (1995)]. The Saccharomyces cerevisiae VPS5 gene is a vacuolar enzyme selection gene that encodes a polypeptide having homology to sorting nexin I.Saccharomyces cerevisiae, which broke the VPS5 gene, is a vacuolar enzyme cell. Missed out was seen
[Journal of Cell Science, 110, 1063 (1997)]。 [Journal of Cell Science, 110, 1063 (1997)].
糸状菌にも形態的に酵母の液胞と類似の細胞内器官が存在しており . 酵母の液胞と同様、 様々な加水分解酵素を有していると考えられる。 ' また糸状菌においても酵母と類似の機構で液胞酵素が分泌酵素から選 別され、 液胞まで輸送されていると予想される。  Filamentous fungi also have intracellular organs that are morphologically similar to yeast vacuoles. Like yeast vacuoles, they are thought to have various hydrolases. 'In filamentous fungi, it is expected that vacuolar enzymes are selected from secretory enzymes and transported to the vacuoles by a mechanism similar to yeast.
糸状菌の液胞の機能解明を目的と して、 樽谷らは、 サッカロミセス To elucidate the function of the vacuole of filamentous fungi, Tarutani et al.
• セレビシェにおいて、 トランスゴルジからプレ液胞コンパートメン トへの小胞輸送に関与する、 液胞酵素選別遺伝子 VPS1の相同遺伝子 vpsAをァスぺノレギノレス . 二 ドランス (Aspergillus nidulans) カ ら単 離し、 該遺伝子破壊株を作製し、 液胞の形態形成に関与していること を示した [Gene, 268, 23 (2001)]。 また、 同じく トランスゴルジから プレ液胞コンパ一トメントへの小胞輸送に関わる液胞酵素選別に関わ ることが知られている、 VPS45の相同遺伝子 vpsBをァスペルギルス ·二 ドランスから単離した （日本農芸化学会誌， 第 7 3卷， 臨時増刊号， ェ 9 9 9年度日本農芸化学会大会講演要旨集， 1 9 9 9年， p . 2 1• In S. cerevisiae, the homologous gene vpsA of the vacuolar enzyme selection gene VPS1, which is involved in vesicle transport from the trans-Golgi to the pre-vacuolar compartment, is isolated from Aspergillus nidulans. A gene-disrupted strain was prepared and shown to be involved in vacuolar morphogenesis [Gene, 268, 23 (2001)]. A VPS45 homologous gene, vpsB, also known to be involved in the selection of vacuolar enzymes involved in vesicle transport from the trans-Golgi to the pre-vacuolar compartment, was isolated from Aspergillus nidulans (Nippon Agriculture) Journal of the chemical Society, Chapter 7 3 Certificates, extra edition No., E 9 9 9 fiscal Japan Society for Bioscience, Biotechnology, and Agrochemistry, Abstracts for the Annual Meeting, 1 9 9 9 years, p. 2 1
2 ) 。 . 2). .
松田らは、 トランスゴルジからプレ液胞コンパー トメントへの小胞 輸送に関与する.スモール GTPase、 VPS21の相同遺伝子 vpsCをァスペルギ ルス .. -ドランスから単離した （例えば、 日本農芸化学会誌， 第 7 3 巻， 臨時増刊号， 1 9 9 9年度日本農芸化学会大会講演要旨集， 1 9 9 9年， p 2 1 3 ) 。 Matsuda et al. Report that vesicles from trans-Golgi to pre-vacuolar compartment . Involved in the transport small GTP ase, VPS21 Asuperugi Angeles homologous gene vpsC of .. - was isolated from Doransu (for example, Japan Agricultural Chemistry Journal, 7 Volume 3, extra edition No., 1 9 9 9 fiscal Japan agrochemical Proceedings of the Annual Meeting of the Congress, 1 1989, p213).
正路らはァスペルギルス · ォリ ゼからサッカ ロ ミセス ' セレビシェ VAM3相同遺伝子を単離 · 同定し、 該遺伝子がサッカロミセス · セレビ シェの vam3遺伝子破壊株およぴ Pepl2/vps6遺伝子破壊株の表現型を相 補したことを示した (第 2回糸状菌分子生物学コンファ レンス要旨集， 2 0 0 2年， p . 2 4 ) 。 Shoji et al. Have isolated and identified the Saccharomyces' cerevisiae VAM3 homologous gene from Asuperugirusu-O Li Ze, the gene is the phenotype of vam3 gene disrupted strain your Yopi P epl2 / vps6 gene disrupted strain of Saccharomyces cerevisiae shell It was shown to be complementary (2nd Abstract of the Conference on Molecular Biology of Filamentous Fungi, 2002, p. 24).
また、 大根田はァスペルギルス · 二ドランスから単離 . 同定された カルボキシぺプチダーゼ Y遺伝子 cpyAに蛍光タンパク質を結合させた 融合タンパク質を、 ァスペルギルス · ォリ ゼの細胞内で発現させ、 該 遺伝子産物がァスペルギルス · ォリゼの液胞に局在することを示し [Fungal Genetics and Biology, 37, 29 (2002)]、 さらに培地中の蛍 光強度が上がる変異株を単離しその表現型を解析した （日本生物工学 会大会要旨集， 2 0 0 1年， p . 2 3 1 ) 。 しかしこの原因遺伝子の 同定には至っていない （第 2回糸状菌分子生物学コンファ レンス要旨 集， 2 0 0 2年， p . 4 9 ) 。 発明の開示  Ohneda also isolated from Aspergillus nidulans. Expressed a fusion protein in which a fluorescent protein was linked to the identified carboxypeptidase Y gene cpyA in Aspergillus oryzae cells, and the gene product was expressed as Aspergillus oryzae. Oryzae was localized in the vacuole [Fungal Genetics and Biology, 37, 29 (2002)], and a mutant strain with increased fluorescence intensity in the medium was isolated and its phenotype was analyzed. Conference Abstracts, 2001, p. 231). However, the causative gene has not been identified (Second Abstract of the 2nd Conference on Molecular Biology of Filamentous Fungi, 2002, p. 49). Disclosure of the invention
ァスペルギルス · ォリゼの、 液胞酵素を細胞内に局在させる活性を 有するポリべプチドをコ一ドする新規な遺伝子、 およぴ該遺伝子を利 用し、 効率的に糸状菌の液胞酵素を培地中に生産する方法が求められ ている。  A novel gene encoding Aspergillus oryzae, which encodes a polypeptide having an activity of localizing vacuolar enzymes to cells, and the use of the gene to efficiently utilize the vacuolar enzymes of filamentous fungi There is a need for a method of producing in a medium.
本発明は、 以下の （ 1 ) 〜 ( 3 4 ) を提供する。  The present invention provides the following (1) to (34).
( 1 ) 配列番号 1 のァミ ノ酸配列を含むポリぺプチド。  (1) A polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
( 2 ) 配列番号 1のァミノ酸配列において 1以上のアミノ酸が欠失、 置換もしくは付加されたァミノ酸配列からなり、 かつ液胞酵素を細胞 内に局在させる活性を有するポリぺプチド。  (2) A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and having an activity of localizing vacuolar enzymes to cells.
( 3 ) 配列番号 1のァミノ酸配列と 60%以上の相同性を有するァミノ 酸配列を含み、 かつ液胞酵素を細胞内に局在させる活性を有するポリ ぺプチド。 (3) Amino having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 A polypeptide comprising an acid sequence and having an activity of localizing vacuolar enzymes to cells.
( 4 ) ( 1 ) 〜 （ 3 ) のいずれか 1項に記載のポリペプチドをコード する DNA。  (4) A DNA encoding the polypeptide according to any one of (1) to (3).
( 5 ) 配列番号 2または 3の塩基配列を含む DNA。  (5) A DNA comprising the nucleotide sequence of SEQ ID NO: 2 or 3.
( 6 ) 配列番号 2または 3の塩基配列と相補的な塩基配列からなる DNA とス ト リ ンジェン トな条件下でハイブリダイズする DNAであり、 かつ液 胞酵素を細胞内に局在させる活性を有するボリぺプチドをコ一ドする DNA。  (6) DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or 3, and has an activity of localizing vacuolar enzymes to cells. DNA encoding a polypeptide having the same.
( 7 ) ( 4 ) 〜 （ 6 ) のいずれか 1項に記載の DNAをベクターに組み込 んで得られる組換えべクター。  (7) A recombinant vector obtained by incorporating the DNA according to any one of (4) to (6) into a vector.
( 8 ) ( 7 ) に記載の組換えベクターを宿主細胞に導入して得られる 形質転換体。  (8) A transformant obtained by introducing the recombinant vector according to (7) into a host cell.
( 9 ) ( 8 ) に記載の形質転換体を培地に培養し、 培養物中に （ 1 ) 〜 （ 3 ) のいずれか 1項に記載のポリペプチドを生成蓄積させ、 該培 養物から、 該ポリぺプチドを採取することを特徴とする該ポリぺプチ ドの製造方法。  (9) The transformant according to (8) is cultured in a medium, and the polypeptide according to any one of (1) to (3) is produced and accumulated in the culture. A method for producing the polypeptide, comprising collecting the polypeptide.
( 1 0 ) 配列番号 2または 3の塩基配列の連続する 20塩基以上の配列 と相補的な配列を有するポリヌク レオチドを用いて、 （4 ) 〜 （ 6 ) のいずれか 1項に記載の DNAまたは （ 1 ) 〜 （ 3 ) のいずれか 1項に記 載のポリぺプチドをコ一ドする mRNAを検出する方法。  (10) The DNA or (6) according to any one of (4) to (6), using a polynucleotide having a sequence complementary to a sequence of 20 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 2 or 3. A method for detecting mRNA encoding the polypeptide according to any one of (1) to (3).
( 1 1 ) 配列番号 2または 3の塩基配列の連続する 15塩基以上の配列 を有する DNAおよび配列番号 2または 3の塩基配列の連続する 15塩基 以上の配列と相捕的な配列を有する DNAを用いて、 （4 ) 〜 （ 6 ) のい ずれか 1項に記載の DNAまたは （ 1 ) 〜'（ 3 ) のいずれか 1項に記載の ポリぺプチドをコ一ドする raRNAを検出する方法。  (11) A DNA having a sequence of 15 or more consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 2 or 3 and a DNA having a sequence complementary to a sequence of 15 or more nucleotides of the consecutive nucleotide sequence of SEQ ID NO: 2 or 3 A method for detecting a raRNA encoding the DNA according to any one of (4) to (6) or the polypeptide according to any one of (1) to '(3) .
( 1 2 ) 液胞酵素を細胞内に局在させる活性を有するポリぺプチドの 機能を抑制させることを特徴とする、 液胞酵素を細胞外に分泌する糸 状菌の作製方法。  (12) A method for producing a filamentous fungus that secretes a vacuolar enzyme extracellularly, comprising suppressing the function of a polypeptide having an activity of localizing the vacuolar enzyme to a cell.
( 1 3 ) 糸状菌のゲノム中の、 液胞酵素を細胞内に局在させる活性を 有するポリペプチドをコードする遺伝子を遺伝子破壊することにより . 該ポリペプチドの機能を抑制させる （ 1 2 ) に記載の方法。 '(13) The activity of localizing vacuolar enzymes in the genome of filamentous fungi The method according to (12), wherein the function of the polypeptide is suppressed by disrupting the gene encoding the polypeptide. '
( 1 4 ) 液胞酵素を細胞内に局在させる活性を有するポリぺプチドを コードする遺伝子のアンチセンス mRNAを転写させることにより、 該ポ リペプチドの機能を抑制させる ( 1 2 ) に記載の方法。 (14) The method according to (12), wherein the function of the polypeptide is suppressed by transcribing an antisense mRNA of a gene encoding a polypeptide having an activity of localizing vacuolar enzymes to cells. .
( 1 5 ) 糸状菌のゲノ ム中の、 液胞酵素を細胞内に局在させる活性を 有するポリぺプチドをコ一ドする遺伝子のプロモーター領域に変異を 導入することにより、 該ポリぺプチドの機能を抑制させる ( 1 2 ) に 記載の方法。 '  (15) By introducing a mutation into the promoter region of a gene encoding a polypeptide having an activity of localizing a vacuolar enzyme to a cell in the genome of a filamentous fungus, The method according to (12), wherein the function is suppressed. '
( 1 6 ) 液胞酵素を細胞内に局在させる活性を有するポリペプチドの ドミ ナン トネガティブ変異体を発現させることにより、 該ポリぺプチ ドの機能を抑制させる （ 1 2 ) に記載の方法。  (16) The method according to (12), wherein the function of the polypeptide is suppressed by expressing a dominant negative mutant of a polypeptide having an activity of localizing a vacuolar enzyme to a cell. .
( 1 7 ) 液胞酵素を細胞内に局在させる活性を有するポリペプチドが ( 1 ) 〜 ( 3 ) のいずれか 1項に記載のポリぺプチドである、 （ 1 2 (17) The polypeptide having an activity of localizing a vacuolar enzyme to a cell is the polypeptide according to any one of (1) to (3), (12)
) 〜 （ 1 6 ) のいずれか 1項に記載の方法。 ) The method according to any one of (16) to (16).
( 1 8 ) 糸状菌を、 ゲノムに突然変異を誘発させた後に培養し、 培地 に赤褐色の色素を分泌する菌株を選択することを特徴とする、 （ 1 ) 〜 （ 3 ) のいずれか 1項に記載のポリペプチドの機能が抑制された糸 状菌の取得方法。  (18) The method according to any one of (1) to (3), wherein the filamentous fungus is cultured after mutagenesis of the genome, and a strain that secretes a reddish brown pigment in a medium is selected. A method for obtaining a filamentous fungus in which the function of the polypeptide according to 1 is suppressed.
(Ί 9 ) 糸状菌を、 ゲノムに突然変異を誘発させた後に重金属を含む 培地に培養し、 重金属に対する耐性を有する菌株を選択することを特 徴とする、 （ 1 〜 （ 3 ) のいずれか 1項に記載のポリペプチドの機 能が抑制された糸状菌の取得方法。  (Ί9) The method according to any one of (1) to (3), wherein the filamentous fungus is cultured in a medium containing heavy metals after mutagenesis of the genome, and a strain having resistance to heavy metals is selected. A method for obtaining a filamentous fungus in which the function of the polypeptide according to claim 1 is suppressed.
( 2 0 ) 糸状菌がァスペルギルス属に属する糸状菌である （ 1 2 ) 〜 (20) The filamentous fungus is a filamentous fungus belonging to the genus Aspergillus (12) ~
( 1 9 ) のいずれか 1項に記載の方法。 (19) The method according to any one of the above (19).
( 2 1 ) ァスペルギルス属に属する糸状菌がァスペルギルス · ォリゼ である ( 2 0 ) に記載の方法。  (21) The method according to (20), wherein the filamentous fungus belonging to the genus Aspergillus is Aspergillus oryzae.
( 2 2 ) ( 4 ) 〜 （ 6 ) のいずれか 1項に記載の DNAのコー ド領域内に (22) In the DNA coding region according to any one of (4) to (6),
1つ以上の DNAを挿入または欠失することにより、液胞酵素を細胞内に 局在させる活性を有するポリべプチドをコ一ドしなくなった DNA。 ( 2 3 ) ( 2 2 ) に記載の DNAを含む組換えベクター。 DNA that does not encode a polypeptide having the activity of localizing vacuolar enzymes to cells by inserting or deleting one or more DNAs. (23) A recombinant vector comprising the DNA according to (22).
( 2 4 ) ( 1 7 ) 〜 （ 2 1 ) のいずれか 1項に記載の方法で得られる 糸状菌。  (24) A filamentous fungus obtained by the method according to any one of (17) to (21).
( 2 5 ) 糸状菌がァスペルギルス属に属する糸状菌である （ 2 4 ) に 記載の糸状菌。  (25) The filamentous fungus according to (24), wherein the filamentous fungus belongs to the genus Aspergillus.
( 2 6 ) ァスペルギルス属に属する糸状菌がァスペルギルス 'ォリゼで ある ( 2 5 ) に記載の糸状菌。.  (26) The filamentous fungus according to (25), wherein the filamentous fungus belonging to the genus Aspergillus is Aspergillus'oryzae. .
( 2 7 ) ( 1 2 ) 〜 （ 2 1 ) のいずれか 1項に記載の方法で得られる 糸状菌を培地に培養し、 培地中に液胞酵素を生成蓄積させ、 該培地か ら液胞酵素を採取することを特徴とする液胞酵素の製造方法。  (27) A filamentous fungus obtained by the method according to any one of (12) to (21) is cultured in a medium, to produce and accumulate a vacuolar enzyme in the medium, and to form a vacuole from the medium. A method for producing a vacuolar enzyme, comprising collecting an enzyme.
( 2 8 ) 培地が固体培地である （ 2 7 ) に記載の製造方法。  (28) The production method according to (27), wherein the medium is a solid medium.
( 2 9 ) 培地が液体培地である請求項 （ 2 7 ) に記載の製造方法。 ( 3 0 ) 液胞酵素がトリぺプチジルぺプチダーゼである （ 2 7 ) 〜 （ (29) The production method according to (27), wherein the medium is a liquid medium. (30) Vacuolar enzyme is triptidyl peptidase (27) ~ (
2 9 ) のいずれか 1項に記載の製造方法。 29) The production method according to any one of the above items 9).
( 3 1 ) ( 2 7 ) 〜 （ 3 0 ) のいずれか 1項に記載の方法により製造 された液胞酵素、 または （ 1 2 ) 〜 （ 2 1 ) のいずれか 1項に記載の 方法で得られる糸状菌を培養して得られる液胞酵素を含む培養物また は培養処理物を、 基質となるタンパク質に作用させることを特徴とす るタンパク加水分解物の製造方法。  (31) A vacuolar enzyme produced by the method according to any one of (27) to (30), or the method according to any one of (12) to (21). A method for producing a protein hydrolyzate, which comprises causing a culture or a culture-treated product containing a vacuolar enzyme obtained by culturing the obtained filamentous fungus to act on a protein serving as a substrate.
( 3 2 ) ( 1 2 ) 〜 （ 2 1 ) のいずれか 1項に記載の方法で得られる 糸状菌を、 基質となるタンパク質を含む培地に培養することを特徴と するタンパク加水分解物の製造方法。  (32) Production of a protein hydrolyzate characterized by culturing the filamentous fungus obtained by the method according to any one of (12) to (21) in a medium containing a protein serving as a substrate. Method.
( 3 3 ) ( 3 1 ) または （ 3 2 ) に記載の方法で製造されたタンパク 加水分解物。  (33) A protein hydrolyzate produced by the method according to (31) or (32).
( 3 4 ) ( 3 3 ) に記載のタンパク加水分解物を含有することを特徴 とする調味料。 以下、 本発明の実施の形態について詳細に説明する。  (34) A seasoning comprising the protein hydrolyzate according to (33). Hereinafter, embodiments of the present invention will be described in detail.
本発明のポリべプチドと しては、 配列番号 1で表されるアミノ酸配 列を含むポリペプチド、配列番号 1で表されるァミノ酸配列において、 1以上のァミノ酸が欠失、 置換もしくは付加されたァミノ酸配列から なり、 かつ液胞酵素を細胞内に局在させる活性を有するポリぺプチド をあげることができる。 配列番号 1で表されるァミ ノ酸配列からなる ポリぺプチドは、 サッカロミセス ' セレピシェの VPS5遺伝子に対する ァスペルギルス ' ォリゼの相同遺伝子 （以下、 Aovps5遺伝子と もいう ) がコードするポリペプチドである。 The polypeptide of the present invention includes a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 and an amino acid sequence represented by SEQ ID NO: 1. Polypeptides comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added and having an activity of localizing vacuolar enzymes to cells can be given. The polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is a polypeptide encoded by a homologous gene of Aspergillus oryzae to the VPS5 gene of Saccharomyces cerevisiae (hereinafter also referred to as Aovps5 gene).
1以上のアミノ酸が欠失、 置換もしくは付加されたァミノ酸配列か らなり、 かつ液胞酵素を細胞内に局在させる活性を有するポリぺプチ トは、 Molecular Cloning: A Laboratory Manual, 3rd Edition, Cola Spring Harbor Laboratory Press (2001) (以下、 モレキュラー · クロ 一ユング第 3版と略す) 、 Current Protocols in Molecular Biology, John Wiley & Sons (1987-2001) (以下、 カレン 卜 - プロ 卜コーノレズ · イン ·モレキュラー 'ノ ィォロジ一と B各す） 、 Nucleic Acids Research, 10, 6487 (1982)、 Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)、 Gene, 34, 315 (1985) , Nucleic Acids Research, 13., 4431 (1985) , Proc. Natl. Acad. Sci. USA, 82, 488 (1985)等に記載の部位特異的変異導入法を 用いて、 例えば配列番号 1で示されるァミノ酸配列を有するポリぺプ チドをコードする DNAに部位特異的変異を導入し、 2,. に後述する方法 で変異を導入した DNAがコードするポリぺプチドを発現させることに より、 取得することができる。  Polypeptides consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and having the activity of localizing vacuolar enzymes to cells are known as Molecular Cloning: A Laboratory Manual, 3rd Edition, Cola Spring Harbor Laboratory Press (2001) (hereinafter abbreviated as Molecular Kroichi Jung, 3rd edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-2001) (hereinafter abbreviated as Current Molecular 'Norology and B'), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Natl. Acad. Sci. USA, 82, 488 (1985), etc., using the site-directed mutagenesis method described in, for example, the amino acid sequence represented by SEQ ID NO: 1. A site-specific mutation was introduced into the DNA encoding the polypeptide having It can be obtained by expressing the polypeptide encoded by the DNA into which the difference has been introduced.
欠失、置換もしくは付加されるァミノ酸の数は特に限定されないが、 上記の部位特異的変異法等の周知の方法により欠失、 置換もしくは付 加できる程度の数であり、 1〜数十個、 好ましくは 1〜20個、 より好 ましくは 1〜10個、 さらに好ましくは 1〜5個である。 また、 ァミノ 酸の欠失、 置換または付加は、 配列番号 1で表されるアミノ酸配列に おいて 1 ケ所に限らず、 2ケ所以上の位置で同時に生じてもよい。 また、 ァミノ酸の欠失または付加が可能なァミノ酸の位置と しては、 例えば配列番号 1で表されるァミノ酸配列の N末端および C末端をあ げることができる。  The number of amino acids to be deleted, substituted or added is not particularly limited, but is a number that can be deleted, substituted or added by a well-known method such as the above-described site-directed mutagenesis, and is 1 to several tens. The number is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5. Further, deletion, substitution or addition of an amino acid is not limited to one position in the amino acid sequence represented by SEQ ID NO: 1, and may occur simultaneously at two or more positions. Examples of the position of the amino acid at which the amino acid can be deleted or added include the N-terminal and the C-terminal of the amino acid sequence represented by SEQ ID NO: 1.
上記の配列番号 1で表されるァミノ酸配列において 1以上のァミノ 酸が欠失、 置換もしくは付加されたとは、 同一配列中の任意かつ 1 も しく は複数のァミ ノ酸配列中の位置において、 1または複数のァミノ 酸の欠失、 置換もしくは付加があることを意味し、 欠失、 置換もしく は付加が同時に生じてもよく、 置換もしくは付加されるァミ ノ酸は天 然型と非天然型とを問わない。 天然型アミノ酸としては、 L一ァラニ ン、 Lーァスパラギン、 Lーァスパラギン酸、 L—グルタミ ン、 L— グルタミ ン酸、 グリ シン、 L—ヒスチジン、 L—イ ソロイシン、 L— ロイシン、 L—リ ジン、 L一アルギニン、 L—メチォニン、 L—フエ 二ルァラユン、 L一プロ リ ン、 Lーセリ ン、 L—スレオニン、 Lー ト リプトフアン、 Lーチロシン、 Lーバリ ン、 L—システィンなどがあ 'げられる。 - 以下に、 相互に置換可能なアミノ酸の例を示す。 同一群に含まれる ァミノ酸は相互に置換可能である。. One or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 An acid is deleted, substituted or added when one or more amino acids are deleted, substituted or added at any and one or more amino acid sequences in the same sequence. And deletion, substitution or addition may occur simultaneously, and the amino acid to be substituted or added may be either natural or non-natural. Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, Examples include L-arginine, L-methionine, L-phenyalarayun, L-prolin, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valin, and L-cysteine. -The following are examples of mutually substitutable amino acids. Amino acids included in the same group can be substituted for each other. .
A群 ： ロイシン、 イ ソロイシン、 ノノレロイシン、 ノ リ ン、 ノルノ リ ン、 ァラニン、 2—アミ ノブタン酸、 メチォニン、 O—メチルセリ ン、 t 一プチノレグリ シン、 t ーブチノレアラニン、 シクロへキシノレァラニン Group A: leucine, isoleucine, nonoleucine, norin, nornoline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-ptynoreglysine, t-butinolealanine, cyclohexinoleranine
B群 ： ァスパラギン酸、 グルタミ ン酸、 ィ ソァスパラギン酸、 ィ ソ グルタミン酸、 2—アミ ノアジピン酸、 2—アミ ノスべリ ン酸 Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosveric acid
C群 ァスパラギン、 グルタ ミン  Group C Asparagine, Glutamine
D群 リジン、 アルギニン、 オル-チン、 2， 4ージアミ ノブタン 酸、 2 3—ジァミ ノプロピオン酸  Group D Lysine, Arginine, Ortin, 2,4-Diaminobutanoic acid, 23-Diaminopropionic acid
E群 プロ リ ン、 - 3—ヒ ドロキシプロ リ ン、 4—ヒ ドロキシプロ リ ン  Group E proline, -3-hydroxyproline, 4-hydroxyproline
F群 セリ ン、 スレ才ニン、 ホモセリ ン  Group F serine, thread nin, homoserine
G群 フエニノレアラニン、 チロシン  Group G pheninoleanine, tyrosine
また 上記の 1以上のァミノ酸が欠失、 置換もしくは付加されたァ ミノ酸配列を有するポリぺプチドが、 液胞酵素を細胞内に局在化させ るためには、配列番号 1記載のアミノ酸配列と、少なく とも 60 %以上、 通常は 80%以上、 特に 95 %以上の相同性を有していることが好ましい。 上記のポリぺプチドが、 液胞酵素を細胞内に局在させる活性を有す ることは、 糸状菌において、 3. に後述する方法で該ポリペプチドの 機能を抑制したときに、 糸状菌の液胞酵素が細胞外に分泌されること により、 確認することができる。 . In order for the polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted or added to localize the vacuolar enzyme to cells, the amino acid set forth in SEQ ID NO: 1 must be used. It is preferable to have at least 60% or more homology with the sequence, usually at least 80%, especially at least 95%. These polypeptides have the activity to localize vacuolar enzymes to cells This can be confirmed by that the vacuolar enzyme of the filamentous fungus is secreted extracellularly when the function of the polypeptide is suppressed in the filamentous fungus by the method described later in 3. .
細胞外に液胞酵素が分泌されることは、 糸状菌を培地で培養し、 培 養後の培地中の液胞酵素を検出することにより確認できる。 液胞酵素 と しては、 例えばトリぺプチジルぺプチダ一ゼ、 力ルポキシぺプチダ ーゼ Yがあげられる。 液胞酵素を検出する方法と しては、 ( a ) 液胞 酵素の酵素活性を測定する方法、 （ b ) 液胞酵素を認識する抗体を用 いてウェスタンブロッテイング等により免疫学的に検出する方法、 ( c )ダリーン蛍光タンパク質（GFP)等のマーカ一タンパク質または FLAG タグ等のェピトープぺプチドと液胞酵素の融合タンパク質を糸状菌で 発現させ、 マーカータンパク質ゃェピトープぺプチドを特異的に認識 する抗体を利用して融合タンパク質を検出する方法 〔Fungal Genet. Biol. , 37, 29 (2002)〕 等があげられる。 例えば、 トリべプチジルぺ プチダーゼは、 文献 [Biochem. Mol. Biol. Int.， 47, 1079 (1999)〕 に記載されたトリぺプチジルぺプチダーゼ I の活性測定方法に準じて、 基質となるァラニン一ァラニン一ブェ-ルァラニン一パラ二トロア- リ ドを含む基質溶液に、 糸状菌の培養液等の試料溶液を加えて、 30°C で 10分間反応させた後、 分光光度計により 384nmの吸光度を測定するこ とにより、 活性を測定できる。 ·  The secretion of vacuolar enzymes outside the cells can be confirmed by culturing the filamentous fungus in the medium and detecting the vacuolar enzymes in the medium after the culture. Examples of vacuolar enzymes include triptidyl peptidase and lipoxypeptidase Y. Methods for detecting vacuolar enzymes include (a) a method for measuring the enzyme activity of the vacuolar enzyme, and (b) immunological detection by Western blotting using an antibody that recognizes the vacuolar enzyme. (C) Expression of a marker protein such as daline fluorescent protein (GFP) or a fusion protein of an epitope peptide and a vacuolar enzyme such as a FLAG tag in a filamentous fungus, and specifically recognizing the marker protein peptide epitope A method for detecting a fusion protein using an antibody [Fungal Genet. Biol., 37, 29 (2002)] and the like. For example, tripeptidyl peptidase can be used as a substrate for alanine as a substrate according to the method for measuring the activity of tripeptidyl peptidase I described in the literature [Biochem. Mol. Biol. Int., 47, 1079 (1999)]. A sample solution such as a culture solution of a filamentous fungus is added to a substrate solution containing alanine-bye-alanine-para-nitrolide, and the mixture is allowed to react at 30 ° C for 10 minutes.The absorbance at 384 nm is then measured by a spectrophotometer. By measuring the activity, the activity can be measured. ·
あるいは、 3. および実施例 3に後述するァスペルギルス · ォリゼ の Aovps5遺伝子破壊株を宿主として、 2. に記載の方法あるいは 3. に記載の相同組換え法で該ポリペプチドを発現させたときに、 AovpsS 遺伝子破壊株が有する、 （ 1 ) 液胞酵素の細胞外への分泌、 （ 2 ) 赤 褐色の色素の分泌、 （ 3 ) 重金属に対する耐性、 （ 4 ) 分生子の形成 の阻害、 等の表現型がもとの表現型に回復することによつても、 該ポ リぺプチドが液胞酵素を細胞内に局在させる活性を有することを確認 することができる。  Alternatively, using the Aovps5 gene-disrupted strain of Aspergillus oryzae described later in 3. and Example 3 as a host, expressing the polypeptide by the method described in 2. or the homologous recombination method described in 3. Expressions of (1) extracellular secretion of vacuolar enzymes, (2) secretion of reddish brown pigment, (3) resistance to heavy metals, (4) inhibition of conidium formation, etc. possessed by the AovpsS gene-disrupted strain. Even when the type is restored to the original phenotype, it can be confirmed that the polypeptide has an activity of localizing vacuolar enzymes to cells.
本発明の DNAと しては、 上記の本発明のポリぺプチドをコードする The DNA of the present invention encodes the above-described polypeptide of the present invention.
DNAがあげられる。 配列番号 1のァミノ酸配列をコードする DNAとして は、 配列番号 2または 3 の塩基配列を含む DNAがあげられる。 配列番号 2の塩基配列は、 ァスペルギルス ' ォリゼのゲノム上の Aovps5遺伝子 の配列であり、 配列番号 3の塩基配列は、 該遺伝子の cDNAのコード領 域の配列である。 DNA. As a DNA encoding the amino acid sequence of SEQ ID NO: 1 Is a DNA containing the nucleotide sequence of SEQ ID NO: 2 or 3. The nucleotide sequence of SEQ ID NO: 2 is the sequence of the Aovps5 gene on the genome of Aspergillus oryzae, and the nucleotide sequence of SEQ ID NO: 3 is the sequence of the cDNA coding region of the gene.
また、 本発明の DNAと しては、 配列番号 2または 3の塩基配列と相補 的な塩基配列からなる DNAとス トリ ンジェン トな条件下でハイブリダ ィズする DNAであり、 かつ液胞酵素を細胞内に局在させる活性を有する ポリペプチドをコードする DNAをあげることができる。 ス トリ ンジェン トな条件下でハイブリダイズする DNAとは、例えば配列番号 2または 3 の塩基配列と相補的な塩基配列からなる DNAまたはその一部の DNA断片 をプローブとして、 コロニーハイプリダイゼーション法、 プラークハ ィプリダイゼーション法あるいはサザンブ口ッ トハイプリダイゼーシ ョン法奪を用いることによ り得られる DNAを意味する。 具体的には、 コ 口-一あるいはプラークの DNAを固定化したフィルターを用いて、 0. 7 〜1. Omol/Lの塩化ナト リ ゥム存在下、 65°Cでプローブとハイプリダイ ゼーシヨンを行った後、 0.：!〜 2倍濃度の SSC溶液 （ 1倍濃度の SSC溶液 の組成は'、 150mmol /L塩化ナトリ ウム、 15mmol/Lタエ'ン酸ナト リ ウムよ りなる） を用い、 65°C条件下でフィルターを洗浄することにより同定 できる DNAをあげることができる。 ハイブリダイゼーションは、 モレキ ユラ一 · クローエング第 3版、 カレント · プロ トコ一/レズ ' イン · モ レキユラ一 · パイォロジ一、 DNA Cloni ng 1 : Core Techni ques, A Pract i cal Approach, Second Edi t i on, Oxford Univers i ty ( 199oノ等 に記載されている方法に準じて行うことができる。 ハイブリダイズ可 能な DNAとして具体的には、 配列番号 1または 3で表される塩基配列と 少なく とも 60 %以上の相同性を有する DNA、 好ましくは 70%以上、 より 好ましくは 80 %以上、 さらに好ましくは 90 %以上、 特に好ましくは 95 %以上、 最も好ましくは 98 %以上の相同性を有する DNAをあげることが できる。  In addition, the DNA of the present invention is a DNA that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or 3, and has a vacuolar enzyme. Examples include DNA encoding a polypeptide having an activity of localizing in a cell. DNA that hybridizes under stringent conditions refers to, for example, a colony hybridization method using, as a probe, a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or 3 or a partial DNA fragment thereof as a probe. DNA obtained by using the plaque hybridization method or the Southern blot hybridization method. Specifically, using a filter on which DNA of plaque-or-plaque was immobilized, hybridization was performed with the probe at 65 ° C in the presence of 0.7 to 1.Omol / L sodium chloride. After that, use a SSC solution with a concentration of 0 :! to 2 times (the composition of a 1% concentration SSC solution is composed of 150 mmol / L sodium chloride and 15 mmol / L sodium tate). DNA that can be identified by washing the filter at 65 ° C can be mentioned. Hybridization is performed by Molecular Yura Closing Third Edition, Current Protocol / Les' in Morekiura Pyrology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (199o, etc.) Specifically, as a hybridizable DNA, at least 60% of the nucleotide sequence represented by SEQ ID NO: 1 or 3 is used. DNA having the above homology, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more homology. Can be done.
1 . 本発明の DNAの調製  1. Preparation of DNA of the present invention
( ,1 ) プロープの調製 以下のようにして得られるァスペルギルス · ォリゼの Aovps5遺伝子 . の部分断片をプローブと して用いることができる。 Preparation of (, 1) probe A partial fragment of the Aovps5 gene of Aspergillus oryzae obtained as follows can be used as a probe.
まず、サッカロミセス 'セレビシェの VPS5遺伝子の塩基配列 (GenBank 登録番号 ： U73512) のコード領域の配列 （配列番号 4 ) を問い合わせ 配列と して、 ァスペルギルス ' エドランス、 ノィロスボラ · クラッサ (Neurospora crassa) 等の各種糸状菌のゲノム配列データベースを相 同性検索プログラム BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993) 〕 を用いて検索することにより、 サッカロ ミセス · セレピシェの VPS5 遗伝子と高い相同性を有するノィロスボラ · クラッサ等の VPS5相同遺 伝子のゲノム配列を見出すことができる。 例えば、 ノィロスボラ · ク ラッサの VPS5相同遺伝子の塩基配列としては配列番号 5をあげること ができる。  First, the base sequence of the Saccharomyces cerevisiae VPS5 gene (GenBank accession number: U73512) was queried for the sequence (SEQ ID NO: 4) of the VPS5 gene. As the query sequence, various filaments such as Aspergillus edulans and Neurospora crassa were used. A homology search program BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] was used to search the genome sequence database of the fungus for high homology with the Saccharomyces cerevisiae VPS5 gene. The genome sequence of the VPS5 homologous gene such as Neurosbora classa with For example, SEQ ID NO: 5 can be mentioned as the nucleotide sequence of the VPS5 homologous gene of Neurosbora classa.
このノィロスボラ · クラッサの VPS5相同遺伝子がコードするァミノ 酸配列から、 縮重プライマーを設計する。 プライマーを設計するアミ ノ酸配列は、 ノィロスボラ · クラッサの VPS5相同遺伝子がコードする アミノ酸配列から任意の 2力所の配列を選ぶことができるが、 ノイロ スポラ · クラッサの VPS5相同遺伝子とサッカロミセス · セレピシェの A degenerate primer is designed based on the amino acid sequence encoded by the Neurospora crassa VPS5 homologous gene. The amino acid sequence for primer design can be selected from any two amino acid sequences from the amino acid sequence encoded by the VPS5 homologous gene of Neurospora crassa.The VPS5 homologous gene of Neurospora crassa and Saccharomyces cerevisiae
VPS5遺伝子がそれぞれコードするァミノ酸配列間で保存性の高い領域 を選択することが望ましい。 例えばノィロスボラ · クラッサの VPS5相 同遺伝子がコードするアミノ酸配列の 181〜 189番目の配列 （Val GlyIt is desirable to select a highly conserved region between the amino acid sequences encoded by the VPS5 genes. For example, the sequence of amino acids 181 to 189 (Val Gly) of the amino acid sequence encoded by the VPS5 homologue of Neurosbora classa
Asp Pro His Lys Val Gly Asp) 、 267〜 274番目の配歹 lj (Pro Pro Glu LysAsp Pro His Lys Val Gly Asp), 267-274th system lj (Pro Pro Glu Lys
Gin Ala Val Gly) をあげることができる。 縮重プライマーは、 選択し た 2箇所のァミノ酸配列について、 より N末側のァミノ酸配列に対応す る全てのコ ドンの塩基配列、 より C末のァミノ酸配列に対応する全ての コ ドンと相補的な塩基配列を、 それぞれ'含む DNAとして設計する。 縮重 の数をへらすため、 コ ドンの 3番目の塩基と してィノシンを利用した り 、 ァスペルギルス · 二 ドランスのコ ドン使用頻度 thiol. Gen. Genet. ,Gin Ala Val Gly). For the two selected amino acid sequences, the degenerate primers have the base sequences of all the codons corresponding to the N-terminal amino acid sequence and all the codons corresponding to the C-terminal amino acid sequence. Are designed as DNAs each containing a complementary nucleotide sequence. In order to reduce the number of degeneracy, inosine was used as the third base of the codon, and the codon usage of Aspergillus dylans thiol. Gen. Genet.,
230, 288 (1991)〕 を利用したりすることもできる。 プライマーの長さ は、 14〜30塩基が好ましい。 縮重プライマーの例と しては、 配列番号230, 288 (1991)]. The length of the primer is preferably 14 to 30 bases. An example of a degenerate primer is SEQ ID NO:
6および 7の塩基配列をそれぞれ有する DNAをあげることができる。 設 計した縮重プライマーは、 例えば、 アプライ ド ' バイオシステムズ社 製の DNA合成装置等を使用して調製することができる。 DNAs having the nucleotide sequences of 6 and 7, respectively, can be mentioned. Setting The measured degenerate primer can be prepared, for example, using a DNA synthesizer manufactured by Applied Biosystems.
上記の縮重プライマ一とァスペルギルス 'オリゼの染色体 DNAあるい は cDNAをテンプレートとして用いた PCRを行う ことにより、 ァスペルギ ルス · ォリゼの Aovps5遺伝子の部分断片を増幅し単離することができ る。 ァスペルギルス . ォリゼの染色体 DNAあるいは cDNAは、 ( 2 ) に後 述する方法で調製できる。 PCRは当業者に周知の条件及び手段を用いて 、行う ことができる。 例えば、 PCRの反応条件と しては、 94°Cで 5分間 の反応の後、 94°Cで 30秒、 58°Cで 30秒、 72°Cで 30秒からなる反応サイ クルを 30サイクル行う条件があげられる。 ただし、 反応サイクル中の アニーリ ングの温度は、 プライマーの長さ、 塩基組成により推定され る TJこ基づいて、 適当なものにする。 なお、 サーマルサイクラ一とし ては、 パーキン · エルマ一 ( Perki n Elmer) 社製 9600、 アステック社 製プログラム . テンプ ' コントロール . システム PC- 700など市販の'サ 一マルサイクラ一を用いることができる。 耐熱性 DNAポリメラーゼと し ては、 Taq DNAポリメラーゼ （宝酒造社製） 、 ExTaq DNAポリメラーゼ (宝酒造社製） などの市販品を用いることができる。 反応液の組成は ポリメラーゼに添付の説明書に従う。  By performing PCR using the above degenerate primer and chromosomal DNA or cDNA of Aspergillus oryzae as a template, a partial fragment of the Aovps5 gene of Aspergillus oryzae can be amplified and isolated. The chromosomal DNA or cDNA of Aspergillus oryzae can be prepared by the method described later in (2). PCR can be performed using conditions and means known to those skilled in the art. For example, PCR reaction conditions include a reaction at 94 ° C for 5 minutes, followed by 30 cycles of a reaction cycle consisting of 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds. The conditions to be performed are given. However, the annealing temperature during the reaction cycle should be appropriate based on the TJ estimated from the primer length and base composition. As the thermal cycler, a commercially available thermal cycler such as Perkin Elmer 9600, Astec program.temp.control.system PC-700, etc. can be used. Commercial products such as Taq DNA polymerase (Takara Shuzo) and ExTaq DNA polymerase (Takara Shuzo) can be used as the heat-resistant DNA polymerase. The composition of the reaction solution follows the instructions attached to the polymerase.
PCRにより増幅した DNA断片を単離し、 ジゴキシゲニン (以下、 DIGと 略す） 、 放射性同位元素等で標識したものをプローブとする。 また、 上記の PCRの反応液中に DIG、 放射性同位元素等で標識したヌクレオチ ドを添加して PCRを行うことにより、 プローブの標識を増幅と同時に行 うこともできる。 以上のようにして得られる、 プローブと して用いる ことのできるァスペルギルス · ォリゼの Aovps5遺伝子の部分断片とし ては、 配列番号 8の塩基配列を有する DNAをあげることができる。 増幅された DNA断片は、適当なプラスミ ドべクターにクローニングし 、 DSQ2000L DNAシークェンサ一 (島津製作所社製) 、 ABI 310ジヱネェ ティ ック · アナライザー (パーキンエルマ一社製) 等の DNAシークェン サーを用いて、 塩基配列を決定し、 ノィロスボラ · クラッサの VPS5相 同遣伝子と相同性を有しているか確認することが好ましい。 ( 2 ) 染色体 DNAライブラリ一または cDNAライブラリ一の調製 A DNA fragment amplified by PCR is isolated, and labeled with digoxigenin (hereinafter abbreviated as DIG), a radioisotope or the like is used as a probe. Further, by adding a nucleotide labeled with DIG, a radioisotope, or the like to the reaction solution of the above PCR and performing PCR, the labeling of the probe can be performed simultaneously with the amplification. Examples of the partial fragment of the Aovps5 gene of Aspergillus oryzae obtained as described above, which can be used as a probe, include a DNA having the nucleotide sequence of SEQ ID NO: 8. The amplified DNA fragment is cloned into an appropriate plasmid vector and used with a DNA sequencer such as DSQ2000L DNA Sequencer (manufactured by Shimadzu Corporation) or ABI310 Genetic Analyzer (manufactured by PerkinElma). Then, it is preferable to determine the nucleotide sequence and confirm whether or not it has homology with the VPS5 syngeneic gene of Neurosbora classa. (2) Preparation of one chromosomal DNA library or one cDNA library
ァスペルギルス ·ォリゼから抽出したゲノム DNAを、 適当な制限酵素 を用いて切断し、 同じ切断末端をもつ制限酵素で切断したベタターに' 挿入する。 ベクターと して例えば λ DASHI I (ス トラタジーン社製） 、 λ FIXI I (ス トラタジーン社製) 等のラムダファ一ジべクター、 pUC118 Genomic DNA extracted from Aspergillus oryzae is cleaved with an appropriate restriction enzyme, and inserted into a beta cleaved with a restriction enzyme having the same cleaved end. Lambda vector vectors such as λ DASHI I (Stratagene), λFIXI I (Stratagene), and pUC118 as vectors
(宝酒造社製) 、 PBR322 (宝酒造社製) 等のプラスミ ドベクターがあ げられる。 ァスペルギルス ·ォリゼのゲノム DNAは、 五味らの方法 C J. Gen. App l . Mi crob io l . , 35, 225， ( 1989)〕 に従って抽出できる。 例えば、 ファージベクター λ DASHI Iへの挿入は、 BamHIで開裂したベ クタ一と BamHIで分解した染色体 DNAとを T4DNAリガーゼを用いて連結 することにより行うことができる。 このようにして得られたゲノム DNA 断片を挿入したべクターを適切なェシヱリ ヒア ' コリ宿主に導入する ことにより染色体 DNAライブラリーが得られる。 There are plasmid vectors such as (Takara Shuzo) and PBR322 (Takara Shuzo). Aspergillus oryzae genomic DNA can be extracted according to the method of Gomi et al., C J. Gen. Appl. Microcrobio l., 35, 225, (1989)]. For example, insertion into the phage vector λ DASHI I can be performed by ligating a vector cleaved with BamHI and chromosomal DNA degraded with BamHI using T4 DNA ligase. A chromosomal DNA library can be obtained by introducing the vector into which the genomic DNA fragment thus obtained has been inserted into an appropriate Escherichia coli host.
cDNAライプラリーは以下のようにして調製できる。 まず、 液体窒素 で凍結したァスペルギルス · ォリゼの菌体を細かく粉砕した後、 グァ 二ジンィソチオシァネートを含んだフエノール又はフエノールーク口 口ホルム溶液でホモジナイズし、 高速遠心により水層と有機層に分離 する。 その後、 水層とイソプロパノールを混合し、 水層に含まれる全 RNAを沈殿させて回収するか、 あるいはショ糖もしくはセシウムクロラ ィ ド密度勾配遠心法により回収する。 もしくは市販の全 RNA抽出キッ ト A cDNA library can be prepared as follows. First, the cells of Aspergillus oryzae frozen in liquid nitrogen are finely pulverized, and then homogenized with phenol or phenolic orifice-form solution containing guanidine isothiocynate, and then centrifuged into an aqueous layer and an organic layer by high-speed centrifugation. To separate. Then, mix the aqueous layer with isopropanol, and collect by precipitating and collecting the total RNA contained in the aqueous layer, or recover by sucrose or cesium chloride density gradient centrifugation. Or a commercially available total RNA extraction kit
(例えば RNeasy ki t , キアゲン社製） を用いることもできる。 この全 RNAをォリ ゴ （dT) セルロースクロマ トグラフィ一にかけて mRNA (即ち 、 po ly ( A) RNA) を精製する。 (For example, RNeasy kit, manufactured by Qiagen) can also be used. The total RNA is subjected to oligo (dT) cellulose chromatography to purify mRNA (ie, poly (A) RNA).
次に、 mRNAから逆転写酵素の存在下に cDNAを合成し、 ファージもし くはプラスミ ドべクターに連結可能なように適当な制限酵素部位を作 り、 これを同様の制限酵素部位をもつファージもしくはプラスミ ドべ クターに連結し、 このようにして得られたベタターで大腸菌を形質転 換して cDNAライブラリ一を調製する。 mRNAから cDNAを合成する方法と して、 ゲートウェイ技術を用いた cDNA合成およびクローニング用スー パースク リ プ ト · プラス ミ ド ' システム （SUPERSCRIPT Plasmi d Syst em with GATEWAY" Technology for cDNA Synthesis and Cloning, ィ ンビ ト口ジェン社製） などの市販キッ トを用いることもできる。 Next, cDNA is synthesized from mRNA in the presence of reverse transcriptase, and an appropriate restriction enzyme site is created so that it can be linked to a phage or a plasmid vector. Alternatively, the cDNA is ligated to a plasmid vector, and Escherichia coli is transformed with the vector obtained in this manner to prepare a cDNA library. As a method for synthesizing cDNA from mRNA, the Superscript Plasmid System for cDNA synthesis and cloning using gateway technology (SUPERSCRIPT Plasmid System) Commercially available kits, such as with GATEWAY "Technology for cDNA Synthesis and Cloning (manufactured by Invitoguchi Gen), can also be used.
( 3 ) Aovps5遺伝子ク ローンの選別  (3) Selection of Aovps5 gene clone
Aovps5遺伝子を含むク ローンの選別は、 具体的には、 プラークハイ ブリダィゼーショ ンあるいはコロニーハイブリダィゼーショ ン 〔モレ キユラ一 クローニング第 3版、 ucleic Acids Res. , 9, 879 (1981) 〕 により行うことができる。 例えば、 プラークを二 トロセルロースあ るいはナイロンメンブレン上に転写し、 真空中で 80°C、 2時間あるいは 紫外線照射によって、 DNAをメンブレン上に固定化する。 この時、 必要 に応じて 0.5mol/l水酸化ナトリ ウム、 1.5mol/l塩化ナトリ ウムを含む アル力リ性溶液を用いた変性および 0.5mol/lトリスー塩酸（pH 7.5)、 3mol/l塩化ナトリ ゥムの溶液を用いた中和を行う。 このメ ンプレンを、 5XSSC；、 50%ホルムァミ ド、 0.1 % N-ラウロイルサルコシン、 0.2%SDS、 1%ブロッキング ' リージヱント （ロシュ社製） からなる溶液中でプロ ーブとメンプレンを 42°C、 6時間インキュベートした後、 42°Cで 0.1% SDSを含む 2XSSCで 5分間、 0.1%SDSを含む 0.1XSSCで 15分間、 順次洗 浄する条件、 あるいは 4 XSSC、 50%ホルムアミ ド、 50 mmol/1 HEPES —水酸化ナト，リ ウム（pH 7.0)、 lOXDenhardt' s溶液 （0.2%フイ コール 400、 0.2°/。ポリ ビニルピロ リ ドン、 0.2%ゥシ血清アルブミ ン） 、 100 IX g/mlサケ精子 DNAからなる溶液中でプローブとメンブレンを、 42°C、 一昼夜インキュベートした後、 0.1%SDSを含む 2 XSSC溶液で室温、 2 分間で 3回洗浄した後、 0.1%SDSを含む 0.1XSSC溶液中で 50°C、 2時 間で 3回洗浄する条件でプローブとハイブリダイゼーションさせる。 洗浄後のメンプレンは放射性同位体で標識したプローブの場合は、 風 乾した後、 一 70°Cで 2時間から一昼夜 X線フィルムに露光させ、 現像し て、 ポジティブクローンを可視化する。 ジゴキシゲニンで標識したプ ローブの場合は、 DIGハイプライム DNAラベリ ング/検出キッ ト I (DIG High Prime DNA Label ing& Detect ion Starter Kitl、 ロシュ社^ ) を用いて発色反応を行い、 ポジティブクローンを検出する。  Specifically, clones containing the Aovps5 gene are selected by plaque hybridization or colony hybridization (Molecular cloning, 3rd edition, ucleic Acids Res., 9, 879 (1981)). be able to. For example, plaques are transferred to nitrocellulose or nylon membrane, and the DNA is immobilized on the membrane by irradiating with UV light at 80 ° C for 2 hours in a vacuum. At this time, if necessary, denaturation using an alkaline solution containing 0.5 mol / l sodium hydroxide and 1.5 mol / l sodium chloride, and 0.5 mol / l tris-hydrochloric acid (pH 7.5), 3 mol / l chloride Neutralize with a solution of sodium. The probe was put in a solution of 5XSSC; 50% formamide, 0.1% N-lauroyl sarcosine, 0.2% SDS, 1% blocking reagent (Roche) at 42 ° C, 6 ° C. After incubating for 2 hours at 42 ° C, wash sequentially with 2XSSC containing 0.1% SDS for 5 minutes, then with 0.1XSSC containing 0.1% SDS for 15 minutes, or 4 XSSC, 50% formamide, 50 mmol / 1 HEPES — From sodium hydroxide, lithium (pH 7.0), lOXDenhardt's solution (0.2% polyester 400, 0.2 ° /. Polyvinyl pyrrolidone, 0.2% ゥ serum albumin), 100 IX g / ml salmon sperm DNA After incubating the probe and the membrane at 42 ° C overnight in a solution, wash with 2XSSC solution containing 0.1% SDS three times for 2 minutes at room temperature, then in 50 ° C in 0.1XSSC solution containing 0.1% SDS. C. Hybridization with probe under the conditions of washing 3 times in 2 hours To be down. After washing the membrane, if the probe is labeled with a radioisotope, air dry and then expose to X-ray film at 170 ° C for 2 hours to 24 hours, develop and visualize the positive clones. For probes labeled with digoxigenin, perform a color reaction using DIG High Prime DNA Labeling & Detection Starter Kit I (Roche ^) to detect positive clones .
( 4 ) 本発明の DNAの単離 上記のようにして選別されたポジティブクローンのプラ一クまたは コロニーからファージ DNAまたはプラスミ ド DNAを抽出 · 単離し、 挿入 断片を適当な制限酵素でべクターから切り出すことで本発明の DNAを 取得することができる。 取得した DNAは、 適当なプラスミ ドベクターに サブクローユングし、 DSQ2000L DNAシークェンサ一 (島津製作所社製 ) 、 ABI .310ジエネエティ ック ' アナライザー (パーキンエルマ一社製 ) 等の DNAシークェンサーを用いて、 該 DNAの塩基配列を決定すること ができる。 Aovp s 5遺伝子のゲノム DNAの配列と cDNAの配列を決定し、 両 者の塩基配列を比較することにより、 ゲノム DNAのィントロンの領域を 決定することができる。 (4) Isolation of the DNA of the present invention The phage DNA or plasmid DNA is extracted and isolated from the positive clones or plasmids selected as described above, and the inserted fragment is cut out from the vector with an appropriate restriction enzyme to obtain the DNA of the present invention. be able to. The obtained DNA is subcloned into an appropriate plasmid vector, and the DNA is sequenced using a DNA sequencer such as DSQ2000L DNA Sequencer (manufactured by Shimadzu Corporation) or ABI .310 Genetic 'Analyzer (manufactured by PerkinElma). The nucleotide sequence of the DNA can be determined. The intron region of the genomic DNA can be determined by determining the sequence of the genomic DNA and the sequence of the cDNA of the Aovps5 gene, and comparing the nucleotide sequences of both.
Aovp s 5遺伝子の塩基配列が決定された後は、 ポリぺプチドのコ一ド 領域を含む領域を適当に選択し、 選択した領域の塩基配列の 5'端 20〜 40塩基の配列を 3'端に含む DNA、選択した領域の塩基配列の 3'端 20〜40 塩基と相補的な配列を 3 '端に含む DNAをそれぞれ DNA合成機で合成する c ァスペルギルス · 才リゼのゲノム DNAまたは cDNAをテンプレートとし、 2種類の合成 DNAをプライマーとして用いた PCRによ り本発明の DNAを増 幅し単離することができる。 PCRは （ 1 ) に記載した条件と同様にして 行うことができる。  After the nucleotide sequence of the Aovps5 gene has been determined, a region containing the coding region of the polypeptide is appropriately selected, and the sequence of 20 to 40 nucleotides at the 5 'end of the nucleotide sequence of the selected region is 3'. Use a DNA synthesizer to synthesize the DNA at the end and the DNA at the 3 'end that is complementary to the base sequence 20 to 40 bases at the 3' end of the selected region using a DNA synthesizer c Genomic DNA or cDNA from Aspergillus cerevisiae The DNA of the present invention can be amplified and isolated by PCR using two types of synthetic DNAs as primers as a template. PCR can be performed under the same conditions as described in (1).
以上のようにして得られる本発明の DNAと して、 配列番号 2の塩基配 列からなるァスペルギルス ·オリゼの Aovp s 5遺伝子のゲノム DNA、 配列 番号 3の塩基配列からなるァスペルギルス · ォリゼの Aovps 5遺伝子の cDNAをあげることができる。  The DNA of the present invention obtained as described above includes the genomic DNA of the Aovps 5 gene of Aspergillus oryzae comprising the nucleotide sequence of SEQ ID NO: 2, and the Aovps 5 of Aspergillus oryzae comprising the nucleotide sequence of SEQ ID NO: 3 Examples include the cDNA of the gene.
また、 このようにして得られた本発明の DNAをプローブとして、 （ 2 ) および （ 3 ) に記載した方法に準じて、 他の糸状菌のゲノム DNAライ ブライ リーまたは cDNAライブラリーを、 上記 （ 3 ) の条件下でのハイ ブリダイゼーショ ンでスク リーニングすることにより、 他の糸状菌の Aovps5相同遺伝子のゲノム DNAまたは cDNAを取得することができる。 こ れらの DNAも本発明の DNAに含まれる。  Further, using the thus obtained DNA of the present invention as a probe, a genomic DNA library or cDNA library of another filamentous fungus is prepared according to the method described in (2) and (3) according to ( By screening by hybridization under the condition of 3), genomic DNA or cDNA of Aovps5 homologous gene of other filamentous fungi can be obtained. These DNAs are also included in the DNA of the present invention.
( 5 ) 本発明の DNAの検出  (5) Detection of DNA of the present invention
本発明の DNAの塩基配列と相補的な配列の連続する 20塩基以上の配 列を含む DNA断片またはオリ ゴ DNAを放射性同位体、 ジゴキシゲニン、 ピオチン等で標識したものをプローブと し、 ハイプリダイゼーション を行う ことにより、 本発明の DNA、 本発明のポリペプチドをコードする mRMを検出することができる。 本棻明の DNAの塩基配列と相捕的な連続 する 20塩基以上の配列を含む DNA断片は、 上記 （4 ) に記載した方法に 準じて、 本発明の DNAの任意の領域を含む領域を適当に選択し、 選択し た領域の塩基配列の 5'端 20〜40塩基の配列を 3'端に含む DNA、選択した 領域の塩基配列の 3 '端 20〜40塩基と相捕的な配列を 3'端に含む DNAを それぞれプライマーと した PCRにより調製することができる。 オリ ゴ DNAは DNA合成装置により調製できる。 プロ'ープの長さは、 検出対象な どに応じて当業者が適宜選択することができるが、 通常、 15〜3000塩 基、 好ましくは 20〜1000塩基の長さである。 Sequences of 20 or more consecutive bases of a sequence complementary to the base sequence of the DNA of the present invention A DNA fragment containing the sequence or oligo DNA labeled with a radioisotope, digoxigenin, biotin, or the like is used as a probe to perform hybridization, thereby obtaining the mRM encoding the DNA of the present invention or the polypeptide of the present invention. Can be detected. A DNA fragment containing a continuous sequence of 20 or more bases complementary to the base sequence of the DNA of the present invention can be obtained by preparing a region containing any region of the DNA of the present invention according to the method described in (4) above. Appropriately selected, DNA containing a sequence of 20 to 40 bases at the 5 'end of the base sequence of the selected region at the 3' end, a sequence complementary to 20 to 40 bases of the base sequence of the selected region at the 3 'end Can be prepared by PCR using, as primers, DNAs containing 3 ′ at the 3 ′ end. Oligo DNA can be prepared using a DNA synthesizer. The length of the prope can be appropriately selected by those skilled in the art according to the detection target and the like, but is usually 15 to 3000 bases, preferably 20 to 1000 bases.
ハイブリダイゼーションは、 電気泳動ゲルあるいはコロニーなどか らゲノム DNAあるいは mRNAを転写した- トロセルロースメンブレンあ るいはナイロンメンプレンに対して、 ( 3 ) に記載したハイプリダイ ゼーシヨンの方法に準じて行うことができる。 また、 基板上にオリ ゴ DNAまたは DNA断片を固定化し、標識した mRNAあるいは DNAとノヽィプリダ ィズさせた後、 ドッ トとして検出する DNAチップ [ Genome Re s .， 639 ( 1996)〕 によつても本発明の DNAまたは本発明のポリぺプチドをコ一ド する mRNAを検出することができる。  Hybridization can be performed on a nitrocellulose membrane or a nylon membrane to which genomic DNA or mRNA has been transcribed from an electrophoresis gel or a colony according to the method of hybridization described in (3). . Alternatively, a DNA chip [GenomeRes., 639 (1996)] is used in which oligo DNA or DNA fragments are immobilized on a substrate, and the resulting DNA is hybridized with labeled mRNA or DNA and then detected as a dot. Can also detect mRNA encoding the DNA of the present invention or the polypeptide of the present invention.
また、本発明の DNAの塩基配列の連続する 15塩基以上の配列を有する DNAおよぴ本発明の DNAの塩基配列の連続する 15塩基以上の配列と相補 的な配列を有する DNAをプライマーと して、 コロニー等から調製したゲ ノム DNAまたは cDNAをテンプレートと した PCRにより、本発明の DNAまた は本発明のポリぺプチドをコ一ドする mRNAを検出することができる。 PCRは （ 1 ) に記載した条件と同様にして行うことができる。  Further, a DNA having a sequence of 15 or more consecutive bases of the DNA of the present invention and a DNA having a sequence complementary to a sequence of 15 or more consecutive bases of the DNA of the present invention are used as primers. By using PCR as a template with genomic DNA or cDNA prepared from a colony or the like, mRNA encoding the DNA of the present invention or the polypeptide of the present invention can be detected. PCR can be performed in the same manner as described in (1).
2 . 本発明のポリペプチドの調製  2. Preparation of the polypeptide of the present invention
本発明のポリぺプチドは、 例えば以下の方法により、 本発明のポリ ぺプチドをコ一ドする DNAを含む組換え体 DNAを宿主細胞に導入した形 質転換体を作製し、 該形質転換体を培.養することによ り、 調製するこ 2004/004789 The polypeptide of the present invention can be prepared, for example, by the following method, by preparing a transformant in which a recombinant DNA containing the DNA encoding the polypeptide of the present invention has been introduced into a host cell, Can be prepared by cultivating 2004/004789
とができる。 具体的な遺伝子操作的手法は、 モレキュラー ' クロー- ング第 3版やカレン ト · プロ トコールズ · イン · モレキュラー · パイ ォロジ一等に記载された方法等を用いることができる。 Can be. As a specific genetic manipulation technique, a method described in Molecular 'Cloning Third Edition, Current Protocols in Molecular, Piology, etc. can be used.
( 1 ) で得られた本発明の DNAから、 本発明のポリペプチドをコード する部分を含む適当な長さの DNAを調製する。 また、 必要に応じて、 本 発明のポリぺプチドをコ一ドする部分の塩基配列を、 宿主細胞の発現 に最適なコ ドンとなるように塩基を置換した DNAを調製する。  From the DNA of the present invention obtained in (1), a DNA of an appropriate length containing a portion encoding the polypeptide of the present invention is prepared. If necessary, a DNA is prepared by substituting the nucleotide sequence of the portion encoding the polypeptide of the present invention so that the nucleotide sequence becomes an optimal codon for expression in a host cell.
該 DNAを適当な発現べクターのプロモーターの下流に揷入すること により、 組換えベクターを作製する。  A recombinant vector is prepared by inserting the DNA downstream of the promoter of an appropriate expression vector.
該組換えべクターを、 該発現べクターに適合した宿主細胞に導入す る。  The recombinant vector is introduced into a host cell compatible with the expression vector.
宿主細胞と しては、 細菌、 酵母、 糸状菌、 動物細胞、 昆虫細胞、 植 物細胞等、 目的とする遺伝子を発現できるものであればいずれも用い ることができる。 '  As the host cell, any bacteria, yeast, filamentous fungi, animal cells, insect cells, plant cells and the like can be used as long as they can express the gene of interest. '
発現べクターと しては、 上記宿主細胞において自立複製可能ないし ' は染色体中への組込が可能で、 宿主細胞中で機能するプロモーターを 含有しているものが用いられる。 また、 形質転換体を選択するための 形質転換マーカーとなる遺伝子を含むことが好ましい。  As the expression vector, those which are capable of autonomous replication in the host cell or capable of integration into the chromosome and which contain a promoter which functions in the host cell are used. Further, it preferably contains a gene serving as a transformation marker for selecting a transformant.
細菌等の原核生物を宿主細胞と して用いる場合は、 本発明のポリべ プチドをコ一ドする DNAを含有する組換えべクターは原核生物中で自 立複製可能であると同時に、 プロモーター、 リボソーム結合配列、 本 発明のポリぺプチドをコ一ドする DNAおよぴ転写終結配列が連結され た構造を含むべクターであることが好ましい。 プロモーター'を制御す る遺伝子が含まれていてもよい。  When a prokaryote such as a bacterium is used as a host cell, the recombinant vector containing the DNA encoding the polypeptide of the present invention is capable of autonomous replication in the prokaryote, and has a promoter, The vector is preferably a vector containing a structure in which a ribosome binding sequence, DNA encoding the polypeptide of the present invention, and a transcription termination sequence are linked. A gene that controls the promoter 'may be included.
原核生物用の発現べクターと しては、 例えば、 pGEMEX-Ι (プロメガ 社製） 、 pQE-30 (キアゲン社製) 、 pKYP200 [Agric. Biol. Chem.， 48, Examples of expression vectors for prokaryotes include pGEMEX-II (promega), pQE-30 (Qiagen), pKYP200 [Agric. Biol. Chem., 48,
669 (1984)] 、 pLSAl [Agric. Biol. Chem. , 53, 277 (1989) ] 、 pGELl669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl
[Proc. Natl. Acad. Sci. , USA, 82> 4306 (1985) 3 , pTrS30 [Escherichia coli JM109/pTrS30(FERM BP - 5407)より調製〕 、 pGEX - 5X-3 (アマシャ ム · バイォサイエンス社製) 、 pET14 (ノバジェン社製） 、 pPROTet.E (クロンテック社製) 、 pRSET C (インビトロジェン社製) 等をあげる ことができる。 [Proc. Natl. Acad. Sci., USA, 82> 4306 (1985) 3, pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pGEX-5X-3 (Amersham Biosciences) ), PET14 (Novagen), pPROTet.E (Manufactured by Clontech), pRSET C (manufactured by Invitrogen) and the like.
プロモーターと しては、 宿主細胞中で機能するものであればいかな るものでもよい。 例えば、 trpプ口モーター (Ptrp) 、 lacプ口モーター 、 pLプロモーター、 Prプロモーター、 Τ7プロモーター等の、 大 β昜菌ゃフ ァージ等に由来するプロモータ一をあげることができる。 また Ptrpを 2 つ直列させたプロモーター (PtrpX 2 ) 、 tacプロモーター、 lacT7プロ モーター、 let Iプロモーターのように人為的に設計改変されたプロモ 一ター等も用いることができる。 Any promoter can be used as long as it functions in the host cell. For example, trp-flop opening motor (P trp), lac flop port motor, p L promoter, Pr promoter, can be mentioned, such as Τ7 promoter, a promoter one derived from large β昜菌Ya off Aji like. Also, artificially designed and modified promoters such as a promoter in which two P trps are connected in series (P trp X 2), a tac promoter, a lacT7 promoter, and a let I promoter can be used.
リボソーム結合酉 S列であるシャインーダルガノ （Shine- Dal garno) 配列と開始コ ドンとの間を適当な距離 （例えば 6〜18塩基） に調節し たプラスミ ドを用いることが好ましい。  It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is the ribosome-binding rooster S row, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
本発明の組換えベクターにおいては、 転写終結配列は必ずしも必要 ではないが、 本発明のポリぺプチドをコ一ドする DNAの直下に転写終結 配列を配置することが好ましい。  In the recombinant vector of the present invention, the transcription termination sequence is not always necessary, but it is preferable to arrange the transcription termination sequence immediately below the DNA encoding the polypeptide of the present invention.
宿主細胞としては、 ェシエリ ヒア属、 セラチア属、 バチルス属、 ブ レビパクテリ ゥム属、コリネパクテリ ゥム属、 ミクロバタテリ ゥム属、 シユードモナス属等に属する微生物、 例えば、 Escherichia coli XL1 - Blue、 Escherichia coli XL2-Blue、 Escherichia coli BL21、 Escherichia coli DH1 Escherichia coli Mし 1000、 Escherichia coli KY3276、 Escherichia coli W1485、 Escherichia coli JM109、 Escherichia coli HB101、 Escherichia coli No.49、 Escherichia coli ,W3110、 Escherichia coli NY49N Escherichia coli GI698、 Escherichia coli TB1、 Serratia f icaria Serratia f ontico丄 a、 Serratia liquef aciens 、 Serratia marcescensN Bacillus subti丄 is、 Bacillus Examples of the host cell include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevipacterium, Corynepacterium, Microbatteryrum, Pseudomonas, and the like, for example, Escherichia coli XL1-Blue, Escherichia coli XL2- Blue, Escherichia coli BL21, Escherichia coli DH1 Escherichia coli M and 1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli, W3110, Escherichia coli NY49 N Escherichia coli GI698, Escherichia coli TB1, Serratia f icaria Serratia f ontico 丄 a, Serratia liquef aciens, Serratia marcescens N Bacillus subti 丄 is, Bacillus
amylol iquef acines、 Brevibacterium ammoniagenes Brevibacterium immariopnilum ATCC14068、 Brevi bacterium saccharolvticum ATCC14066 、 Brevibacterium f lavum ATCC14067 Brevibacterium lactof ermentum ATCC13869、 Corynebacterium glutamicum ATCC13032、 Corynebacterium g_lu^micU2j_ ATCC13869、 Corynebacterium acetoacidophi lum ATCC13870 、 Microbacterium ammoniaDhilum ATCC15354、 Pseudomonas putida 、 Pseudomonas sp. D - 0110等をあげることができる。 amylol iquef acines, Brevibacterium ammoniagenes Brevibacterium immariopnilum ATCC14068, Brevi bacterium saccharolvticum ATCC14066, Brevibacterium f lavum ATCC14067 Brevibacterium lactof ermentum ATCC13869, Corynebacterium glutamicum ATCC 130g, Corynebacterium glutamicum ATCC13032g , Microbacterium ammonia Dhilum ATCC15354, Pseudomonas putida, Pseudomonas sp. D-0110 and the like.
組換えべクターの導入方法と しては、 上記宿主細胞へ DNAを導入する 方法であればいずれも用いることができ、 例えば、 カルシウムイオン を用いる方法 [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)〕 、 プ ロ トプラス ト法 （特開昭 63- 2483942) 、 または Gene, 17, 107 (1982) や Molecular & General Genetics, 168, 111 (1979)に記載の方法等を あげることができる。  Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)], the protoplast method (JP-A-63-2483942), or the method described in Gene, 17, 107 (1982) or Molecular & General Genetics, 168, 111 (1979). Can be.
酵母を宿主細胞と して用いる場合には、 発現ベクターとして、 例え ば、 YEP 13 (ATCC37115) 、 YEp24 (ATCC37051) 、 YCp50 (ATCC37419) 、 pHS19、 pHS15等をあげることができる。  When yeast is used as a host cell, examples of expression vectors include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.
プロモーターと しては、 酵母細胞中で機能するものであればいずれ のものを用いてもよく、 例えば、 へキソースキナーゼ等の解糖系の遺 伝子のプロモーター、 PH05プロモーター、 PGKプロモーター、 GAPプロ モーター、 ADHプロモ ター、 gal 1プロモーター、 gal 10プロモータ 一、 ヒートショ ックポリペプチドプロモーター、 MFa lプロモーター、 CUP 1プロモーター等をあげることができる。  Any promoter can be used as long as it functions in yeast cells. For example, promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP Promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFal promoter, CUP 1 promoter and the like.
百王糸田 S包と し は、 Saccharomyces禺、 Scnizosaccharomyces属、 Kluyveromyces属、 Tr ichpsooron¾ Schwanniomvces属、 Pichiafe、 Candida属等に属する微生物、 {列えば、、 Saccharomyces cerevisiae bchi zo sac char omyces pombe.、 Kluyveromyces
Figure imgf000020_0001
Trichosporon oullulans、 Schwanniomyces alluvius、 Candi^_§_ uti lis等をあり oこ とができる。 ' Hyakuo Itoda S-package is a microorganism belonging to the genus Saccharomycesyu, Scnizosaccharomyces, Kluyveromyces, Trichpsooron¾ Schwanniomvces, Pichiafe, Candida, etc.
Figure imgf000020_0001
Trichosporon oullulans, Schwanniomyces alluvius, Candi ^ ____ utilis, etc. are available. '
組換えべクターの導入方法としては、 酵母に DNAを導入する方法であ ればいずれも用いることができ、 例えば、 エレク ト口ポレーシヨン法 As a method for introducing a recombinant vector, any method for introducing DNA into yeast can be used. For example, an electroporation method can be used.
[Methods Enzymol. , 194. 182 (1990)〕 、 スフエロプラス ト法 [Proc.[Methods Enzymol., 194.182 (1990)], Spheroplast method [Proc.
Natl. Acad. Sci. USA, 75, 1929 (1978)〕 、 酢酸リチウム法 〔J.Natl. Acad. Sci. USA, 75, 1929 (1978)], lithium acetate method [J.
Bacteriology, 153, 163 (1983)〕 、 Proc. Natl. Acad. Sci. USA, 75,Bacteriology, 153, 163 (1983)), Proc. Natl. Acad. Sci. USA, 75,
1929 (1978)に記載の方法等をあげることができる。 1929 (1978).
糸状菌を宿主細胞として用いる場合には、 発現ベクターとして、 例 えば pPTRI (白鶴酒造社製）、 pPTRII (白鶴酒造社製） 、 pAUR316 (宝酒 造社製） 等をあげることができる。 When a filamentous fungus is used as a host cell, the For example, pPTRI (manufactured by Hakutsuru Shuzo), pPTRII (manufactured by Hakutsuru Shuzo), pAUR316 (manufactured by Takara Shuzo) and the like can be mentioned.
プロモーターと しては、 糸状菌株中で機能するものであればいずれ のものを用いてもよく 、例えば amyBプ口モーター、 enoAプ口モーター、 gpdプロモーター、 melOプロモーター、 alcAプロモーター、 prAプロモ 一ター等をあげることができる。  Any promoter may be used as long as it functions in a filamentous strain, and examples thereof include amyB promoter, enoA promoter, gpd promoter, melO promoter, alcA promoter, and prA promoter. Can be given.
宿主細胞と してはァスペルギルス (Aspergillus) 属、 ぺニシリ ウム (Penicillium) 属、 トリ コデルマ (Tricoderma) 属、 フザリ ウム （ Pusarium) 属、 フ ミ コラ (Humicola) 属、 ムユーノレ (Mucor) 属、 リ ゾ ープス (Rhizopus) 属、 モナスカス (Monascus) 属等に属する糸状菌、 例えばァスぺノレギノレス ' ォリゼ、 ァスぺノレギノレス ' - ドランス、 ァス ぺノレギルス ' 二ガー (Aspergillus niger) 、 ァスぺノレギルス ' フイカ ム (Aspergillus f iccum)、 トリコテノレマ-リ 1— ィ、 fr ichoderma r e_e s e i ) 、 リゾープス ' 二べウス (Rhizopus nibeus) をあげることができる 組換えべクターの導入方法と しては糸状菌に DNAを導入する方法で あればいずれも用いることができ、 プロ トプラス ト法 [GENETICS of ASPERGILLUS NIDULANS: EMBO Practical Course Manual, 8 (1988) 〕 等をあげることができる。 Host cells include the genus Aspergillus, the genus Penicillium, the genus Tricoderma, the genus Fusarium, the genus Humicola, the genus Mucor, and lysozoa. Filamentous fungi belonging to the genus Rhizopus, Monascus, etc., such as Aspergillus niger and Aspergillus niger, Aspernoluginus As a method for introducing a recombinant vector, filamentous fungi can be used as a method for introducing a recombinant vector, such as squid (Aspergillus ficcum), trichothenorema li- 1 , fr ichoderma re_e sei), and Rhizopus 'Rhizopus nibeus'. Any method for introducing DNA can be used, and examples include a protoplast method [GENETICS of ASPERGILLUS NIDULANS: EMBO Practical Course Manual, 8 (1988)].
動物細胞を宿主として用いる場合には、 発現ベクターとして、 例え ば、 pEGFP - C2 (クロンテック社製） 、 pAGE107 (特開平 3- 22979;  When an animal cell is used as a host, as an expression vector, for example, pEGFP-C2 (manufactured by Clontech), pAGE107 (JP-A-3-22979;
Cytotechnol. , 3, 133 1990) 、 pAS3- 3 (特開平 2 - 227075) 、 pCDM8 〔 Nature, 329, 840 (1987)〕 、 pCMV-Tagl (ス トラタジーン社製） 、 pcDNA3.1(+) (インビトロジェン社製） 、 pREP4 (インビトロジェン社 製）、 pMSG (アマシャム 'バイオサイエンス社製）、 pAMo〔J. Biol. Chera. , 268, 22782 (1993) 〕 等をあげることができる。 Cytotechnol., 3, 133 1990), pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 (Nature, 329, 840 (1987)), pCMV-Tagl (Stratagene), pcDNA3.1 (+) (Invitrogen PREP4 (Invitrogen), pMSG (Amersham's Bioscience), pAMo [J. Biol. Chera., 268, 22782 (1993)], and the like.
プロモーターと しては、 動物細胞中で機能するものであればいずれ も用いることができ、 例えば、 サイ トメガロウィルス （CMV) の IE ( immediate early) 遺伝子のプロモーター、 SV40の初期プロモーター、 レ トロゥイノレスのプロモーター、 メタロチォネイ ンプロモーター、 ヒ 9 Any promoter can be used as long as it functions in animal cells. For example, the promoter of the IE (immediate early) gene of cytomegalovirus (CMV), the early promoter of SV40, and the promoter of retrodinores Promoter, metallothionein promoter, human 9
一トショ ックプロモーター、 SRctプロモーター等をあげることができ る。 また、 ヒ ト CMVの IE遺伝子のェンハンサーをプロモーターと共に用 いてもよい。 One-shot promoter, SRct promoter and the like. Further, the enhancer of the IE gene of human CMV may be used together with the promoter.
宿主細胞としては、 ヒ トの細胞であるナマルバ (Namalwa) 細胞、 サ ルの細胞である COS細胞、 チャイニーズ · ハムスターの細胞である CH0 細胞、 HBT5637 (特開昭 63- 299) 等をあげることができる。  Examples of the host cell include Namalwa cell, a human cell, COS cell, a Sal cell, CH0 cell, a Chinese hamster cell, and HBT5637 (JP-A-63-299). it can.
動物細胞への組換えべクターの導入方法と しては、動物細胞に DNAを 導入する方法であればいずれも用いることができ、 例えば、 エレク ト 口ポレーシヨ ン法 [Cytotechnology, 3, 133 (1990)〕 、 リン酸カルシ ゥム法 （特開平 2-227075) 、 リポフエクシ.ヨ ン法 [Proc. Natl. Acad. Sci. USA, 8 > 7413 (1987)〕 、 Virology, 52, 456 (1973)等をあげる ことができる。  As a method for introducing a recombinant vector into animal cells, any method can be used as long as DNA can be introduced into animal cells. For example, an electoral poration method [Cytotechnology, 3, 133 (1990) )], Calcium phosphate method (Japanese Patent Application Laid-Open No. 2-227075), Lipofexion method (Proc. Natl. Acad. Sci. USA, 8> 7413 (1987)), Virology, 52, 456 (1973), etc. Can be raised.
昆虫細胞を宿主と して用いる場合には、 例えば Current Protocols in Molecular Biology, John Wi ley & Sons (,1987) Baculovirus Expression Vectors： A Laboratory Manual, W. H. Freeman and Company (1992)、 Bio/Technology, 6, 47 (1988)等に記載された方法によって、 ポリべ プチドを発現することができる。  When insect cells are used as hosts, for example, Current Protocols in Molecular Biology, John Wiley & Sons (1987) Baculovirus Expression Vectors: A Laboratory Manual, WH Freeman and Company (1992), Bio / Technology, 6, 47 (1988) and the like can express the polypeptide.
即ち、 組換え遺伝子導入べクターおよびバキュロウィルスを昆虫細 胞に共導入して昆虫細胞培養上清中に組換えウィルスを得た後、 さら に該組換えウィルスを昆虫細胞に感染させ、 ポリペプチドを発現させ ることができる。  That is, after the recombinant gene transfer vector and the baculovirus are co-transfected into insect cells to obtain a recombinant virus in the culture supernatant of insect cells, the recombinant virus is further infected into insect cells, and the polypeptide is Can be expressed.
該方法において用いられる遺伝子導入べクターと しては、 例えば、 pVL1392、 pVL1393、 PBlueBac4.5 (ともにイ ンビトロジェン社製） 、 PBacPAK9 (クロンテック社製） 等をあげることができる。 Is a transgenic base compactors used in the method, for example, can be exemplified pVL1392, pVL1393, P BlueBac4.5 (both manufactured by Lee Nbitorojen Co.), P BacPAK9 (Clontech) and the like.
パキュロウィルス と しては、 例えば、 ャガ科昆虫に感染するウィル スであるァゥ トグラファ · カリフオル-力 · ヌクレア一 · ポリへドロ シス · ヮ ノレス (Autographa californica nuclear polyhedrosis virus ) 等を用いることができる。  As the paculovirus, for example, a virus that infects nocturnal insects such as Atographa californica-force, nucleus, polyhedrosis, ノ noreth (Autographa californica nuclear polyhedrosis virus) and the like are used. Can be.
昆虫細胞としては、 Spodoptera f rugiperdaの卵巢細胞である Sf 9ヽ As insect cells, Sf9 ヽ, an egg cell of Spodoptera f rugiperda
Sf 21 [Baculovirus Expression Vectors： A Laboratory Manual, W. H. Freeman and Company (1992)〕 、 Tri choplus i a niの卵巣細胞である Hi gh 5 (インビトロジェン社製） 等を用いることができる。 Sf 21 [Baculovirus Expression Vectors: A Laboratory Manual, WH Freeman and Company (1992)], and High 5 (manufactured by Invitrogen), which is an ovarian cell of Trichoplus ia ni, can be used.
組換えウィルスを調製するための、 昆虫細胞への上記組換え遺伝子 導入べク ターと上記パキュ口ウィルスの共導入方法と しては、 例えば、 リ ン酸カルシウム法 (特開平 2- 227075) 、 リ ボフェク シヨ ン法 C Proc. Nat l . Acad. Sc i . USA, 84, 7413 ( 1987)〕 等をあげることができる。 植物細胞を宿主細胞として用いる場合には、 発現べク ターと して、 例えば、 Tiプラスミ ド、 タバコモザイクウィルスベクター等をあげる ことができる。  Examples of the method for co-transferring the above-mentioned recombinant gene introduction vector and the above-mentioned PacuMouth virus into insect cells for preparing a recombinant virus include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075). Acad. ScI. USA, 84, 7413 (1987)]. When a plant cell is used as a host cell, examples of the expression vector include Ti plasmid and tobacco mosaic virus vector.
プロモーターと しては、 植物細胞中で機能するものであればいずれ のものを用いてもよく、 例えば、 力リ フラワーモザイクウィルス （ CaMV ) の 35Sプロモーター、 ィネアクチン 1プロモーター等をあげることが できる。  As the promoter, any promoter can be used as long as it functions in plant cells, and examples thereof include the 35S promoter of potato flower mosaic virus (CaMV) and the inactin 1 promoter.
宿主細胞と しては、 タバコ、 ジャガイモ、 トマ ト、 ニンジン、 ダイ ズ、 アブラナ、 アルフアルファ、 イネ、 コムギ、 ォォムギ等の植物細 胞等をあげることが.できる。  Examples of the host cell include plant cells such as tobacco, potato, tomato, carrot, soybean, oilseed rape, alf alfa, rice, wheat, oats and the like.
組換えベクターの導入方法と しては、植物細胞に DNAを導入する方法 であればいずれも用いることができ、 例えば、 ァグロバタテリ ゥム （ Agrobacterium) (特開昭 59— 140885、 特開昭 60— 700.80、 W094/00977) 、 エレク ト口ポレーシヨ ン法 （特開昭 60- 251887) 、 パーテイクノレガン （ 遺伝子銃） を用いる方法 （特許公報 2606856号、 特許公報 2517813号） 等をあげることができる。  As a method for introducing a recombinant vector, any method for introducing DNA into plant cells can be used. For example, Agrobacterium (Agrobacterium) (JP-A-59-140885, JP-A-60-1985) 700.80, W094 / 00977), an electoral-portion method (JP-A-60-251887), a method using a partake noregan (gene gun) (Patent Publications 2606856 and 2517813), and the like.
以上のようにして得られる本発明の形質転換体を培地に培養し、 培 養物中に本発明のポリべプチドを生成蓄積させ、 該培養物から採取す ることにより、 本発明のポリぺプチドを製造することができる。  The transformant of the present invention obtained as described above is cultured in a medium, the polypeptide of the present invention is produced and accumulated in a culture, and the polypeptide of the present invention is collected from the culture. Peptides can be produced.
本発明の形質転換体を培地に培養する方法は、 宿主の培養に用いら れる通常の方法に従って行うことができる。  The method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
本発明の形質転換体が大腸菌等の原核生物あるいは酵母、 糸状菌等 の真核微生物を宿主と して得られた形質転換体である場合、 該形質転 換体を培養する培地として、 該形質転換体が資化し得る炭素源、 窒素 源、 無機塩類等を含有し、 該形質転換体の培養を効率的に行える培地 であれば天然培地、 合成培地のいずれを用いてもよい。 When the transformant of the present invention is a transformant obtained using a prokaryotic organism such as Escherichia coli or a eukaryotic microorganism such as yeast or filamentous fungus as a host, the transformant is used as a medium for culturing the transformant. Nitrogen, a carbon source that the body can utilize Any of a natural medium and a synthetic medium may be used as long as the medium contains a source, inorganic salts, and the like, and can efficiently culture the transformant.
炭素源と しては、 該形質転換体が資化し得るものであればよく、 グ ルコース、 フラク ト一ス、 スク ロース、 これらを含有する糖蜜、 デン プンあるいはデンプン加水分解物等の炭水化物、' 酢酸、 プロピオン酸 等の有機酸、 エタノール、 プロパノールなどのアルコール類等を用い るこ とができる。  Any carbon source may be used as long as the transformant can be assimilated, and glucose, fructoses, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, and the like can be used. Organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used.
窒素源と しては、 アンモニア、 塩化アンモニゥム、 硫酸アンモニゥ ム、 酢酸アンモ-ゥム、 リン酸アンモ-ゥム等の無機酸もしくは有機 酸のアンモニゥム塩、 その他の含窒素化合物、 ならびに、 ペプトン、 肉エキス、酵母エキス、 コーンスチープリカー、 カゼィン加水分解物、 小麦タンパク質おょぴ小麦タンパク加水分解物、 大豆粕おょぴ大豆粕 加水分解物、各種発酵菌体およびその消化物等を用いることができる。  Nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; other nitrogen-containing compounds; and peptone and meat. Extract, yeast extract, corn steep liquor, casein hydrolysate, wheat protein and wheat protein hydrolysate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used. .
無機塩と しては、 リ ン酸第一カリ ウム、 リン酸第二カリ ウム、 リ ン 酸マグネシウム、 硫酸マグネシウム、 塩化ナトリ ウム、 硫酸第一鉄、， 硫酸マンガン、 硫酸銅、 炭酸カルシウム等を用いることができる。  Inorganic salts include potassium potassium phosphate, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. Can be used.
糸状菌の培養においては、 小麦ふすま、 米糠、 大豆ふすま、 脱脂大 豆タンパク質、 蒸米等を、 炭素源、 窒素源および無機物源と し、 適当 な塩類を補強したものを培地として用いることもできる。  In the cultivation of filamentous fungi, wheat bran, rice bran, soy bran, defatted soy protein, steamed rice, and the like can be used as a carbon source, a nitrogen source, and an inorganic substance source, and those supplemented with appropriate salts can be used as a medium.
培養は、 振と う培養または深部通気攪拌培養などの好気的条件下で 行う。 培養温虔は 15〜40°Cがよく、 培養時間は、 通常 16時間〜 7 日間 である。 培養中の pHは 3. 0〜9. 0に保持することが好,ましい。 pHの調整 は、 無機または有機の酸、 アルカ リ溶液、 尿素、 炭酸カルシウム、 ァ ンモユアなどを用いて行う。  The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days. Preferably, the pH during the cultivation is kept between 3.0 and 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
また、 培養中必要に応じて、 アンピシリンやテトラサイク リ ン等の 抗生物質を培地に添加してもよい。  If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.
プロモーターとして誘導性のプロモーターを用いた組換えベクター で形質転換した微生物を培養するときには、 必要に応じてィンデュー サーを培地に添加してもよい。 例えば、 lacプロモーターを用いた組換 えべクターで形質転換した微生物を培養するときにはイソプロピル - ]3 - D-チォガラタ.トピラノシド等を、 trpプ口モーターを用いた組換え べクターで形質転換した微生物を培養するときにはィン ドールァク リ ル酸等を培地に添加してもよい。 When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with a recombinant vector using the lac promoter, isopropyl- ] When culturing a microorganism transformed with 3-D-thiogalata.topyranoside or the like by a recombinant vector using a trp motor, indoleacrylic acid or the like may be added to the medium.
糸状菌を培養する場合、 通常の液体培地だけではなく固体培地を使 用して培養することもできる。 糸状菌の培養に用いられる固体培地と しては、 通常の寒天培地より水分活性が低い培地を用いることができ る。 該固体培地と しては、 小麦ふすまに水性媒体を浸潤させた小麦ふ すま培地、 蒸米等があげられる。  When culturing filamentous fungi, it is possible to use not only a normal liquid medium but also a solid medium. As a solid medium used for culturing filamentous fungi, a medium having lower water activity than a normal agar medium can be used. Examples of the solid medium include a wheat bran medium in which an aqueous medium is infiltrated into wheat bran, steamed rice, and the like.
糸状菌を、 固体培地で培養する場合は、 糸状菌を植菌後、 固体培地 と糸状菌を充分に混合し、 アルミまたはステンレス製のト レーに薄く 広げて培養室内に静置する。 その後、 温度を通常 15〜50°C、 好ましく は 25〜37°Cに、 湿度を通常 80〜： 100 %、 好ましくは 90〜： 100%に制御し て、 3〜 10日間静置培養する。  When culturing a filamentous fungus in a solid medium, after inoculating the filamentous fungus, thoroughly mix the solid medium and the filamentous fungus, spread it thinly on an aluminum or stainless steel tray, and leave it in the culture chamber. Thereafter, the culture is allowed to stand still for 3 to 10 days at a temperature of usually 15 to 50 ° C, preferably 25 to 37 ° C, and a humidity of usually 80 to 100%, preferably 90 to 100%.
動物細胞を宿主と して得られた形質転換体を培養する培地と しては、 一般に使用されている RPMI 1640培地 〔： Γ. Am. Med. As soc. , 199, 519 (1967) ] 、 Eagl eの醒 (Mimimum Es sent ial Medium) [ Sc i ence, 122, 501 ( 1952)〕 、 Dalbecco改変 Eagl e培地 [Virology, 8, 396 (1959) ] 、 199培地 〔Proc. Soc. Exp. Biol . Med. , 73, 1 (1950)〕 またはこれら 培地に牛胎児血清等を添加した培地等を用いることができる。  As a medium for culturing a transformant obtained using animal cells as a host, a commonly used RPMI 1640 medium [: III. Am. Med. Assoc., 199, 519 (1967)], Awakening of Eagl e (Mimimum Es sential Medium) [Science, 122, 501 (1952)], Dalbecco modified Eagle medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol Med., 73, 1 (1950)] or a medium obtained by adding fetal bovine serum or the like to such a medium.
培養は、 通常 pH6〜8、 30〜40°C、 5 % C02存在下等の条件下で 1〜 7 日 間行う。 また、 培養中必要に応じて、 カナマイシン、 ペニシリ ン等の 抗生物質を培地に添加してもよい。 Culture is usually pH 6-8, carried out between 30~40 ° C, 5% C0 2 1~ under conditions such presence 7 days. If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
昆虫細胞を宿主と して得られた形質転換体を培養する培地と しては. 一般に使用されている TNM- FH培地（ファーミンジヱン社製）、 Sf - 900 II SFM培地 （イ ンビトロジヱン社製） 、 ExCel l400、 ExCe l l405 (いずれも JRHバイォサイエンス社製）、 Graceの昆虫培地 [ Nature, 195, 788 ( 1962) 〕 等を用いることができる。 Insect cells as a medium for culturing a transformant obtained by the host commonly used TNM- FH medium (Faminjiwen Inc.), Sf -. 9 (manufactured by Lee Nbitorojiwen Co.) 00 II SFM medium , ExCel l400, ExCel l405 (all manufactured by JRH Biosciences), Grace insect medium [Nature, 195, 788 (1962)], and the like.
培養は、 通常 pH6〜7、 25〜30°C等の条件下で、 1〜5日間行う。  The cultivation is usually performed under conditions of pH 6 to 7 and 25 to 30 ° C for 1 to 5 days.
また、 培養中必要に応じて、 ゲンタマイ シン等の抗生物質を培地に 添加してもよレ、。 JP2004/004789 If necessary, antibiotics such as gentamicin may be added to the medium during the culture. JP2004 / 004789
植物細胞を宿主として得られた形質転換体は、 細胞と して、 または 植物の細胞や器官に分化させて培養することができる。 該形質転換体 を培養する培地と しては、 一般に使用されているムラシゲ ' アンド ' スターグ（MS)培地、 ホワイ ト（Whi te)培地、 またはこれら培地にォーキ シン、 サイ トカイニン等、 植物ホルモンを添加した培地等を用いるこ とができる。 A transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ. As a culture medium for culturing the transformant, commonly used Murashige 'and' Sturg (MS) medium, white medium, or a plant hormone such as auxin or cytokinin is added to these mediums. An added medium or the like can be used.
培養は、 通常 pH5〜9、 20〜40°Cの条件下で 3〜60日間行う。  Cultivation is usually performed at pH 5-9 and 20-40 ° C for 3-60 days.
また、 培養中必要に応じて、 カナマイシン、 ハイグロマイシン等の 抗生物質を培地に添加してもよい。  If necessary, antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.
上記のとおり、 本発明のポリぺプチドをコ一ドする DNAを組み込んだ 組換え体ベクターを保有する微生物、 動物細胞、 あるいは植物細胞由 来の形質転換体を、 通常の培養方法に従って培養し、 該ポリペプチド を生成蓄積させ、 該培養物より該ポリぺプチドを採取することによ り、 該ポリぺプチドを製造することができる。  As described above, a transformant derived from a microorganism, animal cell, or plant cell having a recombinant vector into which a DNA encoding the polypeptide of the present invention has been incorporated is cultured according to a conventional culture method. By producing and accumulating the polypeptide and collecting the polypeptide from the culture, the polypeptide can be produced.
酵母、 糸状菌、 動物細胞、 昆虫細胞または植物細胞により発現させ た場合には、 糖あるいは糖鎖が付加されたポリぺプチドを得ることが できる。  When expressed by yeast, filamentous fungi, animal cells, insect cells or plant cells, a sugar or a sugar chain-added polypeptide can be obtained.
本発明のポリぺプチドの生産方法としては、 宿主細胞内に生産させ る方法、 宿主細胞外に分泌させる方法、 あるいは宿主細胞外膜上に生 産させる方法があり、 使用する宿主細胞や、 生産させるポリペプチド の構造を変えることにより、 該方法を選択することができる。  The polypeptide of the present invention can be produced in a host cell, secreted out of the host cell, or produced on the outer membrane of the host cell. The method can be selected by changing the structure of the polypeptide to be made.
本発明のポリぺプチドが宿主細胞内あるいは宿主細胞外膜上に生産 される場合、 ポールソンらの方法 〔： Γ. Bi ol . Chem. , 264> 17619 (1989) 〕 、 ロウらの方法 [ Proc. Nat l . Acad. Sc i . USA, 86, 8227 ( 1989)、 Gene s Deve lop. , 4, 1288 ( 1990)〕 、 または特開平 5 - 336963、 W094/23021 等に記載の方法を準用することにより、 該ポリぺプチドを宿主細胞外 に積極的に分泌させることができる。 すなわち、 遺伝子組換えの手法 を用いて、 本発明のポリペプチドの手前にシグナルぺプチドを付加し た形で発現させることにより、 本発明のポリぺプチドを宿主細胞外に 分泌させることができる。 . また、 特開平 2- 227075に記載されている方法に準じて、 ジヒ ドロ葉 酸還元酵素遺伝子等を用いた遺伝子増幅系を利用して生産量を上昇さ せることもできる。 When the polypeptide of the present invention is produced in a host cell or on a host cell outer membrane, the method of Paulson et al. [: III. Biol. Chem., 264> 17619 (1989)] and the method of Lowe et al. Natl. Acad. Sc i. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or the methods described in JP-A-5-336963, W094 / 23021, etc. apply mutatis mutandis. This allows the polypeptide to be positively secreted out of the host cell. That is, the polypeptide of the present invention can be secreted out of the host cell by expressing the polypeptide of the present invention with a signal peptide added in front thereof using a gene recombination technique. . Further, according to the method described in Japanese Patent Application Laid-Open No. 2-227075, the production amount can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like.
また、 公知の方法 C J. B iomo l . 匪， 6, 129 ( 1998)、 Sc i ence, 242, 1162 (1988)、 J. Biochem. , 110, 166 (1991)〕 に準じて、 in vi tro転 写 ·翻訳系を用いて本発明のポリペプチドを生産することができる。 すなわち、 本発明のポリペプチドをコードする DNAを SP6、 T7、 Τ3等の プロモーターの下流につなげ、 それぞれのプロモーター特異的な RNAポ リメラーゼを反応させることにより大量の本発明のポリペプチドをコ 一ドする RNAをィンビトロで合成した後、無細胞系の翻訳系例えばゥサ ギ網状赤血球ライセートゃコムギ胚芽抽出液を用いた翻訳系を利用し て、 本発明のポリペプチドを生産することができる。  In addition, according to known methods C J. Biomol. Marauder, 6, 129 (1998), Science, 242, 1162 (1988), J. Biochem., 110, 166 (1991)] The polypeptide of the present invention can be produced using a tro transcription / translation system. That is, DNA encoding the polypeptide of the present invention is connected downstream of promoters such as SP6, T7, and Τ3, and a large amount of the polypeptide of the present invention is coded by reacting each promoter-specific RNA polymerase. After synthesizing RNA in vitro, the polypeptide of the present invention can be produced using a cell-free translation system, for example, a translation system using a heron reticulocyte lysate wheat germ extract.
本発明の形質転換体により製造されたポリぺプチドを単離精製する ためには、 通常の酵素の単離精製法を用いることができる。 例えば本 発明のポリペプチドが、 細胞内に溶解状態で発現した場合には、 培養 終了後、 細胞を遠心分離により回収し、 水系緩衝液にけん濁後、 超音 波破砕機、 フ レンチプレス、 マン ト ンガウリ ンホモゲナイザー、 ダイ ノ ミル等により細胞を破砕し、 無細胞抽出液を得る。 該無細胞抽出液 を遠心分離することにより得られる上清から、 通常の酵素の単離精製 法、 即ち、 溶媒抽出法、 '硫安等による塩析法、 脱塩法、 有機溶媒によ る沈殿法、ジェチルァミ ノェチル（DEAE)—セファ ロース、 DIAI0N ΗΡΑ-75 In order to isolate and purify the polypeptide produced by the transformant of the present invention, a conventional enzyme isolation and purification method can be used. For example, when the polypeptide of the present invention is expressed in a lysed state in cells, the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, The cells are disrupted using a mantongaulin homogenizer, dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation and purification method can be used, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent. Law, Jechilami Noechil (DEAE)-Sepharose, DIAI0N ΗΡΑ-75
(三菱化成社製） 等のレジンを用いた陰イオン交換ク口マ トグラフィ 一法、 S- Sepharo s e FF ( Pharmac ia社製） 等のレジンを用いた陽イオン 交換クロマ トグラフィー法、 プチノレセファ ロース、 フエ-ノレセファ ロ ース等のレジンを用いた疎水性クロマ トグラフィ一法、 分子篩を用い たゲルろ過法、 ァフイエティークロマトグラフィー法、 クロマ トフォ 一力シング法、 等電点電気泳動等の電気泳動法等の手法を単独あるい は組み合わせて用い、 精製標品を得ることができる。 pRSET系ベクター (インビトロジェン社製） 、 pGEX系ベクター （アマシャム ' バイオサ ィエンス社製） 等、 該ポリペプチドにタグをつけて発現させた場合、 ニッケルレジン、 グルタチオンセファロースなどの適当な担体を用い てァフィ二ティ精製することもできる。 Anion-exchange chromatography using a resin such as (Mitsubishi Kasei), cation-exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), petitinoresepharose, Hydrophobic chromatography using resin such as pheno-recepharose, gel filtration using molecular sieve, affinity chromatography, chromatophoresis, electrophoresis such as isoelectric focusing Using a method such as the method alone or in combination, a purified sample can be obtained. When the polypeptide is tagged and expressed, such as a pRSET-based vector (manufactured by Invitrogen) or a pGEX-based vector (manufactured by Amersham's Biosciences), Affinity purification can also be performed using a suitable carrier such as nickel resin and glutathione sepharose.
また、該ポリぺプチドが細胞内に不溶体を形成して発現した場合は、 同様に細胞を回収後、 破砕し、 遠心分離を行うことにより、 沈殿画分 と してポリぺプチドの不溶体を回収する。 回収したボリぺプチドの不 溶体をポリぺプチド変性剤で可溶化する。 該可溶化液を希釈または透 折し、該可溶化液中のポリぺプチド変性剤の濃度を下げることにより、 該ポリぺプチドを正常な立体構造に戻す。 該操作の後、 上記と同様の 単離精製法により該ポリべプチドの精製標品を得ることができる。 本発明のポリぺプチドが細胞外に分泌された場合には、 培養上清に 該ポリペプチドを回収することができる。 即ち、 該培養物を上記と同 様の遠心分離等の手法により処理することにより培養上清を取得し、 該培養上清から、 上記と同様の単離精製法を用いることにより、 精製 標品を得ることができる。  When the polypeptide is expressed in an insoluble form in the cells, the cells are similarly collected, crushed, and centrifuged to obtain an insoluble form of the polypeptide as a precipitate fraction. Collect. Solubilize the insoluble recovered polypeptide with a polypeptide denaturant. The polypeptide is returned to a normal three-dimensional structure by diluting or diffusing the solubilized solution and reducing the concentration of the polypeptide denaturing agent in the solubilized solution. After this operation, a purified preparation of the polypeptide can be obtained by the same isolation and purification method as described above. When the polypeptide of the present invention is secreted extracellularly, the polypeptide can be recovered in the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above. Can be obtained.
このようにして取得される本発明のポリペプチドと して、 例えば、 配列番号 1で示されるァミノ酸配列を含むポリぺプチドをあげること ができる。  Examples of the polypeptide of the present invention obtained in this manner include a polypeptide containing an amino acid sequence represented by SEQ ID NO: 1.
3 . 液胞酵素の培地中への生産  3. Production of vacuolar enzymes in the medium
( 1 ) 液胞酵素を細胞外に分泌する糸状菌の作製  (1) Preparation of filamentous fungi secreting vacuolar enzymes extracellularly
糸状菌において、 液胞酵素を細胞内に局在させる活性を有するポリ ペプチドの機能を抑制させることにより、 液胞酵素、 例えばトリぺプ チジルぺプチダーゼ、カルボキシぺプチダーゼ、ァミノぺプチダーゼ、 セリ ンプロテアーゼ、 ァスパルチルプロテアーゼが、 好ましくはト リ ぺプチジルぺプチダーゼ、 カルボキシぺプチダーゼが効率的に細胞外 に分泌されるため、 液胞酵素を培地中に生産させることができる。 液胞酵素を細胞内に局在させる活性を有するポリぺプチドとは、 該 ポリぺプチドの機能を抑制させたときに、 液胞酵素のすべてまたはそ の一部が液胞内に正常に局在せず、 細胞外に分泌されるようになるポ リペプチドを意味する。 細胞外に分泌される液胞酵素は前駆体または 成熟体のどちらであってもよい。. このようなポリぺプチドと しては、 液胞酵素の選別や輸送、 液胞の酸性化や形態形成 ·維持に関わる遺伝 子がコードするポリぺプチドがあげられ、 例えばサッカロミセス · セ レピシェの VPS遺伝子群、 PEP遺伝子群、 VAC遺伝子群、 TLG遺伝子群、 VAM遺伝子群、 APG遺伝子群、 CVT遺伝子群おょぴ GRD遺伝子群または動 植物細胞におけるシンタキシンファミ リーおよびソーティング · ネキ シンフアミ リーなどの遺伝子群のいずれかと相同性を有する、 糸状菌 の遺伝子がコ一ドするポリぺプチドがあげられる。 該ポリぺプチドと しては、 ァスペルギルス · ニ ドランスの vpsA遺伝子、 vpsB遺伝子およ ぴ vpsC遗伝子、 ァスペルギルス · ォリ ゼの VAM3相同遺伝子、 Aovps 5遺 伝子等がそれぞれコードするポリペプチド （Aovps5遺伝子がコードす るポリペプチドを以下、 Aovps5ポリペプチドともいう） があげられ、 Aovp s5ポリぺプチドが好ましく用いられる。 このようなポリぺプチド をコードする糸状菌の遺伝子は、 1 . に記載した本発明の丽 Aを取得す る方法と同様にして、 ，取得することができる。 In filamentous fungi, the function of a polypeptide having the activity of localizing vacuolar enzymes to cells is suppressed, so that vacuolar enzymes such as triptidyl peptidase, carboxypeptidase, aminopeptidase, and serine protease are used. Since aspartyl protease, preferably, tripidyl peptidase and carboxypeptidase are efficiently secreted extracellularly, vacuolar enzymes can be produced in the medium. A polypeptide having an activity of localizing a vacuolar enzyme to a cell means that when the function of the polypeptide is suppressed, all or a part of the vacuolar enzyme is normally localized in the vacuole. Means a polypeptide that is not present and is secreted extracellularly. Vacuolar enzymes secreted extracellularly may be either precursors or matures. Such polypeptides include: Examples include polypeptides encoded by genes involved in the selection and transport of vacuolar enzymes, vacuolar acidification, morphogenesis, and maintenance.For example, Saccharomyces cerevisiae VPS genes, PEP genes, VAC genes, A filamentous fungus that is homologous to one of the TLG gene group, VAM gene group, APG gene group, CVT gene group, GRD gene group, or a gene group such as syntaxin family and sorting-nexin family in animal and plant cells. And a polypeptide encoded by the gene. Examples of the polypeptides include polypeptides encoded by the vpsA gene, vpsB gene and vpsC gene of Aspergillus nidulans, the VAM3 homologous gene of Aspergillus oryzae, the Aovps 5 gene, and the like. The polypeptide encoded by the Aovps5 gene is also hereinafter referred to as Aovps5 polypeptide), and Aovps5 polypeptide is preferably used. A filamentous fungal gene encoding such a polypeptide can be obtained in the same manner as in the method for obtaining 丽 A of the present invention described in 1.
糸状菌としては、 ァスペルギルス属、 ぺニシリ ウム属、 トリ コデル マ属、 フザリ ウム属、 フミ コラ属、 ムコール属、 モナスカス属等に属 する糸状菌をあげることができるが、 ァスペルギルス属に属する糸状 菌が好ましく、 特に、 醸造工業で用いられるァスペルギルス属に属す る糸状菌が好ましい。 醸造工業で用いられるァスペルギルス属に属す る糸状菌と しては、 例えば、 ァスペルギルス ' ォリゼ、 ァスペルギル ス · ソーャ、 ァスぺノレギノレス · 二ガー、 ァスぺノレギノレス · カヮチ （ Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, Fusarium, Humicola, Mucor, Monascus, etc., and those belonging to the genus Aspergillus. And a filamentous fungus belonging to the genus Aspergillus used in the brewing industry. Examples of filamentous fungi belonging to the genus Aspergillus used in the brewing industry include, for example, Aspergillus' oryzae, Aspergillus soja, Aspernoreginoles niger, Aspernoreginoles caschi (
Aspergi l lus kawachi i ) 、 ァスぺ /レギノレス . ァヮモリ ( Aspergi lus awamor i ) 等をあげることができる。 Aspergi lus kawachi i), Asugi / Reginoles. Amori (Aspergi lus awamor i) and the like.
液胞酵素を細胞内に局在させる活性を有するポリペプチドの機能を 抑制させる方法と して、 ( a ) 遗伝子破壊をする方法、 ( b ) アンチ センス mRNAを転写させる方法、 （ c ) プロモーター領域に変異を導入 する方法、 U ) 優性不活性 (ドミナントネガティブ) 変異体を発現 させる方法等があげられる。  (A) a method for disrupting a gene, (b) a method for transcribing an antisense mRNA, and (c) a method for suppressing the function of a polypeptide having an activity of localizing a vacuolar enzyme to a cell. A method of introducing a mutation into the promoter region, U) a method of expressing a dominantly inactive (dominant negative) mutant, and the like.
( a ) 遺伝子破壌をする方法では、 まず、 機能を抑制させたいポリ ペプチド Aをコードする DNAを単離し、 該 DNAのポリべプチド Aをコー ドする領域中の制限酵素サイ トを利用して 1つ以上、 通常は数 100bp〜 数 kbの DNAの挿入あるいは欠失を行うことにより構造を破壊し、 ポリべ プチド Aの機能 （本発明の場合は、 液胞酵素を細胞内に局在させる活 性） を有するポリペプチドをコードしなくなった DNA (以下、 遺伝子破 壌用 DNAとよぶ） を作製する。 次いで、 遺伝子破壊用 DNAを含む組換え べクターで糸状菌を形質転換した後、 ゲノム中のポリぺプチド Aをコ 一ドする遺伝子が、 遺伝子破壌用 DNAにより相同組換えをおこした形質 転換体を、 PCRやサザンブロッテイングにより選択する。 (a) In the method for gene disruption, first, DNA encoding polypeptide A whose function is to be inhibited is isolated, and polypeptide A of the DNA is coded. The structure is destroyed by inserting or deleting one or more DNAs, usually several hundred bp to several kb, using the restriction enzyme site in the region to be ligated, and the function of polypeptide A (the present invention) In this case, DNA that no longer encodes a polypeptide having the activity of localizing vacuolar enzymes to cells (hereinafter referred to as DNA for gene disruption) is prepared. Next, after the filamentous fungus is transformed with a recombination vector containing the DNA for gene disruption, the gene encoding polypeptide A in the genome is transformed by homologous recombination with the DNA for gene disruption. The body is selected by PCR or Southern blotting.
ポリぺプチド Aをコードする領域に挿入する DNAと して、形質転換マ 一力一となる遺伝子を用いれば、 選択培地を用いて形質転換体を容易 に選択することができる。 形質転換マーカーとなる遺伝子は、 宿主と する糸状菌の栄養要求性や薬剤耐性と関係する遺伝子で、' 例えば、 sC 遺伝 +、 argB這伝十、 ni aD¾ fe +、 ' pyrGja i ナ、 amdS退 1 子、 ptrA退 ■■ 伝子、 pabaA遺伝子、 ni iA遺伝子、 BenAR遺伝子などがあげられる。 If a gene that can be used for transformation is used as the DNA to be inserted into the region encoding polypeptide A, the transformant can be easily selected using a selection medium. Genes that serve as transformation markers are genes that are related to auxotrophy and drug resistance of the host filamentous fungi, such as sC inheritance +, argB crawling, ni aD¾ fe +, pyr pyrGja na and amdS 1 child, ptrA exclusion ■■ gene, pabaA gene, niiA gene, BenA R gene, etc.
組換えべクターの作製に用いるベクターは、 大腸菌で自律複製でき、 適当なクローユング用制限酵素サイ トを持つ通常のプラスミ ドベクタ 一であればよく、 例えば、 pBl uescript SK I I (+) (ス トラタジーン社 製） 等があげられる。 遺伝子破壊用 DNA.の作製に形質転換マーカー遺伝 子を用いない場合は、 上記のべクターに形質転換マーカー遺伝子を揷 入したベクターを組換えべクターの作製に用いることにより、 形質転 換体を選択することができる。 宿主とする糸状菌は形質転換マーカー 遺伝子が欠損している株を用いる。  The vector used for the construction of the recombinant vector may be any ordinary plasmid vector that can autonomously replicate in E. coli and has an appropriate restriction enzyme site for closing.For example, pBluescript SK II (+) (Stratagene) Manufactured). When a transformation marker gene is not used to prepare the DNA for gene disruption, a transformant is selected by using a vector in which the transformation marker gene has been inserted into the vector described above to prepare a recombinant vector. can do. A filamentous fungus used as a host is a strain deficient in a transformation marker gene.
( b ) アンチセンス mRNAを転写させる方法では、 まず機能を抑制さ せたいポリペプチド Aをコードする DNAを単離する。 次いで、 該 DNAを 2 . に記載の方法に準じて糸状菌用発現べクターのプロモーターの下 流に逆方向に連結し、 通常の遺伝子 Aの mRNAとは相捕的な配列を有す るアンチセンス raRNAを転写する組換えベクターを作製する。 該組換え ベタターで糸状菌を形質転換することにより、 形質転換体において、 遺伝子 Aの raRNAとアンチセンス mRNAのハイプリ ッ ドを形成させて遺伝 子 Aの mRNAからの翻訳を阻害させる。 PC蘭 004/004789 (b) In the method of transcribing antisense mRNA, first, DNA encoding polypeptide A whose function is to be inhibited is isolated. Next, the DNA is ligated in the reverse direction to the downstream of the promoter of the expression vector for filamentous fungi according to the method described in 2., and the DNA having a sequence complementary to the normal gene A mRNA is used. Create a recombinant vector that transcribes the sense raRNA. By transforming the filamentous fungus with the recombinant beta, the transformant forms a hybrid of the raRNA of the gene A and the antisense mRNA, thereby inhibiting the translation of the gene A from the mRNA. PC orchid 004/004789
( C ) プロモーター領域に変異を導入する方法では、 まず機能を抑 制させたいポリペプチド Aをコードするゲノム遺伝子のコード領域の 上流にあるプロモーター領域を含む DNAを単離する。 該 DNAのプロモー ター領域中の制限酵素サイ トを利用して数 l OObp〜数 kbの DNAの揷入ぁ るいは欠失を行う ことにより、 プロモーター機能を破壊した DNAを作製 する。 次いで該 DNAを含む組換えべクターで糸状菌を形質転換した後、 ゲノム中のポリペプチド Aをコードする遺伝子のプロモーター領域が、 プロモーター機能を破壌した DNAによ り相同組換えをおこした形質転 換体を、 PCRやサザンブロッティングにより選択する。 組換えベクター の作製に用いるベクターは、 （ a ) の遺伝子破壊に用いるベクターと 同様のベクターを用いる。 . (C) In the method of introducing a mutation into a promoter region, first, DNA containing a promoter region upstream of the coding region of a genomic gene encoding polypeptide A whose function is to be suppressed is isolated. A DNA having a promoter function disrupted is produced by inserting or deleting a DNA of several lOObp to several kb by using a restriction enzyme site in a promoter region of the DNA. Next, a filamentous fungus is transformed with a recombination vector containing the DNA, and the promoter region of the gene encoding polypeptide A in the genome is transformed by homologous recombination with DNA whose promoter function has been destroyed. Transformants are selected by PCR or Southern blotting. As the vector used for the preparation of the recombinant vector, the same vector as the vector used for the gene disruption in (a) is used. .
( d ) ドミナントネガティブ変異体は、 あるポリペプチドの機能ド メインに欠失、 置換、 付加等の改変を加えることにより作製した、 も との該ポリべプチドが有する機能を失い、 しかも細胞内の正常な該ポ リぺプチドの機能を阻害するような性胃の変異体である。 ドミナント ネガティブ変異体を発現させる方法では、 まず機能を抑制したいポリ ぺプチド Aの ドミナントネガティブ変異体をコードする DNAを作製す る。 該 DNAを用いて、 2 . に記載の方法にしたがって糸状菌用の発現 クターを作製する。 該発現べクターで糸状菌を形質転換して得られる 該形質転換体において、 ドミナントネガティブ変異体を発現させ、 細 胞内の正常なポリべプチド Aの機能を阻害させる。  (d) a dominant negative mutant is a polypeptide prepared by adding a modification such as deletion, substitution, or addition to a functional domain of a certain polypeptide, loses the function of the original polypeptide, and furthermore, A variant of the sex stomach that interferes with the normal function of the polypeptide. In the method of expressing a dominant negative mutant, first, a DNA encoding a dominant negative mutant of polypeptide A whose function is to be suppressed is prepared. Using this DNA, an expression vector for filamentous fungi is prepared according to the method described in 2. above. In the transformant obtained by transforming the filamentous fungus with the expression vector, a dominant negative mutant is expressed to inhibit the function of normal polypeptide A in the cell.
ポリぺプチド Aの機能ドメインは、 例えばタンパク質ファミ リ一の データベース Pfam [ Nucl e i c Ac i ds Res . , 30, 276 (2002)、 Prote ins, 28, 405-420 ( 1997)〕，の検索を行うことにより推察できる。 Pfamの検 索は、 サンガー研究所の Pfaniに関するウェブサイ ト (  For the functional domain of polypeptide A, search the protein family database Pfam [Nucleic Acids Res., 30, 276 (2002), Proteins, 28, 405-420 (1997)], for example. This can be inferred. A search for Pfam can be found on the Sanger Institute website for Pfani (
http : / /www. sanger. ac. uk/ Sof tware/Pf am/) 上で行つ こと力 C?きる。 例えば、 ァスペルギルス · ォリゼの Aovps5ポリぺプチドのアミノ酸 配列 （配列番号 1 ) に対し、 Pfamの検索を行う と、 44番目のリジンか ら 158番目のグルタミン酸までが、 ホスファチジルイノシトール 3-リン 酸等の脂質と結合する機能を有する PXドメィン、 および 159番目のス レ ォニンから C末端のァラニンまでが、 ポリペプチド同士の重合体の形成 に関与するとされているコイルドコィル領域として予測される。 これ らのドメインの改変を行う ことにより ドミナントネガティブ変異体を 作製できる。 http: / / www. sanger. ac. uk / Software / Pf am /) For example, a search for Pfam against the amino acid sequence of Aovps5 polypeptide of Aspergillus oryzae (SEQ ID NO: 1) shows that from lysine 44 to glutamic acid 158, lipids such as phosphatidylinositol 3-phosphate are found. PX domain that has the function of binding to It is predicted that the region from onin to C-terminal alanine is a coiled coil region that is considered to be involved in the formation of a polymer between polypeptides. By modifying these domains, a dominant negative mutant can be produced.
例えば、 サッカロ ミセス · セレピシェの VAM7ポリ ぺプチドの PXドメ イ ンの解析 〔Nat. Cel l B i ol . , 3, 613 (2001) ] から、 Aovps5ポリべ プチドの PXドメイン中で、 脂質との結合に関与するァミノ酸と して、 87番目のアルギニン、 95番目のリジン、 128番目のアルギニン、 129番 目のアルギニンがあげられ、 特に 87番目から 89番目のアミノ酸配列 （ Arg Arg Tyr) が重要と考えられるので、 これらのアミノ酸のいずれか 、 もしくはこれらから選ばれる複数のァミノ酸に欠失や置換等の変異 を導入することにより ドミナントネガティブ変異体を作製できる。 また、 コイルドコイル領域以外の部分を欠失させたコイルドコイル 領域のみからなる変異体も、 正常なポリべプチドの重合体形成を阻害 する ドミナントネガティブ変異体の例と してあげられる。 また、 PXド メイ ンのみからなる変異体、 コイル ドコイル領域の一部または全てを 欠失させた変異体であってもよい。 .  For example, from the analysis of the PX domain of the Saccharomyces cerevisiae VAM7 polypeptide [Nat. Cell Biol., 3, 613 (2001)], the PX domain of the Aovps5 polypeptide showed The amino acids involved in the binding include arginine at position 87, lysine at position 95, arginine at position 128, and arginine at position 129. The amino acid sequence from position 87 to position 89 (Arg Arg Tyr) is particularly important. Therefore, a dominant negative mutant can be prepared by introducing a mutation such as deletion or substitution into any of these amino acids or a plurality of amino acids selected from these amino acids. In addition, a mutant comprising only a coiled coil region in which a portion other than the coiled coil region has been deleted is also an example of a dominant negative mutant that inhibits formation of a normal polypeptide polymer. Further, a mutant comprising only the PX domain or a mutant in which part or all of the coiled coil region is deleted may be used. .
糸状菌において、 液胞酵素を細胞内に局在させる活性を有するポリ ぺプチドの機能が抑制されると、 分生子の形成が阻害される傾向 あ るため、 上記の、 アンチセンス mRNAの転写やドミナントネガティブ変 異体の発現を、 薬物の投与等で誘導できる発現べクターを用いて行え ば； 増殖および分生子の形成過程では遺伝子の機能を抑制せず、 分生 子が形成した後に該遺伝子の機能を抑制することで、 分生子形成能を 損なわず液胞酵素を細胞外に生産できる利点がある。  In filamentous fungi, when the function of the polypeptide having the activity of localizing the vacuolar enzyme to the cell is suppressed, the formation of conidia tends to be inhibited. If the expression of the dominant negative mutant can be performed using an expression vector that can be induced by administration of a drug, etc., the function of the gene is not suppressed in the process of growth and conidiation, and the gene By suppressing the function, there is an advantage that the vacuolar enzyme can be produced extracellularly without impairing the conidial ability.
糸状菌の形質転換は、 糸状菌に DNAを導入する方法であればいずれも 用いることができ、 例えば、 プロ トプラス ト法 [ GENETICS of  For transformation of filamentous fungi, any method can be used as long as DNA can be introduced into filamentous fungi. For example, the protoplast method [GENETICS of
ASPERGILLUS NIDULANS： EMBO Pract i cal Course Manua l , 8 ( 1988)〕 等をあげる事ができる。 ASPERGILLUS NIDULANS: EMBO Practical Course Manual, 8 (1988)].
また、 Aovps5糸状菌の分生子への紫外線照射等でゲノム DNAにランダ ムに突然変異を誘発させた後、 AoVps5遺伝子破壌株に特徴的な表現型 を利用して、 AovP S5ポリペプチドの機能が欠損した変異株 （以下、 AoVps5機能欠損変異株ともいう） を選択することにより、 上記のよ う な遺伝子操作を用いずに、 Aovps 5ポリぺプチドの機能が抑制された糸 状菌を取得することができる。 Further, after mutagenized randomly genomic DNA with UV irradiation or the like to conidia of Aovps5 filamentous fungi, characteristic phenotype to Ao V ps5 gene dashed壌株 By using, AOV PS 5 polypeptide mutants function is deficient in by selecting (hereinafter, also referred to as Ao V ps5 function deficient mutant), without using the Do genetic manipulation will Yo above, Aovps 5 Filamentous fungi in which the function of the polypeptide is suppressed can be obtained.
ァスペルギルス . ォリ ゼの Aovps5遺伝子破壌株は、 寒天培地上で培 養を行う と赤褐色の色素を培地中に分泌する。 したがって、 本性質を 利用することにより AovpS 5ポリぺプチドの機能が欠損した変異株のス ク リーニングが可能である。 The Aovps5 gene disrupted strain of Aspergillus oryzae secretes a reddish brown pigment into the medium when cultured on an agar medium. Therefore, Aovp S 5 polypeptides of the functions are possible scan cleaning mutant strain deficient by utilizing this property.
スク リ ーニングは以下に記載の方法によ り行う。 五味らの方法 〔日 本醸造協会誌， 465 (1989) ) に基づいて、 ァスペルギルス · ォリ ゼの分生子に紫外線を照射することにより、 分生子のゲノム中に変異 を誘発する。 この分生子を寒天培地 （0. 2 %塩化アンモニゥム、 0. 1 % . 硫酸アンモニゥム、 0. 05 %塩化力.リ ゥム、 0. 05 %塩化ナトリ ウム、 0. 1 %リ ン酸ニ水素一力リ ウム、 0. 05 %硫酸マグネシゥム 7水和物、 0. 002 %硫酸鉄 7水和物、 2 %グルコース、 1. 5%寒天、 pH5. 5) 上にまき、 シ ヤーレ本体と蓋を、 密閉状態にならないようにパラフィルムで卷いて 固定し、 30°Cで、 5日以上、 好ましく は 7日以上培養する。 周辺部の培 地が赤褐色になるコロニーを Aovps5機能欠損変異株の候補株と して選 択することができる。 選択した候補株の 2次スクリーニングと して、 上記の候補株を培養した培地中の トリぺプチジルぺプチダーゼ活性を 測定し、 親株に比べて活性が上がっているものを Aovps 5機能欠損変異 株と して選択する。  Screening is performed by the method described below. Based on the method of Gomi et al. [Journal of the Japan Brewing Association, 465 (1989)], the conidia of Aspergillus oryzae are irradiated with ultraviolet rays to induce mutations in the conidia genome. The conidia were transferred to an agar medium (0.2% ammonium chloride, 0.1% ammonium sulfate, 0.05% chloride, lithium, 0.05% sodium chloride, 0.1% dihydrogen phosphate). 1% lithium, 0.05% magnesium sulfate heptahydrate, 0.002% iron sulfate heptahydrate, 2% glucose, 1.5% agar, pH 5.5) Is wrapped and fixed with parafilm so as not to be in a sealed state, and cultured at 30 ° C. for 5 days or more, preferably 7 days or more. A colony whose peripheral medium turns reddish brown can be selected as a candidate strain for Aovps5 function-deficient mutants. As a secondary screening of the selected candidate strains, tryptidyl peptidase activity in the medium in which the above candidate strains were cultured was measured, and those with increased activity compared to the parent strain were identified as Aovps 5 function-deficient mutants. And select.
また、 サッカロ ミセス ' セレピシェの VPS5遺伝子破壌株が親株に比 ベてコバルトやニッケルなどの重金属に対する耐性が向上しているこ とカ 幸艮告されている [ Proc. Nat l . Acad. Sc i . USA, 98, 9660 (2001) 〕 。 したがってァスペルギルス . ォリゼの Aovps 5遺伝子破壊株も、 重 金属に対する耐性を獲得していると予想されるため、 重金属に対する 耐性を指標に A 0 V p s 5機能欠損変異株のスク リーニングが可能である。 重金属の例と して、 クロム、 マンガン、 鉄、 コバルト、 ニッケル、 銅、 亜鉛、 モリブデン、 銀、 カ ドミウム、 スズ、 バリ ウム、 水銀、 鉛等が あげられる。 ' It has also been reported that the SPS strain of Saccharomyces cerevisiae has improved resistance to heavy metals such as cobalt and nickel compared to its parent strain [Proc. Natl. Acad. USA, 98, 9660 (2001)]. Therefore, the Aovps 5 gene-disrupted strain of Aspergillus oryzae is also expected to have acquired resistance to heavy metals. Therefore, it is possible to screen A0 V ps5 function-deficient mutants using the resistance to heavy metals as an index. Examples of heavy metals include chromium, manganese, iron, cobalt, nickel, copper, zinc, molybdenum, silver, cadmium, tin, barium, mercury, and lead. can give. '
これらの重金属を含まないか、 通常の培地に含まれる濃度でしか含 まない合成培地にこれらの重金属のいずれかを何段階か 適当な濃度 で添加した培地に、 親橡と Aov- p s 5遺伝子破壊株を培養し、 親株は生育 できないが Aovps 5遺伝子破壌株が生育できるような選択培地の重金属 の濃度を決定する。 紫外線を照射してゲノム中に変異を誘発した分生 子を、 決定した濃度の重金属を含む選択培地上で培養し、 生育してき たコロニーを AoVp s 5機能欠損変異株の候補株と して選択する。 選択し た候補株の 2次スク リーニングとして、 候補株を培養した培地中のト リぺプチジルぺプチダーゼ活性を測定し、 親株に比べて活性が上がつ ているものを Aovp s 5機能欠損変異株と して選択する。 A parent medium and Aov-ps5 gene were added to a medium in which these heavy metals were not contained or contained only at the concentration that is contained in a normal medium. Culture the disrupted strain and determine the concentration of heavy metals in the selection medium so that the parent strain cannot grow but the Aovps 5 gene-broken strain can grow. Conidia which ultraviolet was irradiated to induce mutations in the genome, cultured on selective media containing heavy metals determined concentration, the colonies grown in the candidate strains of Ao V ps 5 loss-of-function mutant select. As a secondary screening of the selected candidate strain, the activity of triptidyl peptidase in the medium in which the candidate strain was cultured was measured, and those with higher activity compared to the parent strain were identified as Aovps5 function-deficient mutants. Select as stock.
このよ うにして得られる Aovp s 5機能欠損変異株は,遺伝子操作を施し ていないため、 食品、 飲料などの製造に好適である。  The Aovps 5 function-deficient mutant obtained in this manner is suitable for production of foods, beverages, and the like because it has not been subjected to genetic manipulation.
( 2 ) 液胞酵素の製造方法 ' ' 以下に、 （ 1 ) に記載した方法で得られる液胞酵素を細胞外に分泌 する糸状菌を用いた液胞酵素の製造方法について述べる。  (2) Method for producing vacuolar enzyme '' Hereinafter, a method for producing a vacuolar enzyme using a filamentous fungus secreting extracellularly the vacuolar enzyme obtained by the method described in (1) will be described.
該糸状菌を培地に培養し、 液胞酵素を培地中に生産 · 蓄積させ、 該 培地から液胞酵素を採取することができる。  The filamentous fungus is cultured in a medium, the vacuolar enzyme is produced and accumulated in the medium, and the vacuolar enzyme can be collected from the medium.
該糸状菌を培養する方法は、 固体培養、 液体培養、 多孔性膜培養、 ゲル包括固定化培養等があげられる。 菌体の接種の方法は胞子を接種 することもできるが、 Aovp s 5遺伝子破壊株では胞子を形成しないため、 例えば菌糸を滅菌した生理食塩水などに分散させた後に接種すること ができる。  The method for culturing the filamentous fungus includes solid culture, liquid culture, porous membrane culture, gel entrapment immobilization culture, and the like. In the method of inoculating cells, spores can also be inoculated. However, since Aovps 5 gene-disrupted strains do not form spores, they can be inoculated after, for example, dispersing hyphae in sterile physiological saline or the like.
液体培地の場合、 炭素源としてグルコース、 澱粉、 デキス ト リ ン、 フラク トース、 スクロース等、 窒素源と して、 硝酸ナト リ ウム、 塩化 アンモニゥム、 硫酸アンモニゥム、 ポリペプトン、 酵母エキス、 脱脂 大豆タンパク質等、 ミネラル成分と してリ ン酸一ナト リ ウム、 リ ン酸 一カリ ウム、 硫酸マグネシウム、 硫酸鉄等を添加することができる。 培養は、振と う培養または深部通気攪拌培養などの好気的条件下で、 通常 15 ~ 50°C,、 好ましくは 25〜37°Cで通常 1〜7日間行う。 ' 固体培地の場合、培地原料と して水性媒体を湿潤させた小麦ふすま、 蒸米、 脱脂大豆タンパク質等があげられ、 水性媒体と して水、 無機塩' 類を含有する水溶液があげられる。 培養は温度を通常 15〜50°C、 好ま しくは 25〜37°Cに、 湿度は 80〜 100 %、 好ましくは 90〜： 100 %、 通常 3〜 10日間で行う。 In the case of a liquid medium, glucose, starch, dextrin, fructose, sucrose, etc. as a carbon source, sodium nitrate, ammonium chloride, ammonium sulfate, polypeptone, yeast extract, defatted soy protein, etc. as a nitrogen source As a mineral component, sodium phosphate, potassium phosphate, magnesium sulfate, iron sulfate and the like can be added. The cultivation is carried out under aerobic conditions such as shaking culture or deep aeration stirring culture, usually at 15 to 50 ° C, preferably 25 to 37 ° C, usually for 1 to 7 days. In the case of a solid medium, the raw material of the medium includes wheat bran, steamed rice, defatted soybean protein, etc. in which an aqueous medium is moistened, and the aqueous medium includes an aqueous solution containing water and inorganic salts. The cultivation is performed at a temperature of usually 15 to 50 ° C, preferably 25 to 37 ° C, and a humidity of 80 to 100%, preferably 90 to 100%, usually for 3 to 10 days.
. 多孔性膜培養またはゲル包括固定化培養の場合は、 例えば特開平 1 1 - 2 2 5 7 4 6に記載の方法に従い培養することができる。  In the case of a porous membrane culture or a gel entrapment immobilization culture, for example, the culture can be performed according to the method described in JP-A-11-225574.
液胞酵素を細胞外に分泌する能力を有する糸状菌を液体培地で培養 する楫合、 以下に記載の方法に従い、 液胞酵素を単離 * 精製すること ができる。  Kajii in which a filamentous fungus having the ability to secrete a vacuolar enzyme out of a cell is cultured in a liquid medium. The vacuolar enzyme can be isolated and purified according to the method described below.
培養終了後、 培養液から不溶性固形分を、 遠心分離、 膜ろ過等の方 法により分離除去し、 上清を取得する。 該上清から、 通常の酵素の単 離 ·精製法、 即ち、 溶媒抽出法、 硫安等による塩析法、 脱塩法、 有機 溶媒による沈殿法、 ジェチルアミ ノエチル （DEAE) —セファロース、 DIAION HPA-75 (三菱化成社製） 等のレジンを用いた陰イオン交換クロ マ トグラフィ一法、 S- Sepharose FF ( Pharmac ia社製） 等のレジンを用 いた陽イオン交換クロマ トグラフィー法、 プチルセファロース、 フエ 二ルセファロース等のレジンを用いた疎水' i生クロマ トグラフィー法、 分子篩を用いたゲルろ過法、 ァフィ二ティークロマ トグラフィー法、 クロマトフォーカシング法、 等電点電気泳動等の電気泳動法等の手法 を単独あるいは組み合わせて用い、 精製標品を得ることができる。  After completion of the culture, the insoluble solids are separated and removed from the culture by centrifugation, membrane filtration, etc., and the supernatant is obtained. From the supernatant, ordinary enzyme isolation / purification methods, that is, solvent extraction method, salting out method with ammonium sulfate, desalting method, precipitation method with organic solvent, getylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 Anion exchange chromatography using a resin such as (Mitsubishi Kasei Co., Ltd.), cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), butyl sepharose, Hydrophobic i chromatographic method using resin such as lusepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing Can be used alone or in combination to obtain a purified sample.
pRSET系ベクター (ィンビトロジェン社製) 、 pGEX系べクター (アマシ ャム · バイオサイエンス社製） 等のタグを、 液胞酵素につけて発現さ せた場合、 ニッケルレジン、 グノレタチオンセファロースなどの適当な 担体を用いてアブイ -ティ精製することもできる。 When tags such as pRSET-based vector (Invitrogen) and pGEX-based vector (Amersham Biosciences) are attached to vacuolar enzymes and expressed, appropriate tags such as nickel resin and gnoletathion sepharose can be used. Abity purification can also be performed using a carrier.
固体培地で培養する場合、 例えば糸状菌が増殖した固体培地を水系 緩衝液に分散した後、 固液分離して酵素抽出液を取得し、 液体培養に おいて上清から液胞酵素を単離 · 精製した方法と同様の方法により、 該酵素抽出液から液胞酵素を単離 · 精製することができる。  When culturing on a solid medium, for example, after dispersing the solid medium in which filamentous fungi have grown in an aqueous buffer, solid-liquid separation is performed to obtain an enzyme extract, and in liquid culture, vacuolar enzymes are isolated from the supernatant. · The vacuolar enzyme can be isolated and purified from the enzyme extract by the same method as the purified method.
多孔性膜培養またはゲル包括固定化培養の場合、 菌体が生育する膜 または菌体を包埋したゲルと接触させた培地から、 液体培地で培養し た場合と同様にして、 液胞酵素を単離 · 精製できる。 In the case of porous membrane culture or gel entrapment immobilization culture, the membrane on which the cells grow Alternatively, a vacuolar enzyme can be isolated and purified from a medium that has been brought into contact with a gel in which cells have been embedded, as in the case of culturing in a liquid medium.
4 . タンパク加水分解物の調製  4. Preparation of protein hydrolyzate
3 . ( 2 ) に記載した方法等に従って、 液体培地で糸状菌を培養す る場合、 タンパク質を含む原料を該培養物中に添加することにより、 タンパク加水分解物を製造することができる。 または、 タンパク質を 含む原料に、 3 . ( 2 ) に記載した方法で得られる液胞酵素を添加し て混合し、 通常 20°C〜60°C、 好ましくは 30°C〜50°Cにて、 24〜264時間 、 好ましくは 48〜240時間反応させることにより、 タンパク加水分解物 を製造することができる。 反応時の pHは、 本発明の液胞酵素および分 泌酵素が作用できる pHであればよいが、 好ましくは pH3〜8、 より好ま じくは pH5〜8に調整する。  3. When a filamentous fungus is cultured in a liquid medium according to the method described in (2) or the like, a protein hydrolyzate can be produced by adding a protein-containing raw material to the culture. Alternatively, the vacuolar enzyme obtained by the method described in 3. (2) is added to the protein-containing raw material and mixed, and the mixture is usually treated at 20 ° C to 60 ° C, preferably 30 ° C to 50 ° C. By reacting for 24 to 264 hours, preferably for 48 to 240 hours, a protein hydrolyzate can be produced. The pH during the reaction may be any pH at which the vacuolar enzyme and secretory enzyme of the present invention can act, but is preferably adjusted to pH 3 to 8, and more preferably to pH 5 to 8.
この製造方法で用いる液胞酵素と しては、 精製した液胞酵素を用い ることもできるし、 液胞酵素を精製せずに、 上記 3 . に記載した液胞 酵素を細胞外に分泌する糸状菌を培地に培養して得られる、 液胞酵素 を含む培養物または培養処理物を用いることもできる。 培養処理物と しては、 培養物からろ過や遠心分離等により菌体を除去して得られる 溶液、 該溶液の濃縮物、 乾燥物等があげられる。  As a vacuolar enzyme used in this production method, a purified vacuolar enzyme can be used, or the vacuolar enzyme described in 3. above is secreted extracellularly without purifying the vacuolar enzyme. A culture containing a vacuolar enzyme or a culture-treated product obtained by culturing a filamentous fungus in a medium can also be used. Examples of the culture product include a solution obtained by removing cells from the culture by filtration, centrifugation, or the like, a concentrate of the solution, a dried product, and the like.
糸状菌は、 醸造工業で用いられるものであれば、 いかなるものでも よいが、 例えば、 ァスペルギルス · ォリゼ、 ァスペルギルス · ソーャ、 ァスペルギルス . -ガー、 ァスペルギルス . 力ヮチ、 ァスペルギルス • ァヮモリをあげることができる。  The filamentous fungus may be any as long as it is used in the brewing industry, and examples include Aspergillus oryzae, Aspergillus soja, Aspergillus.
タンパク加水分解物の製造方法に用いる原料に含まれるタンパク質 は、 特に限定されないが、 グルタ ミ ン酸含量の高いものが好ましい。 例えば、 .小麦ダルテン、 コーンミールグルテン、 脱脂大豆、 分離大豆 タンパク質等があげられ、 小麦ダルテン、 コーンミールダルテン等が 好ましく用いられる。  The protein contained in the raw material used in the method for producing a protein hydrolyzate is not particularly limited, but a protein having a high glutamic acid content is preferable. Examples include wheat dart, corn meal gluten, defatted soy, isolated soy protein, and the like. Wheat dart, corn meal darten, and the like are preferably used.
また、 タンパク質を含む原料は特に限定されないが、 タンパク質含 量が高いものが好ましい。  The raw material containing the protein is not particularly limited, but those having a high protein content are preferable.
反応終了後、 固液分離により未反応の原料タンパク質、 菌体などを 除去後、 必要に応じて濃縮、 乾燥することによりタンパク加水分解物 を得ることができる。 After the reaction is completed, unreacted raw material proteins, bacterial cells, etc. are separated by solid-liquid separation. After removal, the protein hydrolyzate can be obtained by concentrating and drying as necessary.
得られたタンパク加水分解物はそのままでも調味料として用いるこ ともできるが、 アミノ酸、 核酸、 エキス類、 酸味料、 甘味料等と混合 し、 味、 風味を調整することにより、 うまみが強く風味の良好な調味 料をつく ることができる。  The obtained protein hydrolyzate can be used as it is as a seasoning, but it is mixed with amino acids, nucleic acids, extracts, acidulants, sweeteners, etc. to adjust the taste and flavor to give a strong umami. Good seasoning can be made.
以下に、 実施例により本発明をさらに具体的に説明するが、 本発明 はそれに限定されるものではない。 図面の簡単な説明  Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. BRIEF DESCRIPTION OF THE FIGURES
' 第 1図 サッカロミセス 'セレピシェの VPS5遺伝子がコ" ドするアミ ノ酸配列とノィロスボラ · クラッサの VPS5相同遺伝子がコードするァ ミノ酸配列の比較である。 上段、 下段の配列はそれぞれサッカロミセ ス · セレビシェ、 ノイロスポラ · クラッサのアミノ酸配列を示してい る。 また、 両配列で一致するアミノ酸を中段に示す。 数字は各ポリべ プチド N末端からのァミノ酸残基の位置を示す。 下線は縮重プライマ 一に対応するアミ ノ酸配列を示す。 '  Figure 1. Comparison of the amino acid sequence encoded by the VPS5 gene of Saccharomyces cerevisiae and the amino acid sequence encoded by the VPS5 homologous gene of Neurosbola classa. The upper and lower sequences are Saccharomyces cerevisiae, respectively. The amino acid sequence of Neurospora crassa is shown in the middle row, and the amino acids corresponding to both sequences are shown in the middle row The numbers indicate the positions of amino acid residues from the N-terminus of each polypeptide. The amino acid sequence corresponding to is shown.
第 2図 Aovp s 5遺伝子の破壊に用いる形質転換用 DNAの構造を示す。 第 3図 CT61 18株、 CT9103株おょぴ CT96株のゲノム中の破壌 Aovp s 5遺 伝子の PCRによる検出を示す。 レーン 1〜3はそれぞれ、 Aovp s 5変異株で ある CT61 18株、 CT9103株および CT96株のゲノム匪を、 レーン 4は Aovp s 5 遺伝子破壌用プラスミ ド pDV51を、 レーン 5は野生株 （RIB40株） のゲノ ム DNAをテンプレートと したときの PCR産物を示す。  FIG. 2 shows the structure of a transforming DNA used for disruption of the Aovps5 gene. Fig. 3 shows PCR detection of the shattered Aovps5 gene in the genomes of CT6118 strain, CT9103 strain and CT96 strain. Lanes 1 to 3 show the genomes of Aovps5 mutant strains CT6118, CT9103 and CT96, respectively. Lane 4 shows plasmid pDV51 for Aovps5 gene disruption, and lane 5 shows wild type (RIB40 This shows the PCR product when genomic DNA of the strain was used as a template.
第 4図 Aovp s 5遺伝子破壊株の培養液を用いて製造したタンパク加 水分解物の窒素可溶化率 （ろ液の総窒素 ÷原料に含まれる総窒素 X 1 0 0 ) を示す。 左から親株 （NS4株） 、 Aovps 5遺伝子破壊株 (それぞれ 左から CT61 18株、 CT9103株、 CT96株) の各培養液を用いて製造したタ ンパク加水分解物の、 湿菌体重量 ( g) あたりの窒素可溶化率 ( % ) を 示す。  FIG. 4 shows the nitrogen solubilization rate (total nitrogen in the filtrate 総 total nitrogen in the raw material × 100) of the hydrolyzed protein produced using the culture solution of the Aovps5 gene-disrupted strain. Wet cell weight (g) of protein hydrolyzate produced from each culture of parent strain (NS4 strain) and Aovps 5 gene-disrupted strain (CT61 18 strain, CT9103 strain, CT96 strain from left, respectively) Shows the nitrogen solubilization rate (%) per unit.
第 5図 Aovp s 5遺伝子破壌株の培養液を用いて製造したタンパク加 水分解物の遊離アミノ酸の濃度 （mmol/1) を示す。 左から親垛 （NS4株 ) 、 Aovps5遺伝子破壌株 （それぞれ左から CT6118株、 CT9103株、 CT96 株） の各培養液を用いて製造したタンパク加水分解物の、 湿菌体重量Fig. 5 Protein protein produced using a culture solution of Aovps 5 gene disrupted strain Shows the concentration (mmol / 1) of free amino acids in the hydrolyzate. Wet cell weight of protein hydrolyzate produced from each culture of parent strain (NS4 strain) and Aovps5 gene disrupted strain (CT6118, CT9103, and CT96 strains from left, respectively)
(g) あたりの遊離アミ ノ酸の濃度 （mmol/1) を示す。 発明を実施するための最良の形態 (g) shows the concentration of free amino acid (mmol / 1). BEST MODE FOR CARRYING OUT THE INVENTION
実施例 1 ァスペルギルス 'オリゼの Aovps5のゲノム DNAのクローニン グ Example 1 Cloning of genomic DNA of Aspergillus oryzae Aovps5
( 1 ) ゲノム DNAの調製  (1) Preparation of genomic DNA
ァスペルギルス · ォリゼ RIB 40株 （ATCC番号 ： 42144) の菌体から、 五味らの方法 〔 Gen. Appl. Microbiol. , 35, 225 (1989)〕 に従い、 以下に示すようにゲノム DNAを調製した。  Genomic DNA was prepared from the cells of Aspergillus oryzae RIB 40 strain (ATCC No. 42144) according to the method of Gomi et al. [Gen. Appl. Microbiol., 35, 225 (1989)] as shown below.
150mlの DPY培地 （2%デキス ト リ ン、 2%ポリペプトン、 0, 5%酵母ェ キス、 0.5%リ ン酸一カリ ウム、 0.05%硫酸マグネシウム 7水和物） に ァスペルギルス · ォリゼ RIB 40株を植菌し、 30°Cで 20時間振と う培養 後、 ブフナー漏斗で集菌し、 液体窒素存在下で乳鉢で粉砕した。 これ を 10mlのプロテアーゼ溶液 （50mmol/l EDTA(pH8.0) , 0.5% SDS, Aspergillus oryzae RIB 40 strain in 150 ml of DPY medium (2% dextrin, 2% polypeptone, 0.5% yeast yeast, 0.5% potassium phosphate, 0.05% magnesium sulfate heptahydrate) After inoculating the cells and culturing with shaking at 30 ° C for 20 hours, the cells were collected with a Buchner funnel and ground in a mortar in the presence of liquid nitrogen. This was added to a 10 ml protease solution (50 mmol / l EDTA (pH 8.0), 0.5% SDS,
0. lmg/ml プロティナーゼ K) に懸濁し、 50°Cで 4時間インキュベート した。 この溶液を、 フエノール処理 2回、 フエノール/クロ口ホルム処 理 2回、 クロ口ホルム処理 1回を順次行った。 なお、 これら有機溶媒 による処理は、 溶液と有機溶媒を穏やかに混合し、 上清を分取するこ とにより行った。 次に溶液に 1/10容量の 3 mol/1酢酸ナトリ ウム （PH5.2The cells were suspended in 0.1 mg / ml proteinase K) and incubated at 50 ° C for 4 hours. This solution was sequentially subjected to phenol treatment twice, phenol / cloth form treatment twice, and closform form treatment once. The treatment with these organic solvents was carried out by gently mixing the solution and the organic solvent and collecting the supernatant. Then 1/10 volume of a solution 3 mol / 1 sodium acetate (P H5.2
) 溶液と 2.5倍量の 99.5%エタノールを加え、 一 20°Cで 20分間.静置し、 遠心分離により沈殿を回収した。 沈殿は 70%エタノールでリ ンスした 後、 5mlの TE緩衝液に溶解した。 この溶液に 10 1の 10mg/ml リ ポヌク レアーゼ Aを添加し 37°C、 30分間インキュベー ト後、 フエノール/クロ 口ホルム処理を行った。 回収した上清に 1/10容量の 3 mol/1酢酸ナトリ ゥム (pH5.2) 溶液と 2.5倍量の 99.5%エタノールを加え、 — 20°Cで 20分 間静置し、 遠心分離により沈殿を回収した。 沈殿は 70%エタノールで リ ンスした後、 1mlの TE緩衝液に溶解し、 ァスペルギルス ' ォリゼのゲ ノム DNA溶液と した。 ) The solution and 2.5 volumes of 99.5% ethanol were added, the mixture was allowed to stand at 20 ° C for 20 minutes, and the precipitate was collected by centrifugation. The precipitate was rinsed with 70% ethanol and dissolved in 5 ml of TE buffer. To this solution was added 10 1 of 10 mg / ml liponuclease A, and the mixture was incubated at 37 ° C for 30 minutes, followed by phenol / cloth form treatment. Add 1/10 volume of 3 mol / 1 sodium acetate (pH5.2) solution and 2.5 volumes of 99.5% ethanol to the collected supernatant, and leave them at −20 ° C for 20 minutes and centrifuge. The precipitate was collected. The precipitate was rinsed with 70% ethanol, dissolved in 1 ml of TE buffer, and mixed with Aspergillus oryzae. A nome DNA solution was used.
( 2 ) 糸状菌の VPS5相同遺伝子の検索  (2) Search for VPS5 homologous genes in filamentous fungi
まず、 サッカロミセス ' セレビシェの VPS5遺伝子の塩基配列を公共 データベース GenBankから取得した （登録番号： U73512) 。 取得したサ ッカロミセス ' セレピシェの VPS5遺伝子のコード領域の配列 (配列番 号 4 ) を問い合わせ配列 （クエリー） と して各種糸状菌データベース を検索した。 その結果、 ホワイ トヘッ ド生物医学研究所ゲノ ム研究セ ンターのウェブサイ 1、内のノイロスポラ · クラッサのゲノムデータべ ース ( ht tp： // www-genome, wi . mi t . edu/ annotat ι οη/f ungi /neurospora/ ) から、 サッカロミセス ' セレピシェの VPS5遺伝子と高い相同性 （ E-value=3E-54) を有する遺伝子配列 （配列番号： NCU04137. 1 ) を見出 した。 該塩基配列を配列番号 5に記載した。 サッカロ ミセス ' セレビ シェ VPS5遺伝子と該遺伝子と相同性の高いノイロスポラ · クラッサの 遺伝子がコードするアミノ酸配列との比較を第 1図に示した。 First, the nucleotide sequence of the Saccharomyces cerevisiae VPS5 gene was obtained from the public database GenBank (registration number: U73512). Various filamentous fungi databases were searched using the obtained Saccharomyces cerevisiae VPS5 gene coding region sequence (SEQ ID NO: 4) as a query sequence. As a result, the genomic database of Neurospora crassa (http: // www-genome, // www-genome, wi.mit.edu/annotat ιoη) / from f ungi / neurospora /), Saccharomyces' Serepishe of VPS 5 genes highly homologous (E-value = 3E-54 ) gene sequence (SEQ ID NO:. NCU04137 1) it was Heading a. The nucleotide sequence is shown in SEQ ID NO: 5. FIG. 1 shows a comparison between the Saccharomyces cerevisiae VPS5 gene and the amino acid sequence encoded by the gene of Neurospora crassa having high homology to the gene.
( 3 ) プローブの作製  (3) Preparation of probe
第 1図から比較的保存性が高く、 機能的に保存されている可能性が 高いと予想される領域から、 縮重プライマ一 V5P- Fおよび V5P- Rを設計 した。 該プライマーの塩基配列をそれぞれ配列番号 6および配列番号 7に示す。  As shown in Fig. 1, the degenerated primers V5P-F and V5P-R were designed from the regions that are relatively highly conserved and are likely to be functionally conserved. The nucleotide sequences of the primers are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.
配列番号 6および 7の塩基配列は、 ノィロスボラ · クラッサの VPS5 相同遺伝子がコードするァミノ酸配列の 181〜189番目の配列 （Val Gly Asp Pro Hi s Lys Val Gly Asp) 、 267〜 274番目の配歹 U ( Pro Pro Glu Lys Gin Al a Val Gly) にそれぞれ対応する。  The nucleotide sequences of SEQ ID NOS: 6 and 7 are the amino acid sequence of amino acids 181 to 189 (Val Gly Asp Pro His Lys Val Gly Asp) and the amino acids 267 to 274 of the amino acid sequence encoded by the VPS5 homologous gene of Neurosbora classa. U (Pro Pro Glu Lys Gin Al a Val Gly) respectively.
PCRは上記ォリ ゴヌクレオチドをプライマー、 ァスペルギルス ·オリ ゼのゲノム DNAをテンプレートと して、 GeneAmp PCR Syst em 2400 (ノヽ0 一キン .エルマ一社製) により以下の条件で行った。 まず 94°Cで 5分間 加熱しテンプレー トの DNAを変性させた後、 1サイクル 94°Cで 30秒、 58 °Cで 30秒、 72。Cで 30秒の反応を 30サイクル行った。 反応液を 2 %ァガロ ースゲルで電気泳動した結果、 約 250bpの DNA断片を検出した。 PCR is the above-mentioned O Li Gore-nucleotide primer, Asuperugirusu-cage zero of genomic DNA template, was carried out under the following conditions by the GeneAmp PCR Syst em 2400 (Nono 0 manufactured by one Kin. Elma one company). After heating at 94 ° C for 5 minutes to denature the template DNA, one cycle was performed at 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 cycles. A 30-second reaction at C was performed for 30 cycles. As a result of electrophoresis of the reaction solution on a 2% agarose gel, a DNA fragment of about 250 bp was detected.
( 4 ) プローブの塩基配列決定 ジゴキシゲニン （以下、 DIGと略す） - dUTP (ロシュ · ダイァグノス ティ ック社製） 存在下で PCRを行う以外は、' （ 3 ) に記載の条件で取得 した PCR産物を切り 出し、 ジーンク リーン ' キッ ト (GENECLEAN Kit、 Q パイォジーン社製） によ り精製した。 精製した PCR産物は TAク口一ニン' グによ り、 pT7Blue(R) (ノバジヱン社製） の Tクローニング部位に導入 し、 プラスミ ド pV5PRを取得した。 DSQ2000L DNAシークェンサ一 (島津 製作所社製） によ り、 得られたプラスミ ドの挿入断片の塩基配列を決 定した。 その結果を配列番号 8に示す。 (4) Probe sequencing Digoxigenin (hereinafter abbreviated as DIG)-Except for performing PCR in the presence of dUTP (Roche Diagnostics), the PCR product obtained under the conditions described in (3) is cut out, and the gene clean (GENECLEAN Kit, manufactured by Q Pyrogen). The purified PCR product was introduced into the T cloning site of pT7Blue (R) (manufactured by Novadion) by TAguchi Ichining to obtain plasmid pV5PR. The base sequence of the obtained plasmid insert was determined by DSQ2000L DNA Sequencer (manufactured by Shimadzu Corporation). The result is shown in SEQ ID NO: 8.
( 5 ) ァスペルギルス · ォリゼのゲノム DNAライブラ リーのスク リ ー二 ング  (5) Screening of genomic DNA library of Aspergillus oryzae
( 1 ) で取得したァスペルギルス · ォリゼのゲノム DNAを BamHIで切 . 断し、ラムダファージべクター λ DASH II (ス トラタジーン社製）の BamHI 部位に挿入した。 これをイ ンビトロパッケージングし、 ァスペルギル ス · ォリゼのゲノム DNAライブラ リーと した。 このライブラリ一を用い The genomic DNA of Aspergillus oryzae obtained in (1) was cut with BamHI and inserted into the BamHI site of lambda phage vector λ DASH II (Stratagene). This was packaged in vitro and used as a genomic DNA library for Aspergillus oryzae. Using this library
、 大腸菌 P2392を宿主と し常法に従いプラークを形成させた。 プラーク は Hybond- N+ (アマシャム · バイォサイェンシズ社製） にブロッテイ ン グし、 変性溶液 （1.5 mol/1塩化ナト リ ウム、 0.5 mol/1水酸化ナ ト リ ゥム） 上で 5分間静置後、 中和液 （1.5 mol/1塩化ナ ト リ ウム、 0.5 mol/1 ト リス—塩酸（pH8.0)) 上で静置し、 2XSSC (0.3 mol/1塩化ナト リ ウ ム、 70 mmol/1クェン酸ナト リ ウム） に 5分'間浸し、 嵐乾した。 次にメ . ンブレンを、 緩衝液 （5XSSC、 50%ホルムアミ ド、 0.1%N-ラウロイル サルコシン、 0.2%SDS, 1%プロ ッキング · リージェン ト (ロシュ社製A plaque was formed using E. coli P2392 as a host according to a conventional method. The plaques are blotted onto Hybond-N + (Amersham Biosciences) and denatured (1.5 mol / 1 sodium chloride, 0.5 mol / 1 sodium hydroxide) for 5 min. After standing, let stand on a neutralizing solution (1.5 mol / 1 sodium chloride, 0.5 mol / 1 tris-hydrochloric acid (pH 8.0)), and add 2XSSC (0.3 mol / 1 sodium chloride, (70 mmol / 1 sodium citrate) for 5 minutes and storm-dried. Next, the membrane was added to a buffer solution (5XSSC, 50% formamide, 0.1% N-lauroyl sarcosine, 0.2% SDS, 1% blocking reagent (Roche).
) ) 中で 42°C、 1時間ィ ,ンキュペー ト した後、 （4 ) で作製した DIG標 識プロープを 0.1%(ν/ν)添加した上記緩衝液中で、 メ ンブレンを 42°C、)) At 42 ° C for 1 hour, and then in a buffer containing 0.1% (ν / ν) of the DIG labeling probe prepared in (4), the membrane was heated at 42 ° C.
6時間インキュベー ト した。 上記メ ンブレンを、 2XSS (：、 0.1%SDSで 5 分間、 0.1XSSC、 0.1%SDSで 15分間、 順次洗浄した後、 マレイ ン酸バ ッファー (0.1 mol/1 マレイン酸、 0.15 mol/1塩化ナ ト リ ウム） でリ ンスした。 発色は、 DIGハイプライム DNAラベリ ング /検出キッ ト I (DIGIncubated for 6 hours. After washing the above membrane sequentially with 2XSS (: 0.1% SDS for 5 minutes, 0.1XSSC, 0.1% SDS for 15 minutes), maleic acid buffer (0.1 mol / 1 maleic acid, 0.15 mol / 1 sodium chloride) The color was developed using the DIG high-prime DNA labeling / detection kit I (DIG
High Prime DNA Labeling&Detection Starter Kitl, ロシュ社製High Prime DNA Labeling & Detection Starter Kitl, Roche
) を用いて、 添付の説明書に従い行った。 その結果、 約 5000個のプラ ークからポジティブクローンを 1個取得した。 ) Was performed according to the attached instructions. As a result, about 5000 One positive clone was obtained from the workout.
( 6 ) ァスペルギルス ' オリゼの Aovps5遺伝子のゲノム DNAの塩基配列 決定  (6) Sequence determination of genomic DNA of Aovps5 gene of Aspergillus oryzae
上記 （ 5 ) で得られたポジテイブク ローンから ファージ DNAを常法 に従い精製し、 Sailで切断後、約 3.2kbpの Sail断片を pBluescript SK II (+) (ス トラタジーン社製) の Sail部位に挿入し、 プラスミ ド pVPS5Sを 取得した。 DSQ2000L DNAシークェンサ一 (島津製作所社製) によ り、 ァスペルギルス · ォリゼの Aovps5遺伝子の塩基配列を決定した。 結果 を配列番号 2に示す。  The phage DNA is purified from the positive clone obtained in the above (5) according to a conventional method, cut with Sail, and a 3.2 kbp Sail fragment is inserted into the Sail site of pBluescript SK II (+) (Stratagene). And plasmid pVPS5S. The base sequence of the Aovps5 gene of Aspergillus oryzae was determined using DSQ2000L DNA Sequencer (manufactured by Shimadzu Corporation). The results are shown in SEQ ID NO: 2.
実施例 2 ァスペルギルス ' ォリゼの Aovps5遺伝子の cDNAのクロー二 ング Example 2 Cloning of cDNA of Aovps5 gene of Aspergillus oryzae
( 1 ) ァスペルギルス ' オリゼ RIB 40株の cDNAの調製  (1) Preparation of cDNA of Aspergillus oryzae RIB 40 strain
ァスペルギルス . 才リゼ RIB 40株を DPY培地 60ml (こ接種し 300mlのノ ッフ/レ付きの三角フラスコで、 30。C、 2日間、 150rpmで振と う培養した 。 培養物をろ過し得られた湿菌体 l g を、 液体窒素存在下で、 乳鉢で微 細な粉末と した。  Aspergillus strain RIB 40 strain was inoculated with 60 ml of DPY medium (inoculated and shake-cultured at 300 rpm for 2 days at 150 rpm in a 300 ml Erlenmeyer flask with a notch / resp.). The wet cells lg were turned into a fine powder in a mortar in the presence of liquid nitrogen.
該菌体破碎物から、アールェヌイージー 'ミディ 'キッ ト（RNeasy Midi Kit, キアゲン社製） を用いて全 RNAを取得した。  Total RNA was obtained from the crushed cells using an RNeasy Midi Kit (RNeasy Midi Kit, manufactured by Qiagen).
取得した全 RNAからオリ ゴテックス ' dT30スーパー · mRNA精製キッ ト (01igotex™-dT30<Super> mRNA Purification Kit, 宝酒造社製） を用 い、 添付の説明書に従い、 0.6μ g/mlの mRNA溶液を 100μ 1取得した。 こ の溶液に 10μ 1の 3 mol/1酢酸ナト リ ウム溶液 （pH5.2) と 250 1の99.5 %エタノールを添加し、 激しく攪拌後、 一 20°Cで 2時間静置した。 12000 rpmで 20分間遠心分離後、 沈殿を 200 1の 70%エタノールで洗った後、 6 μ 1のジェチルピ口カーボネー 1、処理水に溶解した。  Using an Origotex dT30 Super mRNA purification kit (01igotex ™ -dT30 <Super> mRNA Purification Kit, manufactured by Takara Shuzo Co., Ltd.), purify a 0.6 μg / ml mRNA solution from the obtained total RNA according to the attached instructions. 100 μl was obtained. To this solution, 10 μl of a 3 mol / 1 sodium acetate solution (pH 5.2) and 2501 of 99.5% ethanol were added, and after vigorous stirring, the mixture was allowed to stand at 120 ° C. for 2 hours. After centrifugation at 12000 rpm for 20 minutes, the precipitate was washed with 200 1 of 70% ethanol, and then dissolved in 6 μl of getyl-piper-carbonate 1 and treated water.
回収した mRNAは、 ゲー ト ウェイ技術を用いた cDNA合成およびク ロー ユング用ス—パースク リ プ 1、 ' プラス ミ ド · システム (SUPERSCRIPT The recovered mRNA is used for cDNA synthesis using gateway technology and Superscript 1 for the Crowjung, 'plasmid system (SUPERSCRIPT).
Plasmid System with GATEWAY™ Technology for cDNA Synthesis andPlasmid System with GATEWAY ™ Technology for cDNA Synthesis and
Cloning, インビトロジェン社製） を用いて第 1鎖 cDNAおよぴ第 2鎖 cDNAの合成を行ない PCRのテンプレー トに供した。 ( 2 ) ァスペルギルス ·ォリゼの Aovps5遺伝子の, cDNAの PCRによる取得 テンプレート配列番号 2に示す塩基配列から設計したそれぞれ配列 番号 9および配列番号 10に示す塩基配列からなるオリ ゴヌクレオチドCloning, Invitrogen) was used to synthesize first-strand cDNA and second-strand cDNA and used for PCR templates. (2) Acquisition of Aspergillus oryzae Aovps5 gene by cDNA PCR Oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 designed from the nucleotide sequence shown in SEQ ID NO: 2 of the template
(プライマ一 VPS5Fおよび VPS5R) を合成した。 PCRは、 上記のテンプレ 一卜 cDNA、 プライマーを用いて、 Geneamp PCR system 2400 (パ一キン エルマ一社製) により行った。 反応は、 94°Cで 5分間加熱しテンプレー トの DNAを変性させた後、 94°Cで 30秒、 56°Cで 30秒、 72°Cで 2分の反応 を 3:0サイクルの条件下で行った。 (Primers VPS5F and VPS5R) were synthesized. PCR was performed by Geneamp PCR system 2400 (manufactured by Parkin Elma) using the template cDNA and primers described above. The reaction is performed by heating at 94 ° C for 5 minutes to denature the DNA of the template, and then the reaction is performed at 94 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 2 minutes in a 3: 0 cycle. Went under.
PCR産物を 0.8%ァガロースゲルで分離した結果、 約 1.5kbpの DNA断片 を検出した。 これを切り出し、 ジーンク リーン 'キッ ト (GENECLEAN Kit 、 Qバイオジーン社製） により精製した。.  As a result of separating the PCR product on a 0.8% agarose gel, a DNA fragment of about 1.5 kbp was detected. This was cut out and purified using Gene Clean 'Kit (GENECLEAN Kit, manufactured by Q Biogene). .
( 3 ) ァスペルギルス · ォリゼの Aovps5遺伝子の cDNAの塩基配列決定 精製した DNA断片は TAクローニングにより、 pT7Blue (R) (ノバジェン 社製） の Tクローニング部位に導入し、 プラスミ ド pVPS5を取得した。 DSQ2000L DNAsequencer (島津製作所社製） により、 ァスペルギルス . ォリゼの Aovps5遺伝子の cDNAの塩基配列を決定した。 結果を配列番号 3に示す。  (3) Determination of nucleotide sequence of cDNA of Aovps5 gene of Aspergillus oryzae The purified DNA fragment was introduced into the T cloning site of pT7Blue (R) (manufactured by Novagen) by TA cloning to obtain plasmid pVPS5. The cDNA sequence of the Aovps5 gene of Aspergillus oryzae was determined using DSQ2000L DNAsequencer (manufactured by Shimadzu Corporation). The result is shown in SEQ ID NO: 3.
実施例 3 AovPs5遺伝子破壊株の作製 Example 3 Preparation of Aov P s5 Gene-Disrupted Strain
本発明のポリペプチドのコード領域中にマーカー遺伝子を挿入して Aovps5遺伝子本来の機能を破壌した Aovps5遺伝子 （以下、 破壌 Aovps5 遺伝子ともいう） を用い、 相同組換えにより、 ァスペルギルス · オリ ゼのゲノ ム上の Aovps5遺伝子が破壌された、 Aovps5遺伝子破壊株を作 製した 以下に方法を記す。  A homologous recombination of Aspergillus oryzae was performed by homologous recombination using an Aovps5 gene (hereinafter, also referred to as a “broken Aovps5 gene”) in which a marker gene was inserted into the coding region of the polypeptide of the present invention to break the original function of the Aovps5 gene. An Aovps5 gene disrupted strain in which the Aovps5 gene on the genome has been broken has been prepared. The method is described below.
プラスミ ド pUNA [Biosci. Biotechnol. Biochem, 61_, 1367 (1997) 〕 のァスペルギルス · 二ドランスの sC遺伝子を含む Kpnl/Pstl断片を 切り出し、 pBluescript II SK (+) (ス トラタジーン社製） の Kpnl/ Pstlサイ トに挿入し、 プラスミ ド pIADを取得した。 sC遺伝子を含む、 pIADの BamHl/BamHI断片を、 pVPS5Sの Aovps5ゲノム遺伝子のコ一ド領 域内に存在する Bglllサイ トに揷入し、破壌 Aovps5遺伝子を有するプラ スミ ド pDV51を作製した。 形質転換用 DNAは、 pDV51を Sailで切断後、 0.8%ァガロースゲルで分 離し、 3.5kbpの断片をジーンクリーン ' キッ トにより精製した。 形質 転換用 DNAの構造を第 2図に示す。 A Kpnl / Pstl fragment containing the sC gene of Aspergillus nidulans of plasmid pUNA [Biosci. Biotechnol. Biochem, 61_, 1367 (1997)] was cut out, and Kpnl / Pstl of pBluescript II SK (+) (Stratagene) was cut out. Inserted into the site to obtain plasmid pIAD. The BamHl / BamHI fragment of pIAD containing the sC gene was inserted into a Bglll site present in the coding region of the Aovps5 genomic gene of pVPS5S to prepare a plasmid pDV51 having the broken Aovps5 gene. The DNA for transformation was obtained by cleaving pDV51 with Sail, separating on a 0.8% agarose gel, and purifying a 3.5 kbp fragment by GeneClean 'kit. Fig. 2 shows the structure of the transforming DNA.
sC遺伝子欠損株であるァスペルギルス 。ォリゼ NS4株 （独立行政法人 酒類総合研究所 RIB No. NS4) を 150mlの DPY培地に植菌し、 30°Cで 20 時間振と う培養した後、 ろ過により菌体を回収し滅菌水で洗浄した。 回収した菌体を 10mlの酵素反応液 〔0.6 mol八硫酸アンモニゥム、 50 mmol/1マレイン酸緩衝液 （pH5, 5) 、 1%ャタラーゼ （宝酒造社製） 〕 に懸濁し 30。Cで 3時間ゆるやかに振と う した。 反応液はミラク ロス 〔力 ルビオケム （CALBI0CHEM) 社製〕 を用いてろ過し、 プロ トプラス トを 回収した。 プロ トプラス ト溶液に等量の緩衝液 A 〔1.2 mol/1ソルビト ール、 50 mmol/1塩化カルシゥム、■ 35 mmol/1塩化ナトリ ウム、 10 mmol/1 ト リスー塩酸 （pH7. 5) 〕 を加え、 沈殿を緩やかに分散した後、 2000rpm 、 8分間遠心分離し沈殿を回収した。 上記洗浄工程を 2回行なった後、 1X107〜1X108個/ mlになるように上記緩衝液を加え懸濁し、 プロ トプ ラス ト懸濁液とした。 ， Aspergillus, an sC gene-deficient strain. Oryzae NS4 strain (National Institute of Liquor Research, RIB No. NS4) was inoculated into 150 ml of DPY medium, shake-cultured at 30 ° C for 20 hours, and the cells were collected by filtration and washed with sterile water. did. The collected cells were suspended in 10 ml of an enzyme reaction solution [0.6 mol ammonium sulphate, 50 mmol / 1 maleate buffer (pH 5.5), 1% catalase (Takara Shuzo)]. Shake gently for 3 hours at C. The reaction solution was filtered using Miracloth (manufactured by Calbiochem) to collect protoplasts. An equal volume of buffer A (1.2 mol / 1 sorbitol, 50 mmol / 1 calcium chloride, ■ 35 mmol / 1 sodium chloride, 10 mmol / 1 tris-hydrochloride (pH 7.5)) was added to the protoplast solution. In addition, the precipitate was gently dispersed and centrifuged at 2000 rpm for 8 minutes to collect the precipitate. After the above washing step was performed twice, the above buffer was added and suspended at a concentration of 1 × 10 7 to 1 × 10 8 cells / ml to prepare a protoplast suspension. ,
200 At 1のプロ小プラス ト懸濁液と 5 gの形質転換用 DNAを混合し、氷 上で 30分間静置した。 次に、 緩衝液 B 〔60%ポリエチレングリ コール 4000、 50 mmol/1塩化カルシウム、 10 mmol/1ト リスー塩酸 （PH7.5) 〕 を 250 1加え穏やかに混合した後、 緩衝液 Bを 250 μ 1加え穏かに混合 し、 さらに緩衝液 Βを 850 1加え穏やかに混合した。 これを 20分間、 室温で静置して DNAを菌体内に導入した。 The 200 At 1 pro-plast suspension was mixed with 5 g of the transforming DNA and allowed to stand on ice for 30 minutes. Next, buffer B [60% polyethylene glycol 4000, 50 mmol / 1 calcium chloride, 10 mmol / 1 bets Risu hydrochloric acid (P H7.5)] were mixed 250 1 added gently, the buffer B 250 μ 1 was added and mixed gently, and 850 1 of buffer 加 え was further added and mixed gently. This was allowed to stand at room temperature for 20 minutes to introduce DNA into the cells.
次に 10mlの緩衝液 Aを加え穏やかに混合後、 2000rpmで 8分間遠心分 離し、 プロ トプラス ト懸濁液を回収した。 プ トプラス トに 500 μ 1の 緩衝液 Βを加え穏やかに混合後、 あらかじめ 45°Cに保温しておいた 5ml の トップアガー (1.2 mol/1ソルビトール、 0.8%ァガロース、 0.2%塩 化アンモニゥム、 0.1%硫酸アンモニゥム、 0.05%塩化力リ ウム、 0.05 %塩化ナトリ ウム、 0.1%リ ン酸ニ水素一力リ ウム、 0.05%硫酸マグネ シゥム 7水和物、 0.002%硫酸鉄 7水和物、 2 %グルコース、 pH5.5) を フ。'口 トプラス ト懸濁液に加えて、 選択培地 （1.2 mol八ソルビトール、 0.2%塩化アンモニゥム、 0.1%硫酸アンモニゥム、 0.05%塩化力 y ゥ ム、 0.05%塩化ナトリ ウム、 0.1%リ ン酸ニ水素一力リ ウム、 0.05%硫 酸マグネシウム 7水和物、 0.002%硫酸鉄 7水和物、 2%グルコース、 1.5 %寒天、 pH5.5) に重層した。 Next, 10 ml of buffer A was added and mixed gently, and then centrifuged at 2000 rpm for 8 minutes to recover a protoplast suspension. Add 500 μl of buffer solution プ to the toplast, mix gently, and incubate 5 ml of top agar (1.2 mol / 1 sorbitol, 0.8% agarose, 0.2% ammonium chloride, 0.1% % Ammonium sulfate, 0.05% sodium chloride, 0.05% sodium chloride, 0.1% sodium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.002% iron sulfate heptahydrate, 2% Glucose, pH 5.5). 'Mouth In addition to the toplast suspension, a selective medium (1.2 mol sorbitol, 0.2% ammonium chloride, 0.1% ammonium sulfate, 0.05% chloride, 0.05% sodium chloride, 0.1% dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.002% iron sulfate Layered over heptahydrate, 2% glucose, 1.5% agar, pH 5.5).
sC遺伝子が欠損した株は、 硫酸化合物を唯一の硫黄源とする培地上 では生育できないので、 該選択培地を用いて、 sC遺伝子が揷入された 破壊 Aovps5遺伝子を含む DNAが導入された形質転換体を選択すること ができる。 30°Cで 7日間培養し、 約 100株の形質転換体を取得した。 取 得された形質転換体は上記選択培地上で 3回継代培養を行った。  Since a strain lacking the sC gene cannot grow on a medium containing only a sulfur compound as a sulfur source, the selective medium is used to transform a strain into which a DNA containing the disrupted Aovps5 gene into which the sC gene has been introduced has been introduced. You can choose your body. After culturing at 30 ° C for 7 days, about 100 transformants were obtained. The obtained transformant was subcultured three times on the above selective medium.
得られた形質転換体から、 AovpS5遺伝子の遺伝子座で破壊 Aovps5遺 伝子の相同組換えが生じた株は、 実施例 1の （ 1 ) 記載の方法により それぞれの形質転換体から取得されたゲノム DNAをテンプレー トと し、 cDNAのクローニングに使用した配列番号 9および配列番号 10に示す塩 基配列からなるオリ ゴヌクレオチドをプライマーと して PCRを行う こ とにより検出することができる。 PCRは、 プログラム · テンプ ' コント ローノレ · システム (Program Temp Control System) PC - 700 (ァステツ ク社製） により行った。 反応,は 94°Cで 3分間加熱しテンプレー トの DNA を変性させた後、 94°Cで 1分 30秒、 56°Cで 1分 15秒、 72°Cで 4分の反応を 30サイクルの条件下で行った。 PCR産物を 0.8%ァガロースゲルで電気 泳動し > 野生型の Aovps5遺伝子 （約 1.6kbp) は.検出されず、 破壌 Aovps5 遺伝子 （約 5kbp) が検出された形質転換体を、 Aovps5遺伝子破壌株と して選択した。 図 3に、 このようにして選択された形質転換体 CT6118 株、 CT9103株、 CT96株のゲノム DNAをテンプレートと して上記の条件下 で PCRを行ったときの、 PCR産物の電気泳動の結果を示す。 From the resulting transformant, strain homologous recombination has occurred in the gene heritage Aovp S 5 destroyed locus gene Aovps5 is obtained from each of the transformants by the method of (1) described in Example 1 The genomic DNA is used as a template, and PCR can be performed by using as a primer the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 9 and SEQ ID NO: 10 used for cloning of the cDNA. PCR was performed using a Program Temp Control System PC-700 (manufactured by Astec). After denaturing the template DNA by heating at 94 ° C for 3 minutes, 30 cycles of 1 minute 30 seconds at 94 ° C, 1 minute 15 seconds at 56 ° C, and 4 minutes at 72 ° C Was performed under the following conditions. The PCR product was electrophoresed on a 0.8% agarose gel.> The wild-type Aovps5 gene (about 1.6 kbp) was not detected. And selected. Figure 3 shows the results of electrophoresis of the PCR products when PCR was performed under the above conditions using the genomic DNA of the transformants CT6118, CT9103, and CT96 selected as a template. Show.
なお、 これらの Aovps5遺伝子破壌株では、 分生子の形成がみられな かった。 また、 寒天培地 (0.2%塩化アンモニゥム、 0.1%硫酸アンモ 二ゥム、 0.05%塩化力リ ウム、 0.05%塩化ナトリ ウム、 0.1%リン酸ニ 水素一カリ ウム、 0.05%硫酸マグネシゥム 7水和物、 0.002%硫酸鉄 7 水和物、 2%グルコース、 1.5%寒天、 pH5.5) にこれらの Aovps5遺伝子 破壊株の菌糸を植え、 シャーレ本体と蓋を、 密閉状態にならないよう にパラフィルムで巻いて固定し、 30°Cで 5日以上培養した結果、 コロニ 一周辺の寒天培地が乳白色から赤褐色に変化した。 したがって、 AovPs 5 遺伝子破壊株は、 上記培養条件下で赤褐色の色素を分泌すると予想さ れる。 No conidium formation was observed in these Aovps5 gene disrupted strains. In addition, agar medium (0.2% ammonium chloride, 0.1% ammonium sulfate, 0.05% lithium chloride, 0.05% sodium chloride, 0.1% potassium monohydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.002% iron sulfate heptahydrate, 2% glucose, 1.5% agar, pH 5.5), plant the hyphae of these Aovps5 gene-disrupted strains, and keep the petri dish body and lid from being sealed. The agar medium around the colony changed from milky white to reddish brown as a result of culturing at 30 ° C for more than 5 days. Therefore, AOV P s 5 gene disruption strain would be expected to secrete reddish brown dye in the culture conditions.
実施例 4 培地中に分泌された液胞酵素の活性測定  Example 4 Measurement of vacuolar enzyme activity secreted into the medium
ァスペルギルス · ォリゼの親株 （NS4株） 、 および Aovps 5遺伝子破壌 株 3株 （CT6118株、 CT9103株および CT96株） をそれぞれ DPY培地 60mlに 接種し 300mlのバッフル付きの三角フラスコで、 30°C、 2日間、 180rpm で振と う培養した。  A parenter strain of Aspergillus oryzae (NS4 strain) and three Aovps 5 gene-broken strains (CT6118, CT9103 and CT96 strains) were inoculated into 60 ml of DPY medium, respectively. The cells were cultured with shaking at 180 rpm for 2 days.
培養液を孔径 0. 45 mのメンブレンフィルターでろ過し菌体を除去 した後、該ろ液の 435 μ 1 に 2415 1の 50ramol/l酢酸パッファーを加え、 2. 85mlの酵素溶液と した。 これを 30°Cで 5分間ィンキュベートした後、 基質溶液 〔8 ramol/1 ァラニンーァラェンーフヱニルァラニン一パラ二 トロアニリ ドを含む 50 mmol八酢酸バッファー （ρΗ4· 5) 〕 を 150 μ 1混 合し基質の分解を開始した。 30°Cで 10分間反応させ、 経時的にサンプ リ ングを行い、 分光光度計により 384nmの吸光度を測定した。 パラニ ト ロア-リ ンのモル吸光係数 （10300) より、 反.応時間 1分あたりのパラ 二'トロア-リ ンの遊離量を計算し、 30°Cで 1分間に 1 μモルのパラ二 トロアユリ ンを遊離する活性を 1単位と した。 該培養液のト リべプチ ジルぺプチダーゼ活性を測定し、 活性値を算出した結果を第 1表に示 す。  The culture was filtered through a 0.45 m membrane filter to remove the cells, and 435 μl of the filtrate was added with 24151 50 ramol / l acetic acid buffer to give 2.85 ml of the enzyme solution. After incubating the mixture at 30 ° C for 5 minutes, the substrate solution (50 mmol octaacetic acid buffer (ρΗ4.5) containing 8 ramol / 1 alanine-araphen-phenylanilane-paranitroanilide) was added in 150 ml. μ1 was mixed to initiate the decomposition of the substrate. The mixture was reacted at 30 ° C for 10 minutes, sampled with time, and the absorbance at 384 nm was measured with a spectrophotometer. From the molar extinction coefficient (10300) of paranitroline, calculate the amount of para2'-troline released per minute of reaction time. The activity to release troyuulin was defined as 1 unit. Table 1 shows the results obtained by measuring the tripeptidyl peptidase activity of the culture solution and calculating the activity value.
第 1表  Table 1
Figure imgf000045_0001
の結果から示されるとおり、 Aovps 5遺伝子破壌株では培地中に液 胞酵素である トリぺプチジルぺプチダーゼが効率よく分泌される。 実施例 5 Aovps 5遺伝子破壌株が生産する酵素によるタンパク加水分 解物の調製
Figure imgf000045_0001
As shown in the results, the Aovps 5 The vesicular enzyme triptidyl peptidase is secreted efficiently. Example 5 Preparation of protein hydrolyzate with enzyme produced by Aovps 5 gene-broken strain
親株 （NS4株） 及ぴ AovPs 5遺伝子破壊株 3株 （CT6118棕、 CT9103株お よび CT96株) を、 それぞれ DPY培地 60mに植菌し 300mlのバッフル付き三 角フラスコで、 30°C、 、 2日間、 180rpmで振と う培養した。 培養後、 吸 引ろ過により菌体と上清に分離した。 菌体は湿菌体の状態でその重量 を測定した。 The parent strain (NS4 strain) and three Aov P s 5 gene-disrupted strains (CT6118 palm, CT9103 strain and CT96 strain) were inoculated respectively in 60 m of DPY medium and placed in a 300 ml baffled triangular flask at 30 ° C. The cells were cultured with shaking at 180 rpm for 2 days. After the culture, the cells were separated into bacterial cells and supernatant by suction filtration. The weight of the cells was measured in a wet cell state.
121°C、 15分間加圧滅菌した 15 %の小麦グルテン (プロミ ック GT、 協 和醱酵工業社製） 分散液 3.0mlに、 孔径 0. 2 mのメ ンブレンフィルタ一 でろ過した上清 30mlを加え、 40°C、 lOOrpmで 48時間ィンキュベートし、 タンパク加水分解物を調製した。 タンパク質を分'解した後、 反応物を 孔径 0. 2 mのメンブレンフィルターでろ過し、 100°C、 10分間加熱処理 を行ない、 窒素可溶化率と遊離アミノ酸量を測定した。 窒素可溶化率 は、 原料に含まれる総窒素に対するろ液の総窒素の割合とする。 総窒 素の測定は、 NA2100 (サーモフィ二ガン社製） により測定した。 測定 値 （％) は湿菌体重量 （g) で標準化し、 3回の実験結果の平均値を算 出した。 結果を第 4図に示す。 遊離アミノ酸量の測定法はオルトフタ ルジアルデヒ ド （0PA) 法 〔Food Chem. , 62, 363 ( 1998)〕 に従った。 測定した遊離アミノ酸量 （nimol /1 ) は湿菌体重量 （g) で標準化し、 3 回の実験結果の平均値を算出した （図 5 ) 。 その結果、 親株と比較し Aovps5遺伝子破壌株の培養液を用いて製造したタンパク加水分解物で は、 湿菌体重量 （g) あたりの窒素可溶化率および遊離アミノ酸量が有 意に増加していることを見出した。 したがって、 Aovps5遺伝子の破壌 株の培養液を用いることにより、 従来より加水分解率の高いタンパク 加水分解物を製造することができる。 産業上の利用可能性  15% wheat gluten (Promic GT, manufactured by Kyowa Hakko Kogyo Co., Ltd.) sterilized by autoclaving at 121 ° C for 15 minutes Supernatant filtered through a membrane filter with a pore size of 0.2 m in 3.0 ml of a dispersion liquid 30 ml was added, and the mixture was incubated at 40 ° C. and 100 rpm for 48 hours to prepare a protein hydrolyzate. After disassembly of the protein, the reaction product was filtered through a membrane filter having a pore size of 0.2 m, and heat-treated at 100 ° C for 10 minutes to measure the nitrogen solubilization ratio and the amount of free amino acids. The nitrogen solubilization rate is the ratio of the total nitrogen in the filtrate to the total nitrogen contained in the raw material. The total nitrogen was measured using NA2100 (manufactured by Thermofinigan). The measured value (%) was normalized by the wet cell weight (g), and the average of the results of three experiments was calculated. The results are shown in FIG. The method for measuring the amount of free amino acids was in accordance with the orthophthalaldehyde (0PA) method [Food Chem., 62, 363 (1998)]. The measured amount of free amino acid (nimol / 1) was standardized by the wet cell weight (g), and the average of the results of three experiments was calculated (FIG. 5). As a result, the protein hydrolyzate produced using the culture solution of the Aovps5 gene disrupted strain significantly increased the nitrogen solubilization rate and the amount of free amino acids per wet cell weight (g) compared to the parent strain. I found that. Therefore, a protein hydrolyzate having a higher hydrolysis rate than before can be produced by using a culture solution of the Aovps5 gene-broken strain. Industrial applicability
本発明は、 Aovps 5遺伝子、 該遺伝子がコードするポリペプチド、 該 遺伝子の機能を抑制させた糸状菌、 および該糸状菌を用いてタンパク 加水分解率が顕著に上昇したタンパク加水分解物の作製方法が提供さ れる。 The present invention relates to the Aovps 5 gene, a polypeptide encoded by the gene, a filamentous fungus in which the function of the gene is suppressed, and a protein using the filamentous fungus. A method for producing a protein hydrolyzate having a significantly increased hydrolysis rate is provided.
「配列表フリーテキス ト J `` Sequence List Free Text J
配列番号 1 一発明者 ： 徳永税、 斎藤知明 Sequence number 1 One inventor: Tokunaga tax, Tomoaki Saito
発明者 ： 北本勝ひこ  Inventor: Katsuhiko Kitamoto
配列番号 6—縮重プライマー V5P - F SEQ ID NO: 6—Degenerate primer V5P-F
配列番号 7—縮重プライマー V5P - R SEQ ID NO: 7—Degenerate primer V5P-R
配列番号 9 一 PCRプライマー VPS 5F SEQ ID NO: 9-PCR primer VPS 5F
配列番号 10— PCRプラィマー VPS 5R SEQ ID NO: 10—PCR primer VPS 5R

Claims

請求の範囲 The scope of the claims
1. 配列番号 1 のァミノ酸配列を含むポリぺプチド。  1. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
2. 配列番号 1 のァミノ酸配列において 1以上のア ミ ノ酸が欠失、 置換もしくは付加されたァミノ酸配列からなり、 かつ液胞酵素を細胞 内に局在させる活性を有するポリぺプチド。  2. A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and having an activity of localizing vacuolar enzymes to cells.
3. 配列番号 1のァミノ酸配列と 60 %以上の相同性を有するァミノ 酸配列を含み、 かつ液胞酵素を細胞内に局在させる活性を有するポリ ぺプチド。  3. A polypeptide comprising an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 and having an activity of localizing vacuolar enzymes to cells.
4. 請求項 1〜 3のいずれか 1項に記載のポリぺプチドをコ一ドす る DNA。  4. A DNA encoding the polypeptide according to any one of claims 1 to 3.
5. 配列番号 2または 3の塩基配列を含む DNA。  5. DNA containing the nucleotide sequence of SEQ ID NO: 2 or 3.
6. 配列番号 2または 3の塩基配列と相捕的な塩基配列からなる DNAとス ト リ ンジェン トな条件下でハイプリダイズする DNAであり、 力、 っ液胞酵素を細胞内に局在させる活性を有するポリペプチドをコード する DNA。  6. DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 or 3, and localizes force and vacuolar enzymes to cells. DNA encoding a polypeptide having activity.
7. 請求項 4〜 6のいずれか 1項に記載の DNAをベクターに組み込 んで得られる組換えべクター。  7. A recombinant vector obtained by incorporating the DNA according to any one of claims 4 to 6 into a vector.
8. 請求項 7に記載の組換えべクターを宿主細胞に導入して得られ る形質転換体。  8. A transformant obtained by introducing the recombinant vector according to claim 7 into a host cell.
9. 請求項 8に記載の形質転換体を培地に培養し、 培養物中に請求 項 1〜 3のいずれか 1項に記載のポリぺプチドを生成蓄積させ、 該培 養物から、 該ポリぺプチドを採取することを特徴とする該ポリぺプチ ドの製造方法。  9. The transformant according to claim 8 is cultured in a medium, the polypeptide according to any one of claims 1 to 3 is produced and accumulated in the culture, and the polypeptide is produced from the culture. A method for producing the polypeptide, comprising collecting the polypeptide.
10. 配列番号 2または 3の塩基配列の連続する 20塩基以上の配列 と相補的な配列を有するポリヌク レオチドを用いて、 請求項 4：〜 6の いずれか 1項に記載の DNAまたは請求項 1〜 3のいずれか 1項に記載 のポリぺプチドをコ一ドする mRNAを検出する方法。 10. The DNA or claim 1 according to any one of claims 4 to 6, using a polynucleotide having a sequence complementary to a continuous sequence of 20 or more nucleotides in the base sequence of SEQ ID NO: 2 or 3. Described in any one of items 1 to 3 A method for detecting mRNA encoding a polypeptide.
11. 配列番号 2または 3の塩基配列の連続する 15塩基以上の配列 を有する DNAおよび配列番号 2または 3の塩基配列の連続する 15塩基 以上の配列と相補的な配列を有する DNAを用いて、請求項 4〜 6のいず れか 1項に記載の DNAまたは請求項 1〜 3のいずれか 1項に記載のポ リぺプチドをコ一ドする mRNAを検出する方法。  11. Using a DNA having a sequence of 15 or more consecutive bases of SEQ ID NO: 2 or 3 and a DNA having a sequence complementary to a sequence of 15 or more consecutive bases of SEQ ID NO: 2 or 3, A method for detecting the DNA according to any one of claims 4 to 6 or the mRNA encoding the polypeptide according to any one of claims 1 to 3.
12. 液胞酵素を細胞内に局在させる活性を有するポリペプチドの 機能を抑制させることを特徴とする、 液胞酵素を細胞外に分泌する糸 状菌の作製方法。  12. A method for producing a filamentous fungus that secretes a vacuolar enzyme extracellularly, which comprises suppressing the function of a polypeptide having an activity of localizing the vacuolar enzyme to a cell.
,  ,
13. 糸状菌のゲノム中の、 液胞酵素を細胞内に局在させる活性を 有するポリペプチドをコードする遺伝子を遺伝子破壌することにより 該ポリぺプチドの機能を抑制させる請求項 1 2に記載の方法。 13. The function of the polypeptide according to claim 12, wherein the function of the polypeptide is suppressed by gene disruption of a gene encoding a polypeptide having an activity of localizing a vacuolar enzyme in a cell of a filamentous fungus. the method of.
14. 液胞酵素を細胞内に局在させる活性を有するポリぺプチドを コードする遺伝子のアンチセンス mRNAを転写させることにより、 該;ポ リぺプチドの機能を抑制させる請求項 1 2に記載の方法。  14. The method according to claim 12, wherein the function of the polypeptide is suppressed by transcribing an antisense mRNA of a gene encoding a polypeptide having an activity of localizing a vacuolar enzyme in a cell. Method.
15. 糸状菌のゲノム中の、 液胞酵素を細胞内に局在させる活性を 有するポリぺプチドをコ一ドする遺伝子のプロモーター領域に変異を 導入することにより、 該ポリべプチドの機能を抑制させる請求項 1 2 に記載の方法。  15. Suppresses the function of the polypeptide by introducing a mutation into the promoter region of the gene encoding a polypeptide having the activity of localizing vacuolar enzymes to cells in the filamentous fungal genome The method according to claim 12, wherein the method is performed.
16. · 液胞酵素を細胞内に局在させる活性を有するポリペプチドの ドミナントネガティブ変異体を発現させることにより、 該ポリべプチ ドの機能を抑制させる請求項 1 2に記載の方法。  16. The method according to claim 12, wherein the function of the polypeptide is suppressed by expressing a dominant negative mutant of a polypeptide having an activity of localizing a vacuolar enzyme to a cell.
17. 液胞酵素を細胞内に局在させる活性を有するポリぺプチドが 請求項 1〜 3のいずれか 1項に記載のポリペプチドである、 請求項 1 2〜 1 6のいずれか 1項に記載の方法。  17. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide having an activity of localizing a vacuolar enzyme to a cell is the polypeptide according to any one of claims 1 to 3. The described method.
18. 糸状菌を、 ゲノムに突然変異を誘発させた後に培養し、 培地 に赤褐色の色素を分泌する菌株を選択することを特徴とする、 請求項 丄〜 3のいずれか 1項に記載のポリぺプチドの機能が抑制された糸状 菌の取得方法。 18. Culture the filamentous fungus after mutagenesis of the genome 4. A method for obtaining a filamentous fungus in which the function of the polypeptide is suppressed, according to claim 3, wherein a strain that secretes a reddish brown pigment is selected.
19. 糸状菌を、 ゲノムに突然変異を誘発させた後に重金属を含む 培地に培養し、 重金属に対する耐性を有する菌株を選択することを特 徴とする、 請求項 1〜 3のいずれか 1項に記載のポリぺプチドの機能 が抑制された糸状菌の取得方法。  19. The method according to any one of claims 1 to 3, wherein the filamentous fungus is cultured in a medium containing heavy metals after mutagenesis of the genome, and a strain resistant to heavy metals is selected. A method for obtaining a filamentous fungus in which the function of the polypeptide is suppressed.
20. 糸状菌がァスペルギルス属に属する糸状菌である請求項 1 2 〜 1 9のいずれか 1項に記載の方法。  20. The method according to any one of claims 12 to 19, wherein the filamentous fungus is a filamentous fungus belonging to the genus Aspergillus.
21. ァスペルギルス属に属する糸状菌がァスペルギルス ' ォリゼ である請求項 2 0に記載の方法。  21. The method according to claim 20, wherein the filamentous fungus belonging to the genus Aspergillus is Aspergillus oryzae.
22. 請求項 4〜 6のいずれか 1項に記載の DNAのコ一ド領域内に 1 つ以上の DNAを揷入または欠失することにより、液胞酵素を細胞内に局 在させる活性を有するポリぺプチドをコードしなくなつた DNA。  22. The activity of localizing vacuolar enzymes to cells by inserting or deleting one or more DNAs in the code region of the DNA according to any one of claims 4 to 6. DNA that no longer encodes the polypeptide it has.
23. 請求項 2 2に記載の DNAを含む組換えベクター。  23. A recombinant vector comprising the DNA according to claim 22.
24. 請求項 1 7〜 2 1のいずれか 1項に記載の方法で得られる糸 状菌。 ·  24. A filamentous fungus obtained by the method according to any one of claims 17 to 21. ·
25. 糸状菌がァスペルギルス属に属する糸状菌である請求項 2 4 に記載の糸状菌。  25. The filamentous fungus according to claim 24, wherein the filamentous fungus is a filamentous fungus belonging to the genus Aspergillus.
26. ァスペルギルス属に属する糸状菌がァスペルギルス 'ォリゼで ある請求項 2 5に記載の糸状菌。  26. The filamentous fungus according to claim 25, wherein the filamentous fungus belonging to the genus Aspergillus is Aspergillus oryzae.
27. 請求項 1 2〜 2 1のいずれか 1項に記載の方法で得られる糸 状菌を培地に培養し、 培地中に液胞酵素を生成蓄積させ、 該培地から 液胞酵素を採取す.ることを特徴とする液胞酵素の製造方法。  27. The filamentous fungus obtained by the method according to any one of claims 12 to 21 is cultured in a medium, to produce and accumulate vacuolar enzymes in the medium, and to collect the vacuolar enzymes from the medium. A method for producing a vacuolar enzyme, comprising:
28. 培地が固体培地である請求項 2 7に記載の製造方法。  28. The method according to claim 27, wherein the medium is a solid medium.
29. 培地が液体培地である請求項 2 7に記載の製造方法。 29. The method according to claim 27, wherein the medium is a liquid medium.
30. 液胞酵素が トリぺプチジルぺプチダーゼである請求項 2 7〜 2 9のいずれか 1項に記載の製造方法。 30. The production method according to any one of claims 27 to 29, wherein the vacuolar enzyme is triptidyl peptidase.
31. 請求項 2 7〜 3 0のいずれか 1項に記載の方法によ り製造さ れた液胞酵素、 または請求項 1 2〜 2 1のいずれか 1項に記載の方法 で得られる糸状菌を培養して得られる液胞酵素を含む培養物または培 養処理物を、 基質となるタンパク質に作用させることを特徴とするタ ンパク加水分解物の製造方法。  31. A vacuolar enzyme produced by the method according to any one of claims 27 to 30 or a filament obtained by the method according to any one of claims 12 to 21. A method for producing a protein hydrolyzate, which comprises causing a culture or a culture product containing a vacuolar enzyme obtained by culturing bacteria to act on a protein serving as a substrate.
32. 請求項 1 2〜 2 1のいずれか 1項に記載の方法で得られる糸 状菌を、 基質となるタンパク質を含む培地に培養することを特徴とす るタンパグ加水分解物の製造方法。  32. A method for producing a protein hydrolyzate, which comprises culturing the filamentous fungus obtained by the method according to any one of claims 12 to 21 in a medium containing a protein serving as a substrate.
33. 請求項 3 1または 3 2に記載の方法で製造されたタンパク加 水分解物。  33. A hydrolyzed protein produced by the method according to claim 31 or 32.
34. 請求項 3 3に記載のタンパク加水分解物を含有することを特 徴とする調味料。  34. A seasoning characterized by containing the protein hydrolyzate according to claim 33.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07274944A (en) * 1994-04-14 1995-10-24 Ajinomoto Co Inc New variant and method for producing protein hydrolysate

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07274944A (en) * 1994-04-14 1995-10-24 Ajinomoto Co Inc New variant and method for producing protein hydrolysate

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HORAZDOVSKY B.F. ET AL.: "A sorting nexin-1 homologue, vps5p, forms a complex with vps17p and is require for recycling the vacuolar proteinsorting receptor", MOL. BIOL. CELL., vol. 8, 1997, pages 1529 - 1541, XP002904705 *
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