JPH0817709B2 - Alkaline protease gene - Google Patents

Description

TECHNICAL FIELD The present invention relates to an alkaline protease gene.

[Conventional technology]

Conventionally, the structure of the alkaline protease gene derived from Aspergillus oryzae, which is one of Aspergillus oryzae, is completely unknown, and the fact is that the gene has not even been isolated.

Alkaline protease is a hydrolase that acts on a protein or a partial hydrolyzate thereof to decompose a peptide bond, and is widely used in medicine, foods and drinks, detergents and the like.

[Problems to be Solved by the Invention]

The object of the present invention is to provide an alkaline protease gene.

[Means for solving the problem]

Therefore, as a result of various studies on the alkaline protease gene derived from Aspergillus oryzae, the present inventors succeeded in isolating and determining the structure of the alkaline protease gene derived from Aspergillus oryzae for the first time, and completed the present invention.

That is, the present invention is an alkaline protease gene encoding the amino acid sequence shown below.

Hereinafter, the present invention will be described in detail.

First, examples of Aspergillus oryzae used as a donor of the gene of the present invention include Aspergillus oryzae (ATCC 20386).

Then, the above microorganism is cultured in exactly the same manner as the method described in Japanese Examined Patent Publication No. 48-38873 to obtain a culture, and the culture is subjected to a conventional method, for example, filtration, centrifugation, etc. of Aspergillus oryzae. Get the body.

To prepare m-RNA from Aspergillus oryzae cells, for example, except that glass beads and phenol are used during cell disruption, see, for example, Molecular Cloning, page 196, cold. Spring Harbor Laboratory (Cold Spr
ing Harbor Laboratory) (1982) and Molecular Genetics Experimental Method, Haruo Ozeki, Rero Shimura, pp. 66-67 (1983) and the like.

To concentrate m-RNA encoding alkaline protease (hereinafter referred to as alkaline protease m-RNA) from the obtained m-RNA, for example, Biomedical Research, Volume 3, 534 to 540. It can be carried out by the method described on page (1982).

At this time, an anti-alkaline protease serum against alkaline protease is used, and the serum is, for example, immunochemistry, Yuichi Yamamura, pp. 43-50 (1973).
It can be obtained by the method described.

For synthesizing c-DNA from alkaline protease m-RNA, for example, Mol. Cell Biol is used.
l.), Volume 2, page 161, (1982) and Gene,
It can be carried out by the method described in Vol. 25, page 263 (1983).

Then, the c-DNA thus obtained is used as a vector D.
NA, for example, plasmid pUC119DNA (Takara Shuzo Co., Ltd.)
Etc. to obtain various recombinant plasmid DNAs,
Using the DNA, for example, E. coli DH1 (ATCC 338
49), E. coli HB101 (ATCC 33694), etc. by the method of Hanahan [DNA Cloning, Volume 1, pp. 109-135 (1985)].
To obtain various transformants.

C-DNA encoding alkaline protease (hereinafter, alkaline protease c-D from the various transformants described above).
NA) to detect hybridization
Selection [Molecula
r Cloning), pages 329-333 and 344-349, Cold Spring Harbor Laboratory (Cold Sprin).
g Harbor Laboratory) (1982)].

Then, the incomplete alkaline protease c-DNA was digested with ³²
Nick translation method using P [Molecular Cloning, 109-112
Page, Cold Spring Harbor Laboratory (1982), and J. Mol. Biol., Vol. 113, No. 23.
7-251 (1977)] and the plasmid pUC119DNA as a vector by the colony hybridization method [protein, nucleic acid, enzyme, Vol. 26, 575-579 (1981)] using the c-DNA as a probe. Created c
-A plasmid DNA containing 1.1 Kb of alkaline protease c-DNA can be obtained from the gene bank library of DNA.

In order to obtain a DNA containing a gene encoding an alkaline protease derived from Aspergillus oryzae (hereinafter referred to as an alkaline protease gene) from the recombinant plasmid DNA thus obtained, a restriction enzyme is added to the plasmid DNA, For example, Eco RI temperature 30-40
After the reaction is carried out at ℃, preferably 37 ℃ for 1 to 24 hours, preferably 2 hours, the resulting reaction solution is subjected to agarose gel electrophoresis [Molecular Cloning (Molecular Cloning
g), p. 150, Cold Spring Harbor Laboratory (1982)
According to the description], a DNA containing an Aspergillus oryzae-derived alkaline protease gene can be obtained.

Then, the base sequence of this alkaline protease gene was determined by the method as shown in item 9 of the example (see FIG. 2), and then the amino acid sequence of the polypeptide translated by the gene having the base sequence was determined. Confirm (see FIG. 3).

The gene encoding the amino acid sequence thus determined is the gene of the present invention.

〔The invention's effect〕

As is clear from the above, according to the present invention, by culturing, for example, a microorganism containing a recombinant DNA in which the alkaline protease gene of the present invention is incorporated, in a medium, the alkaline protease can be efficiently treated in a very short period of time. The present invention is extremely useful industrially because it can be obtained well and the above gene can be used with a sample for protein engineering.

〔Example〕

 Hereinafter, the present invention will be described in more detail with reference to examples.

Example Cloning of alkaline protease c-DNA derived from Aspergillus oryzae (ATCC 20386) of Aspergillus oryzae 1. Acquisition of bacterial cells Spores (1.2 × 10 ⁸ ) of Aspergillus oryzae (ATCC 20386) of Aspergillus oryzae are treated with an alkaline protease production medium. [3% defatted soybeans that have been subjected to 3% (W / V) heating and pressure expansion
(W / V) KH ₂ PO ₄ ] 50 ml, and 120 rpm with a constant temperature shaker (M-100 ⁿ , manufactured by Taiyo Kagaku Kogyo Co., Ltd.) at a temperature of 30 ° C.
The culture was shaken for 45 hours to obtain a culture, and this culture was filtered by a conventional method to obtain 10 g of cells.

2. Acquisition of m-RNA 10 g of the bacterial cell obtained in item 1 was added to 20 ml of a guanidine isothiocyanate solution [6M guanidine isothiocyanate
/37.5mM sodium citrate (pH7.0) /0.75% (W / V) N
-Sodium lauroyl sarcosine / 0.15M β-mercaptoethanol] was added to a cup-type blender (Nippon Seiki Seisakusho Co., Ltd.), and 10 g of glass beads (diameter 0.5 mm) was added to 10,000. 5 at rpm
After the treatment for 1 minute, 10 ml of water-saturated phenol was further added, and the mixture was treated at 10,000 rpm for 10 minutes to crush the cells to obtain a crushed material.

The crushed material thus obtained was centrifuged at 5,000 rpm for 10 minutes using a cooling centrifuge (Hitachi Koki Co., Ltd., 18PR-52) to obtain 20 ml of crushed cell suspension.

Next, tubes for ultracentrifuges (Hitachi Koki
1.2 ml of 5.7 M cesium chloride solution was pre-layered on each of the four cells, and the above-mentioned cell disruption solution was dispensed on each of them in an ultracentrifuge (Hitachi Koki Co., Ltd.).・ Manufactured, SCP55
H) was used to centrifuge at 30,000 rpm for 16 hours at a temperature of 15 ° C. to obtain a precipitate.

The obtained precipitate was washed with cold 70% (V / V) ethanol and then used as a 10 mM Tris buffer solution (10 mM Tris-hydrochloric acid buffer solution (pH 7.4) / 5 mM EDTA / 1% sodium dodecyl sulfate). To the suspension in 4 ml, the same amount of n-butanol and chloroform mixed at a ratio of 4 to 1 (volume ratio) was added and extracted, followed by centrifugation at 3,000 rpm for 10 minutes by a conventional method, and then water. Layer and an organic solvent layer are separated, 4 ml of the above 10 mM Tris buffer is added to this organic solvent layer, and the extraction and separation operations are repeated twice, and the aqueous layer obtained is 1/10 volume of 3M. It was obtained by adding sodium acetate (pH5.2) and twice the amount of cold ethanol and leaving it at a temperature of -20 ° C for 2 hours, and then centrifuging at 8,000 rpm for 20 minutes by a conventional method to precipitate RNA. Dissolve RNA in 4 ml water,
RNA obtained after the above ethanol precipitation procedure
Was dissolved in 1 ml of water to obtain 12 mg of RNA.

To select m-RNA from this RNA, 12 mg of RNA
Was subjected to oligo (dT) -cellulose (New England Biolabs, Inc.) column chromatography.

2.5 ml Terumosyringe (Terumo Co., Ltd.)
0.5 g of resin was swollen with elution buffer [10 mM Tris-hydrochloric acid buffer (pH 7.6) / 1 mM EDTA / 0.1% (W / V) sodium dodecyl sulfate] and then packed in a column. , Binding buffer [10mM Tris-HCl (pH7.6) / 1mM EDTA / 0.4M
NaCl / 0.1% sodium dodecyl sulfate].

The same amount of buffer solution [10 mM Tris-hydrochloric acid (pH
7.6) / 1mM EDTA / 0.8M NaCl / 0.1% sodium dodecyl sulfate] was added, and the mixture was heat-treated at a temperature of 65 ° C. for 10 minutes, quenched in ice, and then applied to an oligo (dT) -cellulose column,
Wash the resin with binding buffer to remove unbound r-RNA and tR
NA was thoroughly washed, and m-RNA was further eluted with an elution buffer to obtain 90 µg of m-RNA.

3. Concentration of alkaline protease m-RNA Next, alkaline protease m-RNA was concentrated by a sucrose density gradient centrifugation method.

A sucrose density gradient of 10-25% (W / V)
40% (W /
V) Sucrose solution [50 mM Tris-HCl buffer (pH 7.5) / 20 mM
NaCl / 1mM EDTA / 40% (W / V) sucrose] 0.5ml, 2.4ml each 25% (W / V), 20% (W / V), 15% (W / V)
And 10% (W / V) sucrose solution were overlaid and left standing at a temperature of 4 ° C. for 24 hours. 50 μg of m-RNA was overlaid on this sucrose density gradient, and SW4 manufactured by Beckman
1 rotor, 30,000 rpm, temperature 18 ℃
It was centrifuged for 18 hours. After centrifugation, 0.
Fractions of 5 ml each were collected, m-RNA was recovered by the ethanol precipitation method, and dissolved in 10 μl of water.

Next, the protein encoded by m-RNA was examined to identify the fraction enriched in alkaline protease m-RNA. 1 μl of fractionated RNA, 9 μl of rabbit reticulocyte lysate (manufactured by Amersham) and 1 μl of [ ³⁵ S] methionine (manufactured by Amersham) were mixed and reacted at 30 ° C. for 30 minutes, and then 150 μl
Of NET buffer 150mM NaCl / 5mM EDTA / 0.02% (W / V) NaN 3/2
0 mM Tris-HCl buffer (pH7.4) /0.05% (W / V) Nonidet P-40 (Bethesda Research Laboratory Co., Ltd.,
Surfactant)], 1 μl of anti-alkaline protease serum (prepared as described below) was further added, and the mixture was allowed to stand at a temperature of 4 ° C. for 18 hours.・ Company) was added and left for 30 minutes at a temperature of 20 ° C.
After centrifugation at 00 rpm for 1 minute, the resin, that is, the above protein A sepharose was collected.

Wash the recovered resin 3 times with 200 μl of NET buffer,
Add 40 μl of sample buffer for SDS-PAGE [62.5
mM Tris-HCl buffer (pH 6.8) / 10% (V / V) glycerol / 2% (W / V) sodium dodecyl sulfate / 5% (V / V) mercaptoethanol / 0.02% (W / V) bromine Phenol blue], boiled at a temperature of 100 ° C for 3 minutes, centrifuged at 12,000 rpm for 1 minute by a conventional method, and the supernatant was recovered. The total amount was 12% (W / V) sodium dodecyl sulfate-polyacrylamide. I put it on the gel.

Gel electrophoresis was performed by the method of Laemmli [Nature, p. 227, p. 680 (1970)], and the gel after electrophoresis was diluted with 10% (V / V) acetic acid at 30%.
Soak for 30 minutes to fix the protein, then soak in water for 30 minutes, then soak in 1M sodium salicylate solution for 30 minutes and dry to obtain a dry gel. X-ray film (Fuji Photo Film Co., Ltd., RX ) Was used for fluorography.

By the above operation, the band of alkaline protease protein was observed on the X-ray film only when the RNA of the fraction containing alkaline protease m-RNA was used.
A fraction enriched in alkaline protease m-RNA could be identified.

4. Preparation of antiserum Rabbit antialkaline protease serum against purified alkaline protease was prepared by the following method.

28 mg of suspension of 0.7 ml of 40 mg / ml alkaline protease solution in an equal amount of Freund's complete adjuvant was administered as an antigen to the palm of a Japanese white rabbit weighing 2 kg, and kept for 2 weeks After that, the same amount of the antigen as the initial dose was administered intradermally on the back, and after 1 week of breeding, the same operation was performed, and further 1 week after breeding, whole blood was collected.

Then, the obtained blood was left at a temperature of 4 ° C. for 18 hours, and centrifuged at 3,000 rpm for 15 minutes by a conventional method.
Anti-alkaline protease serum was obtained as the supernatant.

5. Synthesis of c-DNA The synthesis of c-DNA was performed using a kit manufactured by Amersham.

Using 1.6 μg of the m-RNA obtained as described above, Mol.Ce (Mol.Ce) specified by Amersham was used.
II Biol.), Vol. 2, p. 161 (1982) and Gene (Gen).
e), Vol. 25, p. 263 (1983), the result was 160 ng of double-stranded c-DNA.

To 160 ng of c-DNA thus obtained, Amersham
Methylation of the restriction enzyme Eco RI cleavage site was carried out by the method instructed by Amersham, using a c-DNA cloning kid manufactured by the same company, and further Eco RI linkers were attached to both ends of c-DNA.

6. Preparation of c-DNA bank 100 ng of plasmid pUC119DNA (Takara Shuzo Co., Ltd.)
What was dissolved in 1 μl of water, 1 μl of Med buffer [100 mM
Tris - HCl buffer (pH7.5) / 100mMMgCl ₂ / After 10mM added dithiothreitol / 500 mM NaCl], and further, this, 10 units restriction (manufactured by Takara Shuzo, Co.,) enzyme Eco RI in (1 [mu] l) The mixture was added and cut at a temperature of 37 ° C. for 2 hours.

Next, 1 μl of 1 M Tris-hydrochloric acid buffer (pH 8.0) and 0.3 unit (1 μl) of alkaline phosphatase (Takara Shuzo Co., Ltd.) were added to the digested product, and the temperature was adjusted to 65.
After enzymatic reaction at 1 ° C for 1 hour, dephosphorylation of both ends of the digested product was performed, and 12 µl of water-saturated phenol was added to deproteinize, and 1 µl was added to the recovered aqueous layer.
3M sodium acetate (pH 5.8) and 26 μl of cold ethanol were added respectively, and the mixture was allowed to stand at a temperature of −70 ° C. for 15 minutes, and then a microcentrifuge (Tomy Seiko, MRX-150) was used for 12,000 rp.
DNA was collected by centrifugation at m. for 5 minutes.

The plasmid vector pUC119DNA obtained by cutting with the restriction enzyme Eco RI and dephosphorylating both ends was obtained in this way.
And 100 ng, a mixture of c-DNA 160 ng adjusted in item 5, were suspended in water 8 [mu] l, this ligation buffer 1μl [20mMMgCl ₂ / 66mM Tris - HCl buffer (pH 7.6) / 100 mM ATP / 15 mM dithiothreitol] was added, and 1 unit (1 μl) of T4 DNA ligase (Takara Shuzo Co., Ltd.) was added, and the mixture was allowed to stand at a temperature of 16 ° C. for 16 hours to obtain plasmid vector DNA and c Ligation of DNA was performed to obtain a reaction product.

Using this reaction product, Escherichia coli DH1 strain (ATC) was obtained by the method of Hanahan [DNA Cloning, Vol. 1, pp. 109-135 (1985)].
C33849) was transformed into a c-DNA bank using the plasmid pUC119DNA as a vector.

7. Search for Alkaline Protease c-DNA Fragment Hybridization Selection [Molecular Cloning, p. 329, Cold Spring Harbor Laboratory (198
2)] was performed to search for alkaline protease c-DNA. The details will be described below.

Any E. coli 70 from the c-DNA bank obtained in item 6
The strains were selected and the following operations were performed for each. Molecular Cloning, page 86,
Cold Spring Harbor Laboratory (198
2) By the method described above, 500 μg of each recombinant plasmid DNA was obtained from E. coli in the c-DNA bank. 1 of these
Dissolve 00 μg in 200 μl of water, leave it at 100 ° C for 10 minutes, then transfer to ice and rapidly cool it to 200 μl of 1M.
Sodium hydroxide was added and left at room temperature for 20 minutes to obtain a denaturing solution of recombinant plasmid DNA.

Then, to the denaturing solution thus obtained, 200 μl of the neutralizing solution [1M sodium chloride / 0.3M sodium citrate / 0.5M
[Tris-hydrochloric acid buffer solution (pH 8.0) / 1M hydrochloric acid] was quickly added and mixed, then, transferred to ice and rapidly cooled. This solution was filtered using a circular nitrocellulose filter with a diameter of 5 mm (Millipore Corp., Catalog No. HAWP 025 00), and the denatured recombinant plasmid DNA was adsorbed on the nitrocellulose filter. After air-drying with 6 × SSC (0.9M sodium chloride / 0.09M
It was washed with sodium citrate), air-dried by a conventional method, and then left in a vacuum oven kept at a temperature of 80 ° C. for 2 hours to immobilize the recombinant plasmid DNA on a nitrocellulose filter.

Then, 10 μl of the hybridization solution [100
μg / ml m-RNA (prepared in item 2) / 65% (V / V) deionized formamide / 20 mM l, 4-piperazinediethanesulfonic acid (pH6.4) /0.2% sodium dodecyl sulfate /
0.4 M sodium chloride / 100 μg / ml yeast t-RNA] was dipped in a nitrocellulose filter on which the recombinant plasmid DNA obtained as described above was immobilized, and the mixture was allowed to stand at a temperature of 50 ° C. for 3 hours. Plasmid DNA and m
-Hybridization with RNA was performed. After completion of the reaction, the filter was taken out and washed with 1 ml of washing solution I [10 mM Tris-hydrochloric acid buffer solution (pH 7.6) /0.15 M sodium chloride / 1 mM ED
After washing 9 times with TA / 0.5% sodium dodecyl sulfate], 1 ml of washing solution II [10 mM Tris-hydrochloric acid buffer solution (pH 7.6)]
/0.15M sodium chloride / 1mM EDTA], and washed twice to remove the m-RNA not hybridized with the plasmid DNA fixed on the filter.

Then, this nitrocellulose filter is
1 μl of water, add 10 μg of yeast t-RNA,
It was left standing in hot water at 100 ° C. for 10 minutes, transferred to ethanol cooled with dry ice, and then left standing at room temperature to melt. The thus hybridized m-RNA was peeled off from the nitrocellulose filter. In the obtained aqueous solution, 10
After adding 3 μl of 3M sodium acetate (pH5.2) and 300 μl of cold ethanol and leaving it at a temperature of −70 ° C. for 1 hour to perform ethanol precipitation, a microcentrifuge [Tomy Seiko Co., Ltd., MRX-150] was used. Was used for 5 minutes at 12,000 rpm to collect the hybridized m-RNA.

Then, in the same manner as described in item 3, the recovered protein encoded by the above-mentioned m-RNA is synthesized, and
Using the anti-alkaline protease serum obtained in step 1, it was analyzed whether the synthesized protein was alkaline protease.

As a result, a positive result was obtained for 1 out of 70 strains. Recombinant plasmid DNA (pOA
Aspergillus Orize (ATCC 2038)
It can be concluded that the fragment of alkaline protease c-DNA derived from 6) has been inserted.

Then, 1 μg of the recombinant plasmid pOAP3DNA was treated with the restriction enzyme Eco RI by the method described in item 6, and the mobility pattern was analyzed by agarose gel electrophoresis. The obtained pattern and plasmid pBR322DNA (Takara Shuzo Co., Ltd.
By comparison with a standard pattern of the resulting DNA fragment was digested with restriction enzymes Alu I Ltd.), alkaline protease c which is inserted into a recombinant plasmid pOAP3DNA
-The size of the DNA fragment was found to be 750 bp.

8. Search for large alkaline protease c-DNA From the agarose gel obtained in item 7, an agarose gel containing a 750 bp alkaline protease c-DNA fragment was cut out and placed in a dialysis tube, and 500 μl of TE buffer [10 mM Tris] was added. -After adding a hydrochloric acid buffer solution (pH 8.0) / 1 mM EDTA), the dialysis tube is sealed, and the solution obtained by eluting the DNA from the gel to the buffer solution by electrophoresis is electrophoresed.
An equal volume of water-saturated phenol was added and stirred, the aqueous layer was recovered, and 100 ng of DNA was recovered by ethanol precipitation according to a conventional method. This 100ng of alkaline protease c-DNA
Was labeled with [α- ³² P] dCTP (manufactured by Amersham Co.) by the nick translation method. Nick Translation is directed by Takara Shuzo Co., Ltd., J. Mol. Biol., 113, 237-251 (1977) and Molecular Cloning (Molecular).
Cloning), pp. 109-112 (1982). Using the ³² P-labeled alkaline protease c-DNA fragment thus prepared as a probe, colony hybridization [protein / nucleic acid / enzyme, Vol. 26, No. 575- 579 pages (19
81)], and a colony having alkaline protease c-DNA was obtained. The recombinant plasmid DNA contained in one of the colonies was named pOAP5, and the recombinant plasmid DNA was prepared according to item 7.

E. coli containing the recombinant plasmid DNA was designated as E. coli DH1 (pOAP5). The transformant strain has been deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Microindustrial Research Institute No. 9870 (FERM P-9870).

The recombinant plasmid pOAP5DNA was treated with the restriction enzyme Eco RI in the same manner as in item 6 and subjected to agarose gel electrophoresis. As a result, the size of the inserted DNA fragment was 1,1.
It turned out to be 00bp. Then, in the same manner as in item 8, the 1,100 bp alkaline protease cd
0.1 μg of DNA fragment containing NA was separated and purified. Recombinant plasmid pOAP5DNA with restriction enzymes Afl II, Eco RI, Kpn I, S
Single or double digestion of al I and Xmn I, item 7
By analyzing the size of the appeared fragment in the same manner as described in 1., a restriction enzyme map of the recombinant plasmid pOAP5DNA was prepared, and the map is shown in FIG.

9. Determination of base sequence of alkaline protease c-DNA Each alkaline protease c-DN obtained in item 8
Plasmid A pUC18 and pUC19 DNA obtained by cleaving the A fragment with the same or the same restriction enzyme (Takara Shuzo Co., Ltd.
Cloned). Sequencing was carried out by using the plasmid DNA method [Vector DNA, page 70, Kodansha (19
1986)], and then by using the M13 sequence kit (Takara Shuzo Co., Ltd.) by the dideoxy chain termination method in a conventional manner.

In this way Aspergillus Orize (ATCC 203
The nucleotide sequence of the alkaline protease c-DNA fragment derived from 86) was determined, and the nucleotide sequence corresponding to the mature alkaline protease in the obtained nucleotide sequence is shown in FIG. 2 and the nucleotide sequence shown in FIG. The amino acid sequences of the polypeptides translated from the genes having the are shown in FIG. 3, respectively.

The gene encoding this amino acid sequence is the gene of the present invention.

At the N-terminal of the amino acid sequence deduced from this DNA base sequence, 16 N-terminal amino acid sequences of purified alkaline protease (Gly Leu Thr Thr Glu Lys Ser Ala Pro Tr
p Gly Leu Gly Ser Ile Ser) was confirmed (underlined part in Fig. 3).

From the above findings, it can be concluded that the nucleotide sequence shown in FIG. 2 encodes from the N-terminal portion to the C-terminal portion of mature alkaline protease.

[Brief description of drawings]

FIG. 1 is a diagram showing a restriction enzyme map of a recombinant plasmid pOAP5DNA, and FIG. 2 is a diagram showing a nucleotide sequence of a gene corresponding to a mature alkaline protease described in item 9 of Example. FIG. 3 is a diagram showing the amino acid sequence of a polypeptide translated from the gene having the nucleotide sequence shown in FIG.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical display location C12R 1:69) (72) Inventor Seiji Murakami 399 Noda, Noda, Chiba Prefecture Research Division, Kikkoman Corporation (72 ) Inventor Eiichi Nakano 399 Noda, Noda City, Chiba Prefecture Kikkoman Corporation Research Headquarters (72) Inventor Hiroshi Motai 399 Noda City, Chiba Prefecture Kikkoman Corporation Research Headquarters (72) Inventor Atsushi Masaki Osaka Hirakata City Syoutsutsumi Otani 2-chome 1180 in Midori Cross Central Research Institute Co., Ltd. (72) Inventor Yutaka Ishida 1-1180 Shoutsutsutsumi Otani Hirakata, Osaka Prefecture Midori Cross Research Center Co., Ltd. (72) Inventor Koji Murakami Midori Cross Central Research Institute Co., Ltd., 2-chome Otani, 2-chome, Hirakata-shi, Osaka Prefecture (72) Inventor, Haruhide Kawabe 1 1-chome Research Center, 2-chome, Otsumi-Otani, Hirakata, Hirakata City, Osaka (72) ) Inventor Hirofumi Arimura Cabinet Office Hirakata 招堤 Otani 2-chome 1180 address of 1 Co., Ltd. Green Cross center in the Institute

Claims (1)

[Claims] 1. An alkaline protease gene encoding the amino acid sequence shown below.