JPH022374A - Alkaline protease gene - Google Patents

Abstract

PURPOSE:To perform mass-production of an alkaline protease which is a decomposition enzyme for peptide bond and useful as a drug, food, drink, detergent, etc., by separating an alkaline protease gene from a microorganism belonging to genus Aspergillus and determining the structure of the gene. CONSTITUTION:The inventors have succeeded for the first time in separating an alkaline protease from Aspergillus oryzae and determining the structure of the gene. The restriction endonuclease cleavage map of the alkaline protease gene is shown in the figure (E is EcoRI, A is AflII, K is KpnI, X is XmnI and S is SalI) and the amino acid sequence of the gene is expressed by the formula.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to alkaline protease genes.

[Conventional technology] Conventionally, Aspergillus oryzae (
The structure of the alkaline protease gene derived from Aspergillus oryzae is completely unknown, and the reality is that the gene has not even been isolated.

Alkaline protease is a hydrolase that acts on proteins or partially hydrolyzed products thereof to decompose peptide bonds, and is widely used in medicines, food and drink products, detergents, and the like.

[Problem to be solved by the invention]

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

[Means for Solving the Problems] 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 derived from a microorganism belonging to the genus Aspergillus and defined by the restriction enzyme cleavage map shown below, (wherein E is EcoRI
, A is Aflll, K is Kpnl.

X represents Xmn[, S represents 5ail].
This is an alkaline protease gene encoding the amino acid sequence shown in the figure.

The present invention will be explained in detail below.

First, Aspergillus oryzae used as a donor of the gene of the present invention includes, for example, Aspergillus oryzae (ATCC20386).

Next, the above-mentioned microorganisms are cultured in exactly the same manner as described in Japanese Patent Publication No. 48-38873 to obtain a culture, and from the culture, Aspergillus oryzae is isolated by a conventional method such as; Obtain bacterial cells.

In order to prepare m-RNA from the above-mentioned Aspergillus oryzae cells, for example, other than using glass beads and phenol when disrupting the cells, for example, molecular cloning can be used.
, p. 196, Cold Spring 1 (arbor
Lab.

(1982) and Molecular Genetics Experimental Methods, Yasuo Koseki, Reibe Shimura, pp. 66-67 (1983).

The obtained m-RNA encodes alkaline protease m-Rf'JA (hereinafter referred to as alkaline protease m-
To concentrate RNA), for example, Biomedical Research
ch), Volume 3, pp. 534-540 (1982).

At this time, an anti-alkaline protease serum against alkaline protease is used;
For example, Immunochemistry, Yu Yamamura-1, pp. 43-50 (197
3) Can be obtained by the method described.

To synthesize c-DNA from alkaline protease m-RNA, for example, Mol Cell Hiol (Mo1.C
e1l Biol, ), Volume 2, Page 161 (1982
) and Gene, Vol. 25, p. 263 (1)
983).

The c-DNA thus obtained is then transformed into hector DNA, for example plasmid pUc119 DNA.
A (Takara Shuzo Co., Ltd.), etc., to obtain various recombinant plasmid DNAs, and using the DNAs, for example, Escherichia coli (E, coli) D H1 (ATCC33849)
), E. coli HB 101 (ATC
C33694) etc. using the Hanahan method [DNA Cloning
ning), Vol. 1, pp. 109-135 (1985
)) to obtain various transformed strains.

c-DNA encoding alkaline earth metalase (hereinafter referred to as alkaline protease c-DNA) was obtained from the various transformed strains mentioned above.
To detect DNA), hybridization selection [molecular cloning (
Molecular Cloning), No. 329~
Pages 333 and 344-349, Cold Spring Harbor Laboratory
l+abor Laboratory) (Can be detected by going up the river in 1982.

Then, the incomplete alkaline protease c-DNA was
Nick translation method (Molecular Cloning) using 2p
), pp. 109-112, Cold Spring Harbor Laboratory
abor Laboratory) (1982), and J. Mol.

Biol, ), Vol. 113, pp. 237-251 (19
77)), then colony hybridization method [protein,
Nucleic Acids, Enzymes, Vol. 26, pp. 575-579 (1981
) A 1.1 Kb alkaline protease c-DN was obtained from a c-DNA scene bank library created using plasmid ρUC119 DNA as a vector.
Plasmid DNA containing A can be obtained.

In order to obtain DNA containing a gene encoding alkaline protease derived from Aspergillus oryzae (hereinafter referred to as alkaline protease gene) from the recombinant plasmid DNA thus obtained, restriction enzymes, For example, EcoRI is applied at a temperature of 30 to 40°C, preferably 37°C, for 1 to 24 hours, preferably 2 hours, and the resulting reaction-completed liquid is
Agarose gel electrophoresis [Molecular Cloning, No. 150]
Page, Cold Spring Harbor Laboratory (
Cold Spring Harbor Laborat
ory) (described in 19B2)), Aspergillus
DNA containing the alkaline protease gene derived from S. oryzae can be obtained.

Then, the base sequence of this alkaline protease gene is determined by the method shown in Item 9 of the Examples (see Figure 2), and then the amino acid sequence of the polypeptide translated by the gene having the base sequence is determined. (See Figure 3).

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

〔Effect of the invention〕

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

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Examples Cloning of alkaline protease c-DNA derived from Aspergillus aspergillus 1, Obtaining bacterial cells Aspergillus aspergillus oryzae (ATCC 2038)
6) spores (1.2xlO”@) were added to alkaline protease production medium [3% (W/V) heated and pressure-swelled defatted soybeans, 3% (W/V) K)12PO4) 50
ml, and a constant temperature shaker (manufactured by Taiyo Kagaku Kogyo Co., Ltd., M
-100') using 12Or. p. m. , temperature 30
A culture was obtained by performing shaking culture for 45 hours at °C, and the culture was filtered by a conventional method to obtain a microbial cell log.

2. Obtaining m-RNA 10 g of the bacterial cells obtained in item 1 was added to 20 ml of guanidine isothiocyanate solution (6M guanidine isothiocyanate/37.5mM sodium citrate (pH 7, 0).
) 10.75% (W/V) N-Laul:l It
Lt sarcosine sodium 10. 15M β-mercaptoethanol] was added to a cup-shaped blender (manufactured by Nippon Seiki Seisakusho Co., Ltd.), and 10 g of glass peas (diameter 0.5 mm) were added to the mixture.
0r, p. m, for 5 minutes, and then again for 10 minutes.
ml of water saturated phenol was added and the mixture was heated to 10.00 Or. p
．． The microbial cells were crushed by treatment with m for 10 minutes to obtain a crushed product.

The crushed material obtained in this manner was heated to 5,000 Or
.

The cells were centrifuged for 10 minutes at m, to obtain a bacterial cell disruption solution 20-.

Next, ultracentrifuge tube (manufactured by Hitachi 1 Machine Co., Ltd.)
4. Layer 5.7M cesium chloride solution of 1 and 2- in advance on a wooden plate, dispense the above-mentioned bacterial cell disruption solution on top of it so as to layer it, and place it in an ultracentrifuge (Hitachi 1 machine). Manufactured by 5C
P55H) at a temperature of 15°C130,00Or,p
, m, for 16 hours to obtain a precipitate.

The obtained precipitate was washed with cold 70% (V/V) ethanol, and the precipitate was washed with 10mM) Lys buffer (10mM).
M Tris-HCl buffer (pH 7,4) 15mM EDT
A/ 1% sodium dodecyl sulfate] To the suspension in 4 rnl, a mixture of the same amount of n-butanol and chloroform at a ratio of 4:1 (volume ratio) was added and extracted, and 3. Centrifuge at 000r, 20m for 10 minutes to separate into an aqueous layer and an organic solvent layer, add 4 Ui of the above 10mM) Lys buffer to this organic solvent layer, and repeat the above extraction and separation operations twice. To the resulting aqueous layer was added 1710 amounts of 3M sodium acetate (pH 5,
2) and the one with twice the amount of cold ethanol added to the temperature -
After leaving at 20°C for 2 hours, 8.000
Centrifuge at r, p, m for 20 minutes to precipitate the RNA, dissolve the obtained RNA in 4-ml water, perform the above ethanol precipitation procedure, and then dissolve the obtained RNA in 1 mf water. 12 mg of RNA was obtained.

In order to select m-RNA from this RNA, 12m
g RNA was subjected to oligo(dT)-cellulose column chromatography (manufactured by New England Biolabs).

A 2.5-Thermo syringe (manufactured by Terumo Co., Ltd.) was used as a column, and 0.5 g of resin was mixed with an elution buffer (10 mM Tris-HCl buffer (pH 7,6) / 1 mM EDT).
After swelling with A/0.1% (W/V) sodium dodecyl sulfate], the column was packed with binding buffer (1%
0mM) Squirrel salt # (pH 7,6)/1mM EDT
A/0.4 M NaCl/o, 1% sodium dodecyl sulfate].

Add the same amount of buffer solution [10mM Tris-HCl (pH 7,6), 71mM EDTA/0.8M
NaC1/0.1% sodium dodecyl sulfate] was added, heat treated at a temperature of 65 °C for 10 minutes, quenched in water, applied to an oligo(dT)-cellulose column, and the resin was washed with a binding buffer. Unbound r-RNA and t-
Thoroughly wash the RNA and further m-RN with elution buffer.
A was eluted and 90 ug of m-RNA was obtained.

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

A 10-25% (W/V) Sea IFu density gradient was prepared using a 40% (W/V) sucrose solution (50mM) in a Beckman Rotor SW41 polyaromatic tube.
Hydrochloric acid buffer (pH 7,5)/20mM NaC171m
M EDTA/40% (W/V) sucrose) 0.5+mc
and 2.4d each at 25% (W/V), 2
0% (W/V), 15% (W/V) and 10% (W/V)
It was produced by layering the sucrose solution of V) and leaving it at a temperature of 4°C for 24 hours. On this sucrose density gradient, mRN
A5Qμg is layered and 5W410 manufactured by Be7kuman Co., Ltd.
- 30.00 Or, p, m, , by the usual method using a
Centrifugation was performed at a temperature of 18°C for 18 hours. After centrifugation, the mixture was fractionated into 0.5 ml portions, m-RNA was collected by ethanol precipitation, and dissolved in 10 μl of water.

Next, by examining the proteins encoded by m-RNA, we identified both components enriched in alkaline protease m-RNA. Fractionated RNAIμ! ,
Rabbit reticulocyte lysate (manufactured by Amajam Co., Ltd.) 9
μ! and (ffs3) methionine 1μm (amajam.
150μiON E T ill liquid 150μ
M NaC 115mM EDTAlo, 02% (匈/V
) NaN: +/20mM) Lis-HCl buffer (pH
7,4) 10°05% (W/V) Nonidet P-40 (
Surfactant manufactured by Bethesda Research Laboratory, Inc.)
], 1 μl of anti-alkaline protease serum (prepared as described below) was added, and the mixture was left at 4°C for 18 hours, followed by 10 mg of protein A Sepharose (Pharmacia). The mixture was then centrifuged at 12,0 OOr, pom, for 1 minute in a conventional manner to recover the resin, ie, the protein A Sepharose described above.

The collected resin was added to 200 μl of NETl! Washed three times with street solution and added 40μ to this resin! Sample buffer for 5DS-PAGE (62,5mM) Lis-HCl buffer (p
H6,8) /10% (V/V) Gu'J Cero) Lt/
2% (W/V) Sodium dodecyl sulfate 15% (
V/V) Mercaptoethanol 10.02% (W/V)
Bromophenol blue] was added, boiled for 3 minutes at a temperature of 100°C, centrifuged for 1 minute at 12.00 Or, pom, by a conventional method, the supernatant was collected, and the total volume was reduced to 12% (
W/V) mounted on a sodium dodecyl sulfate-polyacrylamide gel.

Gel electrophoresis was performed using the method of Laemmli (
Nature, pages 227, 68
After electrophoresis, the gel was immersed in 10% (V/V) acetic acid for 30 minutes to fix the proteins, then immersed in water for 30 minutes, and then immersed in 1M sodium salicylate. The gel was immersed in the solution for 30 minutes and dried to obtain a dry gel, which was then subjected to fluorography using an X-ray film (RX, manufactured by Fuji Photo Film Co., Ltd.).

Through the above procedure, an alkaline protease protein band was observed on the X-ray film only when RNA from the fraction containing alkaline protease m-RNA was used, and both fractions enriched in alkaline protease m-RNA were detected. was able to be identified.

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

Alkaline protease solution with a concentration of 40 mg/mf 0.7
-, suspended in an equal volume of Freund's complete adjuvant, was used as an antigen at a weight of 2 kg.
After 2 weeks of rearing, the same amount of antigen as the first time was administered to the dorsal skin of Japanese colored rabbits, and after 1 week of rearing, the same operation was performed, and After one week of rearing, whole blood was collected.

The obtained blood was left at a temperature of 4°C for 18 hours, and then centrifuged for 15 minutes at 3.00 Or, pom, in a conventional manner to obtain anti-alkaline protease serum as a supernatant.

5. c-DNA ρ Synthesis c-DNA was synthesized using a kit manufactured by Amajam Co., Ltd.

Using 1.6 μg of m-RNA obtained as described above, Mol Cell Paiol (
Mo1. Ce1l R4ot, ), Vol. 2, p. 161 (1982) and Gene, Vol. 25, No. 2
As a result of following the method described on page 63 (1983), 1
Two strands of jJc-DNA of 60 ng were obtained.

Using a c-DNA cloning kit manufactured by Amajam Co., Ltd., 160 ng of the c-DNA thus obtained was treated with the restriction enzyme Ec according to the method instructed by Amajam Co., Ltd.
.

Methylation of the R1 cleavage site and further c-DNA
EcoRI linkers were attached to both ends.

6. Construction of c-DNA bank Plasmid pUc119DNA (manufactured by Takara Shuzo Co., Ltd.) 1
00ng dissolved in 8μρ of water, add 1μl of M
ed buffer (100mM Tris-HCl buffer (p
After adding H7,5)/100mM MgCIz/10mM dithiothreitol 1500mM NaC 1:1, 10 units (1μl) of restriction enzyme 1xR1 (manufactured by Takara Shuzo Co., Ltd.) was added thereto, and the mixture was incubated at a temperature of 37°C. °C
The cutting process was carried out for 2 hours.

Next, 1 μl of IM Tris-HCl buffer (pH 8,0) and 0.3 units (1 μl
) of alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.) was added, and enzymatic reaction was performed at a temperature of 65°C for 1 hour to dephosphorylate both ends of the cleaved product. To this, 12 μl of water-saturated phenol was added. After deproteinization using a water filter, 1 μl of 3M sodium acetate (pH 5, 8) was added to the collected aqueous layer.
) and 26 μl of cold ethanol were added, respectively, and the temperature was -7.
Leave at 0″C for 15 minutes, then use a microcentrifuge [■Tomy Seiko,
DNA was recovered by centrifugation using MRX-1501 at 12.00 Or, p, m for 5 minutes.

Digested with the restriction enzyme EcoRI obtained in this way,
and plasmid vector pUc1 with both ends dephosphorylated
19DNA 1100n and CD prepared in item 5
A mixture of 160 μg of NA was suspended in 8 μl of water, and then 1 μl of ligation buffer (
20mM MgC! , /66mM) Lis-HCl buffer (pH 7,6) / 1mM ATP/15mM dithiothreitol) was added to 1 unit (1u
℃) T4 DNA ligase (manufactured by Zenshuzo Co., Ltd.)) was added, and the mixture was left to stand at a temperature of 16°C for 16 hours to perform ligation of plasmid vector DNA and c-DNA to obtain a reaction product.

Using this reaction product, the method of Ilanahan [DNA Cloning, Vol. 1, pp. 109-135 (1985
)) was used to transform Escherichia coli DHI strain (ATCC33849) to create a c-DNA bank using plasmid pUc119 DNA as a vector.

7. Search hybridization selection of alkaline protease c-DNA fragments [Molecular Cloning
), page 329, Cold Spring Harbor
A search for alkaline protease c-DNA was conducted according to Laboratory (1982). This will be explained in detail below.

Any Escherichia coli 7 from the c-DNA bank obtained in item 6
0 stocks were selected and the following operations were performed for each. Molecular Cloning (Molecular C1C1)
o-n1n, No. 86E4, Cold Spring Barber Laboratory (19B2),
500 μg of recombinant plasmid DNA was obtained from E. coli in the c-DNA bank. Of this, 100μg
Water 200μ! The denatured recombinant plasmid DNA was dissolved in water and left at 100°C for 10 minutes, then transferred to water and rapidly cooled. 200 μ2 of 1M sodium hydroxide was added and left at room temperature for 20 minutes. Obtained.

Next, 200 μl of a neutralizing solution [1M sodium chloride 10.3M sodium citrate 10.5M 1-Lis-HCl buffer (pH 8°0)/I
After quickly adding M hydrochloric acid and mixing, the mixture was transferred to ice and rapidly cooled. This solution was filtered using a circular nitrocellulose filter with a diameter of 5 mm (manufactured by Millipore, catalog number 1 (AWP 02500)), and the denatured recombinant plasmid DNA was adsorbed onto the nitrocellulose filter. After air drying in the usual manner,
After washing with 6X5SC (0.9M sodium chloride, 10.09M sodium citrate) and air-drying in a conventional manner, it was heated in a vacuum oven maintained at a temperature of 80"C for 2 hours.
The recombinant plasmid DNA was allowed to stand for a period of time to be fixed on the nitrocellulose filter.

Then 10 μi of hybridization solution (10
0ug/me m-RNA (from 3II in item 2) 765% (V/V) deionized formamide/
20mM 1°4-piverazine diethanesulfone [J (
1) H6,4) 10.2% sodium dodecyl sulfate 1
0.4M sodium chloride/1100u/lri yeast t
The nitrocellulose filter on which the recombinant plasmid DNA obtained as described above was immobilized was immersed in the recombinant plasmid DNA (-RNA) and left at a temperature of 50°C for 3 hours. Hybridization was performed. After the reaction, take out the filter and add washing solution 1 (10mM) Lis-hydrochloric acid buffer (pH 7,6) 10.15M sodium chloride/1mM
EDTAlo, 5% Sodium Dodecyl Sulfate]9
After washing twice, wash twice with 1ml of washing solution U (10mM) Lis-HCl buffer (pH 7,6)/0.15M sodium chloride/1mM EDTA).
m-RNA that did not hybridize with the plasmid DNA immobilized on the filter was removed.

Next, this nitrocellulose filter was
! 10 μg of yeast t-RNA was added thereto, and the mixture was left in hot water at 100°C for 1 minute, then transferred into ethanol cooled with dry ice, and then left at room temperature to dissolve. The thus hybridized m-RNA was peeled off from a nitrocellulose filter. To the resulting aqueous solution, add 10μρ of 3M sodium acetate (pH 5.2).
and 300 μI of cold ethanol, temperature -70
After leaving it at °C for 1 hour and performing ethanol precipitation, the above hybridization was performed using a microcentrifuge (Tomy Seiko, MRX-150) for 5 minutes at 12,000 Or, p, m. The resulting m-RNA was collected.

Next, the protein encoded by the recovered m-RNA is synthesized in the same manner as described in item 3, and the protein encoded by the recovered m-RNA is synthesized.
Using the anti-alkaline protease serum obtained in , it was analyzed whether the synthesized protein was alkaline protease or not.

As a result, a positive result was obtained for 1 out of 70 strains. Recombinant plasmid DN possessed by this strain
It can be concluded that A (designated pOAP3) contains a fragment of alkaline protease c-DNA derived from Aspergillus oryzae (ATCC 20386).

Next, recombinant plasmid pOAP3 DNA 1
μg was treated with the restriction enzyme EcoRI by the method described in item 6, and the mobility pattern was analyzed by agarose gel electrophoresis, and the obtained pattern and plasmid pBR322
By comparing the standard pattern of DNA fragments obtained by digesting DNA (manufactured by Takara Shuzo Co., Ltd.) with the restriction enzyme Alul, the recombinant plasmid pOAP3DN was determined.
Alkaline protease c-D N inserted into A
The size of the A fragment was found to be 750 bp.

8. Search for large alkaline protease c-DNA From the agarose gel obtained in item 7, cut out the agarose gel containing the 750 bp alkaline protease c-DNA fragment, put it in a dialysis tube, and add 500 μf of T.
E buffer (10mM l-lis-hydrochloric acid buffer (pH 8,
After adding 0)/1 mM EDTA), the dialysis tube was sealed, and an equal volume of water-saturated phenol was added to the solution obtained by eluting the DNA from the gel into the buffer solution by electrophoresis, and the mixture was stirred. Then, the aqueous layer was collected, and 1100n DNA was collected by ethanol precipitation according to a conventional method.

This 1100n alkaline protease c-DNA,
(α-”P) was labeled by the nick translation method using dCTP (manufactured by Amajam Co., Ltd.).Nick translation was carried out using J. Mo1. 1st
13, pp. 237-251 (1977) and Molecular Cloning.
g), pp. 109-112 (1982). Using the 32p-labeled alkaline protease c-DNA fragment prepared in this way as a probe,
The c-DNA bank obtained in item 6 was searched using the colony hybridization method [Proteins/Nucleic Acids/Enzymes, Vol. 26, pp. 575-579 (1981)], and colonies containing alkaline protease c-DNA were obtained. . Recombinant plasmid D possessed by one of the colonies
The NA was named pOAP 5, and recombinant plasmid DNA was prepared according to Item 7.

The E. coli containing the recombinant plasmid DNA was transformed into E. coli D H1 (pOAP 5 ).
It was named.

The transformed strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiology Research Institute No. 9870 (FERM P-987
0).

When the recombinant plasmid pOAP5 DNA was treated with the restriction enzyme EcoRI in the same manner as described in Item 6 and subjected to agarose gel electrophoresis, the size of the inserted DNA fragment was found to be 1.100 bp. Then, in the same manner as described in item 8, this 1,100bp
0.1 μg of a DNA fragment containing alkaline protease c-DNA was isolated and purified. The recombinant plasmid pOAP5 DNA was subjected to restriction enzyme map ■, 1 parallel R1, KL.
The recombinant plasmid pOAP was determined by performing single or double digestion of
A restriction enzyme map of 5DNA was prepared, and the map is shown in FIG.

9. Determination of base sequence of alkaline protease c-DNA Each alkaline protease c-D obtained in item 8
Plasmid pUc18 and pUc19 DNA obtained by cutting the NA fragment with the same or the same restriction enzymes
(manufactured by Takara Shuzo Co., Ltd.).

Sea fencing uses 44 methods to extract plasmid DNA.
Vector DNA, p. 70, Kodansha (1986)], the DNA was denatured with alkali, and then the dideoxy chain termination method was carried out using the M13 Sequencing Kit (manufactured by Takara Shuzo Co., Ltd.) in a conventional manner.

In this way, Aspergillus oryzae (ATCC2)
The nucleotide sequence of the alkaline protease c-DNA fragment derived from 0386) was determined, and among the obtained nucleotide sequences, the nucleotide sequence corresponding to mature alkaline protease was
In addition, the amino acid sequence of the polypeptide translated from the gene having the base sequence shown in FIG. 2 is shown in FIG. 3.

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

The N-terminus of the amino acid sequence deduced from this DNA base sequence contains the 16 N-terminal amino acid sequences of purified alkaline protease (Gly Leu Thr Thr Gl
uLys Ser Ala Pro Trp Gly
Leu Gly Ser Ile 5er) was confirmed (underlined part in Figure 3).

From the above findings, it can be concluded that the base sequence shown in Figure 2 encodes the C-terminal rather than the N-terminal portion of mature alkaline protease.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a restriction enzyme map of the recombinant plasmid pOAP5 DNA, and FIG. 2 is a diagram showing the nucleotide sequence of the gene corresponding to the mature alkaline protease described in item 9 of Examples. , FIG. 3 is a diagram showing the amino acid sequence of a polypeptide translated from a gene having the base sequence shown in FIG. 2. Applicant: Food Industry Enzyme Function Conversion Technology Research Association Representative, Patent Attorney: Yusuke Hiraki (Figure 2) GGC AG TG GGC CT GGC GGC AC AG AC AG TC GT AC GGC AC GGC CT GG GT GT GGC TG GGC GGC GGC TT GGC AC AG GGC AG AC CT

Claims (2)

[Claims] (1) An alkaline protease gene derived from a microorganism belonging to the genus Aspergillus and defined by the restriction enzyme cleavage map shown below. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, E is EcoR I, A is AflII, K is Kpn
I, X represents Xmn I, S represents Sal I) (2) The alkaline protease gene according to claim 1, wherein the alkaline protease gene encodes the amino acid sequence shown below. [There is a gene sequence]