JPH0223871A - Prepro type alkaliprotease gene - Google Patents

Abstract

NEW MATERIAL:The title gene prescribed in restriction enzyme cleavage map expressed by formula I (E is Eco RI; A is Afl II; K is Kpn I; X is Xmn I; S is SalI; B is Bgl II; H is Hind III) derived from a microorganism belonging to the genus Aspergillus. USE:An alkali protease is efficiently obtained. PREPARATION:c-DNA is synthesized from an alkaliprotease m-RNA derived from Aspergillus oryzae (ATCC 20386) and various kind of transformed strains are obtained through a vector-DNA. Imperfect alkaliprotease c-DNA is labeled with <32>P and plasmid DNA containing alkali protease c-DNA of 1.5Kb is obtained using the above-mentioned c-DNA as a probe and DNA containing the title gene is obtained from a liquid after completion of reaction of the plasmid DNA with a restriction enzyme by an agalose gel electrophoresis method.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a prepro-type alkaline protease gene.

(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.

[Problems to be Solved by the Invention] Therefore, the present inventors conducted various studies on the alkaline protease gene derived from Aspergillus oryzae with the aim of providing an alkaline protease gene. We succeeded in isolating and determining the structure of a gene for the first time, and completed the present invention.

[Means for Solving the Problem] The present invention is derived from a microorganism belonging to the genus Aspergillus,
This is a prepro-type alkaline protease gene defined by a restriction enzyme cleavage map.

EHBS X KAE (where E is EcoRI, A is Aflll, K is Kp
nI, X is XmnT, S is 5all, B is BglII,
(H indicates Hind m) This Proble-type alkaline protease gene has a DNA sequence encoding the amino acid sequence shown below.

Met　Gin　5er Leu Gly Ala Ala Pro Val Glu Lys Leu Phe Lys Pr.

Gln Glu His His　Gln　Arg Thr Gly Gly Arg Asn Tyr Glu Glu l1e Asp Gly Leu Trp Gly Leu lie Lys Arg lie Leu Pr.

Gin Glu Thr Pro Gly Lys Gly Tie Asp Thr　Thr　Trp Ser　Leu　Glu Asp Leu Pr.

Lys Ile Asn Arg Lys Asn Thr　Thr　Gln Gly Ser 1ie Thr　Leu　Leu Ala Val Leu Arg Arg Ala Tyr Ile Val Glu　Ala　Lys Ala Thr Asn Arg　Arg　Gly Val Gly Tie Lys Phe Ala Glu Asp Val Lys Ser Ala Ser　His　lys 1a Pr.

1y Gln Gln Ser Thr Asp Tyr
Ile Tyr Asp ThrLeu Asp
Gly Phe Asn Trp Ala Ala
Asn Asp 8l Ala Gly A
sn Glu Asn Ser Asp A
La Glylle Thr Val Ala
Ala Ile Gln Lys Ser
To Asn8s Arg Ala Ser
Phe Ser Asn Phe Gly
Lyslle Leu Ser Ala Tr
p lie Gly Ser Ser 5erAl
a Thr Asn Thr Ile Ser Gl
y Thr Ser Met Below, the present invention will be described in detail.

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

Next, the above microorganisms are cultured in exactly the same manner as described in Japanese Patent Publication No. 48-38873 to obtain a culture, and the culture is subjected to conventional methods such as filtration, centrifugation, etc. to isolate Aspergillus oryzae bacteria. Get a body.

To prepare m-RNA from the cells of Aspergillus oryzae, for example, except for using glass beads and phenol when disrupting the cells, for example, "Molecular Cloning J (Molecular CIo
niB), page 196, Cold Spring Bar
Bar Laboratory (Cold Spring Har
bor Laboratory) (1982) and "Molecular Genetics Experimental Methods", Yasuo Koseki, Reibu Shimura, No. 66-67
(1983).

From the obtained m-RNA, z-RNA coats alkaline protease (hereinafter referred to as alkaline protease m-RN).
For example, to concentrate [Biomedical Research (referred to as A)]
rch) J Vol. 3, pp. 534-540 (1982)
It can be carried out by the method described.

At this time, an anti-alkaline protease serum against alkaline protease is used;
For example, "Immunochemistry", Yu Yamamura, pp. 43-50 (19
73) can be obtained by the method described.

To synthesize c-DNA from alkaline protease m-RNA, for example, [Mole Cell Piol] (Mo
1. Ce1l Biol, ), Volume 2, Page 161 (1
982) and 1 Gene, Vol. 25, p. 263 (1983).

The c-DNA thus obtained is then transformed into vector DNA, for example, plasmid plJc119D N
A (manufactured by Takara Shuzo Co., Ltd.) etc. to obtain various recombinant plasmid DNAs, and using the DNAs, for example, Escherichia coli (E coli) D H1 (ATCC33849),
Escherichia coli (E.

coli) HB 101 (ATCC 33694) etc. using the Hanahan method ["DN A Cloning J,
1, pp. 109-135 (1985)) to obtain various transformed strains.

The c-DNA encoding alkaline protease (hereinafter referred to as alkaline protease c-
To detect DNA), hybridization selection ["Molecular Cloning J, Vol.
Pages 29-333 and 344-349, Cold Spring Harbor Laboratory (Cold Spr.
(1982)
year)].

Then, the incomplete alkaline protease c-DNA was
Nick translation method using zp [Molecular Cloning J
ng), pp. 109-112, Cold Spring
Harbor Laboratory (Cold Spring 1)
abor Laboratory) (1982), and “J. Mo1.
ol, ), Vol. 113, pp. 237-251 (1977
)), and then colony hybridization method using the c-DNA as a probe [Protein/Nucleic Acids/Enzymes, Vol. 26, pp. 575-579 (1981
) A 1.5 Kb alkaline protease c was obtained from a c-DNA scene bank library created using plasmid pUc119 DNA as a vector.
- It is possible to obtain plasmid DNA containing DNA.

Then, from the recombinant plasmid DNA thus obtained, a DNA containing a gene encoding alkaline protease derived from Aspergillus oryzae having a prepro region (hereinafter referred to as prepro-type alkaline protease gene) was obtained. Add a restriction enzyme, such as EcoRI, to the plasmid DNA at a temperature of 30°C.
The reaction was carried out at ~40°C, preferably 37°C, for 1 to 24 hours, preferably 2 hours, and the resulting reaction solution was subjected to agarose gel electrophoresis ["Molecular Cloning"]
(Molecular Cloning), No. 150
Page, Cold Spring Harbor Laboratory (
Cold Spring Harbor Laboratory
(1982)], it is possible to obtain DNA containing the prepro-type alkaline protease gene derived from Aspergillus oryzae.

Then, the base sequence of this prepro-type alkaline protease gene is determined by the method shown in Item 11 of Examples (see Figure 5), and then the amino acid sequence of the polypeptide translated by the gene having the base sequence is determined. (See Figure 6).

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

Furthermore, the base sequence of the gene of the present invention is preferably shown by base numbers 53 (A) to 1261 (T) in FIG.

[Effects of the Invention] As is clear from the above, according to the present invention, by culturing, for example, a microorganism in a medium containing a recombinant DNA into which the prepro-type alkaline protease gene of the present invention has been incorporated, an extremely short period of time can be achieved. The present invention is industrially extremely useful because alkaline protease can be obtained efficiently 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.

Example yellow koji mold Aspergillus oryzae (ATCC20386
) Cloning of prepro-type alkaline protease c-DNA derived from Aspergillus oryzae (ATCC20386)
) spores (1.2 x 10'') were inoculated into 50 ml of alkaline protease production medium [3% (W/V) heated and pressure-swelled defatted soybeans, 3% (W/V) KH, Po4). Constant temperature shaker (manufactured by Taiyo Kagaku Kogyo Co., Ltd., M-100'
”) using 12Or, p, m, 4 at a temperature of 30°C.
A culture was obtained by performing shaking culture for 5 hours, 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 a 20 m/m guanidine isothiocyanate solution (6 M guanidine isothiocyanate/37.5 mM sodium citrate (pH 7,0
) 10.75% (W/V) N-Rau0IB/sarcosine sodium 10.15M β-mercaptoethanol) was added to a cup-shaped blender (manufactured by Nippon Seiki Seisakusho Co., Ltd.), and further glass peas (
0.5mm in diameter), 10.0OOr, p, m,
After 5 minutes of treatment, 10 rnl of water-saturated phenol was added and the mixture was heated at 10.00 Or, p, m for 10 minutes.
The microbial cells were crushed by treatment for a minute to obtain a crushed product.

The thus obtained crushed material was collected using a refrigerated centrifuge (manufactured by Hitachi Ichiki Co., Ltd., 18PR-52) at 5.00 Or, p, m.
, centrifuged for 10 minutes to remove 20 rnl of the cell suspension.
I got it.

Next, 1.2 ml of 5.7M cesium chloride solution was layered in each of four ultracentrifuge tubes (manufactured by Hitachi Ichiki Co., Ltd.) in advance, and then the above bacterial cell disruption solution was layered on top of the 5.7M cesium chloride solution. 5cP5, ultracentrifuge (manufactured by Hitachi Ichiki Co., Ltd.)
5H) at a temperature of 15°C, 130,00 Or, 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 Tris buffer (10mM
Tris-HCl buffer (pH 7,4) 15mM EDTA
/ 1% sodium dodecyl sulfate) 4/, and the same amount of n-butanol and chloroform was added in a ratio of 4:1.
(volume ratio) is added and extracted.
Centrifuge for 10 minutes at 3,000 Or, p, m in a conventional manner to separate into an aqueous layer and an organic solvent layer, add the above 1101II Tris buffer 4- to this organic solvent layer, and perform the above extraction and separation operations. To the aqueous layer obtained by repeating the above procedure twice, 1,710 volumes of 3M sodium acetate (pH 5,2) and twice the volume of cold ethanol were added, and the mixture was heated to -20°C.
After leaving for 2 hours at 8゜000r, p,
After centrifugation for 20 minutes at m, to precipitate the RNA, dissolve the obtained RNA in 4 mf of water, perform the above ethanol precipitation operation, dissolve the obtained RNA in 1 mf of water,
12 ■ RNA was obtained.

In order to select m-RNA from this RNA, 12
mg of RNA was subjected to oligo(dT)-cell 0-se (manufactured by New England Hyolabo) column chromatography.

A 2.5 Cotermo syringe (manufactured by Terumo Corporation) was used as a column, and 0.5 g of resin was mixed with elution buffer (10 mM) Lis-HCl buffer (pH 7,6) / 1 mM EDTA.
/ 0.1% (enemy/V) sodium dodecyl sulfate]
After swelling with water, the column was filled with binding buffer (10
mM) Lis-HCl (pH 7,6)/1mM EDT
A/0.4 M NaCl/o, 1% sodium dodecyl sulfate].

Add the same amount of buffer [1101II Tris-HCl (pH 7,6)/1mM EDTA/0.8 to 12μ of RNA.
M NaCl/o, 1% sodium dodecyl sulfate] was added, heat treated at a temperature of 65 °C for 10 min, quenched in water, applied to an oligo(dT)-cellulose column, and the resin was washed with binding buffer. , unbound r-RNA
and t-RNA were thoroughly washed, and further washed with elution buffer.
-RNA was eluted and 90% g of m-RNA was obtained.

3. Concentrated batter of alkaline protea-zem-RNA,
Alkaline protease m-RNA was concentrated by sucrose density gradient centrifugation.

A 10-25% (W/V) sucrose density gradient was applied to a Beckman rotor SW41 polyaromatic tube.
0% (W/V) CytF' solution (50mM)
Hydrochloric acid buffer (pH 7,5)/20mM NaCl 71
mM EDTA/40% (W/V) sucrose) 0.5
- and then 2.4 mj 25% (W/V)
, 20% (W/V), 15% (-c) and 10% (W
/V) (D'i Layer sucrose solution and heat at 4°C for 2 hours.
It was produced by leaving it for 4 hours. 50 μg of m-RNA was layered on this sucrose density gradient, and the mixture was incubated at 30.00
r, p, m, centrifugation was performed at a temperature of 18"C for 18 hours. After centrifugation, fractionation was performed in 0.5 d increments, m-RNA was recovered by ethanol precipitation, and dissolved in 10 μl of water. did.

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) 9μ
! and (353) methionine 1μ! (Manufactured by Amajam)
were mixed and reacted at a temperature of 30°C for 30 minutes,
150μl NET buffer (150mM NaC115
mM EDTAlo, 02% (W/V) NaNz/20
mM Tris-HCl buffer (p) 17.4) 10.05%
(W/V) Nonidet P-40 (manufactured by Bethesda Research Laboratory, surfactant)] and further 1 μl
anti-alkaline protease serum (prepared as described below) was added and left at a temperature of 4°C for 18 hours, 10μ of Protein A Sepharose (manufactured by Pharmacia) was added, and the mixture was incubated at a temperature of 20°C. After being left at C for 30 minutes, the mixture was centrifuged for 1 minute at 12,0 OOr, p, m in a conventional manner to recover the resin, ie, the protein A Sepharose described above.

The recovered resin was washed 3 times with 200 μl of NET buffer, and 40 μl of 5DS-PAGE sample buffer [62.5 mM] Lis-HCl buffer (pH
6,8) / 10% (V/V) Gree-f=O-)L
// 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, p, m 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 [
After electrophoresis, the gel was immersed in 10% (V/V) acetic acid for 30 minutes to fix the proteins, and then immersed in water. The gel was immersed for 30 minutes, further immersed in a 1M sodium salicylate solution for 30 minutes, dried, and the dried gel was subjected to fluorography using fLX&! film (RX, manufactured by Fuji Photo Film Co., Ltd.).

By the above procedure, the alkaline protease protein band was observed on the X-ray film only when both RNA fractions containing alkaline protease m-RNA were used, and the fraction enriched with alkaline protease RNA was detected. I was able to identify it.

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

40mg/m 14 degrees alkaline protease solution 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 intradermally to the back of Japanese white rabbits, and after 1 week of rearing, the same procedure 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 centrifuged for 15 minutes at 3.00 Or, p, m in a conventional manner to obtain anti-alkaline protease serum as a supernatant.

5. Synthesis of c-DNA c-DNA was synthesized using a kit manufactured by Amajam.

1.6 μg of the m-RNA obtained as described above was used as directed by Itea Masham [Mole Cell Paiol J (
Mo1. Ce1l Biol, ), Vol. 2, p. 161 (1982) and 1 Gene, Vol. 25,
As a result of following the method described on page 263 (1983),
160 ng of double-stranded CDNA was obtained.

160 ng of the c-DNA thus obtained was treated with the restriction enzyme EcoR using a c-DNA cloning kit manufactured by Amajam Co., Ltd. according to the method instructed by Amajam Co., Ltd.
(2) Methylation was performed at the cleavage site, and 1-line R1 linkers were attached to both ends of the c-DNA.

6. Construction of c-DNA bank Plasmid pUc119DNA (manufactured by Takara Shuzo Co., Ltd.) 100
ng dissolved in 8 μl of water, add 1 μl of Med
Buffer [100mM Tris-HCl buffer (pH
7,5)/100mM MgCh/10mM dithiothreitol/500mM NaCl), 10 units (1 μm) of restriction enzyme Tatsunami R1 (manufactured by Takara Shuzo Co., Ltd.) was added thereto, and the mixture was incubated at a temperature of 37°C. 2
A time cutting process was performed.

Next, lul of IM Tris-HCl buffer (pH 8,0) and 0.3 units (1 μl
) was added to the alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.) and subjected to enzymatic reaction treatment 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 and removed. After protein analysis, the collected aqueous layer was treated with 3M sodium acetate (pH 5,8) at lμ and 2
Add 6μ2 of cold ethanol to each, leave at -70''C for 15 minutes, and use a microcentrifuge [Tomy Seiko, MRX-
1501, 12.00 Or, p, m.

The DNA was collected by centrifugation for 5 minutes.

Digested with the restriction enzyme EcoRI obtained in this way,
and plasmid vector pUc1 with both ends dephosphorylated
19 D N A 1100n and C prepared in item 5
After suspending a mixture of 160% DNA in 8 μl of water, add 1 μl of ligation buffer (20 mM MgCh/66 mM) Lis-HCl buffer (pH 7,6) / 1 mM TP. /15mM dithiothreitol] to which 1 unit (1
μf! 74 DNA ligase (manufactured by Takara Shuzo Co., Ltd.)) was added, and the mixture was left at a temperature of 16°C for 16 hours to perform ligation of plasmid vector DNA and CDNA to obtain a reaction product.

Using this reaction product, the method of Hanahan ["DNAC Cloning J (DNAC
loning), Vol. 1, pp. 109-135 (198
5) ] to Escherichia coli DHI strain (ATCC33849)
was transformed to create a c-DNA bank using plasmid ρUC119DNA as a vector.

7. Search hybridization selection of alkaline protease c-DNA fragments [“Molecular
Cloning J (Molecular Cloning
), p. 329, Cold Spring Barber Laboratory (1982)), the search for alkaline protease c-DNA was conducted. This will be explained in detail below.

Seventy arbitrary E. coli strains were selected from the c-DNA bank obtained in item 6, and the following operations were performed on each strain.

"Molecular Cloning"
500 μg of recombinant plasmid DNA was obtained from E. coli in the c-DNA bank by the method described in Cold Spring Barber Laboratory (1982). 100μ of this
g was dissolved in 200 μl of water, left at a temperature of 100°C for 10 minutes, then transferred to water and rapidly cooled.
μl of 1M sodium hydroxide was added and allowed to stand at room temperature for 20 minutes to obtain a denatured solution of recombinant plasmid DNA.

Next, add 200μ! to the denatured solution thus obtained. Neutralization solution [1M sodium chloride 10.3M sodium citrate 10.5M Tris-HCl buffer (pH 8,0)/IM
After quickly adding hydrochloric acid and mixing, the mixture was transferred to ice and rapidly cooled. This solution was filtered through a circular nitrocellulose filter with a diameter of 5 mm (manufactured by Millipore, catalog number HAW).
The denatured recombinant plasmid DNA was adsorbed onto a nitrocellulose filter, air-dried using a conventional method, and then filtered using 6X5S.
After washing with C (0.9M sodium chloride 10.09M sodium citrate) and air-drying using a conventional method, the recombinant plasmid DNA was left in a vacuum oven kept at 80°C for 2 hours. It was fixed on a cellulose filter.

Then add 10 μl of hybridization solution (10
00μlml m-RNA (prepared in item 2)
765% (V/V) deionized formamide 720
mM 1.4-piverazine diethanesulfonic acid (pH 6,
4) 10.2% sodium dodecyl sulfate 10.4M sodium chloride/100 u g/wI yeast t-RNA: l
The nitrocellulose filter on which the recombinant plasmid DNA obtained as described above was immobilized was immersed in the solution at a temperature of 50°C.
The filter was left for 3 hours at °C, and the recombinant plasmid DNA on the filter was hybridized with m-RNA. After the reaction, take out the filter and add 1 part of washing solution [10mM Tris-HCl buffer (pH 7,6) 1 part]
0.15M sodium chloride/1mM EDTAlo,
After washing nine times with 5% sodium dodecyl sulfate,
m! Washing solution U (10mM) Lis-HCl buffer (p
H7,6) 10.15 M Sodium Chloride/1mM E
M-R that has not been hybridized with the Blasumido DNA fixed on the filter after washing twice with DTA)
Nitrogenization was removed.

Next, this nitrocellulose filter was
1 of water, 10 μg of yeast t-RNA was added, and the mixture was left in hot water at 100°C for 1 minute, transferred to 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. Add 10 μl of 3M sodium acetate (pH 5,2) to the resulting aqueous solution.
and 300μ! of cold ethanol, temperature -70
After standing at °C for 1 hour and performing ethanol precipitation,
1 using microcentrifugation [Tomy Seiko, MRX-150]
The hybridized m-RNA was recovered by centrifugation at 2.0 OOr, pom, for 5 minutes.

Next, the protein encoded by the recovered m-RNA was synthesized in the same manner as described in item 3, and the anti-alkaline protease serum obtained in item 4 was used to determine whether the synthesized protein was an alkaline protease or not. We analyzed whether or not.

As a result, a positive result was obtained for 1 out of 70 strains. The recombinant plasmid DNA possessed by this strain (
Aspergillus (named pOAP3).

It can be concluded that a fragment of alkaline protease c-DNA derived from S. oryzae (ATCC 20386) was inserted.

Next, recombinant plasmid pOAP3 DNA
1 μg was added to the restriction enzyme EcoRI by the method described in item 6.
The mobility pattern was analyzed by agarose gel electrophoresis, and the obtained pattern and plasmid pBR32
By comparing the standard pattern of the DNA fragment obtained by digesting 2D DNA (manufactured by Takara Shuzo Co., Ltd.) with the restriction enzyme AluI, the recombinant plasmid pOAP3DNA was determined.
Alkaline protease c-DNA inserted into
The size of the fragment was found to be 750 bp.

8. Cut out the agarose gel containing a 750 bp alkaline protease c-DNA fragment from the agarose gel obtained in search item 7 for large alkaline protease c-DNA, and add 500 μl to the dialysis tube.
TE buffer [:10mM] Lis-HCl buffer (p
After adding H8,0) / 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 stir, collect the aqueous layer,
1100n DNA was obtained by ethanol precipitation according to the conventional method.
was recovered. This 1100n alkaline protease c
- DNA, [a-”P) dCTP (7 Masiyam l
) using the nick translation method. Nick translation is based on Takara Shuzo's instructions.
J,Mo1.Biol” (J,Mo1.Biol,)
, Vol. 113, pp. 237-251 (1977) and [
Molecular Cloning J
Cloning), pp. 109-112 (1982). Using the 3Zp-labeled alkaline protease c-DNA fragment prepared in this way as a probe, the c-DNA puncture obtained in item 6 was subjected to colony hybridization [[Proteins, Nucleic Acids, Enzymes], Vol. 26, pp. 575-579 (1981
)), alkaline protease c-DN
A colony with A was obtained. The recombinant plasmid DNA of one of the colonies was named pOAP 5, and the recombinant plasmid DNA was prepared according to Item 7.

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

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 in the method described in item 8, a DNA fragment containing this 1,100 bp alkaline protease c-DNA was obtained.
1 μg was isolated and purified. Recombinant plasmid pOA
P5 DNA was digested with the restriction enzyme AflII, indented into R1,
Recombinant plasmid pOAP5 was determined by performing single or double digestion of pOAP5all and MatakunI and analyzing the size of the resulting fragments in the same manner as described in section 7.
A restriction enzyme map of the DNA was prepared, and the map is shown in FIG.

9. Determination of base sequence of alkaline protease c-DNA Each alkaline protease c-DNA obtained in item 8
Plasmids pUc18 and pUc19 DNA in which the A fragment was cut with the same or the same restriction enzymes (
(manufactured by Takara Shuzo Co., Ltd.). Sea fencing involves converting plasmid DNA into Sakaki's method ([vector DNA,
70, Kodansha (1986)], and then the dideoxy chain termination method was carried out in a conventional manner using M13 Sequencing Kit (manufactured by Takara Shuzo Co., Ltd.).

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

IO, alkaline protease full-length c-DNA search As a result of nucleotide sequencing in item 9, the recombinant plasmid pOAP 5 obtained in item 8 encodes a C-terminal partial host rather than an N-terminal portion of mature alkaline protease. However, the prepro region, which is thought to be located upstream, was missing. Therefore, as in item 8, alkaline protease c-
After isolating the DNA fragment, it was labeled with 32p using the nick translation method, and used as a probe to search the c-DNA bank obtained in item 6 again using the colony hybridization method. Colonies containing protease c-DNA were obtained. The recombinant plasmid possessed by one of the colonies was named pOAPlo, and the E. coli containing the recombinant plasmid was named E. coli D H1 (pOAPlo).
It was named Plo).

According to item 7, recombinant plasmid pOAP10 was prepared.

When the recombinant plasmid pOAP10 DNA was treated with the restriction enzyme EcoRI in the same manner as described in item 7 and subjected to agarose gel electrophoresis, it was found that the inserted DNA was
The size of the A fragment was found to be 1,500 bp. Furthermore, recombinant plasmid pOAP10 DNA
Restriction enzyme map ■, Ashinami RI, K box 1, S sword', Xmn
1. The recombinant plasmid p
A restriction enzyme map of O^PIO DNA was prepared, and the map is shown in FIG.

11. Determination of base sequence of alkaline protease full-length c-DNA Recombinant plasmid pOAP10 obtained in item 10
The alkaline protease c-DNA fragment was cloned into plasmid pUc18 and pUc19 DNA cut with the same or the same restriction enzymes in the same manner as in item 9. Sea fencing was performed using the dideoxy chain termination method as in item 9.

In this way, Aspergillus oryzae (ATCC2)
The base sequence of the alkaline protease c-DNA derived from 0386) was determined, and the entire base sequence is shown in Figure 5, and the amino acid sequence of the polypeptide translated from the gene having the base sequence shown in Figure 5 is shown in Figure 6. They are shown in the figure.

The translated polypeptide consists of 403 amino acids,
16 amino acid sequences at the N-terminus of purified alkaline protease (Gly Leu ThrThr Glu Ly
s Ser Ala Pro Trp Gly Leu
Gly 5er11e 5er) was defeated by the amino acid sequence starting from position 122 (underlined part in Figure 6). Therefore, the amino acid sequence from the N-terminus to the 121st position is a prepro region necessary for secretion of alkaline protease, and 1
The amino acid sequence from positions 22 to 403 is a region encoding mature Arca JJ protease.

Based on the above findings, it can be concluded that the base sequence shown in FIG. 5 encodes the entire polypeptide of alkaline protease including the prepro region.

[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. Figure 4 shows the recombinant plasmid pOAP10D N
This is the restriction enzyme sequence of A. FIG. 5 shows the base sequence of the gene corresponding to the full-length alkaline protease (prepro type) described in Example 111. In the figure, base numbers 53 (A) to 415 (T) represent the prepro region, base numbers 416 (G) to 1261 (T)
indicates the mature alkaline protease gene region. FIG. 6 shows the amino acid sequence of polypeptide translated from the gene having the base sequence shown in FIG. Applicant Food Industry Enzyme Function Conversion Technology Research Association Agent Patent Attorney Yusuke Hiraki Patent Attorney Tadashi Ishii Figure 1 pLJ (+19 2:l c-DNA ξ - puc +19 2222 〇 DNA Figure 2 GGCCTG CAG AGC GAT AGC GCT GCC GCT GGC GGT GAA GTT AGC AAG GCT GCCGGT ACT GTT GTT GAC ACCAAAC CTCGCT GCCACC AACGCT ACT ACC CCT CCT AAC TCC AAC ATC AAC ACC ATC ACC ACC AAC ACCCAG IEAc TAC GTCAAT GGT CAG ACT TAT AGCAGC AAG CGT AACGAT GAG AAC GCT ATC TTCGCT ATI: TCT CTT GAG GACGTC AAG AGT GCC ATCTACGAC GTCGACCAT CAT GTG GAC GGT ATCGCC ACT TCG CTC ACCAGCA AG GCG GTCGAG TCT GAT GCC CAG AAG AGC CCCGGT CAA GGT ACCTCC AACCTCGAT GTCAAG GAT CCCTGG GGT CTG GGCAGCC ACT AGT GCCGGCGAG G G.C.G.
AG GAG TTCGAG GGCCGCAG
CA-T GGCCAT GGCACCAAG
AAG GCCAGCATCCTTATT CTT
GACGGCTTCAACGCT GCA A
TC-AACATG AGCAACGCA TTC
GAG CAG GGTGGCCAA ACCA
GC: CCT GCCAACAAACCGCGCC
AGT TTCAT ATC GGCCCCGCT GCCGTG ACCGTT
AAG GGCAGCCCT AACTT ACC ACC AAC TCC TCG ATC ATT ACT ACC AAC ATC ACC CAT ACC ATT ATC ACT ACC ATC ACC AAC ACC ATT AAC ACC A AC ACC AA ACC CCT TCC ACT ATT ATC ACC AAC AAC ACC ACC ATT AAC ACC ATT CAT ACC AAC AAC ACC ATC AAC ACC ATC AAC AAC GTC ACC AAC AAC AAC AAC ATC AAC ATT AAC ATT ACT ACT ACT ATC ATC ATC GT 5th 1460 1470 1. ! , 80ATAAA
ATCTT GGCmCTAA AAAAAAAAAAAA
AAAAAAAAA 3'Figure 6 (Part 1) Figure 6 (Part 2)

Claims (1)

[Claims] (1) A prepro-type alkaline protease gene derived from a microorganism belonging to the genus Aspergillus and defined by a restriction enzyme cleavage map. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, E is EcoR I, A is AflII, K is Kpn
I, X is Xmn I, S is Sal I, B is BglII,
H indicates HindIII)