JPH0817700B2 - Method for producing alkaline protease - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing an alkaline protease.

[Conventional technology]

Conventionally, the structure of the alkaline protease gene derived from Aspergillus orzae, 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]

An object of the present invention is to provide a method for producing alkaline protease.

[Means for solving the problem]

Therefore, the present inventors have previously variously studied the Aspergillus oryzae-derived alkaline protease gene, and succeeded in isolating and determining the structure of the Aspergillus oryzae-derived alkaline protease gene and prepro-type alkaline protease gene for the first time. Filed a patent application (Japanese Patent Application No. 63-51777 and Japanese Patent Application No. 63-279).
No. 401 and Japanese Patent Application No. 63-170018 and Japanese Patent Application No. 63-280370).

Then, the present inventors, using the prepro-type alkaline protease gene, as a result of various studies to efficiently produce alkaline protease, the recombinant DNA obtained by inserting the prepro-type alkaline protease gene derived from Aspergillus oryzae into the plasmid vector DNA. The present invention has been completed by culturing a medium containing Zygossaccharomyces rouxii, which has the ability to produce alkaline protease, and found that the alkaline protease can be secreted and produced efficiently outside the yeast cells. .

That is, the present invention comprises a recombinant DNA obtained by inserting a prepro-type alkaline protease gene encoding the amino acid sequence shown below into vector DNA, culturing in a medium a microorganism belonging to the genus Tigosaccharomyces having alkaline protease producing ability, A method for producing an alkaline protease, which comprises collecting the alkaline protease from a culture.

Hereinafter, the present invention will be described in detail.

First, examples of Aspergillus oryzae used as a donor of the alkaline protease gene used in the present invention include Aspergillus oryzae (ATCC 2
0386) and the like.

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 the above Aspergillus oryzae cells, for example, "Molecular Cloning", p. 196, except that glass beads and phenol are used during cell disruption. ,
Cold Spring Harbor Laboratory (Cold
Spring Harbor Laboratory) (1982) and "Molecular Genetics Experimental Method", Haruo Koseki, Rero Shimura, pp. 66-67 (1983).
It can be obtained by the method described.

To concentrate m-RNA encoding alkaline protease (hereinafter referred to as alkaline protease m-RNA) from the obtained m-RNA, for example, "Biomedical Research", Vol. 3, 534- 540
It can be performed 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 (19).
73) It can be obtained by the method described.

For synthesizing c-DNA from alkaline protease m-RNA, for example, "Mol.Cell B" is used.
iol.), Volume 2, 161 (1982) and "Gene" (Gen).
e), Vol. 25, p. 263 (1983).

Then, the c-DNA thus obtained is used as a vector D.
NA, for example, plasmid pUC119DNA (manufactured by Takara Shuzo Co., Ltd.) or the like to obtain various recombinant plasmid DNA,
For example, E. coli DH1 (ATCC 3384
9), E. coli HB101 (ATCC 33694) and the like by the method of Hanahan (“DNA Cloning”, Volume 1, pp. 109-135 (198).
5)] 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 ["Molecular Cloning" (Mole
cular Cloning), pages 329-333 and 344-349, Cold Spring Harbor Laboratory (Cold S
pring Habor Laboratory) (1982)].

Then, incomplete alkaline protease c-DNA was digested with ³² P.
Nick translation method ["Molecular
Cloning "(Molecular Cloning), 109-112
Page, Cold Spring Habor Laboratory (1982), and "J. Mol. Biol.", No. 113.
Vol., Pp. 237-251 (1977)] and then c
-From a gene bank library of c-DNA prepared using the plasmid pUC119DNA as a vector by the colony hybridization method using DNA as a probe ["Protein / nucleic acid / enzyme", Vol. 26, pp. 575-579 (1981)].
A plasmid DNA containing 1.5 Kb of alkaline protease c-DNA can be obtained.

Then, to obtain a DNA containing a gene having a prepro region in a gene encoding an alkaline protease derived from Aspergillus oryzae (hereinafter, referred to as a prepro-type alkaline protease gene) from the recombinant plasmid DNA thus obtained, The plasmid D
A restriction enzyme such as EcoRI is allowed to act on NA at a temperature of 30 to 40 ° C., preferably 37 ° C. for 1 to 24 hours, preferably 2 hours, and the obtained reaction-terminated solution is subjected to agarose gel electrophoresis [“Molecular. Cloning "(Molecular Cloning),
P.150, Cold Spring Harbor Laboratory (1982)], a DNA containing the prepro-type alkaline protease gene derived from Aspergillus oryzae can be obtained.

Then, the base sequence of the prepro-type alkaline protease gene was determined by the method as shown in item 11 of the example (the base sequence is shown in FIG. 5), and then the polys that are translated by the gene having the base sequence was analyzed. Determine the amino acid sequence of the peptide (see Figure 6).

Then, in the same manner as described in item 12 of the below-mentioned Example, for example, in various vector DNAs, restriction enzymes Eco RI, Pst I, Sal I, Pvu were added.
II and SspI (both manufactured by Takara Shuzo) at a temperature of 30 to 40 ° C,
Preferably, the vector DNA is cleaved by allowing it to act at 37 ° C. for 1 to 16 hours, preferably 2 hours, and the prepro-type alkaline protease gene-containing DNA and T4 DNA ligase (Takara Shuzo Co., Ltd.) obtained as described above are obtained. ) Etc. are added and ligated by a conventional method to obtain a recombinant DNA.

Any expression vector DNA may be used, for example, by removing the prepro-type alkaline protease gene portion with a recombinant plasmid pZAP101ADNA obtained by recombining the recombinant plasmids pSRT303P and pGAG41DNA described in item 12 of the Example below. Examples of the plasmid vector obtained by

Then, using the recombinant DNA thus obtained, a microorganism belonging to the genus Tigo Saccharomyces, for example,
Chigosaccharomyces rouxii (IFO 1876),
Kimura's method [Recombinant DNA Experimental Technology, pp. 243-253 (198
5)] to obtain a microorganism belonging to the genus Tygosaccharomyces which contains a recombinant DNA in which a prepro-type alkaline protease gene is inserted into a vector DNA and has an alkaline protease secretion-producing ability.

From the thus obtained microorganism belonging to the genus Tigo Saccharomyces, in order to obtain a purified new recombinant DNA, for example, Proc. Natl.
Acad.Sci) 62, 1159-1166 (1969).

Then, the above-mentioned microorganism is cultured in a medium and the alkaline protease is collected from the culture.

As the medium, for example, any one may be used as long as it is used for culturing a microorganism belonging to the genus Tigo-Saccharomyces, for example, glucose, polypeptone, gentisin (Geneticin) in YPD medium consisting of enzyme extract.
Examples include a medium containing [Gibco Laboratories, trade name of antibiotic G418 sulfate].

The culture temperature is, for example, 25 to 35 ° C, preferably 30.
At about C, the culture time is, for example, 6 to 100 hours, preferably about 72 hours.

Then, the culture is subjected to, for example, a centrifugation treatment or a membrane filter treatment at 3,000 rpm for about 2 minutes to obtain a culture solution and bacterial cells, and the obtained culture solution can be used as a crude enzyme solution. The obtained bacterial cells are crushed by, for example, vortex mixer together with glass beads for about 3 minutes to obtain a crude enzyme solution.

Each crude enzyme solution can be used as it is, but if necessary, by ammonium sulfate fractionation, ion exchange chromatography (for example, DEAE-Biogel A, etc.), gel filtration (for example, Ultrogel AcA34, etc.). Purification gives purified alkaline protease.

The physicochemical properties of the alkaline protease thus obtained are “Agr.Biol.Chem” (Agr.Biol.C).
hem.), 37, pp. 2685 to 2694 (1973).

〔The invention's effect〕

As is clear from the above, according to the present invention, containing recombinant DNA in which the prepro-type alkaline protease gene has been incorporated, by culturing in a medium a microorganism belonging to the genus Tigosaccharomyces, in an extremely short period of time, INDUSTRIAL APPLICABILITY The present invention is extremely useful industrially because it can efficiently secrete and produce alkaline protease.

〔Example〕

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

Example Cloning of prepro-type 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 were treated with alkaline protease. Production medium [3% (W / V) defatted soybeans that have been heated and pressure-expanded, 3%
(W / V) KH ₂ PO ₄ ] 50 ml, and 120 rpm at a temperature of 30 ° C. using a constant temperature shaker (M-100 ⁿ , manufactured by Taiyo Kagaku Kogyo Co., Ltd.)
The culture was shaken for a period of time to obtain a culture, and this culture was filtered by a conventional method to obtain 10 g of bacterial 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
-Lauroyl sarcosine sodium / 0 / 15M β-mercaptoethanol] was added to a cup-type blender (manufactured by Nippon Seiki Seisakusho Co., Ltd.), 10 g of glass beads (diameter 0.5 mm) was added, and 10,000 rpm After 5 minutes of treatment, 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 product.

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

Next, ultracentrifuge tube (Hitachi Koki Co., Ltd.) 4
To the book, 1.2 ml of 5.7 M cesium chloride solution was layered in advance, and on top of that, the above-mentioned cell disruption solution was dispensed, respectively, and supercentrifuged (Hitachi Koki Co., SCP55H) The precipitate was obtained by centrifuging at 15 ° C. and 30,000 rpm for 16 hours.

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, and 4 ml of the above 10 mM Tris buffer is added to the organic solvent layer, and the extraction and separation operations are repeated twice to obtain an aqueous layer. 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,
The above-mentioned ethanol precipitation operation was performed, and the obtained RNA 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) column chromatography.

A 2.5 ml Terumo syringe (manufactured by Terumo Corp.) is used as a column, and 0.5 g of resin is an elution buffer [10 mM Tris-HCl buffer (pH 7.6) / 1 mM EDTA / 0.1% (W / V) sodium dodecyl sulfate]. After swelling, packed in a column, binding buffer [10 mM Tris-HCl (pH 7.6) / 1 mM EDTA / 0.4 M 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) is 40% (W / V) in the poly-aroma tube for Beckman rotor SW41.
Sucrose solution [50 mM Tris-HCl buffer (pH 7.5) / 20 mM NaC
0.5 ml of l / 1mM EDTA / 40% (W / V) sucrose], and put 2.4 ml on each, 25% (W / V), 20% (W / V), 15% (W / V) and A 10% (W / V) sucrose solution was overlaid and left at a temperature of 4 ° C. for 24 hours. In this sucrose density gradient,
Overlay 50 μg of m-RNA obtained in item 2 and use SW41 rotor manufactured by Beckman Co., Ltd. by a conventional method at 30,000 rpm and temperature.
Centrifugation was performed at 18 ° C for 18 hours. After the centrifugation operation,
Fractions of 0.5 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 m-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 a temperature of 30 ° C. for 30 minutes, and 150 μl of NET
Buffer [150mM NaCl / 5mM EDTA / 0.02% (W / V) NaN 3 / 20mM
Tris-HCl buffer (pH 7.4) /0.05% (W / V) Nonidet P-40 (manufactured by Bethesda Research Laboratory, surfactant)] was added, and 1 μl of anti-alkaline protease serum (see below) was added. Prepared in step 1) was added, and 10 mg of Putin A Sepharose (manufactured by Pharmacia) was added to the mixture that was left standing at a temperature of 4 ° C for 18 hours, and the temperature was adjusted to 20
What was left for 30 minutes at ℃, 12,000 rpm 1
The resin, that is, the above-mentioned protein A sepharose was recovered by centrifugation for minutes.

Wash the recovered resin 3 times with 200 μl of NET buffer,
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", page 227, page 680 (1970)], and the gel after electrophoresis was diluted with 10% (V / V) acetic acid.
After soaking for 0 minutes to fix the protein, soaking in water for 30 minutes, further soaking in 1M sodium salicylate solution for 30 minutes and drying to obtain a dried gel, X-ray film (Fuji Photo Film Co., RX) Was used for fluorography.

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

4. Preparation of alkaline protease serum Rabbit anti-alkaline 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 thalamus of Japanese white rabbits weighing 2 kg, and two weeks after breeding 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 m-RNA obtained as described above, "Mole Cell Viol" (Mol. Ce
ll Biol.), Volume 2, p. 161 (1982) and "Gene".
(Gene), Vol. 25, p. 263 (1983). As a result, 160 ng of double-stranded c-DNA was obtained.

In this way, the c-DNA 160 ng obtained, using the Amersham c-DNA cloning Kidd, the method of instructing the Amersham perform methylation restriction enzyme Eco RI cleavage site, further Eco across the c-DNA RI linker was attached.

6. Preparation of c-DNA bank 100 ng of plasmid pUC119DNA (manufactured by Takara Shuzo) was added to 8 μl.
Of those dissolved in water, 1 [mu] l of Med buffer - after addition of [100mM Tris-HCl buffer (pH7.5) / 100mMMgCl ₂ / 10mM dithiothreitol / 500 mM NaCl], and further, to this, 10 units ( 1 μl) of restriction enzyme Eco RI (manufactured by Takara Shuzo Co., Ltd.) was added, and a cleavage treatment was performed 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) were added to the cleaved product, and the enzyme reaction was performed at a temperature of 65 ° C. for 1 hour, After dephosphorylation of both ends of the digested product, 12 μl of water-saturated phenol was added to this to deproteinize, and then 1 μl of 3M sodium acetate (pH 5.8) and 26 μl of the recovered aqueous layer were added. Cold ethanol was added to each, and the mixture was allowed to stand at -70 ° C for 15 minutes, and a microcentrifuge (Tomy Seiko, MRX-150) was used at 12,000 rpm.
DNA was collected by centrifugation for 5 minutes.

Thus obtained plasmid vector pUC119DNA 10 cleaved with the restriction enzyme Eco RI and dephosphorylated on both ends
A mixture of 0 ng and 160 ng of c-DNA prepared in item 5 was suspended in 8 μl of water, and 1 μl of X10 was added to the suspension.
Ligation buffer [200 mM MgCl _2/660 mM tris -
Hydrochloric acid buffer (pH 7.6) / 10 mM ATP / 150 mM dithiothreitol] was added to 1 unit (1 μl) of T4 D
NA ligase (manufactured by Takara Shuzo Co., Ltd.) was added and the mixture was allowed to stand at a temperature of 16 ° C. for 16 hours to ligate the above plasmid vector DNA and c-DNA to obtain a reaction product.

Using this reaction, the Hanahan method ["DNA Cloning" (DNA Clonin
g), Vol. 1, pp. 109-135 (1985)].
The strain (ATCC 33849) was transformed with the plasmid pUC119DNA.
Was used as a vector to prepare a c-DNA bank.

7. Search for fragments of alkaline protease c-DNA Hybridization selection ["Molecular Cloning", No. 329
Page, Cold Spring Harbor Laboratory (1982)], the alkaline protease c-DNA was searched. 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", No. 86
Recombinant plasmid DNA (500 µg) was obtained from Escherichia coli in the c-DNA bank by the method described on page, Cold Spring Harbor Laboratory (1982). Of this, 100 μg was dissolved in 200 μl of water, left at 100 ° C for 10 minutes, then transferred to ice and rapidly cooled to 200 μl.
l of 1 M 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 Co., Catalog No. HAWP 025 00), and the denatured recombinant plasmid DNA was adsorbed on the nitrocellulose filter and air dried by a conventional method. After that, it was washed with 6 × SSC (0.9M sodium chloride / 0.09M 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 give a recombinant plasmid. DNA was immobilized 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 1,4-piperazinediethanesulfonic acid (pH 6.4) /0.2% sodium dodecyl sulfate / 0.4 M sodium chloride / 100 μg / ml yeast t-RNA] and the recombinant obtained as described above The nitrocellulose filter on which the plasmid DNA was fixed was dipped and left at a temperature of 50 ° C. for 3 hours to hybridize the recombinant plasmid DNA on the filter with m-RNA. After completion of the reaction, the filter was taken out and 1 ml of the washing solution I [10 mM Tris-HCl buffer (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] was performed twice to remove m-RNA that did not hybridize with the plasmid DNA immobilized on the filter.

Then, this nitrocellulose filter is
1 μl of water, add 10 μg of yeast t-RNA,
The sample was allowed to stand in hot water at 100 ° C. for 10 minutes, transferred to ethanol cooled with dry ice, and then allowed to stand at room temperature to dissolve. 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 −70 ° C. for 1 hour for ethanol precipitation, a microcentrifuge [Tomy Seiko, MRX-150] was used. Was used for 5 minutes at 12,000 rpm to collect the hybridized m-DNA.

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 the plasmid pBR322DNA (Takara Shuzo) were used as the restriction enzyme Alu. By comparing with the standard pattern of the DNA fragment obtained by digesting with I, the recombinant plasmid pO
Alkaline protease c-DNA inserted in AP3 DNA
The size of the fragment was found to be 750 bp.

8. Search for large alkaline protease c-DNA Agarose gel containing 750 bp of alkaline protease c-DNA fragment was cut out from the agarose gel obtained in item 7, put in a dialysis tube, and added to 500 μl of TE buffer [10 mM. Tris-HCl buffer (pH 8.0) / 1 mM EDTA)]
After the addition of, the dialysis tube was sealed, and the solution obtained by elution of the DNA from the gel in the buffer solution by electrophoresis was added with an equal volume of water-saturated phenol and stirred, and the aqueous layer was recovered. According to a conventional method, 100 ng of alkaline protease c-DNA was recovered by ethanol precipitation. This 100ng
Alkaline protease c-DNA of [α- ³² P] dCTP
(Amersham) was used for labeling by the nick translation method. Nick Translation is directed by Takara Shuzo Co., Ltd. (J.Mol.Bi)
ol.), 113, 237-251 (1977) and "Molecular Cloning", 109-1.
The method described on page 12 (1982) was followed. Using the ³² P-labeled alkaline protease c-DNA fragment thus prepared as a probe, the colony hybridization method [“protein / nucleic acid / enzyme”, Vol. 26, Pp. 575-579 (1981)] to obtain a colony having alkaline protease c-DNA. 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 named E. coli DH1 (pOAP 5). 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).

When the recombinant plasmid pOAP5DNA was treated with the restriction enzyme Eco RI in the same manner as in item 6, and agarose gel electrophoresis was performed, the size of the inserted DNA fragment was 1,100.
It turned out to be bp. 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, EcoR I, Kpn I,
A single or double digestion of Sal I and Xmn I was performed, and the size of the fragment that appeared was analyzed in the same manner as described in item 7 to prepare a restriction enzyme map of the recombinant plasmid pOAP5DNA, 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
The A fragment was cloned into the plasmids pUC18 and pUC19 DNA (Takara Shuzo Co., Ltd.) cut with the same or the same restriction enzyme as the margin. Sequencing is a plasmid DN
Method of Sakaki of A ["Vector DNA", p. 70, Kodansha (1986
After the alkaline denaturation according to the above method), it was carried out by the dideoxy chain termination method by an ordinary method using an M13 sequence kit (Takara Shuzo).

In this way, Aspergillus Orize (ATCC2038
The nucleotide sequence of the alkaline protease c-DNA fragment derived from 6) was determined, and the nucleotide sequence of the gene corresponding to the mature alkaline protease among the obtained nucleotide sequences is shown in FIG. 2 and also in FIG. The amino acid sequences of the polypeptides translated from the genes having the nucleotide sequences are shown in FIG. 3, respectively.

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.

10. Search for full-length alkaline protease c-DNA As a result of nucleotide sequence determination in Item 9, the recombinant plasmid pOAP5 obtained in Item 8 encoded the mature alkaline protease from N-terminal to C-terminal. However, it lacked a prepro region that was thought to be located upstream of it. Therefore, after the alkaline protease c-DNA fragment of the recombinant plasmid pOAP5 was isolated in the same manner as in item 8, it was labeled with ³² P by the nick translation method, and this was used as a probe for the c-DNA bank obtained in item 6. Then, a colony hybridization method was performed again to obtain a colony having a longer alkaline protease c-DNA. The recombinant plasmid contained in one of the colonies was named pOAP10, and E. coli containing the recombinant plasmid was transformed into E. coli DH1 (pOA
P10). Purified recombinant plasmid pOAP10DNA, 100 μg, was prepared according to item 7.

When the recombinant plasmid pOAP10DNA was treated with the restriction enzyme Eco RI in the same manner as in item 7, and subjected to agarose gel electrophoresis, the size of the inserted DNA fragment was 1,5.
It turned out to be 00bp. Further recombinant plasmid
Restriction of pOAP10 DNA with restriction enzymes Afl II, Eco RI, Kpn I, Sal I, Xmn
A single or double digestion of I, Bgl II, and Hind III was performed, and the size of the fragment that appeared was analyzed by the same method as described in item 7, to create a restriction enzyme map of the recombinant plasmid pOAP10DNA. The map is shown in FIG.

11. Determination of base sequence of alkaline protease full-length c-DNA The alkaline protease c-DNA fragment appearing from the recombinant plasmid pOAP10 obtained in item 10 is cleaved with the same or the same restriction enzyme as in item 9. The cloned plasmids pUC18 and pUC19 were cloned.
Sequencing was performed by the dideoxy chain termination method as in item 9.

In this way Aspergillus oryzae (ATCC2038
The base sequence of the alkaline protease c-DNA derived from 6) was determined, and FIG. 5 shows the base sequence of the gene corresponding to the full-length alkaline protease (prepro type) described in Example item 11. The amino acid sequences of the polypeptides translated from the gene having the nucleotide sequence shown in FIG. 5 are shown in FIG. 6, respectively. In the figure, base numbers 53 (A) to 415
(T) is the prepro region and has base numbers 416 (G) to 1261.
(T) shows the mature alkaline protease gene region.

The polypeptide translated from the nucleotide sequence shown in FIG. 5 consists of 403 amino acids, and the purified N-terminal 16 amino acid sequence of alkaline protease (Gly Leu ThrThr Glu
Lys Ser Ala Pro Trp Gly Leu Gly Ser Ile Ser) is 12
It coincided with the amino acid sequence from the second amino acid (underlined in Fig. 6). Therefore, the amino acid sequence from the N-terminal to the 121st position is a prepro region required for the secretion of alkaline protease, and the amino acid sequence from the 122nd to 403th position is a region encoding a mature alkaline protease.

From the above findings, it can be concluded that the base sequence shown in FIG. 5 encodes all the polypeptides of alkaline protease including the prepro region.

12. Construction of Recombinant Plasmid pZAP101ADNA To construct the recombinant plasmid pZAP101ADNA in T. saccharomyces rouxii, first, 1 μg of plasmid pUC19DNA (Takara Shuzo) having ampicillin resistance was used as restriction enzymes Eco RI and Pst I (both Takara Shuzo). ) Was used for reaction at a temperature of 37 ° C. for 16 hours for complete cleavage. The total amount of the completely cleaved product was separated by 0.7% agarose gel electrophoresis into a long DNA fragment containing the ampicillin resistance gene by Eco RI- Pst I and a short DNA fragment containing no resistance gene. Eco RI containing ampicillin resistance gene
-The DNA fragment with Pst I was extracted from the gel and subjected to phenol treatment and ethanol precipitation by a conventional method.

Similarly, glyceroaldehyde triphosphate dehydrogenase of Tygosaccharomyces rouxii (hereinafter GAPD
H)) plasmid pTI41
DNA (Hiroshima Faculty Fermentation Engineering Dongjiang Akio obtained from Mr) to a 1μg the same manner as above was completely digested with Eco RI and Pst I, Eco include promoters GAPDH of 0.7 kbp RI-Pst
A DNA fragment according to I was obtained.

A DNA fragment by Eco RI-Pst I containing ampicillin resistance gene of the preceding plasmid pUC19 DNA, GAP from pTI41DNA
Mix with a DNA fragment by Eco RI- Pst I containing a DH promoter, add T4 DNA ligase (Takara Shuzo), and heat at 16 ° C.
The two fragments were ligated by leaving them to stand for 16 hours.

Using this reaction product, the Hanahan method [“DNA Cloning” (DNA Clonin
g), Vol. 1, pp. 109-135 (1985)].
The strain (ATCC 33849) is converted to light, and plasmid DNA is extracted from the obtained light-converted strain by the alkaline method [Recombinant DNA Techniques, pp. 50-51 (1983)], Plasmid pUC
In the DNA fragment of 19 DNA Eco RI- Pst I, the plasmid pT
Plasmid DNA DNA fragments by Eco RI-Rst I of I141DNA is inserted was named PUG41.

1 μg of this plasmid pUG41DNA was transformed into Eco as described above.
Cleavage was performed with RI, alkaline phosphatase was added to this digested product, and an enzymatic reaction treatment was performed at a temperature of 65 ° C. for 1 hour to dephosphorylate both ends of the digested product. Then, phenol treatment and ethanol precipitation were carried out by a conventional method to obtain a DNA fragment by Eco RI of the plasmid pUG41 DNA having both ends dephosphorylated.

On the other hand, 1 μg of the recombinant pOAP10 DNA obtained in item 10 and containing Aspergillus oryzae alkaline protease c-DNA was cleaved with Eco RI as described above, and then subjected to 0.7k agarose gel electrophoresis for 1.5 k.
A DNA fragment by EcoRI containing bp Aspergillus oryzae alkaline protease c-DNA was separated and extracted, and phenol treatment and ethanol precipitation were carried out by a conventional method to obtain the plasmid pUG41DNA.
Mixed with Eco RI fragment.

T4 DNA ligase was added to this by the method described above, and after ligation, Escherichia coli DH1 (ATCC 33849) was transformed by the method of Hanahan, and a plasmid was extracted from the obtained transformant strain by the alkaline method described above. And then Sph I and Bg1
The double-cut DNA patterns of II (both manufactured by Takara Shuzo) were analyzed by agarose gel electrophoresis. By this analysis, recombinant plasmid DNA in which alkaline protease c-DNA was incorporated in the forward direction with respect to GAPDH promoter was selected, and this recombinant plasmid DNA was selected.
It was named pGAP41.

1 μg of the obtained recombinant plasmid pGAP41DNA was cleaved with the restriction enzyme Ssp I (manufactured by Takara Shuzo Co., Ltd.) as described above, followed by phenol treatment and ethanol precipitation by a conventional method.
On the other hand, plasmid pAT1 containing the antibiotic G418 resistance gene
1 μg of 36 DNA (obtained from Mr. Akio Toe, Department of Fermentation Engineering, Faculty of Engineering, Hiroshima University) was cleaved with the restriction enzyme Pvu II (Takara Shuzo) as described above, and separated by 0.7% agarose gel electrophoresis.
After extraction, it was treated with phenol and precipitated with ethanol to obtain a PvuII DNA fragment containing 1.7 Kbp of G418 resistance gene. This Pvu II DNA fragment and the above recombinant plasmid pGAP41DNA Ssp I DNA fragment were mixed, ligated by T4 DNA ligase by the above-mentioned method, and E. coli DH1 (ATCC 33849) strain was transformed with this. Plasmid DNA was extracted from the obtained kanamycin-resistant transformant strain by the above-mentioned alkaline method, and Ssp of the recombinant plasmid pGAP41DNA was extracted.
Pvu I containing G418 resistance gene of the plasmid pAT136 to I site
Recombinant plasmid pGAG41 in which the DNA fragment from I was inserted
I got the DNA.

Then, 1 μg of plasmid pUC19DNA (Takara Shuzo) was cleaved with Ssp I as described above, treated with phenol and precipitated with ethanol, mixed with Pst I linker (Takara Shuzo), ligated with T4 DNA ligase, and transformed into the Escherichia coli. After transforming the DH1 strain, plasmid DNA was extracted from the obtained transformant by the above-mentioned alkaline method, and Ssp I of the plasmid pUC19DNA was extracted.
A plasmid pUCP19DNA having a Pst I linker inserted at the DNA fragment site was obtained. 1 μg of this pUCP19 DNA was digested with the restriction enzyme Sal
DNA digested with Sal I of plasmid pUCP19DNA after digestion with I (Takara Shuzo), phenol treatment and ethanol precipitation
A fragment was obtained. On the other hand, plasmid pSRT303D (obtained from Mr. Osaka University Faculty of Engineering fermentation Engineering Taiji Oshima) DNA1μg the Sa
was cut with l I, separated on a 0.7% agarose gel electrophoresis, then extracted, subjected to phenol treatment and ethanol precipitation Zygosaccharomyces self propagating plasmid My Seth Rukishi pSR1 [THEY mole Baioru "J. Mol. Biol." Section 182
Winding, to obtain a Sal I fragment containing the nucleotide sequence of pp 191-203 (1985) ". A DNA fragment of the obtained plasmid pSRT303DDNA with Sal I and a D of the above plasmid pUCP19DNA with Sal I
After ligating the NA fragment with T4 DNA ligase, the Escherichia coli DH1 strain was transformed, and the plasmid DNA was extracted from the obtained transformant by the alkaline method.
The plasmid pUCP19DNA was inserted at the DNA fragment site of Saal I of DDNA.
A recombinant plasmid pSRT303PDNA in which was inserted was obtained.

The resulting pSRT303PDNA cut with the Pst I, after separating and extracted with 0.7% agarose electrophoresis to obtain a DNA fragment by Pst I comprising the base sequence of the plasmid pSR1DNA performs phenol treatment and ethanol precipitation.

In addition, the recombinant plasmid pGAG41DNA described above was similarly transformed into P
Cleavage with st I, treatment with alkaline phosphatase by the above-mentioned method to remove phosphates at both ends, and then phenol treatment and ethanol precipitation to dephosphorylate both ends of pG
A Pst I fragment of AG41 DNA was obtained. After this fragment and the fragment by Pst I of the previous recombinant plasmid pSRT303PDNAN were mixed and ligated by T4DNA ligase, the E. coli DH1
The strain was transformed, and a plasmid was extracted by the alkaline method from the transformant strain obtained, the recombinant plasmid DNA fragments by Pst I DNA fragment and pGAG41DNA by Pst I was ligated comprising the nucleotide sequence of pSR1DNA of pSRT303PDNA It was named pZAP101A, and the restriction enzyme map of the pZAP101A plasmid DNA is shown in FIG.

13. Transformation of Yeast with Recombinant Plasmid pZAP101ADNA Using the recombinant plasmid pZAP101ADNA, Tigosaccharomyces ruxie (IFO 1876) was transformed by the method of Kimura, and the transformant strain, Tigosaccharomyces rouxii [(Zygosaccharomyces rouxii) IFO 18
76 (pZAP101A)] was obtained.

In addition, Zygosacc
haromyces rouxii) IFO 1876 (pZAP101A)] was submitted to the Institute of Microbial Science and Technology of the Institute of Industrial Science, Microtechnology Research Institute No. 10514 (FEPM).
It has been deposited as P-10514).

14. Transformed strain Tigosaccharomyces rouxii [(Z
ygosaccharomyces rouxii) IFO 1876 (pZAP101A) production of alkaline proteases Tygosaccharomyces rouxii [(Zygosaccharo
myces rouxii) IFO 1876 (pZAP101A) (FERM P-1051
4)] was inoculated into 10 ml of YPD medium (glucose 20 g / l, polypeptone 20 g / l and yeast extract 10 g / l) containing the antibiotic G418 sulfate (100 μg / ml), and shake-cultured at 30 ° C. for 72 hours. Then, 5 ml of the obtained culture was sterilized with a 0.45 μm membrane filter to obtain a culture filtrate.

Regarding the obtained culture filtrate, Anson-Hagiwara modified method [Y. Fukushima Agric. Biol. Chem.] Vol. 49, pp. 1643 to 1648 (198)
5)] was used to measure the alkaline protease activity, the alkaline protease activity was 70 P.U./ml. For comparison, plasmid vector pS was used as a control.
Using a T. saccharomyces ruxie strain having RT303D ((a strain transformed by the method of Kimura using T. saccharomyces ruxie (IFO 1876) and the ma plasmid vector pSRT303D)).
When the culture and the measurement of protease activity were carried out in the same manner as above, it was 0.1 PU / ml.

As is clear from the above, the culture solution obtained by the present invention has a markedly increased protease activity as compared with the control, and thus by culturing the yeast cells belonging to the genus Tigo Saccharomyces according to the present invention, the medium It was found that a large amount of alkaline protease was secreted and produced therein.

[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. FIG. 4 is a restriction map of the recombinant plasmid pOAP10DNA. FIG. 5 shows the nucleotide sequence of the gene corresponding to the full-length alkaline protease (prepro type) described in the item of Example 11. In the figure, base numbers 53 (A) to 415 (T) represent the prepro region, and base numbers 416 (G) to 1261 (T) represent the mature maalkali protease gene region. FIG. 6 shows the amino acid sequence of the polypeptide translated from the gene having the nucleotide sequence shown in FIG. FIG. 7 is a diagram showing a restriction enzyme map of the recombinant plasmid pZAP101ADNA.

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Claims (1)

[Claims] 1. A vector DNA containing a prepro-type alkaline protease gene encoding the amino acid sequence shown below.
A method for producing an alkaline protease, which comprises culturing in a medium a microorganism belonging to the genus Tygosaccharomyces, which contains the recombinant DNA inserted into, and has an alkaline protease-producing ability, and collecting the alkaline protease from the culture.