JPH0787790B2 - METHOD FOR PRODUCING NOVEL GENE DNA AND ALKALINE PROTEASE - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The DNA of the present invention is derived from Bacillus licheniformis and is a DNA used for secretory production of a larger amount of alkaline protease, and the gene DNA is The present invention relates to a method for efficiently producing an alkaline protease using a bacterium belonging to the genus Bacillus transformed with a recombinant DNA containing the same.

[Conventional technology]

Alkaline protease is an enzyme capable of degrading a polypeptide even in a strongly alkaline region, and is widely used by being added to detergents as a cleaning aid.

Conventionally, an alkaline protease is a bacterium with high productivity of the enzyme, for example, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus subtilis, etc. are cultured, and the alkaline protease is recovered from the culture supernatant. Has been manufactured by the method. However, since these microorganisms secrete a large amount of enzymes such as amylase, neutral protease, and levansucyclase, in addition to alkaline protease, to the outside of the cells, it is complicated to recover and purify alkaline protease from the culture supernatant. Requires a process. In addition, there are problems such as production of contaminants that cause allergies when using alkaline protease as a detergent.

[Problems to be solved by the invention]

Microorganisms used for the production of alkaline protease usually carry an alkaline protease gene within the cell.
I have one (1 copy). However, recombinant DNA
The alkaline protease gene was isolated by the method described above, and a recombinant plasmid ligated to a vector plasmid retained in Bacillus bacterium was prepared to transform Bacillus bacterium. You can get the body. When such Bacillus bacterium is cultured, only the productivity of alkaline protease can be specifically increased by the gene amplification effect, and Bacillus bacterium has the property of secreting the protein to the outside of the bacterium. The protease can be recovered and purified very easily. In addition, by using a transformant in which the above recombinant plasmid is introduced into a Bacillus bacterium having a low productivity of proteins other than alkaline protease and substances causing allergies, it is easier to recover and purify alkaline protease from the culture. become.

Further, in order to mass-produce useful proteins derived from heterologous organisms such as human growth hormone, recombinant production using Escherichia coli as a host has been attempted. There is also a problem of endotoxin contamination in the product obtained because proteins and proteins are not secreted outside the cells. On the other hand, Bacillus bacteria have been industrially mass-cultured for a long time, and their safety has been confirmed, and since they have the property of secreting proteins outside the cells, the above-mentioned problems do not occur.

Since Bacillus licheniformis secretes and produces a large amount of alkaline protease extracellularly, the base sequence of the promoter region of the alkaline protease gene, the region involved in extracellular secretion, etc. has a structure suitable for large-scale protein production. Therefore, if such a region is isolated, it can be used for the above purpose.

The alkaline protease gene of Bacillus licheniformis has been described by Jacobs et al. [M. Jacobs et al .: Nucleic Acids Re
s., 13, 8913 (1985)], and the nucleotide sequence has been reported from Bacillus licheniformis NCIB 6816. However, it was cloned because it lacks the promoter region required for expression of alkaline protease. The gene itself cannot produce alkaline protease. Such a gene cannot produce an alkaline protease or a useful protein by taking advantage of the advantages of the alkaline protease gene having a strong expression control region.

In order to solve such problems, the present invention provides an alkaline protease gene having a promoter region capable of expressing an alkaline protease (hereinafter also referred to as the entire region of the promoter),
Another object of the present invention is to provide a method for producing an alkaline protease by culturing a Bacillus bacterium transformed with a recombinant plasmid having this gene inserted therein.

[Means / actions for solving problems]

A gene having a nucleotide sequence having a region required for expression of Bacillus licheniformis alkaline protease gene
DNA cloning is performed, for example, by the following procedure.

Bacillus licheniformis is used as a gene donor. The use of this microorganism is suitable, for example, for producing an alkaline protease having suitable properties as a cleaning aid.

Further, since a strain having a higher alkaline protease production ability can be expected to have a stronger promoter, it is desirable to use a strain having a higher production ability due to mutation treatment or the like. In the present invention, a known strain of Bacillus licheniformis is mutated with nitrosoguanidine as a mutagen to use a mutant strain having a high protease-producing ability.

To clone the alkaline protease gene from the selected alkaline protease-producing strain, the chromosomal DNA of this producing strain is cut with a restriction enzyme to fragment and then ligated to the vector plasmid retained by Bacillus subtilis. There is a method of transforming a low-protease producing strain of, and selecting a strain having enhanced protease-producing ability from the obtained transformants.
However, it seems that the Bacillus licheniformis alkaline protease gene has a special base sequence in its periphery, and thus it cannot be cloned by this method.

In the present invention, in order to overcome this problem, Esherichia
Recombinant using Escherichia coli as a host and DN homologous to Bacillus licheniformis alkaline protease gene
The alkaline protease gene of Bacillus licheniformis was cloned by the colony hybridization method using A.

Homologous to the Bacillus licheniformis alkaline protease gene used for colony hybridization.
The following can be used as DNA.

That is, the amino acid sequence of the Bacillus licheniformis alkaline protease has already been revealed (M.
Ottensen, I.Svendsen: Methods in Enzymology, 19 , 199, A
Academic Press, New York (1979)], so it has a base sequence corresponding to a sequence of about 5 to 6 consecutive amino acids at any position, and usually consists of about 14 to 18 bases. Single-stranded oligonucleotides can be used as homologous DNA. However, since there are 1 to 6 codons corresponding to one amino acid depending on the type of amino acid, the above-mentioned oligonucleotide usually has a plurality of types. Therefore, the above-mentioned amino acid sequence has a corresponding type of oligonucleotide. It is desirable to select the area where the For example, "A corresponding to the amino acid sequence from the N-terminal to the 134th to 138th (Met-Lys-Gln-Ala-Val)"
TGAA (X) CA (Y) GC (Z) GT (X, Y: mutually identical or different A or G, Z: T, C, A or G) "16 kinds of 14 bases And a mixture of oligonucleotides. Oligonucleotide synthesis is commercially available DNA
Using a synthesizer or known solid-state phosphotriester method [H. Ito et al .: Nucleic Acids Res., 10 , 1755 (1982)]
And so on. The synthesized DNA is purified by high performance liquid chromatography, etc., and then γ
^The 5'end is labeled with ³² P using ³² P-ATP and T4 polynucleotide kinase. On the other hand, chromosomal DNA is prepared from Bacillus licheniformis cells. Chromosomal DNA is lysed after lysozyme treatment and sodium decylsulfate is added to lyse the cells for chloroform lysis.
Marmur's method [J. Marmur: J. Mol. Biol., 3 , 208 (1961)]
It is prepared by This chromosomal DNA is cut with a restriction enzyme to fragment. The restriction enzyme used here does not require any special one, but a restriction enzyme having one recognition site in the vector used for ligating the fragments to construct a recombinant plasmid is suitable. For example, when using the Escherichia coli plasmid pBR322 as a vector, EcoR
Examples include restriction enzymes such as I HindIII, BamHI, pstI, PvuII. However, for example, BglII and BamHI are It is a restriction enzyme that recognizes a base sequence different from that of Since it becomes a sticky end consisting of 4 bases that can form a pair of A / T and G / C with each other, they can be ligated to each other when T4 DNA ligase is allowed to act. It does not matter if the restriction enzyme that cuts differs from the restriction enzyme that cuts the vector plasmid. Also, in the case of a restriction enzyme such as PvuII or SmaI that cuts both strands of DNA at the same position to generate a blunt end, different restriction enzymes can be used in combination. On the other hand, vector plasmids carried in E. coli, such as
Cleavage pBR322, PUC19, etc. with the restriction enzyme used to cleave the chromosomal DNA or with a restriction enzyme that produces a cleaved end that can be ligated to each other to make linear, and then treat with alkaline phosphatase to remove the phosphate at the 5'end. Let go. This dephosphorylation treatment prevents the plasmid from returning to the original circular DNA by T4 DNA ligase added later.
Then, the chromosomal DNA fragment and the dephosphorylated linear plasmid are mixed, and T4 DNA ligase is added to react with each other to form a phosphate bond between both DNAs to prepare a circular recombinant plasmid. Chromosomal DNA fragments cleaved with restriction enzymes were fractionated by agarose gel electrophoresis, and the DNA was transferred to a nitrocellulose filter and hybridized with the ³² P-labeled oligonucleotide previously synthesized. Southern hybridization method [EM Southern: J. Mo, which detects chromosomal DNA fragments having nucleotide sequences homologous to oligonucleotides by soybean and autoradiography.
l.Biol., 98 , 503 (1975)], the length of the fragment in which the alkaline protease gene is present is determined, and then the chromosomal DNA fragment is fractionated in advance and the fragment of this length is concentrated before use. By doing so, the colony hybridization described later can be efficiently performed. Concentration of fragments of a specific length is carried out by, for example, fractionating a chromosomal DNA fragment by agarose gel electrophoresis, cutting out a gel corresponding to the migration position of the fragment of the desired length, and subjecting it to electrophoresis elution (T. Mani
atis et al .: Molecular Cloning, p.164, Cold Spring Har
bor Laboratory (1982)] and the like to elute the DNA fragment.

A recombinant plasmid obtained by ligating a chromosomal DNA fragment and a vector plasmid is transferred to a host microorganism Escherichia coli that has been made easy to accept foreign DNA by, for example, MnCl ₂ -CaCl ₂ treatment, and a trait that retains the recombinant plasmid. Get a transformant. The transformant expresses the drug resistance gene present in the vector plasmid used, that is,
For example, it can be selected by utilizing the fact that Escherichia coli transformed with a recombinant plasmid prepared by ligating a chromosomal DNA fragment cleaved with HindIII to pBR322 can grow on a medium containing ampicillin. Colony hybridization [M. Grunstein, J. Walls: Methods in Enzymology, 68 , 37] was selected from the large number of transformants (usually 3,000 to 10,000) thus selected.
9, Academic Press Inc., New York (1979)], a transformant carrying an alkaline protease gene is selected. As a probe for detecting the alkaline protease gene-carrying strain, when the oligonucleotide previously synthesized and labeled with ³² P is used, the film at the position corresponding to the colony of the alkaline protease gene-carrying strain is detected by autoradiography. Is exposed to the ³² P radiation of the hybridized probe and detected as a black signal. The corresponding alkaline protease gene-carrying strain is obtained by replicating from a nitrocellulose filter on which colonies are formed before performing colony hybridization to another new nitrocellulose filter.

On the other hand, however, the alkaline protease gene of Bacillus licheniformis can also be cloned by carrying out colony hybridization using the following DNA.

That is, the alkaline protease gene of Bacillus amyloliquefaciens, which produces an alkaline protease having 70% homology with the alkaline protease of Bacillus licheniformis, has homology even in the nucleotide sequence with the alkaline protease gene of Bacillus licheniformis. Presumed to be expensive. The nucleotide sequence of the alkaline protease gene of one strain of Bacillus amyloliquefaciens ATCC23844 strain has already been clarified.
(J. Wells et al: Nucleic Acids Res., 11 , 7911 (1983)
Therefore, in this base sequence, a continuous base sequence of about 14 to 18 is arbitrarily selected from the region corresponding to the mature protein portion of the alkaline protease, and a single-stranded chain having the same sequence as this is selected. Is synthesized and cloned with the alkaline protease gene of Bacillus amyloliquefaciens in exactly the same manner as the cloning of the alkaline protease gene of Bacillus licheniformis described above. This gene is then used to clone the Bacillus licheniformis alkaline protease gene suspected of being homologous to this gene.

Cloning of the Bacillus licheniformis alkaline protease gene, except that the Bacillus amyloliquefaciens alkaline protease gene is used as a probe for colony hybridization,
Basically, it is carried out in the same manner as the cloning of the Bacillus licheniformis alkaline protease gene. That is, the chromosome DN of Bacillus licheniformis
Transformation obtained by transforming Escherichia coli LE392 with a recombinant plasmid obtained by ligating a DNA fragment containing a DNA fragment containing A or a protease fragment with a restriction enzyme and a vector plasmid with T4 DNA ligase. From the body, a transformant carrying an alkaline protease gene is selected by colony hybridization. A probe used for colony hybridization is a plasmid extracted from a transformant having an alkaline protease gene of Bacillus amyloliquefaciens, cleaved with a restriction enzyme, and fractionated by agarose gel electrophoresis. A fragment containing the alkaline protease gene was prepared, and the nickel translocation [PWJRigby et al .: J. Mol. Biol., 113 ,
237 (1977)], labeled with ³² P is used. In this case, the DNA of the probe, namely the Bacillus
Since the base sequences of the amyloliquefaciens alkaline protease gene and the Bacillus licheniformis alkaline protease gene are not completely the same, the stability of the hybrid formed by hybridization has exactly the same base sequence. Since the temperature is lower than that of DNA hybridized, it is necessary to lower the hybridization temperature to a temperature lower than the normal temperature of 42 ° C and to perform it at around 25 to 30 ° C.

Thus, the fact that the transformant selected by colony hybridization carries the alkaline protease gene means that a plasmid is extracted from this transformant and suitable restriction enzyme, for example, chromosomal DNA cleavage Cleavage with the restriction enzyme used, the labeled DNA used for colony hybridization as a probe, Southern
It is confirmed by carrying out hybridization and detecting a signal-providing fragment by autoradiography.

Furthermore, to determine whether the alkaline protease gene present on this recombinant plasmid contained the entire region, the recombinant plasmid was cleaved with a restriction enzyme to cut out the inserted chromosomal DNA fragment, and this fragment was isolated by a Bacillus bacterium. A vector plasmid that is retained, for example, pUB110 or a shuttle vector that is retained by both Bacillus bacteria and Escherichia coli, such as pHY300PLK, is ligated to transform a protease low-producing strain of Bacillus subtilis, and a recombinant plasmid is obtained. This can be confirmed by observing whether the introduced transformant has a high protease-producing ability.

In the present invention, as this protease low-producing strain, Bacillus subtilis RM125 (T. Uozumi et al: Molec. Gen. Genet.,
152 , 65 (1977)] is mutated with N-ethyl-N'-nitro-N-nitrosoguanidine (NTG) to obtain Bacillus subtilis PW10 (Microtechnical Research Institute No. 9176). This strain does not form a halo (cloudy ring) formed by the action of protease, even if it is inoculated on a Nutrient agar plate (casein plate) containing 1% casein and cultured overnight at 37 ° C. A transformant having a high protease-producing ability can be confirmed by forming a halo.

Transformation of Bacillus subtilis with a recombinant plasmid is a protoplast method of forming protoplasts by lysozyme treatment and incorporating DNA in the presence of polyethylene glycol [S. Chang, SN Cohen: Molec. Gen. Gene.
t., 168 , 111 (1979)] or a method using a competent cell in which DNA is easily accepted by growing in a minimal glucose medium [D. Dubnan, RDAbelson: J. Mo.
l.Biol., 56 , 209 (1971)] and the like.

When introduced into Bacillus subtilis, the protease-producing ability is expressed, and the produced protease has the same properties as the alkaline protease of Bacillus licheniformis, for example, the same molecular weight and immunological properties, the entire alkaline protease gene The region is determined to have been cloned. However, when the protease-producing ability is not expressed even when transferred to Bacillus subtilis, a part of the gene is missing, and therefore colony hybridization is performed again to clone the missing part.

At this time, if the chromosomal DNA fragment used to create the recombinant plasmid was cleaved with a different type of restriction enzyme from the one used at the beginning, the alkaline protease gene cloned here would be cloned. Colony hybridization can be performed using a fragment containing a part as a probe, and a transformant carrying a gene containing a region adjacent to this fragment, that is, a region lacking the alkaline protease gene can be efficiently screened. can do.

When adjacent gene fragments are obtained in this way, this fragment can be ligated to the gene fragment obtained first to construct a gene having the entire region of the alkaline protease gene. Of course, the above operation is not always necessary when the entire region of the alkaline protease gene is contained in the fragment inserted into the recombinant plasmid obtained later.

The fact that the cloned gene or the reconstituted gene contains the entire region of the alkaline protease gene as described above means that the gene is ligated to a vector plasmid retained in Bacillus bacterium, and low production of Bacillus subtilis protease is produced. It can be confirmed by transforming the strain and expressing the ability to produce alkaline protease as an index.

The recombinant plasmid containing the alkaline protease gene thus obtained is further reduced in size, if necessary, by removing a region unnecessary for expression of the alkaline protease. Such reduction of the recombinant plasmid is effective for reducing the labor of determining the base sequence and reducing the load on the host bacterium carrying the recombinant plasmid to allow efficient gene expression.

The nucleotide sequence of the DNA fragment containing the entire region of the alkaline protease gene is the method of Clewell and Helinski from the bacterial cells in the logarithmic growth phase of Escherichia coli LE392 or Bacillus subtilis carrying the recombinant plasmid containing the fragment [DB Clewell, DR Helinski. : Proc.Natl.Acad.Sci., U
SA, 62 , 1159 (1969)] and the like, the recombinant plasmid is extracted, purified, and cleaved with a restriction enzyme to recover a fragment containing an alkaline protease gene, and the M13 dideoxy thie termination method [J.Messing: Methods in Enzym
ol., 101 , 20, Academic Press, New York (1983); F. Sang
er: Science, 214 , 1205 (1981)] and the like.

In addition, the Bacillus bacterium when introducing the recombinant plasmid is not particularly limited, but a strain having a low productivity of amylase, levan eucyclase, neutral protease, or the like, or a strain having a low productivity of a causative agent of allergy It is preferable to produce a transformant using the above and to culture the transformant, because it enables easier recovery and purification of the alkaline protease.

A gene DN that forms the entire region of the alkaline protease gene
A conventional method may be used for producing an alkaline protease using a Bacillus bacterium transformed by transfecting a recombinant plasmid to which A is ligated. That is, carbon source, nitrogen source,
Inorganic ions, if necessary, using a medium containing a trace amount of organic nutrient sources such as vitamins, pH and temperature under aerobic conditions, while culturing until the desired alkaline protease is accumulated while appropriately adjusting the temperature, good.

The alkaline protease can be recovered from the culture medium by a usual method. That is, after completion of the culture, the precipitate produced by adding an organic solvent such as ethanol or acetone or an inorganic salt such as ammonium sulfate to the supernatant obtained by removing the cells by a method such as centrifugation may be recovered.

In order to secrete and produce useful proteins derived from heterologous organisms such as growth hormone by utilizing the cloned Bacillus licheniformis alkaline protease gene, the entire region of the alkaline protease gene promoter, the ribosome binding region and the structural gene DNA having a memorized sequence corresponding to the amino acid sequence of the useful protein in a recombinant plasmid containing a region involved in the secretion of
A recombinant plasmid in which the fragment is ligated adjacent to the downstream region of the region involved in secretion is prepared, and the transformant obtained by introducing into a Bacillus bacterium may be cultured.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention should not be limited thereto.

The restriction enzymes and DNA modifying enzymes such as T4 polynucleotide kinase, T4 DNA ligase, alkaline phosphatase, and DNA polymerase I used in the present invention are all commercially available, for example, purchased from Takara Shuzo Co., Ltd., Toyobo Co., Ltd., etc. be able to. Although vector plasmids are also commercially available, in the present invention, in addition to commercially available products, those extracted and purified from Escherichia coli or Bacillus subtilis carrying the plasmid are used.

Example 1 Cloning of the alkaline protease gene of Bacillus amyloliquefaciens (1) Synthesis of probe oligonucleotide and labeling with ³² P The nucleotide sequence of the alkaline protease gene of Bacillus amyloliquefaciens ATCC23844 was prepared by JA Wells et al. [JA Wells et al .: Nucleic Acid
s Res., 11 , 7911 (1983)]. 8 of the reported nucleotide sequences from the N-terminus of the mature protein of alkaline protease
ATCA in the region corresponding to the 13th amino acid
An oligonucleotide consisting of 16 bases having a base sequence of CAAATTAAAGCC was selected as a probe and synthesized by the solid phase phosphotriester method. Five kinds of 3'-chlorophenyl phosphate protected deoxydinucleotides (GC, AA, TT, CA and AT) manufactured by Pharmacia PL Biochemicals and fully protected deoxymononucleotide manufactured by Dojindo Laboratories were used for the synthesis of oligonucleotides. (A) and polystyrene support-protected nucleoside manufactured by Wako Pure Chemical Industries (C-
Resin) was used.

0.6m of a 1M solution of zinc bromide in dichloromethane-isopropanol (85:15, V / V) in 5.5mg (0.55µmol) of C-resin
l was added and reacted at room temperature (20 to 30 ° C) for 3 minutes, then the reaction solution was removed and 1 ml of dichloromethane-isopropanol (8
The resin was washed with 5:15 V / V). 2 more steps
After repeating 1 time, the resin was further added 3 times with 1 ml of dichloromethane-isopropanol (85: 15V / V), 3 times with 1 ml of 0.5M triethanolamine-acetic acid (pH 7.5), 2 times with 1 ml of dry pyridine, It was washed twice with 1 ml of dry tetrahydrofuran successively. After drying the resin after washing under reduced pressure,
55 μl of 100 mM GC solution (dissolved in 0.45 M trimethylbenzenesulfonylnitrotriazolide-dry pyridine solution)
In addition, a base was added to the C-resin by reacting at 37 ° C. for 40 minutes. Then, the reaction solution was removed, washed with 1 ml of dry pyridine, and then dissolved in a capping solution (dry tetrahydrofuran: acetic anhydride: dry pyridine = 7: 1: 2, V / V) at 33.3 mg / m 2.
l 0.6 ml of dimethylaminopyridine was added and reacted at room temperature for 5 minutes to acetylate the unreacted 5'-hydroxyl group. After removing the reaction solution, the resin was washed 4 times with 1 ml of dichloromethane-isopropyl alcohol (85:15 V / V). Through the steps so far, a GCC-resin having 3 bases bound to the resin was obtained. A series of steps up to this point is one cycle, and a resin to which a base is added in each cycle is used as a starting material for the next cycle, and a predetermined dinucleotide or mononucleotide, that is, AA, A, TT, AA, CA, CA, By sequentially repeating the reaction of adding AT, ATCACAAATTAAAG
CC-resin was synthesized.

The thus synthesized oligonucleotide-immobilized resin was washed three times with 1 ml of dry pyridine, and then 300 μl of 0.5M tetramethylguanidine-0.5M nitrilobenzaldoxime in 50% dioxane was added thereto, and the mixture was incubated at 37 ° C. After keeping for 18 hours, the oligonucleotide was cleaved from the resin and the phosphate group was deprotected. Next, the reaction solution was recovered, and the resin was mixed with a washing solution which was washed twice with 500 μl of 50% pyridine and dried under reduced pressure. To this, 28% ammonia water 0.6 ml
Was added and the mixture was kept at 55 ° C. for 6 hours to deprotect the base. Ammonia was removed under reduced pressure, extraction was further performed with an equal volume of ether, and the mixture was concentrated to about 100 μl, to which 2.6 μl of 2M triethylamine-acetic acid (pH 7.0) was added. This solution was subjected to high performance liquid chromatography using a Solvax ODS column (manufactured by DuPont), fractionated by a linear concentration gradient of 10-40% acetonitrile in 50 mM triethylamine-acetic acid (pH 7.0) solution, and The fraction corresponding to the peak was collected. The collected fractions are dried under reduced pressure and then 1 ml of 80%
Acetic acid was added and kept at room temperature for 20 minutes to deprotect the 5'end, and acetic acid was removed under reduced pressure to obtain purified oligonucleotide 160.
μg was obtained.

Γ ³² was added to 96 ng of the purified oligonucleotide thus obtained.
Add P-ATP (5000 Ci / m mol Amersham) 100 μCi, T4 polynucleotide kinase 2.2 unit and kinase buffer (50 mM Tris-HCl buffer pH 7.5, 10 mM MgCl ₂ , 10 mM dithiothreitol, concentration is final concentration) Reaction liquid 30
The µl was kept at 37 ° C for 60 minutes, and the 5'end was labeled with ³² P.
20 μg of yeast tRNA and 75 μl of ethanol were added to the reaction solution to precipitate the oligonucleotide, and the precipitate collected by centrifugation was washed with ethanol, dried, and then dried with 100 μl of TE.
It was dissolved in a buffer solution (10 mM Tris-base buffer pH 8.0, 1 mM EDTA) to obtain a solution (0.3 μCi / μl) of a 5′-end labeled oligonucleotide.

(2) Preparation of chromosomal DNA 20 ml of new broth (meat extract 0.5 wt%, polypeptone S 1 wt%, NaCl 0.5 wt%, pH 7.0) is inoculated with Bacillus amyloliquefaciens ATCC23844 one platinum loop ear from the agar slope. Then, preculture was performed overnight at 37 ° C. Then, 5 ml of the same was inoculated into 500 ml of the same medium and cultured for about 2.5 hours, and the obtained cells were collected by centrifugation. 50 ml of this fungus body
After washing with 150 mM NaCl / 100 mM EDTA solution (pH 8), the cells were suspended in 25 ml of the same solution, 10 mg of lysozyme (Sigma) was added, and the mixture was kept at 37 ° C for 30 minutes. Then add 2.5 ml of 20% sodium dodecyl sulphate (SDS) and mix gently, then 60
The cells were lysed at 10 ° C for 10 minutes. After cooling the lysate to room temperature,
6.6 ml of 5M sodium perchlorate and 31.6 ml of chloroform-isoamyl alcohol (24: 1, V / V) mixed solution were added, and the mixture was mixed gently to make a uniform emulsion. 10,000 this
Centrifugation was performed at rpm for 10 minutes to separate the aqueous layer, the intermediate layer, and the organic layer into three layers, and the aqueous layer was collected. To this water layer was added 2 volumes of ethanol, and the resulting filamentous precipitate was wound on a glass rod,
The precipitate was washed with 70% and 99.5% ethanol successively and dried under reduced pressure. Add 15 ml of 0.1 x SSC (1 x SSC is 1
Equivalent to 50 mM NaCl, 15 mM trisodium citrate, 0.1 ×
The SSC number represents the concentration ratio of each component to 1 × SSC.
That is, 0.1 × SSC is a solution having a concentration 1/10 that of 1 × SSC. 10x after dissolution in (following this indication method)
SSC (1.5 ml) was added to this, and 5 mg / ml RNase A (manufactured by Sigma) in which mixed DNase was inactivated by heat treatment at 90 ° C for 10 minutes.
165 μl was added and kept at 37 ° C. for 30 minutes to decompose contaminating RNA. To remove RNase A, add equal volumes of phenol-chloroform (1: 1, V / V, 0.1M Tris-hydrochloric acid buffer saturated with pH 7.5) and mix gently to form a uniform emulsion, then centrifuge. Separation was performed and the separated aqueous layer was collected. An equal volume of chloroform-isoamyl alcohol (24:
1, V / V) was added, extraction was performed in the same manner as above, and the operation of collecting the aqueous layer was repeated twice. The water layer thus obtained has 2
A filamentous precipitate produced by adding a double volume of ethanol was recovered in the same manner as above, washed with ethanol and dried. this
Chromosomal DNA solution (5.6 mg of DNA) by dissolving in 10 ml of TE buffer
Got

(3) Chromosome containing alkaline protease gene
Calculation of length of DNA fragment of 60 μg of chromosomal DNA obtained in the above (2) and 120 units
600 μl of reaction solution containing HindIII and HindIII reaction buffer (10 mM Tris-hydrochloric acid buffer pH 7.5, 7 mM MgCl ₂ , 60 mM NaCl, the final concentration) was kept at 37 ° C. for 3 hours to cleave the DNA, then 2.5 times A quantity of ethanol was added to precipitate the DNA.
The precipitate was recovered by centrifugation and dissolved in 180 μl of TE buffer to obtain a HindIII fragment solution of chromosomal DNA. This solution
15 μl was fractionated by agarose gel electrophoresis. At this time,
As a molecular weight marker for calculating the length of a DNA fragment, 1 μg of DNA obtained by cleaving λ-fage DNA with HindIII was electrophoresed. After the electrophoresis, the gel was immersed in a 1 µg / ml ethidium bromide solution to stain the DNA, and then 0.2 times the volume of 3 times the gel.
Treated sequentially with 5N HCl, 0.5N NaOH-1M NaCl and 0.5M Tris-HCl buffer (pH 7.5) -3M NaCl for 15 minutes, twice each
The DNA was denatured. A nitrocellulose filter was brought into close contact with this gel, and the DNA was transferred to the nitrocellulose filter by leaving it for about 16 hours while supplying 20 × SSC from the bottom of the gel through paper. After drying this filter, it was treated at 80 ° C. for 2 hours under reduced pressure to fix the DNA to the filter.

The filter was placed in a plastic bag and the pre-hybridization solution [5 x Denhardt solution (1 x Denhardt solution
0.02wt% equol, 0.02wt% bovine serum albumin, 0.02
(wt% polyvinylpyrrolidone), 5 × SSC, 50 mM phosphate buffer pH 6.5, 100 μg / ml denatured salmon semen DNA] 20 ml is added, the vinyl bag is sealed and kept at 55 ° C. overnight for prehybridization. I went. Then, the prehybridization solution was removed from the plastic bag, and 4 ml of the hybridization solution [the composition is the same as that of the prehybridization solution] and the oligonucleotide labeled with ³² P obtained in (1) above After heat treating 2 μCi at 95 ° C. for 5 minutes, a rapidly cooled solution was added and the vinyl bag was sealed. After thoroughly mixing the solution in the bag, the mixture was kept at 37 ° C. for about 16 hours for hybridization. Then, the filter was taken out, washed in about 200 ml of 4 × SSC-0.1% SDS solution at room temperature for 3 times for 10 minutes each, dried, and then adhered to the X-ray film on the filter and exposed at −80 ° C. for 16 hours. . The blackened part of the developed X-ray film is compared with the migration position of the molecular weight marker, and the chromosome where the alkaline protease gene exists
The length of the DNA fragment was calculated to be 2.8 kb (kb is an abbreviation for kilobase pair, the same applies hereinafter).

(4) Fractionation of chromosomal DNA fragment Solution of HindIII fragment of chromosomal DNA prepared in (3) above
150 μl (DNA 50 μg) was fractionated by agarose gel electrophoresis, stained with ethidium bromide, and then the gel was irradiated with an ultraviolet lamp to give a fragment of 1.8 to 4.5 kb calculated from the position of the fragment of the λ-charge DNA of the molecular weight marker. The gel piece at the corresponding position was cut out.

After putting this gel piece in a dialysis membrane and filling it with 2 ml of TE buffer and sealing it, electrophoresis is performed at 100 V for 1 hour in Tris-borate buffer (90 mM Trizma base, 90 mM boric acid, 4 mM EDTA, pH 8.3). The DNA was then eluted from the gel. Collect the eluate and add 2.5 volumes of ethanol to precipitate the DNA,
The precipitate was recovered by centrifugation, dried, and then dissolved in 40 μl of TE buffer to obtain about 8 μ of a chromosomal DNA fragment of 1.8 to 4.5 kb.

(5) Preparation of vector plasmid Escherichia coli LE392 carrying plasmid pBR322
(ATCC33572) to 1 NZYM medium [NZ amine type A
(Wako Pure Chemical Industries) 1wt%, Polypeptone S0.5wt%, NaCl
0.5 μg / ml ampicillin was added to 0.5 wt% 10 mM MgCl ₂ , pH 7.2], and the cells were cultured at 37 ° C. up to 10 ⁹ cells / ml. To this, 100 mg of chloramphenicol was added, and further cultured for 16 hours. Clewell and Helinski from cells collected by centrifugation
Method of DBClewell, DR Helinski: Proc.Natl.Acad.Sc
i., USA, 62 , 1159 (1969)] and extraction of crude plasmid fraction and CsCl-ethidium bromide equilibrium density gradient centrifugation (Ultracentrifuge 85P for Hitachi separation, rotor RP83T, 33,0).
Fractionation was carried out at 00 rpm, 40 hours, 20 ° C.) to obtain a plasmid fraction. This fraction was extracted with an equal volume of isopropanol to remove ethidium bromide, and then dialyzed twice against TE buffer 1 for 1 hour to give purified plasmid pBR322.
Obtained 1.5 mg.

20 μg of this plasmid pBR322 and 40 units of HindIII
After reacting at 37 ° C. for 3 hours and cleaving, the precipitate was precipitated with ethanol and dried, and dissolved in 200 μl of 10 mM Tris-hydrochloric acid buffer pH 8.0. 0.2 unit of Escherichia coli alkaline phosphatase was added thereto and reacted at 65 ° C for 30 minutes. After the reaction
An equal volume of phenol / chloroform (1: 1, V / V) was added, and the mixture was shaken for 5 minutes for extraction. The two layers were separated by centrifugation and the aqueous layer was collected. This aqueous layer was extracted again with phenol / chloroform and then twice with an equal volume of chloroform / isoamyl alcohol (24: 1, V / V). 500 μl of ethanol was added to the aqueous layer to precipitate the DNA, the precipitate was recovered by centrifugation, dried, and then dissolved in 100 μl of water. Thus, a linear vector plasmid pBR322 cleaved with HindIII and dephosphorylated at the 5'end was obtained.

(6) Preparation of Recombinant Plasmid About 1.8 to 4.5 kb chromosomal DNA fragment obtained in (4) above
μg (20 μl) was mixed with 2 μg (10 μl) of the linear vector plasmid pBR322 cleaved with HindIII and dephosphorylated at the 5'end obtained in (5) above, and mixed with 0.8
Unit T4 DNA ligase and ligase buffer [66 mM
Reaction solution 4 to which Tris-hydrochloric acid buffer solution pH 7.6, 6.6 mM MgCl ₂ , 10 mM dithiothreitol, 2 mM ATP, the final concentration was added)
0 μl was kept at 5 ° C. for 16 hours, and both DNAs were ligated to prepare a recombinant plasmid.

(7) Transformation of Escherichia coli with recombinant plasmid and selection of alkaline protease gene-carrying strain Escherichia coli LE392 was inoculated into 5 ml of NZYM medium and 0.2 ml of the preculture liquid was inoculated into 20 ml of NZYM medium. Then 3
It was cultured at 7 ° C for 2.5 hours. Collect the cells by centrifugation and 10 ml
After washing with 10 mM NaCl, 10 ml of Mn-Ca solution (70 mM MnCl ₂ , 3
After suspending in 0 mM CaCl ₂ , 40 mM sodium acetate (pH 5.6) for 15 minutes on ice, the cells were collected by centrifugation and 0.5 ml of Mn-
The cells were suspended in a Ca solution to obtain a DNA-acceptable cell. To 0.4 ml of this suspension, 30 μl of the recombinant plasmid solution obtained in (6) (about 4.5 μg of DNA) was added and mixed, and the mixture was kept in ice for 60 minutes and then heated at 37 ° C. for 2 minutes. Cells were allowed to incorporate the recombinant plasmid. Add NZYM medium 1.
After adding 6 ml and shaking at 37 ° C. for 30 minutes, 100 μl was applied to each plate of NZYM agar plate containing 25 μg / ml of ampicillin and nitrocellulose filter. This plate was cultured overnight at 37 ° C. to obtain 7900 colonies of transformants that had become resistant to ampicillin. next,
Using this filter as a master filter, a new nitrocellulose filter was brought into close contact with it, and a replica was taken. Replace both filters with a new agar plate (25 μg / ml
NZYM medium containing ampicillin) and cultured overnight at 37 ° C., and the plate with the master filter was stored.
On the other hand, the replica filter was transferred to an NZYM agar plate containing 200 μg / ml chloramphenicol, and the culture was continued at 37 ° C. overnight.

Next, this filter was sequentially kept on paper moistened with the following four kinds of solutions for 10 minutes each, and DNA in the bacterial cells was eluted and adsorbed on the filter. As four kinds of solutions,
0.5N NaOH 1M Tris-hydrochloric acid buffer pH 8.0 0.5M Tris-hydrochloric acid buffer pH 8.0-1M NaCl 2 × SSC was used.
The filter thus treated was dried, and the DNA was fixed to the filter by the same operation as the above (3).

Then, using this filter, obtained in (1) above.
Pre-hybridization using the oligonucleotide labeled with ³² P as a probe in the same manner as in (3) above,
As a result of performing hybridization and autoradiography, one signal was detected on the autoradiogram. The transformant corresponding to this signal was taken from the master filter to obtain a transformant carrying a recombinant plasmid containing the alkaline protease gene.

(8) Identification of Recombinant Plasmid Containing Alkaline Protease Gene When a plasmid was prepared from the transformant obtained in (7) by the same method as pBR322, a plasmid having a length of 13 kb was obtained. (See FIG. 1). pAMP3 was cleaved with the restriction enzymes shown in FIG.
The restriction enzyme map shown in Fig. 1 was created based on the results of calculating the size of the fragments detected by fractionation by agarose gel electrophoresis.
Two R322 molecules and a HindIII fragment of two chromosomal DNAs (2.8kb and
(1.4 kb) was ligated.

Of the two chromosomal DNA fragments, the 2.8 kb fragment (hatched area in Fig. 1) hybridizes with the probe oligonucleotide by Southern hybridization, and matches the length calculated in (3) above. From this, it was determined that this fragment contained the alkaline protease gene.

Escherichia coli LE392 (pAMP3) carrying the recombinant plasmid pAMP3 has been deposited with the Institute of Microbial Science and Technology, National Institute of Advanced Industrial Science and Technology as No. 9175.

Example 2 Cloning of Bacillus licheniformis alkaline protease gene (1) Preparation of probe DNA fragment and labeling with ³² P The recombinant plasmid pAMP3 obtained in Example 1 was cleaved with HindIII into a 2.8 kb fragment. Bacillus amyloliquefaciens alkaline protease gene is present, so this fragment was used as a probe.

After 30 μg of pAMP3 was cleaved by reacting with 60 units of HindIII at 37 ° C. for 3 hours, 2.5 volumes of ethanol was added to precipitate DNA. Collect the precipitate by centrifugation and
It was dissolved in μl of TE buffer and fractionated by agarose gel electrophoresis. When the gel was stained with ethidium bromide and irradiated with an ultraviolet lamp, three bands were detected. Of these, the middle band having a length of 2.8 kb was cut out, the DNA was eluted by the electrophoresis method in the same manner as in Example 1 (4), and the DNA was recovered by ethanol precipitation. After drying the DNA
It was dissolved in 60 μl of TE buffer to obtain a DNA fragment solution of about 6 μg.

Α ³² P-dCTP was added to 10 μl of this DNA fragment solution (about 1 μg of DNA).
(410Ci / mmol, Amersham) 50μCi, 120ng / ml DNase
2.5 μl of I (Boehringer Mannheim Yamanouchi) and nickel translation buffer [50 mM Tris-hydrochloric acid buffer pH 7.8, 5 mM MgCl ₂ , 10 mM _2- mercaptoethanol, 15 μg / ml bovine serum albumin, 0.6 mM dATP, 0.6 mM dG
TP, 0.6 mM dTTP, concentration is final concentration)
1 was incubated at 27 ° C. for 30 minutes, 5 units of DNA polymerase I was added, and at 14 ° C. for an additional 60 minutes, nickel translocation [PWJRigby et al .: J. Mol. Biol., 113 , 2
37 (1977)] and synthesized DNA labeled with ³² P.

20 μg of yeast tRNA and 125 μl of ethanol were added to this reaction product to precipitate the DNA, the DNA was recovered by centrifugation, dried, dissolved in 100 μl of TE buffer, and probe DNA labeled with ³² P. (0.2 μCi / μl) was obtained.

(2) Preparation of gene donor A publicly known strain of Bacillus licheniformis was searched and IFO12196 strain was selected as a strain having high protease production ability.

Bacillus licheniformis IFO12196 to N-ethyl-N '
-Nitro-N-nitrosoguanidine was used as a mutagen to carry out a mutagenesis treatment that is usually performed, and new agar (meat extract 0.5 wt%, peptone 1) containing casein 1 wt% was used.
The mutant strain HP19611, which forms a large halo on wt%, NaCl 0.5 wt%, agar 1.5 wt%, pH 7.0) was induced to serve as a donor of the alkaline protease gene. Bacillus licheniformis HP19611 has been deposited with the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology, as Microbiology Research Institute No. 9177.

(3) Preparation of chromosomal DNA The chromosomal DNA of Bacillus licheniformis HP19611 was prepared in exactly the same manner as in Example 1 (2), and 9.7 mg of chromosomal DNA was prepared.
Got

(4) Chromosome containing the alkaline protease gene
Calculation of length of DNA fragment 60 μg of chromosomal DNA obtained in (3) above and 120 units
EcoRI and EcoRI reaction buffer (100 mM Tris-hydrochloric acid buffer pH
7.5, 7 mM MgCl ₂ , 50 mM NaCl, 7 mM _2- mercaptoethanol, 0.01 wt% bovine serum albumin, concentration is final concentration] 600 μl of reaction solution was kept at 37 ° C. for 3 hours to cleave DNA, and then 2.5 times amount of ethanol was added. Was added to precipitate the DNA.
The precipitate was recovered by centrifugation and dissolved in 180 μl of TE buffer to obtain an EcoRI fragment solution of chromosomal DNA. This solution 15
Electrophoresis, DNA transfer to a nitrocellulose filter, and immobilization of the microliter were performed in exactly the same manner as in Example 1 (3).

Hybridization was performed using the filter thus prepared. Pre-hybridization solution [50% formamide, 5 x Denhardt's solution, 5 x SSC, 50 mM phosphate buffer pH stored in a plastic bag
6.5, 1wt% glycine, 500μg / ml denatured salmon semen DNA] 20ml
Was added, the mixture was sealed, and the mixture was kept overnight at 42 ° C. for prehybridization.

Then, the prehybridization solution was removed from the plastic bag, and the hybridization solution [50% formamide, 1 × Denhardt's solution, 5 × SSC, 20 mM phosphate buffer pH
6.5, 100 μg / ml denatured salmon semen DNA, 10% sodium dextran sulphate] 4 ml and ³² P-labeled probe DNA 2 μCi obtained in (1) above was heat-treated at 95 ° C for 5 minutes and then rapidly cooled to add vinyl. The bag was sealed. After thoroughly mixing the solution in the bag, the mixture was kept at 25 ° C. for 16 hours for hybridization.

After that, take out the filter and take about 200 ml of 2 x SSC-0.1.
After washing in a SDS solution at room temperature for 10 minutes three times, and then drying, autoradiography was carried out in the same manner as in Example 1 (3). From the position of the signal on the autoradiogram, the length of the chromosomal DNA fragment containing the Bacillus licheniformis alkaline protease gene was calculated to be 3.4 kb.

(5) Fractionation of chromosomal DNA fragment 150 μL EcoRI fragment solution of chromosomal DNA obtained in (4) above
The same treatment as in Example 1 (4) was performed using 1 (50 μg of DNA) to give about 5 μg (25 μl) of a 3-4 kb chromosomal DNA fragment.
Got

(6) Preparation of vector plasmid 20 μg of plasmid pBR322 obtained in Example 1 (5)
Was cleaved by reacting with 40 units of EcoRI at 37 ° C for 3 hours. After that, the same processing as in Example 1 (5) was performed to obtain Ec
A linear vector plasmid pBR322 cleaved with oRI and dephosphorylated at the 5'end was obtained.

(7) Preparation of recombinant plasmid About 4 μg of 3-4 kb chromosomal DNA fragment obtained in (5) above
(20 μl) and the linear vector plasmid cleaved with EeoRI obtained in (6) above and dephosphorylated at the 5 ′ end
2 μg (10 μl) of pBR322 was mixed and treated with T4 DNA ligase in the same manner as in Example 1 (6) to prepare a recombinant plasmid.

(8) Transformation of Escherichia coli with recombinant plasmid and selection of alkaline protease gene-carrying strain 30 μl of the recombinant plasmid solution obtained in (7) above (DN
A (about 4.5 μg) was used in the same manner as in Example 1 (7).
Transforming the treated E. coli LE392,
5800 colonies of transformants resistant to ampicillin were obtained. A filter on which colonies were formed was used in Example 1 (7).
After fixing DNA by treating in the same manner as in (4) above,
Hybridization was performed under the same conditions as described above, and 6 signals were detected on the autoradiogram.

Six transformants corresponding to these signals were separated from the master filter, and recombinant plasmids carried by these transformants were identified in the same manner as in Example 1 (8). A 3.4 kb EcoRI fragment that hybridizes with the alkaline bacterium protease gene of Bacillus amyloliquefaciens as a probe was inserted. Recombinant plasmid possessed by one of these strains
It was named pLP3 (see FIG. 2). Escherichia coli LE392 (pLP3), which carries this plasmid,
No. 178 has been deposited with the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology.

The 3.4 kb EcoRI fragment contained in the recombinant plasmid pLP3 was
Vector plasmid pUB110 retained in Bacillus
Since the expression of alkaline protease was not observed even when it was ligated to and introduced into Bacillus subtilis, it was determined that this fragment contained only a part of the alkaline protease gene. In addition, recombinant plasmid pLP3 was added to EcoRI.
Cleaved with BglII and subjected to Southern hybridization in the same manner as in (4) above, the probe hybridizes to the 1.2 kb fragment in the shaded area shown in FIG. 2, so the EcoRI site of this fragment It can be seen that there is a portion lacking the alkaline protease gene in the adjacent region. Therefore, in order to clone this missing portion again, a recombinant plasmid was prepared again and colony hybridization was performed.

Chromosomal DNA of Bacillus licheniformis HP19611 20 μg
40 units of BglII and BglII reaction buffer [10 mM T
ris- HCl buffer pH7.5,7mMMgCl _2, 100mM NaCl, 7mM2-
Reaction solution containing mercaptoethanol and the final concentration]
200 μl and 37 ° C for 3 hours to cut the DNA, and then at 65 ° C
After heat treatment for 10 minutes, 500 μl of ethanol was added to precipitate DNA. The precipitate was recovered by centrifugation, dried and dissolved in 100 μl of water to prepare a chromosomal DNA fragment.

On the other hand, the vector plasmid is pBR322 20 μg and 40 units of BamHI and BamHI reaction buffer [10 mM Tris-hydrochloric acid buffer pH 8.0, 7 mM MgCl ₂ , 100 mM NaCl, ₂ mM _2- mercaptoethanol, 0.01 wt% bovine serum albumin, the final concentration is 200 μl of the reaction solution containing the above] was kept at 37 ° C. for 3 hours to cut the DNA, and then the same treatment as in Example 1 (5) was performed to prepare BamHI.
A linear vector plasmid pBR322, which had been cleaved with and dephosphorylated at the 5'end, was prepared.

4 μg of the chromosomal DNA fragment thus prepared and Bam
2 μg of vector plasmid pBR322 cleaved with HI and dephosphorylated at the 5'end was mixed and treated with T4 DNA ligase in the same manner as in Example 1 (6) to prepare a recombinant plasmid.

20 μl of this recombinant plasmid solution (3 μg of DNA) was used to transform Mn-Ca-treated Escherichia coli LE392 in the same manner as in Example 1 (7) to obtain 7900 colonies of ampicillin-resistant transformants.

The filter on which colonies were formed was treated in the same manner as in Example 1 (7), and then hybridized in the same manner as in (4) above. However, for the probe, the recombinant plasmid pLP3 was cleaved with EcoRI and BglII, and the above (1)
A 1.2 kb fragment obtained by fractionation in the same manner as in (1) was subjected to hybridization at 42 ° C. using DNA labeled with ³² P by nick translation. The filter was washed in 2 x SSC-0.1% SDS solution and then in 0.1 x SSC-0.1% SDS solution at 42 ° C.
It was washed twice for 15 minutes each.

In this way, one signal was detected on the autoradiogram. The transformant corresponding to this signal was separated from the master filter, and the recombinant plasmid contained therein was fixed in the same manner as in Example 1 (8) to obtain the 9.6 kb plasmid shown in the restriction map of FIG. It was shown that this recombinant plasmid was named pLP35. Escherichia coli LE392 (pLP35) containing this recombinant plasmid pLP35 has been deposited with the Institute of Microbial Science and Technology, National Institute of Advanced Industrial Science and Technology as No. 9179.

When Southern hybridization to the recombinant plasmid pLP35 is performed using the above-mentioned 1.2 P EcoRI-BglII fragment labeled with ³² P as a probe, it hybridizes to the fragment containing the hatched portion in FIG. It can be seen that the region adjacent to the EcoRI site, which was lacking, was included.

Example 3 Confirmation of Expression of Bacillus licheniformis Alkaline Protease Gene To confirm that the recombinant plasmid pLP35 obtained in Example 2 contains the entire region of the alkaline protease gene, a vector retained in the genus Bacillus. Bacillus subtilis PW10 was transformed by ligating to the plasmid, and the productivity of protease by the transformant was examined.

The recombinant plasmid pLP35 obtained in Example 2 contains the third
It is presumed that the alkaline protease gene exists across the fragment (1.2 kb) in the shaded area in the figure and the 2.0 kb EcoRI fragment adjacent to it in the counterclockwise direction (moisturizing part in FIG. 3).

However, among these fragments, one end of the hatched fragment is one in which the BglII cutting end of the chromosomal DNA and the BamHI cutting end of the vector plasmid pBR322 are ligated, so that specific cleavage with a restriction enzyme is difficult. is there. To this end, a recombinant plasmid pLP3 containing the same fragment as
A recombinant plasmid for expressing alkaline protease was prepared from pLP35.

As a vector plasmid retained in the genus Bacillus, a shuttle vector pHY300PLK (a product of Takara Shuzo) retained in both Escherichia coli and Bacillus was used. p
HY300PLK has two drug resistance genes and expresses both ampicillin resistance and tetracycline resistance in Escherichia coli and tetracycline resistance in Bacillus bacteria. An outline of the procedure for producing a recombinant plasmid for expressing alkaline protease is shown in FIG.

Recombinant plasmid pLP3 30 μg, 60 units EcoRI, 60
Reaction solution containing unit BglII and EcoRI reaction buffer 30
After 0 μl was kept at 37 ° C. for 3 hours to cut the plasmid, 750 μl of ethanol was added to precipitate the DNA. The precipitate was recovered by centrifugation, dissolved in 150 μl of TE buffer, and the fragments were fractionated by agarose gel electrophoresis in the same manner as in Example 2 (1) to obtain about 4 μg of 1.2 kb fragment.

On the other hand, shuttle vector pHY300PLK 5 μg, 10 units
50 μl of the reaction mixture containing EcoRI, 10 units of BglII and EcoRI reaction buffer was kept at 37 ° C. for 3 hours to cut the plasmid, and after heat treatment at 65 ° C. for 10 minutes, 125 μl of ethanol was added to precipitate the DNA. It was The precipitate was recovered by centrifugation, dried and dissolved in 25 μl of water to obtain a vector DNA solution.

0.25 μg of the above 1.2 kb fragment was mixed with 1 μg of vector DNA, and 20 μl of a reaction solution containing 0.4 unit of T4 DNA ligase and a ligase buffer was kept at 5 ° C. for 16 hours to ligate both DNAs.

10 μl of the DNA solution thus obtained (DNA 0.625 μm
g) was used to transform Mn-Ca-treated Escherichia coli LE392 in the same manner as in Example 1 (7).

Ten strains were selected from the obtained ampicillin-resistant transformants, the plasmids were extracted, and the fragments generated by digestion with EcoRI and BglII were examined by agarose gel electrophoresis. A kb fragment was detected. PH of one of these strains
It was named LP301 (see FIG. 4).

Then, 10 μg of the recombinant plasmid pHLP301 was cleaved by reacting with 20 units of EcoRI at 37 ° C for 3 hours, heat-treated at 65 ° C for 10 minutes, and 2.5 times volume of ethanol was added to the DNA.
Was allowed to settle. The precipitate is collected by centrifugation and dried 50
It was dissolved in μl of water to obtain pHLP301 cleaved with EcoRI.

On the other hand, recombinant plasmid pLP35 10 μg, 20 units Ec
100 µl of a reaction solution containing oRI, 20 units of PvuII and EcoRI reaction buffer was kept at 37 ° C for 3 hours to cut the DNA. pL
Cleavage of P35 with EcoRI produced three fragments of 5.0 kb, 2.6 kb, and 2.0 kb, and the fragment now required is the 2.0 kb fragment (hatched portion in FIG. 4). pLP35 to EcoRI and Pv
When uII is allowed to act simultaneously, two unnecessary fragments of the three EcoRI fragments are further cleaved by PvuII, resulting in a fragment with one end blunt ended, which is equivalent to pHLP301 cleaved with EcoRI. Since it is not ligated, the efficiency of ligation of the necessary 2.0 kb fragment is increased. DN disconnected in this way
The solution A was heat-treated at 65 ° C. for 10 minutes, and 250 μl of ethanol was added to precipitate the DNA. The precipitate was recovered by centrifugation, dried and then dissolved in 50 μl of ice.

10 μl (2 μg of DNA) of both DNA solutions thus obtained
40 μl of the reaction mixture containing 0.8 units of T4 DNA ligase and ligase buffer was mixed for 16 hours at 5 ° C
A is linked.

The thus obtained DNA was used to transform Bacillus subtilis PW10, which is a low protease producing strain. Transformation was performed by the protoplast method as follows.

Bacillus subtilis PW10 with New Orient Broth 5m
0.3 ml of the preculture liquid inoculated into 1 l and cultured overnight was inoculated into 30 ml of a fresh broth and incubated at 37 ° C for 1.5 hours, and then the cells were collected by centrifugation to obtain 2.5 ml of SMMP [2 x SMM 1: 1 mixture of Penassay broth (manufactured by Difco) at 4 times concentration, 2 × SMM is 1M sucrose, 0.04M maleic acid, 0.04
MMgCl ₂ , pH 6.5) and add 5 mg of lysozyme.
Hold at 2 ° C for 2 hours to form protoplasts. Protoplasts were collected by centrifugation, washed once with 4 ml of SMMP, and then suspended in 1.5 ml of SMMP to obtain a protoplast suspension.

To 0.5 ml of this protoplast suspension was added the connection D obtained above.
20 μl of NA solution (2 μg of DNA) and 1.5 ml of 40% polyethylene glycol 4000 dissolved in 2 × SMM were added, and gently mixed for 2 minutes to allow DNA to be incorporated into protoplasts. Next, 5 ml of SMMP was added thereto and mixed, and then the protoplasts were collected by centrifugation, suspended in 1 ml of SMMP, and kept at 30 ° C. for 1.5 hours.

This protoplast solution was added to DM3 regeneration medium containing 20 μg / ml tetracycline (0.5 M sodium succinate pH 7.3, 0.5 w
t% glucose, 0.5wt% casamino acid, 0.5wt% yeast extract, 0.01wt% bovine serum albumin, 0.35wt% dipotassium phosphate, 0.15wt% monopotassium phosphate, 20mMMgCl ₂ , 0.8wt%
100 μl of each agar was applied on the agar, and the mixture was kept at 37 ° C. for 2 days to form a colony.

Approximately 3000 strains of tetracycline-resistant transformants thus obtained were replicated on a casein plate containing 20 μg / ml of tetracycline (a novel broth agar plate containing 1 wt% casein) and cultured at 37 ° C. for 16 hours. Four halo-forming strains were obtained.

Extraction of the plasmids from each of these 4 strains revealed that they all possessed a plasmid having a length of 8.1 kb. The plasmid possessed by one of these strains was used as pHLP351.
(See Fig. 4), EcoRI, BglII, SmaI, EcoRI
The length of the fragment generated by digestion with + BglII, EcoRI + SmaI and BglII + SmaI was examined to obtain the restriction map shown in FIG. 4, and it was confirmed that the planned expression plasmid was prepared.

As described above, since Bacillus subtilis PW10 carrying the recombinant plasmid pHLP351 forms a halo on the casein plate, the gene fragment inserted into pHLP351 contains the entire region of the alkaline protease gene. It is determined that

Recombinant plasmid pH carrying alkaline protease gene
Reduction of LP351 The chromosomal DNA fragment inserted into the recombinant plasmid pHLP351 obtained in Example 3 extends to 3.2 kb.

On the other hand, Bacillus licheniformis alkaline protease has a molecular weight of about 27,500 daltons, and a gene directing the production of this protein was considered to be sufficient if only 2 kb including the region necessary for secretion and the promoter region is necessary. The fragment that was thought to be deleted was deleted to reduce the plasmid, and two types of reduced plasmids were prepared. An outline of the procedure for plasmid reduction is shown in FIGS. 5 and 6.

(1) Preparation of recombinant plasmid pHLP352 and introduction into Bacillus subtilis Recombinant plasmid pHLP351 5 μg, 20 units of SmaI and SmaI reaction buffer [10 mM Tris-hydrochloric acid buffer pH8.0, 7 m
MMgCl ₂ , 20 mM KCl, 7 mM _2- mercaptoethanol, 0.01
wt% bovine serum albumin, concentration is final concentration]
After keeping 50 μl at 37 ℃ for 3 hours, 20 units of BglI
I and 5 μl of 10 times concentrated BglII reaction buffer
Cleavage the plasmid by reacting at 37 ° C for 3 hours and
After heating for 1 minute, add 125 μl of ethanol and add D
NA was precipitated.

On the other hand, 5 μg of the vector plasmid pHY300PLK was treated in the same manner as above to cut and precipitate the DNA.

Collect both DNA precipitates by centrifugation and collect 25 μl each
Dissolved in water and mixed with 10 μl of each, and add 0.8 unit T
40 μl of a reaction solution containing 4 DNA ligase and a ligase buffer was kept at 5 ° C. for 16 hours to ligate DNA.

Escherichia coli L treated with Mn-Ca in the same manner as in Example 1 (7) using 5 μl of this ligated DNA solution (0.5 μg of DNA)
E392 was transformed to obtain 373 ampicillin-resistant transformants.

From these 48 strains, plasmids were extracted and cleaved with SmaI and BglII to obtain 7 strains harboring plasmids producing fragments of 4.9 kb and 2.0 kb.

The plasmid carried by one of these strains was used as pHLP352.
(See FIG. 5). The chromosomal DNA fragment inserted into this plasmid is the chromosome inserted into pHLP352.
This is a deletion of a 1.2 kb fragment (open double-lined portion in FIG. 5) generated by cleavage of a DNA fragment with EcoRI and SmaI.

Escherichia coli LE carrying this recombinant plasmid
392 (pHLP352) has been deposited at the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology, as Microorganism Research Institute No. 9180.

Then, using 0.2 μg of the above-mentioned recombinant plasmid pHLP352, Bacillus subtilis PW10 was transformed by the protoplast method in the same manner as in Example 3 to obtain 23 strains of tetracycline-resistant transformants.

When the transformants of these 23 strains were inoculated on a tetracycline-containing casein plate and cultured at 37 ° C for 16 hours, all the strains formed halos.

Further, it was confirmed that the plasmids possessed by 5 of these strains all possess pHLP352.

Bacillus subtilis PW10 possessing pHLP352 (pHLP35
2) has been deposited with the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology, as Microbiology Research Institute No. 9181.

(2) Preparation of recombinant plasmid pULP354 Vector plasmid pUB110 5 μg, 20 unit BamHI, 20 unit P
50 μl of reaction solution containing vuII and BamHI reaction buffer at 37 ° C
DNA was cleaved by holding for 3 hours, heat-treated at 65 ° C. for 10 minutes, and 125 μl of ethanol was added to precipitate. The precipitate was recovered by centrifugation and dissolved in 25 μl of water.

10 μl of this solution and 10 μl of the solution of pHLP351 cleaved with SmaI and BglII in the above (1) were mixed, and T
4 DNA ligase was allowed to act to carry out DNA ligation reaction.

Using 0.2 μg of the obtained DNA, Bacillus subtilis PW10 was transformed by the protoplast method in the same manner as in Example 3. Protoplasts were regenerated in DM3 regeneration medium containing 150 μg / ml of kanamycin to obtain 229 strains resistant to kanamycin (Km ^r ).

These 229 transformants were replicated on a casein plate containing 10 μg / ml kanamycin and cultured at 37 ° C. for 16 hours to obtain 4 halo-forming strains.

Examining the plasmids carried by these 4 strains all 6.
It carried a 3 kb plasmid. The plasmid possessed by one of these strains was designated as pULP354 (see FIG. 6).

Chromosomal DNA fragment inserted in this plasmid (6th
The shaded portion in the figure) is the same as the chromosomal DNA fragment inserted in the recombinant plasmid pHLP352 obtained in (1) above.

Bacillus subtilis PW10 (pULP35 that owns pULP354
In 4), it has been deposited with the Institute of Microbial Science and Technology, National Institute of Advanced Industrial Science and Technology, as Microbiology Research Institute No. 9182.

Example 5 Production of alkaline protease by Bacillus subtilis transformant carrying reduced recombinant plasmid Bacillus licheniformis HP19611, Bacillus subtilis PW10, Bacillus subtilis PW10 (pHY300PLK) carrying vector plasmid pHY300PLK, reduced recombinant plasmid pHLP352 Bacillus subtilis PW10
(PHLP352), Bacillus subtilis PW10 carrying the vector plasmid pUB110 (pUB110) and Bacillus subtilis PW10 carrying the reduced recombinant plasmid pULP354.
Each of (pULP354) was inoculated into a 100 ml Erlenmeyer flask containing 20 ml of Nutrient Broth and shake-cultured at 37 ° C. for 24 hours and 48 hours. 20 μg / ml tetracycline was added to the medium of the strain having pHY300PLK and pHLP352,
In addition, the medium of strains harboring pUB110 and pULP354 contains 10
μg / ml kanamycin was added.

The obtained culture solution was centrifuged, and the protease activity of the supernatant was measured. The protease activity was determined by the method of Uehara et al. [H. Uehara et al .: J. Bacteri] using casein as a substrate.
ol., 119 , 82 (1974)], pH 7.0 and pH 10.
It was measured at 0, and the activity was expressed by setting the amount of enzyme that releases 1 μg of tyrosine per minute as 1 unit. The results are shown in Table 1.

As shown in Table 1, both of the two transformants carrying the reduced recombinant plasmid produced significantly higher alkaline proteases than the host Bacillus subtilis PW10 and the strain carrying the vector plasmid. .
In particular, Bacillus subtilis (pULP354) showed higher alkaline protease productivity than the DNA donor Bacillus licheniformis HP19611.

Next, using the culture supernatant of Bacillus subtilis PW10 (pHLP352), Bacillus subtilis PW10 (pULP354) and Bacillus licheniformis HP19611 as an antigen, an antiserum against a commercially available alkaline protease, subtilisin Carlsberg (manufactured by Sigma) was used. When the identification of the protease by the immuno-double diffusion method was carried out, the precipitation lines of the proteases produced by each strain were fused to each other, and it was confirmed that the proteases produced by these transformants were Bacillus licheniformis type. Was messed up.

In addition, it has been inserted into the reduced recombinant plasmid pHLP352.
When a 2.0 kb chromosomal DNA fragment was used as a probe and Southern hybridization was performed on a fragment obtained by cleaving the chromosomal DNA of Bacillus licheniformis HP19611 with SmaI and BglII, hybridization was observed with the 2.0 kb fragment. And the insert of pHLP352 is Bacillus licheniformis HP1.
It was confirmed to be derived from 9611 chromosomal DNA.

From the above results, it was revealed that the 2.0 kb chromosomal DNA fragment inserted into the reduced recombinant plasmid pHLP352 or pULP354 contains the entire alkaline protease region of Bacillus licheniformis HP19611.

Example 6 Determination of the nucleotide sequence of the alkaline protease gene of Bacillus licheniformis HP19611 The nucleotide sequence of the alkaline protease gene of Bacillus licheniformis HP19611 is the same as that of the insert fragments of the two recombinant plasmids pLP3 and pLP35 obtained in Example 2. It was determined from the sequence.

The transformant Escherichia coli LE39 obtained in Example 2
2 (pLP3) and Escherichia coli LE392 (pLP35) were cultured in the same manner as in Example 1 (5) to obtain purified plasmids of pLP3 1.2 mg and pLP35 0.7 mg, respectively.

pLP3 30 μg, 60 units EcoRI, 60 units BglI
Add 300 μl of reaction solution containing I and EcoRI reaction buffer to 37 ℃
After the DNA was cleaved by keeping it for 3 hours, 750 μl of ethanol was added to precipitate the DNA. The precipitate was recovered by centrifugation, dissolved in 150 μl of TE buffer, and used in Example 2 (1).
Fractionation by agarose gel electrophoresis in the same manner as in 1.2 kb Eco
About 4 μg of RI-BglII fragment was obtained.

On the other hand, after 300 μl of a reaction solution obtained by adding 60 units of SmaI and SmaI reaction buffer to 30 μg of pLP35 and kept at 37 ° C. for 3 hours, 60 units of BglII and 30 μl of 10 times concentration of BglII reaction buffer were added and further 37 The DNA was cleaved by holding at 3 ° C for 3 hours. Thereafter, fractionation was carried out in the same manner as in the case of pLP35, and 0.8 kb
About 2 μg of aI-EcoRI fragment was obtained.

The nucleotide sequences of both fragments thus obtained were analyzed by the M13 dideoxy-thie termination method [J. Messing: Meth
ods in Enzymol., 101 , 20, Academic Press, New York (19
83); F. Sanger: Science, 214 , 1205 (1981)].

Said two fragments and their further restriction enzymes San3AI, H
The fragment obtained by digesting with aeIII and AluI is M13 phage mp18
Or connect it to mp19, Escherichia coli JM109
(Purchased as an accessory to the M13 sequencing kit described below) was transformed to obtain a large number of recombinant phages.

On the other hand, 15 μl of the preculture liquid which was cultured overnight at 37 ° C. in 2 × TY medium (1.6 wt% bactotryptone, 1 wt% yeast extract, 0.5 wt% NaCl, pH 7.4) was added to 1.5 ml of 2 × TY medium. The recombinant plaques of the above-mentioned recombinant phage were transplanted into the
After culturing at 5 ° C. for 5 hours, centrifugation was performed to obtain a supernatant containing fare particles.

20 ml of polyethylene glycol 6000-2.5M in 1 ml of this supernatant
0.2 ml of NaCl was added and the mixture was allowed to stand for 15 minutes to precipitate the charge. The precipitate was collected by centrifugation, suspended in 100 μl of TE buffer, added with 50 μl of phenol (saturated with TE buffer) and mixed well to extract DNA. The aqueous layer was recovered by centrifugation, and 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were added thereto to precipitate the DNA. The precipitate was recovered by centrifugation, dried and then dissolved in 30 μl of TE buffer to obtain a single-stranded forage DNA solution (DNA amount: 1 to 2 μg).

Using this single-stranded DNA, M13 sequence kit (Takara Shuzo) and α ³² P-dCTP (410 Ci / mmol, Amersham), a complementary strand was synthesized. Formamide dye solution [95
Vol% formamide, 0.1wt% xylene cyanol, 0.1w
3% after stopping the reaction by adding t% bromphenol blue]
It was boiled for a minute, rapidly cooled, and electrophoresed using an 8% polyacrylamide gel. After electrophoresis, the gel was dried and autoradiographed.The base sequence of each fragment was determined by analyzing the base sequence on the autoradiogram.Then, they were ligated and the base sequence of the alkaline protease gene was determined. It was determined. The base sequence obtained as a result is shown in FIG.

According to the results obtained here, there is a region encoding the polypeptide from the 224th position to the 1360th position. In this region, Bacillus licheniformis NCIB68 reported by Jacobs et al.
There were 16 bases and 7 base differences. That is,
268 in the alkaline protease gene of HP19611 strain
The 733th, 793th, 793th, 795th, 820th, 842nd and 910th bases are respectively A → C, G → A, G → A, A → G, G → A, T → in the gene of NCIB6816 strain. A,
C → T. In addition, stop codon (from 1361th
The 1388th A in the downstream region of the 1363th TAA) is deleted in the NCIB6816 strain gene. And the biggest difference is
The 115 base pairs from the 1st to the 115th, which were lacking in the gene cloned from NCIB6816 strain, were added to the alkaline protease gene of HP19611 strain cloned by the present invention. Since the alkaline protease is not expressed in the NCIB6816 strain gene lacking this region, and the alkaline protease is expressed in the HP19611 gene in which this region is added as shown in Example 5, this region is It is considered to be an essential region for gene expression. In addition, a sequence (GGAGG) that seems to be a ribosome binding region required for initiation of protein synthesis
Were found from 209th to 213th, Metssenjier RN
Sequences believed to be the −35 and −10 regions required for initiation of A synthesis are 149 to 154 (TTAACA) and 17 respectively.
It is found from the 2nd to the 177th (TATATT). Therefore, the promoter region required for the expression of the Bacillus licheniformis alkaline protease gene is
It exists in the region up to the 208th position, and the DNA containing the entire promoter region retains the ability to express alkaline protease even if it lacks a part of this region in addition to the DNA containing the entire region. DNs that include areas like
Say A. In addition, a terminator structure having a sequence capable of forming a stem-loop structure found at the transcription termination site is found at positions 1389 to 1419.

In addition, the amino acid sequence derived from the sequence from the 539th sequence in the region encoding the 224th to 1360th polypeptides is excluded from the literature [M. Ottersen, I. Sve.
ndsen: Methods in Enzymology, 19 , 199, Academic Press,
New York (1979)], which is identical with the amino acid sequence of subtilisin Carlsberg reported in 53.
The 9th to 1360th regions are considered to be the regions encoding the mature protein. Therefore, it seems that the 224th to 538th regions are involved in secretion.

〔The invention's effect〕

A recombinant plasmid having an alkaline protease gene containing the entire region of the promoter required for the expression of the Bacillus licheniformis alkaline protease gene and a recombinant Bacillus bacterium transformed with this recombinant plasmid were obtained. During the production of the protease, it became possible to carry out culture so as to specifically enhance the alkaline protease production ability, and along with this, it was possible to easily purify the alkaline protease.

In addition, a gene containing a promoter region of the alkaline protease gene and a region involved in protein secretion was isolated, and its nucleotide sequence was clarified. Has a nucleotide sequence corresponding to the amino acid sequence of a useful protein
It becomes possible to construct a recombinant plasmid in which DNA fragments are ligated, and by culturing a transformant in which this recombinant plasmid is introduced into a highly safe Bacillus bacterium, it is possible to safely secrete large-scale production of the useful protein. It is possible to carry out

[Brief description of drawings]

Figure 1 is a restriction map of recombinant plasmid pAMP3, 2
The figure shows the restriction map of the recombinant plasmid pLP3, Fig. 3 shows the restriction map of the recombinant plasmid pLP35, and Fig. 4 is a schematic diagram explaining the construction method of the recombinant plasmid pHLP351.
Fig. 6 is a schematic diagram for explaining the construction method of the recombinant plasmid pHLP352, Fig. 6 is a schematic diagram for explaining the construction method of the recombinant plasmid pULP354, and Fig. 7 is Bacillus licheniformis HP.
D containing the entire region of the 19611 alkaline protease gene
It is a figure which shows the base sequence of NA. In FIGS. 1 to 6, the single-lined portion indicates the region of the vector plasmid, the double-lined portion indicates the region derived from the chromosomal DNA, and the arrow indicates the region where the drug resistance gene is present on the vector plasmid.

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Claims (1)

[Claims] 1. A novel gene DNA of Bacillus licheniformis alkaline protease gene, which has a promoter region having the following nucleotide sequence and having an ability to express alkaline protease.