JPH0824586B2 - Novel DNA and method for producing protein using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a DNA base sequence involved in protein production. The present invention particularly relates to a DNA base sequence containing a neutral protease gene that is secreted and produced in a large amount in Bacillus bacteria and a method for producing the neutral protease.

2. Description of the Related Art Neutral protease is one of the most useful enzymes currently produced industrially, and its use is in a wide range of fields such as food manufacturing, cosmetics, leather tanning, and detergent for dry cleaning. Conventionally, a neutral protease is a culture medium containing a neutral protease which is produced by culturing bacteria such as Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus saccharicus, and Bacillus chinenifarmis that produce high production of the enzyme. It is obtained by collecting liquids and purifying the enzyme. Bacteria belonging to the genus Bacillus secrete and accumulate a large amount of extracellular proteins such as amylase, alkaline protease, and levansucrase in the culture filtrate in addition to the enzyme. Therefore, great effort is required to remove impurities during production of the enzyme. It was a requirement.

Problems to be Solved by the Invention By producing a neutral protease using the method of recombinant DNA utilizing the DNA nucleotide sequence revealed by the present invention, it is possible to improve the productivity in the production of the enzyme and to purify it. It is possible to simplify the operation extremely easily. That is, when a protein produced by a gene cloned by a gene recombination method is produced, a large number of genes can be present in the microbial cell, so that the production amount of the target protein is dramatically increased as compared with the conventional production method. In addition, the enzyme protein can be recovered and purified very easily.

Recent knowledge of molecular biology has made it possible to allow a host bacterium to produce a heterologous protein by linking a gene derived from a heterologous organism with an appropriate vector and introducing it into the host bacterium. When a bacterium belonging to the genus Bacillus is used as a host bacterium, the bacterium has a property of secreting a large amount of protein outside the microbial cell,
It has no pathogenicity and has been used in the fermentation industry for a long time (Debabov, “The Molecular Biology of t
he Bacilli " 1 332 (1982), Dubnau, DA ed Academic Pre
From ss), it is of great industrial significance from the viewpoint of protein production by microorganisms to create a vector system having high secretory ability by using the genetic information involved in the secretion of such secretory proteins of microorganisms. That is, the gene is expressed thereby, the protein is secreted in a large amount outside the bacterial cell, that is, in the culture medium, and the protein can be recovered from the culture filtrate in a simple process.

Furthermore, the successful cloning of the neutral protease gene has enabled the determination of its DNA base sequence, which was previously completely unknown. As a result, it was possible to analyze the neutral protease at the gene level, and it became possible to improve the activity and stabilization of the neutral protease at the DNA level.

As will be described later, the neutral protease gene of the genus Bacillus obtained in the present invention has information relating to secretion commonly found in proteins secreted outside the cells. The neutral protease of the genus Bacillus is considered to have a very strong promoter region, a ribosome binding region, and genetic information involved in secretion because it is secreted and produced in a large amount. The DNA base sequence obtained by the present invention naturally includes the base sequences of such promoter region, ribosome binding region and region involved in secretion.
By utilizing these regions existing in the A base sequence, it is possible to secrete and produce not only neutral protease but also heterologous proteins in large quantities. This is extremely significant in industrially producing useful proteins. From such a viewpoint, the inventors of the present invention for the first time in the world determined the DNA base sequence and amino acid sequence of the neutral protease gene secreted outside the bacterium of Bacillus bacteria by the method of the present invention,
The structure of the promoter region, ribosome binding region, DNA sequence of the region involved in secretion and the structural gene of neutral protease were clarified. Also, by using the recombinant DNA containing the obtained DNA base sequence, we succeeded in secreting the neutral protease to the outside of the cells 50 times as much as in the conventional method, and completed a group of the present invention.

The term "DNA base sequence containing a neutral protease gene" as used in the present invention means that the order of the base sequences is specified according to the common sense of those skilled in the art according to the direction from the 5'end to the 3'end. Is. In addition, by incorporating a DNA fragment corresponding to the neutral protease gene into an appropriate vector DNA or chromosomal DNA of the host bacterium, high production of the enzyme protein becomes possible, and at the same time, it becomes possible to create Bacillus bacteria having their secretory ability. .

 The present invention will be described in detail below.

(DNA Base Sequence of the Present Invention) The DNA base sequence of the present invention is involved in the production of neutral protease. And the DNA of this sequence is Bacillus amyloliquefacien (Bacillus amyloliquefacien
s) It can be easily obtained from an F strain having the DNA base sequence of the present invention on its chromosome. The DNA base sequence of the present invention containing a neutral protease gene is derived from the chromosomal DNA of the enzyme-producing strain and contains a region necessary for expression of the gene. The region relating to gene expression includes a promoter region containing "-35" and "-10" regions, which are regions recognized and bound by RNA polymerase, and a nucleotide sequence for binding mRNA synthesized by RNA polymerase to ribosome. There is a ribosome binding region that specifies It is known that the base sequences of these regions are extremely important for the efficiency of gene expression, but not only the distance between them, that is, the number of bases is also very important (Moran, Jr. et al. , Mol.Gen.Genet. 1
86 339 (1982)). It is considered that the DNA base sequence of the present invention contains all of these regions and is essential for the expression of the neutral protease gene.

The DNA nucleotide sequence of the present invention is also shown in FIG. 1, and the “−35” region and the “−10” region found in the promoter region of this sequence are generally common to TTGCAG and TATTATA, respectively, in the present invention. Sequence (Sueji Horinouchi, protein / nucleic acid / enzyme 28 1468 (1983)).
In the ribosome binding region, AAAAGGGGG in the present invention.
Is a sequence completely complementary to the ribosomal RNA of Bacillus subtilis (AACGGAGG (Mclaughlin JR, Biol. Chem.
It differs from 256 1283 (1983) by only one base. From these results, the neutral protease gene has a very strong promoter region and ribosome binding region.

The terminator region, which indicates the end of genetic information, is also important for the efficiency of gene expression. It is considered that the mRNA that started in the promoter region ends in the terminator region located at the end of the gene. In the case of the present invention, the presence of a DNA base sequence that defines the higher-order structure of DNA considered to be a terminator region is recognized at the end of the neutral protease gene (Fig. 1).

The DNA base sequence of the present invention contains a structural gene that defines the protein of the neutral protease itself. In addition, it is generally known that proteins secreted extracellularly have a unique secretory amino acid sequence known as a signal sequence (protein, nucleic acid, enzyme 26 2044).
(1981)). The neutral protease specified by the DNA base sequence of the present invention is also efficiently secreted outside the microbial cell, and therefore the DNA base sequence is also involved in the extracellular secretion of the enzyme. It contains a region containing genetic information. The DNA base sequence of the present invention is shown in the drawing. Further, the structural gene and the region involved in secretion define the corresponding amino acid by the DNA base sequence, but the DNA base sequence obtained by modifying the DNA without changing this amino acid is the DNA base sequence.
It is considered to be exactly the same as the base sequence. DNA of the present invention
Although the DNA containing the nucleotide sequence was obtained from the chromosomal DNA of Bacillus bacterium, the DNA nucleotide sequence of the neutral protease gene was clarified for the first time in the world by the present invention. Although it has become possible to obtain a DNA having the DNA base sequence by synthesizing DNA by a usual method,
This case is naturally included in the scope of the present invention.

(Recombinant DNA) Recombinant DNA containing the DNA base sequence of the present invention means any recombinant DNA in which the DNA base sequence of the present invention and the vector DNA are bound. At this time, chromosomal DNA is obtained by recombination within the host cell with chromosomal DNA of the host bacterium by utilizing not only extranuclear genes such as plasmids, phages and cosmids that can be retained in bacteria but also DNA containing the DNA base sequence of the present invention. When incorporated, it also falls within the scope of recombinant DNA of the present invention. In the present invention, when a plasmid or a phage retained in bacteria is used as the vector DNA, it is possible to allow a large number of DNAs containing the DNA base sequence to be present in the cells, which is purposeful for the purpose of the present invention. .
That is, since a large amount of the neutral protease is present in the cells, a large amount of the neutral protease can be produced, and since a large amount of DNA can be recovered, it is easy to improve the neutral protease at the DNA level. As the vector DNA in the present invention, any vector DNA usually used in gene recombination experiments can be used. For example, when Bacillus subtilis is used as the host bacterium, the plasmid pU is used.
B110, pC194, etc., ρ11, φ105, etc., are suitable as phages, and pBR322, pUC12, pUC13, etc. are suitable for plasmids, and λ phage is suitable for phages when Escherichia coli is used as the host bacterium.

The recombinant DNA of the present invention can be introduced into not only Bacillus bacteria but also all bacteria such as Escherichia coli, yeast, actinomycetes, fungi, etc. by appropriately selecting vector DNA.

Recombinant containing the neutral protease gene of the present invention
Bacteria into which DNA is introduced include these strains. When transforming a Bacillus bacterium with the recombinant DNA, a method using competent cells (Anagno
stopoulos, C.and Spizizen J., J.Bacteriol. 81 741 (19
61)) and the protoplast method (Chang, S. and Cohen,
SN, Mol. Gen. Genet. 168 11 (1878)). The CaCl ₂ method is suitable as a method for transforming E. coli (Maniati
s T. et al. “Molecular cloning” p247 (1982) Cold Sp-
ring Harbor Laboratory).

By culturing, the bacterium transformed with the recombinant DNA of the present invention produces a protein corresponding to the recombinant DNA in an extremely large amount as compared with the conventional method. In this case, protein refers to neutral protease, but DN generated by changing part of the DNA base sequence of recombinant DNA
The protein corresponding to the A base sequence is naturally included.

The recombinant DN of the present invention is used in the protein production method of the present invention.
It also includes the case of producing the protein both inside and outside the cells of the bacterium transformed with A. For example, a method of producing the protein in the cells of a strain obtained by transforming the recombinant DNA obtained by removing the region involved in secretion in the DNA base sequence of the recombinant DNA is also a method of the present invention. included. However, when industrially producing a neutral protease, it is desirable that the enzyme be secreted outside the microbial cell of the producing bacterium, that is, in the culture solution, for ease of recovery and purification of the enzyme protein. The present inventors used the recombinant DNA containing the DNA nucleotide sequence obtained in the present invention to transform Bacillus subtilis and culturing the resulting strain, which is now industrially used for the production of neutral protease. We succeeded in secretory accumulation and accumulation of the neutral protease in the culture solution in 50 times larger amount than that of the existing strain Bacillus amyloliquefaciens. Also, 95% of the proteins secreted in the culture solution were neutral proteases, but this value is a very good value from the viewpoint of reducing contamination of heterologous proteins compared to 60% by the conventional method. From this result, it became clear that the neutral protease obtained by this method can be purified very easily.

A method for obtaining a DNA base sequence containing the neutral protease gene of Bacillus amyloliquefaciens of the present invention by the following specific examples, a method for determining the base sequence of the DNA base sequence of the present invention, and a recombinant DNA containing the DNA base sequence A method for obtaining bacteria to obtain bacteria and a method for producing a neutral protease using the transformed strain will be described, but the present invention is not limited to this example.

Example 1 (Chromosomal DNA from Bacillus amyloliquefaciens
And cleavage with restriction enzymes) Bacillus amyloliquefaciens F strain (ATCC2335
0) 2 liters of broth medium (Difco Nutrient Broth)
After culturing at 37 ℃ for 15 hours, the cells were collected, and Saito-Miura
Method (Saito, H., and Miura, KI., Biochem.Biophys.ac
According to ta 72 , 619 (1963)), 10 mg of purified chromosomal DNA was obtained. 500 μg of this chromosomal DNA was reacted with 100 units of the restriction enzyme Sau3A (Takara Shuzo) at 37 ° C. for 5 minutes. The composition of the reaction system is 10 mM Tris-HCl buffer (pH 7.5), 7 mM MgC
l ₂ , 100 mM NaCl. After the reaction, test 1 μg of DNA 1
As a result of examination by% agarose gel electrophoresis, it was confirmed that this reaction product was a partial cleavage product of the donor chromosome mainly consisting of a DNA fragment of 2 kb-8 kb in size. Then, the remaining whole amount of this reaction product was electrophoresed on 0.7% low-melting point agarose gel for 100 V for 3 hours to cut out a gel of about 1.5 kb-9 kb portion, purified by phenol extraction and chloroform extraction, and ethanol-precipitated to recover DNA. The recovered DNA was dissolved in 200 μl of 50 mM Tris-HCl buffer (pH 7.5) and subjected to the following experiment as a donor chromosome fragment.

Example 2 (ligation and transformation of donor chromosomal DNA fragment with vector) The donor chromosomal DNA fragment obtained in Example 1 is a restriction enzyme B.
It was completely cleaved with amHI (Takara Shuzo) and ligated to the plasmid pUB110 in which the terminal phosphate ester was hydrolyzed with large E. coli alkaline phosphatase (Worthigton).

BamHI treatment of pUB110 was carried out by incubating 100 μg of pUB110 and 50 units of BamHI (Takara Shuzo) at 37 ° C. for 4 hours. The composition of the reaction system is 10 mM Tris-HCl buffer (pH 8.
0), 7 mM MgCl ₂ , 100 mM NaCl, 2 mM 2-mercaptoethanol, 0.01% bovine serum albumin. Obtained Ba
The mHI-cut pUB110 was extracted with phenol three times and recovered by ethanol precipitation. The recovered pUB110 was then reacted by incubating 5 units of Escherichia coli alkaline phosphatase (BAPF manufactured by Worthington) and 0.1 M Tris-hydrochloric acid buffer (pH 8.0) at 65 ° C. for 4 hours. After that, phenol extraction and ethanol precipitation were performed to recover pUB110. Recovery pUB110
Was dissolved in 100 μl of 50 mM Tris-HCl buffer (pH 7.5).

The thus obtained BamHI- and phosphatase-treated pUB110 was ligated to the donor chromosomal DNA fragment obtained in Example 1 using T ₄ ligase (Takara Shuzo). The composition of the reaction system is 50 μl of donor chromosome fragment, 20 μl of pUB110, T ₄
Ligase 5 units, 66 mM Tris-HCl buffer (pH 7.5),
6.6 mM MgCl ₂ , 10 mM dithiothreitol and 1 mM ATP (adenosine triphosphate). The reaction was carried out at 15 ° C for 4 hours.
As a result of performing 1% agarose gel electrophoresis using a portion after the reaction, it was confirmed that the vector pUB110 was bound to the donor chromosomal DNA to form a recombinant DNA molecule.

Transformation with the recombinant DNA molecule thus obtained was carried out by the Chang's protoplast method (Chang, S. and Cohe
n, SN, Mol. Gen. Genet. 168 111 (1978)). Kanamycin sulfate was added to the protoplast regeneration medium to a final concentration of 100 μg / ml. The Bacillus subtilis 1A274 strain (Ohio University Bacillus Stock Center conserved strain) was used as the host bacterium for cloning.

The kanamycin-resistant strain obtained by transformation was 0.
It was subcultured to TBAB agar medium (manufactured by Difco) containing 8% casein-40 μg / ml kanamycin and cultured at 37 ° C. for 14 hours to examine the presence or absence of halo formation around colonies. As a result of examining about 10,000 kanamycin resistant strains, one strain (# 15
0) formed a significantly large halo around the colony.

Example 3 (Preparation of Recombinant DNA Molecule from Transformant and Confirmation of Containing Neutral Protease Gene of Donor Strain) Transformant # 150 forming large halo obtained in Example 2
The large halo-forming strain obtained by isolating a single colony of the above was cultured at 37 ° C. for 14 hours in 50 ml of Pen assay medium (manufactured by Difco), and then collected. Collected cells were treated with 50 mM Tris-HCl buffer (pH
7.5), 5mM EDTA, 50mM NaCl and then the alkaline method (Birnboim, HCand Poly, J., Nucleic acidres. 7 1513
(1979)) to prepare a plasmid. The obtained plasmid was treated with restriction enzymes EcoRI, BglII, and BamHI and then examined by 1% agarose gel electrophoresis.
The plasmid was cut at one site with glII and the size was 6.2 kb
And was not cleaved with BamHI. Since pUB110 used as a vector has a size of 4.5 kb and is cleaved at one site with EcoRI, BBglII and BamHI, the plasmid obtained from the large halo-forming transformant contains a plasmid of about 1.7 kb at the BamHI site of pUB110. The plasmid was found to be a recombinant DNA molecule inserted. This recombinant plasmid was named pNP150.

The recombinant plasmid pNP150 thus obtained
Used the competent method to transform Bacillus subtilis 1A20 strain (Ohio University Bacillus Stock Center stock strain). Transformation of competent method is Anagnostopou
los-Spizizen method (Anagnostopoulos, C., and Spizie
n, JJBacteriol. 81 741 (1961). 0.8% of the culture solution (1 ml) after incorporating about 5 μg of recombinant DNA molecule
Casein-40 μg / ml 50 on TBAB agar containing kanamycin
μl was plated. 100% of the obtained kanamycin-resistant transformants were large halo-forming strains. Then, the transformant MT-0150 (FERM BP-425) was cultured at 37 ° C. for 8 hours using Pen assay medium, and the protease activity of the culture supernatant was measured. The measurement method was based on the casein decomposition method ("Experimental Agricultural Chemistry" p284 Asakura Publishing (1678)).
As a result of the measurement, it was revealed that this transformant secreted 50 times as much neutral protease as Bacillus amyloliquefaciens (Table 1). The culture supernatant was used as an antigen, and as a result of an immune double diffusion method using an antiserum against neutral protease of Bacillus amyloliquefaciens and Bacillus subtilis, the neutral protease secreted by the transformant was Bacillus. It was found to be of the amyloliquefaciens type. Furthermore MT-015
Neutral protease secreted by 0 and extracellular neutral proteases of both Bacillus amyloliquefaciens and Bacillus subtilis are recovered from the culture,
After purification, the amino-terminal amino acid sequence was determined. As a result, the neutral protease produced by the transformant MT-0150 was completely the same as that of Bacillus amyloliquefaciens. Therefore, this is the same as the mature protein of the neutral protease gene derived from Bacillus amyloliquefaciens gene, and the region involved in the secretion of the neutral protease gene of Bacillus amyloliquefaciens crosses Bacillus species. It turned out to work properly.

Example 4 (Large-scale preparation of neutral protease gene of Bacillus amyloliquefaciens and determination of DNA base sequence) Bacillus subtilis MT-0150 strain having the DNA base sequence of the present invention obtained in Example 3 was added to a broth medium ( Method of Gryczan (Gryczan, TJJBacteriol. 134 ) from cells cultured at 37 ° C. for 14 hours using 5 l of Difco's Nutriient Broth.
318 (1978)) and the purified plasmid (pNP150) 2 having the DNA base sequence of the present invention.
to obtain mg. Regarding the obtained insert DNA fragment containing the neutral protease of pNP150, the method of Maxam-Gilbert (Maxam, A., Gil
The nucleotide sequence was determined according to bert, W.Proc.Natl.Acad.Sci.USA 74 560 (1977)).

FIG. 1 shows the DNA base sequence obtained as a result and its description. As shown here, the complete amino acid sequence of the neutral protease of Bacillus amyloliquefaciens was revealed for the first time.

Further upstream, the promoter region required for gene expression, that is, the so-called −35 and −10 regions required for RNA polymerase recognition and DNA binding can be seen. In addition, a ribosome binding region is found downstream of the promoter region, and there is an open reading frame that encodes a protein from the protein synthesis initiation point several bases downstream. A part of the open reading frame upstream from the amino terminus of the neutral protease is excised during secretion, and this region contains an important part involved in protein secretion.

A DNA base sequence capable of forming a secondary structure considered to be a terminator is found downstream of the stop codon indicating the end of the open reading frame. This confirmed that the recombinant paired plasmid pNP150 containing the neutral protease gene contained all of the gene.

Example 5 (Production of Neutral Protease Using Transformant) Bacillus subtilis MT-0150 having the DNA nucleotide sequence of the present invention obtained in Example 3 was used in 5 l of a broth medium (nutrient broth manufactured by Difco) 37 Cultivated at ℃ for 14 hours. When the protease activity of the culture supernatant was measured by the method described in Example 3, a high activity was shown, and 95% or more thereof was neutral protease. The host bacterium, Bacillus subtilis, produces almost no protease under these conditions.
MT-0150 showed a value 50 times higher than that of Bacillus amyloliquefaciens, which is the donor of DNA (Table 1).

Purification of neutral protease was carried out by a conventional method, and the culture supernatant was collected and subjected to ammonium sulfate precipitation (75% saturation) and acetone precipitation (30-70% fraction), followed by CM-Sepharose (Pharmacia Fine Chemicals) column chromatography. It was As a result of CM-Sepharose, a uniform protein was obtained. Conventional strains are often contaminated with amylase, levansucrase and the like, and it was impossible to obtain a homogeneous enzyme protein by such an operation. At present, industrially, the enzyme was produced by a method of culturing Bacillus amyloliquefaciens and recovering a neutral protease from the supernatant, but the method of the present invention improved productivity 50 times at a stroke. Was approved.

Unit: unit One unit is defined as the activity that decomposes milk casein per 1 ml of the enzyme solution and increases the absorbance at 275 nm by 0.01 per minute.

The culture was carried out at 37 ° C for 14 hours. The medium is a broth medium. The reaction was carried out at 30 ° C. Neutral protease is 1 mM DF in the reaction solution
It was determined by adding P (Difuruolopyroca-rbonate) to inhibit the alkaline protease activity. Alkaline protease was added in the reaction solution to 20 mM EDTA (Ethylendiam
inetetraacetate) was added to inhibit the neutral protease activity.

[Brief description of drawings]

FIG. 1 is a diagram showing the sequence of DNA bases containing the neutral protease gene of Bacillus amyloliquefaciens. Of these, 179th to 184th include the −35 region, the same 203th to 208th the −10 region, the same 236th to 243rd the ribosome binding region, and the same 251st to 913rd the region involved in secretion. The DNA base sequences are generally 914th to 1813th, and the DNA base sequences encoding the mature protein are generally 1820th to 1851st.
Numbers indicate the DNA base sequences containing the terminator region. In FIG. 1, A is adenine, T is thymine, and G is
Indicates guanine, C indicates cytosine, and is located below these sequences in the promoter region. Indicates the -35 region, ====== indicates the -10 region, and ++++++
+ Indicates the ribosome binding region, Begins the region involved in secretion, Is the mature protein coding region, *** is the protein transcription termination codon, Indicates the terminator region, respectively.

 ─────────────────────────────────────────────────── ───Continuation of the front page Judgment panel Judge Hirayama Koji Judge Judge Masaki Hirota Judge Ken Ugi Ken (56) References JP59-59190 (JP, A) JP60-91980 (JP, A)

Claims (2)

[Claims] 1. A Bacillus amyloliquefaciens (Ba) having an amino acid sequence of a precursor protein as shown below.
cillus amyloliquefaciens) A DNA base sequence encoding a promoter region, a ribosome binding region, a structural gene and a terminator region of a gene encoding a neutral protease of F strain. 2. The DNA base sequence according to claim 1, wherein one strand of the DNA base sequence has the following order. (In the above, A is adenine, T is thymine, G is guanine, and C is cytosine.)