JPH0771497B2 - New plasmid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel plasmid useful as a cloning vector for highly expressing α-amylase in a host cell.

[Problems to be Solved by Conventional Techniques and Inventions] Bacillus, a bacterium of the genus Bacillus
subtilis) secretes and produces a large amount of α-amylase, protease and the like outside the cells, and is therefore widely used industrially as an enzyme-producing bacterium used for fermentation, detergents and the like. In addition, this Bacillus subtilis does not produce endotoxin, etc., and since it does not parasitize or coexist as a pathogenic microorganism in humans and animals, it is safer than E. coli, and next to E. coli as a host strain for gene recombination. Genetic analysis is in progress.

In the case of Bacillus subtilis, α-amylase, which is a typical extracellular enzyme of Bacillus subtilis, is classified into three types, amyR1, amyR2, and amyR3, because the control sites that control gene expression are different depending on the nucleotide sequence. The amyR2 type regulatory site is said to have the highest activity. In the case of Bacillus subtilis,
It is classified into Marburg type (hereinafter referred to as M type), Natto type (hereinafter referred to as N type), and amylosaccharicus type (hereinafter referred to as S type) according to its starch-degrading activity.

And, there are many reports on the clonig of these α-amylases and their genetic mechanisms, and their structures have been clarified. For example, cloning of amylase gene (amyR1-M type) and gene structure by sequencing (Maria Yang, Nucleic Acids Research, Vol. 11, Number)
2,237-249 (1983)) and α-amylase highly expressing strain B.
α-amylase regulatory gene amyR2 derived from subtilis NA64
And the structural gene amyE have been reported to be further cloned into the multicopy plasmid pUB110 (Yamazaki et al, J. Bacteriol., 156,327-337 (198
3)).

The difference between M-type, N-type, and S-type α-amylase is reported to be due to translation termination occurring at different sites of the α-amylase gene (Yamane.
et al, J. Biochem., 96, 1849-1858 (1984)). Furthermore, the α-amylase gene of B. subtilis 2633 was transformed into E. coli (E
recombinant plasmid pAM26 containing the α-amylase gene into B. subtilis 6160
It has been reported that α-amylase is highly expressed due to the amyR3 type promoter when introduced into E. coli (Emori et al, Agric. Biol. Chem., 52 (2), 399-406,1).
988).

Also Bacillus thuringiensis
The terminator obtained from the crystal protein (cry) gene of Bacillus licheniformis (B. lichenif
ormis) -derived penicillinase gene, etc., and examined its function in the host. As a result, the terminator contributes to the stability of mRNA (Proc.Natl.Acad.Sci., USA, Vol.83,3
233-3237, May (1986)) or the human growth hormone is linked to the downstream of the promoter of Bacillus amyloliquefaciens neutral protease.
It has been reported that, when a terminator of a neutral protease gene is further linked downstream thereof, the expression and secretion thereof is increased as compared with the case where the terminator is not present (Journal of Biotechnology, 8, 123-134 (1988)).

However, in order to enhance the expression of α-amylase,
Even when the α-amylase gene having the amyR2 type control site, which is said to have the strongest activity, was cloned into pUB110, which is a multi-copy plasmid of Bacillus subtilis, the plasmid copy number was about 40 to 50 copies. Nevertheless, α-
The amylase activity is only about 20 times that of the chromosome donor.

Therefore, an object of the present invention is to provide a novel plasmid useful for introducing α-amylase into a host cell and efficiently producing a large amount of α-amylase in the host cell.

[Structure of the Invention] The present inventors have found that a specific nucleotide sequence containing a stem-and-loop structure existing downstream of an α-amylase structural gene significantly affects α-amylase activity,
The present invention has been completed. That is, the present invention has a control gene amyR derived from Bacillus subtilis, an α-amylase structural gene amyE on the downstream side of this regulatory gene, and a base sequence containing a stem-and-loop structure on the downstream side of this structural gene. A circular plasmid having a base sequence containing a stem-and-loop structure represented by the following formula [I] The above problem is solved by a novel plasmid represented by (in the formula, an arrow indicates a site capable of having a stem-and-loop structure).

Further, the present invention is a Bacillus subtilis-derived control gene amyR2 or a Bacillus subtilis-derived control gene amyR3 having a sequence that can have a stem-and-loop structure upstream of the α-amylase structural gene amyE, and this control gene A circular plasmid having a downstream α-amylase structural gene amyE and a nucleotide sequence containing a downstream stem-and-loop structure of the structural gene, wherein the stem-and-loop structure is at least the following formula [II ] The above problem is solved by a novel plasmid having a base sequence containing a stem-and-loop structure represented by (in the formula, an arrow indicates a site capable of having a stem-and-loop structure). In order to enhance the α-amylase activity, it is preferable that the base sequence containing the stem-and-loop structure has the base sequence represented by the formula [I].

In the present specification, the representation of nucleic acid in the nucleotide sequence means IUPA
The notation adopted by C shall be followed.

The plasmid control gene amyR of the present invention is not particularly limited as long as it is derived from Bacillus subtilis, and may be any of the amyR1 type, amyR2 type and amyR3 type. These regulatory genes are present in known strains and function as promoters. The control gene amyR1 is, for example, B. subtilis.
168 and B. subtilis 6160 strains. Regulatory gene am
yR2 is, for example, B.natto IAM1212, B.subtilis N7, B.sub.
tilis 1A412 strain (The Bacillus Genetic of Ohio University
It is available from the stock center) etc. The regulatory gene amyR3 is, for example, B. subtilis 2633, B. subtil
is T2N26 strain
No., No. 3345 deposited) etc. Among these control genes amyR, the control genes amyR2 and amyR3 having high α-amylase activity are preferable. Regulatory gene amyR2
Differs from the other regulatory genes amyR1 and amyR3 in that the following base sequence of the stem-and-loop structure is located upstream of the α-amylase structural gene. (In the formula, an arrow indicates a site that can have a stem-and-loop structure) (Yamazaki et al, J. Bacterio
l., 156, 327-337 (1983)).

The features and nucleotide sequences of the control genes amyR1, amyR2, and amyR3 have been reported in many documents, and for example, the control gene amyR1 is described in the document “Nucleic Acids R
esearch ”Vol. 11, No. 2, 237-249 (1983), etc., for the regulatory gene amyR2, see“ Journal of Bacteriology ”.
Vol.156, No.1, p327-337 (183), etc. can be referred to, and the regulatory gene amyR3 can be referred to in the literature “Nucleic Acids Researc
h ”Vol. 16, No. 14, 7178 (1988), etc. can be referred to.

The α-amylase structural gene am is located downstream of the control gene.
yE exists in close proximity. α-amylase structural gene amyE
May be the M type structural gene amyEm,
A structural gene encoding an α-amylase consisting of 477 amino acids is preferable in terms of α-amylase activity. This 477
Structural genes encoding α-amylase composed of amino acids include the N-type structural gene amyEn and the S-type structural gene amyEs.

Then, a base sequence containing the stem-and-loop structure represented by the above formula [I] exists downstream of the α-amylase structural gene amyE. The presence of this stem-and-loop structure significantly enhances α-amylase activity.

The regulatory gene is amyR2 or amyR3 type, and α-
Amylase structural gene is α-amylase structural gene amy
E, particularly in the case of the structural gene amyEn and structural gene amyEs encoding α-amylase consisting of 477 amino acids, etc., at least a nucleotide sequence having a stem-and-loop structure represented by the above formula [II], particularly the above formula [ A base sequence containing a stem-and-loop structure represented by I] is preferable. The base sequence of this stem-and-loop structure seems to function as a terminator.

The control genes other than the control gene amyR, which constitutes the control site, include the gene pap that simultaneously controls the production of amylase and protease, and the antibiotic tunicamycin (tunicam).
ycin) Resistance gene tmr encoding resistance, gene sacU
And operators are included.

Furthermore, a Shine-and-Dalgarno (SD) nucleotide sequence may be present upstream of the translation initiation codon ATG, and the regulatory site of the regulatory genes amyR2 and amyR3 is a Pribnow box (Pribnow box) of approximately -10 region. And its homologous base sequence TAAAAT (TTAAAT in the case of amyR1), about -35 region and its homologous base sequence TTGATA. An open reading frame may be present downstream of the translation initiation codon.

The structural gene amyE is close to the translation stop codons TAA, TAG, and TGA. In addition, a spacer may be interposed between the stop codon and the stem-and-loop structure.

FIG. 1 shows the novel plasmid pMD490 of the present invention (23 February 1989).
The restriction enzyme map of Microtechnology Research Institute No. 10559) deposited at the Institute of Microbial Science and Technology on the date is shown in FIG. 2, and the restriction enzyme map of another novel plasmid pTUB125 of the present invention is shown in FIG. In addition,
These restriction maps are used for the vector pUB110.
The EcoRI site above is shown as the starting point. In the above restriction enzyme map starting from the EcoRI site, the molecular weight of the fragment falls within the range of about ± 0.2 Kb.

The novel plasmid of the present invention has the following features.

(1) Molecular weight by agarose gel electrophoresis: (A) The plasmid pMD490 has a molecular weight of about 8.8 Kb, and (B) the plasmid pTUB125 has a molecular weight of about 8.1 Kb.

(2) Cleavage by restriction enzyme: (A) EcoRI cleavage site is present in plasmids pMD490 and 2.6 Kb. Restriction enzyme EcoRI
It is cut into two fragments of about 2.6KB and about 6.2Kb.

It is cleaved by the restriction enzyme BclI into two fragments of about 2.1 Kb and about 6.7 Kb.

(B) EcoRI cleavage sites are present in plasmids pTUB1250 and 1.9Kb. Restriction enzyme EcoRI
It is cleaved into two fragments of about 1.9 Kb and about 6.2 Kb.

(3) Drug resistance: All plasmids show resistance to kanamycin. Therefore, the host into which the plasmid of the present invention has been introduced can grow in a medium containing 15 μg / ml of kanamycin, for example, LB medium.

(4) Copy number: The copy number of each of the plasmids of the present invention is about 40-50.

In pTUB125, the nucleotide sequence including the stem-and-loop structure in the downstream region after the translation termination codon TGA is as shown in the following formula [III].

When the novel plasmid of the present invention is introduced into a host, it is possible to produce 100 times or more α-amylase of an α-amylase gene-donating strain having one copy of α-amylase gene in its chromosome.

It should be noted that, among the fragments cloned into pTUB125, a circular plasmid in which 0.9 Kb has been deleted from the end containing the stem-and-loop structure represented by the above formula [III], that is, represented by the above formula [I] A circular plasmid having a base sequence containing a stem-and-loop structure has high α-amylase activity. On the other hand, when 1.1 Kb is deleted from the downstream end of the above formula [III], the base sequence of the stem-and-loop structure represented by the above formula [I] is deleted and the α-amylase activity remarkably increases. descend.

As a host into which the novel plasmid of the present invention is introduced, Bacillus subtilis that secretes and produces a large amount of α-amylase outside the cells is preferable.

The novel plasmid of the present invention is, among the strains belonging to the conventionally known Bacillus subtilis, a DNA fragment of a strain having a regulatory gene amyR and an α-amylase structural gene amyE, a structural gene amyE and the above formula [I].
Alternatively, it can be prepared by ligating with a DNA fragment of a strain having a base sequence containing a stem-and-loop structure represented by [II].

More specifically, among the novel plasmids of the present invention, plasmid pTUB125 having a regulatory gene amyR3, a structural gene amyEs, and a nucleotide sequence containing a stem-and-loop structure represented by the above formula [I] is B. It can be prepared by incorporating the chromosomal DN of the subtilis T2N26 strain (which has been deposited with the Institute of Microbial Science and Technology as Micromachine Research Institute No. 3345) into a multicopy plasmid.

Extraction of the chromosomal DNA from the B. subtils T2N26 strain is carried out by culturing the strain, collecting the cells, and then using a conventional method, for example, Saito Miura method (H. Saito, et al., Biochim. Biophys.Acta., 72,619). (196
3)). The extracted chromosomal DNA contains the regulatory gene amyR3, the structural gene amyEs, and the base sequence of the stem-and-loop structure represented by the above formula [I].

Then, in order to clone the α-amylase chromosomal DNA, the chromosomal DNA is partially digested with a restriction enzyme. Partial digestion with a restriction enzyme can be performed by a conventional method. Also, partially digested chromosomal DNA is isolated. Chromosomal DNA
Can be isolated by extraction with an organic solvent such as phenol, chloroform, isoamyl alcohol or a mixed solvent thereof, and precipitation with a lower alcohol such as ethanol or isopropanol.

On the other hand, a multicopy plasmid having an antibiotic resistance marker is used as a cloning vector, the cloning vector is digested and digested with a restriction enzyme, and the isolated chromosomal DNA is ligated with a DNA ligase, preferably T4 DNA ligase. Known cloning vectors are
The B. subtilis strain was cultured in a medium containing antibiotics, and the bacterial cells were subjected to an alkaline method (Molecular cloning, Cold Spring Harbor L
Various known vectors extracted by Aboratory, T. Maniatis, p90, (1982) can be used, but for example, pUB110 having a kanamycin resistance gene is preferable. pUB110 is B.su
btilis IE6 strain (The Bacillus Genetics of Ohio University
It is a publicly known multicopy plasmid having a molecular weight of 4.5 Kb, which is obtained by extraction from the Tok Center) and is sold by Sigma, USA.

The ligated DNA is introduced into a host, and a transformant strain having the α-amylase gene is screened. The host is not particularly limited as long as it is an α-amylase deficient strain, and can be appropriately selected from strains such as Bacillus subtilis, Escherichia coli, and yeast. Of the above hosts, the B. subtilis IA289 strain (available from The Bacilus Genetic stock center of Ohio University) is advantageous. Introduction of DNA into a host can be performed, for example, by protoplast transformation (S. Chang et a
l., Molec. Gen. Genet., 168, 111 (1979)) and the like. The screening is carried out by culturing in a medium containing an antibiotic and selecting a strain showing resistance to the antibiotic and amylase-producing Amy ⁺ as a colony.

The plasmid pTUB125 thus prepared was digested with restriction enzyme E
Of the fragments cleaved with coRI, the approximately 6.2 Kb fragment was approximately 1.6 Kb from the downstream portion of the α-amylase gene and the translation termination site.
It is a fragment that includes the downstream.

Among the novel plasmids of the present invention, the regulatory genes amyR2 and 4
PMD490, which is a plasmid having a structural gene coding for α-amylase consisting of 77 amino acids and the stem-and-loop structure represented by the above formula [I], comprises the above plasmid pTUB125 and B. subtilis 1A412 strain (University of Ohio T
chromosomal DNA (available from he Bacillus Genetic stock center) is digested by digesting the plasmid pDCA100 with the above-mentioned multicopy plasmid pUB110 with restriction enzymes, and the resulting fragments are recombined with T4 DNA ligase. It can be manufactured by connecting them.

The plasmid pDCA100 can be prepared in the same manner as the above plasmid pTUB125 except that the B. subtilis 1A412 strain is used instead of the B. subtilis T2N26 strain. The chromosomal DNA extracted from the B. subtilis 1A412 strain contains the regulatory gene amyR2 and the structural gene amyEm. The molecular weight of the plasmid pDCA100 is about 7.5 Kb,
It is cleaved with the restriction enzyme EcoRI into a fragment of about 2.6 Kb and a fragment of about 4.9 Kb. The fragment of about 2.6 Kb is a fragment of the upstream part of the α-amylase gene containing the restriction gene amyR2.

Then, the strain containing plasmid pTUB125 and plasmid pDCA1
The strain containing 00 is cultured, the plasmids are extracted, and each is digested with the restriction enzyme EcoRI. The approximately 6.2 Kb fragment of pTUB125 and the approximately 2.6 Kb fragment of pDCA100 are then separated, purified and isolated.

Then, the above fragments were ligated by incubating with DNA ligase, preferably T4 DNA ligase, and introduced into the same host as the α-amylase deficient strain by the protoplast transformation method or the like, in the same manner as described above. By screening a strain showing antibiotic resistance and amylase-producing Amy ⁺ , a plasmid pMD490 containing a chimeric α-amylase gene can be obtained.

In addition, separation of fragments digested and cleaved with restriction enzymes,
For purification, conventionally known separation and purification methods, for example, a method utilizing solubility difference such as salting out and solvent precipitation method, dialysis method, ultrafiltration method, gel filtration method, SDS-agarose gel electrophoresis method, SDS -Methods that mainly use the difference in molecular weight, such as polyacrylamide gel electrophoresis, and electroelut
ion) method, a method utilizing a charge difference such as ion exchange chromatography, a method utilizing an affinity such as affinity chromatography, a method utilizing a hydrophobic difference such as reverse phase high performance liquid chromatography, and a combination thereof. Methods are available.

The nucleotide sequence of the novel plasmid of the present invention can be determined by a conventional nucleotide sequencing method such as the Maxam-Gilbert method (Maxam).
-Gilbert, Methods Enzymol., 65,499-560 (1980)),
Dideoxy chain termination method (Sanger et al, Proc. Natl. Acad. Sc
i., USA 74,5463-5467 (1977)).

In the above example, the case of the plasmids pTUB125 and pMD490 has been described as an example, but the gene corresponding to the control gene amyR, the structural gene amyE, and the base sequence containing the stem-and-loop structure represented by the formula [I] is used. A plasmid comprising a combination of various genes can be constructed by treating with a strain containing the same as above.

Further, the plasmid in which the base sequence containing the stem-and-loop structure represented by the formula [II] is deleted is the stem-and-loop represented by the formula [I] among the plasmids obtained as described above. -Cut downstream of the nucleotide sequence containing the loop structure with a restriction enzyme, such as restriction enzymes Hind III or Kpn I,
It can be constructed by deleting a predetermined region, removing a DNA fragment, and ligating ends by a conventional method. For the deletion, for example, exonuclease III, S ₁ nuclease, Bal31 exonuclease and the like can be used.
When there is no recognition site for the restriction enzymes Hind III, Kpn I, etc.
The DNA is ligated, the synthetic DNA is cut with a restriction enzyme Hind III, etc.,
It may be deleted with the above-mentioned exonuclease III or the like. In addition, the deleted end is treated by a conventional method, for example, blunt-ended with nuclease and modified with Klenow fragment,
You may connect with a ligase.

[Effects of the Invention] As described above, according to the novel plasmid of the present invention, since it has a nucleotide sequence containing a stem-and-loop structure,
When introduced into a host, α-amylase can be efficiently produced in large quantities in the host cell.

[Examples] Hereinafter, the present invention will be described in more detail based on Examples.

Example 1 Construction of plasmid pTUB125 (1) Chromosome extraction B. subtilis T2N26 strain (deposited at the Institute of Microbial Science and Technology as Micromachine Research Institute No. 3345) was LB medium (Bactotryptone 1). Wt%, yeast extract
The cells were cultured for 16 hours in 10 ml (containing 0.5% by weight and 0.5% by weight of sodium chloride) and centrifuged at a temperature of 4 ° C. and a rotation speed of 3000 rpm for 20 minutes to collect the cells. Chromosomal DNA was extracted from the obtained bacterial cells by the Saito-Miura method.

(2) Partial digestion with the restriction enzyme Sau3AI In order to clone the α-amylase gene, the chromosomal DNA obtained was restricted with the restriction enzyme Sau3AI (TAKARA Shuzo Co., Ltd.).
Then, it was partially digested as follows. That is, 3 μg of the obtained chromosomal DNA was suspended in 100 μm of a buffer solution consisting of 10 mM Tris-HCl (pH 7.5), 7 mM magnesium chloride and 100 mM sodium chloride, 10 U (unit) of restriction enzyme Sau3AI was added, and the temperature was adjusted to 37 Incubated at 1 ° C for 1-5 minutes. Then, after adding 100 μl of phenol in an amount equal to that of the sample and stirring, the mixture was centrifuged at a rotation speed of 15000 rpm for 5 minutes, the upper layer was collected in a container (Eppeldorf tube), and extracted with phenol. Precipitate the upper layer with ethanol and spin at 15,000 rpm
After centrifugation for 20 minutes, the supernatant was discarded, the mixture was dried and suspended in 100 μm of a TE buffer solution (10 mM Tris-hydrochloride-1 mM EDTA (pH 7.5)).

(3) Preparation of Bacillus subtilis plasmid pUB110 The B. subtilis IE6 strain, which is a retaining strain of the plasmid pUB110 used as a vector, is the Bacillus Gen of Ohio University.
Obtained from etic stock center. This B. subtilis IE6
The strain was cultured in 1 LB medium supplemented with kanamycin (Km) at a final concentration of 15 μg / until the OD600nm reached 0.5-0.8, and the plasmid vector pUB110 was extracted from the obtained cells by the alkaline method. did.

(4) Digestion and digestion of vector DNA with restriction enzyme BamHI 5 μg of plasmid vector pUB110 was added with 10 mM Tris-HCl (pH 8.0), 7 mM magnesium chloride, 100 mM sodium chloride, 2 mM 2-mercaptoethanol, 0.01% by weight. Of bovine serum albumin were suspended in a buffer solution.
Add 10 U (units) of the restriction enzyme BamHI (TAKARA SHUZO CO., LTD.), React at a temperature of 300 ° C for 2 hours, add 1 U (unit) of alkaline phosphatase, and heat at a temperature of 65 ° C for 30
Heated for minutes. Phenol extraction was performed 3 times, ethanol precipitation was performed, and the precipitate was suspended in 100 µ TE buffer solution to obtain vector DNA.

(5) Ligation of chromosomal DNA-Sau3AI partial digest and vector DNA 1 μg vector DNA, chromosomal DNA-Sau3AI partial digest 1 μm
g was suspended in a buffer solution consisting of 66 mM Tris-HCl (pH 7.6), 6.6 mM magnesium chloride, 10 mM DTT and 0.1 mM ATP, 350 U (unit) of T4 DNA ligase was added, and the temperature was 16 ° C.
And reacted for 16 hours.

(6) Introduction into Bacillus subtilis host and screening of α-amylase gene The DNA obtained in (5) above is an α-amylase-deficient strain Bs.
ubtilis IA289 strain (The Bacillus Geneti of Ohio University
(available from the c stock center) by the protoplast transformation method described above, and kanamycin resistant Km ^r , amylase productivity A on a DM3 plate containing 150 μg / ml of kanamycin and 1% by weight of soluble starch.
A strain showing my ⁺ was screened. One amylase-producing strain was obtained from about 5000 Km ^r strains.

From the strain thus obtained, a plasmid pTUB125 containing an amylase gene having an amyR3 type control site was obtained.

This plasmid pTUB125 has a chromosomal gene fragment of about 3.6 Kb cloned in it, digested with various restriction enzymes and
As a result of DNA sequencing, the presence of an S-type α-amylase gene consisting of 477 amino acids having amyR3 type restriction site was confirmed. The plasmid pTUB125 had the nucleotide sequence represented by the above formula [III]. Figure 2 shows the restriction map of plasmid pTUB125.

Example 2 Preparation of plasmid pDCA100 B. subtilis T2N used in step (1) of Example 1 above
Instead of 26 strains, B. subtilis 1A412 strain (Th
Plasmid pDCA100 was prepared by the same procedure as the above-mentioned plasmid pTUB125 except that it was available from e Bacillus Genetic stock center.

This plasmid pDCA100 has a chromosomal gene fragment of approximately 3.0 Kb cloned into it, digested with various restriction enzymes and
As a result of the DNA sequence, the presence of the M-type α-amylase gene having an amyR2 type control site and consisting of 561 amino acids was confirmed. The plasmid pDCA10 is shown in FIG.
A restriction map of 0 is shown.

Construction of plasmid pMD490 (1) Strain containing plasmid pTUB125 and plasmid pDCA1
The strain containing 00 was cultured in 1000 ml each, and the plasmid was extracted by the alkaline method.

(2) Digestion with restriction enzyme EcoRI 10 μg of plasmid pTUB125 was added to 100 mM Tris-HCl (pH
7.5), 7 mM magnesium chloride, 50 mM sodium chloride, 7 mM 2-mercaptoethanol, and 0.01% by weight of bovine serum albumin. Then, 50 U (unit) of restriction enzyme EcoRI (manufactured by TAKARA Shuzo Co., Ltd.) was added and the digestion was carried out at a temperature of 37 ° C. for 3 hours, whereby it was digested into a fragment of about 1.9 Kb and a fragment of about 6.2 Kb. .

Of the above fragments, the fragment containing the downstream portion of the α-amylase structural gene and the downstream portion thereof, that is, the stem-and-loop structure was a fragment of about 6.2 Kb.

When the plasmid pDCA100 was similarly digested with the restriction enzyme EcoRI, it was digested into a fragment of about 2.6 Kb and a fragment of about 4.9 Kb. Of the above fragments, a fragment containing the amyR2 type control site and the upstream portion of the α-amylase structural gene,
It was a fragment of about 2.6 Kb.

Therefore, the above fragments were separated by 0.8% by weight agarose gel electrophoresis and electroeluted using a dialysis membrane.
The desired fragment was isolated by the Ution) method. In addition,
Agarose gel electrophoresis and electroelution were performed according to a conventional method.

Then, the approximately 6.2 Kb fragment of pTUB125 and the approximately 2.6 Kb fragment of pDCA100 were suspended in a buffer solution consisting of 66 mM Tris-HCl (pH 7.6), 6.6 mM magnesium chloride, 10 mM DTT, and 0.1 mM ATP (100 ml). Turbid, 700 U (unit) of T4 DNA ligase (TA
KARA SHUZO CO., LTD. Was added and incubated at a temperature of 16 ° C. for 16 hours.

The resulting ligated sample was introduced into the host B. subtilis 1A289 strain by the protoplast transformation method, and in the same manner as above, kanamycin resistant Km ^r , and a strain showing amylase-producing Amy ⁺ were screened to obtain plasmid pMD490. . This plasmid pMD490 contains the restriction enzyme EcoR
It was cut by I to about 2.6 Kb and about 6.2 Kb. Plasmid p
MD490 had a base sequence represented by the above formula (III). Figure 1 shows the restriction map of plasmid pMD490.
FIG. 4 shows a process diagram for constructing plasmid pMD490 from plasmid pDCA100 and plasmid pTUB125.

The restriction enzymes Sau3AI, BamHI, E
For the activity of coRI, the amount of enzyme that completely decomposes 1 μg of λDNA in 1 hour at 37 ° C. in 50 μl of each enzyme reaction solution was set to 1 U. With respect to the activity of alkaline phosphatase, the enzyme activity of releasing 1 μmole of p-nilophenol in 1 minute at a temperature of 25 ° C. and pH 8.0 was defined as 1 U using p-nitrophenyl phosphate as a substrate. In addition, the activity of T4 DNA ligase was determined using 6μg / 20μ of λDNA-Hind III degradation product at
The enzymatic activity of 90% or more ligation at 30 ° C for 30 minutes was defined as 1U.

Example 3 Preparation of Strain Introducing Deletion Mutant Plasmid To confirm the usefulness of the stem-and-loop structure,
Of the plasmid pTUB125 obtained in Example 1, the above formula (III)
The nucleotide sequence containing the stem-and-loop structure represented by was deleted as follows. Figure 5 shows the plasmid pTUB
The process drawing which constructs plasmid pTUB125HK and deletion mutant pTUB125HKd from 125 is shown.

(1) Ligation of synthetic DNA to pTUB125 First, pTUB125 was treated with 10 mM Tris-HCl (pH 7.5), 7 mM magnesium chloride, 60 mM sodium chloride, 7 mM 2-.
The cells were suspended in a buffer solution containing mercaptoethanol, and the cleavage site derived from the plasmid pUB110 in the plasmid pTUB125 was cleaved with restriction enzymes Sph I and Pvu II.
A DNA fragment was isolated in the same manner as in step (4) above to obtain vector DNA.

On the other hand, synthetic DNA having cleavage sites for restriction enzymes Hind III and Kpn I was synthesized using a DNA synthesizer (Model 381A) manufactured by Applied Biosystems. This synthetic DNA has the following base sequence.

Then, 1 μg of the above DNA fragment and 30 ng of synthetic DNA were used in Example 1.
350 U (unit) of T4 DNA as in step (5)
It was ligated with ligase.

(2) Introduction into Bacillus subtilis Host and Screening Ligation was performed in the same manner as in step (6) of Example 1 above.
DNA was introduced into the B. subtilis 1A289 strain of Bacillus subtilis, and a strain showing kanamycin-resistant Km ^r and amylase-producing Amy ⁺ was screened to obtain a plasmid pTUB125HK. It was confirmed in advance that the obtained plasmid pTUB125HK was cleaved at the recognition sites of the restriction enzymes Hind III and Kpn I in the above synthetic DNA.

(3) Cleavage with restriction enzymes Kpn I and Hind III Plasmid pTUB125HK 10 μm was added to 10 mM Tris-HCl (pH 7.
5), suspended in 100 mM of 7 mM magnesium chloride and 7 mM 2-mercaptoethanol buffer solution, added 200 U (unit) of the restriction enzyme Kpn I, and incubated at a temperature of 37 ° C. for 4 hours. The TE buffer solution 450 was separated by agarose gel electrophoresis and subjected to phenol extraction and ethanol precipitation in the same manner as in step (2) of Example 1 above.
suspended in μ.

Then, a buffer solution of 10 mM Tris-HCl (pH 7.5), 7 mM magnesium chloride, 60 mM sodium chloride, and 100 U (unit) of the restriction enzyme Hind III were added. Also at a temperature of 37 ° C
After incubating for a period of time and cutting,
Phenol extraction and ethanol precipitation were performed in the same manner as in step (2) above, and 50 mM Tris-HCl (pH 8.0), 5 mM was added.
Of magnesium chloride and 10 mM of 2-mercaptoethanol were suspended in 100 μm of a buffer solution.

(4) Deletion of 3'end 180 U (unit) of exonuclease III was added, and digestion treatment time was 1 minute, 2 minutes, 3 minutes, 3.5 minutes, 4 minutes, 5 minutes, 5.5
Samples were sampled every 10 minutes every 6 minutes, 6 minutes, and 7 minutes, and injected into a buffer solution of 30 mM sodium acetate (pH 5.0), 100 mM sodium chloride, 1 mM zinc acetate, 10% glycerol at 100 ° C. Processed for minutes. Then 10 U for each sample
(Unit) Mung Bean Nucl
ease) was added and the end blunting treatment was carried out at a temperature of 37 ° C. for 25 minutes, followed by phenol extraction and ethanol precipitation treatment, washing and drying in the same manner as in step (2) of Example 1.

For each sample, 67 mM potassium phosphate (pH 7.4), 6.7 mM magnesium chloride, 1 mM 2-mercaptoethanol and 3 mM.
50 μl of a buffer solution of 3 μM dNTPs was added, 2 U (unit) of Klenow Fragment was added, and the mixture was treated at 37 ° C. for 10 minutes to end-modify. Then, ethanol precipitation treatment is carried out in the same manner as described above, followed by washing and drying,
It was suspended in 20 μm of TE buffer solution.

(5) Ligation To each DNA 15μ obtained in the above step (4), 66 mM Tris-HCl (pH 7.6), 6.6 mM magnesium chloride, 10 mM
DTT, 0.1 mM ATP buffer solution 5 μ, sterile water 25 μ and 1650 U (unit) of T4 DNA ligase were added, and the temperature was 16 ° C.
The cells were incubated for 16 hours and the ends were ligated.

(6) Introduction into Bacillus subtilis Host and Screening Ligation was performed in the same manner as in step (6) of Example 1 above.
The DNA was introduced into the B. subtilis 1A289 strain of Bacillus subtilis, and a strain showing kanamycin-resistant Km ^r and amylase-producing Amy ⁺ was screened, and plasmid pTUB125H with a different degree of deletion was screened.
A strain containing Kd (d represents a deletion No.) was obtained.

In addition, the nucleotide sequence of each plasmid was determined by the nucleotide sequencing method, and 1.1 Kb of the plasmid was deleted from the downstream of the synthetic DNA, that is, a strain containing the plasmid pTUB125HK16 in which the nucleotide sequence represented by the above formula [I] was not deleted. , A plasmid lacking 1.3 Kb from the downstream of synthetic DNA, that is, the above-mentioned formula [I]
Plasmid pTUB125HK15 in which the nucleotide sequence represented by is deleted
Was selected.

α-amylase productivity Five obtained plasmids pDCA100, pTUB125, pMD490,
α of strains containing pTUB125HK, pTUB125HK16, pTUB125HK15
-Amylase productivity was investigated as follows.

After culturing the strain containing each plasmid in 5 ml of LB medium for 12 hours, add 1% to 100 ml of LB medium (500 ml Sakaguchi flask is used).
The cells were inoculated and cultivated with reciprocal shaking for 12 hours under the conditions of a temperature of 37 ° C and a rotation speed of 120 rpm. Then, 1 ml of the culture solution was centrifuged at 15,000 rpm for 5 minutes, and the supernatant was used as an enzyme solution.

The α-amylase activity in the enzyme solution was measured according to the method of H. Fuwa (J. Biochem., 42, 583 (1954) using soluble starch as a substrate. The α-amylase activity was used as a substrate in 1 minute. The enzyme activity of digesting 1% of (Starch) was defined as 1 U (unit), and the results are shown in the table and FIG.

From the table, it was revealed that the plasmid having the base sequence containing the stem-and-loop structure represented by the above formula [I] highly expresses α-amylase.

[Brief description of drawings]

1 is a restriction map of the novel plasmid pMD490 of the present invention, FIG. 2 is a restriction map of another novel plasmid pTUB125 of the present invention, FIG. 3 is a restriction map of the plasmid pDCA100, and FIG. 4 is a plasmid pDCA100. Fig. 5 is a process diagram for constructing plasmid pMD490 from plasmid PTUB125, and Fig. 5 shows plasmid pTUB125 to plasmid pTUB125HK.
FIG. 6 is a process diagram for constructing the deletion mutant plasmid pTUB125HKd, and FIG. 6 is a graph showing the results of α-amylase productivity in each example.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1: 125)

Claims (3)

[Claims] 1. A ring having a Bacillus subtilis-derived regulatory gene amyR, an α-amylase structural gene amyE on the downstream side of the regulatory gene, and a base sequence containing a stem-and-loop structure on the downstream side of the structural gene. A plasmid having a base sequence including a stem-and-loop structure represented by the following formula (I) (In the formula, an arrow indicates a site capable of having a stem-and-loop structure), a novel plasmid. 2. A control gene amyR2 derived from Bacillus subtilis having a sequence capable of having a stem-and-loop structure upstream of the α-amylase structural gene amyE or a control gene amyR3 derived from Bacillus subtilis having no said sequence, and this control gene. Of the α-amylase structural gene amyE on the downstream side of, and a circular plasmid having a base sequence containing a stem-and-loop structure on the downstream side of the structural gene, wherein the stem-and-loop structure is at least the following formula ( II) AAAGAAACCA TCTATGATGG TTTCTTT (II) (in the formula, an arrow indicates a site that can have a stem-and-loop structure), which has a base sequence containing a stem-and-loop structure and is novel. Plasmid. 3. A base sequence containing a stem-and-loop structure on the downstream side of the α-amylase structural gene amyE has the formula (I).
The novel plasmid according to claim 2, which has a nucleotide sequence represented by: