JP2681634B2 - New bacterial strain with enhanced thermostable liquefied amylase production capacity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION I. Purpose of the invention (Industrial applications)   INDUSTRIAL APPLICABILITY The present invention enhances the production capacity of thermostable liquefied amylase.
Bacterial strain, its breeding method, and the
The present invention relates to a method for producing millase. (Conventional technology)   Thermostable liquefied amylase in starch utilization industry
Is a useful and essential enzyme whose source is exclusively
Many are required for those produced by microorganisms, especially bacteria.
Strains are used. However, regarding enzyme productivity
Are required to be further improved.   Enhancing enzyme productivity through genetic engineering techniques
Examples include Japanese Patent Publication 58-11998 (Maruo et al.).
This is a saccharified amylor of Bacillus subtilis
Introducing multiple regulatory genes to improve productivity of ze
Therefore, the thermostable liquefied amylase, which is the object of the present invention,
About the enzyme produced by Bacillus licheniformis
As a result, it has been desired to create effective and stable strains. (Problems to be solved by the invention)   As mentioned above, it is essential for the starch utilization industry.
The present invention relates to a thermostable liquefied amylase which is an enzyme.
Are more stable and stable than the conventional ones.
Aim to provide the strain and the breeding method.
Yet another objective is to use the strain to
It is intended to provide a method for performing enzyme production. B. Structure of the invention (Means to solve the problem)   In the present invention, Bacillus licheniformis
A suitable thermostable liquefied amylase-producing bacterium belonging to, for example, Ba
cillus licheniformis No.74 or its mutant
Bacillus licheniform
is) TY002 strain (Microtechnology Research Institute, No. 9222) is selected and stained
Remains involved in the production of thermostable liquefied amylase from body DNA
Cloning the gene and transferring this gene to an appropriate vector,
For example, ligation to the plasmid pUB110
Liquefied amylase-producing strain B. licheniformis TY002 strain
Quality change, that is, self-cloning,
Introduction of recombinant plasmids
Obtain a gene-enhanced strain.   In addition, especially for hosts used for self-cloning,
Use the TY002 strain, a thermophilic liquefied amylase high-producing mutant strain
By doing so, it is possible to obtain extremely high productivity at an extremely high level.
come.   Next, culture is performed using this constructed strain to preserve the bacteria.
In addition to the existing thermostable liquefying amylase production capacity,
Amplification of the gene newly integrated on the intracellular chromosome
Achieve the purpose of increasing production of thermostable liquefied amylase by effect
Is what you do.   As mentioned above, the present invention provides Bacillus licheniform
Thermostable liquefied amylase inheritance cloned from Miss
Larvae to TY002, a mutant strain with high production of thermostable liquefied amylase
By transforming, heat-resistant enzyme which is useful for production
Of highly liquefied amylase
The strains that can
Providing means that can contribute to the glucose manufacturing industry from flour
Is what you do. However, using recombinant genes
In the molecularly bred strains,
Stability in the host can be a problem.   The strain B. licheniformis TY002 created in the present invention
Even in the case of (pBA61) -20 strain, when culturing this strain
Is an eye that prevents the recombinant plasmid from falling out of the host cells.
The appropriate drug, eg pUB110 in a vector
Kanamycin, if used, throughout the culture period
And always needed to be added. In addition, the recombinant gene
In the recombinant in which
Culture due to intramolecular deletion of recombinant gene during subculture of strain
A decrease in activity is also a problem.   Strains that improve this point are more useful industrial means.
Is to give. Therefore, in the present invention,
Then, the recombinant plasmid pBA61 was transformed with
Recombinant plasmid pBA61 introduced from the recombinant TY002 strain
Strain B. lichenifor introduced on the TY002 strain chromosomal DNA
Isolate the mis TY002-57I strain to further improve the above problems
It was created as a strain.   The construction and transformation of the recombinant plasmid of the present invention will be described below.
Replacement, self-cloning, and heat-resistant liquids from these
The production of modified amylase will be described in more detail. Cloning of thermostable liquefied amylase gene   As a thermostable liquefied amylase-producing bacterium, Bacillus
Choose a suitable one from the licheniformis
However, we have found that Bacillus licheniform
is No.74 or its mutant Bacillus lichenifo
Choose rmis TY002, Saito and Miura et al. (H.Saito & K.Miu
ra: Biochim.Biophys.Acta72, 619 (1963))
Chromosomal DNA was prepared. Cut this with the restriction enzyme EcoR I
And sucrose density gradient centrifugation to cut off about 2 Kb or more.
The pieces were separated.   On the other hand, the plasmid pBR322 for E. coli was transformed with the restriction enzyme EcoRI
Disconnect with T_FourE. coli HB101 strain after ligation with ligase
The properties were changed according to the conventional method. Ampicillin (20
μg / ml), tetracycline (20 μg / ml), strept
Contains mycin (100 μg / ml) and soluble starch (1%)
Smear it onto Luria broth (LB) agar plate medium containing
Roll showing starch degrading (halo) in elementary starch reaction
Knees were detected to obtain transformed cells.   Recombinant plasmids carried by these transformed cells
When extracted and examined by agarose gel electrophoresis, 3.02
Two types of DNA inserts, Kb and 9.44 Kb, were detected.
The recombinant plasmid containing the former DNA fragment was constructed into pEA6.
Named.   The restriction enzyme map of pEA6 is shown in Fig. 1.   Production of Escherichia coli [E.coli HB101 (pEA6)] carrying pEA6
The thermostable liquefied amylase produced by
Heat-resistant liquefaction type produced by illus licheniformis TY002 strain
As a result of comparison with amylase, the exact same heat resistance behavior
Since the formula was confirmed, the recombinant plasmid pEA6 was TY002.
Cloned the thermostable liquefied amylase gene of the strain
I was able to confirm.   Next, mass-culture E. coli HB101 (pEA6) strain
pEA6 was prepared.   Protoplast pUB110, a known vector of Bacillus subtilis
The TY002 strain was transformed according to the
Fruits and mosquitoes of Bacillus subtilis 1012 strain
Namycin resistant transformants were obtained.   Therefore, the cloned thermostable liquefied amylase gene was cloned.
Self-cloning offspring into DNA donor strain TY002
Therefore, the vector pUB110 which can transform TY002 strain at high rate
Recloned.   That is, pEA6 (Fig. 1) and pUB110 were digested with the restriction enzyme Ec.
Cut with oR I and T_FourAfter ligation with ligase, Bacillus subtilis (Ba
cillus subtilis 1012 strain), Akamatsu and Sekiguchi et al.
amatsu & J. Sekiguchi: Agr. Biol. Chem.46, 1617 (198
2)) Transformation according to the protoplast transformation method
The regenerated colonies with kanamycin and soluble starch.
Replicate to LB agar plate containing
Colonies that form halos in the reaction
A number of transformed cells were obtained.   Extract recombinant plasmids with these transformed cells
And then electrophoresed on an agarose gel to obtain a 3.02 Kb insert DNA.
The one carrying the fragment was selected and named pBA61. p
A map of the restriction enzyme cleavage points of BA61 is shown in FIG.   Large amount from culture of Bacillus subtilis 1012 (pBA61) strain
PBA61 was prepared.   The inserted DNA fragments of pEA6 and pBA61 are
Chromosome DN of Bacillus licheniformis TY002 strain
It was confirmed that it was derived from A.   In addition, E. coli HB101 (pEA6) and Bacillus subti
Thermostable liquefied amylase produced by lis 1012 (pBA61)
Is a heat-resistant liquefied amylor produced by DNA donor strain TY002
Equivalent in molecular weight, heat resistance and immunology
Was also confirmed. Increase of thermostable liquefied amylase by introducing pBA61 into TY002 strain
Creation of production stock   Akamatsu and Sekiguchi to Bacillus licheniformis TY002 strain
Et al. (T. Akamatsu & J. Sekiguchi; Agr. Biol. Che
m., 46 1617 (1982))
pBA61 was transformed.   Regenerated colonies were transferred to LB agar containing kanamycin.
Numerous traits that show kanamycin resistance when cultured in a plate medium
Converted cells were obtained.   Shake it in LB medium containing soluble starch.
Examine productivity of thermostable liquefied amylase
We selected strains with higher productivity than the two strains.   Extract recombinant plasmids from these transformed cells
And run on agarose gel electrophoresis to determine the full chain length of pBA61.
Confirm the presence of the
Bacillus licheniformis TY
It was named 002 (PBA61) -20.   The mycological properties of this transformed cell are:
Increased millase productivity and kanamycin resistance
B. licheniformis T
It is exactly the same as the Y002 strain, and it produces the heat-resistant liquefied
Millase also has its molecular weight, thermostability and immunological properties.
Was confirmed to be equivalent to that of the DNA-donating bacterium. Thermostable liquefaction type A by integrating pBA61 into the chromosome of TY002 strain
Creation of high-producing millase   Product corresponding to gene by introduction of recombinant plasmid
In order to increase the production of
Instability due to such problems may be a problem.   Self-cloning of recombinant plasmid pBA61 into TY002 strain
In the subcultured strain, the internal deletion of pBA61 was also observed in the subcultured strain.
Loss of enzyme productivity that may be lost may be observed.
You.   In the present invention, the recombinant plasmid pBA61 of the TY002 strain is
By incorporating chromosomal DNA, a thermostable liquefied amylase
Create stable strains with increased productivity.   That is, pBA61 was transformed with the protoplast transformant of Akamatsu et al.
The TY002 strain was transformed and regenerated by the method described above.
Replica the knee onto LB agar plate containing kanamycin.
For transformants showing kanamycin resistance,
Control the production of thermostable liquefied amylase and the presence or absence of plasmids
Stable heat-resistant liquefaction about twice that of the TY002 strain
Type amylase producing ability and plasmid inside the cell
We succeeded in the isolation of the transformed cells in which was not detected.   This transformed cell was transformed into Bacillus licheniformis.
(Bacillus licheniformis) TY002-57I strain,
Deposited at the Institute of Microbial Science and Technology, the deposit number is
It is No. 9224.   The kanamycin resistance of this strain is due to the pU used in the vector.
Derived from the kanamycin resistance gene of B110, and
This kanamycin resistance gene is present on the chromosomal DNA of this strain.
In Southern blotting
It was confirmed.   The mycological properties of this strain are kanamycin resistant and
And high productivity of thermostable liquefied amylase
It is exactly the same as the 002 strain.   Therefore, pBA61 was not integrated into the chromosomal DNA in this strain.
Stabilizes the production of thermostable liquefied amylase.
It turned out to have been done.   Furthermore, a heat-resistant liquefied amylor produced by this strain
Ze is a heat-resistant liquefied type produced by the TY002 strain that is a DNA-donor bacterium.
Amylase and its molecular weight, heat resistance and immunological properties
It was also confirmed that the quality was the same. Form using different strains of Bacillus licheniformis
Quality change   Exactly the same as when using the TY002 strain as a host, the TY002 strain
The parent strain of No.74 strain and TY002 strain as well as No.74 strain
Transformation using the mutant strain YB304 as a host,
From the transformed strain to chromosomal DNA similar to that of the TY002-57I strain,
Attempt to isolate strains containing the recombinant plasmid pBA61
However, from the transformed strains of No. 74 strain and YB304 strain,
Isolate strains with recombinant plasmid pBA61 integrated into DNA
I couldn't do it. Therefore, the method shown here
This recombinant plasmid pBA619 was transformed into Bacillus licheni.
TY0 is frequently recombined into Formis chromosomal DNA
It was considered to be a characteristic of the 02 strain. Recombinant heat-resistant liquefied amylor with various strains as hosts
Comparison of production   Cloned by Bacillus licheniformis
Using a thermostable liquefied amylase-producing gene
Production of recombinant thermostable liquefied amylase from various strains
The amounts were compared. Of these, B. subtilis as a host
Have been published in several publications (Gregory L.G. et al .;
J. Bacteriol.,166 p.635 (1986), Stephen A.O.et a
l.; Gene,twenty three p.267 (1983) and Richard P.P. et al.; B
iochem.Biophys.Res.Comm,122 p.175 (1984))
However, those strains are the same as the strains used in the present invention.
But also the production of recombinant thermostable liquefied amylase
Regarding the amount, there is no clear statement that can be directly compared. There
In the present inventors, the same B. subtilis Marburg strain as described in the literature
B. subtilis 1012 strain and B. subtilis M15 (pur
B6, metB5, amyE) strains were tested for comparison. B.subti
The lis 1012 strain has an ability to produce a non-thermostable liquefied amylase.
B. subtilis M15 (purB6, metB5, am
The yE) strain is a strain lacking amylase-producing ability.
Considered to be a strain that has no essential difference from the strain described
It is.   The results of comparing several cultures are summarized in Table 3.
However, Bacillus
・ Created by self-cloning using licheniformis
The strain produced is superior to that of any other strain.
It was rank.   In addition, among them, high production of thermostable liquefied amylase
It is best to use the foreign strain TY002 as a recombinant host.
Is also effective and proved to have extremely high productivity
did. Furthermore, in the TY002-57I strain, pBA61 was in a plasmid state.
Productivity surpassing the TY002 (pBA61) -20 strain introduced in
Indicated.   Therefore, the recombinant plasmid should be integrated into the chromosome.
With, the recombinant gene is stabilized, and the heat-resistant liquefied type is stable
TY002-57I strain with enhanced amylase production is industrially
It was clear that it was a more useful strain. Thermostable liquefied amylase activity assay   Measurements used to measure general α-amylase activity
According to the method, it was performed as follows. In addition, according to this measurement method
The unit is the unit of starch liquefaction according to JIS-method,
Corresponds to 1: 1. [Preparation of reagents] Substrate:   1% soluble starch / 50 mM acetate buffer (pH 6.0) Substrate preparation method:   Purify 1 g (dry weight) of soluble starch (for biochemistry, Wako)
Weigh, suspend in 50 to 70 ml of distilled water, and dissolve by heating. cooling
Then, add 5 ml of 1M acetate buffer (pH 6.0) to make 100 ml. Iodine diluent:   Dilute iodine stock solution (1% iodine solution) 200 times when using
I do. Preparation method of iodine stock solution:   5 g of iodine and 50 g of potassium iodide in 50 ml of distilled water
After dissolution, make up to 500 ml. Enzyme diluent:   2 mM-CaCl / 10 mM-NaCl / 2 mM-acetate buffer (pH 6.0) [Enzyme reaction]   Add 1.0 ml of substrate solution to a test tube (φ1.5 x 10.5 cm), and add 40
After preheating at ℃ for 5 minutes, add 100 μl of enzyme solution to this, and
Incubate for 10 minutes at 0 ℃. After the reaction, add 100 μl of the reaction mixture to 0.
Add to a test tube (φ1.5 x 10.5 cm) containing 1.0 ml of 1N-HCl.
Well, stop the reaction. 500 μl of the test tube (φ1.8 ×
1.3 cm) and add 5.0 ml of diluted iodine solution to develop color
And measure the absorbance at 660 nm (optical path length: 10 MM, control:
Distilled water). [Activity calculation method]   Under the above conditions, reduce 1% iodine coloration per minute
The amount of enzyme is 1 unit. α-amylase activity (u / ml) = ((E_b-E) / E_b) X 100 x 1/10 x dilution rate E_b: Blank absorbance, E: Reaction absorbance   However, (E_b-E) / E_b= 0.2 to 0.2 Measure within the range. (Example)   Next, the present invention will be described in more detail by way of examples.
However, the present invention is not limited to this example.
Needless to say. Example 1 (1) Preparation of chromosomal DNA   Bacillus licheniformis TY002 strain
Lipton 10g, Bacto yeast extract 5g, Sodium chloride 5g,
A medium consisting of 1 L of distilled water and pH 7.0 (hereinafter abbreviated as LB medium)
In 1000 ml, logarithmic growth phase (OD₆₆₀≒ 0.5)
The cells were collected by centrifugation (5 g of wet cells) and stored frozen at -80 ° C.
Was. After thawing the frozen cells, the method of Saito and Miura et al.
DNA was extracted and purified according to the procedure described above, and 1.9 mg (purity OD₂₆₀/ OD
₂₈₀= 2.047) chromosomal DNA was obtained. (2) Insertion of chromosomal DNA fragment into vector   100 μg of the TY002 strain DNA obtained in (1) was treated with restriction enzyme E
Process 600 μl of reaction solution containing 100 units of coR I at 37 ℃ for 1 hour
After heating at 65 ° C for 5 minutes, perform sucrose density gradient centrifugation.
Then, a DNA fragment of about 2 Kb was fractionated.   On the other hand, the widely known E. coli vector
Sumido pBR322 5 μg was completely erased with the same restriction enzyme EcoR I.
And heated at 65 ° C. for 5 minutes.   Next, 10 μg of the former and 5 μg of the latter were mixed, and the linked enzyme T_Four
100 μl of reaction mixture containing 0.5 units of ligase was added at 16
A continuous reaction was carried out for a time. Heat the reaction mixture at 65 ℃ for 5 minutes
After that, collect by DNA ethanol precipitation and add 20 μl TEN
Tuffer (10 mM-Tris-HCl buffer pH 7.5; 10 mM-NaCl)
Thorium; 1 mM-EDTA) was used for transformation. (3) Transformation with recombinant plasmid   4 μl of the DNA solution obtained in (2) was added to Escherichia coli
Hear coli HB101 strain (pro,leu B, B₁,lac Y,hsd R,hsd
M,ara,gal kz,xyl,mtl,sup 44, F^-,endo I^-,rec A^-)
To 200 μl of competent cells prepared according to standard method
Then, transformation was carried out. Add 4 ml of LB medium to this.
E. After shaking culture at 37 ° C for 3 hours, dilute appropriately and
Sillin 20 μg / ml, streptomycin 100 μg / ml and acceptable
Apply to LB agar medium containing 1% soluble starch and apply at 37 ℃ for 2
After culturing for a day, ampicillin and Streptomyces
Transformant 1.7 × 10^FourIodine damp from inside
10 colonies showing halo were obtained in the reaction. (4) Detection of inserted DNA fragment in recombinant plasmid and
Large-scale preparation of recombinant plasmids   Amylase production positive 10 strains obtained in (3)
Picillin 20 μg / ml, tetracycline 20 μg / ml, and suture
Inoculate 3 ml of LB medium containing 100 μg / ml of treptomycin.
After shaking and culturing at 37 ° C for 16 hours, the grown cells were rapidly
Lasmid separation method (D.S.Holmes & M.Quigley: Anal.Bioch
em.114, 193 (1981)) and isolate and
After digestion with the restriction enzyme EcoR I, use agarose gel electrophoresis.
When the inserted DNA fragment was detected, 3.0 out of 5 strains was detected.
2Kb DAN fragment, and the remaining 5 strains have 9.44Kb DNA fragment
Each was detected. The insert DNA fragment of the former (3.02Kb)
The recombinant plasmid having this was named pEA6.   Escherichia coli HB101 (pEA6) strain was treated with M9 medium (Kaza
Minoic acid 0.4%, disodium hydrogen phosphate dodecahydrate 1.7%,
0.3% potassium dihydrogen phosphate, 0.05% sodium chloride,
Ammonium chloride 0.1%, glucose 0.4%, calci chloride
Um 0.1 mM, magnesium sulfate 1 mM, thiamine hydrochloride 40 μ
g / ml, pH7.2), using the standard method for the bacterial cells obtained by 1 L culture
Lysisate treatment followed by lysis, followed by phenol extraction
Deproteinization by the method of cesium chloride density gradient centrifugation
Separate the plasmid by separation, and use it for ethanol precipitation after dialysis.
Was recovered to obtain 1.2 mg of purified plasmid DNA (OD₂₆₀/ OD
₂₈₀= 1.91). (5) Map of restriction enzyme cleavage points of pEA6   For the inserted DNA fragment of pEA6, cut the restriction enzyme by a conventional method.
Table 1 shows the results of examining the cut site.   Furthermore, due to the combination of two and three types of restriction enzymes
After digestion, agarose gel electrophoresis is used to
Position and size are measured and the inserted DNA fragment is cleaved with a restriction enzyme.
A point map was prepared and shown in FIG. (6) Heat-resistant liquefied form produced by Escherichia coli harboring pEA6
Properties of amylase   Escherichia coli HB101 (pEA6) strain and Bacillus
・ Richeniformis strain TY002 containing 1% soluble starch
Culture in LB medium for 3 days at 37 ℃ with shaking
Sonicate the culture solution to break up small cells and centrifuge.
The supernatant was prepared. On the other hand, for TY002 strain, culture medium is centrifuged.
A supernatant was prepared by removing bacterial cells by separation. For both samples
The optimum reaction temperature and thermal stability were compared. Sand
Then, both samples were incubated at 40 ℃, 70 ℃, 80 ℃, and 90 ℃.
Optimal working temperature of thermostable liquefied amylase that undergoes elementary reaction
And both samples at 40 mM in the presence of 10 mM calcium chloride.
After heat treatment for 10 minutes at each temperature of ℃, 70 ℃, 80 ℃ and 90 ℃
The residual activity of the strains was measured and the heat stability was examined.
The optimum working temperature of the enzyme produced by is about 90 ℃,
Heat resistance is 100% remaining after heat treatment at 80 ℃ for 10 minutes.
The active form was obtained, and both showed exactly the same mode of action.   The origin of the inserted DNA fragment of pEA6 was determined by standard methods.
Southern transfer and hybridization
As a result of examination, it was confirmed that it was derived from the TY002 strain.   From these, the recombinant plasmid pEA6 is the TY002 strain.
Of the heat-resistant liquefied amylase-producing chromosomal DNA fragment of
It became clear that the training was done. (7) Form of TY002 strain by vector plasmid pUB110
Quality change   Use the known vector of Bacillus subtilis, plasmid pUB110
Then, according to the protoplast transformation method (described above),
Transformation of tyrus licheniformis strain TY002
When it became 1.2 × 10⁶High frequency transformation of / g DNA
It was revealed that it would be done. This is done for comparison
Of Bacillus subtilis (Bacillus subtilis strain 1012)
It was at the same level as frequency. (8) Reclamation of thermostable liquefied amylase-producing DNA fragment
Rowing   The plasmid pU which is a known vector of Bacillus subtilis in (7)
B110 is frequently added to Bacillus licheniformis TY-002 strain
It became clear that it will be introduced once.   Tolerance from the TY002 strain inserted into the recombinant plasmid pEA6
Thermophilic liquefied amylase-producing DNA fragment self-produced in TY002 strain
For cloning, insert this DNA fragment at high frequency.
Incorporate into a vector that can be transformed into TY002 strain by
Is most desirable. Therefore, recloning to pUB110
became. Ie 10 μg pEA6 and pUB110 respectively
Digested with the restriction enzyme EcoR I and heat-treated at 65 ℃ for 5 minutes.
After the treatment, both were mixed and the ligation enzyme T_FourIncludes 0.5 units of ligase
100 μl of the added reaction solution was ligated at 15 ° C for 16 hours.
Was. Heat the reaction mixture at 65 ° C for 5 minutes and then precipitate with ethanol.
DNA is collected by and dissolved in 20 μl TEN buffer,
Bacillus subtilis 1012 strains (leu A8,met B5,hsr ME
(M⁺)) According to the protoplast transformation method (described above).
And transformed.   Co-regenerated with regeneration medium containing 100 μg / ml kanamycin
Ronnie contains 10 μg / ml kanamycin and 1% soluble starch
Replicate to Nutrient Agar plate with
Cultivated at ℃ for 24 hours and formed halo by iodine starch reaction
The colonies that survive were detected. Shows kanamycin resistance
Among the 750 strains, 53 heat-resistant liquefied amylase acid-positive strains
was detected. Contains 10 strains of kanamycin 10 μg / ml
The cells were cultivated in 3 ml of LB medium with shaking at 37 ° C for 16 hours.
Mini-screening method for bacterial plasmids (D.S. Goldfar
b, R.L.Rodoriguez & R.H.Doi: Proc.Natl.Acad.Sci.USA
79, 5886 (1982)) isolation of recombinant plasmid
And digest with restriction enzyme EcoR I, and then agarose gel electrophoresis
As a result of examination in
A 3.02 Kb insert DNA fragment of the same size incorporated into pEA6
A piece was detected. This recombinant plasmid was named pBA61
Then, 1 L of Bacillus subtilis 1012 (pBA61) in LB medium
Incubate and centrifuge after lysing with lysozyme by the usual method.
The supernatant obtained by the separation was extracted with phenol three times and chlorinated.
Isolate the plasmid by cesium density gradient centrifugation,
After dialysis, collect by ethanol precipitation, purified plasmid DN
A 200 μg was obtained.   The inserted DNA fragment of pBA61 was digested with restriction enzymes by a conventional method.
As a result of examining the cleavage site, it was inserted into the EcoR I site of vector pUB110.
The inserted DNA fragment is the recombinant plasmid described in (5).
DN inserted into EcoR I site of vector pBR322 with pEA6
Shown to have exactly the same restriction sites as the A fragment
Then, the inserted DNA fragment of pEA6 was left as it was, and the EcoRI part of pUB110 was
It became clear that it was inserted in the position. (9) By integrating pBA61 into the chromosomal DNA of TY002 strain
Construction of a thermostable liquefied amylase high-producing strain DNA 0.9 of pBA61
μg according to the protoplast transformation method (described above)
Introduced into TY002 strain (self-cloning)
Colonies regenerated in regeneration medium containing 100 μg / ml
On LB agar plate containing 10 μg / ml kanamycin.
96 strains of transformants replicating and showing kanamycin resistance
I got   20 ml of LB medium containing 1% soluble starch was added to all isolates.
Using a 100 ml Erlenmeyer flask dispensed,
Incubate at 7 ℃ for 72 hours with shaking (176 rpm), and then centrifuge the supernatant.
Then, the thermostable liquefied amylase activity was measured.
Three strains showing about twice the enzyme productivity of the 002 strain were isolated. This
These thermostable liquefied amylase high-producing transformants were transformed into
16 hours in 3 ml LB medium containing 10 μl / ml kanamycin
Bacteria obtained by culturing with shaking for between
Isolate the plasmid according to the cleaning method (above),
After digestion with restriction enzyme EcoR I, prepare by agarose gel electrophoresis.
As a result, 2 out of 3 strains were vector plasmids.
Detects pUB110 band and 3.02 kb insert DNA fragment band
Then, pBA61 was introduced into the TY002 strain, and
It became clear that the ability to produce the enzyme was doubled.
Was. Of these two strains, one promising strain was named TY002 (pBA61) -20
Named.   On the other hand, the band of vector pUB110 from the other strain was also 3.
No band of 02 kb inserted DNA fragment was detected at all.
This strain was named TY002-57I strain.   TY002 strain, TY002 strain transformed with pUB110
110) and TY002-57I strains
After culturing at a sex concentration of 5 ml of LB medium at 37 ° C for 16 hours
TY002 strain was 5 μg / ml, TY002
(PUB110) strain 200 μg / ml, and TY002-57I strain 100
Growth was observed at each concentration of μg / ml. These results
Et al., The acquisition of kanamycin resistance in the TY002-57I strain was
It was not due to mutation, but pBA61 was stained by the TY-002 strain.
This confirmation is thought to be due to integration into the chromosomal DNA.
Therefore, chromosomal DNA was isolated from the TY002-57I strain and
Southern transfer after digestion with elementary EcoR I or BgL II
Hybridization using pUB110 as a probe.
As a result of the analysis, pBA61 was found to be chromosome D of the TY002-57I strain.
It became clear that it was incorporated into NA.   That is, the TY002-57I strain is present on the chromosomal DNA of the own bacterium.
In addition to the existing thermostable liquefaction type amylase production gene,
In addition, pBA61 was integrated into the chromosome, and it was a heat-resistant liquefaction derived from autologous bacteria.
Type amylase gene, both of these genes
Production of thermostable liquefied amylase is approximately doubled
It is obvious that it has been strengthened.   In addition, the enzyme productivity of the TY002-57I strain is extremely stable.
Was. Extra-chromosomal transformation of pBA61 strain TY002 (cell
TY002 (pBA61) -20 strain cultivated and bred
For the production of thermostable liquefied amylase,
Those who applied kanamycin selection pressure (10 μg / ml)
Gives favorable results, but in the case of the TY002-57I strain,
There is no need to do so, for example, simply
Selective pressure applied until isolation of one cell or subsequent seed culture
Is enough.   Table 2 shows that pBA61 was cloned into TY002 by self-cloning.
Heat resistance of strain TY002-57I incorporated into chromosomal DNA of strain
The productivity of liquefied amylase was investigated by flask culture.
In comparison with the TY002 strain, the fruit and the thermostability of the enzyme
Shown. The TY002-57I strain has about twice as much enzyme as the TY002 strain.
It has the productivity and the heat resistance is the same as the TY002 strain.
Became clear.   Furthermore, the enzyme produced by the TY002-57I strain is
And immunochemically equivalent to TY002 strain
, But the molecule by SDS-polyacrylamide gel electrophoresis
Dose (60 Kd) and microoctaloni with rabbit antiserum
-Confirmed by law. Example 2.   E. coli, B. subtilis and B. licheniformis strains
Production of thermostable amylase when
Cultivate each recombinant bacterium in LB medium with 1% soluble starch.
Feed and compare. B. subtilis 1012 strain among the compared strains
Is a non-thermostable amylase-producing bacterium, B. subutilis M15
The strain is a non-amylase-producing strain. Also B.licheniformi
s YB304 strain is a mutant strain derived from No. 74 strain like TY002 strain.
is there.   The results of several cultures are summarized in Table 3.
・ When using licheniformis as a host
It was confirmed that the alternative thermostable amylase was produced. When
Using the thermostable amylase high-producing mutant strain TY002 as a host
Show that extremely superior production is possible when
Further, the TY002-57I strain constructed according to the present invention is pBA6.
TY002 (pBA61) -2 in which 1 is introduced as a plasmid
The productivity exceeded that of the 0 strain. Example 3.   Thermostable liquefied amylase constructed in (9) of Example 1.
Shake the high-producing strain TY002-57I in the following medium at 37 ° C for 70 hours.
Culturing by shaking and measuring the production of the enzyme over time, TY002
It is shown in FIG. 3 in comparison with the strain. Medium: LB medium containing 1% soluble starch Medium volume: 20 ml medium / 100 ml volume-Erlenmeyer flask Culture conditions: 37 ℃, 176st / min.   The growth rate of both strains does not change, but TY002-57I strain is TY
Shows twice as much production of thermostable liquefied amylase as 002 strain
Was. Example 4.   Thermostable liquefied amylase constructed in (9) of Example 1.
High-producing strain TY002-57I strain is cultured in an industrial production medium and
The productivity of the raw material was investigated.   In other words, this strain is soybean flour 3%, corn steep
Liquor 3%, Lactose 2%, Potassium dihydrogen phosphate 0.3%,
Magnesium sulfate heptahydrate 0.03%, potato starch 1 or
500 ml Erlen containing 30 ml medium consisting of 3% and pH 7.0
Inoculate a Mayer flask and incubate for 3 days at 37 ℃ in a sugar-opening culture (21
0 rpm).   The results are shown in Fig. 4 (A and B).   In the figure, A is an example of potato starch 1% and B is an example of 3%.   Also, 5.3% sucrose was added in place of potato starch.
An example of the medium is shown in C.   In any medium, the TY002-57I strain is the TY002 strain.
It showed about twice as much enzyme production as. C. The invention's effect   According to the present invention, a thermostable liquefied amylase raw material
More stable and stable Bacillus licheniform
Miss strain was created, and using this strain, effective
A method for producing various enzymes has been established.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a restriction enzyme cleavage point map of pEA6. FIG. 2 shows the restriction enzyme map of pEA61. FIG. 3 shows the growth degree (dotted line) and the enzyme production status (solid line) in LB medium containing 1% of soluble starch. In the figure, the horizontal axis represents the culture time, the left vertical axis represents the growth rate (OD ₆₆₀ ), the right vertical axis represents the enzyme activity (u / ml), and the curves ○ and ● indicate TY002.
Strains, □, ■ marks indicate TY002-57I strain. FIG. 4 shows the production status of the enzyme in the industrial medium,
A is potato starch 1%, B is 3%, and C is sucrose 5.3%. The horizontal axis is the number of days of culture, the vertical axis is the enzyme activity (u / ml),
In the curve, ● indicates the TY002 strain and ■ indicates the TY002-57I strain.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication (C12N 9/30 C12R 1:10) (C12N 15/09 C12R 1:10) (72) Inventor Bunji Maruo 1-12-6 Kitaurawa, Urawa-shi, Saitama Examiner Ken Ukai (56) References J. Bacteriol. , 166 (2) (1986) P. 635-643 Gene, 23 (1983) P. 267-276 Biochem. Biophys. Res. Commun. 122 (1) (1984) P. 175-183 J.I. Bacteriol. , 168 (2) (1986) P. 973-981

Claims (1)

(57) [Claims] Bacillus licheni
of thermostable liquefying amylase-producing bacterium belonging to formis), cloning a thermostable liquefying amylase-producing chromosomal DNA fragment, and transforming this into a heat-resistant liquefying amylase high-producing mutant strain TY002, Bacillus licheniformis cells, which have enhanced thermostable liquefaction-type amylase production capacity and have a stably enhanced thermostable liquefaction-type amylase production capacity by integrating the introduced recombinant plasmid into the chromosome, Bacillus licheniformis TY002-57I (Microtechnology Research Institute, No. 9224), Bacillus licheniformis cells.