JPS62104580A - Dna-expressing heat-resistant enzyme - Google Patents

Abstract

PURPOSE:The titled DNA that is prepared by allow the structural gene of a specific enzyme to include a codon expressing one or more molecules of cysteine, thus being used in mass-production of heat-resistant alpha-amylase. CONSTITUTION:Cultured cell bodies of Bacillus subtilis are treated with a restriction enzyme to obtain a structural gene of alpha-amylase containing a codon corresponding to cysteine. The component A is treated with an endonuclease, then the DNA segment is bonded to a linker. Further, the product is treated with a ligase, the resultant recombinant plasmid is introduced into Bacillus subtilis 207-25 strain to give a trans-formed strain (B). The strain B is cultured for preferential segregation to obtain a heat-resistant amylase-producing strain, Bacillus subtilis pTUB 616 (C). Finally, a heat-resistant enzyme-manifesting DNA in which the 44th aminoacid from the initiation cokon, ATG (Met) in DNA sequence is a molecule of cysteine is obtained from the cell bodies of the strain C.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to highly thermostable enzyme-expressing DNA.

More specifically, the present invention relates to DNA that can be expressed to contain two or more molecules of cysteine per molecule of enzyme protein.

In general, Bacillus stearothermophilus is heat resistant α
-It is well known that amylase is produced in large quantities.

The present inventors previously transferred the thermostable α-amylase structural gene of Bacillus stearothermophilus to Escherichia
When the mutant strain was successfully cloned into a coli bacterium and cultured, it was possible to produce a large amount of heat-stable α-amylase.

(Japanese Patent Application No. 59-148634) Next, the Escherichia coli mutant strain obtained here will be explained.

(Creation of thermostable α-amylase-producing Escherichia coli) Escherichia coli in which the thermostable α-amylase structural gene used in the present invention has been cloned can be created as follows. The strain was FERM under the name Escherichia coli C600-pTUE 107.
It has been deposited with the Institute of Fine Technology as P-7670.

Bacillus stearothermophilus
stearothermophilus) A 631
．． IAM 11003. ATCC7954 is known as a moderately thermophilic bacterium that secretes thermostable α-amylase outside the bacterial body. Chromosomal DNA was isolated from the cultured bacterial cells of this bacterial cell, and this was partially digested with the restriction enzyme Sau 3 A.
The digested product is subjected to sucrose density gradient ultracentrifugation to separate chromosome fragments of approximately 2 kb or more.

Separately, commercially available plasmid pBR322 (Takara Shuzo Co., Ltd. product)
(Fig. 1) was cut with the restriction enzyme Ram HI, the previous chromosome fragment was mixed with this, T 4 DNA ligase was added, and ligation was carried out.
It was added to a suspension of 600 cultured bacterial cells and subjected to transformation treatment [Lederberg, E, M, and N. Cohen (Lederberg, E, M, and S.
N. Cohen) (1974) Journal Bacteriology (J.

Bacteriol) 119.1072-1074)
be done.

A transformed strain that is resistant to ampicillin and produces α-amylase is isolated, and this strain is cultured to obtain plasmid PTLIE 107 from the cultured cells. The physical map (restriction enzyme cleavage map) of plasmid PTUE 107 is shown in Figure 2, where the white part is derived from vector ρBR322, the black dot part is a chromosome fragment, and the size of this cloned fragment is is about 7.6kb
It is.

The transformant obtained here is an excellent mutant Escherichia coli that secretes thermostable α-amylase well outside the cells, and a large amount of plasmid pTLIE107 can be obtained from the cultured cells.

The chromosomal fragment (black dot in Figure 2) derived from Bacillus stearothermophilus A631 of plasmid pTUE 107 contains all of the promoter, SD sequence, signal region, and α-amylase structural gene. I understand.

Subsequently, the present inventors conducted extensive research based on the idea that productivity could be increased by allowing Bacillus subtilis to produce thermostable α-amylase, and Bacillus stearotherm, which was cloned in Escherichia coli. Heat resistance α of filament
They transferred the structural gene for amylase to Bacillus subtilis and succeeded in producing heat-stable α-amylase.

(Japanese Patent Application No. 60-121537) Next, the Bacillus subtilis mutant strain obtained here will be explained.

(Creation of thermostable α-amylase-producing Bacillus subtilis) Escherichia coli C600-PTUE 107, FE
Plasmid pTUE from cultured cells of RM P-7670
107 is isolated and partially cleaved with Sau 3 A.

On the other hand, the known Bacillus subtilis vector pUB 110 (Journal of Bacteriology Vol 156. No.
, pp. 1327-337) was nicked with Bam 81, treated with BA Pa5e (bacterial alkaline phosphatase) to prevent religation, and this and the Sau 3 A partial digest of plasmid pTUE 107 were combined with DNA ligase. This treatment solution was used to incubate Bacillus subtilis strain 207-25 (Journal of Bacteriology Vol. 156, No. 1327).
- p. 337) and transformed.

The transformed strain is Bacillus subtilis pTUB 6, which is kanamycin resistant and produces α-amylase.
07 is isolated, this strain is cultured, and plasmid pTUB 607 (Fig. 3) is obtained from the cultured cells.

By cleaving this plasmid pTUB 607 with Hinf I, a promoter-free fragment of the α-amylase structural gene derived from Bacillus stearothermophilus A631, ie, the α-amylase gene, is obtained.

Then, after cutting off both ends with Bal 31 and ligating a Hind m linker to this fragment, the Bacillus subtilis secretion vector pTUB 285 (Gene Vol. 134.1-8 pages 1
985) with llind m to cut out the β-lactamase portion, and the remaining vector portion, ie, the portion corresponding to the downstream of the promoter, SD sequence, and signal region of PTUE 285, is ligated and inserted using DNA ligase.

The resulting recombinant plasmid was transformed into Bacillus subtilis 20
7-25 strain by protoplast transformation method.

A transformed strain that is resistant to kanamycin and produces α-amylase is isolated, and this strain is cultured to obtain plasmid pTUB 610 (FIG. 4) from the cultured cells.

This plasmid ρTUB 610 is again introduced into Bacillus subtilis strain 207-25 by the protoplast transformation method.

The transformed strain is Bacillus subtilis ρTUB 6, which is kanamycin resistant and produces α-amylase.
Separate 10.

Then, the chromosome fragment portion (No.4) derived from Bacillus stearothermophilus 8631 of plasmid pTUB 610 obtained from Bacillus subtilis pTUB 610
The shaded area in the figure) is the α-amylase structural gene, and the promoter, SD sequence, and signal region are PTLIB 28
It is derived from 5.

Bacillus subtilis pTUB 610 at 37℃
As a result of culturing for hours and measuring the thermostability of α-amylase in the supernatant, Escherichia coli C600-pTUE
It had the same thermostability as α-amylase produced by FERM P-7670 and FERM P-7670.

In addition, LG- of Bacillus subtilis PTUB 610
24 in broth (containing 50 μg/raQ of kanamycin)
The α-amylase activity of the supernatant after incubation for an hour was 2-3 times higher than that of the supernatant of the same culture of Bacillus subtilis pTUB 607.

Thus, the α-amylase structural gene of the chromosomal fragment derived from Bacillus stearothermophilus 8631 was obtained by t(in) plasmid pTUB607 obtained in large quantities by culturing Bacillus subtilis PTUB607.
It is now possible to obtain a large amount by cutting with fI.

Therefore, the present inventors conducted intensive research to further improve heat resistance by modifying this α-amylase structural gene. They learned that if a codon that allows the expression of one molecule of cysteine is present, heat resistance will be further increased.

The present invention relates to a highly thermostable enzyme-expressing DNA comprising a structural gene for an enzyme having at least one molecule of cysteine in which a codon for expressing at least one other molecule of cysteine is present.

When modifying the α-amylase structural gene, if one more molecule of cysteine can be expressed, the thermostability will increase.

Figure 5 shows a plasmid pTUB 6 created to express two molecules of cysteine in the α-amylase structural gene.
13 and plasmid ρTUB 616 were respectively inserted into Bacillus subtilis to produce α-amylase,
FIG. 2 is a diagram showing the heat resistance of the champion when plasmid pTUB 607 containing the original α-amylase structural gene without any modification was inserted into Bacillus subtilis to produce α-amylase. For the reaction, each α-amylase was added to an aqueous solution of 9
After heat treatment at 0°C for 15 to 60 minutes, residual activity was measured using starch as a substrate.

From Figure 5, the temperature is 90°C when two molecules of cysteine are expressed.
Even after 60 minutes of treatment, there was 50% residual activity, but one molecule of cysteine had only 30% residual activity after the same treatment.

In order to have a codon that expresses one more molecule of cysteine in the α-amylase structural gene, you can convert the existing codon to a cysteine codon. You may also add

Next, a method for creating a cysteine expression codon at the end of the W synthetic gene will be explained.

(Modification of thermostable α-amylase structural gene) [Preparation method 1] Plasmid pTUB 607 is isolated from cultured cells of Bacillus subtilis pTtJB 607, and this plasmid pTUB 607 is cut with 1linf I.

α- derived from Bacillus stearothermophilus 8631
A promoter-free fragment of the amylase structural gene, ie, the α-amylase gene, is obtained.

The entire DNA of this α-amylase structural gene has been published [R, Nakajima, T, Imanaka and Nis Aiba].
ndS, Aiba) (1985) Journal of Bacteriology (J, Bactariol)
163.401-406), the 35th codon GCC (Ala) counting from the translation initiation codon GTG (Net) is the mature a-amylase Ni.
It is the 2-terminus, and contains only one codon corresponding to cysteine at position 363 counting from the 2-terminus.

Next, the α-amylase structural gene fragmented with Hinf I is treated with endonuclease BAL 31 to remove DNA from both ends.

This kind of scratching process is performed after the scratching is performed by using the Hind I [linker (1) CAA GCT T
G (1) When Gin Ala is combined, TO on the right end is combined with T or C,
This is to form a TGT or TGC codon and to express cysteine.

Therefore, the cleavage reaction by endonuclease BAL 31 is carried out until the free DNA T or C is exposed.

The position of T or C may be any position as long as the enzyme function of α-amylase is not impaired; for example, in pTUB 616, the translation initiation codon GTG
The 35th codon GCC (At) counting from (Mat)
This is the case of T in the 34th DNA immediately before a) (NH2 terminus of mature α-amylase).

The reaction progresses depending on the relationship between enzyme concentration, temperature, and time; for example, in the case of pTUB 616, the enzyme concentration is 10
If the conditions are u/100 μQ, temperature is 0° C., and time is about 30 minutes, the probability that the third DNA of the 34th codon counting from the translation initiation codon GTG (Net) will be T becomes quite high.

After ligating a 1(ind m linker) to the cut fragment, the Bacillus subtilis secretion vector pTUB 285 was added to Hind.
The β-lactamase portion was excised by cutting with III and the remaining vector portion, ie, the portion corresponding to the downstream of the PTUB 285 promoter, SD sequence, and signal region, is ligated with DNA ligase.

The resulting recombinant plasmid was transformed into Bacillus subtilis 20
7-25 strain by protoplast transformation method.

A transformed strain was isolated that was resistant to kanamycin and produced α-amylase, and Bacillus subtilis pTUB 616, which produced heat-stable α-amylase, was obtained.

Plasmid PTutl 616 was obtained from Bacillus subtilis pTLIB 616'O cultured cells, from which the α-amylase structural gene was isolated and subjected to DNA analysis (by the dideoxy chain terminator method).
The DNA sequence shown in Figure 7 was obtained. As can be seen from this DNA sequence, in addition to the original cysteine, there is a translation initiation codon A.
It was confirmed that the 44th amino acid counting from TG (Met) was cysteine.

[Preparation method 2] Same as preparation method 1 Bacillus subtilis PTUB 60
Plasmid PTUB 607 is isolated from the cultured bacterial cells of No. 7 and digested with tlinf I to obtain a promoter-free fragment of the α-amylase structural gene derived from Bacillus stearothermophilus A631, that is, the α-amylase gene. The DNA sequence corresponding to the signal peptide derived from Bacillus stearothermophilus A631 remains in this obtained fragment. This signal sequence has a codon TGC corresponding to cysteine at the 26th position counting from the translation initiation codon GTG (Met). When expressed in Bacillus stearothermophilus A631, this 26th codon TGC is expressed as part of a signal peptide when α-amylase is secreted as an enzyme protein, and this signal peptide is cleaved by signal peptidase. and does not exit the cell.

Therefore, the 26th cysteine did not come out of the bacterial cell as part of the enzyme protein. This Hinf-cleaved fragment was cleaved from both ends with endonuclease BAL 31, and the translation start codon GTG
(Met) until just before the 26th codon.

After such a cutting process, Hind In linker
and the codon TG corresponding to the 26th cysteine
The α-amylase gene can be expressed in a C-linked form. In other words, the DNA of the original signal peptide
The codons that were part of the α-amylase structural gene are expressed as if they were part of the α-amylase structural gene.

In this way, cysteine can be caused to exist by adding a codon corresponding to cysteine present near the α-amylase structural gene.

The position to be excised by the endonuclease BAL 31 may be any position in the DNA closer to the GTG side than the 25th codon counting from the translation start codon GTG (Met). 23rd TTG (L
This is the case where the DNA is deleted up to the 22nd DNA, which is the one before eu).

The reaction proceeds depending on the relationship between enzyme concentration and time, as in the case of PTLIB 6162, for example, pTUB 613.
In the case of 5 to 10 units of enzyme/100μQ, temperature O
~5℃ If the time is about 15 to 30 minutes, the 23rd TT counting from the translation start codon GTG (Met)
The probability of cutting down to the 22nd DNA, one before G (Leu), is quite high.

After ligating a Hindm linker to the cut fragment, the Bacillus subtilis secretion vector pTUB 285 was added to the fragment.
The β-lactamase portion is excised by cutting with m, and the vector is ligated to the remaining portion of the vector, that is, the portion corresponding to the downstream of the promoter, SD sequence, and signal region of pTUB 285, using DNA ligase.

The resulting recombinant plasmid was transformed into Bacillus subtilis 20
7-25 strain by protoplast transformation method.

A transformed strain was isolated that was resistant to kanamycin and produced α-amylase to obtain Bacillus subtilis pTUB 613, which produced heat-stable α-amylase.

Plasmid ρTUB 613 was obtained from cultured cells of Bacillus subtilis pTUB 613, from which the α-amylase structural gene was isolated and subjected to DNA analysis (by the dideoxy chain terminator method) to obtain the DNA sequence shown in FIG. As can be seen from this DNA sequence, in addition to the original cysteine, there is a translation initiation codon ATG (
It was confirmed that the 49th codon, counting from Net), corresponds to cysteine.

As shown in FIG. 5, the α-amylases produced by each Bacillus subtilis into which both plasmids were inserted were highly thermostable α-amylases.

Highly thermostable α-amylase producing Bacillus subtilis is cultured in liquid. As a medium, any medium used for culturing Bacillus subtilis may be used, but as a carbon source, starch, liquefied starch, glucose, glycerin,
Molasses, ita molasses, etc. Nitrogen sources include various protein decomposition products, soybean flour, meat extract, peptone, urea, nitrates, ammonium salts, yeast extract, corn staple liquor, and other nutrients such as biotin and trace amounts. Metals and the like are used as appropriate.

Cultivation is carried out at 37-45° C. with aeration and stirring.

After 20 to 30 hours of culture, the highly thermostable α-amylase in the medium reaches its maximum accumulation, so the culture is stopped and the culture solution is centrifuged to remove the bacterial cells.

Highly thermostable α-amylase can be isolated from the obtained culture filtrate by general enzyme purification methods such as salting out, dialysis, ion exchange resin treatment, chromatography, and affinity chromatography.

The highly thermostable α-amylase obtained here has higher heat resistance than the thermostable α-amylase produced by Bacillus stearothermophilus, and is suitable for use in high-temperature liquefaction of starch.

Next, creation examples, creation examples, manufacturing examples, and test examples of the present invention will be shown.

Creation example 1. Creation of mutant Escherichia coli Bacillus stearothermophilus A631, IAM
11003. ATCC7954 in LG medium (bactodrypton log, yeast extract 5g, NaCQ 5g, glucose 2g dissolved in water IQ water and adjusted to pH 7.5)
The bacteria were cultured overnight at 60°C, collected by centrifugation, washed, and the resulting bacterial cells were collected using Miura's method ((1964)).
Biochim Pioffice Acta
Bi0pys, Acta), 72.619-629)
The chromosomal DNA was separated using Tris-HCl and E.
It was dissolved in DTA buffer, restriction enzyme Sau 3 A (Takara Shuzo Co., Ltd. product) was added, and the mixture was digested at 37°C. The digested product was subjected to sucrose density gradient ultracentrifugation to separate and obtain chromosome fragments of approximately 2 kb or more.

A schematic cleavage map of the commercially available plasmid pBR322 (Takara Shuzo Co., Ltd. product) is shown in Figure 1.
322 is 4.4 kb and is ampicillin resistant (Ampr
) and tetracycline resistance (Tc'), and is cleaved at one site by the restriction enzyme Bam HI.

pBR322 cut at one site with Bag HI and about 2 g
Chromosome fragments larger than kb were mixed and ligated by adding T4 DNA ligase. [Weiss B, Sabron Ni C, Live, Farid, G C Richardson C C (weiss.

B,, 5abLoi, A, J,, Live,
T., R., Fareed, G., C.

Richardson, C.C.) (1968) Journal of Sub-Biological Chemistry (J.
Biol, Chew) 243, 4543-4555]
The treatment solution was added to a bacterial cell suspension of Escherichia coli C600, and the calcium/low temperature treatment method [radar barb,
E.M. and N.N. (Lederbe)
rg, E, M,, and S.

N, Cohen) (1974) Journal Bacteriol (J, Bacteriol) 119.10
72-1074).

The resulting transformed bacterial cells were transferred to a selective medium containing 1% soluble starch, 50 μg/m Q ampicillin, and 1.5% agar L medium (10 g Bactodorypton, 5 g yeast extract).
, 5 g of NaCQ was dissolved in IQ water and adjusted to pH 7.5), cultured at 37°C, and the iodine solution was sprayed on the ampicillin-resistant strains that appeared, and the strains that did not react with iodine were - It was possible to isolate the strain as an amylase-secreting strain.

The resulting α-amylase-producing bacterial strain was isolated and cultured overnight at 37°C in L medium containing 100 μg of ampicillin/IIIQ. The culture solution was centrifuged to collect the bacteria. After washing, the isolated bacterial bodies were extracted with alkali. Birnboim H.C. and J. Dawley (Birnboim.

H, C, and L Doly) (1979) Nucleic Acid Research (Nucleic Ac
1d Res, ) 7.1513), the plasmid was isolated and explored, and a novel plasmid was obtained. This plasmid was named plasmid pTUE107.

Plasmid pTUE 107 has a size of 12.0 kb, and its physical map (restriction enzyme cleavage map) is shown in FIG. Here, the white part is vector pBR32
2, the black dotted part is a chromosome fragment part, and each abbreviation is a restriction enzyme.

This plasmid pTUE 107 was labeled with 32p,
Bacillus stearothermophilus A631 strain chromosome D
NA, Escherichia coli strain C600 chromosomal DNA and α-amylase high producing strain Bacillus subtilis (Ba
cillus 5ubtilis) NA 64 chromosomal DNA and Southern hybridization [Southern N.M.
n.

E, M.) (1975) Journal of Sub-Molecular Biology (J, Mo1. Biol),
98.503-517), it did not hybridize at all with the chromosomal DNA of Escherichia coli and Bacillus subtilis, but it specifically hybridized with the chromosomal DNA of Bacillus stearothermophilus strain 8631 at 6 locations. It was confirmed that the cloned gene was derived from Bacillus stearothermophilus A631.

Furthermore, the obtained plasmid pTUE 107 was added to a bacterial cell suspension of Escherichia coli C600 and introduced into the bacterial cell by a calcium low temperature treatment method.

Add the transformant obtained here to the above-mentioned selection medium,
After culturing at 37°C, strains with high α-amylase titer were selected from the grown strains, and Escherichia coli C600-p
TUE 107 and FERM P-7670 were obtained.

Creation example 2. Bacillus that produces thermostable α-amylase
Creation of Escherichia coli C600-pTIJ obtained in Creation Example 1
E107, FERM P-7670 in L medium (Bactodrypton 10gIQ, yeast extract 5gIQ, NaC
Q 5g/Q, ampicillin 100μg/mQ, pH
7 to 7.5) at 37°C overnight, harvested and washed, plasmid PTUE 107 was obtained from the obtained bacterial cells by alkaline extraction method, and partially degraded with Sau 3 A as shown in Fig. 2. got something.

Separately, plasmid pUB 110 was nicked with Bag HI and treated with BAPase to prevent recombination with the same plasmid.

pUB 110 cut at one site with Ram HI and the Sau 3 A partial digest obtained from the plasmid ρTIE 107 were mixed and ligated by adding T 4 DNA ligase.

The treatment solution was added to a protoplast suspension of Bacillus subtilis strain 207-25 cultured cells, and introduced into the cells by the protoplast transformation method.

The plasmid-introduced bacterial cells were grown in SNMP medium [a 4-fold concentration solution of Penacei broth and SMM buffer (0.5M sucrose, 0.02M maleic acid, 0.02M MgCρ2, P
After centrifuging and washing a 2x concentrated solution of H6, 5) in an equal room, gently suspend it again in SNMP medium, and add it to the SNMP medium while gently mixing.
Culture was performed at 0°C for 1.5-2.0 hours. Thereafter, regeneration medium containing 150 μg/IIIQ of kanamycin (3% agar solution 200% IIIQ, IM sodium succinate solution 500%
+nQ 5% Casamino Acid Solution Loom Q, 20% Glucose Solution 25m f), 1.5% Potassium Phosphate,
100ra Q of solution containing 3.5% dipotassium phosphate,
10% yeast extract solution 50m Q, IMMgCQ2 solution 2011IQ, 2% bovine serum albumin solution 20rm Q
, kanamycin (150 mg), and kanamycin-resistant strains were isolated. Furthermore, this kanamycin-resistant strain was plated on LG medium containing 1% soluble starch, and α-amylase producing strains were selected. This bacterium was named Bacillus subtilis pTUB 607.

The transformed strain is Bacillus subtilis pTUB 6, which is kanamycin resistant and produces α-amylase.
07 is isolated, this strain is cultured, and plasmid pTUB 607 (Fig. 3) is obtained from the cultured cells.

By cleaving this plasmid pTUB 607 with Hinf I, a promoter-free fragment of the α-amylase gene derived from Bacillus stearothermophilus A631, ie, the α-amylase gene, is obtained. Next, cut off both ends with Bal 31, and add Hi to this fragment.
After joining the nd m linker, the Bacillus subtilis secretion vector ρ
By cleaving TUB 285 with Hind m, β
- the remaining part of the vector from which the ragtamase part has been cut out,
That is, it is ligated and inserted into the promoter, SD sequence, and downstream portion of the signal region of ρTU8285 using DNA ligase.

The resulting recombinant plasmid was transformed into Bacillus subtilis 20
7-25 strain by protoplast transformation method.

The transformed strain is resistant to kanamycin, and a strain producing α-amylase is isolated, this strain is cultured, and plasmid pTUB 610 (FIG. 4) is obtained from the cultured cells.

This plasmid pTUB 610 is again introduced into Bacillus subtilis strain 207-25 by the protoplast transformation method.

The transformed strain is Bacillus subtilis ρTLIB, a strain that is kanamycin resistant and produces α-amylase.
Separate 610.

Creation Example Creation of Bacillus subtilis that produces highly thermostable α-amylase Bacillus subtilis pTUB 607 obtained in Creation Example 2
plasmid ρTUB 607 (
Figure 3) is obtained.

By cutting plasmid pTUB 607 with Hinf I, Bacillus stearothermophilus A6
The α-amylase gene derived from No. 31, that is, a promoter-free fragment of the α-amylase gene is obtained.

Next, 10 u/100/AQ of Bal 31 was added, the reaction was carried out at 0°C for 20 minutes, both ends were cut off, and a Hind m linker was attached to this fragment. After that, the Bacillus subtilis secretion vector pTUB 285 was cut with 1lind m. The remaining part of the vector from which the β-lactamase part was excised, namely the promoter of pTU8285, S
D sequence and the portion corresponding to the downstream of the signal region are linked and inserted using DNA ligase.

The resulting recombinant plasmid was transformed into Bacillus subtilis 207.
-25 strain by protoplast transformation method.

A transformed strain that was resistant to kanamycin and produced α-amylase was isolated, and Bacillus subtilis PTIJB 613, a strain that produced highly thermostable α-amylase, was selected from among these strains.

Bacillus subtilis PTUB 613 was cultured in LG medium (containing 10 μg/ml of kanamycin) at 37° C. overnight, harvested and washed, and plasmid pTUB 613 was obtained from the resulting bacterial cells by an alkaline extraction method.

This plasmid PTIJB 613 was subjected to DNA analysis (dideoxy chain terminator method), and the highly thermostable α-amylase structural gene of the DNA sequence shown in FIG. 6 was confirmed.

Bacillus subtilis pTU similarly obtained in Creation Example 2
B 607 was cultured, and plasmid pTUB was extracted from the cultured cells.
607 (Figure 3) is obtained.

By cleaving this plasmid pTLIB 607 with Hinf engineering, a promoter-free fragment of the α-amylase gene derived from Bacillus stearothermophilus A631, ie, the α-amylase gene, is obtained.

Next, 10 u/100 pQ of Bal 31 was added, the mixture was reacted at 0°C for 30 minutes, both ends were excised, a Hind III linker was ligated to this fragment, and the Bacillus subtilis secretion vector pTtlB 285 was cut with ) find m. The remaining portion of the vector from which the β-lactamase portion was excised, namely the promoter and SD sequence of pTU8285.

It is linked and inserted into a portion corresponding to the downstream of the signal region using DNA ligase.

The obtained recombinant plasmid was transformed into Bacillus subtilis 207.
-25 strain by protoplast transformation method.

A transformed strain that was resistant to kanamycin and produced α-amylase was isolated, and Bacillus subtilis pTUB 616, a strain that produced highly thermostable α-amylase, was selected from among these strains.

Bacillus subtilis PTLIB 616 was grown in LG medium (
The cells were cultured overnight at 37° C. (containing 10 μg/ml of kanamycin), harvested and washed, and plasmid PTUB 616 was obtained from the resulting bacterial cells by an alkaline extraction method.

This plasmid PTUB 616 was subjected to DNA analysis (dideoxy chain terminator method), and the highly thermostable α-amylase structural gene having the DNA sequence shown in FIG. 7 was confirmed.

Production Example 1 Composition of LG medium Bactodrypton Log/Q yeast extract 5g/Q NaCQ 5g/u Glucose 2gIQ Kanamycin 10μg/m12 ρ11 7-7.5 Bacillus subtilis PTUB 613 in the above medium 10
It was inoculated into a 500 m Q slope flask containing 0IIIQ, and cultured with shaking at 37°C overnight to obtain a seed culture.

Seed culture solution 200+aQ was added to a jar fermenter containing medium 20Q with the same composition as above, and the mixture was incubated at 37℃ for 48 hours.
Culture was carried out with aeration for hours.

The resulting culture solution was centrifuged to remove the bacterial cells and a culture filtrate 19Q was obtained.

12 kg of ammonium sulfate was added to this culture filtrate 19Q to cause salting out and precipitate. Furthermore, this precipitate was collected by centrifugation, dissolved in about IQ of water, insoluble materials were removed, the pH was adjusted to 7.0 with aqueous ammonia, and diatomaceous earth was added and filtered.

The filtrate was placed in a dialysis tube and dialyzed at room temperature against 1 and 10-'' mol of Tris-HCl buffer (pH 7.5).

Furthermore, 2. OX 1. Containing 10-3 moles of calcium acetate. OX, 10-2 moles of Tris salt N! It was brought into contact with DEAE-cellulose equilibrated with a buffer solution (PH 7,5). Contaminants in the dialysis fluid were adsorbed to DEAE-cellulose, and the α-amylase active fraction was recovered as a non-adsorbed fraction. It was brought into contact with DEAE-cellulose again and the non-adsorbed portion was collected. Then, 1.
OX 10-2M Tris-HCl buffer (P!■7.
The enzyme protein was adsorbed onto biogel P100 equilibrated in step 5), and the enzyme protein was eluted with the same buffer.

From this eluate, both fractions with α-amylase activity were collected and freeze-dried to obtain approximately 35 g of a crude enzyme preparation of highly thermostable α-amylase (ρTUB 613 α-amylase).

Production Example 2゜Same as Production Example 1, Bacillus subtilis pTU861
About 40 g of highly thermostable α-amylase (pT
A crude enzyme preparation (UB α-amylase) was obtained.

Bacillus subtilis ρTU separately obtained in Test Example Creation Example
B 607 (a thermostable α-amylase producing bacterium) was cultured to obtain a crude enzyme preparation of thermostable α-amylase (pTUB 607 α-amylase).

Here, pTUB 613α-amylase, pTUB
A thermostability test was conducted on 616α-amylase and pTUB 607 α-amylase.

The reaction was carried out using an aqueous solution of each α-amylase at 90°C for 15 to 6 hours.
After heat treatment for 0 minutes, residual activity was measured using starch as a substrate.

From Figure 5, PTUB 613 α-amylase and pT
UB616 α-amylase (expressed with 2 molecules of cysteine) has 50% residual activity even after being treated at 90°C for 60 minutes, but pTUB 607 α-amylase (expressed with 1 molecule of cysteine) has 30% residual activity after the same treatment. There was only residual activity.

[Brief explanation of drawings]

Figure 1 shows the restriction enzyme cleavage map of plasmid ρBR322, Figure 2 shows the restriction enzyme cleavage map of plasmid ρTOE 107, Figure 3 shows the restriction enzyme cleavage map of plasmid PTUB607, and Figure 4 shows the restriction enzyme cleavage map of plasmid pTUB607.
Figure 5 shows the restriction enzyme cleavage map of pTUB 610.
613α-amylase, PTUB 616α-amylase, and pTUB 607α-amylase.
FIG. 7 is a diagram showing the DNA sequence of the highly thermostable α-amylase structural gene of plasmid pTUB 616. Agent Patent Attorney 1) Parent Male Hinii

Claims (4)

[Claims] (1) A highly thermostable enzyme-expressing DN in which a codon expressing at least one other molecule of cysteine is present in the structural gene of an enzyme that has at least one molecule of cysteine in the molecule.
A. (2) Claim 1, wherein the structural gene of the enzyme having one molecule of cysteine is the structural gene of α-amylase.
Highly thermostable enzyme expression DNA described in . (3) The highly thermostable enzyme-expressing DNA according to claim 1, wherein the structural gene of the enzyme having one molecule of cysteine is a structural gene of thermostable α-amylase. (4) The structural gene for an enzyme containing one molecule of cysteine is the heat-resistant α produced by Bacillus stearothermophilus.
- The highly thermostable enzyme-expressing DNA according to claim 1, which is a structural gene of amylase.