JP3220471B2 - Recombinant DNA, transformant containing the same, and method for producing glucose dehydrogenase using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an amino acid at a specific position represented by the amino acid sequence of DNA encoding glucose dehydrogenase (hereinafter referred to as "GDH") derived from Bacillus megaterium by other amino acids. An improved recombinant DN capable of replicating in Escherichia coli in which the improved DNA obtained by the substitution is incorporated into a DNA introduction vector for Escherichia coli.
A, a transformant containing the same and a method for producing GDH using the same.

[0002]

2. Description of the Related Art GDH [EC 1.1.1.47]
It is used as an important enzyme in the fields of clinical testing and the food industry as an enzyme for quantifying glucose. Conventionally, G
Microorganisms that produce DH include Bacillus megaterium and Bacillus cereus.
bacillus) such as Bacillus sp.
137199).

[0003] GDH derived from Bacillus megaterium is known as a tetrameric enzyme consisting of the same subunit having a molecular weight of about 30,000. GDH has the property of being reversibly dissociated and deactivated on the alkaline side. However, it is known that it is not deactivated when the ionic strength is high.

[0004]

However, in order to use GDH as an enzyme for measuring glucose, it is desired to have more stable properties, and even if the ionic strength is low, it is affected by pH. It has been desired to produce GDH which is difficult and has excellent stability against heat at lower cost.

[0005] Recently, a method for producing GDH using a recombinant obtained by incorporating a GDH gene derived from Bacillus megaterium into Escherichia coli has been disclosed [European Journal of Biochemistry (Eur. J. Bio).
chem. 174, 485-490, (198)
8)].

However, G derived from Bacillus megaterium
It was shown that a plurality of DH genes were present, and transformants were used to produce GDH using them. However, the vector used was not suitable for mass production of GDH, and GDH was used. No improvements have been made to further stabilize.

[0007] In order to produce GDH at a lower cost, the present inventors have already incorporated a GDH gene derived from Bacillus megaterium into a high expression vector pKK223-3 to obtain a transformant, and obtained the transformant in a nutrient medium. GDH was successfully produced by culturing, and further studied, and the DDH encoding GDH derived from Bacillus megaterium was
When a transformant obtained by substituting an amino acid at a specific position represented by the amino acid sequence of NA with another amino acid and incorporating the improved DNA into Escherichia coli was cultured in a nutrient medium,
It has succeeded in producing a large amount of GDH having higher thermal stability than conventional GDH in a culture (Japanese Patent Laid-Open No. 2-8).
6779).

[0008]

In the present invention, the above-mentioned invention was further studied, and DNA obtained by substituting one set of specific amino acids with another set of specific amino acids was incorporated into Escherichia coli. When the transformant was cultured in a nutrient medium, it was possible to produce GDH having excellent thermostability. Furthermore, it was possible to produce GDH which exists in the form of a tetramer at any pH and is thermostable. The inventors have found what can be done and completed the present invention.

In order to prepare a GDH recombinant DNA derived from Bacillus megaterium, it is first necessary to prepare a recombinant DNA encoding GDH. As the strain to be used therefor, any Bacillus megaterium having GDH-producing ability can be used, but Bacillus megaterium IAM1030 and Bacillus megaterium IWG3 isolated from soil are preferably used.

Among them, a strain of Bacillus megaterium IWG3 obtained from soil was identified as follows.

Tests of mycological properties are described in Ruth E. Gordon, Ruth EG
ordon: The Genus Bacillus
(1973)], and the classification method was Bergey's Manual of Deterinary Bacteriology.
minative Bacteriology) (No. 8
Edition) and the aforementioned Genus Bacillus
According to

A. Form (1) The size of the cells is 1.1 to 1.6 μ × 3.0 to
It is a bacillus at 5.0μ. Glucose nutrient medium (gl
When cells grown on euconusentient agar are stained with fuchsin, the cells are granular. (2) There is no mobility. (3) Spores are formed, and the size is 1.0 to 1.3 μ ×
2.0-2.5μ, oval or cylindrical. Spores do not swell. Neutral or semi-fine. (4) Gram stainability is positive.

B. Physiological properties (1) Reduction of nitrate: negative (2) Denitrification: negative (3) VP test: negative. The pH of the broth is 4.6-5.0 after 7 days of culture. (4) Indole formation: negative (5) Starch hydrolysis: negative (6) Use of citrate: positive (7) Use of inorganic nitrogen source: Use both ammonium salt and nitrate. (8) Pigment production: A brown water-soluble pigment is produced in a tyrosine medium. (9) Urease: weakly positive (10) Catalase: positive (11) Attitude to oxygen: aerobic (12) Generation of acid and gas from saccharides: arabinose, xylose, glucose, fructose, galactose, maltose, sucrose, lactose , Trehalose, mannitol, inosit, glycerin, starch, but not gas. No acid or gas is produced from mannose or sorbite. (13) Growth on 7% NaCl medium: does not grow (14) Growth at 45 ° C .: grows (15) Growth at 65 ° C .: does not grow (16) Sabouraud Dextrose (Saboura)
(17) Deamination of phenylalanine: positive (18) Liquefaction of gelatin: positive (19) Degradability of casein: positive (20) Degradability of tyrosine: positive (21) Egg yolk Reaction: negative

[0014] The above properties are described by the Burgess Manual of Defective Native Bacteriology (No. 8).
When this strain is searched according to the classification method of the above edition, the strain is classified as Bacillus because it forms spores with Gram-positive aerobic bacilli.

Regarding the species, (1) the size of the vegetative cells is 1.0 to 1.6 μ × 3.0
~ 5.0μ, and the inside of cells is granular in a glucose medium. (2) The spore sac does not swell, and the spore formation site is located at the center or slightly from the end. (3) Generating acid from glucose, VP test is negative. (4) Does not grow under anaerobic conditions. (5) Sabouraud Dextrose (Sabourau
d dextrose) medium. (6) An acid is produced from arabinose, xylose and mannitol. (7) The yolk reaction is negative. It was identified as Bacillus megaterium from properties such as
The present inventors have established this strain as Bacillus megaterium IWG.
No. 3.

Hereinafter, the present invention will be described in detail with reference to examples.

【Example】

Preparation of Transformant A (1) Purification of GDH Bacillus megaterium IWG3 is inoculated into 2XTY broth, collected after culturing, crushed, crushed, and centrifuged to remove the supernatant. After concentration, 105 μg of GDH crude enzyme powder obtained by freeze-drying was dissolved in 15 ml of imidazole buffer (20 mM, pH 6.5) containing 10% of glycerol, adsorbed on DEAE-Sephadex A-50, and then concentrated in sodium chloride. Elution is performed with a gradient (0.1M-0.5M), and the active fraction is collected and desalted and concentrated. Next, TSK-Gel D
Molecular weight fractionation was performed by high performance liquid chromatography using EAE-3-SW as a carrier, and further TSK-gel G
It was adsorbed and eluted by high performance liquid chromatography using 3000-SW as a carrier to obtain an electrophoretically uniform active fraction (about 5 mg in terms of protein mass).

(2) Determination of amino acid sequence at the amino terminal of GDH The amino acid sequence at the amino terminal of the purified enzyme protein obtained by the above (1) was determined by ABI [Applied Biosystems (A)
[Plied Biosystem]], a peptide sequencer Gas Phase 470A, and a sequence of 29 amino acid residues from the N-terminus (SEQ ID NO:
2) was determined. The underlined portion indicates the sequence used for probe synthesis.

(3) Synthesis of DNA probe The amino acid sequence shown in FIG.
Select sequences at several locations, and among the possible DNA base sequences on the gene estimated from these amino acid sequences, estimate the DNA base sequence with reference to the codon usage frequency of Bacillus subtilis,
The base sequence of one 38-mer DNA probe (SEQ ID NO: 3) was determined. DNA synthesis was performed by ABI, using a synthesizer (Synthesizer) model 381A.
This was performed using

(4) Extraction and cleavage of total DNA from Bacillus megaterium The method of Saito, Miura et al.
Actor (Biochim. Biophys. Act)
a), Vol. 72, p. 619 (1963)], and total DNA was extracted and purified from Bacillus megaterium IWG3. Take 240 μg of this DNA, and use the restriction enzyme EcoR
The reaction was carried out with 150 units of I and BglII at 37 ° C. for 3 hours. The entire amount of the reaction solution was subjected to 1% agarose gel electrophoresis, a portion containing DNA corresponding to a size of 3 to 4 Kb was cut out, and a DNA fragment was eluted from the gel by an electric extraction method. Next, the eluate was sequentially extracted with an equivalent amount of phenol and phenol / chloroform. Ethanol was added to the obtained aqueous layer to precipitate DNA, which was then dissolved in 100 μl of TE buffer.

(5) Insertion of DNA Fragment into Vector Although pBR322 was used as a vector, 20 μg of pBR322 was used for insertion of a DNA fragment.
-Linear vector D obtained by complete digestion with BamHI
NA was dissolved in 200 μl of TE buffer and used. The binding to the DNA fragment obtained in the above step (4) was carried out by combining the solution obtained in the step (4) with the linear vector DNA solution.
This was performed by mixing at a ratio of 0: 1 and allowing T4 DNA ligase to react at 14 ° C. overnight.

(6) Bacillus megaterium DNA
Preparation of library The recombinant DNA obtained in the above step (5) was transformed into host Escherichia coli (Escherichia).
ia coli) introduced into C600, and ampicillin 50
Colonies that had grown on L-broth agar containing μg / ml were collected and the B. megaterium IWG3 D
It was called NA library.

(7) Selection and Separation of GDH Clone from DNA Library The DNA probes obtained in the above step (3) were each isolated by the method of Inglia et al. [Nucleic Acids Research (Nucleic Acids Research).
s. ), Vol. 9, and T4 polynucleotide kinase according to pages from 1627 to 1642 (1982)] γ- ³² P-A
Labeled with TP.

Next, the Escherichia coli obtained in the above step (6) was grown as a colony on an L-broth agar medium containing 50 μg / ml of ampicillin, and transferred to an Amersham nylon membrane by a replica method. The cells were lysed, denatured with alkali DNA, neutralized with hydrochloric acid, and then hybridized with the probe. Hybridization was performed at a 6-fold concentration of SSC (0.15 M NaCl, 0.0
15M sodium citrate, pH 7.0), 5 times concentration of Denhardt solution (0.02% ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.5% SDS, bovine thymus DNA20
Prehybridization was performed at 45 ° C. for 3 hours using μg / ml (final concentration) and about 5 × 10 ⁵ cpm / ml of the labeled DNA probe, followed by overnight hybridization at 45 ° C.

Thereafter, using a 5-fold concentration SSC at 45 ° C.
The membrane was washed twice with SSC at a concentration of 5 times (including 0.1% SDS) twice at 45 ° C. and twice with SSC at a concentration of 4 times. Thereafter, the nylon membrane is dried and subjected to autoradiography (condition: -8
0 ° C, overnight). As a result, three hybridization-positive colonies were found. Therefore, after performing liquid culture on the positive colonies, the burnboim (Bi
rnboim) et al. [Nucleic Acids Res., 7
Volume, pp. 1513 to 1523 (1979)].

The obtained plasmid DNA was digested with the restriction enzyme Ec.
After cutting with oRI and SalI and performing agarose gel electrophoresis, labeled DNA probe and Southern (Son
then) hybridization [Journal of Molecular Biology (J. Mol. Bi)
ol), Vol. 98, pp. 503-517 (1975)]. As a result, it was found that the DNA probe strongly hybridized to a DNA fragment of about 3.6 Kb generated by digestion with EcoRI and SalI. The three strains thus isolated were shown to have the same plasmid, and this plasmid was designated as pGDAl as a candidate for a GDH clone.

(8) Identification of GDH Clone and Determination of DNA Nucleotide Sequence A 930 bp DNA fragment generated by cutting EcoRI and Sau3AI from plasmid pGDA1 was used according to the method of Sanger et al.
Of National Academy Science USA (Proc. Natl. Acad. Sci. U.S.A.).
S. A. 74, 5463-5467 (197).
7)] and the DNA base sequence was determined.

As a result, the GD obtained in the above step (2)
A nucleotide sequence encoding an amino acid sequence completely identical to the amino acid terminal amino acid sequence of H was found.
It was found to contain part of the DH gene. For the plasmid pGDA1, a restriction enzyme map shown in FIG. 1 was created based on the results of restriction enzyme digestion. When the DNA base sequence at the downstream site in the gene reading direction was determined from the base sequence already determined, it was shown that a base sequence encoding a protein consisting of 261 amino acids represented by SEQ ID NO: 4 was present.

From the above results, it is presumed that the DNA fragment derived from Bacillus megaterium IWG3 in the plasmid pGDA1 completely contains the GDH structural gene.

(9) Expression of GDH gene In order to express the cloned GDH gene in Escherichia coli, gene expression was attempted from a DNA fragment derived from Bacillus megaterium IWG3 in plasmid pGDA1 according to the following steps.

10 μg of the plasmid pGDA1 was
After digestion with RI and PvuII, the fragment was subjected to 1% agarose electrophoresis, and a fragment having a size of about 1.5 Kb was recovered. 1 μg of the obtained fragment was added to dATP, dGTP, dCTP, dTTP.
TP was added at a final concentration of 1 mM each, 4 units of DNA polymerase Klenow fragment was added, and 10 mM Tris-HCl buffer (pH 7.5) was added.
The reaction was carried out at 30 ° C. for 20 minutes in 20 μl of a reaction solution containing 7 mM MgCl 2 and 1 mM dithiothreitol. As a result, the DNA fragment having blunt ends at both ends was purified. To about 0.5 μg of the DNA fragment, a PstI linker and 10 units of T4 DNA ligase were added, and a 66 mM Tris-HCl buffer solution (pH
7.5) In a 20 μl reaction mixture of 5 mM MgCl 2, 5 mM dithiothreitol, 1 mM ATP, 14 ° C.
Allowed to react overnight. After the reaction, the DNA fragment was purified, and
After digestion with I, this fragment was treated with mung bean nuclease (M
ung bean nuclease) 1 unit,
40 mM sodium acetate (PH 4.5), 100 mM
The reaction was carried out at 30 ° C. for 30 minutes in 50 μl of a reaction solution of NaCl, 2 mM ZnCl 2 and 10% glycerol. By this operation, the protruding end of BanII was made blunt, and an EcoRI linker was ligated in the same manner as described above. After the reaction, the DNA fragment is purified, and EcoRI and Ps
Both ends were cut with tI and recovered as an EcoRI-PstI fragment.

The expression vector pK used in this example
K223-3 is Brosius.
J. ) Et al. [Proceedings of National Academy Sciences USA (Proc.
tl. Acad. Sci. U. S. A. ), 81, 6
929-6933 (1984)], and has a tac promoter as a promoter.

After the expression vector pKK223-3 was digested with restriction enzymes EcoRI and PstI, the recovered Ec
The mixture was mixed with the oRI-PstI fragment, and a binding reaction was performed with T4 DNA ligase. Escherichia coli JM105 was transformed with the reaction solution, and 50 μg of ampicillin was used.
/ Ml, colonies growing on an L-broth agar medium containing isopropyl-β-D-thiogalactopyranoside (IPTG) were selected.

In order to confirm the expression of GDH in the obtained colonies, a colony assay using a dye conjugate method was performed. The colony was replicated on a filter paper, and a lysozyme solution prepared by adjusting lysozyme to a concentration of 1 mg / ml in 50 mM Tris-hydrochloric acid (pH 7.5) and 10 mM EDTA buffer was added to the colony on the filter paper, and incubated at 30 ° C. for 20 minutes. A 1% Triton solution was added, and the mixture was left at room temperature for 5 minutes.
Further, a buffer for heat treatment [50 mM phosphate buffer (pH
6.5) 2M-NaCl, 50 mM-EDTA] was added, and heat treatment was performed at 60 ° C. for 20 minutes.

Next, a substrate mixture [20 mM Tris-HCl (pH 8.0), 1 M NaCl, 100 mM glucose, 0.5 mM phenazine ethosulfate (PE
S), 0.5 mM 3- (4 ', 5'-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT), 50 μM-NAD], and 3
Leave at 7 ° C for 5 minutes in the dark. As a control experiment, the one in which glucose in the above substrate mixture was removed was used. The reaction was stopped by adding a 10% acetic acid solution. For selection of colonies, those whose colonies turned blue-purple were selected. As a result of the colony assay, a large number of positive colonies were obtained. Plasmid DNA was extracted from one of these strains, named pGDA2, and the structure expected by cleavage with the restriction enzyme, FIG. 2, was confirmed.

The plasmid was introduced into Escherichia coli JM105 by transformation to obtain a high GDH-expressing strain Escherichia coli JM105 / pGDA2. This strain is FERM BP by the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology.
-2584. Preparation of transformant B

Using Bacillus megaterium IAM1030 in place of Bacillus megaterium IWG3, the same procedure as in the above (1) to (9) for preparing the transformant was carried out,
The high GDH expression plasmid DNA was extracted and named pGDA3. Next, this plasmid was transformed to obtain a high GDH expression strain Escherichia coli JM105 / pGDA3.

The Bacillus megaterium IAM10
Amino acid sequence of GDH obtained from SEQ ID NO: 30 (SEQ ID NO:
5) shows that the serine at position 22 from the N-terminus is alanine, the aspartic acid at position 43 is glutamic acid, and the position 79 at position 79 are compared with the DNA amino acid sequence of GDH derived from Bacillus megaterium IWG3 shown in SEQ ID NO: 4. It was found that only alanine was replaced by serine and leucine at position 95 was replaced by methionine.

That is, the amino acid sequence of DNA encoding GDH derived from Bacillus megaterium is summarized in (SEQ ID NO: 1). GD from Bacillus megaterium IWG3
Conversion of base sequences corresponding to amino acids 96, 252 and 253 from the N-terminus of H DNA

The site-specific mutation technique [Zora et al. (M.Z
oller, M .; Smith), Nucleic Acids Res.
10, 6487 (1982)] to prepare a mutant gene (hereinafter, referred to as E96V gene) in which the base sequence GAA (glutamic acid) corresponding to position 96 from the N-terminus of the GDH gene is converted to GTA (valine). . Further N
Mutant gene obtained by converting the base sequence GAA corresponding to position 96 from the terminal a (glutamic acid) to the AAA (Li di down) (hereinafter,
It is called E96K gene. ) Was adjusted. Next, the base sequence CAG (glutamine) corresponding to position 252 is converted to CTG (leucine), and the base sequence TAC (tyrosine) corresponding to position 253 is converted to GAA (glutamic acid), which is referred to as a mutant gene (hereinafter referred to as Q252L-Y253E gene). ) Was adjusted.

Further, positions 96 and 252 were obtained by a recombination method using a restriction enzyme site which was present only once in the reading using the transformant containing the E96K gene and the transformant containing the Q252L-Y253E gene. And a mutant enzyme gene obtained by converting positions 253 (hereinafter referred to as E96K-Q25
It is called 2L-Y253E gene. ) Got.

Preparation of Mutant Double-Stranded Gene Fragments Each of the above-mentioned mutation-treated DNAs (E96V gene, Q252L
-Y253E gene and E96K-Q252L-Y25
10 μg of a 10-fold concentration of a reverse transcriptase buffer [70 mM Tris-HCl buffer (pH 7.5), 70 m
M magnesium chloride, 0.5M sodium chloride, 20m
M dithiothreitol] 2 μl of a solution containing 10 μl and 20 pmol of a primer and 74 μl of distilled water are added to give 85
After incubating for 5 minutes at 40 ° C. for 15 minutes, 13 μl of 10 mM dNTP and 1 μl (20 units) of reverse transcriptase were added to carry out the reaction at 37 ° C. After 1 hour, phenol extraction was carried out, followed by ethanol precipitation. The precipitate was dissolved, digested with restriction enzymes EcoRI and PstI, agarose gel electrophoresis was performed, and a mutant double-stranded gene fragment was recovered from the gel according to a conventional method. .

Selection of heat-resistant GDH gene-bearing strain The mutated double-stranded gene fragment obtained in the preceding step was inserted into the EcoRI and PstI sites of the expression vector pKK223 to transform Escherichia coli JM103. For colonies grown on Petri dishes,
The enzyme activity was examined by a replica print method using filter paper. Hook the strain showing strong color development on the filter paper from the master plate.

Next, each of the strains selected by the above method was inoculated into 5 ml of 2XTY medium and cultured with shaking at 37 ° C. for 18 hours. After washing, the cell suspension was subjected to ultrasonic treatment and centrifuged to obtain a supernatant.

Determination of base sequence of mutant enzyme gene and identification of mutation Plasmid DNA was prepared from the mutant enzyme gene-bearing strain,
The nucleotide sequence of the gene fragment was determined according to a conventional method, the mutation point was clarified, and the change in the amino acid sequence of the enzyme protein was confirmed.

That is, 96 amino acids from the N-terminus of the amino acid sequence of GDH natural DNA derived from Bacillus megaterium IWG3
Improved recombinant DN in which glutamic acid at the position is changed to valine
A, pGDA2F-30, pGDA2F-40 which is an improved recombinant DNA in which glutamine at position 252 is changed to leucine and tyrosine at position 253 is changed to glutamic acid, and glutamic acid at position 96 from the N-terminus to alanine;
Each improved recombinant DNA of pGDA2F-50, which is an improved recombinant DNA in which glutamine at position 52 was changed to leucine and tyrosine at position 253 to glutamic acid, was obtained.

Next, each transformant obtained by transforming Escherichia coli JM103 with each plasmid (Escherichia coli JM103 / pGDA2F-30, Escherichia coli JM103 / pGDA2F-40, and Escherichia coli JM103 / pGDA2F-50) ) Was cultured in 2XTY broth medium at 37 ° C. for 18 hours,
After cell collection, the cell suspension was subjected to ultrasonic treatment, and after centrifugation, the obtained supernatant was purified to homogeneity by electrophoresis using DEAE-Sepharose column chromatography. The following tests were performed on this enzyme.

Escherichia coli JM103 / pGDA
GDH obtained from 2F-50 was treated with 30M in the presence and absence of 2M NaCl in each buffer.
PH stability at 20 ° C. for 20 minutes was examined (p
H4-6 are 75 mM acetate buffer, pH 6-7.5 and pH11-12 are 75 mM phosphate buffer, pH 7.5
8.5 is 75 mM Tris-HCl buffer, PH 8.5-1
0.5 uses 75 mM glycine-NaOH buffer).
The result is shown in FIG.

Escherichia coli JM103 / pGDA
GDH obtained from 2F-50 was converted to Na at each temperature.
In the absence of Cl, 50 mM phosphate buffer (pH
The temperature stability when treated in 6.5) for 20 minutes was examined. The result is shown in FIG.

Escherichia coli JM103 / pGDA
GDH obtained from 2F-40 was converted to Na at each temperature.
In the absence of Cl, 50 mM phosphate buffer (pH
The temperature stability when treated in 6.5) for 20 minutes was examined. The results are also shown in FIG.

As is clear from FIGS. 3, 4 and 5, the specific amino acid represented by the amino acid sequence encoding GDH, that is, glutamic acid at position 96 from the N-terminus corresponds to alanine, and glutamine at position 252 to leucine. 253
By substituting glutamic acid for the tyrosine at position 2,
When the ionic strength is low, GD maintains activity at any pH and is more stable to heat than natural type.
It can be seen that H is produced.

As is apparent from FIG. 5, the substitution of glutamic acid at position 96 with valine improves the heat resistance of GDH as compared with the natural type.

Further, it can be seen that the heat resistance of GDH is improved by substituting glutamine at position 252 with leucine and replacing tyrosine at position 253 with glutamic acid, as compared with the natural type.

The transformant Escherichia coli was inoculated into 100 ml of 2XTY broth containing 50 μg / ml of ampicillin, cultured at 37 ° C. for 13 hours with shaking, added with IPTG (final concentration 0.1 mM), and centrifuged 2 hours later. And washed with 50 mM phosphate buffer (pH 6.5) containing 2 M NaCl, suspended in 10 ml of the same buffer, crushed with an ultrasonic crusher, and centrifuged to separate the supernatant. Obtained. On the other hand, Bacillus megaterium IWG
3 inoculated in 100 ml of 2XTY broth,
The cells were cultured with shaking for hours. Next, cells are collected in the same manner as above, and after washing,
After sonication, the mixture was centrifuged, and the supernatant was used as an enzyme solution.

GDH enzyme activity was measured according to the following method. D-glucose 0.1 M, NAD 20
The enzyme solution is added to 75 mM Tris-HCl buffer containing mM and reacted at 30 ° C. in a photometer cell, and the increase in absorbance at 340 nm is measured. 1 μmol per minute of reaction
The enzyme activity for producing NADH of e was defined as 1 unit, and the specific activity was shown as the number of units per 1 mg of protein in the enzyme solution.

As a result, Escherichia coli JM103
/ Specific activity of GDH obtained from pGDA2F-50 (p
H6.5) is 12.7 u / m in the presence of 2M NaCl.
g and 9.20 u / mg in the absence. Tsumari, Escherichia coli JM103 / pGDA2F-
It is considered that GDH produced from No. 50 exists as a tetramer regardless of ionic strength and shows activity.

Escherichia coli JM103 / pGDA
The specific activity of GDH obtained from 2F-40 was 2M NaC
1 showed 7.67 u / mg, and 0.96 u / mg in the absence. That is, GDH produced from Escherichia coli JM103 / pGDA2F-40.
Is considered to dissociate and show no activity when the ionic strength is low.

The specific activity (pH 6.5) of GDH obtained from Bacillus megaterium IWG3 was 510 u / mg in both the presence and absence of 2M NaCl. However, inactivity at pH 9.0 was 2M N
475 u / mg was shown in the presence of aCl, but no activity was shown in the absence. That is, GDH produced from L. megaterium IWG3 is considered to dissociate and show no activity in a state of low ionic strength.

A transformant was obtained in the same manner using Bacillus megaterium IAM1030 instead of Bacillus megaterium IWG3, and GDH was produced. The same results as described above were obtained.

[0060]

According to the present invention, there is provided an improved DNA obtained by replacing the amino acid at a specific position represented by the amino acid sequence of the DNA encoding GDH derived from Bacillus megaterium with another amino acid, and further improving the GDH level. Improved recombinant D capable of replicating in Escherichia coli integrated into an expression vector
By culturing a transformant containing NA, it is possible to supply a large amount of GDH which is present in a tetramer form at any pH without being affected by ionic strength and is stable to heat. Things.

[Sequence list]

[0061]

[0062]

[0064]

[0065]

[Brief description of the drawings]

FIG. 1 shows a restriction map of plasmid pGDA1.

FIG. 2 shows a restriction map of plasmid pGDA2.

FIG. 3. pGDA2F-40, an improved recombinant DNA
Fig. 2 shows the pH stability of GDH produced by culturing a transformant into which GDH has been incorporated. In the figure, --- indicates the results in the presence of 2M NaCl,
-Indicates the result in the absence of NaCl.

FIG. 4. pGDA2F-50, an improved recombinant DNA
Fig. 2 shows the pH stability of GDH produced by culturing a transformant into which GDH has been incorporated. In the figure,-indicates the results in the presence of 2M NaCl,-
●-shows the results in the absence of NaCl.

FIG. 5: pGDA2F-3, an improved recombinant DNA
G produced by culturing a transformant incorporating 0, pGDA2F-40 and pGDA2F-50.
It shows the temperature stability of DH. In the figure,-
■-: G obtained from Bacillus megaterium IWG3
DH,-□-indicates pGDA2F-30,-●-indicates pGDA2F-40, and −- indicates p
The case of GDA2F-50 is shown.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 9/04 C12R 1:19) C12R 1:19) 1:11) (C12N 15/09 ZNA C12N 15/00 ZNAA C12R 1 : 11) C12R 1:11) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C12N 1/21 C12N 9/04 BIOSIS (DIALOG) WPI (DIALOG) JICST file (JOIS)

Claims (3)

(57) [Claims] 1. Glutamate at position 96 from the N-terminus to lysine, glutamine at position 252 to leucine and 253 from position N of the DNA encoding Bacillus megaterium-derived glucose dehydrogenase represented by SEQ ID NO: 1 in the sequence listing.
A recombinant DNA capable of replicating in Escherichia coli, in which tyrosine at position 1 is replaced with glutamic acid and integrated into an Escherichia coli introduction vector. 2. Escherichia coli into which the recombinant DNA of claim 1 has been introduced. 3. A method for producing glucose dehydrogenase, comprising culturing the transformant according to claim 2 in a nutrient medium, producing glucose dehydrogenase in the culture, and collecting glucose dehydrogenase from the culture.