JP4195065B2 - Novel glucose dehydrogenase and method for producing the dehydrogenase - Google Patents

Description

The present invention relates to a novel glucose dehydrogenase and a method for producing the same, a DNA encoding the enzyme, a recombinant vector containing the DNA encoding the enzyme, a transformant transformed with the recombinant vector, and the enzyme A glucose sensor using an enzyme electrode containing the enzyme, a transformant or a microorganism, and a glucose assay kit.

Development of biosensors using enzymes that react specifically with specific substrates has been actively conducted regardless of the industrial field. Among them, in particular, a glucose sensor, which is one of biosensors, has been actively developed in the medical field mainly for a measurement method and a device using the method.
The glucose sensor has a history of about 40 years since the first report of a biosensor combining glucose oxidase and an oxygen electrode (Non-patent Document 1) was made in 1962 by Clark and Lyons.
Thus, the history since adopting glucose oxidase as an enzyme in a glucose sensor is long. This is because glucose oxidase has a high substrate specificity for glucose, is excellent in thermal stability, can be mass-produced for an enzyme, and is less expensive to produce than other enzymes.
The high substrate specificity leads to the advantage that an accurate measurement can be performed without causing an error in the measurement value because the enzyme does not react with sugars other than glucose.
In addition, the excellent thermal stability can prevent the problem that the enzyme is denatured by heat and the enzyme activity is deactivated, leading to the advantage that accurate measurement can be performed for a long period of time.
However, glucose oxidase has a high substrate specificity, excellent thermal stability, and can be produced at a low cost. However, glucose oxidase is affected by dissolved oxygen as described below and has a problem that measurement results are affected.
On the other hand, in addition to glucose oxidase, a glucose sensor using glucose dehydrogenase (hereinafter also referred to as “glucose dehydrogenase”) has been developed. Enzymes have also been discovered from microorganisms.
For example, glucose dehydrogenase derived from the genus Bacillus (EC1.1.1.17) and glucose dehydrogenase derived from the genus Cryptococcus (EC1.1.1.119) are known.
The former glucose dehydrogenase (EC1.1.1.17) is an enzyme that catalyzes the reaction of β-D-glucose + NAD (P) ⁺ → D-δ-gluconolactone + NAD (P) H + H ⁺ . Glucose dehydrogenase (EC 1.1.1.119) is an enzyme that catalyzes the reaction of D-glucose + NADP ⁺ → D-δ-gluconolactone + NADPH + H ⁺ , and the aforementioned microorganism-derived glucose dehydrogenase is already commercially available. ing.
These glucose dehydrogenases have the advantage that they are not affected by the dissolved oxygen of the measurement sample. This means that even when measuring in an environment where the oxygen partial pressure is low or when measuring a high-concentration sample that requires a large amount of oxygen, it is possible to measure accurately without affecting the measurement results. It leads to the advantage of being able to.
However, glucose dehydrogenase is not affected by dissolved oxygen, but has a problem that its thermal stability is poor and its substrate specificity is inferior to that of glucose oxidase.
Therefore, it has been desired to provide an enzyme that compensates for both drawbacks of glucose oxidase and glucose dehydrogenase.
In addition, this inventor has reported the research result about glucose dehydrogenase in the nonpatent literatures 2-4 using the sample extract | collected from the soil near a hot spring.
However, identification of strains having the ability to produce the enzyme has not been performed at the research stage.
L. c. Clark, J. et al. and Lyonas, C.I. “Electrode systems for continuous monitoring in cardiovascular surgery.” Ann, n. y. Acad. Sci. 105: 20-45 Sode, K .; , Tsugawa, W .; Yamazaki, T .; Watanabe, M .; Ogasawara, N .; , And Tanaka, M .; (1996) Enzyme Microb. Technol. 19, 82-85. Yamazaki, T .; , Tsugawa, W .; , AndSode, K .; (1999) Appli Biochemi and Biotec. 77-79 / 0325 Yamazaki, T .; , Tsugawa, W .; , AndSode, K .; (1999) Biotec Lett. 21, 199-202

The present invention has the properties to compensate for the disadvantages of both glucose oxidase and glucose dehydrogenase, which are conventionally known, has high substrate specificity, excellent thermal stability, can be produced at low cost, and has the effect of dissolved oxygen in the measurement sample. It is an object to provide an enzyme that is not affected.
Another object of the present invention is to provide a method for producing the enzyme, a protein utilizing the characteristics of the enzyme, and a novel microorganism that produces the enzyme.
Another object of the present invention is to provide a DNA encoding the enzyme, a recombinant vector containing the DNA encoding the enzyme, and a transformant transformed with the recombinant vector.
Moreover, this invention makes it a subject to provide the glucose sensor using the enzyme electrode containing the said enzyme, a transformant, or the said microorganisms, and the glucose assay kit containing the said enzyme.
The present inventor succeeded in isolating Burkholderia cepacia, which produces an enzyme suitable for the above-mentioned purpose, from soil near a hot spring, and reached the present invention.

That is, the present invention is as follows.
(1) Glucose belonging to the genus Burkholderia, cultivating a microorganism having an ability to produce glucose dehydrogenase in a medium, and collecting glucose dehydrogenase from the medium or / and the microorganism cells A method for producing a dehydrogenase.
(2) The method for producing glucose dehydrogenase according to (1), wherein the microorganism is Burkholderia cepacia.
(3) The method for producing glucose dehydrogenase according to (1) or (2), wherein the glucose dehydrogenase has the following properties.
[1] Action:
Catalyses the dehydrogenation of glucose.
[2] It consists of subunits having a molecular weight of about 60 kDa and a molecular weight of about 43 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions.
[3] Gel filtration chromatography using TSK gel G3000SW (manufactured by Tosoh Corporation) shows a molecular weight of about 380 kDa.
[4] Optimum reaction temperature:
Around 45 ° C. (Tris-HCl buffer, pH 8.0).
(4) The method for producing glucose dehydrogenase according to (3), wherein the subunit having a molecular weight of about 43 kDa is an electron transfer protein.
(5) The method for producing glucose dehydrogenase according to (4), wherein the electron transfer protein is cytochrome C.
(6) A glucose dehydrogenase that can be produced by a microorganism belonging to the genus Burkholderia.
(7) The glucose dehydrogenase according to (6), wherein the microorganism is Burkholderia cepacia.
(8) The glucose dehydrogenase according to (6) or (7), wherein the glucose dehydrogenase has the following properties.
[1] Action:
Catalyses the dehydrogenation of glucose.
[2] It consists of subunits having a molecular weight of about 60 kDa and a molecular weight of about 43 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions.
[3] Gel filtration chromatography using TSK gel G3000SW (manufactured by Tosoh Corporation) shows a molecular weight of about 380 kDa.
[4] Optimum reaction temperature:
Around 45 ° C. (Tris-HCl buffer, pH 8.0).
(9) The glucose dehydrogenase according to (8), wherein the subunit having a molecular weight of about 43 kDa is an electron transfer protein.
(10) The glucose dehydrogenase according to (9), wherein the electron transfer protein is cytochrome C.
(11) The glucose dehydrogenase according to any one of (8) to (10), wherein the subunit having a molecular weight of about 60 kDa comprises the amino acid sequence of amino acid numbers 2 to 12 of SEQ ID NO: 3.
(12) The glucose dehydrogenase according to any one of claims 8 to 11, wherein the N-terminus of the 43 kDa subunit has the amino acid sequence of SEQ ID NO: 5.
(13) The glucose dehydrogenase of (11), wherein the subunit having a molecular weight of about 60 kDa is a protein shown in the following (A) or (B):
(A) A protein having the amino acid sequence of SEQ ID NO: 3.
(B) A protein having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 3 and having glucose dehydrogenase activity.
(14) The glucose dehydrogenase according to (6), which has activity peaks at around 45 ° C. and around 75 ° C., respectively.
(15) A cytochrome C which is a subunit of glucose dehydrogenase according to (10) and has the amino acid sequence of SEQ ID NO: 5.
(16) A DNA encoding a part of cytochrome C of (15) and having the base sequence set forth in SEQ ID NO: 8.
(17) A DNA encoding a part of cytochrome C of (15) and having a base sequence of base numbers 2386 to 2467 of the base sequence set forth in SEQ ID NO: 1.
(18) A DNA encoding the cytochrome C signal peptide of (15) and comprising the nucleotide sequence of nucleotide numbers 2386 to 2451 of the nucleotide sequence of SEQ ID NO: 1.
(19) A peptide peptide of cytochrome C, which has the amino acid sequence of amino acid numbers 1 to 22 among the amino acid sequences of SEQ ID NO: 4.
(20) A protein having the following properties.
[1] The glucose dehydrogenase of (6) can be constituted as a subunit.
[2] Has glucose dehydrogenase activity.
[3] SDS-polyacrylamide gel electrophoresis under reducing conditions shows a molecular weight of about 60 kDa.
[4] Optimum reaction temperature:
Around 75 ° C. (Tris-HCl buffer, pH 8.0).
(21) The protein according to (20), which comprises the amino acid sequence of amino acid numbers 2 to 12 in SEQ ID NO: 3.
(22) The glucose dehydrogenase according to (21), wherein the protein is a protein shown in the following (A) or (B).
(A) A protein having the amino acid sequence of SEQ ID NO: 3.
(B) A protein having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 3 and having glucose dehydrogenase activity.
(23) The protein shown in the following (A) or (B).
(A) A protein having the amino acid sequence of SEQ ID NO: 3.
(B) A protein having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 3 and having glucose dehydrogenase activity.
(24) DNA encoding the protein shown in the following (A) or (B).
(A) A protein having the amino acid sequence of SEQ ID NO: 3.
(B) A protein having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 3 and having glucose dehydrogenase activity.
(25) The DNA of (24), which is the DNA shown in the following (a) or (b).
(A) DNA containing a base sequence consisting of base numbers 764 to 2380 among the base sequence of SEQ ID NO: 1.
(B) a protein that hybridizes under stringent conditions with a nucleotide sequence consisting of nucleotide numbers 764 to 2380 or a probe that can be prepared from this nucleotide sequence of the nucleotide sequence of SEQ ID NO: 1 and has glucose dehydrogenase activity DNA encoding
(26) A recombinant vector containing the DNA of (24) or (25).
(27) The recombinant vector according to (26) comprising a base sequence encoding the signal peptide of (18) and the β-subunit.
(28) A transformant transformed with the DNA of (24) or (25) or the recombinant vector of (26) or (27).
(29) A method for producing glucose dehydrogenase, comprising culturing the transformant of (28), producing glucose dehydrogenase as an expression product of the DNA, and collecting the glucose dehydrogenase. (30) Burkholderia cepacia KS1 strain (FERM BP-7306).
(31) An enzyme electrode comprising the glucose dehydrogenase of any one of (6) to (14), the protein of any of (20) to (23), the transformant of (27), or the strain of (30) Glucose sensor using
(32) A glucose assay kit comprising the glucose dehydrogenase of any one of (6) to (14) or the protein of any one of (20) to (23).
(33) A protein having the amino acid sequence of SEQ ID NO: 2.
(34) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2.
(35) The DNA of (34) comprising the nucleotide sequence consisting of nucleotide numbers 258 to 761 among the nucleotide sequence of SEQ ID NO: 1.
(36) A DNA comprising the DNA of (34) or (35) and the DNA of (24) or (25) in this order.
(37) The DNA of (36) comprising a base sequence consisting of base numbers 258 to 2380 among the base sequence of SEQ ID NO: 1.
(38) A recombinant vector containing the DNA of (36) or (37).
(39) The recombinant vector according to (38), comprising a base peptide encoding the signal peptide of (18) and the β-subunit.
(40) A transformant transformed with the DNA of (36) or (37) or the recombinant vector of (38) or (39).
(41) A method for producing glucose dehydrogenase, comprising culturing the transformant of (40), producing glucose dehydrogenase as an expression substance of the DNA of (36) or (37), and collecting this.

Hereinafter, the present invention will be described in detail.
<1> Novel strain producing the glucose dehydrogenase of the present invention The enzyme of the present invention (hereinafter sometimes referred to as “the present enzyme” or “GDH”) can be produced by bacteria belonging to the genus Burkholderia. The Burkholderia bacterium used in the present invention is not particularly limited as long as it is a bacterium belonging to the genus Burkholderia having the ability to produce this enzyme, but the Burghorderia cepacia, in particular, the Burkholderia cepacia KS1 strain is preferable. This strain is a novel strain isolated from the soil near the hot spring by the present inventors as shown in the examples described later, and was identified as Burkholderia cepacia from its mycological properties. Conventionally, it is not known that microorganisms belonging to the genus Burkholderia can produce glucose dehydrogenase. This strain was named KS1 strain. This strain was established on September 25, 2000, at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan 305-8586). Deposited as BP-7306.
In addition, the present inventors have established a strain other than the Burkholderia cepacia KS1 strain in the Institute for Fermentation (Institute for Fermentation, Osaka, IFO) or the RIKEN Microbial System Storage Facility (Japan Collection of Microorganisms, JCM). Several strains of the same deposit that was deposited with Burkholderia cepacia were collected and measured for glucose dehydrogenase activity. As a result, it was confirmed that all strains were active.

<2> Glucose dehydrogenase of the present invention The glucose dehydrogenase of the present invention is a bacterium belonging to the genus Burkholderia that has the ability to produce glucose dehydrogenase, such as the Burkholderia cepacia KS1 strain, which is usually used for culturing microorganisms. It is collected in a known manner since it is produced and accumulated in the culture product or cells by culturing in a nutrient medium, preferably a medium to which glucose or a substance containing glucose is added in order to enhance enzyme production ability. can do. Further, the production method of the present enzyme will be specifically described by taking the Burkholderia cepacia KS1 strain as an example. First, the Burkholderia cepacia KS1 strain is cultured in a suitable nutrient medium, for example, a medium containing a suitable carbon source, nitrogen source, inorganic salts, glucose or a substance containing these, and the enzyme is cultured in the culture product. Accumulate production throughout the body.
As the carbon source, any substance that can be assimilated can be used, and examples thereof include D-glucose, L-arabisose, D-xylose, D-mannose, starch, and various peptones. As a nitrogen source, organic and inorganic nitrogen compounds such as yeast extract, malt extract, various peptones, various meat extracts, corn steep liquor, amino acid solution, ammonium salt, or substances containing these can be used. As the inorganic salt, various salts such as phosphate, magnesium, potassium, sodium, calcium and the like are used. If necessary, various inorganic and organic substances necessary for the growth of bacteria or enzyme production, for example, antifoaming agents such as silicone oil, sesame oil, various surfactants, and vitamins can be added to the medium.
The culture form may be liquid culture or solid culture, but liquid culture is usually preferable.
The enzyme of the present invention can be obtained from the culture medium or / and the cells of the culture thus obtained. The enzyme in the microbial cell is obtained as a microbial cell extract by crushing or dissolving the microbial cell.
Glucose dehydrogenase in the culture product or bacterial cell extract can be purified by appropriately combining chromatography using ion exchangers, gel filtration carriers, hydrophobic (hydrophobic) carriers, and the like.
The activity of this enzyme can be measured by the same method as the activity measurement of a known glucose dehydrogenase. Specifically, for example, it can be measured by the method shown in the examples described later.

Next, the physicochemical properties of the novel glucose dehydrogenase of the present invention will be shown.
(1) Action:
Catalyses the dehydrogenation of glucose.
(2) It consists of subunits having a molecular weight of about 60 kDa and a molecular weight of about 43 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions.
(3) Gel filtration chromatography using TSK gel G3000SW (manufactured by Tosoh Corporation) shows a molecular weight of about 380 kDa.
(4) Optimal reaction temperature:
Around 45 ° C. (Tris-HCl buffer, pH 8.0).
The glucose dehydrogenase has an activity peak near 45 ° C. under the above conditions, but also has an activity peak near 75 ° C. (see FIG. 3A). Thus, GDH which shows an active peak in two temperature ranges is not known.
The molecular weight and optimum temperature can be measured by the methods described in the examples below.
The glucose dehydrogenase of the present invention is composed of two separate polypeptides, an α subunit having a molecular weight of about 60 kDa and a β subunit having a molecular weight of about 43 kDa (hereinafter, this glucose dehydrogenase is referred to as “multimeric enzyme”). However, the present inventors have further studied in detail about two subunits.
The β subunit was found to be cytochrome C (shown in Examples below).
A protein containing only the α subunit exhibits the following physicochemical properties.
(1) The multimeric enzyme can be constituted as a subunit.
(2) It has glucose dehydrogenase activity.
(3) In SDS-polyacrylamide gel electrophoresis under reducing conditions, the molecular weight is 60 kDa.
(4) Optimal reaction temperature:
Around 75 ° C. (Tris-HCl buffer, pH 8.0).
The optimum temperature can be measured by the method described in Examples below.
In addition, since this protein itself has an enzyme activity, this protein will be used in other words as a peptide enzyme or an enzyme as appropriate according to the content of the explanation.

A specific embodiment of the peptide enzyme of the present invention includes a protein having the amino acid sequence of SEQ ID NO: 3. The peptide enzyme may be a protein having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 3 as long as it has GDH activity. Although SEQ ID NO: 3 shows an amino acid sequence that can be encoded by the base sequence of SEQ ID NO: 1, the N-terminal methionine residue may be missing after translation.
In addition, a specific embodiment of the multimeric enzyme of the present invention includes a multimer containing a protein in which the α subunit has the amino acid sequence of SEQ ID NO: 3. The multimeric enzyme is a protein having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 3 as long as it has GHD activity. It may be a multimer containing.
In the present invention, “one or more” means 1 to 10, preferably 1 to 5, and particularly preferably 1 to 3.
The present inventors have confirmed the presence of a γ subunit in addition to the α subunit or the β subunit.
In the examples described later, the γ subunit was removed from the culture supernatant or bacterial cell extract at the stage of purifying the enzyme of the present invention, and the γ subunit was not confirmed in the purified enzyme. However, as shown in the Examples, when the γ subunit was expressed together with the α subunit, a higher enzyme activity was obtained than when only the α subunit was expressed. From this, it was suggested that the γ subunit is a protein that has some relationship when the α subunit is produced in the microorganism. In either case, if the specific activity of the α subunit (enzyme activity per protein) is the same, the enzyme activity reflects the amount of enzyme, so low enzyme activity means that the amount of α subunit as an enzyme is low. It shows that there are few. On the other hand, the generated α subunit is protected by the γ subunit, or the α subunit as a protein is fully expressed, but since there is no γ subunit, it is a three-dimensional structure that can exhibit enzyme activity. The enzyme activity may have been lowered as a result without taking the structure. In any case, high enzyme activity can be obtained by expressing the γ subunit together with the α subunit.

<3> DNA of the present invention
The DNA of the present invention can be obtained from a microorganism containing the DNA of the present invention, such as Burkholderia cepacia. In the process of completing the present invention, the DNA of the present invention was isolated from burkholderia cepacia chromosomal DNA, but the present invention revealed the nucleotide sequence and the amino acid sequence encoded by the nucleotide sequence. Therefore, it can also be obtained by chemical synthesis based on these sequences. Also. It can also be obtained from chromosomal DNA such as Burkholderia cepacia by hybridization or PCR using an oligonucleotide prepared based on the sequence as a probe or primer.
The DNA of the present invention encodes a protein having the amino acid sequence shown in SEQ ID NO: 3, as well as amino acids in which one or more amino acid residues are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 3. It may encode a protein having a sequence and having GDH activity.
Specific examples of the DNA of the present invention include DNA containing a base sequence consisting of base numbers 764 to 2380 in the base sequence of SEQ ID NO: 1. Of the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence consisting of nucleotide numbers 764 to 2380 encodes the α subunit of GDH having the amino acid sequence of SEQ ID NO: 3.
In addition, the DNA of the present invention hybridizes under stringent conditions with a nucleotide sequence consisting of nucleotide numbers 764 to 2380 or a probe that can be prepared from this nucleotide sequence of the nucleotide sequence of SEQ ID NO: 1, and has GDH activity. It may be DNA encoding a protein.
In addition, it is estimated that the base sequence which consists of base numbers 258-761 among the base sequences of sequence number 1 codes (gamma) subunit. Its amino acid sequence is shown in SEQ ID NO: 2. By including the structural gene of the γ subunit in the upstream region of the α subunit, when the α subunit is produced by the microorganism, the γ subunit is first expressed and present as a protein, so that the α It is thought that subunits can be produced. Therefore, the DNA of the present invention may contain DNA encoding the amino acid sequence of SEQ ID NO: 2 in addition to the DNA.
A DNA encoding a protein substantially identical to the protein having the amino acid sequence shown in SEQ ID NO: 3 as described above can be obtained by a method such as site-specific mutagenesis or mutation treatment. The GDH activity of the protein encoded by the DNA into which the mutation has been introduced can be measured, for example, as follows.
Glucose as a substrate was added to 10 mM potassium phosphate buffer (pH 7.0) containing 594 μM methylphenazine methosulfate (mPMS) and 5.94 μM 2,6-dichlorophenolindophenol (DCIP) as a substrate. Incubate at 37 ° C. The change in absorbance of DCIP at 600 nm is traced using a spectrophotometer, and the enzyme reaction rate is determined by the rate of decrease in the absorbance.
Furthermore, among the base sequence of SEQ ID NO: 1, it is presumed that the base sequence after base number 2386 encodes the β-subunit. Moreover, it is estimated that the base sequence of base number 2386-2451 codes the signal peptide of (beta) -subunit. The deduced amino acid sequence of the signal peptide is the amino acid sequence of amino acid numbers 1 to 22 of SEQ ID NO: 4. The signal peptide is a peptide required when a protein synthesized in the ribosome is secreted through the membrane, and it has been found that it consists of 15 to 30 hydrophobic amino acid residues. Therefore, since the amount of protein contained in the culture supernatant increases due to the presence of the signal peptide, it is a peptide that acts effectively in the protein production method.

The method for obtaining the DNA of the present invention is exemplified below.
Chromosomal DNA is isolated and purified from microorganisms such as Burkholderia cepacia, and then the chromosomal DNA is cleaved using sonication, restriction enzyme treatment, etc., and linear expression vector is closed by DNA ligase. To construct a recombinant vector. After the obtained recombinant vector is transferred to a host microorganism capable of autonomous replication of the vector, the transformant is screened using the expression of the marker of the vector and the enzyme activity as an index, and a group containing a gene encoding GDH. A microorganism carrying the replacement vector is obtained. The recombinant vector possessed by the obtained microorganism is expected to contain at least a base sequence encoding the α subunit. In addition, if the cloned fragment has a sufficient size, it is highly likely that a nucleotide sequence encoding the γ subunit is also included.
Subsequently, the microorganism holding the recombinant vector can be cultured, the recombinant vector can be isolated and purified from the cells of the cultured microorganism, and the gene encoding GDH can be collected from the expression vector. For example, chromosomal DNA that is a gene donor is specifically collected as follows.
A culture solution obtained by stirring and culturing the gene-donating microorganism for 1 to 3 days, for example, is collected by centrifugation, and then lysed to prepare a GDH gene-containing lysate. As a method for lysis, for example, treatment is performed with a lytic enzyme such as lysozyme, and a protease, other enzyme, or a surfactant such as sodium lauryl sulfate (SDS) is used in combination as necessary. Further, it may be combined with a physical crushing method such as freeze-thawing or French press treatment.
In order to separate and purify DNA from the lysate obtained as described above, a conventional method is used, for example, by appropriately combining methods such as deproteinization treatment by phenol treatment or protease treatment, ribonuclease treatment, and alcohol precipitation treatment. be able to.
A method of cleaving DNA separated and purified from microorganisms can be performed by, for example, ultrasonic treatment, restriction enzyme treatment, or the like. Type II restriction enzymes that act on specific nucleotide sequences are suitable. The restriction enzyme may be a restriction enzyme that generates an end compatible with the cut end of the vector, or an arbitrary restriction enzyme may be used to make the cut end blunt and ligated to the vector.
As a vector for cloning, a vector constructed for gene recombination from a phage or plasmid that can autonomously grow in a host microorganism is suitable. Examples of the phage include Lambda gt10 and Lambda gt11 when Escherichia coli is used as a host microorganism. Examples of plasmids include pBR322, pUC18, pUC118, pUC19, pUC119, pTrc99A, pBluescript, or SuperCosI which is a cosmid when Escherichia coli is used as a host microorganism.
At the time of cloning, a vector fragment can be obtained by cleaving the above-mentioned vector with the restriction enzyme used for cleaving the microbial DNA that is the gene donor encoding GDH described above. It is not necessary to use the same restriction enzyme as that used in the above. The method for binding the microbial DNA fragment and the vector DNA fragment may be a method using a known DNA ligase. For example, after ligation between the sticky end of the microbial DNA fragment and the sticky end of the vector fragment, an appropriate DNA ligase may be used. A recombinant vector of a microbial DNA fragment and a vector DNA fragment is prepared by use. If necessary, after ligation, it can be transferred to a host microorganism and a recombinant vector can be prepared using in vivo DNA ligase.
The host microorganism used for cloning is not particularly limited as long as the recombinant vector is stable, can autonomously grow, and can express a foreign gene. In general, Escherichia coli DH5α, XL-1 Blue MR, or the like can be used.
As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method or an electroporation method using calcium treatment can be used.
It can be confirmed that the cloned fragment obtained by the above method encodes GDH by decoding the base sequence of the fragment by a conventional method.
If the recombinant vector is recovered from the transformant obtained as described above, the DNA of the present invention can be obtained.
GDH is produced by culturing a transformant carrying the DNA of the present invention or a recombinant vector containing the DNA, producing GDH as an expression product of the DNA, and collecting this from the cell or culture medium. can do. At this time, the DNA of the present invention may be a DNA encoding an α subunit, but the expression efficiency can be increased by further expressing the γ subunit together with the α subunit.
Examples of microorganisms that produce GDH include enterobacteriaceae such as Escherichia coli, Gram-negative bacteria such as Pseudomonas and Gluconobacter, Gram-positive bacteria including Bacillus bacteria such as Bacillus subtilis, and Saccharomyces cerevisiae. Yeasts such as Aspergillus niger and the like, but are not limited thereto, and any host microorganism suitable for heterologous protein production can be used.
Transfer from a recombinant vector carrying the GDH gene once selected to a recombinant vector that can be replicated in a microorganism is performed by collecting DNA, which is a GDH gene, from the recombinant vector carrying the GDH gene by a restriction enzyme or PCR method. It can be easily carried out by linking with other vector fragments. Moreover, the transformation of microorganisms by these vectors is carried out by, for example, a competent cell method by calcium treatment in Escherichia bacteria, a protoplast method in Bacillus bacteria, a KU method or KUR method in yeast, and a micromanipulation method in filamentous fungi. It can be carried out. Also, electroporation can be widely used.
The selection as to whether or not the target recombinant vector is transferred to the host microorganism may be made by searching for a microorganism that simultaneously expresses the drug resistance marker of the vector holding the target DNA and GDH activity. For example, a microorganism that grows in a selective medium based on a drug resistance marker and generates GDH may be selected.
For the culture form of the transformant, the culture conditions may be selected in consideration of the nutritional physiological properties of the host. Industrially, aeration and agitation culture is advantageous.
As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, lactose, molasses, pyruvic acid and the like are used. The nitrogen source may be any available nitrogen compound. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.
The culture temperature can be appropriately changed within the range in which the bacteria grow and GDH is produced, but is preferably about 20 to 42 ° C. Although the culture time varies slightly depending on the conditions, the culture may be completed at an appropriate time in consideration of the time when GDH reaches the maximum yield, and is usually about 12 to 72 hours. The pH of the medium can be appropriately changed within the range in which the bacteria grow and produce GDH, but is preferably in the range of about pH 6.0 to 9.0.
Although the culture solution containing the cells producing GDH in the culture can be collected and used as it is, generally, when GDH is present in the culture solution according to a conventional method, filtration, centrifugation, etc. It is used after separating the GDH-containing solution and the microbial cells. When GDH is present in the microbial cells, the microbial cells are collected from the obtained culture by means of filtration or centrifugation, and then the microbial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme. Further, if necessary, a chelating agent such as EDTA and a surfactant are added to solubilize GDH and separated and collected as an aqueous solution.
The GDH-containing solution obtained as described above is precipitated by, for example, vacuum concentration, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. You just have to let them know. Heat treatment and isoelectric point treatment are also effective purification means. Then, it can be purified by appropriately combining gel filtration with an adsorbent or gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography. By performing the above, purified GDH can be obtained.
A purified enzyme preparation can be obtained by separation and purification by column chromatography. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE), but may contain a γ subunit.
The purified enzyme obtained as described above can be pulverized and distributed, for example, by freeze drying, vacuum drying, spray drying, or the like.
In addition, the amino acid sequence of the β subunit can be determined in the same manner as the amino acid sequence of the α subunit shown in Examples below, and DNA encoding the β subunit can be isolated. In addition, β subunit can be produced using the obtained DNA. Furthermore, a multimeric enzyme can be produced using a DNA encoding an α subunit and a DNA encoding a β subunit.

<4> Glucose sensor of the present invention The glucose sensor of the present invention includes the enzyme of the present invention (the multimeric enzyme or peptide enzyme, or the multimeric enzyme or peptide enzyme containing a γ subunit), or the transformant of the present invention. Alternatively, the microorganism of the present invention (Burkholderia cepacia KS1 strain) is used as an enzyme electrode. As the electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and the enzyme of the present invention is immobilized on this electrode. Immobilization methods include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc., or ferrocene or a derivative thereof. It may be fixed in a polymer or adsorbed and fixed on an electrode together with a representative electron mediator, or a combination thereof. Typically, after the glucose dehydrogenase of the present invention is immobilized on a carbon electrode using glutaraldehyde, glutaraldehyde is blocked by treatment with a reagent having an amine group.
The concentration of glucose can be measured as follows. Put the buffer in a thermostatic cell and add a mediator to maintain a constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate, or the like can be used. An electrode on which the enzyme of the present invention is immobilized is used as a working electrode, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCl electrode) are used. After a constant voltage is applied to the carbon electrode and the current becomes steady, a sample containing glucose is added and the increase in current is measured. The concentration of glucose in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.

<5> Glucose assay kit of the present invention The saccharide assay kit of the present invention comprises the enzyme of the present invention (the multimeric enzyme or peptide enzyme, or the multimeric enzyme or peptide enzyme containing a γ subunit). And The glucose assay kit of the present invention contains the enzyme of the present invention in an amount sufficient for at least one assay. Typically, the kit contains, in addition to the enzyme of the present invention, buffers necessary for the assay, mediators, standard solutions such as glucose for creating a calibration curve, and directions for use. The enzyme according to the invention can be provided in various forms, for example as a lyophilized reagent or as a solution in a suitable storage solution.

Hereinafter, the present invention will be described more specifically with reference to examples.
Example 1 Acquisition of Bacteria Having Ability to Produce Glucose Dehydrogenase [Screening]
The microorganisms of the present invention were obtained by obtaining soils near various hot springs in Japan, and selecting bacteria exhibiting glucose dehydrogenase activity from bacteria using glucose as a nutrient source.
The results of examining the morphological properties, growth characteristics, and physiological characteristics of this strain are shown below.
Mycological properties;
Gram staining Negative cell shape Motility with gonococcal polar flagella Number of positive fragments> 5
Optimal growth temperature 45 ° C
Oxidase Negative catalase Production of positive acetoin Production of negative H ₂ S Production of negative indole Acid negative arginine dihydrolase Negative urease Negative glucose Negative β-glucosidase Negative protease Negative β-galactosidase Positive lysine carboxylase Negative ornithine carboxylase Negative nitrate reduction Positive [Utilization]
Glycerol positive erythritol negative D-arabinose negative L-arabinose positive ribose positive D-xylose positive L-xylose negative adonitol positive β-methyl-xyloside negative galactose positive D-glucose positive D-fructose positive D-mannose positive L-sorbose negative rhamnose negative Dulcitol positive inositol positive mannitol positive sorbitol positive α-methyl-D-mannosid negative α-methyl-D-glucoside negative N-acetyl-glucosamine positive amygdalin (Amygdaline) negative arbutin negative esculin negative salobiose negative maltose negative lactose negative lactose negative lactose negative Negative trehalose positive inulin negative melethitose negative D-raffinose negative Don (Amidon) Negative glycogen Negative xylitol Positive β-gentiobiose Negative D-Thuranose Negative D-Lixose Negative D-Tagatose Negative D-Fucose Negative L-Fucose Negative D-Arabitol Positive L-Arabitol Positive Gluconic acid Positive 2-Ketogluconic acid Positive 5-ketogluconic acid negative capric acid positive adivic acid positive malic acid positive citric acid positive phenylacetate positive [oxidative]
Glycerol negative erythritol negative D-arabinose negative L-arabinose positive ribose positive D-xylose positive L-xylose negative adonitol positive β-methyl-xyloside negative galactose positive D-glucose positive D-fructose positive D-mannose positive L-sorbose negative rhamnose negative Dulcitol positive inositol positive mannitol positive sorbitol positive α-methyl-D-mannosid negative α-methyl-D-glucoside negative N-acetyl-glucosamine negative amygdalin (Amygdaline) negative arbutin negative esculin positive salicin negative cellobiose positive maltose positive lactose positive lactose Negative trehalose positive inulin negative melethitose negative D-raffinose negative Don (Amidon) Negative glycogen Negative xylitol Negative β-gentiobiose Positive D-Thuranose Negative D-Lixose Negative D-Tagatose Negative D-Fucose Positive L-Fucose Negative D-arabitol Positive L-arabitol Positive gluconic acid Negative 2-Ketogluconic acid Negative 5-Ketogluconic acid Negative When the taxonomic position of the KS1 strain having the above mycological properties is examined with reference to Bergey's Manual of Detergent Bacteriology , A strain belonging to the genus Burkholderia and being Burkholderia cepacia.
The genus Burkholderia was conventionally classified as the genus Pseudomonas, but is now divided into the genus Burghorderia (Yabuchi, E., Kosako, Y., Oyaizu, H., Yano, I., Hotta). H., Hashimoto, Y., Ezaki, T. and Arakawa, M., Microbiol. Immunol. Vol 36 (12), 1251-1275, 1992; International Journal of Systemic Bacteriology-3, Ap.
In addition, the present inventors have deposited strains other than the Burkholderia cepacia KS1 strain to the Institute for Fermentation (Institute for Fermentation, Osaka) or the Institute of Physical and Chemical Research Microbiology System (Japan Collection of Microorganisms, JCM). We obtained several strains of Burkholderia cepacia and measured glucose dehydrogenase activity, and confirmed that it had the same activity. The glucose dehydrogenase activity was measured by the method described in Example 2 described later. Table 1 shows the relative activities when the enzyme activity of the water-soluble fraction of the KS1 strain is defined as 100.

Example 2 Extraction of Glucose Dehydrogenase <1> Cultivation of Bacteria The usual aerobic culture conditions are used for the culture conditions of this bacterium. The culture was performed at 34 ° C. for 8 hours in 7 L of a medium containing the following components in 1 L of a culture solution.
Polypeptone 10g
Yeast extract 1g
NaCl 5g
KH ₂ PO ₄ 2g
Glucose 5g
Einol (ABLE Co. Tokyo Japan) 0.14g
Total, distilled water 1L
pH adjustment 7.2
7 L of the main culture was centrifuged at 9,000 × g for 10 minutes at 4 ° C. to obtain about 60 g of cells.
<2> Preparation of roughly purified fraction 60 g of cells were dispersed in 10 mM potassium phosphate buffer (pH 6.0), and a pressure difference of 1,500 Kg / cm ₂ was applied with a French press (Otake, Tokyo, Japan). The cell membrane was destroyed. The cell extract was centrifuged at 8,000 × g for 10 minutes to remove cell solids. Further, the supernatant was ultracentrifuged at 69,800 × g for 90 minutes at 4 ° C. to obtain about 8 g of a membrane fraction as a precipitate.
<3> The purified membrane fraction of the enzyme was redispersed with 10 mM potassium phosphate buffer (pH 6.0) so that the final concentration of Triton-X100 was 1%. And it stirred slowly at 4 degreeC overnight. After ultracentrifugation (4 ° C., 69,800 g, 90 minutes), the solubilized membrane fraction was recentrifuged at 15000 × g for 15 minutes at 4 ° C. to obtain a supernatant.
A 10 mM potassium phosphate buffer (pH 8.0) containing the same amount of 0.2% Triton-X100 was added to the solubilized membrane fraction. After dialysis, the solution was supplied to a DEAE-TOYOPEARL column (22 mm ID × 20 cm Tosoh Tokyo Japan) which was equalized with 10 mM potassium phosphate buffer (pH 8.0) containing 0.2% Triton-X100. The protein was eluted with a linear gradient such that the concentration of NaCl in 10 mM potassium phosphate buffer (pH 8.0) was 0-0.15M. The flow rate was 5 ml / min. GDH was eluted at a NaCl concentration of about 75 mM. Fractions having GDH activity were collected and dialyzed overnight against 10 mM potassium phosphate buffer (pH 8.0 4 ° C.) containing 0.2% Triton-X100.
Furthermore, the dialyzed enzyme solution was passed through a DEAE-5PW column (8.0 mm ID × 7.5 cm Tosoh, Tokyo, Japan). The column was previously equilibrated with 10 mM potassium phosphate buffer (pH 6.0) containing 0.2% Triton-X100. The protein was eluted with a linear gradient such that the concentration of NaCl in 10 mM potassium phosphate buffer (pH 8.0) was 0-100 mM. The flow rate was 1 ml / min. The fraction with GDH activity eluted at a NaCl concentration of about 20 mM. Fractions having GDH activity were collected and desalted overnight with 10 mM potassium phosphate buffer (pH 8.0) containing 0.2% Triton-X100 to obtain the purified enzyme.
In addition, the activity measurement of GDH was performed according to the following method through the present Example and the following Examples.
As the electron acceptor, 2,6-dichlorophenolindophenol (DCIP) and phenandimethosulphate (PMS) were used. The reaction was carried out at a predetermined temperature in a polyethylene tube. 5 μl of enzyme solution was added to 20 μl of 25 mM Tris HCl buffer pH 8.0 containing 0.75 mM PMS and 0.75 mM DCIP. This mixture was allowed to stand for 1 minute in advance. The reaction was initiated by the addition of 1 μl of 2M glucose (final concentration: 77 mM) and allowed to incubate for 2 minutes. The sample was then cooled by adding 100 μl ice cold distilled water or 120 μl 7.5M urea. The reduction reaction of the electron acceptor based on the dehydrogenation of glucose was traced using a cell for ultra-trace measurement (100 μl) and a spectrophotometer (UV160, Shimadzu Corporation, Kyoto, Japan) that can be measured using this cell. That is, the discoloration due to DCIP reduction was measured over time at 600 nm, which is the absorption wavelength of DCIP. The molar extinction coefficient of DCIP (22.23 mM × cm ⁻¹ ) was used. One unit (U) of enzyme was defined as the amount that oxidizes 1 μM glucose per minute under standard assay conditions. Protein concentration was measured by the Raleigh method.

Example 3
The purified enzyme was subjected to Native PAGE electrophoresis. The electrophoresis conditions were performed on an 8-25% polyacrylamide gradient gel using a Tris-Aline buffer system containing 1% Triton-X100. The gel was stained with silver nitrate. As protein markers, thyroglobulin: 669 kDa, ferritin: 440 kDa, catalase: 232 kDa, aldolase: 158 kDa, bovine serum albumin (buvin Serum Albumin): 67 kDa Chymotrypsinogen A: 25 kDa protein was used.
In addition, activity staining was performed on the Native PAGE gel. This gel was incubated for 30 minutes in the following solution. At the active site of GDH, nitroblue tetrazolium was reduced, formazan was produced, and the color developed dark purple.
200 mM glucose 0.1 mM nitroblue tetrazolium 0.3 mM phenazine methosulfate 20 mM Tris-HCl buffer (pH 8.0)
From the result of silver staining of Native PAGE, it was estimated that this enzyme is a single enzyme and its molecular weight is about 400 kDa. In addition, when the gel was activity-stained, activity was observed at a site having the same mobility as that of silver staining (see Fig. 1. In the figure, lane 1 was silver-stained for the molecular weight standard marker protein, and lane 2 was the enzyme of this enzyme. Silver staining, lane 4 shows activity staining of the enzyme). When the enzyme was heat-treated at 70 ° C. for 30 minutes, the remaining activity remained, and it was separated into proteins having an activity near the molecular weight of 85 kDa (see FIG. 1. In the figure, lane 3 was heat-treated at 70 ° C. for 30 min. In addition, lane 5 shows the active staining of the enzyme that has been heat-treated at 70 ° C. for 30 minutes). This suggests that this enzyme consists of subunits.

Example 4
The purified enzyme solution was subjected to electrophoresis by SDS-PAGE. SDS-PAGE was performed in a gradient gel of 8-25% polyacrylamide using Tris-Tricine buffer. The gel protein was stained with silver nitrate. Separation and development were performed automatically by the Phas System (Pharmacia). The molecular mass was measured by the relative mobility of the standard protein. This enzyme was separated into proteins of about 60 kDa and 43 kDa by SDS-PAGE electrophoresis (see FIG. 2. FIG. 2 is an electrophoresis photograph. In the figure, lane 1 is silver nitrate staining of molecular weight standard marker protein, lane 2). Indicates silver nitrate staining of the enzyme). Therefore, it was suggested that this enzyme was linked to a 60 kDa α-subunit and a 43 kDa β-subunit, and was predicted to form an octamer in which four of them were bound.
After transferring the β-subunit, which is a 43 kDa protein separated by SDS-PAGE, onto a polyvinylidene fluoride membrane, the amino acid sequencer (Shimadzu Corporation, PPSQ-10) was used to determine the N-terminal amino acid sequence of the β-subunit. went. As a result, it was revealed that the N-terminal amino acid sequence of this protein is composed of 16 residues consisting of the amino acid sequence of SEQ ID NO: 5.
In addition, the result of heat-treating this enzyme at 70 ° C. for 30 minutes is shown in lane 3 in FIG. From the result of SDS-PAGE, it can be assumed that this enzyme was changed to a single polypeptide having a molecular weight of 60 kDa after heat treatment.

Example 5
Gel filtration chromatography of this enzyme was performed. As the gel, TSK gel G3000SW (manufactured by Tosoh Corporation) was used, and the gel column was (8.0 mm ID × 30 cm Tosoh, Tokyo, Japan) and 0.3 M NaCl in 10 mM potassium phosphate buffer (PH 6.0) and 0 Equilibrated with a solution containing 1% Triton-X100. Fractions (125 μl) were collected. Seven protein markers were used to determine the molecular weight of the purified enzyme. As protein markers, thyroglobulin: 669 kDa, ferritin: 440 kDa, catalase: 232 kDa, aldolase: 158 kDa, bovine serum albumin (buvin Serum Albumin): 67 kDa Chymotrypsinogen A: 25 kDa was used.
It was confirmed that the molecular weight of this enzyme is about 380 kDa.

Example 6
The optimum temperature of the purified enzyme was examined.
The reaction was started after incubating in Tris-HCl buffer, pH 8.0 for 1 minute at a preset temperature. Activity was measured at a given reaction temperature. The optimum temperature was found around 45 ° C. (see FIG. 3A). Moreover, although activity was low compared with 45 degreeC vicinity, the peak was seen also at 75 degreeC vicinity.
In addition, in order to examine the thermal stability of the present enzyme, the remaining enzyme activity was measured at 45 ° C. after standing at each temperature for 30 minutes (see FIG. 3B).

Example 7
The optimum temperature and thermal stability of the peptide enzyme constituting a single oligopeptide having a molecular weight of 60 kDa when this enzyme was heat-treated at 70 ° C. for 30 minutes were examined.
This peptide enzyme showed a higher optimum temperature than the non-heat-treated enzyme, and also showed thermal stability. There are no reports of enzymes exhibiting such temperature dependence.
The reaction was started after incubating in Tris-HCl buffer, pH 8.0 for 1 minute at a preset temperature. Activity was measured at a given reaction temperature. The optimum temperature was found around 75 ° C. (see FIG. 4A).
Moreover, in order to investigate the thermostability of this enzyme, after standing at each temperature for 30 minutes, the residual enzyme activity was measured at 70 degreeC (refer FIG.4 (b)).

Example 8
In order to investigate the role of each subunit, spectrophotometric analysis of GDH before and after heat treatment was performed. FIGS. 5 (a) and 5 (b) show the absorption of oxidized and reduced GDH before and after heat treatment (in the presence of glucose). The wavelength of oxidized GDH before heat treatment, which is the original GDH, showed a characteristic peak at 409 nm, and in the presence of glucose it moved to 417 nm and two additional peaks were seen at 523 nm and 550 nm ( FIG. 5a). In contrast, no characteristic peak at 409 nm was observed after heat treatment (FIG. 5b), and no significant difference was observed between oxidized and reduced forms.
The absorption wavelength of the oxidized GDH before heat treatment and the original GDH is Gluconobacter sp. Alternatively, Acetobacter sp. The absorption wavelength was similar to that of alcohol dehydrogenase and aldehyde dehydrogenase made of the dehydrogenase cytochrome complex (see the following literature: Adachi, O., Tayama, K., Shinagawa, E., Matsushita, K. and Ameyama, M. (1978) Agr.Biol.Chem., 42, 2045-2056.; Adachi, O., Miyagawa, E., Matsushita, K. and Ameyama, M. (1978) Agr.Biol.Chem., 42, 2331-2340; Ameyama, M. and Adachi, O., (1982) Methods Enzymol., 89, 450-457; Adachi, O., Tayama, K., Shinag Awa, E., Matsushita, K. and Ameyama, M. (1980) Agr. Biol. Chem., 44, 503-515; Ameyama, M. and Adachi, O. (1982) Methods Enzymol., 89, 491-. 497).
As a result, it was suggested that the oligomer complex of the present GDH may contain cytochrome. Therefore, the observed cytochrome C-like wavelength is attributed to the β subunit and can be said to have been lost during the heat treatment. Therefore, it can be said that the β subunit is composed of cytochrome C.

Example 9
The band containing the β subunit obtained by electrophoresis in Example 4 was cut out, and the amino acid sequence was analyzed by a peptide sequencer (Shimadzu Corporation, PPSQ-10). As a result, the N-terminal 16-residue amino acid shown in SEQ ID NO: 5 was obtained. The sequence could be obtained.
An attempt was made to amplify a gene region encoding the peptide sequence from the N-terminal 16-residue amino acid sequence by PCR. That is, the forward-side base sequence (SEQ ID NO: 6) corresponding to the N-terminal 5 residues of the 16-residue peptide chain and the reverse-side base sequence (SEQ ID NO: 7) corresponding to the antisense chain of the C-terminal 5 residues Two PCR primers with) were designed. When PCR was performed on the genome of the KS1 strain using this set of PCR primers according to a conventional method, a gene fragment of about 50 bp was amplified. The base sequence was determined according to a conventional method. As a result, 58 base sequences including one PCR primer set were decoded. Among these, when analyzing 18 bases excluding the PCR primer, the gene sequence (sequence) corresponding to the 11th Arg from the 6th Pro from the N-terminal side of the β-subunit N-terminal 16 residues. No. 8) was found, and it was revealed that this amplified gene fragment contained a gene fragment of β subunit.
The β-subunit was found to be present after the 22 amino acid residues following the α-subunit. This is because the amino acid sequence at the N-terminus of the purified β-subunit determined in Example 4 matches the 5 amino acid residues translated by the nucleotide sequence of nucleotide numbers 2452 to 2466 in SEQ ID NO: 1. From this, it is based on the fact that they are found to be the same.
Moreover, it is estimated that the base sequence of base number 2386-2451 in sequence number 1 is a signal peptide of (beta) -subunit. The amino acid sequence encoded by this base sequence corresponds to amino acid numbers 1 to 22 of the amino acid sequence of SEQ ID NO: 4.

Example 10
This purified enzyme and commercially available NAD coenzyme GDH (abbreviated as NAD-GDH) are each 100 U / L in 50 mM potassium phosphate buffer (pH 7.5) containing 0.1% Triton X-100 and 1 mM CaCl _2. And mixed. This solution was placed in a high-temperature bath at 60 ° C., and the residual activity was measured.

本酵素は現在市販されているＧＤＨ酵素に比べて、驚異的な熱安定性があることが確認できた。本酵素は市販のＮＡＤ−ＧＤＨとは全く別の新規な酵素であることが判明した。

It was confirmed that this enzyme has an amazing thermostability compared to the currently available GDH enzyme. This enzyme was found to be a completely new enzyme different from commercially available NAD-GDH.

Example 10 Isolation of gene encoding GDH α subunit <1> Preparation of chromosomal DNA from Burkholderia cepacia KS1 strain A chromosomal gene was prepared from Burkholderia cepacia KS1 strain according to a conventional method. That is, the strain was shaken overnight at 34 ° C. using TL liquid medium (polypeptone 10 g, yeast extract 1 g, NaCl 5 g, KH ₂ PO ₄ 2 g, glucose 5 g; 1 L, pH 7.2). Proliferated cells were collected by a centrifuge. The cells were suspended in a solution containing 10 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% SDS, 100 μg / ml proteinase K, and treated at 50 ° C. for 6 hours. After adding an equal amount of phenol-chloroform and stirring at room temperature for 10 minutes, the supernatant was recovered by a centrifuge. To this, sodium acetate was added to a final concentration of 0.3 M, and twice the amount of ethanol was overlaid to precipitate chromosomal DNA in the intermediate layer. This was scooped using a glass rod, washed with 70% ethanol, and then dissolved in an appropriate amount of TE buffer to obtain a chromosomal DNA solution.
<2> Determination of N-terminal amino acid sequence of GDH α subunit GDH purified in the same manner as in Example 2 was concentrated by freeze-drying and developed using SDS-electrophoresis using 12.5% polyacrylamide. The α subunit was separated. The α subunit thus obtained was transferred to a polyvinylidene fluoride membrane, and then the N-terminal amino acid sequence was determined with an amino acid sequencer (manufactured by Shimadzu Corporation, PPSQ-10). As a result, it was revealed that this enzyme contains a peptide sequence composed of 11 residues consisting of amino acid numbers 2 to 12 in the amino acid sequence of SEQ ID NO: 3.
<3> Cloning of Gene Encoding α Subunit 1 μg of the DNA prepared in <1> was subjected to limited digestion with the restriction enzyme Sau3AI. This was treated with CIAP (calf intestinal alkaline phosphatase). On the other hand, SuperCosI (obtained from Stragene), which is a cosmid, was treated with BamHI, and a DNA fragment obtained by subjecting the α-15 strain-derived chromosomal DNA fragment to limited digestion with Sau3AI was incorporated into SuperCosI by T4 DNA ligase. Escherichia coli XL-1 Blue MR (obtained from Stragene) was transformed with the obtained recombinant DNA. Transformants were selected from LB agar medium containing 10 μg / ml neomycin and 25 μg / ml ampicillin according to the antibiotic resistance on SuperCosI, neomycin resistance and ampicillin resistance. The obtained transformant was cultured in an LB liquid medium. These transformed cells were collected, suspended in a reagent for measuring GDH activity, and clones were selected using the dehydrogenase activity for glucose as an indicator. As a result, a clone showing one strain of glucose dehydrogenase activity was obtained.
<4> Subcloning A DNA fragment containing the target gene was prepared from the cosmid SuperCosI containing the gene encoding the α subunit obtained in <3>. The inserted gene fragment was excised from the cosmid with the restriction enzyme NotI. This DNA fragment was treated with the restriction enzyme XbaI, and these fragments were incorporated into plasmid pUC18 digested with XbaI. Escherichia coli DH5αMCR strain was transformed with plasmid pUC18 containing each inserted fragment, and colonies produced on an LB agar medium containing 50 μg / ml of ampicillin were collected. The obtained transformant was cultured in a liquid LB medium, and the GDH activity of each cell was examined in the same manner as in <3>. As a result, a strain showing GDH activity in one transformant was obtained. When a plasmid was extracted from this transformant and the inserted DNA fragment was analyzed, an inserted fragment of about 8.8 kbp was confirmed. This plasmid was designated as pKS1.
<5> Determination of base sequence The base sequence of the inserted DNA fragment of pKS1 was determined according to restriction enzyme analysis and conventional methods. As a result, a DNA sequence encoding the N-terminal amino acid sequence of the α subunit revealed in <2> was confirmed in this inserted DNA fragment, and an open reading frame containing this sequence was found. The determined nucleotide sequence and the amino acid sequence that can be encoded by the nucleotide sequence are as shown in SEQ ID NOs: 1 and 3. The molecular weight of the protein determined from the amino acid sequence was 59,831 Da, which almost coincided with the molecular weight of 60 kDa determined by SDS-PAGE of the Burkholderia cepacia KS1 strain α subunit.
When the base sequence of the α subunit was determined, a vector was prepared using the structural gene of the α subunit, and a transformant was further produced using the vector.
First, a gene to be inserted into a vector was prepared as follows.
Using a genomic fragment derived from the KS1 strain as a template, amplification was performed by PCR reaction so as to include a desired restriction enzyme site. The following set of oligonucleotide primers was used in the PCR reaction.
(forward)
5'-cccaagcttg ggccgatacc gatacgca-3 '(SEQ ID NO: 9)
(reverse)
5′-gagaagcttt ccgcacggtc agacttcc-3 ′ (SEQ ID NO: 10)
The gene amplified by PCR was digested with the restriction enzyme HindIII, and then inserted into the HindIII site which is the cloning site of the expression vector pFLAG-CTS (SIGMA). The resulting plasmid was named pFLAG-CTS / α.
Escherichia coli DH5α MCR strain was transformed with the plasmid pFLAG-CTS / α, and colonies produced on an LB agar medium containing 50 μg / ml of ampicillin were collected.
Further, when the open reading frame was searched for the upstream of the α subunit for the pKS1 insert fragment, a structural gene composed of 507 bases newly encoding a polypeptide composed of 168 amino acid residues described in SEQ ID NO: 2 (Base numbers 258 to 761 in SEQ ID NO: 1) were found. This structural gene was thought to encode the γ subunit.
Since the existence of a region encoding the γ subunit was found upstream of the coding region of the α subunit, a recombinant vector containing a gene having a polycistron structure in which the γ subunit and the α subunit are continuous was prepared. A transformant into which the vector was introduced was constructed.
First, a gene to be inserted into a vector was prepared as follows.
A genomic fragment derived from the KS1 strain in which the structural gene of the γ subunit and the structural gene of the α subunit are continuous was used as a template to amplify by PCR reaction so as to include a desired restriction enzyme site. The following set of oligonucleotide primers was used in the PCR reaction.
(forward)
5′-catgccatgg cacacaacga caacact-3 ′ (SEQ ID NO: 11)
(reverse)
5'- cccaagcttg ggtcagactt ccttcttcag c-3 '(SEQ ID NO: 12)

The 5 ′ end of the gene amplified by this PCR was digested with NcoI and 3 ′ end with HindIII, and then inserted into NcoI / HindIII, which is the cloning site of vector pTrc99A (Pharmacia). The resulting plasmid was named pTrc99A / γ + α.
Escherichia coli DH5αMCR strain was transformed with the plasmid pTrc99A / γ + α, and colonies produced on an LB agar medium containing 50 μg / ml of ampicillin were collected.

Example 11 Production of GDH α subunit by recombinant E. coli α subunit using Escherichia coli DH5α MCR strain transformed with the respective plasmids of pKS1, pFLAG-CTS / α and pTrc99A / γ + α Unit production. Each transformant was inoculated into 3 ml of LB medium containing 50 μg / ml of ampicillin, cultured at 37 ° C. for 12 hours, and the cells were collected by a centrifuge. The cells were disrupted with a French press (1500 kgf), and then the membrane fraction (10 mM potassium phosphate buffer pH 6.0) was separated by ultracentrifugation (4 ° C., 160,400 × g, 90 minutes).

Example 12 Glucose assay First, GDH activity was confirmed using each of the membrane fractions. Specifically, visual determination was performed using a 10 mM potassium phosphate buffer (pH 7.0) containing 594 μM methylphenazine methosulfate (mPMS) and 5.94 μM 2,6-dichlorophenolindophenol (DCIP). The results are as follows. The number of + represents the degree of change from blue to colorless.
Transformant culture membrane fractionation with pFLAG-CTS / α +
Transformant culture membrane fraction with pKS1 ++
Transformant culture membrane fractionation with pTrc99A / γ + α ++
GDH activity of the transformant culture membrane fraction with pFLAG-CTS / α incorporating only the α subunit is the lowest, and the GDH activity of the transformant culture membrane fraction with pTrc99A / γ + α that efficiently constructs the vector is the highest. showed that.
Although the α subunit is expressed even in a transformant using a vector containing only the structural gene of the α subunit, it is more efficient by using a vector in which the structural gene of the γ subunit is combined with the structural gene of the α subunit. The α subunit could be obtained.
Glucose was assayed using the glucose dehydrogenase of the present invention. The enzyme activity of the glucose dehydrogenase (α subunit) of the present invention was measured at various concentrations of glucose. The measurement of GDH activity was performed in 10 mM potassium phosphate buffer (pH 7.0) containing 594 μM methylphenazine methosulfate (mPMS) and 5.94 μM 2,6-dichlorophenolindophenol (DCIP). The absorbance change at 600 nm of DCIP when glucose was added as a substrate and incubated at 37 ° C. as an enzyme sample and substrate was traced using a spectrophotometer, and the rate of decrease in the absorbance was taken as the enzyme reaction rate. Using the GDH of the present invention, glucose could be quantified in the range of 0.01 to 1.0 mM.

Example 13 Production and evaluation of glucose sensor 20 mg of carbon paste was added to the glucose dehydrogenase of the present invention (25 units) obtained in Example 2 and freeze-dried. After this was mixed well, it was filled only on the surface of the carbon paste electrode already filled with about 40 mg of carbon paste, and was polished on a filter paper. This electrode was treated in 10 mM MOPS buffer (pH 7.0) containing 1% glutaraldehyde for 30 minutes at room temperature, and then treated in 10 mM MOPS buffer (pH 7.0) containing 20 mM lysine for 20 minutes at room temperature. To block glutaraldehyde. This electrode was equilibrated in a 10 mM MOPS buffer (pH 7.0) at room temperature for 1 hour or longer. The electrode was stored at 4 ° C.
Using the electrode as a working electrode, Ag / AgCl as a reference electrode and a Pt electrode as a counter electrode, the response current value due to the addition of glucose was measured. The reaction solution was a 10 mM potassium phosphate buffer solution containing 1 mM methoxy PMS, and measurement was performed by applying a potential of 100 mV.
The concentration of glucose was measured using the prepared enzyme sensor. Using the enzyme sensor to which the glucose dehydrogenase of the present invention was immobilized, glucose could be quantified in the range of 0.05 mM to 5.0 mM (FIG. 6).

Example 14 Production and evaluation of glucose sensor by GDH obtained from transformant 10 U of α subunit (249 U / mg protein) of the present invention obtained in Example 12 was added with 50 mg of carbon paste and lyophilized. I let you. After this was mixed well, it was filled only on the surface of the carbon paste electrode already filled with about 40 mg of carbon paste, and was polished on a filter paper. This electrode was treated in 10 mM MOPS buffer (pH 7.0) containing 1% glutaraldehyde for 30 minutes at room temperature, and then treated in 10 mM MOPS buffer (pH 7.0) containing 20 mM lysine for 20 minutes at room temperature. To block glutaraldehyde. This electrode was equilibrated in a 10 mM MOPS buffer (pH 7.0) at room temperature for 1 hour or longer. The electrode was stored at 4 ° C.
Using the electrode as a working electrode, Ag / AgCl as a reference electrode and a Pt electrode as a counter electrode, the response current value due to the addition of glucose was measured. The reaction solution was a 10 mM potassium phosphate buffer containing 1 mM methoxy PMS, and glucose aqueous solutions of various concentrations were measured at 25 ° C. and 40 ° C. while applying a potential of 100 mV.
When the concentration of glucose was measured using the prepared enzyme sensor, it was confirmed that a current corresponding to each concentration was obtained.

INDUSTRIAL APPLICABILITY According to the present invention, a novel glucose dehydrogenase that has high substrate specificity, can be produced at low cost, and is not affected by dissolved oxygen in a measurement sample, and particularly excellent in thermal stability, and a method for producing the enzyme Was able to provide. A new strain of Burkholderia cepacia producing this enzyme was also obtained. If an enzyme electrode containing the present enzyme and strain is used, a glucose sensor effective for glucose measurement can also be provided.
In addition, since the present invention has revealed a glucose dehydrogenase gene, a peptide capable of efficiently expressing the gene, and a DNA encoding the peptide, GDH can be produced in a large amount by recombinant DNA technology based on the gene. Can be prepared.

It is a figure which shows the molecular weight by Native PAGE electrophoresis of this invention enzyme. It is an electrophoresis photograph which shows the molecular weight by SDS-PAGE electrophoresis of the enzyme of this invention. It is a figure which shows the optimal reaction temperature (a) of this invention enzyme, and the thermal stability (b). It is a figure which shows the optimal reaction temperature (a) and thermostability (b) of the peptide enzyme which comprises only (alpha) subunit of this invention enzyme. The spectrophotometric analysis (a) of the enzyme of the present invention in the absence and presence of glucose before heat treatment and the spectrophotometric analysis (b) of the enzyme of the present invention in the absence of glucose and in the presence of glucose after heat treatment are shown. FIG. It is a figure which shows the response with respect to glucose of each temperature using the glucose sensor by GDH obtained from a transformant.

Claims (13)

Glucose dehydrogenase characterized by culturing a microorganism having the ability to produce glucose dehydrogenase in a medium, and collecting glucose dehydrogenase from the same medium or / and the microorganism cells. The glucose dehydrogenase comprises an α subunit having a molecular weight of 60 kDa and a β subunit having a molecular weight of 43 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions, and the α subunit But,
(A) a protein having the amino acid sequence of SEQ ID NO: 3, or (B) having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 3, And a protein having glucose dehydrogenase activity, wherein the β subunit is
A method for producing glucose dehydrogenase, wherein the N-terminus is an electron transfer protein having the amino acid sequence of SEQ ID NO: 5. The method for producing glucose dehydrogenase according to claim 1, wherein the microorganism is Burkholderia cepacia. The method for producing glucose dehydrogenase according to claim 1 or 2, wherein the glucose dehydrogenase has the following properties.
(1) Action:
Catalyses the dehydrogenation of glucose.
(2) Gel filtration chromatography using TSK gel G3000SW (manufactured by Tosoh Corporation) shows a molecular weight of 380 kDa.
(3) Optimal reaction temperature:
45 ° C. (Tris-HCl buffer, pH 8.0). The method for producing glucose dehydrogenase according to any one of claims 1 to 3, wherein the electron transfer protein is cytochrome C. A glucose dehydrogenase that can be produced by a microorganism belonging to the genus Burkholderia, which glucose dehydrogenase has an α subunit having a molecular weight of 60 kDa and a molecular weight of 43 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions. Β subunits, and the α subunit is
(A) a protein having the amino acid sequence of SEQ ID NO: 3, or (B) having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 3, And a protein having glucose dehydrogenase activity, wherein the β subunit is
A glucose dehydrogenase, which is an electron transfer protein having an amino acid sequence of SEQ ID NO: 5 at the N-terminus. The glucose dehydrogenase according to claim 5, wherein the microorganism is Burkholderia cepacia. The glucose dehydrogenase according to claim 5 or 6, wherein the glucose dehydrogenase has the following properties.
(1) Action:
Catalyses the dehydrogenation of glucose.
(2) Gel filtration chromatography using TSK gel G3000SW (manufactured by Tosoh Corporation) shows a molecular weight of 380 kDa.
(3) Optimal reaction temperature:
45 ° C. (Tris-HCl buffer, pH 8.0). The glucose dehydrogenase according to any one of claims 5 to 7, wherein the electron transfer protein is cytochrome C. The glucose dehydrogenase according to any one of claims 5 to 8, which has activity peaks at 45 ° C and 75 ° C, respectively. A cytochrome C, which is the β subunit of glucose dehydrogenase according to claim 8. The following properties:
(1) The glucose dehydrogenase according to claim 5 can be constituted as a subunit.
(2) It has glucose dehydrogenase activity.
(3) In SDS-polyacrylamide gel electrophoresis under reducing conditions, the molecular weight is 60 k.
Da is shown.
(4) Optimal reaction temperature:
75 ° C. (Tris-HCl buffer, pH 8.0)
Have
(A) a protein having the amino acid sequence of SEQ ID NO: 3, or (B) having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 3, And protein which is protein which has glucose dehydrogenase activity. The enzyme electrode containing the glucose dehydrogenase of any one of Claims 5-9. A glucose sensor using the enzyme electrode according to claim 12 .

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