JP2009207441A - Glucose dehydrogenase and gene encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a glucose dehydrogenase having salt tolerance, wide pH stability, intrinsic substrate specificity and intrinsic coenzyme dependence, to provide a technology for mass producing the enzyme by an genetic engineering technique in which a gene encoding the enzyme is isolated and the enzyme having objective physicochemical properties is obtained as a recombinant and a saccharide sensor using the properties of the enzyme. <P>SOLUTION: The protein is (a) a protein containing an amino acid sequence composed of a specific sequence or (b) a protein containing an amino acid sequence in which at least one modification selected from deletion, substitution, insertion and addition of one or a plurality of amino acids is carried out. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to glucose dehydrogenase and its gene. More particularly, the present invention relates to a glucose dehydrogenase that has salt tolerance as well as broad pH stability and inherent substrate specificity and that is specifically dependent on NADP ⁺ , and the gene encoding the enzyme, and The present invention relates to a saccharide sensor containing the enzyme.

  Glucose dehydrogenase catalyzes a reaction that oxidizes glucose to produce gluconolactone. Due to such catalytic activity, glucose dehydrogenase is used for detection of saccharides, constituent materials for biofuel cells, and the like, and is an enzyme having high utility value in various fields such as food chemistry, clinical chemistry and biochemistry. And these glucose dehydrogenases are known to exist widely from bacteria and yeast to mammals, and glucose dehydrogenases derived from various organisms have been reported (for example, Patent Document 1). , See Non-Patent Documents 1 to 4.)

  However, it is known that enzymes, not limited to glucose dehydrogenase, have a very strong property of selecting a catalytic action. For example, since an enzyme exhibits catalytic activity under mild conditions in which a living body can survive, the activity of the enzyme is affected by the presence of factors that change the structure of the protein constituting the enzyme. In other words, enzyme activity is known to be affected by physical conditions such as temperature, and chemical conditions such as pH and salt concentration, and its use is limited due to its low stability. there were.

Thus, in recent years, several glucose dehydrogenases exhibiting stability against heat, a wide range of pH, and salt concentration have been reported. For example, glucose dehydrogenase derived from Thermoplasma acidophilum , which is a thermoacidophilic archaeon (see, for example, Non-Patent Document 1), Haloferax Mediterranei, a halophilic archaea mediterranei ) -derived glucose dehydrogenase (see, for example, Non-Patent Document 2), a thermoacidophilic archaeon Sulfolobus solfataricus- derived glucose dehydrogenase (for example, Non-Patent Document 3) For example). Glucose dehydrogenase from Thermoplasma acidophilum has been reported to be thermostable and stable to organic solvents and urea. In addition, glucose dehydrogenase derived from Haloferrax Mediterranei exhibits salt and heat stability and is resistant to a basic pH region (pH 8.8). It has been reported that the thermal stability depends on the salt concentration. And the glucose dehydrogenase derived from Sulfolobus solfataricus also shows salt and heat stability, and also has resistance to the basic pH region. In particular, it has been reported to show the highest activity under pH 9.0, 77 ° C., 20 mM magnesium ion, manganese ion or calcium ion.

  In addition, as described above, since the enzyme has a very strong property of selecting a catalytic action, its substrate specificity is very strict. Enzymes generally act only on certain compounds because of their catalytic action by complementary binding of their reaction sites and substrates. Therefore, it is industrially useful because it can selectively identify only specific molecules in multi-components and catalyze only specific reactions. On the other hand, it also depends on the specific substrate specificity and coenzyme dependency of the enzyme. The use is limited.

For example, many glucose dehydrogenases are abbreviated as oxidized nicotinamide adenine dinucleotide (hereinafter abbreviated as “NAD ⁺ ”) and oxidized nicotinamide adenine dinucleotide phosphate (hereinafter “NADP ⁺ ”). ) As a coenzyme. The aforementioned enzymes also exhibit catalytic activity using both NAD ⁺ and NADP ⁺ as coenzymes. However, glucose dehydrogenase derived from bacteria belonging to the genus Cryptococcus (see, for example, Patent Document 1) and glucose dehydrogenase derived from Saccharomyces bulderi (see, for example, Non-Patent Document 4). As described above, there are also reports of industrially particularly useful ones that require only NADP ⁺ as a coenzyme. However, the stable pH range of glucose dehydrogenase derived from Cryptococcus bacteria is as narrow as 6.0 to 7.5, and heat resistance is low, and there is no report on substrate specificity other than glucose, and it can be used for a wide range of applications. It was not a thing. Furthermore, the glucose dehydrogenase derived from Saccharomyces brudeli has a problem that its specificity for NADP ⁺ is relatively low, 10 times that of NAD ⁺ .

  In addition, with the diversification of enzyme utilization technologies, the current range of enzyme options required by the market is expanding to match the application. However, known glucose dehydrogenases have not been able to sufficiently meet market demands. Therefore, development of a novel glucose dehydrogenase that is highly stable against chemical factors such as pH and salt concentration, and has a unique substrate specificity and coenzyme dependency different from known glucose dehydrogenases Was desired.

Japanese Patent Publication No. 63-109773 Leon D. Smith et al., “Purification and characterization of glucose dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum”, The Biochemical Journal, 1989 Aug, 261, No. 3, pp. 973-977 Maria-Jose Bonete et al., “Glucose dehydrogenase from the halophilic Archaeon Haloferax mediterranei: enzyme purification, characterization and N-terminal sequence. N-terminal sequence) ”, FEBS Letters, 1996, 383, 227-229 Paola Giardina et al., “Glucose dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus”, The Biochemical Journal, November 1986, 239, 3 No., 517-522 Johannes P. van Dijken et al., “Novel pathway for alcoholic fermentation of delta-gluconolactone in the yeast Saccharomyces bulderi”, Journal of Bacteriology, February 2002 184, No. 3, pp. 672-678

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a glucose dehydrogenase having salt tolerance, wide pH stability, inherent substrate specificity, and inherent coenzyme dependency. The present invention also provides a technique for mass production of the enzyme by genetic engineering techniques by isolating the gene encoding the enzyme, obtaining the enzyme having the desired physicochemical properties as a recombinant. Objective. Furthermore, this invention aims at provision of the saccharide | sugar sensor using the property of the said enzyme.

As a result of repeated studies to solve the above problems, the present inventors have focused on halophilic archaea that require a high salt concentration for growth, and the halobacterium varismortis which is a kind of such halophilic archaea. ( Halobacterium vallismortis ) discovered a nucleic acid molecule that appears to encode glucose dehydrogenase. Furthermore, when recombinants were produced using genetic engineering techniques, they were found to be glucose dehydrogenases that catalyze glucose dehydrogenation. Furthermore, it has also been found that such an enzyme has an inherent substrate specificity and coenzyme dependency that it has salt tolerance, a wide range of pH stability, and is also reactive with specific sugars other than glucose. Based on these findings, the present inventors have completed the present invention.

That is, in order to achieve the above object, the following inventions [1] to [10] are provided.
[1] A protein of the following (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (b) at least one modification selected from deletion, substitution, insertion and addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 A protein comprising an amino acid sequence and having the following physicochemical properties (1) to (3):
(1) Oxidized nicotinamide adenine dinucleotide phosphate is specifically required as a coenzyme and catalyzes a dehydrogenation reaction that produces gluconolactone from glucose (2) catalyzes a dehydrogenation reaction of ribose (3) [2] The protein of [1] above having one or more physicochemical properties selected from the following (4) to (7), wherein the pH tolerance is stable at pH 7.0 to 8.8 and the optimum pH is 8.8.
(4) The salt tolerance is stable at a salt concentration of 0 to 2.0 M, and the optimum salt concentration is 1.0 M. (5) Catalyze dehydrogenation of sucrose (6) Catalyze dehydrogenation of xylose ( 7) The protein according to [1] or [2], which is a recombinant, which catalyzes the dehydrogenation reaction of fucose.
[4] A nucleic acid molecule encoding the protein of any one of [1] to [3] above.
[5] The nucleic acid molecule according to [1] above, comprising the following polynucleotide (c) or (d).
(C) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1,
(D) a polynucleotide that hybridizes with a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 under stringent conditions and encodes a protein having the following physicochemical properties (1) to (3): (1) oxidation Type nicotinamide adenine dinucleotide phosphate is specifically required as a coenzyme and catalyzes the dehydrogenation reaction to produce gluconolactone from glucose (2) catalyzes the dehydrogenation reaction of ribose (3) pH tolerance, [6] The nucleic acid molecule according to [5], wherein the protein has one or more physicochemical properties selected from the following (4) to (7): stable at pH 7.0 to 8.8 and having an optimum pH of 8.8. .
(4) The salt tolerance is stable at a salt concentration of 0 to 2.0 M, and the optimum salt concentration is 1.0 M. (5) Catalyze dehydrogenation of sucrose (6) Catalyze dehydrogenation of xylose ( 7) A recombinant vector containing the nucleic acid molecule of any one of [4] to [6] above, which catalyzes the dehydrogenation reaction of fucose.
[8] A transformant containing the recombinant vector of [7] above.
[9] A method for producing glucose dehydrogenase, comprising culturing the transformant of [8] and collecting a protein having an ability to catalyze a dehydrogenation reaction of glucose from the obtained culture.

According to the configuration of [1] to [9] above, it is possible to provide a novel NADP ⁺ dependent glucose dehydrogenase. The NADP ⁺ -dependent glucose dehydrogenase provided by the present invention has a unique substrate specificity capable of reacting with a small amount of saccharides with high substrate selectivity, and a unique coenzyme that depends specifically on NADP ⁺ as a coenzyme. Shows specificity. And it shows stability and salt resistance in a wide pH range, particularly in a basic range. Therefore, the present invention can be applied to a technique requiring a dehydrogenation reaction of sugars in various industrial fields such as medical, food, and environmental fields. Furthermore, since the amino acid sequence and base sequence of the NADP ⁺ -dependent glucose dehydrogenase of the present invention have been clarified, it is possible to mass-produce a low-cost industrially genetic engineering technique as a recombinant. became.

[10] A biosensor for detecting saccharides, wherein the protein according to any one of [1] to [3] above is immobilized on an electrode.
[11] The biosensor for saccharide detection according to [10], wherein the saccharide is one or more saccharides selected from glucose and ribose.

According to the configuration of [10] to [11] above, a biosensor for detecting saccharides utilizing the catalytic ability of the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be provided. Can be used in various fields. The NADP ⁺ -dependent glucose dehydrogenase of the present invention exhibits substrate specificity for trace saccharides other than glucose and has high substrate selectivity, and therefore can be suitably used for detection of these trace saccharides. In particular, the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be suitably used for detection of saccharides in brewed foods having a high salt concentration from the viewpoint of salt tolerance.

  Hereinafter, the present invention will be described in detail.

(Properties of NADP ⁺ -dependent glucose dehydrogenase of the present invention)
The physicochemical properties of the NADP ⁺ glucose dehydrogenase of the present invention are as follows.

(1) Action As shown below, it catalyzes a reaction that produces gluconolactone in a NADP ⁺ dependent manner using glucose as a substrate.
D-glucose + NADP ⁺ → D-glucono-δ-lactone + NADPH + H ⁺

The feature of the NADP ⁺ glucose dehydrogenase of the present invention is that NADP ⁺ is specifically required as a coenzyme and exhibits its catalytic activity. Here, “requires NADP ⁺ specifically as a coenzyme” means that when glucose dehydrogenation activity is measured under the same conditions containing NAD ⁺ and NADP ⁺ at the same concentration as a coenzyme in the reaction solution, It shows that the activity when NADP ⁺ is measured as a coenzyme is more than 3 times, preferably 5 times or more, more preferably 10 times or more than the activity when NAD ⁺ is measured as a coenzyme.

The NADP ⁺ glucose dehydrogenase of the present invention exhibits a unique substrate specificity different from that of known glucose dehydrogenases. In addition to glucose, it is also reactive to monosaccharides and disaccharides containing pentose and hexose other than glucose, and in particular, it is preferable to be reactive to deoxysugar. Specifically, it exhibits reactivity with respect to ribose in addition to glucose. Furthermore, it is preferable to show reactivity with at least one of sucrose, xylose and fucose. The activity with respect to these is preferably 40 or more, more preferably 60 or more, and particularly preferably 80 or more in the relative activity when the activity with respect to glucose is 100. However, the catalytic activity for mannose is preferably 10 or less in relative activity when the activity for glucose is 100.

(2) pH stability The NADP ⁺ glucose dehydrogenase of the present invention exhibits stable activity in the range of pH 7.0 to 8.8, and the optimum pH is pH 8.8. Furthermore, it is preferable that the activity can be maintained even in an acidic region up to about pH 5.0. And, it is preferable that the activity can be maintained even in the basic region from about pH 10 to 11, and it is particularly preferable that the activity can be maintained in the range from about pH 4.0 to 12, but it is not limited thereto.

(3) Salt tolerance The NADP ⁺ glucose dehydrogenase of the present invention exhibits salt tolerance. There are no particular limitations on the type of salt that exhibits salt resistance, but it is preferably a salt containing a monovalent cation, and examples thereof include NaCl and KCl. For example, the salt tolerance against NaCl shows a stable activity in the range of 1.0 to 2.0 M, and the optimum salt concentration is 1.0 M. Furthermore, it can exhibit activity even in the absence of salts, and is active in a wide salt concentration range of 0 to 2.0 M.

(4) Molecular Weight The glucose dehydrogenase of the present invention may have a different molecular weight depending on the amino acid composition, but preferably has a molecular weight of about 40 kDa.

The origin of the NADP ⁺ glucose dehydrogenase of the present invention is not limited as long as it has the physicochemical properties of the glucose dehydrogenase of the present invention. For example, it is derived from an organism having the ability to produce glucose dehydrogenase having the aforementioned physicochemical properties, and is typically a bacterium. Preferably, it is an archaebacteria, more preferably a bacterium capable of growing in an extreme environment (for example, Shioda, the Dead Sea of Israel), for example, a halophilic bacterium. Here, halophilic bacteria are bacteria that require the presence of a relatively high concentration of salt for optimal growth. A halophilic bacterium is classified into a low halophilic bacterium, a moderate halophilic bacterium, and a highly halophilic bacterium according to the difference in the optimal growth salt concentration. In the present invention, any of these may be used, but in particular, it is preferably derived from highly halophilic bacteria. Specific examples include bacteria belonging to the genus Halobacterium , and particularly preferred is Halobacterium vallismortis .

As described above, the NADP ⁺ glucose dehydrogenase of the present invention is preferably a protein derived from halophilic bacteria. And since halophilic bacteria-derived proteins generally exhibit salt tolerance, they have the property of being hardly insoluble. Furthermore, it is preferable that it has the property that it is difficult to aggregate due to the high composition ratio of acidic amino acids, and that even if it is denatured, it can rewind to the natural structure and has high stability.

(Amino acid sequence of NADP ⁺ glucose dehydrogenase of the present invention)
In addition, the NADP ⁺ glucose dehydrogenase of the present invention is preferably exemplified by those containing the amino acid sequence of SEQ ID NO: 2, although not limited thereto. Furthermore, as long as the properties of the above-mentioned NADP ⁺ glucose dehydrogenase are maintained, the amino acid sequence of SEQ ID NO: 2 may contain an amino acid sequence having a modified site in which a specific amino acid is modified. . “Modification” means that a modification in which at least one of one or more amino acids is deleted, substituted, inserted, or added in the amino acid sequence of the protein that is the basis of the modification has occurred. "A modification comprising at least one of deletion, substitution, insertion and addition of one or more amino acids" means a known DNA recombination technique for a gene encoding a protein serving as a basis for the modification, a point mutation introduction method Etc. means that as many amino acids as can be deleted, substituted, inserted or added are deleted, substituted, inserted or added, including combinations thereof. For example, such a variant may maintain homology of 70% or more, preferably 80% or more, more preferably 90% or more, at the amino acid level with respect to the amino acid sequence represented by SEQ ID NO: 2. it can.

Such a variant can be obtained by screening a protein having the above-mentioned physicochemical properties from mutants generated by natural or artificial mutation. Alternatively, it can also be obtained by modifying using a nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention described below. There is no restriction | limiting in particular as a method to modify | change a nucleic acid molecule, The mutagenesis technique for preparation of the modification protein known to those skilled in the art can be utilized. For example, known mutagenesis techniques such as site-directed mutagenesis, PCR abrupt induction that introduces a point mutation using PCR, or transposon insertion mutagenesis can be used. A commercially available mutagenesis kit (for example, QuikChange (registered trademark) Site-directed Mutagenesis Kit (manufactured by Stratagene), etc.) may be used, and a DNA synthesis method such as a conventional phosphoramidite method may be used. It can also be prepared by constructing nucleic acid molecules with the desired modifications.

Those skilled in the art can easily predict modifications that retain the enzymatic activity of the NADP ⁺ -dependent glucose dehydrogenase of the present invention upon modification of the amino acid sequence. Specifically, for example, in the case of amino acid substitution, the protein structure From the viewpoint of retention, the amino acid can be substituted with an amino acid having properties similar to the amino acid before substitution in terms of polarity, charge, hydrophilicity, hydrophobicity, and the like. Such substitutions are well known to those skilled in the art as conservative substitutions. For example, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine and tryptophan are classified as nonpolar amino acids, and thus have similar properties to each other. Examples of the uncharged amino acid include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Examples of acidic amino acids include aspartic acid and glutamic acid. Examples of basic amino acids include lysine, arginine, and histidine. Amino acid substitutions within each of these groups are permissible as protein function is maintained. Further, for convenience such as subsequent purification and immobilization on a solid phase, those having His-tag, FLAG-tag, etc. added to the N- or C-terminus of the amino acid sequence are also preferably exemplified. Such Tag peptide can be introduced by a conventional method. Moreover, the cut | disconnected type which cut | disconnected the amino acid residue of the C terminal side or the N terminal side may be sufficient as long as it does not cause the loss of the enzyme activity of this invention. Furthermore, chemical modification such as glucosylation may be added.

Since the amino acid sequence of the NADP ⁺ -dependent glucose dehydrogenase of the present invention has been clarified, the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be produced based on such sequence information. For example, a glucose dehydrogenase capable of retaining the above physicochemical properties is expressed by a hybridization method using as a probe a DNA prepared based on a base sequence encoding part or all of the amino acid sequence shown in SEQ ID NO: 2. A nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be prepared from the genomic DNA of the bacterium that can be used. Similarly, by PCR using a part of the base sequence encoding the amino acid sequence of SEQ ID NO: 2 as a primer, bacterial genomic DNA capable of expressing glucose dehydrogenase capable of retaining the above-mentioned physicochemical properties is used as a template. Nucleic acid molecules encoding the NADP ⁺ dependent glucose dehydrogenase of the present invention can be prepared. Furthermore, a nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be chemically synthesized using a DNA synthesis method such as a conventional phosphoramidite method. Then, the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be produced by the gene recombination technique described in detail below for the obtained nucleic acid molecule.

Here, the genomic DNA used as a template for hybridization and PCR is a genomic DNA derived from a bacterium capable of expressing glucose dehydrogenase capable of retaining the above-mentioned physicochemical properties. Preferred are archaebacteria, more preferably bacteria that can grow in extreme environments, such as halophilic bacteria, in particular genomic DNA derived from highly halophilic bacteria. For example, a genomic DNA derived from a genus Halobacterium , particularly a Halobacterium vallismortis is preferable. However, the present invention is not limited to this. Since the full-length amino acid sequence has been clarified in the present specification, it becomes possible to construct more specific probes and primers for the NADP ⁺ -dependent glucose dehydrogenase of the present invention. This improves the hybridization accuracy and PCR amplification accuracy. Therefore, on the condition that the glucose dehydrogenase capable of retaining the above-mentioned physicochemical properties can be expressed, a PCR amplification product could not be obtained in Study Example 2 below, Halobacterium vallismortis , Halobacterium Halobacterium pharaonis , Halobacterium sodomense, Halobacterium saccharovorum , Halobacterium volcanii , Halobacterium salinarium , Halobacterium salinarium um halobium (Halobacterium halobium), Halobacterium, Kuchiruburumu genomic DNA from (Halobacterium cutirubrum) may be used as the target.

Then, in the production of NADP ⁺ dependent glucose dehydrogenase of the present invention, the probe used in the case of utilizing the hybridization, an oligonucleotide containing NADP ⁺ dependent glucose dehydrogenase complementary to sequences of the invention Yes, it can be prepared based on conventional methods. For example, a chemical synthesis method based on the phosphoramidite method or the like, or a restriction enzyme fragment or the like can be used when a target nucleic acid has already been obtained. As such a probe, based on the base sequence of the nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention, 10 or more, preferably 15 or more, more preferably about 20 to 50 of this base sequence is continuous. An oligonucleotide consisting of these bases is exemplified. The probe may be appropriately labeled as necessary, and examples of such a label include a radioisotope and a fluorescent dye.

Further, in the preparation of NADP ⁺ dependent glucose dehydrogenase of the present invention, the primers used in the case of using PCR is an oligonucleotide comprising a NADP ⁺ dependent glucose dehydrogenase complementary to sequences of the invention Can be prepared based on conventional methods. For example, a chemical synthesis method based on the phosphoramidite method or the like, or a restriction enzyme fragment or the like can be used when a target nucleic acid has already been obtained. When preparing a primer based on a chemical synthesis method, it is designed based on the sequence information of the target nucleic acid prior to synthesis. The primer can be designed using, for example, primer design support software so as to amplify a desired region. After synthesis, the primer is purified by means such as HPLC. Moreover, when performing chemical synthesis, it is also possible to use a commercially available automatic synthesizer. Such a primer is designed on the basis of the base sequence of the nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention, and is designed with a desired amplification region interposed therebetween, preferably 10 or more, preferably 15 or more, more preferably Examples are oligonucleotides consisting of about 20-50 bases.

  Here, the term “complementary” means that the probe or primer and the target nucleic acid molecule can specifically bind according to the base pairing rule to form a stable duplex structure. Here, not only perfect complementarity, but only a few nucleobases follow base pairing rules as long as the probe or primer and the target nucleic acid molecule are sufficient to form a stable duplex structure with each other. Even partial complementarity that fits is acceptable. The number of bases must be long enough to specifically recognize the target nucleic acid molecule. However, if the length is too long, a nonspecific reaction is induced, which is not preferable. Accordingly, the appropriate length is determined depending on many factors such as target nucleic acid sequence information such as GC content, and hybridization reaction conditions such as reaction temperature and salt concentration in the reaction solution.

Furthermore, the NADP ⁺ -dependent glucose dehydrogenase of the present invention can also be produced by chemical synthesis techniques. For example, the amino acid sequence shown in SEQ ID NO: 2 can be prepared by synthesizing all or part of the amino acid sequence using a peptide synthesizer and reconstructing the resulting polypeptide under appropriate conditions.

(Nucleic acid molecule encoding NADP ⁺ -dependent glucose dehydrogenase of the present invention)
Nucleic acid molecules encoding the NADP ⁺ dependent glucose dehydrogenase of the present invention include those encoding all NADP ⁺ dependent glucose dehydrogenases having the aforementioned physicochemical properties. For example, it is a polynucleotide encoding a protein containing the amino acid sequence shown in SEQ ID NO: 2, and one specific example is a polynucleotide containing the base sequence shown in SEQ ID NO: 1. Here, the polynucleotide in the present invention includes both DNA and RNA, and when it is DNA, it is not necessarily double-stranded if it is single-stranded.

Furthermore, as long as it retains the above-mentioned properties of the NADP ⁺ -dependent glucose dehydrogenase of the present invention, it contains a polynucleotide that hybridizes with the polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 under stringent conditions. Are also included in the present invention. Such a polynucleotide can be prepared by using a known mutation introduction technique. For example, a known mutagenesis technique such as a site-directed mutagenesis method, a PCR abrupt induction method that introduces a point mutation using PCR, or a transposon insertion mutagenesis method can be used. A commercially available mutagenesis kit (for example, QuikChange (registered trademark) Site-directed Mutagenesis Kit (manufactured by Stratagene), etc.) may be used, and a DNA synthesis method such as a conventional phosphoramidite method may be used. Can be performed by constructing a nucleic acid molecule encoding NADP ⁺ -dependent glucose dehydrogenase with the desired modification, or by allowing an exonuclease to act on a polynucleotide having the base sequence of SEQ ID NO: 1. Such polynucleotides include those in which one or more bases are deleted, substituted, added or inserted in the base sequence of SEQ ID NO: 1. Preferred examples include those in which a base sequence encoding a His-Tag sequence is added to the 3 ′ or 5 ′ end of the base sequence.

  Here, the stringent condition means that DNAs having the identity of 60% or more, preferably 70%, more preferably 80% or more, particularly preferably 90% or more in the base sequence are preferentially hybridized. The condition to obtain. Stringency can be prepared by appropriately changing the salt concentration and temperature during the hybridization reaction and washing. For example, the conditions for Southern hybridization described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, and the like.

  More specifically, hybridization is exemplified in 50% (v / v) formamide, 5 × SSC at 42 ° C. for 16 hours. Here, 1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0. Further, sodium dodecyl sulfate (SDS) may be contained in an amount of 0.1 to 1.0% (v / v), and denatured non-specific DNA may be contained in an amount of 0 to 200 μl. The washing conditions include washing for 5 minutes at 5 ° C. in 2 × SSC, 0.1% SDS, and washing for 30 minutes to 4 hours at 65 ° C. in 0.1 × SSC, 0.1% SDS. . Also, those skilled in the art can easily understand these equivalent conditions.

The nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be prepared based on such sequence information since the nucleotide sequence of the nucleic acid molecule of the present invention has been clarified. For example, bacterial genomic DNA capable of expressing glucose dehydrogenase capable of retaining the aforementioned physicochemical properties by a hybridization method used as a DNA probe prepared based on a part or all of the base sequence shown in SEQ ID NO: 1. From the above, a nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be prepared. Similarly, by PCR using a part of the nucleotide sequence of SEQ ID NO: 1 as a primer, the NADP ⁺ of the present invention can be obtained using a bacterial genomic DNA capable of expressing glucose dehydrogenase capable of retaining the aforementioned physicochemical properties as a template. Nucleic acid molecules encoding dependent glucose dehydrogenase can be prepared. Furthermore, a nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be chemically synthesized using a DNA synthesis method such as a conventional phosphoramidite method. Details have been described above.

(Recombinant vector of the present invention)
The recombinant vector of the present invention can be constructed by incorporating a nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention into an appropriate vector. The vector that can be used is not particularly limited as long as it can incorporate foreign DNA and can replicate autonomously in a host cell. Accordingly, the vector contains a sequence of at least one restriction enzyme site into which a nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be inserted. For example, plasmid vectors (pEX system, pUC system, pBR system, etc.), phage vectors (λgt10, λgt11, λZAP, etc.), cosmid vectors, virus vectors (vaccinia virus, baculovirus, etc.) and the like are included.

The recombinant vector of the present invention is incorporated so that the nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention can express its function. Therefore, other known base sequences necessary for the functional expression of the nucleic acid molecule may be included. For example, a promoter sequence, a leader sequence, a signal sequence, a ribosome binding sequence and the like can be mentioned. As the promoter sequence, for example, when the host is Escherichia coli, lac promoter, trp promoter and the like are preferably exemplified. However, the present invention is not limited to this, and a known promoter sequence can be used. Furthermore, the recombinant vector of the present invention can also include a marking sequence that can confer phenotypic selection in the host. Examples of such marking sequences include sequences encoding genes such as drug resistance and auxotrophy. Specifically, kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene and the like are exemplified.

Insertion of a nucleic acid molecule or the like encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention into a vector can be achieved by, for example, cleaving the gene of the present invention with an appropriate restriction enzyme, and the appropriate vector restriction enzyme site or multiple cloning. A method of inserting and linking to a site can be used, but is not limited thereto. For ligation, a known method such as a method using DNA ligase can be used. Commercially available ligation kits such as DNA Ligation Kit (Takara Bio Inc.) can also be used.

(Transformant of the present invention)
The transformant of the present invention can be constructed by transforming a suitable cell with a recombinant vector containing a nucleic acid molecule encoding the NADP ⁺ -dependent glucose dehydrogenase of the present invention. Here, the host cell is not particularly limited as long as it is a host cell that can efficiently express the NADP ⁺ -dependent glucose dehydrogenase of the present invention. Prokaryotes can be preferably used, and in particular, E. coli can be used. In addition, Bacillus subtilis, Bacillus bacteria, Pseudomonas bacteria, and the like can also be used. As E. coli, for example, E. coli DH5α, E. coli BL21, E. coli JM109 and the like can be used. Furthermore, eukaryotic cells can be used without being limited to prokaryotes. For example, yeasts such as Saccharomyces cerevisiae , insect cells such as Sf9 cells, animal cells such as CHO cells and COS-7 cells, and the like can be used. As a transformation method, a known method such as a calcium chloride method, an electroporation method, a liposome transfection method, a microinjection method, or the like can be used.

(Method for producing NADP ⁺ -dependent glucose dehydrogenase of the present invention)
The method for producing the NADP ⁺ -dependent glucose dehydrogenase of the present invention comprises culturing the transformant of the present invention described above, and collecting a protein having an activity of catalyzing a sugar dehydrogenation reaction from the obtained culture. To do. That is, the method includes a culture step of culturing the transformant of the present invention described above and a recovery step of recovering the protein expressed in the culture step. Thus, by expressing in an appropriate host, the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be mass-produced at a low cost.

  The culturing step is carried out by inoculating the transformant of the present invention in an appropriate medium and culturing according to a conventional method. For the culture of the transformant of the present invention, the culture conditions may be selected in consideration of the nutritional physiological properties of the host cells. The medium to be used is not particularly limited as long as it contains nutrients that can be assimilated by the host cell and can efficiently express the protein in the transformant. Therefore, a medium containing a carbon source, a nitrogen source and other essential nutrients necessary for the growth of the host cell is preferable, regardless of whether it is a natural medium or a synthetic medium. Examples of the carbon source include glucose, dextran, starch, and the like, and examples of the nitrogen source include ammonium salts, nitrates, amino acids, peptone, and casein. As other nutrients, inorganic salts, vitamins, antibiotics, and the like can be included as desired. When the host cell is Escherichia coli, LB medium, M9 medium and the like can be suitably used. Moreover, although there is no restriction | limiting in particular also about a culture | cultivation form, A liquid medium can be utilized suitably from a viewpoint of mass culture.

  Selection of a host cell carrying the recombinant vector of the present invention can be performed by, for example, the presence or absence of expression of a marking sequence. For example, when a drug resistance gene is used as the marking sequence, it can be performed by culturing in a drug-containing medium corresponding to the drug resistance gene.

The purification step may be performed by recovering, that is, isolating and purifying the NADP ⁺ -dependent glucose dehydrogenase of the present invention from the culture of the transformant obtained in the above-described culture step. Depending on the fraction in which the enzyme of the present invention is present, a technique according to a general protein isolation and purification method may be applied. Specifically, when the glucose dehydrogenase of the present invention is produced outside the host cell, the culture solution is used as it is, or the host cell is removed by means of centrifugation, filtration or the like, and the culture supernatant is obtained. obtain. Subsequently, the enzyme of the present invention can be isolated and purified by appropriately selecting a known protein purification method for the culture supernatant. For example, known isolation and purification techniques such as ammonium sulfate precipitation, dialysis, SDS-PAGE electrophoresis, gel filtration, various types of chromatography such as hydrophobicity, anion, cation, and affinity chromatography can be applied alone or in appropriate combination. . In particular, when affinity chromatography is used, it is preferable to express the enzyme of the present invention as a fusion protein with a tag peptide such as His Tag and use the affinity for such tag peptide. In addition, when the NADP ⁺ -dependent glucose dehydrogenase of the present invention is produced in the host cell, the host cell is recovered by means of centrifugation, filtration or the like. Subsequently, the host cells are disrupted by an enzymatic disruption method such as lysozyme treatment or a physical disruption method such as ultrasonic treatment, freeze-thawing, and osmotic shock. After crushing, the solubilized fraction is collected by means such as centrifugation or filtration. The obtained solubilized fraction can be isolated and purified by treating in the same manner as in the case where it can be produced extracellularly. Here, since the NADP ⁺ dependent glucose dehydrogenase obtained in the present invention has a wide range of pH stability, it is useful and convenient to use pH treatment in combination with the aforementioned isolation and purification steps. The host cell and culture supernatant obtained from the culture contain various proteins derived from the host cell. However, by carrying out pH treatment, contaminating proteins derived from host cells are denatured and condensed and precipitated. In contrast, the enzyme of the present invention is resistant to a wide range of pH and does not denature, so it can be easily separated from host-derived contaminating proteins by centrifugation or the like. In addition, even when the culture solution is used as it is or when a crude extract is used, since other proteins are inactivated by performing pH treatment, particularly treatment at basic pH, the present invention is substantially effective. It can be used as an enzyme solution containing only NADP ⁺ -dependent glucose dehydrogenase. Therefore, even when the NADP ⁺ -dependent glucose dehydrogenase of the present invention is produced by a genetic engineering technique, other proteins derived from the host can be easily removed. Therefore, there is an advantage that the degree of purification can be improved and a highly reliable enzyme can be produced. The performance of the isolated and purified protein can be confirmed by analysis of its physicochemical properties and sequence, for example, by the methods described in Examples 4 to 7.

(Sugar detection method)
The NADP ⁺ dependent glucose dehydrogenase of the present invention can be used for the detection of saccharides in a sample. The detection of saccharides can be performed by measuring the activity of the enzyme that catalyzes the dehydrogenation reaction in a NADP ⁺ dependent manner with respect to the saccharide substrate. The activity of the enzyme can be performed using any method known as a method for measuring the activity of an enzyme that catalyzes a dehydrogenation reaction of a saccharide in a NADP ⁺ dependent manner. For example, the enzyme of the present invention is reacted with a sample to be measured in the presence of NADP ⁺ , and a change in the amount of NADPH produced by the catalytic reaction of the enzyme is detected with a change in absorbance at 340 nm. The change in absorbance can be used as the activity of the enzyme, and the presence of saccharide can be detected by the change in the amount of NADPH. For example, glucose dehydrogenase activity can be measured by directly quantifying the produced NADPH in a catalytic reaction that produces gluconolactone and NADPH from glucose and NADP ⁺ . Examples of the reaction solution include a sugar substrate such as glucose, 2 mM NAD ⁺ or NADP ⁺ , 25 mM MgCl ₂ , 1 M NaCl, and the enzyme solution of the present invention in a 50 mM phosphate buffer (pH 8.8). To make 200 μl. However, this is a standard condition and can be changed as appropriate. The reaction solution can be assayed for activity by measuring the absorbance at 340 nm at 37 ° C and determining the increase in absorbance. The absorbance can be measured using a known microplate reader (Molecular Device). Can be used. And if appropriate test conditions are selected, this change is linearly proportional to the enzyme activity to be measured. At this time, by preparing a standard curve prepared in advance with a sugar substrate solution having a standard concentration within the target concentration range, the sugar substrate concentration can be determined based on the obtained absorbance change value.

Here, as a sample, all samples in which the presence of saccharides, in particular, saccharides that can be a substrate of the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be used. Examples include biological samples derived from organisms such as blood, urine and saliva, brewing of miso and soy sauce, sake, food samples such as fermented foods, environmental samples and the like, but are not limited thereto. Moreover, the sample which performed the appropriate process to these samples as needed may also be included. Here, the NADP ⁺ -dependent glucose dehydrogenase of the present invention exhibits substrate specificity for trace saccharides other than glucose and has high substrate selectivity, and thus is suitably used for detection of these trace saccharides. The In particular, it can be suitably used for detecting trace saccharides in brewed foods such as soy sauce, particularly ribose. In general, in the food field, saccharides are detected using a differential refractive index method. However, in the case of brewed foods such as soy sauce, glucose can be detected but trace saccharides other than glucose (for example, ribose). , Fucose, sucrose, xylose, etc.) were difficult to detect. In addition, detection by electrochemical detection methods was difficult because the detection was hindered by a large amount of amino acids contained in the brewed food. Furthermore, the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be suitably used for detection of saccharides in samples having a high salt concentration, for example, brewed foods, from the viewpoint of salt tolerance.

(Biosensor for detecting sugar)
The present invention provides a biosensor for saccharide detection using the NADP ⁺ dependent glucose dehydrogenase of the present invention. The saccharide sensor is configured by providing a working electrode having glucose dehydrogenase immobilized on an electrode material and a counter electrode thereof. If necessary, a three-electrode system may be configured by providing a reference electrode. As the electrode, carbon, gold, platinum or the like can be used. Immobilization of the enzyme on the electrode material can be performed by a known method. For example, it is possible to use a carrier binding method that is immobilized through physical adsorption, ionic bond, or covalent bond. In addition, a cross-linking method in which cross-linking and fixing is performed with a reagent having a divalent functional group such as glutaraldehyde can be used. Furthermore, polysaccharides such as alginic acid and carrageenan, gels having a network structure such as polyacrylamide, and semi-permeable It is also possible to use a comprehensive method that closes and immobilizes the membrane. The coenzyme NADP ⁺ required for the catalytic activity of the glucose dehydrogenase of the present invention, an oxidase (such as diaphorase) having the ability to oxidize NADPH, which is a reduced form of NADP ⁺ , an electron mediator (ferrocene, dichloroindophenol) Etc.) is also configured to be fixed on the electrode material as required.

In the measurement of saccharides, for example, when a sample to be measured is brought into contact, the sugar substrate in the sample reacts with the NADP ⁺ -dependent glucose dehydrogenase of the present invention immobilized on the working electrode, and then the electron mediator is reduced. The A voltage is applied to the electrode system to oxidize the reductant of the electron acceptor, and the sugar substrate in the sample can be detected by the change in the obtained oxidation current value. At this time, the sugar substrate concentration can be obtained based on the obtained oxidation current value by preparing a standard curve prepared in advance with a sugar substrate solution having a standard concentration within the target concentration range.

Here, as a sample, all samples in which the presence of saccharides, in particular, saccharides that can be a substrate of the NADP ⁺ -dependent glucose dehydrogenase of the present invention can be used. Examples include biological samples derived from organisms such as blood, urine and saliva, brewing of miso and soy sauce, sake, food samples such as fermented foods, environmental samples and the like, but are not limited thereto. Moreover, the sample which performed the appropriate process to these samples as needed may also be included. In particular, as described above, the NADP ⁺ -dependent glucose dehydrogenase of the present invention is suitable for detection of saccharides in brewed foods such as soy sauce, so that a biosensor for detecting saccharides in brewed foods such as soy sauce It is particularly preferably used.

[Examination Example 1] Examination of cloning of nucleic acid molecule encoding glucose dehydrogenase Cloning of nucleic acid molecule encoding glucose dehydrogenase (hereinafter referred to as "glucose dehydrogenase gene") from highly halophilic bacteria. Went. Here, PCR conditions and degenerate primers for gene cloning using PCR were examined.

First, degenerate primers for gene cloning were designed based on the amino acid sequences of two known glucose dehydrogenases. Specifically, the amino acid sequence (SEQ ID NO: 3) of glucose dehydrogenase derived from Halobacterium mediterranei and the amino acid sequence of glucose dehydrogenase derived from Thermoplasma acidophilum (SEQ ID NO: 3) The homologous region between 4) and 4) was selected and designed based on this amino acid sequence. At this time, two types of primer sets (a first primer set and a second primer set) were synthesized depending on the difference in the selected homologous region. The selected homologous region is indicated by an arrow in FIG. In FIG. 1, it is abbreviated as “Hme GDH”, a glucose dehydrogenase derived from Halobacteria Mediterranei, and this is derived from Haloferax mediterranei described in the above [Background Art] section. It is the same as glucose dehydrogenase. Also. Glucose dehydrogenase derived from Thermoplasma acidophilum is abbreviated as “Tac GDH”.

Primer sequence information is as follows.
<First primer set>
Primer 1: 5'-TTNGTNTTNGGNCAYGARGC-3 '(SEQ ID NO: 5)
Primer 2: 5'-CCNARNARNRYNMCNACNCCRTT-3 '(SEQ ID NO: 6)
<Second primer set>
Primer 1b: 5′-GTNGTNCCNHYNGTNMGNMGNCC-3 ′ (SEQ ID NO: 7)
Primer 2: 5'-CCNARNARNRYNMCNACNCCRTT-3 '(SEQ ID NO: 6)

  As the template DNA, genomic DNA extracted and purified from Thermoplasma acidophilum and Halobacterium Mediterranei, which was the basis of the primer set design described above, was used.

  A PCR reaction solution was prepared using the three types of PCR reagents described above. In other words, 50 pmol of each of the above primers and 200 ng of template DNA, prepared according to the manufacturer's instructions using Multiplex-PCR Kit (Takara Bio), LA-Taq PCR Kit (Takara Bio) Three types were examined: those prepared and PrimeStar PCR Kit (manufactured by Takara Bio Inc.). In the PCR temperature program, 35 cycles of amplification reaction were performed, with heat denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 2 minutes as one cycle.

  The reaction solution after the PCR amplification reaction was subjected to agarose gel electrophoresis, and then the amplification product band was visualized by ethidium bromide staining of the gel to confirm the success or failure of cloning of the glucose dehydrogenase gene.

The results are shown in FIG.
In FIG. 2, lanes 1 to 4 are the results of gene cloning using PCR amplification using Multiplex-PCR Kit. Lanes 1 and 2 are results of using the genomic DNA of Halobacterium Mediterranei as a template. Lane 1 shows the results of the first primer set, and Lane 2 shows the results of the second primer set. Lanes 3 to 4 show the results of using Thermoplasma acidophilum genomic DNA as a template. Lane 3 shows the results with the first primer set, and Lane 4 shows the results with the second primer set.
In FIG. 2, lanes 5 to 8 are the results of gene cloning using PCR amplification by LA-Taq PCR Kit. And lanes 5-6 are the results of using the genomic DNA of Halobacterium Mediterranei as a template, lane 5 shows the results with the first primer set, and lane 6 shows the results with the second primer set. Lanes 7 to 8 show the results of using Thermoplasma acidophilum genomic DNA as a template. Lane 7 shows the results with the first primer set, and Lane 8 shows the results with the second primer set.
In FIG. 2, lanes 9 to 12 are the results of gene cloning using PCR amplification by PrimeStar PCR Kit. And lanes 9-10 are the results of using the genomic DNA of Halobacterium Mediterranei as a template, lane 9 shows the results with the first primer set, and lane 10 shows the results with the second primer set. Lanes 11 to 12 show the results of using Thermoplasma acidophilum genomic DNA as a template. Lane 11 shows the results of the first primer set, and Lane 12 shows the results of the second primer set.
In FIG. 2, lane M is a molecular weight marker.

  From the results shown in FIG. 2, it was confirmed that a single amplification product derived from the target gene was obtained when the first primer set was used as a degenerate primer and the Multiplex-PCR Kit was used as a PCR reagent (lane). 1 and 3). Therefore, it was confirmed that the glucose dehydrogenase gene could be cloned by the combination of such primer and PCR. On the other hand, when the second primer set was used, the glucose dehydrogenase gene could not be cloned significantly (lanes 2 and 4). It turned out to be influenced by. It has also been found that the success or failure of cloning depends on the PCR reagent. In particular, since the Multiplex-PCR Kit has an effect of reducing non-specific reaction products, it is understood that significant glucose dehydrogenase gene cloning has been achieved by reagent components for reducing such an effect. The

[Experimental Example 2] Cloning of glucose dehydrogenase gene Cloning of the glucose dehydrogenase gene was examined from highly halophilic bacteria. Here, in gene cloning using PCR, highly halophilic bacteria to be cloned were examined.

In this example, an attempt was made to clone a glucose dehydrogenase gene from a bacterium belonging to the genus Halobacterium , which is a highly halophilic archaea. Specifically, Halobacterium varismortis ( Halobacterium vallismortis : NBRC 14741, NBRC 14955), Halobacterium pharaonis ( Halobacterium pharaonis : NBRC 14720), Halobacterium sodomense (NRBC 14740), Halobacterium・ Saccharborum ( Halobacterium saccharovorum : NRBC 14717), Halobacterium volcanii (NRBC 14742), Halobacterium salinarium (NRBC 14718), Halobacterium halobium (NRBC 14716), Halobacterium bacterium-Kuchiruburumu (Halobacterium cutirubrum: NRBC 14715) was examined. These microorganisms were all sold by the National Institute for Product Evaluation Technology.

  As the primer, the first primer set confirmed as a suitable primer for glucose dehydrogenase gene cloning in Examination Example 1 was used.

  As template DNA, genomic DNA extracted and purified from the aforementioned bacteria was used. In addition, genomic DNA from Halobacterium Mediterraney (NRBC 14739) and Thermoplasma acidophilum, which were the basis for primer preparation, was also used as a template and used as a positive control.

  The PCR reaction solution was prepared according to the manufacturer's instructions using Multiplex-PCR Kit (manufactured by Takara Bio Inc.) containing 50 pmol of each of the above primers and 200 ng of template DNA. In the PCR temperature program, 35 cycles of amplification reaction were performed, with heat denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 2 minutes as one cycle.

  The reaction solution after the PCR amplification reaction was subjected to agarose gel electrophoresis, and the presence or absence of glucose dehydrogenase gene cloning was confirmed by confirming the amplification product band.

The results are shown in FIG.
In FIG. 3, lane 1 is Halobacterium barismortis, lane 2 is Halobacterium pharaonis, lane 3 is Halobacterium sodomense, lane 4 is Halobacterium saccharborg, lane 5 is Halobacterium volcani, lane 6 shows the result from the genomic DNA derived from Halobacterium salinum, lane 7 shows the result from Halobacterium halobium, and lane 8 shows the result from the genomic DNA derived from Halobacterium cutyllum.
In FIG. 3, lanes 9 and 10 are positive controls, lane 9 shows the results from Thermoplasma acidophilum, and lane 10 shows the results from the genomic DNA derived from Halobacterium Mediterranei.
In FIG. 3, lane M is a molecular weight marker.

  From the result of FIG. 3, it was confirmed that an amplification product that seems to be a glucose dehydrogenase gene can be obtained from Halobacterium varismortis (lane 1). Here, bands were also confirmed in lanes 2 and 4 to 8, but these were all junk. Therefore, the amplification product that was thought to be derived from the glucose dehydrogenase gene was confirmed only in Halobacteria varismortis, and this was the only successful example. In addition, since the amplification of the glucose dehydrogenase gene was confirmed from the thermoplasma acidophilum and the genomic DNA of Halobacterium Mediterranei based on the primer design, it is considered that there is no problem in the PCR amplification process.

Example 1 Cloning and Sequencing of Glucose Dehydrogenase Gene from Halobacteria varismortis Based on the results of Examination Examples 1 and 2, cloning of glucose dehydrogenase gene from genomic DNA of halobacteria varismortis Both were performed and their sequencing was performed.

  Gene cloning was performed using PCR. As the primer, the first primer set confirmed as a primer for cloning glucose dehydrogenase gene in Examination Example 1 was used. As the template DNA, genomic DNA extracted and purified from Halobacterium varismortis in which the amplification product was confirmed by PCR using the first primer set in Examination Example 2 was used.

  The PCR reaction solution was prepared according to the manufacturer's instructions using Multiplex-PCR Kit (manufactured by Takara Bio Inc.) containing 50 pmol of each of the above primers and 200 ng of template DNA. In the PCR temperature program, 35 cycles of amplification reaction were performed, with heat denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 2 minutes as one cycle.

  The reaction solution after the PCR amplification reaction was subjected to agarose gel electrophoresis, and the presence or absence of glucose dehydrogenase gene cloning was confirmed by confirming the amplification product band.

The results of agarose gel electrophoresis are shown in FIG.
In FIG. 4 (A), lane 1 shows the result from the genomic DNA derived from Halobacterium varismortis. Lane 2 is a negative control and shows the result of PCR performed in the same manner without template DNA. Lane M is a molecular weight marker.
From the result of FIG. 4 (A), an amplification product that was considered to be a glucose dehydrogenase gene was confirmed from the halobacterium varismortis at about 700 bp (lane 1).

  Subsequently, the band of the amplified fragment was cut out and purified from the gel after electrophoresis using Gel purification Kit (GE Healthcare) according to the manufacturer's instructions. The purified amplified fragment of about 700 bp was subcloned into pUC18 using Cloning kit (manufactured by Takara Bio Inc.), and its base sequence was determined. The obtained partial sequence information is shown as SEQ ID NO: 8 in the sequence listing.

  Next, in order to clone the unamplified region, inverse PCR was performed to determine the entire nucleotide sequence of the glucose dehydrogenase gene. First, two kinds of primers (primers 3 and 4) for inverse PCR were synthesized. The primer was designed based on the base sequence of the amplification region of about 700 bp obtained above. Specifically, it was designed to be complementary to both ends of the amplified region, but to extend a DNA chain from the amplified region to the sequence of the unamplified region, contrary to normal PCR. Here, the region on the gene selected in the primer design is indicated by an arrow in FIG.

Primer sequence information is as follows.
Primer 3: 5'-GTCCCGTTTGGAGACTCGACGACACCGACG-3 '(SEQ ID NO: 9)
Primer 4: 5'-GCGTTCTCAACAATAGAGGCACTCGATGCA-3 '(SEQ ID NO: 10)

Template DNA for inverse PCR was prepared as follows. First, genomic DNA of Halobacteria varismortis was cleaved with the restriction enzyme Alu I, and then the DNA fragment was purified using Gel purification Kit (GE Healthcare) according to the manufacturer's instructions. The purified DNA fragment is subjected to intramolecular ligation using Ligation Kit (manufactured by Takara Bio Inc.) and then purified using Gel purification Kit (manufactured by GE Healthcare) according to the manufacturer's instructions. Template DNA for PCR was prepared.

  A PCR reaction solution was prepared according to the manufacturer's instructions using a Multiplex-PCR Kit (manufactured by Takara Bio Inc.) containing 50 pmol of each of the primers 3 and 4 and 200 ng of template DNA. In the PCR temperature program, 35 cycles of amplification reaction were performed with heat denaturation at 94 ° C. for 30 seconds, annealing at 60 ° C. for 30 seconds, and extension reaction at 72 ° C. for 90 seconds as one cycle.

  The reaction solution after the PCR amplification reaction was subjected to agarose gel electrophoresis and the amplification product band was confirmed to confirm the amplification of the unamplified region of the glucose dehydrogenase gene.

The results of agarose gel electrophoresis are shown in FIG.
In FIG. 4 (B), lane 1 shows the results when a template prepared from the genomic DNA derived from Halobacterium varismortis for inverse PCR is used as a template. Lane 2 is a negative control, shows the results of PCR was performed in the same manner as the template without cutting the genomic DNA from Harobakuterimu Barris malty scan the restriction enzyme Alu I. Lane M is a molecular weight marker.

  Subsequently, the band of the amplified fragment was cut out and purified from the gel after electrophoresis using Gel purification Kit (GE Healthcare) according to the manufacturer's instructions. The purified amplified fragment was subcloned into pUC18 using a Cloning kit (manufactured by Takara Bio Inc.), and its base sequence was determined. And the determined base sequence was connected, the whole base sequence was determined, and the deduced amino acid sequence was obtained. The base sequence of the full-length glucose dehydrogenase gene derived from Halobacterium varismortis and its deduced amino acid sequence are shown as SEQ ID NOS: 1 and 2 in the sequence listing and also in FIG. Hereinafter, the glucose dehydrogenase gene derived from Halobacteria varismortis obtained in this example is referred to as “hva dgh gene”, and the glucose dehydrogenase encoded thereby is referred to as “Hva GDH”. .

Furthermore, FIG. 6 shows an amino acid sequence alignment with a known halophilic bacteria-derived glucose dehydrogenase. In FIG. 6, the glucose dehydrogenase of Haloarcula marismortui (SEQ ID NO: 11) is “Hma GDH” and the glucose dehydrogenase (SEQ ID NO: 3) derived from Halobacterium mediterranei is “Hme GDH”. And Haloquadratum walsbyi- derived glucose dehydrogenase (SEQ ID NO: 12) is abbreviated as “Hwa GDH”.

[Example 2] Preparation of recombinant vector In order to express the hva dgh gene obtained in Example 1 in E. coli cells, a recombinant vector used for transformation was constructed.

The hva dgh gene to be inserted into the vector was prepared as follows using PCR. First, a PCR primer was designed based on the base sequence of the full-length hva dgh gene determined in Example 2, and at this time, the hva dgh gene was designed to be amplified in a form including an additional sequence for insertion into a vector. . Specifically, a primer designed to add an Nde I site to the start codon portion of the structural gene of the hva dgh gene (primer 5) and a primer designed to add a Hind III site immediately after the stop codon (primer 6 ) Was synthesized.

Primer sequence information is as follows.
Primer 5: 5'-AGATATACATATGCATGCAATCGCCGTCAA-3 '(SEQ ID NO: 13)
Primer 6: 5'-CTAAAGCTTATACCCTGAAATCTACGT-3 '(SEQ ID NO: 14)

  As the template DNA, genomic DNA extracted and purified from Halobacterium varismortis was used as in Example 1.

  A PCR reaction solution was prepared according to the manufacturer's instructions using a Multiplex-PCR Kit (manufactured by Takara Bio Inc.) containing 50 pmol of each of the primers 1 and 2 and 200 ng of template DNA. The PCR temperature program consists of heat denaturation at 94 ° C for 30 seconds, heat denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 15 seconds, and extension reaction at 72 ° C for 60 seconds. 35 cycles of amplification reaction and a final extension reaction at 72 ° C. for 10 minutes.

The amplified fragment after PCR amplification reaction, was digested with Nde I and Hind III, and inserted a protein expression vector pET22b and Nde I and Hind III sites of lac promoter downstream of the respective pET23b. The resulting plasmids were named hva dgh / pET22b and hva dgh / pET23b, respectively.

In addition, a recombinant vector for expressing glucose dehydrogenase fused with a histidine tag in a host cell was also prepared. First, the hva dgh gene to be inserted into the vector was prepared as follows using PCR. The PCR primer was designed based on the base sequence of the full-length hva dgh gene determined in Example 2 so that it could be amplified including additional sequences for insertion into a vector and histidine tag. Specifically, a primer (primer 5) designed to add an Nde I site to the start codon portion of the hva dgh gene, and a stop codon are deleted, and a Hind III site is added immediately thereafter. The designed primer (Primer 7) was synthesized.

Primer sequence information is as follows.
Primer 5: 5'-AGATATACATATGCATGCAATCGCCGTCAA-3 '(SEQ ID NO: 13)
Primer 7: 5'-CCGCAAGCTTTACCCTGAAATCTACGTACG-3 '(SEQ ID NO: 15)

  A PCR reaction solution was prepared according to the manufacturer's instructions using a Multiplex-PCR Kit (manufactured by Takara Bio Inc.) containing 50 pmol of each of the primers 5 and 7 and 200 ng of template DNA. In the PCR temperature program, heat denaturation at 94 ° C. for 30 seconds, heat denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 15 seconds, and extension reaction at 72 ° C. for 60 seconds are defined as one cycle. 35 cycles of amplification reaction and a final extension reaction at 72 ° C. for 10 minutes were performed.

The amplified fragment after PCR amplification reaction, was digested with Nde I and Hind III, and inserted into the Nde I and Hind III sites of lac promoter downstream of the protein expression vector pET22b. The obtained plasmid was designated as hva dgh-his tag / pET22b.

[Example 3] Expression of recombinant Hva GDH The hva dgh gene obtained in Example 2 was expressed in E. coli cells to produce recombinant Hva GDH.

(Plasmid hva dgh / pET22b-synthesis in E. coli cells)
Escherichia coli BL21 (DE3) strain was transformed with the plasmid hva dgh / pET22b obtained in Example 2. The obtained transformant was cultured in an LB medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 50 μg / ml of ampicillin until OD ₆₀₀ = 0.6. Next, to increase the expression level, IPTG having a final concentration of 1 mM was added and further cultured for 4 hours. After incubation, the cells were collected by centrifugation and suspended in 50 mM phosphate buffer (pH 7.0). Subsequently, the microbial cells were crushed by ultrasonic crushing treatment, and then the microbial cell residues were removed to obtain a cell-free extract. This cell-free extract was treated with SDS and then subjected to 12.5% SDS-polyacrylamide gel electrophoresis. After electrophoresis, the expression of the recombinant protein was confirmed by visualizing the protein by Coomassie Brilliant Blue (hereinafter abbreviated as “CBB”) staining (Quick CBB Kit, manufactured by Wako Pure Chemical Industries, Ltd.) (FIG. 7A). ), Lane 1). Moreover, the expression of the recombinant protein was confirmed by the same procedure except that the culture was performed without adding IPTG (FIG. 7 (A), lane 2).

  From the result of FIG. 7A, a protein band derived from Hva GDH could be confirmed at the position of the estimated molecular weight (about 40 kDa) (lane 1). In addition, the result of electrophoresis in the absence of IPTG could not confirm protein expression (lane 2), and it was confirmed that the protein band confirmed in lane 1 was derived from the expression product.

(Plasmid hva dgh-his tag / pET22b-affinity column chromatography)
Recombinant Hva GDH expressed in E. coli cells was separated and purified using an affinity column. First, the plasmid hva dgh-his tag / pET22b obtained in Example 2 described above was transformed into E. coli according to the same procedure as described above, and then the transformant was cultured. Hva GDH fused with His Tag was expressed. After culture, the cells were collected by centrifugation and suspended in a phosphate buffer (20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.4). Subsequently, the cells were subjected to ultrasonic disruption treatment (10 sets of ultrasonic treatment for 30 seconds and ice-cooled for 30 seconds), and the protein was purified by affinity column chromatography.

Details of the column chromatography are as follows.
1. Wash the column (Ni Sepharose 6 Fast Flow: manufactured by GE Healthcare) with 25 ml of pure water.
2. Inject 2.5 ml of 0.1 M NiSO ₄ with a syringe.
3. Wash with 25 ml of pure water.
4. Equilibrate the column with 50 ml of starting buffer (phosphate buffer containing 10 mM imidazole).
5. Add sample.
6. Wash with 50 ml of starting buffer.
7). Inject 30 ml of elution buffer containing 100-500 mM imidazole into phosphate buffer (20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.4) to elute the protein.
8). Desalted with desalting column and concentrated with centricon.

  To the purified protein solution, add 2 volumes of lysate (2 × SDS sample buffer: 125 mM Tris-HCl, pH 6.8, 4% SDS, 5% 2-mercaptoethanol, 20% glycerol, 0.01% bromophenol. Blue) was added and incubated at 85 ° C. for 3 minutes. Subsequently, a part thereof was subjected to polyacrylamide gel (Ato) electrophoresis, and the protein was visualized by CBB staining (FIG. 7 (B), lane 2). In addition, a protein in which the aforementioned plasmid hva dgh / pET22b was expressed in E. coli was also subjected to electrophoresis (FIG. 7B, lane 1). This corresponds to lane 1 in FIG. Lane M shows a protein size marker.

  From the results shown in FIG. 7B, separation and purification of Hva GDH were confirmed by affinity column chromatography (lane 2).

(Plasmid hva gdh / pET23b-cell-free protein synthesis)
Using the plasmid hva gdh / pET23b obtained in Example 2 described above as a template DNA, the protein was tested within the test using a cell-free protein synthesis system (Rapid Translation System (RTS), manufactured by Roche Diagnostics). Synthesized. Subsequently, the synthesized protein was fluorescently labeled using a translation protein labeling system (FluoroTect (registered trademark) GreenLys in vitro Translation Labeling System, manufactured by Promega). Subsequently, the protein solution after fluorescent labeling was added to a double volume of lysate (2 × SDS sample buffer: 125 mM Tris-HCl, pH 6.8, 4% SDS, 5% 2-mercaptoethanol, 20% glycerol, 0.01 % Bromophenol blue), and kept at 85 ° C. for 3 minutes. A part of the solution after the incubation was collected and subjected to polyacrylamide gel (Ato) electrophoresis. The fluorescence signal on the gel after electrophoresis was detected with a fluorescence image analyzer (FluorImager 595: GE Healthcare) (FIG. 7C).

  From the results shown in FIG. 7C, a protein band derived from Hva GDH could be confirmed at the position of the estimated molecular weight (about 40 kDa) even in the cell-free protein synthesis system.

[Example 4] Activity measurement of recombinant Hva GDH-1
The glucose dehydrogenase activity of recombinant Hva GDH was measured. In this example, coenzyme dependency was confirmed.

The glucose dehydrogenase activity was measured by measuring NADH and NADPH produced by the catalytic reaction of glucose dehydrogenase by measuring the absorbance at a wavelength of 340 nm and setting it as the glucose dehydrogenase activity. That is, glucose dehydrogenase activity can be measured by directly quantifying the produced NADH or NADPH in a catalytic reaction that produces gluconolactone and NADH or NADPH from glucose and NAD ⁺ or NADP ⁺ .

Here, the enzyme solution of Hva GDH purified by affinity column chromatography in Example 3 was used as an activity measurement target. The reaction solution was 25 mM glucose, 2 mM NAD ⁺ or NADP ⁺ , 25 mM MgCl ₂ , 1 M NaCl, and enzyme solution (enzyme amount: 1 ng) in 50 mM phosphate buffer (pH 8.8). To 200 μl. Subsequently, this reaction solution was subjected to an activity evaluation test by measuring the absorbance at 340 nm at 37 ° C. and determining the increase in absorbance. The absorbance was measured by using a microplate reader (manufactured by Molecular Devices) and measuring the time course of the absorbance at 340 nm every 30 seconds. At this time, as a control, the activity was also measured when the reaction was carried out by adding only NADP ⁺ without adding the substrate.

  The results are shown in FIG. FIG. 8 is a graph with absorbance on the vertical axis and time on the horizontal axis.

From the results shown in FIG. 8, it was confirmed that Hva GDH is a NADP ⁺ -dependent dehydrogenase that depends specifically on NADP ⁺ as a coenzyme and that uses glucose as a substrate.

[Example 5] Activity measurement of recombinant Hva GDH-2
The glucose dehydrogenase activity of recombinant Hva GDH was measured. In this example, the influence of the salt concentration on the activity was confirmed.

In this example, as in Example 4, the enzyme solution of Hva GDH purified by affinity column chromatography in Example 3 was used as the measurement target. Then, in order to confirm the influence of the salt concentration on the glucose dehydrogenase activity, the glucose dehydrogenase activity of Hva GDH when the NaCl concentration of the enzyme reaction solution was changed was measured. Specifically, the NaCl concentration of the reaction solution of Example 4 was changed in the final concentration range of 0, 1.0, and 2.0 M, and glucose dehydrogenase in the presence of coenzyme NADP ⁺ was otherwise obtained in the same manner as in Example 4. Activity was measured. At this time, as a control, the activity was also measured when the reaction was carried out at a NaCl concentration of 1.0 M without adding a substrate.

  The results are shown in FIG. FIG. 9 is a graph with absorbance on the vertical axis and time on the horizontal axis. From the results of FIG. 9, the optimum reaction concentration of Hva GDH with NaCl was 1.0 M final concentration.

[Example 6] Activity measurement of recombinant Hva GDH-3
The glucose dehydrogenase activity of recombinant Hva GDH was measured. In this example, the effect of pH on activity was confirmed.

In this example, as in Example 4, the enzyme solution of Hva GDH purified by affinity column chromatography in Example 3 was used as the measurement target. And in order to confirm the influence which pH has on an enzyme activity, the glucose dehydrogenase activity of Hva GDH when the pH of an enzyme reaction liquid was changed was measured. Specifically, the pH of the reaction solution of Example 4 was changed in the range of pH 5.0, 7.0, and 8.8, and glucose dehydrogenase activity in the presence of coenzyme NADP ⁺ was measured in the same manner as in Example 4 except that. did. At this time, as a control, the activity was also measured when the reaction was carried out at pH 8.8 without adding a substrate.

  The results are shown in FIG. FIG. 10 is a graph with absorbance on the vertical axis and time on the horizontal axis. From the results shown in FIG. 10, it was found that Hva GDH shows activity in the range of pH 7.0 to 8.8.

[Example 7] Activity measurement of recombinant Hva GDH-4
The glucose dehydrogenase activity of recombinant Hva GDH was measured. In this example, the substrate specificity of Hva GDH was confirmed.

In this example, as in Example 4, the enzyme solution of Hva GDH purified by affinity column chromatography in Example 3 was used as the measurement target. And in order to confirm the substrate specificity of Hva GDH, the reactivity with respect to various saccharides was examined. Here, the reactivity with respect to six types of monosaccharides (glucose, fucose, sucrose, ribose, xylose, mannose) was measured in the same manner as in Example 4 to measure glucose dehydrogenase activity in the presence of the coenzyme NADP ⁺ . At this time, as a control, activity was also measured when the reaction was carried out without adding a substrate.

  The results are shown in FIGS. 11 (A) and 11 (C). FIG. 11A is a graph with absorbance on the vertical axis and time (minutes) on the horizontal axis. From the results shown in FIG. 11 (A), it was confirmed that Hva GDH recognizes glucose, fucose, sucrose, and ribose as substrates among the saccharides confirmed in this example and exerts its catalytic activity. Moreover, although it was lower than the activity with respect to the above-mentioned four saccharides also with respect to xylose, it was confirmed that it recognizes as a substrate. On the other hand, it was confirmed that mannose was hardly recognized as a substrate.

  FIG. 11C is a graph showing the relative activity for each sugar substrate when the activity for glucose is 100. The substrate sample number is plotted on the horizontal axis, the relative activity on the vertical axis, and the activity of Hva GDH is represented by a white bar graph. Sample number 1 is mannose, sample number 2 is xylose, sample number 3 is ribose, sample number 4 is fucose, sample number 5 is sucrose, and sample number 6 is glucose.

[Comparative Example 1] Substrate specificity of known Thermoplasma acidophilum- derived glucose dehydrogenase The substrate specificity of a known glucose dehydrogenase, Thermoplasma acidophilum- derived glucose dehydrogenase "Tac GDH") was confirmed.

The substrate specificity of Tac GDH (Sigma Aldrich, catalog No. G5909), a known glucose dehydrogenase, was confirmed. In the same manner as in Example 6, glucose dehydrogenase activity in the presence of the coenzyme NADP ⁺ was measured.

  The results are shown in FIG. 11 (B) and FIG. 11 (C). FIG. 11B is a graph with absorbance on the vertical axis and time (minutes) on the horizontal axis, as in FIG. 11A. From the result of FIG. 11 (B), it was confirmed that Tac GDH recognizes glucose and fucose as substrates among the saccharides confirmed in this example and exerts its catalytic activity. Moreover, although it was lower than the activity with respect to the above-mentioned two saccharides also with respect to xylose, it was confirmed that it recognizes as a substrate. On the other hand, it was confirmed that ribose, sucrose, and mannose are hardly recognized as substrates.

  FIG. 11C is a graph showing the relative activity for each sugar substrate when the activity for glucose is 100. The sample number is plotted on the horizontal axis, the relative activity on the vertical axis, and the activity of Tac GDH is represented by a shaded bar graph. Sample number 1 is mannose, sample number 2 is xylose, sample number 3 is ribose, sample number 4 is fucose, sample number 5 is sucrose, and sample number 6 is glucose.

From the results of Example 7 and Comparative Example 1, it was found that the Hva GDH of the present invention has a substrate selectivity different from that of the known enzyme Tac GDH in that it can react with ribose and sucrose as substrates. Further, regarding substrate selectivity for other known enzymes, glucose dehydrogenase having reactivity with ribose in addition to glucose is not known. For example, glucose dehydrogenase derived from the thermophilic and halophilic archaeon Halobacterium Mediterranei (Maria-Jose Bonete et al., FEBS Letters, 1996, Volume 383, Volume 227). ˜229) and glucose dehydrogenase derived from Sulfolobus solfataricus (Paola Giardina et al., The Biochemical journal, 1986, Vol. 239, No. 3, pp. 517-522) are not reactive with ribose It has been known. Therefore, it was found that the Hva GDH of the present invention is a novel enzyme having physicochemical properties different from those of known glucose dehydrogenases.

The present invention relates to a NADP ⁺ -dependent glucose dehydrogenase and a method for using the same, and can be used in various industrial fields such as medical, food, and environmental fields.

Diagram showing the region of homology between the amino acid sequences of known glucose dehydrogenases selected for the design of degenerate primers for Hva GDH cloning The figure which shows the result of examination example 1 which examined the cloning (selection of primer and PCR reagent) of Hva GDH The figure which shows the result of the examination example 2 which examined the cloning of Hva GDH (selection of object extreme thermophilic halobacteria) (A) is a figure which shows the result (partial amplification) of Example 1 which cloned Hva GDH from Halobacterium varismortis genomic DNA, (B) is cloning Hva GDH from Halobacterium varismortis genomic DNA. The figure which shows the result (amplification of an unamplified area | region) of Example 1 The figure which shows the full length base sequence and deduced amino acid sequence of the hva gdh gene of this invention Diagram showing amino acid sequence alignment with known glucose dehydrogenase (A) is a figure which shows the result of Example 3 which confirmed the synthesis | combination (recombinant protein expression system by E. coli) of this invention, (B) is the synthesis | combination (His Tag) of Hva GDH of this invention. (C) shows the results of Example 3 in which the synthesis of Hva GDH of the present invention (cell-free protein synthesis system) was confirmed. Illustration The figure which shows the result of Example 4 which confirmed the activity (coenzyme dependence) of recombinant Hva GDH The figure which shows the result of Example 5 which confirmed the activity (salt tolerance) of recombinant Hva GDH The figure which shows the result of Example 6 which confirmed the activity (pH stability) of recombinant Hva GDH. (A) is a figure which shows the result of Example 7 which confirmed the activity (substrate specificity) of recombinant Hva GDH, (B) of the comparative example 1 which confirmed the activity of the known glucose dehydrogenase. It is a figure which shows a result, (C) is a figure which shows the result of Example 7 which confirmed the activity of recombinant Hva GDH, and the comparative example 1 which confirmed the activity of the known glucose dehydrogenase.

Claims (11)

The following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (b) at least one modification selected from deletion, substitution, insertion and addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 A protein comprising an amino acid sequence and having the following physicochemical properties (1) to (3):
(1) Oxidized nicotinamide adenine dinucleotide phosphate is specifically required as a coenzyme and catalyzes a dehydrogenation reaction that produces gluconolactone from glucose (2) catalyzes a dehydrogenation reaction of ribose (3) pH tolerance is stable at pH 7.0 to 8.8, and optimum pH is 8.8 The protein according to claim 1, which has one or more physicochemical properties selected from the following (4) to (7).
(4) The salt tolerance is stable at a salt concentration of 0 to 2.0 M, and the optimum salt concentration is 1.0 M. (5) Catalyze dehydrogenation of sucrose (6) Catalyze dehydrogenation of xylose ( 7) Catalyze dehydrogenation of fucose   The protein according to claim 1 or 2, which is a recombinant.   The nucleic acid molecule which codes the protein as described in any one of Claims 1-3. A nucleic acid molecule comprising the following polynucleotide (c) or (d):
(C) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1,
(D) a polynucleotide that hybridizes with a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 under stringent conditions and encodes a protein having the following physicochemical properties (1) to (3): (1) oxidation Type nicotinamide adenine dinucleotide phosphate is specifically required as a coenzyme, catalyzing the reaction of oxidizing glucose to produce gluconolactone (2) catalyzing the dehydrogenation of ribose (3) pH tolerance, Stable at pH 7.0 to 8.8, optimal pH is 8.8 The nucleic acid molecule according to claim 5, wherein the protein has one or more physicochemical properties selected from the following (4) to (7).
(4) The salt concentration tolerance is stable at a salt concentration of 0 to 2.0 M, and the optimum salt concentration is 1.0 M. (5) Catalyze dehydrogenation of sucrose (6) Catalyze dehydrogenation of xylose (7) Catalyze dehydrogenation of fucose   A recombinant vector containing the nucleic acid molecule according to any one of claims 4 to 6.   A transformant containing the recombinant vector according to claim 7.   A method for producing glucose dehydrogenase, comprising culturing the transformant according to claim 8 and collecting a protein having an ability to catalyze a dehydrogenation reaction of glucose from the obtained culture.   A biosensor for detecting a saccharide, wherein the protein according to any one of claims 1 to 3 is immobilized on an electrode.   The biosensor for detecting a saccharide according to claim 10, wherein the saccharide is one or more saccharides selected from glucose and ribose.