JP2017112860A - Novel glucose dehydrogenase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide FAD-GDH useful for glucose measurement and to provide applications thereof.SOLUTION: The present invention provides an enzyme agent for glucose measurement containing glucose dehydrogenase as an active ingredient, the enzyme agent having (1) an action: catalyzing a reaction to oxidize a hydroxyl group of glucose in the presence of electron acceptors and produce glucono δ-lactone; (2) substrate specificity: having the reactivity to D-xylose of 15% or less when the reactivity to D-glucose is 100%; (3) pH stability: being stable at pH 4 to 6; and (4) amino acid sequences: including the amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence 80% identical to the amino acid sequence.SELECTED DRAWING: None

Description

  The present invention relates to a novel glucose dehydrogenase (glucose dehydrogenase). Specifically, the present invention relates to a flavin adenine dinucleotide (FAD) -dependent glucose dehydrogenase (E.C.1.1.99.10) derived from the genus Aspergillus and its use.

  The number of diabetic patients is increasing year by year, and diabetic patients, particularly those who are dependent on insulin, need to monitor blood glucose levels on a daily basis and control blood sugar levels. In recent years, it has become possible to check the blood glucose level of diabetic patients with an auto-blood glucose meter that can easily and accurately measure in real time using an enzyme. Glucose oxidase (EC1.1.3.4) and PQQ-dependent glucose dehydrogenase (EC1.1.5.2) (see, for example, Patent Documents 1 to 3) have been developed for glucose sensors (for example, sensors used in self blood glucose meters). However, oxygen reactivity, reactivity to maltose and galactose became problems. In order to solve this problem, FAD-dependent glucose dehydrogenase (hereinafter abbreviated as “FAD-GDH”) has been developed (see, for example, Patent Documents 4 and 5 and Non-Patent Documents 1 to 4).

  Generally, at the time of a diabetes determination test, not only an oral glucose tolerance test but also an oral xylose tolerance test and a transvenous xylose tolerance test are performed. FAD-GDH is generally known to react with xylose, and when FAD-GDH is used, there is a problem in that it affects blood glucose levels during the above-mentioned load test.

JP 2000-350588 A JP 2001-197888 A JP 2001-346687 A International Publication No. 2004/058958 Pamphlet International Publication No. 2007/139013 Pamphlet International Publication No. 2015/060150 Pamphlet

Studies on the glucose dehydrogenase of Aspergillus oryzae. I. Induction of its synthesis by p-benzoquinone and hydroquinone, T.C.Bak, and R. Sato, Biochim. Biophys. Acta, 139, 265-276 (1967). Studies on the glucose dehydrogenase of Aspergillus oryzae.II.Purification and physical and chemical properties, T.C.Bak, Biochim.Biophys. Acta, 139, 277-293 (1967). Studies on the glucose dehydrogenase of Aspergillus oryzae. III. General structural properties, T.C.Bak, Biochim. Biophys. Acta, 146, 317-327 (1967). Studies on the glucose dehydrogenase of Aspergillus oryzae. IV. Histidyl residue as an active site, T.C.Bak, and R. Sato, Biochim. Biophys. Acta, 146, 328-335 (1967).

  Although FAD-GDH has a problem of reactivity to xylose, it has excellent substrate specificity and is unlikely to be affected by dissolved oxygen in the measurement sample like glucose oxidase. Has been. In putting FAD-GDH into practical use, the reactivity with xylose becomes a problem as described above. In view of such circumstances, it is an object of the present invention to provide a novel FAD-GDH that is highly practical, particularly for glucose sensors, and uses thereof. In addition, although FAD-GDH having low reactivity to xylose has been reported (Patent Document 6), characteristics such as pH stability which are particularly important when applied to a glucose sensor have not been clarified, and its practical use. Target value is unknown.

  In order to solve the above problems, the present inventor conducted large-scale screening for a wide range of microorganisms. As a result, a novel FAD-GDH having low reactivity to xylose was successfully obtained. When the characteristics of the FAD-GDH were examined, it was found that the FAD-GDH was excellent in pH stability, suitable for glucose sensor applications, and highly practical.

  As a result of further studies, the inventors succeeded in identifying the amino acid sequence and gene sequence of a novel FAD-GDH that was successfully obtained. When the identified amino acid sequence was searched as a query sequence in a public database, it was identical to the sequence registered as glucose oxidase. The fact that it was registered as glucose oxidase rather than FAD-GDH indicates that the protein is known as a substance, while an unexpected new use of the protein has been found.

  As described above, the inventors' studies have revealed that a protein known as glucose oxidase actually functions as FAD-GDH and is suitable for glucose sensor applications.

The following invention is based on the above results and considerations.
[1] The following features:
(1) Action: catalyzes a reaction in which glucose hydroxyl group is oxidized to produce glucono-δ-lactone in the presence of an electron acceptor;
(2) Substrate specificity: The reactivity to D-xylose is 15% or less when the reactivity to D-glucose is 100%;
(3) pH stability: stable at pH 4-6;
(4) Amino acid sequence: The amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence that is 80% or more identical to the amino acid sequence;
An enzyme agent for measuring glucose, comprising as an active ingredient glucose dehydrogenase comprising
[2] The enzyme agent for glucose measurement according to [1], wherein the amino acid sequence of the glucose dehydrogenase is 85% or more identical to the amino acid sequence shown in SEQ ID NO: 1.
[3] The enzyme agent for glucose measurement according to [1], wherein the amino acid sequence of the glucose dehydrogenase is an amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1.
[4] The enzyme agent for glucose measurement according to any one of [1] to [3], which is an enzyme derived from Aspergillus kawachi or Aspergillus awamori.
[5] The enzyme agent for glucose measurement according to [4], wherein Aspergillus kawachi is IFO 4308 strain.
[6] A glucose measurement method comprising measuring glucose in a sample using the glucose dehydrogenase defined in any one of [1] to [5].
[7] A reagent for measuring glucose, comprising the glucose dehydrogenase defined in any one of [1] to [5].
[8] A glucose measurement kit comprising the glucose measurement reagent according to [7].
[9] A glucose sensor comprising a glucose dehydrogenase defined in any one of [1] to [5].

The result of the analysis by SDS-PAGE of the enzyme (purified enzyme) refine | purified from the culture solution of Aspergillus awamori No.1731 stock | strain. M represents a molecular weight marker (200, 116, 97.2, 66.4, 44.3 KDa from the top), and the lane number is a fraction number when separated with Superdex 200. PH stability of the purified enzyme. After predetermined treatment (37 ° C., 1 hour), the residual activity was measured. Results of BLAST analysis using the N-terminal amino acid sequence as a query sequence.

1. Terminology The term “isolated” is used herein interchangeably with “purified”. The term “isolated” is used to distinguish it from its natural state, ie, one that exists in nature. An artificial operation of isolation results in an “isolated state” that is different from the natural state. What has been isolated is clearly and critically different from the natural product itself.

  The purity of the isolated enzyme is not particularly limited. However, if application to a use requiring high purity is planned, it is preferable that the purity of the isolated enzyme is high.

2. Glucose-measuring enzyme agent The first aspect of the present invention provides an enzyme agent for glucose measurement. The active ingredient of the enzyme agent of the present invention is glucose dehydrogenase (hereinafter also referred to as “the present enzyme”) having the following characteristics. First, this enzyme catalyzes the following reaction, that is, the reaction of oxidizing the hydroxyl group of glucose in the presence of an electron acceptor to produce glucono-δ-lactone. On the other hand, this enzyme has excellent substrate specificity and acts selectively on D-glucose. Specifically, this enzyme has low reactivity with D-xylose. Specifically, the reactivity to D-xylose when the reactivity to D-glucose is 100% is 15% or less. Preferably, the reactivity is 12% or less.

  On the other hand, this enzyme has extremely low reactivity with maltose and D-galactose. The reactivity to maltose when the reactivity to D-glucose is 100% and the reactivity to D-galactose when the reactivity to D-glucose is 100% are both 5% or less, preferably 3% or less It is. More preferably, the reactivity is 1% or less. Even more preferably, the reactivity is substantially 0% (ie there is no substantial reactivity to maltose and galactose).

  The present enzyme having excellent substrate specificity as described above is preferable as an enzyme for accurately measuring the amount of glucose in a sample. That is, according to this enzyme, the target glucose level can be measured more accurately even when impurities such as D-xylose, maltose or D-galactose are present in the sample. Therefore, it can be said that this enzyme is suitable for applications in which the presence of such contaminants is expected or concerned (typically, measurement of the amount of glucose in blood), and there are various applications including such applications. It can be said that it is applicable to various uses, that is, versatility is high. In addition, the reactivity and substrate specificity of this enzyme can be measured and evaluated by the method shown in the below-mentioned Example.

  The origin of this enzyme, that is, the producer of this enzyme is Aspergillus kawachii or Aspergillus awamori. As long as the present enzyme having the above characteristics can be produced, the producing bacteria is not limited. A specific example of the producing strain is IFO strain 4308 (produces the same FAD-GDH as Aspergillus awamori No. 1731 strain used in the examples). The strain is stored as the NBRC 4308 strain at the National Institute of Technology and Evaluation Biotechnology Center (NBRC), and can be sold through a predetermined procedure.

  The producing bacterium may be a wild strain (separate from nature and not subjected to mutation / modification treatment such as gene manipulation) or a mutant strain. A transformant obtained by introducing the gene of this enzyme into a host microorganism may be used as a production bacterium.

  A further feature of this enzyme is its excellent pH stability. Specifically, this enzyme is stable at pH 4.0 to 6.0. That is, if the pH of the enzyme solution to be treated is within this range, the activity of 70% or more, preferably 80% or more, more preferably 90% or more is maintained after treatment at 37 ° C. for 1 hour.

  In one embodiment, the polypeptide chain constituting the enzyme consists of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence equivalent to the amino acid sequence. The “equivalent amino acid sequence” here is partially different from the amino acid sequence shown in SEQ ID NO: 1, but the difference does not substantially affect the function of the protein (here, glucose dehydrogenase activity). Refers to an array. Therefore, an enzyme having a polypeptide chain consisting of an equivalent amino acid sequence exhibits glucose dehydrogenase activity. “Glucose dehydrogenase activity” means the activity of catalyzing the reaction of oxidizing the hydroxyl group of glucose to produce glucono-δ-lactone. The degree of the activity is particularly limited as long as the function as glucose dehydrogenase can be exhibited. Not. However, it is preferably about the same as or higher than the enzyme having a polypeptide chain consisting of the amino acid sequence shown in SEQ ID NO: 1.

  “Partial difference in amino acid sequence” typically means deletion, substitution, or addition, insertion of one to several amino acids, or a combination thereof. This means that a mutation (change) has occurred in the amino acid sequence. The difference in the amino acid sequence here is allowed as long as the glucose dehydrogenase activity is retained (there may be some variation in activity). As long as this condition is satisfied, the positions where the amino acid sequences are different are not particularly limited, and differences may occur at a plurality of positions. The “plurality” herein is, for example, a number corresponding to less than about 30% of all amino acids, preferably a number corresponding to less than about 20%, more preferably a number corresponding to less than about 10%, Even more preferred is a number corresponding to less than about 5%, most preferred a number corresponding to less than about 1%. The equivalent protein is, for example, about 80% or more, preferably about 85% or more, more preferably about 90% or more, still more preferably about 95% or more, more preferably about 98% or more, and the amino acid sequence of SEQ ID NO: 1. More preferably, it has about 99% or more identity.

  Preferably, equivalent amino acid sequences are obtained by creating conservative amino acid substitutions at amino acid residues that are not essential for glucose dehydrogenase activity. As used herein, “conservative amino acid substitution” refers to substitution of a certain amino acid residue with an amino acid residue having a side chain having the same properties. Depending on the side chain of the amino acid residue, a basic side chain (eg lysine, arginine, histidine), an acidic side chain (eg aspartic acid, glutamic acid), an uncharged polar side chain (eg glycine, asparagine, glutamine, serine, threonine, tyrosine) Cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), aromatic side chains (eg tyrosine, phenylalanine, Like tryptophan and histidine). A conservative amino acid substitution is preferably a substitution between amino acid residues within the same family.

  By the way, the identity (%) of two amino acid sequences or two nucleic acids (hereinafter, “two sequences” is used as a term including them) can be determined, for example, by the following procedure. First, two sequences are aligned for optimal comparison (eg, a gap may be introduced into the first sequence to optimize alignment with the second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as the molecule at the corresponding position in the second sequence, it can be said that the molecule at that position is the same. The identity of two sequences is a function of the number of identical positions common to the two sequences (ie, identity (%) = number of identical positions / total number of positions × 100), preferably the optimal alignment Take into account the number and size of gaps required for conversion.

  Comparison of two sequences and determination of identity can be accomplished using a mathematical algorithm. Specific examples of mathematical algorithms available for sequence comparison are described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68; Karlin and Altschul (1993) Proc. Natl. There is an algorithm modified in Acad. Sci. USA 90: 5873-77, but it is not limited to this. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10. In order to obtain a nucleotide sequence equivalent to the nucleic acid molecule of the present invention, for example, a BLAST nucleotide search may be performed with score = 100 and wordlength = 12, using the NBLAST program. In order to obtain an amino acid sequence equivalent to this enzyme, for example, a BLAST polypeptide search may be performed using the XBLAST program with score = 50 and wordlength = 3. To obtain a gap alignment for comparison, the Gapped BLAST described in Altschul et al. (1997) Amino Acids Research 25 (17): 3389-3402 can be used. When utilizing BLAST and Gapped BLAST, the default parameters of the corresponding programs (eg, XBLAST and NBLAST) can be used. Please refer to http://www.ncbi.nlm.nih.gov for details. Examples of other mathematical algorithms that can be used for sequence comparison include the algorithm described in Myers and Miller (1988) Comput Appl Biosci. 4: 11-17. Such an algorithm is incorporated in the ALIGN program available for example on a GENESTREAM network server (IGH Montpellier, France) or an ISREC server. When using the ALIGN program for comparison of amino acid sequences, for example, a PAM120 residue mass table can be used, with a gap length penalty = 12 and a gap penalty = 4.

  The identity of two amino acid sequences, using the Gloss program of the GCG software package, using a Blossom 62 matrix or PAM250 matrix, gap weight = 12, 10, 8, 6, or 4, gap length weight = 2, 3 Or 4 can be determined. In addition, the degree of homology between two nucleic acid sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com) with gap weight = 50 and gap length weight = 3. it can.

  The enzyme may be part of a larger protein (eg, a fusion protein). Examples of sequences added in the fusion protein include sequences useful for purification, such as multiple histidine residues, and additional sequences that ensure stability during recombinant production.

  This enzyme having the amino acid sequence can be easily prepared by genetic engineering techniques. For example, it can be prepared by transforming a suitable host cell (for example, E. coli) with DNA encoding the present enzyme and recovering the protein expressed in the transformant. The recovered protein is appropriately purified according to the purpose. Thus, if this enzyme is obtained as a recombinant protein, various modifications are possible. For example, if a DNA encoding this enzyme and another appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, the peptide consists of a recombinant protein linked to any peptide or protein. This enzyme can be obtained. In addition, modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur. By the modification as described above, extraction of recombinant protein, simplification of purification, addition of biological function, and the like are possible.

  This enzyme can be obtained from a bacterium producing this enzyme. Specifically, for example, a step of culturing Aspergillus kawachi IFO 4308 strain (step (1)) and a step of recovering glucose dehydrogenase from the culture medium and / or cells after the culture (step (2)) are performed. To produce this enzyme.

  The culture method and culture conditions are not particularly limited as long as the target enzyme is produced. That is, on the condition that glucose dehydrogenase is produced, a method and culture conditions suitable for the culture of the microorganism to be used can be appropriately set. Hereinafter, examples of the culture conditions include a medium, a culture temperature, and a culture time.

  Any medium can be used as long as the microorganism to be used can grow. For example, carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein Nitrogen sources such as hydrolysates, bran and meat extracts, and further added with inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts and zinc salts can be used. In order to promote the growth of the microorganisms to be used, vitamins, amino acids and the like may be added to the medium. The pH of the medium is adjusted to, for example, about 3 to 8, preferably about 5 to 7, and the culture temperature is usually about 10 to 50 ° C., preferably about 25 to 35 ° C. for 1 to 15 days, preferably 2 to Incubate under aerobic conditions for about 5 days. As the culture method, for example, a shaking culture method or an aerobic deep culture method using a jar fermenter can be used.

  After culturing under the above conditions, glucose dehydrogenase is recovered from the culture solution or the cells (step (2)). When recovering from the culture solution, for example, the culture supernatant is filtered (for example, filtration using diatomaceous earth as a filter aid), the insoluble matter is removed by centrifugation, etc., and then concentrated by ultrafiltration membrane, ammonium sulfate precipitation, etc. The target enzyme can be obtained by performing separation and purification by appropriately combining salting-out, dialysis, various types of chromatography, and the like. On the other hand, when recovering from the microbial cells, the target enzyme can be obtained by crushing the microbial cells by, for example, pressure treatment, ultrasonic treatment, bead treatment, etc., followed by separation and purification in the same manner as described above. . After the cells are collected from the culture solution in advance by filtration, centrifugation, or the like, the above series of steps (crushing, separating, and purifying the cells) may be performed. In each purification process, in principle, fractionation is performed using glucose dehydrogenase activity as an index, and the process proceeds to the next step. However, this does not apply when appropriate conditions can be set by preliminary tests.

  It is also possible to obtain this enzyme as a recombinant. That is, the present enzyme may be produced using a transformant into which the gene of the present enzyme has been introduced. In the production method of this embodiment, first, a transformant into which the gene for this enzyme has been introduced is prepared. In the present specification, the term “gene of the present enzyme” refers to a nucleic acid obtained from the present enzyme when expressed, and includes a nucleic acid having a base sequence corresponding to the amino acid sequence of the present enzyme. Such a nucleic acid is also included in which a sequence that does not encode an amino acid sequence is added. Codon degeneracy is also considered.

  For the gene of this enzyme, reference to the sequence information disclosed in this specification or the attached sequence listing, standard genetic engineering techniques, molecular biological techniques, biochemical techniques, chemical synthesis, PCR methods (for example, Wrap PCR) or a combination thereof can be prepared in an isolated state.

  In order to prepare a transformant that can be used for the production of the enzyme, an expression vector containing the gene for the enzyme is prepared. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting a nucleic acid inserted therein into a target such as a cell.

  An appropriate vector is selected in consideration of the purpose of use, the host cell and the like. M13 phage or a modified form thereof, λ phage or a modified form thereof, pBR322 or a modified form thereof (pB325, pAT153, pUC8, etc.), etc., as a vector using E. coli as a host, pYepSec1, pMFa, pYES2 Examples of vectors using insect cells as hosts include pAc and pVL, and examples of vectors using mammalian cells as hosts include pCDM8 and pMT2PC.

  “Expression vector” refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and allowing expression in the cell. Expression vectors usually contain a promoter sequence necessary for the expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. An expression vector containing a selectable marker can also be used. When such an expression vector is used, the presence / absence (and extent) of introduction of the expression vector can be confirmed using a selection marker.

  Standard recombinant DNA techniques (for example, Molecular Cloning, Third Edition, 1.84, Cold, etc.) include insertion of the gene of this enzyme into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. A known method using a restriction enzyme and DNA ligase, which can be referred to Spring Harbor Laboratory Press, New York.

  As host cells, it is preferable to use microorganisms such as Escherichia coli (Escherichia coli) and budding yeast (Saccharomyces cerevisiae) from the viewpoint of ease of handling, but the recombinant DNA can be replicated and the gene of this enzyme is Any host cell capable of expression can be used. Examples of E. coli include E. coli BL21 (DE3) pLysS when T7 promoter is used, and E. coli JM109 otherwise. Examples of budding yeast include budding yeast SHY2, budding yeast AH22, or budding yeast INVSc1 (Invitrogen).

  An expression vector containing the gene for this enzyme is introduced into a host cell to obtain a transformant. A transformant can be obtained by transfection or transformation using the above expression vector. For example, the calcium chloride method (J. Mol. Biol., Vol. 53, pp. 159 (1970)), the Hanahan method (Journal of Molecular Biology, Vol. 166, Vol. 557). (1983), SEM (Gene, 96, 23 (1990)), Chung et al. (Proceedings of the National Academy of Sciences of the USA, 86) Vol., P. 2172 (1989)), calcium phosphate coprecipitation, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al., Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1984)) and the like.

  The transformant prepared as described above is cultured under conditions that produce a protein encoded by the gene introduced therein (ie, the gene of the present enzyme) (step (i)). Culture conditions for transformants are known for various vector host systems, and those skilled in the art can easily set appropriate culture conditions. Following the culture step, the produced protein (ie, glucose dehydrogenase) is recovered (step (ii)). The recovery and subsequent purification may be performed in the same manner as in the case of the above-described embodiment (production method including steps (1) and (2)).

  The degree of purification of the enzyme is not particularly limited. For example, the enzyme can be purified to have a specific activity of 10 to 1000 (U / mg), preferably 50 to 500 (U / mg). The final form may be liquid or solid (including powder).

  In addition to the active ingredient (the present enzyme), the enzyme agent of the present invention may contain excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline and the like. As the excipient, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

3. Glucose Measuring Method The present invention also provides a glucose measuring method using the present enzyme. In the glucose measuring method of the present invention, the amount of glucose in a sample is measured using an oxidation-reduction reaction by this enzyme. The present invention can be applied to various uses in which changes due to this reaction can be used.

  The present invention is used, for example, for measurement of blood glucose level, measurement of glucose concentration in food (such as seasonings and beverages), and the like. Moreover, you may utilize this invention in order to investigate a fermentation degree in the manufacturing process of fermented foods (for example, vinegar) or fermented drinks (for example, beer and liquor).

4). Glucose measuring reagent, kit, sensor The present invention also provides a glucose measuring reagent containing the enzyme. The reagent is used in the glucose measurement method of the present invention described above. Serum albumin, proteins, surfactants, saccharides, sugar alcohols, inorganic salts, and the like may be added for the purpose of stabilizing the glucose measuring reagent and activating it during use.

  A reagent for measuring glucose can also be used as a component of the measurement kit. In other words, the present invention also provides a kit (glucose measurement kit) containing the glucose measurement reagent. The kit of the present invention contains the above-mentioned reagent for glucose measurement as an essential component. In addition, a reaction reagent, a buffer solution, a glucose standard solution, a container and the like are included as optional elements. The glucose measurement kit of the present invention usually includes an instruction manual.

  A glucose sensor can be constructed using this enzyme. That is, this invention also provides the glucose sensor containing this enzyme. In a typical structure of the glucose sensor of the present invention, an electrode system including a working electrode and a counter electrode is formed on an insulating substrate, and a reagent layer containing the present enzyme and mediator is formed thereon. A measurement system that also includes a reference electrode may be used. If such a so-called three-electrode measurement system is used, the potential of the working electrode can be expressed based on the potential of the reference electrode. The material of each electrode is not particularly limited. Examples of the electrode material for the working electrode and the counter electrode are gold (Au), carbon (C), platinum (Pt), and titanium (Ti). As the mediator, a ferricyan compound (such as potassium ferricyanide), a metal complex (such as a ruthenium complex, an osmium complex, or a vanadium complex), a quinone compound (such as pyrroloquinoline quinone), or the like is used. The structure of the glucose sensor and the electrochemical measurement method using the glucose sensor are detailed in, for example, the actual bioelectrochemistry-practical deployment of biosensors and biobatteries-(published in March 2007, CMC Publishing). .

1. Screening from microorganisms Using as a sample a culture solution obtained by culturing 13,000 strains including stocks obtained from public institutions and strains obtained from the natural world, the following conditions, namely, high glucose dehydrogenase activity, maltose Those satisfying that no reaction, low reactivity to xylose, and no glucose oxidase activity were selected by the following method.

Method for measuring glucose dehydrogenase activity (measuring solution)
100 mmol / L PIPES cont. 0.1% (w / v) Triton X-100 pH 7.0: 24mL
3 mmol / L 1-Methoxy PMS (Phenazine methanesulfate): 2mL
6.6 mmol / L NTB (Nitrotetrazorium blue): 1mL
1 mol / L glucose: 3mL

(Measurement procedure)
After dispensing 20 μL of each sample into a 96-well plate, 200 μL of measurement reagent was added per well, and after 60 minutes at 37 ° C., the absorbance at 570 nm was measured with a plate reader. Among the above measuring solutions, glucose was changed to maltose or xylose and the same measurement was performed, and the reactivity to maltose and xylose was also confirmed.

Method for measuring glucose oxidase activity (measuring solution)
100 mmol / L PIPES cont. 0.1% (w / v) Triton X-100 pH 7.0: 23mL
5g / dL phenol reagent: 0.5mL
25u / mL PO “Amano” 3 (Amano Enzyme Inc.) solution: 3mL
0.5g / dL 4-aminoantipyrine test solution: 0.5mL
1 mol / L glucose: 3mL

(Measurement procedure)
After dispensing 20 μL of each sample into a 96-well plate, 200 μL of the measurement reagent was added per well, and after 60 minutes at 37 ° C., the absorbance at 500 nm was measured with a plate reader.

As a result of the examination, a glucose dehydrogenase produced by Aspergillus awamori No. 1731, which does not react with maltose, has low reactivity with xylose and is not glucose oxidase, was found. The following table shows the reactivity to maltose and xylose and glucose oxidase (GO) activity for Aspergillus awamori No. 1731 strain. The reactivity to maltose and xylose is relative value when the reactivity to glucose is 100% (measured value when maltose (or xylose) is used as substrate / measured value when glucose is used as substrate x 100) Expressed in Glucose oxidase (GO) was expressed as a relative value to glucose dehydrogenase (measured value of glucose oxidase (GO) activity / measured value of glucose dehydrogenase activity × 100). For comparison, results of glucose oxidase (derived from Aspergillus niger), PQQ-dependent glucose dehydrogenase (derived from Acinetobacter calcoaceticus), and FAD-dependent glucose dehydrogenase (derived from Aspergillus oryzae) are also shown.

  It can be seen that the glucose dehydrogenase produced by Aspergillus awamori No. 1731 strain is significantly less reactive to maltose and xylose than the existing PQQ-dependent glucose dehydrogenase and FAD-dependent glucose dehydrogenase.

2. Preparation of purified enzyme About glucose dehydrogenase produced by Aspergillus awamori No.1731 found by the above examination, in order to obtain the purified enzyme, Aspergillus awamori No.1731 was cultured at 30 ° C in solid culture using wheat bran. And cultured for 5 days. An enzyme was extracted from the obtained culture culture cake to obtain a crude enzyme solution.

  The crude enzyme solution was purified (salting out, hydrophobic bond chromatography, ion exchange chromatography, gel filtration chromatography) to obtain a purified enzyme. The purified enzyme was analyzed by gel filtration (using Superdex 200 manufactured by GE Healthcare) and SDS-PAGE. The results of SDS-PAGE are shown in FIG. The fraction with the highest glucose dehydrogenase activity (No. 34) was used in subsequent experiments.

尚、式中のPMSはフェナジンメタンスルフェート（Phenazine methanesulfate）を表し、NTBはニトロテトラゾリウムブルー（Nitrotetrazorium blue）を表す。反応（１）において、グルコースの酸化に伴って還元型PMSが生成し、更に反応（２）において還元型PMSによるNTBの還元により生成したDiformazanを570nmの波長で測定する。 3. Confirmation of substrate specificity of purified enzyme 2. The substrate specificity of the purified enzyme obtained in (1) was examined.
(Activity measurement method)
FAD-GDH catalyzes the reaction of oxidizing the hydroxyl group of glucose in the presence of an electron acceptor to produce D-glucono-δ-lactone. The FAD-GDH activity was measured using the following reaction system.

In addition, PMS in a formula represents phenazine methanesulfate (Phenazine methanesulfate) and NTB represents nitrotetrazolium blue (Nitrotetrazorium blue). In reaction (1), reduced PMS is generated as glucose is oxidized, and Diformazan generated by reduction of NTB by reduced PMS in reaction (2) is measured at a wavelength of 570 nm.

尚、式中のVtは総液量を、Vsはサンプル量を、20.1はDiformazanの0.5μ molあたりの吸光度係数（cm² / 0.5μ mol）を、1.0は光路長（cm）を、dfは希釈倍数をそれぞれ表す。 The enzyme activity is calculated by the following calculation formula.

In the formula, Vt is the total liquid volume, Vs is the sample volume, 20.1 is the absorbance coefficient (cm ² /0.5 μmol) per 0.5 μmol of Diformazan, 1.0 is the optical path length (cm), and df is Each dilution factor is indicated.

100 mmol / L PIPES-NaOH buffer pH 7.0 2.4 mL containing 0.1% (w / v) Triton X-100, 0.3 mL of 1 mol / L D-glucose solution, 0.2 mL of 3 mmol / L PMS solution, and 6.6 mmol / L NTB solution (0.1 mL) was mixed and incubated at 37 ° C. for 5 minutes, and then the enzyme solution (0.1 mL) was added to start the reaction. Diformazan having absorption at 570 nm is generated as the enzymatic reaction proceeds. The increase in absorbance at 570 nm per minute was measured, and FAD-GDH activity was measured. The results are shown in the table below. In addition, glucose was changed into maltose or xylose and measured in the same manner, and the reactivity to maltose and xylose was confirmed. For comparison, the results of glucose dehydrogenase produced by Aspergillus oryzae (GDH “Amano” 8 Amano Enzyme Co., Ltd.) are also shown.

  Glucose dehydrogenase produced by Aspergillus awamori No. 1731 is less reactive to xylose than glucose dehydrogenase produced by Aspergillus oryzae, and does not react with maltose, and has properties suitable for blood glucose measurement. Turned out to be.

4). Confirmation of pH stability of purified enzyme 2. The pH stability of the purified enzyme obtained in (1) was examined. The enzyme solution was prepared with various pH buffers so as to be 1 U / mL, followed by heating at 37 ° C. for 1 hour. Then, it is diluted with a buffer used for measurement, and the above 3. Residual activity was measured by the same method.

  The measurement results are shown in FIG. High stability was exhibited in the range of pH 4 to pH 6 (residual activity was 90% or more). That is, it was proved that the pH stability was excellent, it was suitable for glucose sensor applications and was highly practical.

5. Determination of N-terminal amino acid and amino acid sequence
The band obtained by separating fraction No. 34, which is the active peak of Aspergillus awamori No. 1731 by SDS-PAGE, was blotted to a PVDF membrane according to a conventional method, and then subjected to N-terminal amino acid analysis. Sequence information of “SGSQYDYIVVGGGTSQLVVA (SEQ ID NO: 3)” was obtained with a 66 KDa protein (FIG. 1). When BLAST analysis provided by NCBI was performed using the same sequence as a query sequence, it was found that there is a high possibility of being a glucose oxidase family. The BLAST analysis results are shown in FIG.

  On the other hand, several glucose oxidase families derived from filamentous fungi are known, and are known to have a common motif on the C-terminal side. Among them, the antisense primer “NGGNADNACNSWRGCRTCNACNACNCKNAVRTT (SEQ ID NO: 4)” was designed from the sequence information “N, V / L, RVVDASV, L / M / I / F, P” of the highly homologous portion. Using this primer and a primer designed based on the N-terminal amino acid sequence, genomic DNA was amplified as a template by PCR, and the nucleotide sequence was determined according to a conventional method. Furthermore, the sequence of the undetermined portion was determined from the obtained partial sequence by the inverse PCR method according to a conventional method, and the full-length base sequence (SEQ ID NO: 5) was obtained. Since the obtained sequence (SEQ ID NO: 5) contains an intron sequence, the intron portion was determined using intron prediction software FGENESH (http://linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind). The gene sequence (SEQ ID NO: 2) of the novel FAD-GDH was obtained by prediction, and the amino acid sequence (SEQ ID NO: 1) of the novel FAD-GDH was determined from the obtained gene sequence.

  When the determined amino acid sequence (SEQ ID NO: 1) was searched with BLAST (registered trademark), the sequence registered as glucose oxidase of Aspergillus kawachi (glucose oxidase [Aspergillus kawachii IFO 4308, Accession No. GAA92291.1]) and 100% identical. That is, it was found that a protein known as glucose oxidase actually functions as FAD-GDH.

  The glucose dehydrogenase of the present invention has low reactivity with xylose and is excellent in pH stability. The glucose dehydrogenase of the present invention is particularly suitable for use in a glucose sensor, and its practicality is high.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (9)

The following features:
(1) Action: catalyzes a reaction in which glucose hydroxyl group is oxidized to produce glucono-δ-lactone in the presence of an electron acceptor;
(2) Substrate specificity: The reactivity to D-xylose is 15% or less when the reactivity to D-glucose is 100%;
(3) pH stability: stable at pH 4-6;
(4) Amino acid sequence: The amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence that is 80% or more identical to the amino acid sequence;
An enzyme agent for measuring glucose, comprising as an active ingredient glucose dehydrogenase comprising   The enzyme agent for glucose measurement according to claim 1, wherein the amino acid sequence of the glucose dehydrogenase is an amino acid sequence that is 85% or more identical to the amino acid sequence shown in SEQ ID NO: 1.   The enzyme agent for glucose measurement according to claim 1, wherein the amino acid sequence of the glucose dehydrogenase is an amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1.   The enzyme agent for glucose measurement according to any one of claims 1 to 3, which is an enzyme derived from Aspergillus kawachi or Aspergillus awamori.   The enzyme agent for glucose measurement according to claim 4, wherein Aspergillus kawachi is IFO 4308 strain.   A glucose measuring method, wherein glucose in a sample is measured using the glucose dehydrogenase defined in any one of claims 1 to 5.   A reagent for measuring glucose, comprising the glucose dehydrogenase defined in any one of claims 1 to 5.   A glucose measurement kit comprising the glucose measurement reagent according to claim 7.   A glucose sensor comprising a glucose dehydrogenase as defined in any one of claims 1-5.

Images