Novel glucose dehydrogenase

Abstract

The present invention addresses the problem of providing an FAD-GDH that has high heat stability and, in particular, improved practical usability for a glucose sensor.　Provided is a novel FAD-GDH having the following characteristics: (1) function: catalyzing a reaction of oxidizing a hydroxyl group of glucose in the presence of an electron acceptor to form glucono-δ-lactone; (2) heat stability: after a thermal treatment at 50°C for 20 minutes, showing a relative residual activity of 60% or higher; and (3) molecular weight: approximately 68 kDa (measured by SDS-PAGE after removing sugar chains).

Description

[Technical field]

[0001] The present invention relates to a novel glucose dehydrogenase, specifically to a flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (EC 1.1.99.10) that is highly thermostable and useful for applications such as glucose measurement, and uses thereof. [Background technology]

[0002] The number of diabetic patients is increasing year by year, and diabetic patients, especially those who are insulin-dependent, need to monitor their blood glucose levels on a daily basis to control their blood glucose. In recent years, it has become possible to check the blood glucose levels of diabetic patients using self-blood glucose measuring devices that can measure blood glucose easily and accurately in real time using enzymes. 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 measuring devices), but their reactivity to oxygen and reactivity to maltose and galactose have become 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). Various improvements to FAD-GDH have been considered (see, for example, Patent Documents 6 to 8). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2000-350588 A [Patent Document 2] JP 2001-197888 A [Patent Document 3] JP 2001-346587 A [Patent Document 4] International Publication No. 2004 / 058958 Brochure [Patent Document 5] International Publication No. 2007 / 139013 パンフレット

Patent document 6

Patent document 7

Patent document 8

Non-licensed literature

[0004] [Non-licensed document 1] Studies on the glucose dehydrogenase of Aspergillus oryzae. I. Induction of its synthesis by p-benzoquinone and hydroquinone, TC Bak, and R. Sato, Biochim. Biophys. Acta, 139, 265-276 (1967). [Non-licensed document 2] Studies on the glucose dehydrogenase of Aspergillus oryzae. II. Purification and physical and chemical properties, TC Bak, Biochim. Biophys. Acta, 139, 277-293 (1967). [Non-licensed document 3] Studies on the glucose dehydrogenase of Aspergillus oryzae. III. General enzymatic properties, TC Bak, Biochim. Biophys. Acta, 146, 317-327 (1967).

Non-licensed Document 4

[0005] Enzymes are proteins and are prone to a decrease in activity due to heat. The decrease in activity is directly linked to measurement accuracy, etc. In blood glucose measurement (self-monitoring of blood glucose (SMBG) and continuous blood glucose monitoring (CGM)), it is desirable for the enzyme used therein to have high thermal stability, but FAD-GDH is generally less stable than glucose oxidase (GO). Although there have been attempts to improve the thermal stability of FAD-GDH (for example, Patent Document 7), there is still a high need for improved thermal stability. If FAD-GDH with excellent thermal stability could be used, a highly practical glucose sensor that makes use of the advantages of FAD-GDH would be constructed. Therefore, the object of the present invention is to provide FAD-GDH with high thermal stability and improved practicality, especially for use as a glucose sensor, and its uses, etc. On the other hand, it is desirable to improve the electrode reactivity of the enzyme in order to increase the sensitivity and accuracy of the measurement. The object of the present invention is also to meet this demand. [Means for solving the problem]

[0006] In order to solve the above problems, the present inventors set up their own evaluation index, and proceeded with the search for novel FAD-GDHs targeting a wide variety of microorganisms, focusing on one gene present on the genome of Aspergillus cristatus. The expression product (protein) of the gene is registered as a hypothetical protein in the NCBI database (GenPept) (ACCESSION: ODM22452.1, DEFINITION hypothetical protein SI65_00040 [Aspergillus cristatus].). As a result of the inventors' investigation, it was surprisingly found that the protein exhibited glucose dehydrogenase activity. In addition, as a result of detailed investigation of its properties, it was found to have excellent thermal stability, good electrode reactivity, and high activity in the neutral pH range, making it a preferable enzyme for a glucose sensor. Based on the above results, the following invention is provided. [1] A glucose dehydrogenase having the following characteristics: (1) Function: Catalyzes the oxidation of the hydroxyl group of glucose to produce glucono-δ-lactone in the presence of an electron acceptor; (2) Thermostability: the relative residual activity after heat treatment at 50°C for 20 minutes is 60% or more; (3) Molecular weight: approximately 68 kDa (after removal of glycosylation, determined by SDS-PAGE). [2] A glucose dehydrogenase having the following characteristics: (1) Function: Catalyzes the oxidation of the hydroxyl group of glucose to produce glucono-δ-lactone in the presence of an electron acceptor; (2) Amino acid sequence: Contains the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence that is 62% or more identical to said amino acid sequence. [3] The glucose dehydrogenase according to [2], whose amino acid sequence is at least 70% identical to the amino acid sequence shown in SEQ ID NO:2. [4] The glucose dehydrogenase according to [2], whose amino acid sequence is at least 90% identical to the amino acid sequence shown in SEQ ID NO:2. [5] The glucose dehydrogenase according to any one of [1] to [4], further having the following enzymatic and chemical properties: (4) Substrate specificity: the reactivity to maltose is 5% or less when the reactivity to D-glucose is taken as 100%; (5) Optimum pH: 8.0; (6) Optimum temperature: 55℃; (7) pH stability: stable at pH 5.5 to 7.5. [6] The glucose dehydrogenase described in any one of [1] to [5], which is an enzyme derived from Aspergillus cristatus. [7] A method for measuring glucose, comprising measuring glucose in a sample using the glucose dehydrogenase according to any one of [1] to [6]. [8] A reagent for measuring glucose, comprising the glucose dehydrogenase according to any one of [1] to [6]. [9] A glucose measurement kit comprising the glucose measurement reagent according to [8].

[10] A glucose sensor comprising the glucose dehydrogenase according to any one of [1] to [6].

[11] An enzyme preparation comprising the glucose dehydrogenase according to any one of [1] to [6]. [Brief description of the drawings]

[0007] [Figure 1] Optimal pH of A. cristatus-derived FAD-GDH. [Diagram 2] Optimum temperature of A. cristatus derived FAD-GDH. [Diagram 3] The pH stability of FAD-GDH from A. cristatus is shown in comparison with that of FAD-GDH from A. oryzae strain BB-56. [Figure 4] Thermal stability of A. cristatus-derived FAD-GDH. Residual activity after heat treatment was compared with that of A. oryzae BB-56-derived FAD-GDH. [Diagram 5]Electrode reactivity of FAD-GDH derived from A. cristatus, compared with that of FAD-GDH derived from A. oryzae strain BB-56. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] 1. Terminology In this specification, the term "isolated" is used interchangeably with "purified". In the case of a product produced without the intervention of artificial manipulation, the term "isolated" is used to distinguish it from a natural state, i.e., a state that exists in nature, and in the case of a product produced with the intervention of artificial manipulation, it is used to distinguish it from a product that has not undergone an isolation or purification process. In the former case, the artificial operation of isolation results in an "isolated state" that is a state different from the natural state, and the isolated product is clearly and decisively different from the natural product itself. On the other hand, in the latter case, typically, impurities are removed or the amount thereof is reduced by the isolation or purification process, and the purity is increased. The purity of the isolated enzyme is not particularly limited. However, if the application to a use requiring high purity is planned, it is preferable that the purity of the isolated enzyme is high.

[0009] 2. Glucose dehydrogenase The first aspect of the present invention provides a glucose dehydrogenase. The glucose dehydrogenase of the present invention (hereinafter also referred to as "the present enzyme") has the following characteristics. First, the present 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, the present enzyme has excellent thermostability and maintains high activity even after heat treatment at 50°C for 20 minutes. That is, the relative residual activity (enzyme activity after heat treatment expressed as a relative value (%) when the enzyme activity before heat treatment is taken as 100%. In other words, the ratio of activity after heat treatment to activity before heat treatment) is high. The relative residual activity of the present enzyme is, for example, 60% or more, preferably 70% or more, and more preferably 75% or more (specific examples are 75%, 78%, and 80%). A method for evaluating thermostability is shown in the Examples section.

[0010] In blood glucose measurement (self-monitoring of blood glucose (SMBG) and continuous glucose monitoring (CGM)), it is desirable for the enzyme to have high thermal stability. With this enzyme, which has high thermal stability, it is expected that the measurement accuracy will be improved. Therefore, it can be said that this enzyme has excellent practicality as an enzyme for glucose sensors.

[0011] In one embodiment, the molecular weight of the enzyme after removal of the glycosylation chain is about 68 kDa (see the Examples below). The molecular weight is measured by SDS-PAGE.

[0012] The present enzyme can be defined by its amino acid sequence. That is, in one embodiment, the polypeptide chain constituting the present enzyme consists of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence equivalent to said amino acid sequence. Here, the "equivalent amino acid sequence" refers to an amino acid sequence that is partially different from the amino acid sequence shown in SEQ ID NO: 2, but the difference does not substantially affect the function of the protein (here, glucose dehydrogenase activity). 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, but the degree of the activity is not particularly limited as long as it can exert the function of glucose dehydrogenase. However, it is preferable that the activity is the same as or higher than that of an enzyme having a polypeptide chain consisting of the amino acid sequence shown in SEQ ID NO: 2.

[0013] A "partial difference in the amino acid sequence" is caused, for example, by the deletion or substitution of one or more or several amino acids among the amino acids constituting the amino acid sequence, the addition or insertion of one or more or several amino acids to the amino acid sequence, or any combination thereof. A partial difference in the amino acid sequence is permissible as long as the glucose dehydrogenase enzyme activity is maintained (the activity may vary slightly). As long as this condition is met, the position at which the amino acid sequence differs is not particularly limited. Furthermore, differences in the amino acid sequence may occur at multiple positions (locations).

[0014] The number of amino acids that cause a difference in the amino acid sequence is, for example, a number corresponding to less than about 38% of all amino acids constituting the amino acid sequence, preferably a number corresponding to less than about 30%, more preferably a number corresponding to less than about 20%, even more preferably a number corresponding to less than about 10%, even more preferably a number corresponding to less than about 5%, and most preferably a number corresponding to less than about 1%. Thus, an equivalent protein has an identity with the amino acid sequence of SEQ ID NO:2 of, for example, about 62% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, even more preferably about 95% or more, and most preferably about 99% or more.

[0015] A typical example of a "partial difference in the amino acid sequence" is a mutation (change) in the amino acid sequence caused by the deletion and / or substitution of 1 to 50 (preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and even more preferably 1 to 3) amino acids among the amino acids that make up the amino acid sequence; the addition and / or insertion of 1 to 50 (preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and even more preferably 1 to 3) amino acids to the amino acid sequence; or a combination thereof.

[0016] Preferably, an equivalent amino acid sequence is obtained by making a conservative amino acid substitution at an amino acid residue that is not essential for glucose dehydrogenase activity. Here, "conservative amino acid substitution" refers to replacing an amino acid residue with an amino acid residue having a side chain of similar properties. Amino acid residues are classified into several families according to their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues in the same family.

[0017] Incidentally, the identity (%) of two amino acid sequences or two nucleic acids (hereinafter, the term "two sequences" is used to include both) can be determined, for example, by the following procedure. First, the two sequences are aligned to enable optimal comparison (for example, gaps may be introduced into the first sequence to optimize the 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 a molecule at the corresponding position in the second sequence, the molecule at that position can be said to be identical. The identity of two sequences is a function of the number of identical positions shared by the two sequences (i.e., identity (%) = number of identical positions / total number of positions × 100), and preferably, the number and size of gaps required for optimization of the alignment are also taken into account.

[0018] Comparison of two sequences and determination of identity can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68 and modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST program and XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain a nucleotide sequence equivalent to the nucleic acid molecule of the present invention, for example, a BLAST nucleotide search can be performed with the NBLAST program with score = 100 and wordlength = 12. To obtain an amino acid sequence equivalent to the present enzyme, for example, a BLAST polypeptide search can be performed with the XBLAST program with score = 50 and wordlength = 3. To obtain a gapped alignment for comparison, Gapped BLAST described in Altschul et al. (1997) Amino Acids Research 25(17):3389-3402 can be used. When using BLAST and Gapped BLAST, the default parameters of the corresponding program (e.g., XBLAST and NBLAST) can be used. For details, see http: / / www.ncbi.nlm.nih.gov. Examples of other mathematical algorithms that can be used for comparing sequences 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 the GENESTREAM network server (IGH Montpellier, France) or the ISREC server. When using the ALIGN program to compare amino acid sequences, for example, the PAM120 residue mass table can be used, with gap length penalty=12 and gap penalty=4.

[0019] The identity of two amino acid sequences can be determined using the GAP program in the GCG software package, using a Blossom 62 matrix or a PAM250 matrix with a gap weight of 12, 10, 8, 6, or 4, and a gap length weight of 2, 3, or 4. The homology of two nucleic acid sequences can also be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), with a gap weight of 50 and a gap length weight of 3.

[0020] The enzyme may be part of a larger protein, such as a fusion protein, in which additional sequences may be added to aid in purification, such as multiple histidine residues, or additional sequences to ensure stability during recombinant production.

[0021] The present enzyme having the above amino acid sequence can be easily prepared by genetic engineering techniques. For example, it can be prepared by transforming a suitable host cell (e.g., Escherichia coli) with DNA encoding the present enzyme (a specific example of the sequence is shown in SEQ ID NO: 1) and recovering the protein expressed in the transformant. The recovered protein is purified appropriately depending on the purpose. By obtaining the present enzyme as a recombinant protein in this way, various modifications are possible. For example, by inserting the DNA encoding the present enzyme and other suitable DNA into the same vector and producing a recombinant protein using the vector, the present enzyme consisting of a recombinant protein linked to any peptide or protein can be obtained. In addition, sugar chains and / or lipids may be added, or modifications may be made to cause N-terminal or C-terminal processing. The above-mentioned modifications make it possible to simplify the extraction and purification of the recombinant protein, or to add biological functions.

[0022] The enzyme can be further characterized by the following enzymatic properties (substrate specificity, optimum pH, optimum temperature, pH stability).

[0023] This enzyme has excellent substrate specificity and selectively acts on D-glucose. More specifically, this enzyme has extremely low reactivity with maltose. Specifically, when the reactivity with D-glucose is taken as 100%, the reactivity with maltose is 5% or less. Preferably, the reactivity is 3% or less, and more preferably, the reactivity is 1% or less.

[0024] On the other hand, the reactivity of this enzyme to D-xylose is also low. When the reactivity to D-glucose is taken as 100%, the reactivity to D-xylose is 15% or less. Preferably, the reactivity is 13% or less, and more preferably, the reactivity is 12% or less.

[0025] The enzyme having such excellent substrate specificity is preferable as an enzyme for accurately measuring the amount of glucose in a sample. That is, the enzyme can measure the amount of glucose more accurately even if the sample contains impurities such as maltose and D-xylose. Therefore, the enzyme is suitable for applications where the presence of such impurities in the sample is expected or feared (typically, measurement of the amount of glucose in blood). In addition, as shown in the examples below, the enzyme has excellent electrode reactivity and is particularly useful for glucose sensor applications. Enzymes for self-monitoring of blood glucose (SMBG) are required to have high electrode reactivity. This is because low electrode reactivity requires more enzyme, which causes problems such as an increase in blank. Since the enzyme has excellent electrode reactivity, the amount of enzyme used can be reduced. Therefore, it can be said that the enzyme is highly practical for use in glucose sensors. On the other hand, the enzyme can be applied to various applications including glucose sensor applications, that is, it is highly versatile. The reactivity and substrate specificity of the enzyme can be measured and evaluated by the method shown in the examples below.

[0026] The optimum pH is 8.0. The optimum pH is determined based on the results measured in, for example, 100 mM Britton-Robinson buffer.

[0027] The optimum temperature is 55° C. The optimum temperature can be evaluated based on the results of measurements under the condition of pH 6.5 (for example, using a 0.05 M PIPES-NaOH buffer solution).

[0028] The present enzyme also has excellent pH stability and exhibits high activity in the weakly basic to neutral range. Specifically, the present enzyme is stable at pH 5.5 to 7.5. That is, if the pH of the enzyme solution used for treatment is within this range, the enzyme maintains 80% or more, preferably 90% or more, and more preferably 95% or more of its activity after treatment at 37°C for 1 hour.

[0029] In self-monitoring of blood glucose (SMBG), potassium ferricyanide is generally used as a mediator, but the use of mediators that enable measurement at lower potentials (e.g., thionine, 1-methoxy-5-methylphenazinium methylsulfate, toluidine blue) is being considered. In the case of these mediators (thionine, etc.), the reaction occurs near neutral. Therefore, the present enzyme, which shows the above pH stability, is also suitable for SMBG reaction conditions using these mediators. On the other hand, the reaction in continuous glucose monitoring (CGM) occurs under blood pH (pH 7.4±0.05). Since the present enzyme is stable at pH 5.5 to 7.5, it is also suitable for CGM reaction conditions. Furthermore, it can be said that the present enzyme is also suitable for measuring glucose in basic samples (e.g., basic foods).

[0030] The source of this enzyme, that is, the bacteria that produces this enzyme, is Aspergillus cristatus. There are no limitations on the type of bacteria that can produce this enzyme with the above characteristics. A specific example of a bacteria that produces this enzyme is the Aspergillus cristatus E4 CGMCC 7.193 strain (BMC Genomics 17, 428 (2016)). This strain is stored at the CGMCC (China General Microbiological Culture Collection Center).

[0031] The producing bacteria may be a wild type strain (a strain isolated from nature that has not been subjected to mutation or modification treatment such as genetic engineering) or a mutant strain. The producing bacteria may be a transformant obtained by introducing the gene for the present enzyme into a host microorganism.

[0032] 3. Uses of glucose dehydrogenase A further aspect of the present invention relates to uses of the present enzyme. In this aspect, first, a glucose measurement method using the present enzyme is provided. In the glucose measurement method of the present invention, the amount of glucose in a sample is measured by utilizing an oxidation-reduction reaction caused by the present enzyme. The present invention can be applied to various uses in which the change caused by this reaction can be utilized.

[0033] The present invention can be used, for example, to measure blood glucose levels (self-monitoring of blood glucose (SMBG) and continuous glucose monitoring (CGM)), to measure glucose contained in body fluids other than blood (e.g., tears, saliva, interstitial fluid, urine, etc.), to measure the glucose concentration in foods (condiments, beverages, etc.), etc. The present invention may also be used to examine the degree of fermentation in the production process of fermented foods (e.g., vinegar) or fermented beverages (e.g., beer and sake).

[0034] The present invention also provides a glucose measurement reagent containing the present enzyme. The reagent is used in the glucose measurement method of the present invention. For the purpose of stabilizing the glucose measurement reagent and activating it during use, serum albumin, proteins, surfactants, sugars, sugar alcohols, inorganic salts, etc. may be added.

[0035] The glucose measurement reagent can also be a component of a measurement kit. In other words, the present invention also provides a kit (glucose measurement kit) containing the above-mentioned glucose measurement reagent. The kit of the present invention contains the above-mentioned glucose measurement reagent as an essential component. In addition, the kit may contain reaction reagents, buffer solutions, glucose standard solutions, containers, and the like as optional components. Note that the glucose measurement kit of the present invention is usually accompanied by an instruction manual.

[0036] A glucose sensor can be constructed using this enzyme. That is, the present invention also provides a glucose sensor containing this enzyme. In a typical structure of the glucose sensor of the present invention, an electrode system having a working electrode and a counter electrode is formed on an insulating substrate, and a reagent layer containing the present enzyme and a mediator is formed thereon. More specifically, the reagent layer is usually coated on the working electrode. The present invention can also be applied to a face-to-face glucose sensor in which the working electrode and the counter electrode face each other. On the other hand, a measurement system also including a reference electrode may be used. If such a so-called three-electrode measurement system is used, it is possible to express the potential of the working electrode based on the potential of the reference electrode. The material of each electrode is not particularly limited. Examples of electrode materials for the working electrode and the counter electrode include gold (Au), carbon (C), platinum (Pt), and titanium (Ti). As the mediator, a ferricyanide compound (potassium ferricyanide, etc.), a metal complex (ruthenium complex, osmium complex, vanadium complex, etc.), a quinone compound (pyrroloquinoline quinone, etc.), etc. are used. The structure of the glucose sensor and the electrochemical measurement method using the glucose sensor are described in detail, for example, in "Bioelectrochemistry in Practice - Practical Development of Biosensors and Biobatteries" (published in March 2007 by CMC Publishing).

[0037] The enzyme of the present invention can also be provided in the form of an enzyme preparation. The enzyme preparation of the present invention may contain, in addition to the active ingredient (the enzyme of the present invention), an excipient, a buffer, a suspending agent, a stabilizer, a preservative, a physiological saline solution, etc. Examples of the excipient include starch, dextrin, maltose, trehalose, lactose, sorbitol, D-mannitol, sucrose, glycerol, sugar esters, etc., and derivatives thereof, and D-glucose derivatives. Examples of the buffer include phosphates, citrates, acetates, etc. Examples of the stabilizer include propylene glycol, ascorbic acid, etc. Examples of the preservative include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. Examples of the preservative include ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, etc. Examples of the use of the enzyme preparation include measurement of glucose, blood sugar level, and fermentation degree. EXAMPLES

[0038] In order to find a novel FAD-GDH with high thermal stability and improved practicality, particularly for use in glucose sensors, the following investigations were carried out.

[0039] 1. Obtaining the enzyme gene and expressing the recombinant enzyme An independent evaluation index was set, and an exhaustive search was carried out using NCBI BLAST. As a result, a gene that can be expected to exhibit FAD-GDH activity was found on the genome of Aspergillus cristatus. A synthetic gene was prepared for the gene, and DNA amplification was performed by PCR using it as a template. The amplified DNA was ligated to a filamentous fungus expression vector (pUC19 inserted with an α-amylase modified promoter, a terminator of FAD-GDH derived from A. oryzae, and the pyrG gene), and introduced into the A. oryzae RIB40 strain by the protoplast-PEG method. The gene consists of the nucleotide sequence of SEQ ID NO: 1, and its expression product is registered in the NCBI database (GenPept) as a hypothetical protein (ACCESSION: ODM22452.1, DEFINITION hypothetical protein SI65#00040 [Aspergillus cristatus]). The amino acid sequence of the protein is shown in SEQ ID NO: 2.

[0040] 2. Measurement of FAD-GDH Activity of Transformants The obtained transformants were liquid-cultured at 30°C for 3 days. The culture supernatant was subjected to the following FAD-GDH activity measurement method to evaluate the presence or absence of FAD-GDH activity. As a result, it was confirmed that the transformants exhibited FAD-GDH activity.

[0041] <FAD-GDH Activity Measurement Method> The measurement principle is that when GDH is applied to D-glucose and phenazine methosulfate (hereinafter abbreviated as "PMS"), glucose is oxidized while PMS is reduced. Reduced PMS reduces Nitrotetrazorium blue (hereinafter abbreviated as "NTB"). When NTB is reduced by reduced PMS, a diformazan dye is formed. The enzyme activity is measured by detecting this diformazan dye at 570 nm. The experimental procedure is as follows. First, 2.6 mL of 50 mmol / L PIPES-NaOH buffer pH 6.5 (containing 0.5% TritonX-100), 1 mL of 1.0 mmol / L D-glucose, 1 mL of 6.6 mmol / L NTB solution, and 2 mL of 3.0 mmol / L PMS solution are placed in a spectrophotometer cell and preheated at 37°C for 10 minutes. 0.1 mL of FAD-GDH solution is added and mixed, and the mixture is allowed to react at 37°C. After the start of the reaction, measure the absorbance A3 (after 3 minutes) and A5 (after 5 minutes) of the reaction solution at a wavelength of 570 nm at 3 and 5 minutes. Separately, as a blank, perform the same procedure using 50 mmol / L PIPES-NaOH buffer pH 6.5 (containing 0.1% TrironX-100, 0.1% BSA, 1 mmol / L CaCl2) instead of the FAD-GDH solution, and measure the absorbance Ab3 (after 3 minutes) and Ab5 (after 5 minutes). Substitute the values ​​of A3, A5, Ab3, and Ab5 into the following formula to calculate the FAD-GDH activity value.

number

[0042] 3. Purification of A. cristatus-derived FAD-GDH The culture supernatant of A. cristatus was subjected to ammonium sulfate fractionation, hydrophobic interaction chromatography, and ion exchange chromatography to obtain purified A. cristatus-derived FAD-GDH.

[0043] 4. Molecular weight estimation of purified A. cristatus FAD-GDH by SDS-PAGE The purified A. cristatus-derived FAD-GDH was subjected to SDS-PAGE after cleaving the glycan with endoglycosidase H (EndoH, NEW ENGLAND BioLabs). As a result, a band was obtained at approximately 68 kDa. N-terminal sequencing of the approximately 68 kDa band confirmed that this band was A. cristatus-derived FAD-GDH. This revealed that A. cristatus-derived FAD-GDH is approximately 68 kDa.

[0044] 5. Substrate specificity of A. cristatus FAD-GDH 5-1. Method In the above "FAD-GDH activity measurement method", the substrate (D-glucose) in the reaction solution is changed to maltose or xylose, and the activity value for each substrate is measured. The activity value obtained is divided by the activity value when glucose is used as the substrate to calculate the reactivity for each substrate (ratio to glucose %).

[0045] 5-2.Results The evaluation results are shown in Table 1. It was confirmed that the ratio of maltose to glucose was less than 1%, and the ratio of xylose to glucose was 11.5%. [Table 1]

[0046] 6. Optimum pH of A. cristatus FAD-GDH 6-1. Method The optimal pH of purified A. cristatus-derived FAD-GDH was examined as follows. Reaction solutions were prepared in the range of pH 4.0 to 8.5 by using 100 mmol / L Britton-Robinson buffer instead of 50 mmol / L PIPES-NaOH in the reaction solution, and FAD-GDH activity at each pH was measured according to the "FAD-GDH activity measurement method."

[0047] 6-2.Results The optimum pH of A. cristatus-derived FAD-GDH was confirmed to be 8.0 (Figure 1).

[0048] 7. Optimum temperature of A. cristatus FAD-GDH 7-1. Method The optimal temperature for purified A. cristatus-derived FAD-GDH was examined as follows: Pre-heating and reaction temperatures were changed from 37°C to 20°C to 60°C, and FAD-GDH activity at each temperature was measured according to the "FAD-GDH activity measurement method."

[0049] 7-2.Results The optimum temperature for A. cristatus-derived FAD-GDH was confirmed to be 55°C (Fig. 2).

[0050] 8. pH stability of A. cristatus FAD-GDH 8-1. Method The pH stability of purified A. cristatus-derived FAD-GDH was examined as follows. An enzyme solution of purified A. cristatus-derived FAD-GDH was prepared using 100 mmol / L Britton-Robinson buffer so that the GDH activity value was 5 U / mL, and the enzyme solution was treated at 37°C for 1 hour in the range of pH 4.0 to 8.0, and the activity was evaluated. In addition, A. oryzae BB-56 strain-derived FAD-GDH (see WO 2007 / 139013) was treated in the same manner and used for comparison.

[0051] 8-2.Results The relative activity of A. oryzae BB-56 strain-derived FAD-GDH was 95% or more at pH 5.0-6.0, i.e., it was stable in the pH range of 5.0-6.0 (Fig. 3). On the other hand, the relative activity of A. cristatus-derived FAD-GDH was 95% or more at pH 5.5-7.5, i.e., it was stable in the pH range of 5.5-7.5 (Fig. 3). In other words, A. cristatus-derived FAD-GDH was found to be more resistant to alkali than A. oryzae BB-56 strain-derived FAD-GDH.

[0052] 9. Thermostability of A. cristatus FAD-GDH 9-1. Method An enzyme solution of purified A. cristatus-derived FAD-GDH was prepared using 100 mM sodium phosphate buffer (pH 7.0) so that the GDH activity value was 5 U / mL, and was heated at 50°C for 20 minutes. The ratio of GDH activity after heating to that before heating (residual activity rate) was evaluated. In addition, A. oryzae BB-56-derived FAD-GDH was treated in the same way and used for comparison.

[0053] 9-2.Results The residual activity of FAD-GDH derived from A. oryzae BB-56 strain was 49%, whereas that of FAD-GDH derived from A. cristatus was 80% (Fig. 4), indicating that it is an enzyme with excellent thermostability.

[0054] 10.Electrochemical reactivity of A. cristatus-derived FAD-GDH 10-1. Method To examine the electrode reactivity of purified A. cristatus-derived FAD-GDH, the reagents were mixed so that each component had the following concentration, and glucose was quantified by chronoamperometry, plotting the current value after 5 seconds at 0.4 V. Glucose was also quantified under the same conditions for A. oryzae BB-56-derived FAD-GDH for comparison. 25~100 U / mL A. cristatus-derived FAD-GDH or A. oryzae BB-56-derived FAD-GDH 600mg / dL glucose 100mM Potassium Ferricyanide 50mM KCl 100mM sodium phosphate buffer (pH 7.0)

[0055] 10-2.Results As shown in Figure 5, the electrode reactivity of A. cristatus-derived FAD-GDH was significantly improved compared to that of A. oryzae BB-56-derived FAD-GDH (1.3-fold or more when the amount of enzyme was 50 U / mL).

[0056] 11. Summary We have succeeded in obtaining a novel FAD-GDH (FAD-GDH derived from A. cristatus) that has excellent thermal stability, high electrode reactivity, and high activity in the neutral pH range. [Industrial Applicability]

[0057] The glucose dehydrogenase of the present invention has excellent thermal stability and is useful, for example, as an enzyme for use in a glucose sensor for a blood glucose meter. In addition, from the standpoints of electrode reactivity and pH stability, the glucose dehydrogenase of the present invention is suitable for use in a glucose sensor for a blood glucose meter.

[0058] The present invention is not limited to the above-mentioned embodiments and examples. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent publications, patent publications, etc. specified in this specification are all incorporated by reference.

Claims

1. Glucose dehydrogenase, an enzyme derived from Aspergillus cristatatus, having the following characteristics: (1) Function: Catalyzes the reaction of oxidizing the hydroxyl group of glucose in the presence of an electron acceptor to produce glucono-δ-lactone; (2) Thermostability: The relative residual activity after heat treatment at 50°C for 20 minutes is 60% or more; (3) Molecular weight: Approximately 68 kDa (after removal of glycosylation, determined by SDS-PAGE).

2. A glucose dehydrogenase having the following characteristics: (1) Function: Catalyzes the reaction of oxidizing the hydroxyl group of glucose in the presence of an electron acceptor to produce glucono-δ-lactone; (2) Amino acid sequence: Contains the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence that is 90% or more identical to said amino acid sequence.

3. The glucose dehydrogenase according to claim 1 or 2, further having the following enzymatic chemical properties: (4) Substrate specificity: the reactivity to maltose is 5% or less when the reactivity to D-glucose is taken as 100%; (5) Optimal pH: 8.0; (6) Optimum temperature: 55℃; (7) pH stability: Stable at pH 5.5 to 7.

5.

4. The glucose dehydrogenase according to claim 2, which is an enzyme derived from Aspergillus cristatus.

5. A method for measuring glucose in a sample using the glucose dehydrogenase according to any one of claims 1 to 4.

6. A reagent for measuring glucose, comprising the glucose dehydrogenase according to any one of claims 1 to 4.

7. A glucose measuring kit comprising the glucose measuring reagent according to claim 6.

8. A glucose sensor comprising the glucose dehydrogenase according to any one of claims 1 to 4.

9. An enzyme preparation comprising the glucose dehydrogenase according to any one of claims 1 to 4.

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