Mutant flavin adenine dinucleotide-dependent glucose dehydrogenase and method for producing same

Abstract

Provided is a mutant flavin adenine dinucleotide-dependent glucose dehydrogenase that is improved in the reactivity with a mediator, in particular, the reactivity with a mediator that is a ruthenium compound. The mutant flavin adenine dinucleotide-dependent glucose dehydrogenase, wherein: (1) in the amino acid sequence of a protein which has the amino acid sequence represented by SEQ ID NO:1 except that histidine at the 404-position is substituted by another amino acid such as aspartic acid, or a protein which has an amino acid sequence with an identity of 80% or more to SEQ ID NO: 1 and has a glucose dehydrogenase activity, the amino acid at the 404-position in SEQ ID NO: 1 or an amino acid at a position equivalent thereto is substituted by another amino acid; and (2) the molecular weight, including sugar chain, of the protein is approximately 70-180 kDa.

Description

[Technical Field]

[0001] The present invention relates to a mutant glucose dehydrogenase. More specifically, the present invention relates to a mutant glucose dehydrogenase derived from a microorganism of the genus Aspergillus and having improved reactivity to a mediator, DNA encoding the mutant glucose dehydrogenase, an expression vector carrying the DNA, a transformant transformed with the expression vector, a method for producing the mutant glucose dehydrogenase, a method for measuring glucose using the mutant glucose dehydrogenase, and a glucose sensor. [Background technology]

[0002] Self-monitoring of blood glucose (SMBG) is important for diabetic patients to manage their own blood glucose levels and utilize it in their treatment. Many methods for SMBG have been put into practical use, with electrochemical sensing being advantageous in terms of reducing the amount of sample required, shortening measurement time, and miniaturizing the device. Sensing techniques for blood glucose measurement utilize enzymes that use blood glucose as a substrate. An example of such an enzyme is glucose dehydrogenase (EC 1.1.1.47). In recent years, among glucose dehydrogenases (hereinafter also referred to as GDHs), flavin adenine dinucleotide-dependent glucose dehydrogenase (hereinafter also referred to as FADGDH) derived from Aspergillus oryzae has been put to practical use as an enzyme for glucose sensors due to its advantages of high selectivity for glucose and high thermal stability. Patent Documents 1 and 2 report the enzymatic properties of Aspergillus oryzae FADGDH.

[0003] Glucose sensors using FADGDH measure glucose by allowing electrons generated during the process of oxidizing glucose and converting it into D-glucono-δ-lactone to flow to the electrode via a mediator. Potassium ferricyanide is commonly used as a mediator in combination with FADGDH. However, potassium ferricyanide has the problem of being easily affected by reducing substances in the blood and also easily changing to its reduced form due to the influence of environmental moisture.

[0004] An example of a more stable mediator is a ruthenium complex. While ruthenium complexes have excellent storage stability, the problem is that Aspergillus oryzae-derived GDH cannot transfer electrons to the ruthenium complex. To overcome this problem, Patent Document 3 reports a glucose sensor in which Aspergillus oryzae-derived GDH and a ruthenium complex are combined with phenazine methosulfate (hereinafter also referred to as PMS). This method allows electrons obtained in the oxidation reaction between glucose and GDH to be transferred to the ruthenium complex via PMS, making it possible to quantify glucose concentration by measuring the current response value. However, PMS is easily reduced by the influence of light and moisture in the environment, so storage stability remains an issue.

[0005] Meanwhile, attempts to improve the enzyme have also been reported. Non-Patent Document 1 reports a glucose dehydrogenase mutant that can transfer electrons directly to a ruthenium complex by introducing a mutation into a specific amino acid in FADGDH derived from Aspergillus flavus, produced in an Escherichia coli expression system. This method also reports a glucose sensor that uses a glucose dehydrogenase mutant in which the histidine at position 403 is substituted with aspartic acid and a ruthenium complex. However, the linear relationship between glucose concentration and current response value is poor, and this method has not yet been put to practical use. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-289148 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-237210 [Patent Document 3] Patent No. 5584740 [Non-patent literature]

[0007] [Non-Patent Document 1] Bioelectrochemistry,2018,Vol.123:p62-69 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention was made in view of the current state of the prior art, and aims to establish a mutant FADGDH derived from a microorganism of the genus Aspergillus that has improved reactivity to a mediator, an industrial method for producing the mutant FADGDH, and to provide a glucose sensor for measuring blood glucose levels using the mutant FADGDH. [Means for solving the problem]

[0009] In order to solve the above problems, the present inventors conducted extensive research and, as a result, produced a mutant similar to that described in Non-Patent Document 1 in an Escherichia coli expression system based on FADGDH derived from Aspergillus oryzae, which has the same amino acid sequence as FADGDH derived from Aspergillus flavus. However, they discovered a new problem: the reactivity to dichloroindophenol (hereinafter also referred to as DCPIP) was significantly reduced.

[0010] To solve the above-mentioned problems, we have found that mutant FADGDH derived from Aspergillus oryzae can be produced using an Aspergillus oryzae expression system and that by adding a sugar chain to the protein surface, it is possible to produce mutant FADGDH with improved reactivity to ruthenium complexes and DCPIP. Based on these findings, the present invention has been completed as a result of further research and improvements, and representative aspects of the present invention are as follows.

[0011] Section 1. A mutant flavin adenine dinucleotide-dependent glucose dehydrogenase, which has an amino acid sequence that is 80% or more identical to SEQ ID NO: 1 and is a protein in which the amino acid at or a position equivalent thereto in an amino acid sequence encoding a protein having glucose dehydrogenase activity has been substituted with another amino acid, the molecular weight of the protein including the sugar chain is approximately 70 to 180 kDa, and the protein is derived from a microorganism of the genus Aspergillus. Section 2. Item 1. A mutant flavin adenine dinucleotide-dependent glucose dehydrogenase according to Item 1, which is a protein having an amino acid sequence with 90% or more identity to SEQ ID NO: 1 and in which the amino acid at position 404 or a position equivalent thereto in an amino acid sequence encoding a protein having glucose dehydrogenase activity has been substituted with another amino acid. Section 3. Item 3. The mutant flavin adenine dinucleotide-dependent glucose dehydrogenase according to Item 1 or 2, which is a protein having an amino acid sequence that is 95% or more identical to SEQ ID NO: 1 and encoding a protein having glucose dehydrogenase activity, in which the amino acid at position 404 or a position equivalent thereto is substituted with another amino acid. Section 4. Item 4. The mutant flavin adenine dinucleotide-dependent glucose dehydrogenase according to any one of Items 1 to 3, which is a protein in which the amino acid at position 404 or a position equivalent thereto in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid. Section 5. Item 5. The mutant flavin adenine dinucleotide-dependent glucose dehydrogenase according to any one of Items 1 to 4, which is a protein having glucose dehydrogenase activity and which comprises an amino acid sequence in which one or several amino acids are further deleted, substituted, added, or inserted at a position other than position 404 or a position equivalent thereto in the amino acid sequence of SEQ ID NO: 1. Section 6. Item 6. The mutant flavin adenine dinucleotide-dependent glucose dehydrogenase according to any one of Items 1 to 5, wherein the amino acid substitution at histidine at position 404 or a position equivalent thereto is aspartic acid or glutamic acid. Section 7. Aspergillus microorganisms are Aspergillus oryzae or Aspergillus flavus. Aspergillus nidulans, Item 7. A mutant flavin adenine dinucleotide-dependent glucose dehydrogenase according to any one of Items 1 to 6. Section 8. A DNA encoding the mutant flavin adenine dinucleotide-dependent glucose dehydrogenase according to any one of Items 1 to 7. Section 9. A vector comprising the DNA according to item 8. Section 10. A transformant transformed with the vector according to item 9. Section 11. Item 11. The transformant according to Item 10, wherein the transformed host is a microorganism of the genus Aspergillus. Section 12. Item 12. A method for producing a mutant flavin adenine dinucleotide-dependent glucose dehydrogenase, comprising the steps of culturing the transformant according to Item 10 or 11 and collecting a protein having glucose dehydrogenase activity from the culture medium. Section 13. Item 8. A method for measuring glucose, which uses the mutant flavin adenine dinucleotide-dependent glucose dehydrogenase according to any one of Items 1 to 7. Section 14. Item 14. The glucose measurement method according to Item 13, further comprising using 2,6-dichlorophenolindophenol (DCPIP). Section 15. Item 8. A glucose sensor comprising the mutant flavin adenine dinucleotide-dependent glucose dehydrogenase according to any one of Items 1 to 7. Section 16. Item 16. The glucose sensor according to Item 15, comprising a ruthenium compound as a mediator. Section 17. Ruthenium compounds include hexaamine ruthenium (III) chloride and ruthenium (bipyridine). (II), (4,4'-dimethyl-2,2'-bipyridine)ruthenium(II), (4,4'-diphenyl-2,2'-bipyridine)ruthenium(II), (4,4'-diamino-2 ,2'-bipyridine)ruthenium(II)ruthenium(II), (4,4'-dihydroxy-2,2'-bipyridine)ruthenium(II)ruthenium(II), (4,4'-dicarboxy-2,2'-bipyridine)ruthenium(II)ruthenium(II), (4,4'-dibromo-2,2'-bipyridine)ruthenium(II)ruthenium(II), (5,5'-dimethyl-2,2'-bipyridine)ruthenium(II)ruthenium(II), (5,5'-diphenyl-2,2'-bipyridine) Item 17. The glucose sensor according to Item 16, wherein the ruthenium is at least one selected from the group consisting of ruthenium(II) ruthenium(II), (5,5'-diamino-2,2'-bipyridine) ruthenium(II) ruthenium(II), (5,5'-dihydroxy-2,2'-bipyridine) ruthenium(II) ruthenium(II), (5,5'-dicarboxy-2,2'-bipyridine) ruthenium(II) ruthenium(II), and (5,5'-dibromo-2,2'-bipyridine) ruthenium(II). [Effects of the Invention]

[0012] The mutant FADGDH of the present invention can be applied to a glucose sensor that uses a ruthenium complex as a mediator. It can also be applied to an activity measurement method that uses only DCPIP. Furthermore, the mutant FADGDH can be efficiently produced by using a transformant transformed with a recombinant vector containing a gene encoding the mutant FADGDH of the present invention. [Brief explanation of the drawings]

[0013] [Figure 1] This shows the results of SDS-PAGE of a cell extract containing the H404D mutant prepared in an E. coli expression system. [Figure 2] The results of SDS-PAGE of the H404D mutant of the purified enzyme prepared in the Aspergillus oryzae expression system are shown. [Figure 3] FIG. 1 shows the response value measured at an electrode using the H404D mutant of the purified enzyme prepared in an Aspergillus oryzae expression system. DETAILED DESCRIPTION OF THE INVENTION

[0014] The following describes in detail aspects of the present invention.

[0015] FADGDH is an enzyme that selectively oxidizes glucose using flavin adenine dinucleotide (hereinafter also referred to as FAD) as a coenzyme.

[0016] In the present invention, amino acid sequences are represented by one or three alphabetical letters. The positions of amino acid mutations are represented as follows: For example, "H404D" means that histidine (His) at position 404 is replaced with aspartic acid (Asp). The amino acids in SEQ ID NO: 1 are numbered starting with methionine (Met) as 1.

[0017] One embodiment of the present invention is an Aspergillus-derived FADGDH having a molecular weight of about 70 to 180 kDa including a sugar chain, and is a protein represented by the following (a) or (b): (a) a protein having GDH activity in which the amino acid at position 404 or a position equivalent thereto in the amino acid sequence set forth in SEQ ID NO: 1 has been substituted with another amino acid; (b) a protein having an amino acid sequence with 80% or more identity to SEQ ID NO: 1 and having glucose dehydrogenase activity, in which the amino acid at the position equivalent to position 404 of SEQ ID NO: 1 has been substituted with another amino acid.

[0018] In the above (a), SEQ ID NO: 1 is the amino acid sequence of a mutant FADGDH derived from Aspergillus oryzae, in which G at position 163 is substituted with R and V at position 551 is substituted with C, thereby improving thermostability. The amino acid sequence of SEQ ID NO: 1 is disclosed in Patent Document 1 or Patent Document 2.

[0019] In the present invention, the identity of amino acid sequences refers to a value obtained by comparison using GENETYX software. Furthermore, in the present invention, for example, a position equivalent to position 404 of SEQ ID NO: 1 in a certain amino acid sequence is determined to be equivalent by comparing the primary structure (e.g., alignment) of the sequence using GENETYX software (GENETYX CORPORATION) and the position corresponds to position 404 of SEQ ID NO: 1. If necessary, knowledge of the three-dimensional structure may also be referenced. In the present invention, GENETYX WIN Version 6.1 was used as the GENETYX software.

[0020] In the above (b), the amino acid sequence identity with SEQ ID NO: 1 is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 100%. Furthermore, the amino acid sequence may contain an amino acid sequence in which one or several amino acids have been deleted, substituted, added, or inserted at a position other than position 404 or a position equivalent thereto, as long as the sequence retains glucose dehydrogenase activity.

[0021] In the present invention, the amino acid substitution at histidine at position 404 or a position equivalent thereto is preferably a substitution with aspartic acid or glutamic acid, particularly preferably with aspartic acid.

[0022] The FADGDH of the present invention is derived from a microorganism of the genus Spergillus, including, but not limited to, Aspergillus oryzae and Aspergillus flavus.

[0023] The mutant FADGDH produced in the present invention using an Aspergillus microorganism as a host has a molecular weight including its sugar chain (measured by SDS-PAGE) of about 70 to 180 kDa, preferably about 80 to 170 kDa, and more preferably about 90 to 160 kDa. The Aspergillus microorganism used as a host is not particularly limited, but Aspergillus oryzae is preferred.

[0024] The method for producing the mutant FADGDH of the present invention is not particularly limited, but it can be produced by the procedure shown below.

[0025] The amino acid sequence constituting mutant FADGDH can be modified by conventional techniques for modifying genetic information. Specifically, DNA carrying the genetic information for a modified protein can be prepared by converting specific bases in the DNA carrying the genetic information for the protein, or by inserting or deleting specific bases. Specific methods for converting the base sequence in DNA include the use of commercially available kits (e.g., Transformer Mutagenesis Kit; Clonetech, EXOIII / Mung Bean Deletion Kit; Stratagene, Quick Change Site Directed Mutagenesis Kit; Stratagene, etc.) or polymerase chain reaction (PCR).

[0026] The DNA encoding the mutant FADGDH of the present invention hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 2 and encodes a protein having glucose dehydrogenase activity. Here, stringent conditions refer to conditions under which highly identical nucleic acids hybridize at a temperature ranging from the Tm of a perfectly matched hybrid to a temperature 15°C lower than the Tm, preferably 10°C lower. Specifically, the stringent conditions refer to conditions under which hybridization occurs at 68°C for 20 hours in a general hybridization buffer (5xSSC (0.75 M NaCl, 0.075 M Na citrate; pH 7.0), 1% blocking reagent, 0.02% SDS, 0.1% N-lauryl sarcosine). In the present invention, a base sequence having an identity of 80% or more with the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 2 is preferred, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and even more preferably 100%.

[0027] One embodiment of the present invention is a vector containing the above-mentioned DNA, a transformant transformed with the vector, a method for culturing the transformant and collecting a protein having GDH activity, and a method for producing a protein having GDH activity.

[0028] For example, the mutant FADGDH gene is inserted into an expression vector such as a plasmid, and an appropriate host such as Escherichia coli or a filamentous fungus is transformed with the gene. The resulting transformant is cultured, and the cells are collected from the culture by centrifugation or the like. The cells are then disrupted mechanically or enzymatically using lysozyme or the like, and, if necessary, the cells are degraded by the addition of a chelating agent such as EDTA, polyethylene glycol mono-p-isooctylphenyl ether (Triton-100), polyoxyethylene terephthalate (POTA), or the like. The mutant FADGDH can be solubilized by adding a surfactant such as isosorbitan monolaurate (Tween-20) to obtain a water-soluble fraction containing the mutant FADGDH. Alternatively, the expressed mutant FADGDH can be secreted directly into the culture medium by using an appropriate host-vector system.

[0029] The mutant FADGDH-containing solution obtained as described above can be precipitated by, for example, vacuum concentration, membrane concentration, salting out with ammonium sulfate, sodium sulfate, or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, or acetone. Heat treatment or isoelectric focusing is also an effective purification method. Purified mutant FADGDH can also be obtained by gel filtration using an adsorbent or gel filtration agent, adsorption chromatography, ion exchange chromatography, or affinity chromatography. The purified mutant FADGDH preparation is preferably purified to the extent that it shows a single band in electrophoresis (SDS-PAGE).

[0030] These can be carried out, for example, according to the following documents: (a) Yoshifumi Nishimura and Shigeo Ohno (eds.); Protein Experimental Protocols, Vol. 1 Functional Analysis, Vol. 2 Structural Analysis (Shujunsha), pp. 50-52 (b) Tadaomi Takenawa (ed.); Protein Handbook, p. 22-47 Alternatively, the method can be carried out as exemplified below.

[0031] The DNA carrying the genetic information for the protein to be produced is transferred into a host microorganism in a state linked to a vector.

[0032] Suitable vectors are those constructed for genetic recombination from phages or plasmids that can replicate autonomously in the host microorganism. Examples of phages include Lambda gt10 and Lambda gt11 when Escherichia coli is used as the host microorganism. Examples of plasmids that can be used with Escherichia coli as the host microorganism include pBR322, pUC19, pKK223-3, and pBluescript. Among these, those containing a promoter upstream of the cloning site that can be recognized in Escherichia coli, such as pBluescript, are preferred.

[0033] Suitable host microorganisms are not particularly limited as long as they are capable of stabilizing recombinant vectors, autonomously replicating, and expressing foreign genes. When Escherichia coli is used, examples that can be used include E. coli W3110, E. coli C600, E. coli HB101, E. coli JM109, and E. coli DH5α.

[0034] Methods for transferring a recombinant vector into a host microorganism include, for example, transferring recombinant DNA in the presence of calcium ions when the host microorganism belongs to the genus Escherichia. Electroporation may also be used. Alternatively, commercially available competent cells (e.g., Competent High DH5α; manufactured by Toyobo) may be used. When yeast is used as the host, the lithium method or electroporation method is used, and when filamentous fungi are used, the protoplast method is used.

[0035] In the present invention, methods for obtaining DNA encoding FADGDH include the following: Using the genome sequence information of Aspergillus oryzae, the predicted FADGDH gene can be identified. Next, mRNA is prepared from Aspergillus oryzae cells, and cDNA is synthesized. Using the resulting cDNA as a template, the FADGDH gene is amplified by PCR, and the gene and the vector are ligated to each other at the blunt or sticky ends of both DNAs using DNA ligase or similar to construct a recombinant vector. The recombinant vector is then transferred into a replicable host microorganism, and a recombinant microorganism containing the gene encoding FADGDH is obtained using a marker on the vector.

[0036] The microorganisms that are transformants obtained as described above can stably produce large amounts of FADGDH when cultured in a nutrient medium. Recombinants can be selected by searching for microorganisms that simultaneously express the vector marker and GDH activity. For example, microorganisms that grow in a selective medium based on a drug resistance marker and produce FADGDH can be selected.

[0037] The nucleotide sequence of the FADGDH gene was determined by the dideoxy method described in Science, Vol. 214, p. 1205 (1981). The amino acid sequence of FADGDH was deduced from the nucleotide sequence determined as described above.

[0038] Once the FADGDH gene has been selected, it can be transferred from the recombinant vector carrying it to a recombinant vector that can replicate in other microorganisms by recovering the DNA of the FADGDH gene from the recombinant vector carrying the FADGDH gene using restriction enzymes or PCR, and then ligating it with another vector fragment. Furthermore, transformation of other microorganisms with these vectors can be performed using competent cell methods using calcium treatment, electroporation, protoplast methods, etc.

[0039] Furthermore, as long as the FADGDH gene of the present invention has GDH activity, it may have a DNA sequence in which some of the amino acid residues in the translated amino acid sequence of the gene are deleted or substituted, or it may have a DNA sequence in which other amino acid residues are added or substituted.

[0040] The culture conditions for the host microorganism (transformant) can be selected taking into consideration the nutritional and physiological properties of the host. In most cases, liquid culture is used, and aerated and agitated culture is advantageous for industrial use. However, in terms of productivity, it may be more advantageous to use a filamentous fungus as the host and perform solid culture.

[0041] Nutrient sources for the medium can be a wide variety of those commonly used in microbial cultivation. Carbon sources can be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, and pyruvic acid. Nitrogen sources can be any usable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, and alkaline extract of soybean meal. Other nutrient sources include salts of phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, as well as specific amino acids and specific vitamins, which can be used as needed.

[0042] The culture temperature can be varied as appropriate within the range in which the microorganisms grow and produce FADGDH, but is preferably about 20 to 37°C. The culture time varies somewhat depending on the conditions, but the culture can be completed at an appropriate time when the FADGDH yield reaches its maximum, and is usually about 6 to 48 hours. The pH of the medium can be varied as appropriate within the range in which the microorganisms grow and produce FADGDH, but is preferably in the range of about pH 6.0 to 9.0.

[0043] Although the culture solution containing the FADGDH-producing bacterial cells in the culture can be collected and used as is, it is generally used after separating the FADGDH-containing solution from the microbial cells by filtration, centrifugation, etc., according to standard methods when FADGDH is present in the culture solution. When FADGDH is present within the bacterial cells, the bacterial cells are collected from the resulting culture by means of filtration or centrifugation, and then disrupted mechanically or enzymatically using lysozyme or the like. If necessary, a chelating agent such as EDTA and a surfactant such as polyethylene glycol mono-p-isooctylphenyl ether (Triton-100) or polyoxyethylene sorbitan monolaurate (Tween-20) are added to solubilize the FADGDH, which is then separated and collected as an aqueous solution.

[0044] The FADGDH-containing solution obtained as described above can be precipitated by, for example, vacuum concentration, membrane concentration, salting out with ammonium sulfate, sodium sulfate, or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, or acetone. Heat treatment or isoelectric focusing is also an effective purification method. Purified FADGDH can then be obtained by gel filtration using an adsorbent or gel filtration agent, adsorption chromatography, ion exchange chromatography, or affinity chromatography.

[0045] For example, a purified enzyme preparation can be obtained by separation and purification using gel filtration using Sephadex gel (GE Healthcare Biosciences) or column chromatography using DEAE Sepharose CL-6B (GE Healthcare Biosciences) or Octyl Sepharose CL-6B (GE Healthcare Biosciences). The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

[0046] In the present invention, the activity of GDH using DCPIP is measured under the following conditions.

[0047] [Test example] <Reagents> 50 mM PIPES buffer pH 6.5 (containing 0.1% Triton X-100) 2.0 mM 2,6-dichlorophenolindophenol (DCPIP) solution 1M D-glucose solution

[0048] 20.5 ml of the above PIPES buffer solution, 3.0 ml of DCPIP solution, and 5.9 ml of D-glucose solution are mixed to prepare a reaction reagent.

[0049] <Measurement conditions> 0.1 ml of FADGDH solution, which had been preheated at 37°C for 5 minutes, was added to 3 ml of the reaction reagent and gently mixed. Then, the absorbance change at 600 nm was recorded for 5 minutes using water as a control in a spectrophotometer controlled at 37°C. The absorbance change per minute (ΔOD TEST As a blank test, a solvent that dissolves FADGDH is added to the reagent mixture instead of the FADGDH solution, and the change in absorbance per minute (ΔOD BLANK From these values, GDH activity is calculated according to the following formula (1). Here, one unit (U) of GDH activity is defined as the amount of enzyme that reduces 1 micromole of DCPIP per minute in the presence of 200 mM D-glucose.

[0050]

number

[0051] In the formula, 3.0 is the volume (ml) of the reaction reagent + enzyme solution, and 16.3 is the millimolar extinction coefficient (cm) under these activity measurement conditions. 2 / µmol), 0.1 is the volume of the enzyme solution (ml), and 1.0 is the optical path length of the cell (cm).

[0052] In the present invention, hexaamineruthenium(III) chloride / 3-(4,5-dimethyl bromide The activity of GDH using (-2-thiazolyl)-2,5-diphenyltetrazolium (hereinafter also referred to as Ru / MTT) is measured under the following conditions.

[0053] [Test example] <Reagents> 50 mM phosphate buffer pH 6.5 (containing 0.1% Triton X-100) 30mM hexaamine ruthenium(III) chloride solution 10mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide solution 1M D-glucose solution 10.5 ml of the above phosphate buffer solution, 10.0 ml of hexaamine ruthenium (III) chloride solution , 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide solution 3ml D-glucose solution 5.9 ml are mixed to form a reaction reagent.

[0054] <Measurement conditions> 3 ml of the reaction reagent was preheated at 37°C for 5 minutes. 0.1 ml of the FADGDH solution was added and gently mixed. Then, the absorbance change at 565 nm was recorded for 5 minutes using a spectrophotometer controlled at 37°C with water as the control. The absorbance change per minute (ΔOD TEST For the blank test, instead of the FADGDH solution, a solvent that dissolves FADGDH was added to the reagent mixture, and the change in absorbance per minute (ΔOD BLANK ) is measured. From these values, FADGDH activity is calculated according to the following formula (2). Here, one unit (U) of GDH activity is defined as the amount of enzyme that reduces 1 micromole of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide per minute in the presence of 200 mM D-glucose.

[0055]

number

[0056] In the formula, 3.0 is the volume (ml) of the reaction reagent + enzyme solution, and 20.0 is the millimolar extinction coefficient (cm 2 / µmol), 0.1 is the volume of the enzyme solution (ml), and 1.0 is the optical path length of the cell (cm).

[0057] glucose sensor The present invention also features a glucose sensor using the mutant FADGDH described above. Electrodes include carbon, gold, and platinum electrodes. A capillary structure is typically formed between a reference electrode and a working electrode, and the capillary is then filled with an enzyme and a mediator. Methods for immobilizing the mutant FADGDH of the present invention on an electrode include using a crosslinking reagent, encapsulating it in a polymer matrix, dialysis, a photocrosslinkable polymer, a conductive polymer, or a redox polymer. Alternatively, the mutant FADGDH may be immobilized in a polymer or adsorbed onto the electrode together with an electron mediator, such as ferrocene or its derivatives, or a combination of these methods may be used. Hydrophilic polymers are preferred, including carboxymethylcellulose, polyvinyl alcohol, polystyrene sulfonic acid, chondroitin sulfate, hyaluronic acid, gum arabic, and carrageenan. Typically, the mutant FADGDH of the present invention is immobilized on a carbon electrode using glutaraldehyde, followed by treatment with a reagent containing an amine group to block the glutaraldehyde.

[0058] Glucose concentration can be measured as follows. A buffer solution is placed in a thermostatic cell and maintained at a constant temperature. Examples of mediators include those capable of accepting electrons from FAD, the coenzyme of FADGDH, and donating electrons to a color-forming substance or an electrode. Examples include ferricyanide salts, phenazine ethosulfate, phenazine methosulfate, phenylenediamine, N,N,N',N'-tetramethylphenylenediamine, 1-methoxy-phenazine methosulfate, 2,6-dichlorophenolindophenol, 2,5-dimethyl-1,4-benzoquinone, 2,6-dimethyl-1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone, nitrosoaniline, ferrocene derivatives, osmium compounds, and ruthenium compounds. Among these, ruthenium compounds are particularly preferred.

[0059] The ruthenium compounds include hexaamine ruthenium (III) chloride, (bipyridyl (4,4'-dimethyl-2,2'-bipyridine)ruthenium(II), (4,4'-diphenyl-2,2'-bipyridine)ruthenium(II), (4,4'-diamino-2,2'-bipyridine)ruthenium(II)ruthenium(II), (4,4'-dihydroxy-2,2'-bipyridine)ruthenium(II)ruthenium(II), (4,4'-dicarboxy-2,2'-bipyridine)ruthenium(II)ruthenium(II), (4,4'-dibromo-2,2'-bipyridine)ruthenium(II)ruthenium(II), (5 Examples include (5,5'-dimethyl-2,2'-bipyridine)ruthenium(II)ruthenium(II), (5,5'-diphenyl-2,2'-bipyridine)ruthenium(II)ruthenium(II), (5,5'-diamino-2,2'-bipyridine)ruthenium(II)ruthenium(II), (5,5'-dihydroxy-2,2'-bipyridine)ruthenium(II)ruthenium(II), (5,5'-dicarboxy-2,2'-bipyridine)ruthenium(II)ruthenium(II), (5,5'-dibromo-2,2'-bipyridine)ruthenium(II), etc. Hexaamine ruthenium(III) chloride is particularly preferred.

[0060] An electrode with the mutant FADGDH of the present invention immobilized thereon is used as the working electrode, and a counter electrode (e.g., a platinum electrode) and a reference electrode (e.g., an Ag / AgCl electrode) are used. A preferred method is amperometry, in which a constant voltage is applied to the carbon electrode, and after the current reaches a steady state, a glucose-containing sample such as blood or urine is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared using glucose solutions of standard concentrations. [Example]

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0062] Example 1: Construction of an expression vector for the H404D mutant for expression in E. coli For the FADGDH derived from Aspergillus oryzae, the sequence described in International Patent Publication WO2009 / 119728, i.e., a sequence obtained by introducing the G163R+V551C mutation into the sequence cloned from the cDNA of Aspergillus oryzae TI strain, was used. The amino acid sequence of the FADGDH mutant is shown in SEQ ID NO: 1, and the DNA sequence encoding this amino acid sequence is shown in SEQ ID NO: 2. The recombinant plasmid pAOGDH containing the gene encoding FADGDH of SEQ ID NO: 2 was transformed into commercially available Escherichia coli competent cells (E. coli). DH5α (TOYOBO) was transformed and plated on LB agar medium (1.0% polypeptone, 0.5% yeast extract, 1.0% NaCl, 1.5% agar; pH 7.3) containing ampicillin, and then cultured overnight at 30°C. The resulting transformants were then inoculated into LB liquid medium (1% polypeptone, 0.5% yeast extract, 1.0% NaCl; pH 6.5) containing ampicillin (50 mg / ml; Nacalai Tesque) and cultured overnight at 30°C with shaking.

[0063] The plasmid was extracted and purified from the resulting bacterial cells by miniprep using MagExtractor (registered trademark)-Plasmid- (manufactured by Toyobo Co., Ltd.) to prepare the pAOGDH plasmid. Using pAOGDH as a template, a synthetic oligonucleotide of SEQ ID NO: 3 and its complementary synthetic oligonucleotide of SEQ ID NO: 4, which were designed to replace histidine at position 404 with another aspartic acid, were inserted into the pAOGDH using QuickChange (registered trademark) Site-Directed PCR. PCR was carried out using a Mutagenesis Kit (STRATAGENE).

[0064] Next, commercially available Escherichia coli competent cells (E. coli DH5α; manufactured by TOYOBO) were transformed with the PCR product and cultured on LB agar medium containing ampicillin at 37°C for 16 hours. A single colony was then inoculated into LB liquid medium containing ampicillin and cultured overnight with shaking at 30°C. One milliliter of the culture medium was then inoculated, and the plasmid was extracted by standard methods. The relevant site in the extracted plasmid was identified using a DNA sequencer (ABI PRISM® 3700 DNA Analyzer; manufactured by Perkin-Elmer). The plasmid containing the FADGDH mutant in which position 404 was replaced with D was named pAOGDH-H404D.

[0065] Example 2: Preparation of crude enzyme solution containing H404D mutant using E. coli expression system E. coli JM109 transformed with pAOGDH-H404D obtained in Example 1 was cultured on LB agar medium containing ampicillin at 30°C for 16 hours to obtain a single colony. The single colony of JM109 / pAOGDH-H404D was then inoculated into 5 ml of LB liquid medium containing ampicillin and cultured with shaking at 30°C for 16 hours. A portion of the culture was centrifuged to collect bacterial cells, which were then disrupted with glass beads in 50 mM phosphate buffer (pH 6.0) to prepare a crude enzyme solution. Figure 1 shows the results of SDS-PAGE. The expression levels of wild-type FADGDH before mutation and the H404D mutant were compared by SDS-PAGE and confirmed to be equivalent. The molecular weight of both proteins is approximately 63 kDa.

[0066] Example 3: Reactivity of crude enzyme solution containing H404D mutant using E. coli expression system Using the crude enzyme solution prepared in Example 2, GDH activity was measured using the above-mentioned DCPIP and Ru / MTT. The results are shown in Table 1. When DCPIP was used, the GDH activity of the H404D mutant was reduced to 4% of that of the wild-type, which was taken as 100%. This demonstrated that the reactivity of the H404D mutant to DCPIP was significantly reduced. On the other hand, when Ru / MTT was used, the GDH activity of the H404D mutant was increased to 327% of that of the wild-type, which was taken as 100%. This demonstrated that the reactivity of the H404D mutant to ruthenium was significantly improved when Ru / MTT was used as a mediator.

[0067] [Table 1]

[0068] Example 3: Construction of the gene for the H404D mutant for expression in Aspergillus oryzae The construction of the koji mold expression plasmid, selection of the recombinant host, and transformation were performed according to the method described in WO 2016 / 114334. Specifically, the koji mold expression plasmid was pTNE-AomFADGDH, the recombinant host was the Aspergillus oryzae NS4 strain, and transformation was performed using the protoplast-PEG method. The H404D mutation was introduced into pTNE-AomFADGDH using the same primers (SEQ ID NO: 3 and SEQ ID NO: 4) used in Example 1 and the QuickChange® Site-Directed Mutagenesis Kit. The relevant site in the plasmid was identified using a DNA sequencer, and the plasmid containing the FADGDH mutant with a D substitution at position 404 was designated pTNE-AomFADGDH-H404D. Next, Aspergillus oryzae NS4 was transformed with pTNE-AomFADGDH-H404D by the protoplast-PEG method, and the resulting transformants were cultured in DP medium. The transformant with the highest productivity was selected. For comparison, a transformant expressing wild-type AomFADGDH was generated by the same method.

[0069] Example 4: Preparation of H404D mutant using the Aspergillus oryzae expression system The transformant obtained in Example 3 was inoculated into 60 mL of sterilized DP liquid medium in a 500 mL Sakaguchi flask and cultured with shaking at 30°C for 2 days to prepare a preculture solution. Next, the preculture solution was inoculated into 7.0 L of YPM medium (5% yeast extract, 2% soybean peptone, 5% maltose) in a 10 L jar fermenter and cultured for 3 days at a culture temperature of 35°C, an agitation speed of 400 rpm, an aeration rate of 6.0 L / min, and an internal pressure of 0.2 MPa. The culture solution was then filtered through a filter cloth, and the filtrate was collected.

[0070] The filtrate was concentrated using a 30,000 molecular weight cutoff UF membrane (Millipore), and the buffer was exchanged by successively adding phosphate buffer (50 mM, pH 6.0) to the concentrate. Ammonium sulfate was then gradually added to the concentrate at 40% (w / v) and stirred at room temperature for 30 minutes. Afterwards, excess precipitate was removed using a filter aid. The filtrate was then loaded onto a 200 mL PS Sepharose FastFlow (GE Healthcare) column equilibrated with 50 mM potassium phosphate buffer (pH 6.0) containing 40% (w / v) ammonium sulfate. Proteins were eluted by stepwise exchange with 50 mM phosphate buffer (pH 6.0). The eluted fraction was then concentrated using a 10,000 molecular weight cutoff hollow fiber membrane (Spectrum Laboratories), and the buffer was exchanged by successively adding phosphate buffer (50 mM, pH 6.0) to the concentrate. The enzyme solution was then passed through a DEAE Sepharose Fast Flow (GE Healthcare) column equilibrated with 50 mM phosphate buffer (pH 6.0) to obtain a purified enzyme. Figure 2 shows the results of SDS-PAGE analysis of the purified enzyme. The molecular weight of the H404D mutant was approximately 70-180 kDa, while that of the wild-type enzyme was approximately 90-200 kDa.

[0071] Example 5: Reactivity of H404D mutant using the Aspergillus oryzae expression system The GDH activity of the H404D mutant prepared in Example 4 was measured using DCPIP in the same manner as in Example 1. The results are shown in Table 2. The GDH activity of the H404D mutant when DCPIP was used was 48%, assuming that the wild-type GDH activity was 100%. The reactivity to DCPIP was more than 10-fold higher than that of the H404D mutant prepared in the E. coli expression system of Example 2.

[0072] [Table 2]

[0073] Example 6: Evaluation of response values ​​in electrochemical sensors of H404D mutants using a koji mold expression system Price First, a solution (pH 7.0) having the following composition was prepared as a glucose measurement reagent. 1mM sodium citrate (pH 7.0) 45mM hexaamine ruthenium(III) chloride 0.4mg / ml FADGDH

[0074] Five microliters of 0.5% CMC (carboxymethylcellulose) was dropped onto the working, counter, and reference electrodes of a three-electrode disposable chip (DEP-CHIP; size: 12.5 × 4 × 0.3 mm, carbon: EP-PP, BioDevice Technologies, Inc.) and dried at 50°C for 10 minutes. Next, 5 μL of the above composition solution was dropped onto the CMC-immobilized area and heated at 50°C for 10 minutes to form a sensor chip. This sensor chip was connected to a potentiostat / galvanostat (HSV-110, Hokuto Denko) via a dedicated socket. Five microliters of a 0-500 mg / dL standard glucose solution was added to the composition on the electrodes. A voltage of +0.42 V was applied, and the current was monitored. The current value 3 seconds after application was taken as the response value. Figure 3 shows the relationship between the concentration of the glucose standard solution and the response value.

[0075] The results of the measurements showed that the H404D mutant exhibited a glucose concentration-dependent response, demonstrating that hexaamine ruthenium (III) chloride could be used as a mediator. The wild type used as a control did not give any response values ​​at each glucose concentration, indicating that hexaamine ruthenium (III) chloride could not be used as a mediator. [Industrial Applicability]

[0076] The mutant FADGDH of the present invention is expected to be widely used in the fields of medicine and diagnosis when applied to reagents and sensors for measuring blood glucose levels.

Claims

1. A glucose sensor comprising: (a) a first electrode; (a) A mutant flavin adenine dinucleotide-dependent glucose dehydrogenase, which is expressed by Aspergillus oryzae and has an amino acid sequence having 90% or more identity with SEQ ID NO: 1 and in which the amino acid at position 404 or a position equivalent thereto in the amino acid sequence encoding a protein having glucose dehydrogenase activity is substituted with aspartic acid or glutamic acid, the molecular weight of the protein including the sugar chain is about 70 to 180 kDa, and the protein is derived from a microorganism of the genus Aspergillus. (b) Ruthenium compounds

2. 2. The glucose sensor according to claim 1, wherein (a) the mutant flavin adenine dinucleotide-dependent glucose dehydrogenase is a mutant protein expressed by Aspergillus oryzae, which has an amino acid sequence having 95% or more identity with SEQ ID NO: 1 and in which the amino acid at position 404 or a position equivalent thereto in an amino acid sequence encoding a protein having glucose dehydrogenase activity is substituted with aspartic acid or glutamic acid.

3. 3. The glucose sensor according to claim 1 or 2, wherein (a) the mutant flavin adenine dinucleotide-dependent glucose dehydrogenase is derived from Aspergillus oryzae, Aspergillus flavus, or Aspergillus nidulans.

4. A glucose sensor comprising: (a) a first electrode; (a) A mutant flavin adenine dinucleotide-dependent glucose dehydrogenase, which is a mutant protein expressed by Aspergillus oryzae, in which histidine at position 404 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid, the molecular weight of the protein including the sugar chain is about 70 to 180 kDa, and the protein is derived from the genus Aspergillus. (b) Ruthenium compounds

5. 5. The glucose sensor according to claim 4, wherein (a) the mutant flavin adenine dinucleotide-dependent glucose dehydrogenase is a mutant protein expressed by Aspergillus oryzae, in which histidine at position 404 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid.

6. (b) The ruthenium compound is hexaamine ruthenium chloride (III), (bipyridine) ruthenium (II), (4,4'-dimethyl-2,2'-bipyridine) ruthenium (II), (4,4'-diphenyl-2,2'-bipyridine) ruthenium (II), (4,4' -diamino-2,2'-bipyridine)ruthenium(II)ruthenium(II), (4,4'-dihydroxy-2,2'-bipyridine)ruthenium(II)ruthenium(II), (4,4'-dicarboxy-2,2'-bipyridine)ruthenium(II)ruthenium(II), (4,4'-dibromo-2,2'-bipyridine)ruthenium(II)ruthenium(II), (5,5'-dimethyl-2,2'-bipyridine)ruthenium(II)ruthenium(II), (5,5'-diphenyl-2,2'-bipyridine) The glucose sensor according to any one of claims 1 to 5, wherein the sensor is at least one selected from the group consisting of ruthenium(II) ruthenium(II), (5,5'-diamino-2,2'-bipyridine) ruthenium(II) ruthenium(II), (5,5'-dihydroxy-2,2'-bipyridine) ruthenium(II) ruthenium(II), (5,5'-dicarboxy-2,2'-bipyridine) ruthenium(II) ruthenium(II), and (5,5'-dibromo-2,2'-bipyridine) ruthenium(II).

7. A method for manufacturing a glucose sensor, comprising the following steps (a) to (d): (a) transforming a host, Aspergillus oryzae, with a vector containing DNA encoding a protein having an amino acid sequence with 90% or more identity to SEQ ID NO: 1 and having glucose dehydrogenase activity, in which the amino acid at position 404 or a position equivalent thereto is substituted with aspartic acid or glutamic acid; (b) culturing the transformant obtained in step (a) and collecting a protein having glucose dehydrogenase activity from the culture medium. (c) purifying the protein having glucose dehydrogenase activity obtained in step (b) to obtain a mutant flavin adenine dinucleotide-dependent glucose dehydrogenase. (d) immobilizing the mutant flavin adenine dinucleotide-dependent glucose dehydrogenase obtained in step (c) on an electrode;

8. A method for measuring glucose using the following (a) and (b): (a) A mutant flavin adenine dinucleotide-dependent glucose dehydrogenase expressed by Aspergillus oryzae, which has an amino acid sequence having 90% or more identity with SEQ ID NO: 1 and is a protein in which the amino acid at position 404 or a position equivalent thereto is substituted with aspartic acid or glutamic acid in the amino acid sequence encoding a protein having glucose dehydrogenase activity, and which has a molecular weight including the sugar chain of about 70 to 180 kDa and is derived from a microorganism of the genus Aspergillus. (b) 2,6-dichlorophenolindophenol (DCPIP)

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