JP2012191882A - Method for improving stability of flavin adenine dinucleotide-dependent glucose dehydrogenase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a modified flavin adenine-dependent glucose dehydrogenase improved in stability by overcoming defects of a known flavin adenine-dependent glucose dehydrogenase.SOLUTION: The modified FADGDH improved in stability is obtained by substituting an amino acid sequence in FADGDH with another amino acid by protein engineering method. A protein in which an amino acid at a position 339 in a specific amino acid sequence is substituted with another amino acid is provided. Alternatively, in the amino acid sequence having an amino acid sequence having ≥60% homology with at least either one of specific sequences and encoding a protein having glucose dehydrogenase activity, a protein in which the amino acid at the position 339 in the specific sequence is substituted with another amino acid is also provided.

Description

The present invention improves the stability obtained by substituting the amino acid sequence of Aspergillus oryzae-derived flavin adenine dinucleotide-dependent glucose dehydrogenase (hereinafter, flavin adenine dinucleotide-dependent glucose dehydrogenase is also referred to as FADGDH) with other amino acids. The modified FADGDH.
The present invention also relates to a gene encoding the modified FADGDH, a vector containing the gene, a transformant transformed with the vector, and a method for producing modified FADGDH by culturing the transformant.
Furthermore, the present invention relates to various applications of the modified FADGDH to glucose measuring reagents and the like.

Self-monitoring of Blood Glucose (SMBG) is important for diabetic patients to manage their blood glucose level and utilize it for treatment. With regard to SMBG, practical use is progressing in many ways, and electrochemical sensing is advantageous in terms of reducing the amount of specimen, reducing measurement time, and downsizing the apparatus.

  As a sensing technique in blood glucose measurement technology, an enzyme using glucose in blood as a substrate is used. An example of such an enzyme is glucose oxidase (EC 1.1.3.4). Glucose oxidase has the advantage of high selectivity to glucose and high stability to heat. However, a glucose sensor using glucose oxidase is measured when electrons generated in the process of oxidizing glucose and converting it to D-glucono-δ-lactone flow to the electrode through a mediator. There is a problem that the measured value is affected because it is easily passed to dissolved oxygen in the blood.

  In order to avoid such problems, pyrroloquinoline quinone-dependent glucose dehydrogenase (hereinafter also referred to as PQQGDH) (EC 1.1.5.2 (formerly EC 1.1.9.17)) is used as an enzyme for glucose sensor. It is used. PQQGDH is advantageous in that it is not affected by dissolved oxygen, but it also has a drawback in that the accuracy of the measured value is impaired because it acts on sugars other than glucose such as maltose and lactose.

Therefore, flavin adenine dinucleotide-dependent glucose dehydrogenase is expected to be applied as an enzyme for glucose sensors as a glucose dehydrogenase (hereinafter also referred to as GDH) that is not affected by dissolved oxygen and has excellent substrate specificity. FADGDH is described in Non-Patent Documents 1 to 6, and has been known for a long time.
Further, Patent Document 1 describes a material property of FADGDH derived from Aspergillus terreus and a glucose sensor using FADGDH. Patent Document 2 describes an Aspergillus terreus gene sequence and an amino acid sequence. Patent Document 3 describes the gene sequence, amino acid sequence, and production method of FADGDH derived from Aspergillus oryzae. Patent Document 4 describes a modified FADGDH with improved thermal stability obtained by substituting a part of the amino acid sequence of Aspergillus oryzae-derived FADGDH with another amino acid. Patent Document 5 describes modified FADGDH with improved thermal stability and / or xylose activity obtained by substituting the amino acid sequences of FADGDH derived from Aspergillus oryzae and FADGDH derived from Aspergillus terreus.

WO2004 / 058958 WO2006 / 101239 JP2007-289148 JP2008-237210A WO2008 / 059777

Biochim Biophys Acta. 1967 Jul 11; 139 (2): 265-76. Biochim Biophys Acta. 1967 Jul 11; 139 (2): 277-93. Biochim Biophys Acta. 146 (2): 317-27 Biochim Biophys Acta. 146 (2): 328-35 J Biol Chem (1967) 242: 3665-3672. Appl Biochem Biotechnol (1996) 56: 301-310

  An object of the present invention is to provide an enzyme that can be used as a reagent for measuring blood glucose, which is advantageous in practical use as compared with known enzymes for glucose sensors. More specifically, it is to provide a modified FADGDH with improved stability obtained by substituting the amino acid sequence of FADGDH with another amino acid by genetic engineering techniques.

Since known FADGDH disclosed in Patent Document 3 or Patent Document 4 is not affected by dissolved oxygen and has high selectivity for glucose, it can be used as a next-generation glucose sensor enzyme that changes to glucose oxidase or PQQGDH. Application is expected. In particular, Patent Document 4 describes modified FADGDH that maintains stability in heat and activity in a wide pH range by substituting a part of the amino acid sequence of Aspergillus oryzae-derived FADGDH with another amino acid.
In the manufacturing process of the blood glucose sensor strip, FADGDH may be heat-dried on the strip. In such a case, if the thermal stability of FADGDH is high, inactivation of the enzyme is reduced during the heat treatment, which is beneficial. The modified FADGDH described in Patent Document 4 has greatly improved thermal stability as compared with that before modification. However, depending on the heat drying treatment conditions, there is a risk of inactivation even with the modified FADGDH, and it is necessary to further improve the heat resistance.

  As a result of intensive studies to overcome the disadvantages of the modified FADGDH, the present inventors have further improved stability by substituting specific amino acids of the modified FADGDH described in Patent Document 4 with other amino acids. The site to be made was found and completed in the present invention.

That is, the present invention has the following configuration.
[Claim 1]
A protein represented by any one of the following (a) to (c).
(A) A protein in which the amino acid at position 339 is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 1.
(B) A protein in which the amino acid at position 339 is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 2.
(C) an amino acid sequence having an amino acid sequence having 60% or more homology with at least one of SEQ ID NO: 1 or SEQ ID NO: 2 and encoding a protein having glucose dehydrogenase activity, "position 339 of SEQ ID NO: 1" Or a protein in which an amino acid at a position equivalent to “position 339 of SEQ ID NO: 2” is substituted with another amino acid.
[Section 2]
The protein according to Item 1, further comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added (inserted) at a position other than the position where the amino acid is substituted, and has glucose dehydrogenase activity protein.
[Section 3]
The protein according to Item 1, wherein the amino acid substitution at position 339 P or an equivalent position thereof is A, C, I, L, V.
[Claim 4]
The protein according to Item 1, which is obtained by substituting P at position 339 with A, C, I, L, or V, and having improved stability.
[Section 5]
Item 5. A gene encoding the protein according to any one of Items 1 to 4.
[Claim 6]
Item 6. A vector comprising the gene according to Item 5.
[Claim 7]
A transformant transformed with the vector according to Item 6.
[Section 8]
Item 8. A method for producing a protein having glucose dehydrogenase activity, comprising culturing the transformant according to Item 7 and collecting a protein having glucose dehydrogenase activity.
[Claim 9]
Item 5. A glucose assay kit comprising the protein according to any one of items 1 to 4.
[Section 10]
Item 5. A glucose sensor comprising the protein according to any one of Items 1 to 4.
[Section 11]
Item 5. A glucose measurement method comprising the protein according to any one of Items 1 to 4.

  According to the present invention, a novel modified FADGDH having improved stability, which is useful as an enzyme for clinical tests, can be created, and the FADGDH can be produced industrially in large quantities.

Comparison of FADGDH amino acid sequence (SEQ ID NO: 1) derived from Aspergillus oryzae wild strain and FADGDH amino acid sequence (SEQ ID NO: 3) derived from Aspergillus tereus wild strain. Comparison of pH stability of FADGDH (Mut1) and P339L before modification.

  The present invention is described in detail below.

Flavin adenine dinucleotide-dependent glucose dehydrogenase is an enzyme that selectively oxidizes glucose using flavin adenine dinucleotide (hereinafter, flavin adenine dinucleotide is also referred to as FAD) as a coenzyme.

In the present specification, the amino acid sequence is represented by one or three letters of the alphabet. The position of amino acid mutation is expressed as follows. For example, “P339L” means that proline (Pro) at position 339 (339th from the N-terminus) is replaced with leucine (Leu). “339L” indicates that the amino acid at position 339 (or 339) is Leu (L) regardless of the presence or absence of mutation.
In SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, amino acid notation is numbered with methionine (Met) as 1.

One embodiment of the present invention is a protein represented by the following (a) or (b).
(A) A protein in which the amino acid at position 339 is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 1.
(B) A protein in which the amino acid at position 339 is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 2.

Here, SEQ ID NO: 1 is the amino acid sequence of FADGDH derived from the wild strain Aspergillus oryzae, SEQ ID NO: 2 is G at position 163 in the amino acid sequence of SEQ ID NO: 1 as R, and V at position 551 C is an amino acid sequence of a modified FADGDH whose thermal stability is improved by substituting each of C.
These amino acid sequences are known from Patent Document 3 or Patent Document 4.

  The amino acid substitution at P at position 339 or an equivalent position thereof is not particularly limited, but is preferably any one of the group consisting of P339A, P339C, P339I, P339L and P339V. More preferred are P339A, P339I, P339L and P339V. Most preferably, it is P339L.

Another embodiment of the present invention is a protein represented by the following (c).
(C) an amino acid sequence having an amino acid sequence having 60% or more homology with at least one of SEQ ID NO: 1 or SEQ ID NO: 2 and encoding a protein having glucose dehydrogenase activity, "position 339 of SEQ ID NO: 1" Or a protein in which an amino acid at a position equivalent to “position 339 of SEQ ID NO: 2” is substituted with another amino acid.

  Examples of the “amino acid sequence having an amino acid sequence having 60% or more homology with at least either SEQ ID NO: 1 or SEQ ID NO: 2 and encoding a protein having glucose dehydrogenase activity” include, for example, SEQ ID NO: 3 or SEQ ID NO: The amino acid sequence of FADGDH from Aspergillus terreus wild strain shown in 4 is exemplified. This amino acid sequence is known from US Pat.

In the present specification, the homology of amino acid sequences means a value compared with GENETYX software. For example, when the amino acid sequence of FADGDH derived from the wild Aspergillus oryzae strain (SEQ ID NO: 2) and the amino acid sequence of FADGDH derived from the wild Aspergillus tereus strain (SEQ ID NO: 3) were compared and analyzed using GENETYX software, 63.0 % Homology (see FIG. 1).
In the present specification, for example, the position equivalent to position 339 of SEQ ID NO: 2 in a certain amino acid sequence is a position corresponding to position 339 of SEQ ID NO: 2 when the primary structure (for example, alignment) of the sequence is compared with GENETYX software. Is judged as equivalent. You may refer to the knowledge of a three-dimensional structure etc. further as needed.
Specifically, from the comparison data of the alignment between the amino acid sequence of FADGDH derived from the wild strain of Aspergillus oryzae and the amino acid sequence of FADGDH derived from the wild strain of Aspergillus terreus, the position equivalent to position 339 of the present invention is aspergillus Corresponds to P at position 359 of FADGDH derived from wild Teleus strain.
The GENETYX software used was GENETYX WIN Version 6.1 sold by GENETYX CORPORATION.

In the above (c), the homology of the amino acid sequence with at least either SEQ ID NO: 1 or SEQ ID NO: 2 is preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95%. That's it.

  In the protein represented by any of the above (a) to (c), one or several amino acids were further deleted, substituted or added (inserted) at a position other than the position where the amino acid was substituted. A protein having an amino acid sequence and having glucose dehydrogenase activity is also an embodiment of the present invention.

  The protein of the present invention is a modified FADGDH having improved stability than before modification.

  For example, in the above protein, a protein in which the amino acid substitution at position P at 339 or an equivalent position thereof is L is more stable than FADGDH before modification.

In the present application, the thermal stability is a residual ratio of the activity value after the heat treatment when the activity value before the heat treatment is 100%. This is the residual rate in Table 1, Table 2, and Table 3. It is judged that the higher the residual rate, the better the thermal stability than before the modification.

In the present application, the pH stability is the residual ratio of the activity value after 16 hours when the activity value immediately after dissolving the enzyme in various pH buffers is defined as 100%. It is judged that the higher the residual ratio, the better the pH stability than before the modification.

  In addition, the protein of the present invention has an amino acid substitution at position 339 as long as it has GDH activity in the above protein and has improved stability. The site may have one or more (for example, several) amino acid mutations. The mutation may be one or several substitutions, deletions, insertions, or combinations thereof.

The modified FADGDH of the present invention can be produced by various known means. Below, the manufacturing method of modified FADGDH which improved stability obtained by substituting the amino acid sequence of FADGDH derived from the wild strain of Aspergillus oryzae represented by SEQ ID NO: 1 is exemplified. Although a manufacturing method is not specifically limited, It can manufacture in the procedure as shown below.
As a method for modifying the amino acid sequence constituting FADGDH derived from the wild Aspergillus oryzae strain, a commonly performed method for modifying genetic information is used. That is, DNA having genetic information of a modified protein is created by converting a specific base of DNA having genetic information of the protein, or by inserting or deleting a specific base. As a specific method for converting a base sequence in DNA, for example, a commercially available kit (Transformer Mutagenesis Kit; Clonetech, EXOIII / Mung Bean Selection Kit; manufactured by Stratagene, Quick Change Site Directed; Alternatively, the polymerase chain reaction (PCR) can be used.

The gene that is the basis for modification of the modified FADGDH of the present invention is not particularly limited, but it is desirable to use FADGDH derived from the genus Aspergillus. More preferably, it is FADGDH derived from Aspergillus oryzae or Aspergillus terreus.
Examples of FADGDH derived from Aspergillus oryzae include the protein represented by SEQ ID NO: 1 or SEQ ID NO: 2.
Examples of FADGDH derived from Aspergillus terreus include SEQ ID NO: 3 or the protein represented by SEQ ID NO: 4.

  One embodiment of the present invention is a gene encoding the protein described in any of the above.

  Examples of the gene encoding FADGDH derived from Aspergillus oryzae represented by SEQ ID NO: 1 and SEQ ID NO: 2 include the nucleotide sequences represented by SEQ ID NO: 5 and SEQ ID NO: 6. Examples of the gene encoding FADGDH derived from Aspergillus terreus of SEQ ID NO: 3 and SEQ ID NO: 4 include the nucleotide sequences represented by SEQ ID NO: 7 and SEQ ID NO: 8.

  The gene encoding the modified FADGDH of the present invention hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence set forth in SEQ ID NO: 5, 6, 7 or 8, and exhibits FADGDH activity. DNA encoding a protein having the same. Here, the stringent condition refers to a condition in which nucleic acids having high homology, for example, hybridize at a temperature ranging from Tm of a perfectly matched hybrid to a temperature 15 ° C., preferably 10 ° C. lower than the Tm. Specifically, for example, it refers to conditions for hybridization in a general hybridization buffer solution at 68 ° C. for 20 hours. In the present invention, 50% or more homology, preferably 80% or more homology, more preferably 90% or more homology with the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5, 6, 7 or 8. More preferably, a base sequence encoding an amino sequence having a homology of 95% or more is considered to correspond to a stringent condition.

  The gene of the present invention may include a gene in which the codon usage is changed, for example, for the purpose of improving the expression of FADGDH. Specifically, when substituting proline at position 339 in SEQ ID NOs: 1 and 2 with leucine, the CCTs from the 1015th to the 1017th positions in the nucleotide sequences described in SEQ ID NO: 5 and SEQ ID NO: 6 are replaced with TTA, TTG, CTT, CTC , CTA, or CTG may be modified. When substituting proline at position 357 in SEQ ID NO: 3 with leucine, the CCC from 1075th to 1077th in the base sequence described in SEQ ID NO: 7 is changed to any of TTA, TTG, CTT, CTC, CTA, and CTG. Also good. When substituting proline at position 335 of SEQ ID NO: 4 with leucine, the CCC from the 1003rd to 1005th positions in the base sequence described in SEQ ID NO: 8 is replaced with any of TTA, TTG, CTT, CTC, CTA, and CTG. It may be modified.

  In one embodiment of the present invention, a vector having the above gene, a transformant transformed with the vector, culturing the transformant, and collecting a protein having glucose dehydrogenase activity are obtained. It is a production method.

  For example, the above FADGDH gene is inserted into an expression vector (many things such as plasmids are known in the art), and an appropriate host (many things such as E. coli are known in the art) Transform. After culturing the obtained transformant and collecting the bacterial cells from the culture solution by centrifugation or the like, the bacterial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, such as EDTA A water-soluble fraction containing FADGDH can be obtained by adding a chelating agent or a surfactant and solubilizing. Alternatively, the expressed FADGDH can be secreted directly into the culture medium by using an appropriate host vector system.

  The FADGDH-containing solution obtained as described above is precipitated by, for example, vacuum concentration, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. You just have to let them know. Heat treatment and isoelectric point treatment are also effective purification means. Further, purified FADGDH can be obtained by performing gel filtration with an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

These can proceed according to the following literature, for example.
(A) Protein Experiment Protocol Volume 1 Functional Analysis, Volume 2 Structural Analysis (Syujunsha) Supervised by Yoshifumi Nishimura and Shigeo Ohno (b) Revised Protein Experiment Notes Extraction and separation and purification (Yodosha) Masato Okada, Kaoru Miyazaki Editing It can also be carried out by the method exemplified below.

  The prepared DNA having the genetic information of the protein is transferred into the host microorganism in a state of being linked to the vector.

  Suitable vectors are those constructed for gene recombination from phages or plasmids that can grow independently in a host microorganism. Examples of the phage include Lambda gt10 and Lambda gt11 when Escherichia coli is used as the host microorganism. Examples of plasmids include pBR322, pUC19, and pBluescript when Escherichia coli C600, Escherichia coli HB101, Escherichia coli DH5α, and the like are used as hosts. When yeast such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis is used as a host, pAUR101, pAUR224, pYE32 and the like are exemplified. When molds such as Aspergillus oryzae are used as hosts, pUSA, pUSE and the like are exemplified.

  As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, a method for transferring the recombinant DNA in the presence of calcium ions can be employed. An electroporation method may be used. Furthermore, commercially available competent cells (for example, competent high DH5α; manufactured by Toyobo) may be used. When yeast is used as the host, the lithium method or electroporation method is used. When filamentous fungi are used, the protoplast method or the like is used.

  In the present invention, methods for obtaining a gene encoding FADGDH include the following methods. Using the genome sequence information of Aspergillus oryzae, the predicted FADGDH gene can be found. Next, mRNA is prepared from Aspergillus oryzae cells and cDNA is synthesized. Using the cDNA thus obtained as a template, the FADGDH gene is amplified by the PCR method, and this gene is bound and closed with a DNA ligase or the like at the blunt end or sticky end of both DNAs to construct a recombinant vector. After the recombinant vector is transferred to a replicable host microorganism, a recombinant microorganism containing a gene encoding FADGDH is obtained using the vector marker.

  The microorganism which is a transformant obtained as described above can stably produce a large amount of GDH by being cultured in a nutrient medium. Selection of a recombinant may be performed by searching for a microorganism that simultaneously expresses a marker of the vector and FADGDH activity. For example, a microorganism that grows in a selective medium based on a drug resistance marker and generates GDH may be selected.

  The base sequence of the FADGDH gene was decoded by the dideoxy method described in Science, Vol. 214, 1205 (1981). The amino acid sequence of FADGDH was estimated from the base sequence determined as described above.

  As described above, transfer from a recombinant vector carrying the FADGDH gene once selected to a recombinant vector capable of replicating in another microorganism can be carried out by using a restriction enzyme or PCR method from the recombinant vector carrying the FADGDH gene. It can be easily carried out by collecting the DNA that is the FADGDH gene and binding it to other vector fragments. Further, for transformation of other microorganisms with these vectors, a competent cell method using calcium treatment, an electroporation method, a protoplast method, or the like can be used.

  The FADGDH gene of the present invention may have a DNA sequence in which a part of each amino acid residue of the translated amino acid sequence of the gene is deleted or substituted as long as it has glucose dehydrogenase activity. It may have a DNA sequence in which other amino acid residues are added or substituted.

  As a method for modifying a gene encoding FADGDH derived from a wild strain, a commonly performed technique for modifying genetic information is used. That is, a DNA having genetic information of a modified protein is created by converting a specific base of DNA having genetic information of the protein, or by inserting or deleting a specific base. As a specific method for converting bases in DNA, for example, a commercially available kit (Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Selection Kit; manufactured by Stratagene, QuickChange SiteDirected MutagenStained MutaseNeste, etc.) Use of the chain reaction method (PCR) is mentioned.

  The culture form of the host microorganism as a transformant may be selected in consideration of the nutritional physiological properties of the host. In many cases, it is advantageous to use liquid culture and industrially perform aeration and agitation culture. However, in consideration of productivity, it may be advantageous to use a filamentous fungus as a host and perform the solid culture.

  As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, lactose, molasses, pyruvic acid and the like are used. The nitrogen source may be any available nitrogen compound. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

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

  Although the culture solution containing the cells producing FADGDH in the culture can be collected and used as it is, generally, when FADGDH is present in the culture solution by filtration, centrifugation, etc. It is used after separating the FADGDH-containing solution and the microbial cells. When FADGDH is present in the microbial cells, the microbial cells are collected from the obtained culture by means of filtration or centrifugation, and then the microbial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme. Further, if necessary, a chelating agent such as EDTA and a surfactant are added to solubilize FADGDH and separated and collected as an aqueous solution.

  The FADGDH-containing solution obtained as described above is precipitated by, for example, vacuum concentration, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. You just have to let them know. Heat treatment and isoelectric point treatment are also effective purification means. Thereafter, purified FADGDH can be obtained by performing gel filtration with an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

  For example, gel filtration using Sephadex gel (manufactured by GE Healthcare Bioscience), DEAE Sepharose CL-6B (manufactured by GE Healthcare Bioscience), octyl Sepharose CL-6B (manufactured by GE Healthcare Bioscience) And purified by column chromatography such as) to obtain a purified enzyme preparation. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

In the present invention, FADGDH activity is measured under the following conditions.
[Test example]
<Reagent>
50 mM PIPES buffer pH 6.5 (including 0.1% Triton X-100)
24 mM PMS solution 2.0 mM 2,6-dichlorophenolindophenol (DCPIP) solution 1M D-glucose solution 20.5 ml of the above PIPES buffer, 1.0 ml of DCPIP solution, 2.0 ml of PMS solution, 5.9 ml of D-glucose solution To make a reaction reagent.

<Measurement conditions>
Pre-warm 3 ml of reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 ml of FADGDH solution and mixing gently, the absorbance change at 600 nm was recorded for 5 minutes with a spectrophotometer controlled at 37 ° C. with water as a control, and the absorbance change per minute (ΔOD _TEST) ). In the blind test, a solvent for dissolving FADGDH is added to the reagent mixture instead of the FADGDH solution, and the change in absorbance per minute (ΔOD _BLANK ) is measured in the same manner. From these values, the GDH activity is determined according to the following formula. Here, 1 unit (U) in 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.

Activity (U / ml) =
{− (ΔOD _TEST −ΔOD _BLANK ) × 3.0 × dilution ratio} / {16.3 × 0.1 × 1.0}

In the formula, 3.0 is the amount of the reaction reagent + enzyme solution (ml), 16.3 is the molar molecular extinction coefficient (cm ² / micromol) under the conditions for this activity measurement, and 0.1 is the enzyme solution Amount (ml), 1.0 indicates the optical path length (cm) of the cell.

In the present invention, the measurement of the thermal stability of FADGDH is obtained according to the following calculation formula.

Residual rate (%) = ((activity value after heat treatment) / (activity value before heat treatment)) × 100

In the present invention, the FADGDH pH stability is measured according to the following calculation formula.

Residual rate (%) = ((activity value after 16 hours of pH treatment) / (activity value immediately after pH treatment)) × 100

Glucose Assay Kit The present invention also features a glucose assay kit comprising a modified FADGDH according to the present invention. The glucose assay kit of the present invention comprises a modified FADGDH according to the present invention in an amount sufficient for at least one assay. Typically, the kit contains, in addition to the modified FADGDH of the present invention, buffers necessary for the assay, mediators, a glucose standard solution for creating a calibration curve, and directions for use. The modified FADGDH according to the invention can be provided in various forms, for example as a lyophilized reagent or as a solution in a suitable storage solution.

Glucose Sensor The present invention also features a glucose sensor that uses a modified FADGDH according to the present invention. As the electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and the enzyme of the present invention is immobilized on this electrode. Immobilization methods include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc., or ferrocene or a derivative thereof. It may be fixed in a polymer or adsorbed and fixed on an electrode together with a representative electron mediator, or a combination thereof may be used. Typically, the modified FADGDH of the present invention is immobilized on a carbon electrode using glutaraldehyde, and then treated with a reagent having an amine group to block glutaraldehyde.

  The glucose concentration can be measured as follows. Put buffer in constant temperature cell and maintain at constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate, or the like can be used. An electrode on which the modified FADGDH of the present invention is immobilized is used as a working electrode, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCl electrode) are used. After a constant voltage is applied to the carbon electrode and the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.

  Hereinafter, the present invention will be specifically described by way of examples.

Example 1 Production of Modified FADGDH Gene A recombinant plasmid comprising a gene (SEQ ID NO: 6) encoding a modified FADGDH (sometimes referred to herein as Mut1) having improved thermal stability of SEQ ID NO: 2. A commercially available Escherichia coli competent cell (E. coli DH5α; manufactured by TOYOBO) was transformed with pAOGDH-M76, and LB agar medium (1.0% polypeptone, 0.5% yeast extract, 1.0%) containing ampicillin was transformed. % NaCl, 1.5% agar; pH 7.3) and then incubated at 30 ° C. overnight. The obtained transformant was ingested with LB liquid medium (1% polypeptone, 0.5% yeast extract, 1.0% NaCl; pH 6.5) containing ampicillin (50 mg / ml; manufactured by Nacalai Tesque), Cultured with shaking at 30 ° C. overnight. A plasmid was prepared from the obtained cells by a conventional method.

Example 2 Modification of Proline at Position 339 Using the pAOGDH-M76 used in Example 1 as a template, a synthetic oligonucleotide of SEQ ID NO: 9 designed to replace the proline at position 339 with another amino acid and a synthetic oligo complementary thereto Nucleotides were subjected to PCR using QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE). Subsequently, a commercially available E. coli competent cell (E. coli DH5α; manufactured by TOYOBO) was transformed with the PCR product, and cultured on an LB agar medium containing ampicillin at 37 ° C. for 16 hours. Thereafter, the single colony having the modified FADGDH was ingested into an LB liquid medium containing ampicillin and cultured overnight at 30 ° C. with shaking. Thereafter, 1 ml of the culture solution was ingested, and the plasmid was extracted by a conventional method. The site of the extracted plasmid was identified using a DNA sequencer (ABI PRISM ™ 3700 DNA Analyzer; manufactured by Perkin-Elmer). PAOGDH-M76-P339L, a plasmid having a modified FADGDH in which the 339 position is substituted with L, and pFAOGDH-M76-P339V, a plasmid having the modified FADGDH, in which the 339 position is substituted with V, and a modified FADGDH in which the 339 position is substituted with an A PAOGDH-M76-P339A, a plasmid having modified FADGDH in which position 339 is replaced with C is replaced with pAOGDH-M76-P339C, and a plasmid having modified FADGDH in which position 339 is replaced with I is pAOGDH-M76-P339I Named.

Example 3 Preparation of crude enzyme solution containing modified FADGDH in which proline at position 339 was substituted with other amino acid and comparison of thermal stability pAOGDH-M76-P339L, pAOGDH-M76-P339V, pAOGDH- obtained in Example 2 E. coli DH5α · transformed with M76-P339A, pAOGDH-M76-P339C, and pAOGDH-M76-P339I was cultured on an LB agar medium containing ampicillin at 30 ° C. for 16 hours to obtain a single colony. Thereafter, a single colony of modified FADGDH was ingested into 5 ml LB liquid medium containing ampicillin and cultured with shaking at 30 ° C. for 16 hours. Bacteria obtained by centrifugation are collected from a part of the culture solution, and a crude enzyme solution is prepared by crushing the cells using glass beads in 50 mM phosphate buffer (pH 6.0). did. After adjusting to 100 U / ml using the prepared crude enzyme solution, thermal stability was measured by the activity measuring method described above. Table 1 shows the results. The residual ratio after treatment at 50 ° C. for 15 minutes was 83% for FADGDH (Mut1) before modification, whereas the residual ratio for P339L was 91%, the residual ratio for P339V was 90%, and the residual ratio for P339A 88%, the residual ratio of P339C was improved to 87%, and the residual ratio of P339I was increased to 85%.
Table 1 is a comparison of the thermal stability of FADGDH (Mut1) before modification and P339L, V, A, C, I modification.

Example 4 Preparation of modified FADGDH preparation and comparison of thermal stability Cultured cells were collected from the culture solution of modified FADGDH prepared in Example 3 by centrifugation, and then 50 mM phosphoric acid. It was suspended in a buffer (pH 6.5), subjected to nucleic acid removal, and centrifuged to obtain a supernatant. A saturated amount of ammonium sulfate was dissolved therein to precipitate the target protein, and the precipitate collected by centrifugation was redissolved in 50 mM phosphate buffer (pH 6.5). Then, gel filtration using a G-25 Sepharose column, hydrophobic chromatography using a Phenyl-Sepharose column (the elution conditions are both 25% saturated to 0% ammonium sulfate concentration gradients are used to extract peak fractions), and further a G-25 Sepharose column Ammonium sulfate was removed by gel filtration with a modified FADGDH preparation.

A preparation was prepared at 100 U / ml, and the thermal stability of modified FADGDH in which proline at position 339 was replaced with leucine was measured by the above method. Table 2 shows the results. The residual ratio after treatment at 50 ° C. for 15 minutes was 85% for FADGDH (Mut1) before modification, whereas the residual ratio for P339L was improved to 91%.
Table 2 is a comparison of the thermal stability of FADGDH (Mut1) and the P339L variant when treated for 15 minutes at various temperatures.

A preparation was prepared at 100 U / ml, and the thermal stability of modified FADGDH in which proline at position 339 was replaced with leucine was measured by the above method. Table 3 shows the results. The residual ratio after treatment at 50 ° C. for 30 minutes was 73% for FADGDH (Mut1) before modification, whereas the residual ratio for P339L was improved to 85%.
Table 3 is a comparison of the thermal stability of FADGDH (Mut1) and the P339L variant when treated at 50 ° C. for various times.

Example 5 Comparison of pH stability of modified FADGDH preparations The pH stability of the preparations obtained in Example 4 was measured. Buffers in the range of pH 4.5 to 8.5 (pH 4.5 to 5.5: 0.1 M acetate buffer, pH 5.5 to 7.5: 0.1 M phosphate buffer, pH 7.5 to 9.0: 0.1 M Tris-HCl buffer) was prepared, and each GDH was diluted with these to an enzyme concentration of 1 U / ml. The diluted solution was incubated at 25 ° C. for 16 hours, and the residual activity was evaluated from the measured values before and after the incubation. A graph showing the activity remaining rate after incubation with respect to the activity before incubation is shown in FIG. As shown in FIG. 2, it was confirmed that the modified P339L had improved stability at pH 8, and the pH stability range was expanded.
FIG. 2 is a comparison of the pH stability of the FADGDH preparation (Mut1) before modification and the P339L modified preparation.

  By using the modified FADGDH with improved stability according to the present invention, it is possible to reduce the amount of enzyme used in the preparation of glucose measurement reagents, glucose assay kits, and glucose sensors and to improve the measurement accuracy. It is great to contribute to industry.

Claims (11)

A protein represented by any one of the following (a) to (c).
(A) A protein in which the amino acid at position 339 is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 1.
(B) A protein in which the amino acid at position 339 is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 2.
(C) an amino acid sequence having an amino acid sequence having 60% or more homology with at least one of SEQ ID NO: 1 or SEQ ID NO: 2 and encoding a protein having glucose dehydrogenase activity, "position 339 of SEQ ID NO: 1" Or a protein in which an amino acid at a position equivalent to “position 339 of SEQ ID NO: 2” is substituted with another amino acid.   The protein according to claim 1, further comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added (inserted) at a position other than the position where the amino acid is substituted, and has glucose dehydrogenase activity. Protein.   The protein according to claim 1, wherein the amino acid substitution at position 339 or P or an equivalent position thereof is A, C, I, L or V. The protein according to claim 1, which is obtained by substituting P at position 339 with A, C, I, L, V, and having improved stability.   The gene which codes the protein in any one of Claims 1-4.   A vector comprising the gene according to claim 5.   A transformant transformed with the vector according to claim 6.   A method for producing a protein having glucose dehydrogenase activity, comprising culturing the transformant according to claim 7 and collecting a protein having glucose dehydrogenase activity.   A glucose assay kit comprising the protein according to claim 1.   The glucose sensor containing the protein in any one of Claims 1-4. The glucose measuring method containing the protein in any one of Claims 1-4.