JP6455134B2 - Novel glucose dehydrogenase - Google Patents

Description

The present invention relates to glucose dehydrogenase (also referred to as “GDH”). The present invention also uses a flavin adenine dinucleotide-dependent glucose dehydrogenase (also referred to as “FADGDH”), a method for producing FADGDH, and FADGDH using flavin adenine dinucleotide (also referred to as “FAD”) as a coenzyme. The present invention relates to a glucose measurement method, a glucose sensor, a glucose assay kit, 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. In recent years, a simple self-blood glucose meter using an electrochemical biosensor has been widely used for SMBG. A biosensor is an apparatus in which an electrode and an enzyme reaction layer are formed on an insulating substrate.

FAD-dependent glucose dehydrogenase (EC 1.1.9.10.10) is an enzyme mainly used for blood glucose concentration measurement and catalyzes the following reaction.
D-glucose + electron acceptor (oxidized form)
→ D-glucono-δ-lactone + electron acceptor (reduced form)

  Examples of enzymes for quantifying blood glucose include glucose dehydrogenase and glucose oxidase (also referred to as “GO”) (EC 1.1.3.4). It has been pointed out that the method using GO is easily affected by dissolved oxygen in the measurement sample, and that dissolved oxygen affects the measurement result. On the other hand, pyrroloquinoline quinone-dependent glucose dehydrogenase (also referred to as “PQQGDH”) (EC 1.1.5.2 (formerly EC 1.1.9.17)) is also affected by dissolved oxygen. Although it acts on sugars other than glucose such as maltose and lactose, it is not suitable for accurate blood glucose measurement. Flavin-binding glucose dehydrogenase (also referred to as “FADGDH”) is not affected by dissolved oxygen and hardly acts on maltose. A representative example is the FADGDH derived from Mucor fungus described in Patent Document 1.

  By the way, the environment where the glucose sensor is used is indoors, and it is rare that a general user who uses the glucose sensor performs strict temperature management when using the sensor strip. Accordingly, the temperature to which a glucose sensor stored by an individual is exposed is wider due to climate change (eg, 0 ° C. to 40 ° C.).

  In general, it is known that the reactivity of an enzyme tends to decrease as the reaction conditions become lower, and FADGDH is no exception. Further, it is assumed that the winter season is used at a considerably low temperature (for example, 2 to 10 ° C.). Based on this finding, a glucose sensor used in a situation where the room temperature is low may show a lower blood glucose level than actual.

  In fact, in the industry paper “Medical and Testing Equipment / Reagents”, “SBGing of blood glucose measuring device and POCT compatible blood glucose measuring device” (2009 32 (6) P.707-713) On the other hand, when the environmental temperature conditions are low temperature range and high temperature range, there are many devices whose measured values fall outside the allowable range of ISO15197, and if the measured values show extremely low or high values, it may cause a medical accident. It has been pointed out.

On the other hand, in the manufacturing process of the blood glucose sensor chip, a heat drying process may be performed, and a process of evaporating and drying the GDH solution on the reaction layer is often performed. Such heating treatment is carried out at a temperature of 50 ° C. or higher, which is effective for improving the efficiency of production. However, it is generally known that proteins are denatured by heat, and an enzyme that does not have sufficient heat stability has a risk of significant heat inactivation.

In addition, sensor strips after fabrication usually have a warranty period of up to two years at a temperature of about room temperature, and it is difficult to imagine that high stability of the enzyme itself is desired in order to satisfy such requirements. Absent. Enzymes with excellent thermal stability generally have a stable steric structure and can be said to be more suitable for long-term storage under harsh conditions.

In order to improve the measurement accuracy derived from the reactivity of FADGDH, patent documents 3 to 5 have been filed as FADGDH which is hardly affected by the reaction temperature. However, FADGDH described in these patent documents lacks thermal stability and leaves room for improvement.
For example, FADGDH described in Patent Document 3 has a residual activity of 0 to 40% at 50 ° C. according to the examples, and a significant deactivation at temperatures after 50 ° C. is expected.
In addition, FADGDH described in Patent Document 4 has no examples relating to thermal stability, and no mention is made of enzyme properties at temperatures exceeding 40 ° C., so that it has sufficient stability at temperatures around 50 ° C. or higher. It is strongly suggested that this is not done.
Further, FADGDH described in Patent Document 5 found by the present inventors has a residual activity of less than 80% at 50 ° C., and has not reached 90% or more of the residual activity which is considered to be substantially stable.

JP2013-116102A Japanese Patent No. 4556164 WO2013-031664 WO2013-147206 Japanese Patent Application No. 2014-021623

  An object of the present invention is to provide FADGDH that is less affected by blood glucose measurement at a low temperature and has improved storage stability in a sensor by FADGDH having excellent low-temperature reactivity and excellent thermal stability. That is.

  As a result of diligent research to achieve the above object, the present inventors have obtained FADGDH having high low-temperature reactivity and superior thermal stability to the conventionally known temperature-dependent improved FADGDH. The headline and various characteristics of the FADGDH were investigated, and the present invention was completed.

That is, the invention represented by the following is provided.
Item 1.
A flavin-binding glucose dehydrogenase that satisfies the following (a) to (c):
(A) It is comprised with the polypeptide in any one of following [1]-[3].
[1] Polypeptide having the amino acid sequence shown in SEQ ID NO: 1 [2] Amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 A polypeptide having glucose dehydrogenase activity [3] having an amino acid sequence having an identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 1, and having glucose dehydrogenase activity Polypeptide (b) having an activity at 5 ° C. of 35% or more when the activity at 25 ° C. is defined as 100%.
(C) The residual activity after treatment at 50 ° C. for 15 minutes is 90% or more.
Item 2.
The flavin-binding glucose dehydrogenase according to Item 1, which satisfies at least one of the following (d) and (e):
(D) pH stability: The residual enzyme activity after treatment of 2 U / mL enzyme at 25 ° C. for 16 hours is 90% or more in the range of at least pH 4.5 to pH 7.0.
(E) Optimum pH: The highest activity is shown between pH 6.0 and 6.7, and in that range, the activity at pH 6.3 (phosphate buffer) is 80%, compared with the case where the activity is 100%. % Relative activity.
Item 3.
Item 3. The glucose dehydrogenase according to Item 1 or 2, wherein the polypeptide portion has a molecular weight of about 55 kDa.
Item 4. The glucose dehydrogenase according to any one of Items 1 to 3, which is a filamentous fungus belonging to the genus Metalidium.
DNA of any of the following (A) to (H):
(A) DNA encoding the amino acid sequence represented by SEQ ID NO: 1,
(B) DNA consisting of the base sequence shown in SEQ ID NO: 2,
(C) DNA consisting of a base sequence having identity to the base sequence represented by SEQ ID NO: 2 of 80% or more and encoding a polypeptide having glucose dehydrogenase activity,
(D) a DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence represented by SEQ ID NO: 2 and that encodes a polypeptide having glucose dehydrogenase activity;
(E) In the base sequence shown in SEQ ID NO: 2, one or several bases are substituted, deleted, inserted, added and / or inverted, and have glucose dehydrogenase activity DNA encoding a polypeptide,
(F) DNA comprising the base sequence represented by SEQ ID NO: 3,
(G) a polymorphism having a glucose dehydrogenase activity, comprising a nucleotide sequence having an identity of 80% or more with the nucleotide sequence represented by SEQ ID NO: 3 and being spliced during transcription and translation in a eukaryotic host. DNA encoding the peptide,
(H) a polyamino acid sequence comprising one or several amino acid residues substituted, deleted, inserted, added or inverted in the amino acid sequence represented by SEQ ID NO: 1 and having glucose dehydrogenase activity DNA encoding a peptide.
Item 6.
Item 6. A vector incorporating the DNA of Item 5.
Item 7.
A transformant into which the vector according to Item 5 has been introduced.
Item 8.
Item 6. A method for producing the flavin-binding glucose dehydrogenase according to Item 1, wherein the microorganism containing the DNA according to Item 5 is cultured, and the obtained culture solution is purified.
Item 9.
Item 9. The method for producing a flavin-binding glucose dehydrogenase according to Item 8, comprising a step of purifying a culture solution obtained by culturing the transformant according to Item 7.
Item 10.
A method for measuring a glucose concentration, comprising causing the flavin-binding glucose dehydrogenase according to Item 1 to act on glucose.
Item 11.
A glucose assay kit comprising the flavin-binding glucose dehydrogenase according to Item 1.
Item 12.
A glucose sensor comprising the flavin-binding glucose dehydrogenase according to Item 1.

  According to the present invention, it is possible to provide FADGDH that hardly affects blood glucose measurement at a low temperature and has improved storage stability in a sensor. In addition, efficient flavin-binding GDH using Escherichia coli, yeast, vigor and the like as host cells is provided.

The reaction temperature dependence of Metalidium sp. F2114 origin GDH is shown. 1 shows the thermal stability of GDH derived from Metalidium sp. F2114 strain. The relative activity in each pH of Metalidium sp. F2114 origin GDH is shown. The activity residual rate after 16-hour processing in each pH of Metalidium sp. F2114 origin GDH is shown.

Hereinafter, the present invention will be described in detail.
One of the embodiments of the present invention is a flavin-binding glucose dehydrogenase that satisfies the following (a) to (c):
(A) It is comprised with the polypeptide in any one of following [1]-[3].
[1] Polypeptide having the amino acid sequence shown in SEQ ID NO: 1 [2] Amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 A polypeptide having glucose dehydrogenase activity [3] having an amino acid sequence having an identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 1, and having glucose dehydrogenase activity Polypeptide (b) having an activity at 5 ° C. of 35% or more when the activity at 25 ° C. is defined as 100%.
(C) The residual activity after treatment at 50 ° C. for 15 minutes is 90% or more.

1. Flavin-binding glucose dehydrogenase (FADGDH) activity The enzyme of the present invention is flavin-binding GDH (FADGDH) that requires flavin as a prosthetic group. GDH is an enzyme having physicochemical properties that catalyzes a reaction in which a hydroxyl group of glucose is oxidized to produce glucono-δ-lactone in the presence of an electron acceptor. In this document, this physicochemical property is referred to as glucose dehydrogenase activity, and unless otherwise specified, “enzyme activity” or “activity” means the enzyme activity. The electron acceptor is not particularly limited as long as it is capable of transferring electrons in a reaction catalyzed by GDH. For example, 2,6-dichlorophenolindophenol (DCPIP), phenazine methosulfate (PMS), 1-methoxy-5-methylphenadium methyl sulfate, a ferricyan compound, and the like can be used.

  Various methods are known as methods for measuring glucose dehydrogenase activity. In this document, DCPIP is used as an electron acceptor, and the activity is measured by using the change in absorbance of the sample at a wavelength of 600 nm before and after the reaction as an index. Is used. The specific reagent composition and measurement conditions are as follows unless otherwise specified.

Method for measuring glucose dehydrogenase activity <Reagent>
1. 100 mM phosphate buffer pH 6.5 containing 300 mM D-glucose (including 0.1% Triton X-100)
2.1.6 mM 2,6-dichlorophenolindophenol (DCPIP) solution 20 mL of phosphate buffer containing D-glucose and 10 mL of DCPIP solution are mixed to obtain a reaction reagent.

<Measurement conditions>
Prewarm 3 mL of reaction reagent at 37 ° C. for 5 minutes. Add 0.1 mL of GDH solution and mix gently, then record the absorbance change at 600 nm for 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control. The measurement results are plotted with the reaction time as the X-axis and the absorbance as the Y-axis, and the change in absorbance per minute (ΔOD _TEST ) is measured from the linear portion (that is, after the reaction rate becomes constant). In the blind test, a change in absorbance per minute (ΔOD _BLANK ) is similarly measured by adding a solvent that dissolves GDH to the reagent mixture instead of the GDH solution. From these values, the GDH activity is determined according to the following formula. Here, 1 unit (U) in GDH activity is the amount of enzyme that reduces 1 micromole of DCPIP per minute in the presence of D-glucose at a concentration of 1M.

Activity (U / mL) =
{− (ΔOD _TEST −ΔOD _BLANK ) × 3.1 × dilution ratio} / {0.85 × 0.1 × 1.0}

In the formula, 3.1 is the amount of the reaction reagent + enzyme solution (mL), 0.85 is the molar molecular extinction coefficient (cm ² / micromol) under the conditions for this activity measurement, and 0.1 is the solution of the enzyme solution. Amount (mL), 1.0 indicates the optical path length (cm) of the cell. In this document, unless otherwise indicated, enzyme activity is measured according to the measurement method described above.

2. Low temperature reactivity The FADGDH of the present invention has high low temperature reactivity. The low temperature reactivity in the present invention is evaluated as a relative value of the enzyme activity at 5 ° C compared to the enzyme activity at 25 ° C. The enzyme reactivity at each temperature is as described in 1. above. According to the method for measuring flavin-binding glucose dehydrogenase (FADGDH) activity described in 1., the reaction temperature is changed to 5 ° C. or 25 ° C. The enzyme activity at 5 ° C. of the FADGDH of the present invention is 35% or more, preferably 37% or more, more preferably 39% or more, more preferably 40% or more, compared with the enzyme activity at 25 ° C.

3. Polypeptide FADGDH of the present invention comprises the above-mentioned 1. And 2. And further comprising any one of the following polypeptides [1] to [3].
[1] Polypeptide having the amino acid sequence shown in SEQ ID NO: 1 [2] Amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 A polypeptide having glucose dehydrogenase activity [3] having an amino acid sequence having an identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 1, and having glucose dehydrogenase activity Polypeptide having

  The amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence of FADGDH (hereinafter also referred to as MesGDH) derived from Metallicium sp. F2114 strain, as shown in Example 1 described later.

  The polypeptide of [2] above has substitution, deletion, insertion and / or addition of one or several amino acid residues in the amino acid shown in SEQ ID NO: 1 as long as it retains glucose dehydrogenase activity ( Hereinafter, these are collectively referred to as “mutation”.) A polypeptide having an amino acid sequence.

  When the mutation is an amino acid substitution, the type of substitution is not particularly limited, but conservative amino acid substitution is preferred from the standpoint that it does not significantly affect the FADGDH phenotype. “Conservative amino acid substitution” refers to substitution of an amino acid residue with an amino acid residue having a side chain similar in nature to the side chain. Depending on the side chain of the amino acid residue, a basic side chain (eg lysine, arginine, histidine), an acidic side chain (eg aspartic acid, glutamic acid), an uncharged polar side chain (eg glycine, asparagine, glutamine, serine, threonine, tyrosine) Cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), aromatic side chains (eg tyrosine, phenylalanine, (Tryptophan, histidine). Therefore, it is preferable to substitute between amino acid residues in the same family.

  One or several mutations include restriction enzyme treatment, treatment with exonuclease, DNA ligase, etc., site-directed mutagenesis or random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New (York) and the like can be carried out by introducing a mutation into DNA encoding GDH. Variants can also be obtained by other methods such as ultraviolet irradiation. Variants also include naturally occurring variants (eg, single nucleotide polymorphisms) such as those based on individual differences in microorganisms that retain GDH, differences in species or genera.

  From the viewpoint of maintaining the activity, it is preferable that the mutation is present at a site that does not affect the active site or the substrate binding site.

  The polypeptide [3] is a polypeptide comprising an amino acid sequence having an identity of 80% or more compared to the amino acid sequence represented by SEQ ID NO: 1. Preferably, the identity between the amino acid sequence of FADGDH of the present invention and the amino acid sequence represented by SEQ ID NO: 1 is 85% or more, more preferably 88% or more, still more preferably 90% or more, and still more preferably 93% or more, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. Such a polypeptide comprising an amino acid sequence having a certain identity or more can be prepared based on the known genetic engineering techniques as described above.

  In one embodiment, it is preferable from the viewpoint that the polypeptide of [3] does not have an amino acid sequence that completely matches the amino acid sequence shown in SEQ ID NO: 1 with improved properties. From the same viewpoint, the polypeptide (c) is preferably a polypeptide obtained by recombinant expression. A polypeptide obtained by recombinant expression is expected to have more favorable characteristics because it differs from a naturally occurring polypeptide in sugar chain bonding and the like.

  Various methods are known as methods for calculating the identity of amino acid sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). In this document, the National Biotechnology Information Center (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: // www. ncbi. nlm. nih. The amino acid sequence identity is calculated by using default (initial setting) parameters in gov / BLAST /.

  In addition, the polypeptide which comprises the said FADGDH may have deleted the signal peptide part. Using SignalP 4.1 (http://www.cbs.dtud./default.) Of The Technical University of Denmark's The Center for Biological Sequence Analysis (CBS) According to the estimation, the first to the 16th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 is predicted as the signal sequence. Therefore, it is speculated that deleting this part does not have an adverse effect on the enzyme properties.

4). Thermal stability The FADGDH of the present invention is excellent in thermal stability. The heat stability in the present invention means that FADGDH is added to 50 mM potassium phosphate buffer (pH 6.0) and 1. Are added so as to be 2 U / ml by the FADGDH activity measurement method described in 1., and the remaining enzyme activity after maintaining at each measurement target temperature for 15 minutes is compared and evaluated.
Under the above-mentioned conditions, the residual enzyme activity at each measurement target temperature of FADGDH of the present invention is 78% or higher at 55 ° C. or lower (that is, a temperature range of 0 ° C. to 55 ° C.).
Moreover, on the said conditions, the residual enzyme activity in each measuring object temperature of FADGDH of this invention shows 90% or more in 50 degrees C or less (namely, temperature range of 0 degree C-50 degree C).

5). Various characteristics other than thermal stability FADGDH of the present invention preferably has at least one of the following three characteristics shown in 5-1 to 5-3, more preferably two or more of the following characteristics: More preferably, it has all three characteristics. The FADGDH of the present invention may have the characteristics shown in the above 5-1 to 5-3 in any combination.

5-1. The molecular weight of the molecular weight polypeptide moiety is 55 kDa as measured by SDS-PAGE.
The term “55 kDa” means that a range in which a person skilled in the art normally determines that there is a band at a position of 55 kDa when molecular weight is measured by SDS-PAGE is included. “Polypeptide moiety” means FADGDH in a state where sugar chains are not substantially bound. When the FADGDH of the present invention produced by a microorganism is a sugar chain-binding type, it is treated with heat treatment or a sugar hydrolase so that the sugar chain is removed (ie, “polypeptide portion”). be able to. The state in which the sugar chains are not substantially bound allows the presence of sugar chains unavoidably remaining in the sugar chain-binding FADGDH treated by heat treatment or sugar hydrolase. Therefore, when FADGDH is not inherently a sugar chain-binding type, it itself corresponds to a “polypeptide moiety”.

  Various means are known as means for removing sugar chains from sugar chain-binding FADGDH. In this document, as shown in the examples described later, sugar chain-bound FADGDH was denatured by heat treatment at 100 ° C. for 10 minutes, and then N-glycosidase Endo H (manufactured by New England Biolabs) was used. A method of treating at 37 ° C. for 1 hour or longer can be used.

  When a sugar chain is bound to FADGDH of the present invention, its molecular weight is not particularly limited as long as it does not negatively affect glucose dehydrogenase activity, substrate specificity, specific activity and the like. For example, the molecular weight of the FADGDH of the present invention in a state in which sugar chains are bound is preferably 60 to 120 kDa as measured by SDS-PAGE. Glycan-linked FADGDH is preferable from the viewpoint of making the enzyme more stable and increasing water solubility and facilitating solubility in water.

  The measurement of the molecular weight by SDS-PAGE can be performed using a commercially available molecular weight marker using a general method and apparatus.

5-2. Optimum active pH
Shows the highest activity between pH 6.0 and 6.7, and in that range shows relative activity of 80% or more compared to the case where the activity at pH 6.3 (phosphate buffer) is 100%. about.
Preferably, the highest activity is exhibited at pH 6.3 (phosphate buffer).

5-3. pH stability Stable at least over the entire range of pH 4.5-7.0.
The pH stability in the present invention is a comparison between the enzyme activity under the same pH condition before the treatment after treating the 2 U / mL enzyme at 25 ° C. for 16 hours under a specific pH condition. To evaluate.
It is preferable that the residual enzyme activity in each measurement object pH of FADGDH of this invention is 90% or more in the whole range of pH 4.5-7.0 at least.

6). Origin The origin of the FADGDH of the present invention is not particularly limited, but in one embodiment, the FADGDH of the present invention is preferably a filamentous fungus belonging to the genus Metalidium. Furthermore, it is preferably derived from a Metarhidium sp. F2114 (Metalhispium sp. F2114) strain. Metallicium sp. F2114 strain is a strain stored in a microbial library of Hypha Genesis Inc., and can be obtained therefrom.

  The FADGDH of the present invention includes not only FADGDH directly isolated from microorganisms, but also those obtained by modifying the isolated FADGDH by a protein engineering method or by modifying a genetic engineering method. . For example, it may be a modified enzyme imparted with characteristics by modifying the amino acid sequence revealed based on the enzyme obtained from the above-mentioned Metalidium sp. F2114 strain.

  The FADGDH of the present invention is preferably isolated FADGDH or purified FADGDH. Moreover, FADGDH of this invention may exist in the state melt | dissolved in the solution suitable for the said preservation | save, or the freeze-dried state (for example, powder form). “Isolated” when used in connection with the FADGDH of the present invention means a state that does not substantially contain components other than the enzyme (for example, contaminating proteins derived from host cells, other components, culture fluid, etc.). Say. Specifically, for example, the isolated enzyme of the present invention has a contaminant protein content of less than about 20% by weight, preferably less than about 10%, more preferably less than about 5%, even more preferably. Is less than about 1%. On the other hand, the FADGDH of the present invention may be present in a solution (for example, a buffer) suitable for storage or measurement of enzyme activity.

7). DNA encoding flavin-binding glucose dehydrogenase
One of the embodiments of the present invention is the above item 1.. The DNA encoding FADGDH, specifically, one of the following DNAs (A) to (H).
(A) DNA encoding the amino acid sequence represented by SEQ ID NO: 1,
(B) DNA consisting of the base sequence shown in SEQ ID NO: 2,
(C) DNA consisting of a base sequence having identity to the base sequence represented by SEQ ID NO: 2 of 80% or more and encoding a polypeptide having glucose dehydrogenase activity,
(D) a DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence represented by SEQ ID NO: 2 and that encodes a polypeptide having glucose dehydrogenase activity;
(E) In the base sequence shown in SEQ ID NO: 2, one or several bases are substituted, deleted, inserted, added and / or inverted, and have glucose dehydrogenase activity DNA encoding a polypeptide,
(F) DNA comprising the base sequence represented by SEQ ID NO: 3,
(G) a polymorphism having a glucose dehydrogenase activity, comprising a nucleotide sequence having an identity of 80% or more with the nucleotide sequence represented by SEQ ID NO: 3 and being spliced during transcription and translation in a eukaryotic host. DNA encoding the peptide,
(H) a polyamino acid sequence comprising one or several amino acid residues substituted, deleted, inserted, added or inverted in the amino acid sequence represented by SEQ ID NO: 1 and having glucose dehydrogenase activity DNA encoding a peptide.

  In the above (A), “a DNA encoding an amino acid sequence of a protein (sometimes referred to as a polypeptide or FADGDH)” refers to a DNA obtained when the protein is expressed, that is, an amino acid of the protein A DNA having a base sequence corresponding to the sequence. Therefore, DNA that differs depending on codon degeneracy is also included.

  In the above (B) to (G), SEQ ID NO: 2 is the cDNA sequence of GDH derived from Metallicium sp. F2114 strain having a total chain length of 1782 bp. Further, SEQ ID NO: 3 is the gene sequence of GDH derived from Metallicium sp. F2114 strain, and the region of 348 bp to 416 bp and 1640 bp to 1697 bp counted from the beginning is an intron.

  In the above (C) and (G), the DNA of the present invention is preferably a protein having an amino acid sequence encoded by the DNA as long as it is a protein having glucose dehydrogenase activity (preferably, the low-temperature reactivity described in 2 above). And / or as long as the thermal stability of 4. above is satisfied), the identity with the base sequence shown in SEQ ID NO: 2 in (C) is the same as the base shown in SEQ ID NO: 3 in (G). The identity with the sequence is 80% or more (preferably 85% or more, more preferably 88% or more, still more preferably 90% or more, still more preferably 93% or more, still more preferably 95% or more, particularly preferably 98% or more, most preferably 99% or more).

  Various methods are known for calculating the identity of base sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). In this document, base sequence identity values (%) are calculated by using default (initial setting) parameters in the National Biotechnology Information Center (NCBI) homology algorithm BLAST.

  In the above (D), the DNA of the present invention is preferably (as long as the protein encoded by it is a protein having glucose dehydrogenation activity (preferably, the low-temperature reactivity described in 2. above and / or the heat described in 4. above). DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 2 may be used as long as the stability is satisfied. Here, “stringent conditions” generally refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such stringent conditions are known to those skilled in the art, and can be set with reference to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York).

  In this document, “stringent conditions” refers to the following conditions. 50% formamide, 5 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0) as a hybridization solution, 1 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)) is used.

  DNA that hybridizes under such conditions may include those in which a stop codon has occurred in the middle or those that have lost activity due to mutations in the active center, but these are incorporated into commercially available active expression vectors. It can be easily removed by expressing it in a suitable host and measuring the enzyme activity by a known method.

In the above (E) and (H), “several” is not particularly limited, but is, for example, a number corresponding to less than about 20% of the total number of amino acids or the total number of bases, preferably less than about 15%. It is a corresponding number, 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%. More specifically, the number of amino acid residues or bases to be mutated is, for example, 2 to 160, preferably 2 to 120, more preferably 2 to 80, still more preferably 2 to 40, More preferably, it is 2-10, and still more preferably 2-5.

  In a preferred embodiment, the DNA encoding FADGDH of the present invention is DNA present in an isolated state. Here, “isolated DNA” refers to a state separated from other components such as nucleic acids and proteins that coexist in the natural state. However, the isolated DNA may contain some other nucleic acid components such as a nucleic acid sequence adjacent in the natural state (for example, a promoter region sequence and a terminator sequence). For example, an “isolated” state in the case of chromosomal DNA is preferably substantially free of other DNA components that coexist in the natural state. On the other hand, the “isolated” state in the case of DNA prepared by genetic engineering techniques such as cDNA molecules is preferably substantially free of cell components, culture medium, and the like. Similarly, the “isolated” state in the case of DNA prepared by chemical synthesis is preferably substantially free of precursors (raw materials) such as dNTPs, chemical substances used in the synthesis process, and the like. In addition, unless it is clear that a different meaning is expressed, when simply described as “DNA” in this specification, it means DNA in an isolated state. The DNA of the present invention also includes DNA (cDNA) complementary to the above DNAs (A) to (H).

  The DNA of the present invention can be produced and obtained by a chemical DNA synthesis method based on the sequence information disclosed in this specification or the attached sequence listing (particularly SEQ ID NO: 2). For example, a standard gene It can be easily prepared by using an engineering method, a molecular biological method, a biochemical method, or the like (see Molecular Cloning 3d Ed, Cold Spring Harbor Lab. Press (2001), etc.). As a chemical DNA synthesis method, a solid phase synthesis method by a phosphoramidite method can be exemplified. An automatic synthesizer can be used for this synthesis method.

  In addition, as a method for obtaining DNA used for GDH production of the present invention, a DNA strand is chemically synthesized or a synthesized partially overlapping oligo DNA short chain is connected by using a PCR method. It is also possible to construct a DNA encoding the full length of the GDH of the present invention. The advantage of constructing full-length DNA in combination with chemical synthesis or PCR is that the codons used can be designed over the entire length of the gene in accordance with the host into which the gene is introduced. A plurality of codons encoding the same amino acid are not used uniformly, and the frequency of use varies depending on the species. In general, codons contained in genes that are highly expressed in a given species contain many codons that are frequently used in that species, and conversely, the presence of codons that are used infrequently becomes a bottleneck for genes with low expression levels. There are many examples that prevent high expression. In the expression of a heterologous gene, many examples have been reported so far in which the expression level of the heterologous protein has been increased by replacing the gene sequence with a codon that is frequently used in the host organism. It is expected to be effective in increasing the expression level of heterologous genes.

  For the above reasons, it is desirable to modify (optimize) the DNA encoding the GDH of the present invention into a codon more suitable for the host cell into which it is introduced (that is, a codon frequently used in the host). The codon usage frequency of each host is defined by the ratio of each codon used in all genes present on the genome sequence of the host organism, and is expressed, for example, by the number of times used per 1000 codons. In addition, the codon usage frequency can be approximately calculated from the sequence of a plurality of typical genes in an organism whose entire genome sequence has not been elucidated. The data on the frequency of codon usage in the host organism to be recombined uses, for example, the genetic code frequency database published on the website of Kazusa DNA Research Institute (http://www.kazusa.or.jp). Or you can refer to the literature describing the codon usage in each organism, or you can determine the codon usage data for the host organism you are using. By referring to the obtained data and the gene sequence to be introduced and replacing the codon used in the gene sequence that is not frequently used in the host organism with a codon that encodes the same amino acid and is frequently used Good. As such a frequently used codon, for example, when the host is E. coli K12, Gly is GGT or GGC, Glu is GAA, Asp is GAT, Val is GTG, and Ala is GCG and Arg are CGT or CGC, Ser is AGC, Lys is AAA, Ile is ATT or ATC, Thr is ACC, Leu is CTG, Gln is CAG, Pro is CCG, and the like.

  Specifically, as a standard genetic engineering technique, a cDNA library is prepared according to a conventional method from an appropriate source microorganism in which the FADGDH of the present invention is expressed, and the DNA sequence of the present invention ( For example, it can be carried out by selecting a desired clone using an appropriate probe or antibody peculiar to the nucleotide sequence of SEQ ID NO: 2 [Proc. Natl. Acad. Sci. USA. 78, 6613 (1981) etc.].

  The source microorganism for preparing the cDNA library is not particularly limited as long as it is a microorganism that expresses FADGDH of the present invention, but is preferably a microorganism classified into the genus Metalidium.

  Isolation of total RNA from microorganisms, isolation and purification of mRNA, acquisition of cDNA and its cloning, etc. can all be carried out according to conventional methods. The method for screening the DNA of the present invention from a cDNA library is not particularly limited, and can be performed according to a usual method. For example, for a polypeptide produced by cDNA, a method for selecting a corresponding cDNA clone by immunoscreening using the polypeptide-specific antibody, a plaque high using a probe that selectively binds to a target nucleotide sequence Hybridization, colony hybridization, etc., and combinations thereof can be selected as appropriate.

  In obtaining DNA, a PCR method or a modified DNA or RNA amplification method thereof can be suitably used. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method, particularly the 5'-RACE method [M. A. Frohman, et al. , Proc. Natl. Acad. Sci. USA. , 8, 8998 (1988)].

  Primers used for the PCR method can be appropriately designed and synthesized based on the nucleotide sequence of SEQ ID NO: 2. In addition, isolation and purification of the amplified DNA or RNA fragment can be carried out according to a conventional method as described above, for example, by gel electrophoresis, hybridization or the like.

  By using the DNA of the present invention, the FADGDH of the present invention can be easily and stably produced in large quantities.

7). Vector The vector of the present invention is the above-mentioned 7. The vector into which the DNA encoding FADGDH of the present invention described in the above is incorporated. Here, the “vector” is a nucleic acid molecule (carrier) capable of transporting a nucleic acid molecule inserted therein into a target such as a cell, and can replicate the DNA of the present invention in an appropriate host cell. The type and structure are not particularly limited as long as it can be expressed. That is, the vector of the present invention is an expression vector. As the type of vector, an appropriate vector is selected in consideration of the type of host cell. Specific examples of the vector include a plasmid vector, a cosmid vector, a phage vector, a virus vector (an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a herpes virus vector, etc.) and the like. It is also possible to use a vector suitable for using a filamentous fungus as a host or a vector suitable for self-cloning.

  When Escherichia coli is used as a host, for example, M13 phage or a variant thereof, λ phage or a variant thereof, pBR322 or a variant thereof (pB325, pAT153, pUC8, etc.) can be used. When yeast is used as a host, pYepSec1, pMFa, pYES2, etc. can be used. When a filamentous fungus is used as a host, pUN1, pUSC and the like can be used. When insect cells are used as hosts, for example, pAc and pVL can be used. When mammalian cells are used as hosts, for example, pCDM8 and pMT2PC can be used, but the present invention is not limited thereto. .

  Expression vectors usually contain a promoter sequence necessary for expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. An expression vector containing a selectable marker can also be used. When such an expression vector is used, the presence or absence of the expression vector (and the degree thereof) can be confirmed using a selection marker. Insertion of the DNA of the present invention into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. are performed by standard recombinant DNA techniques (for example, Molecular Cloning, Third Edition, 1.84). , Cold Spring Harbor Laboratory Press, New York, which is a well-known method using restriction enzymes and DNA ligase).

8). Transformant The present invention relates to a transformant in which the DNA of the present invention is introduced into a host cell. The means for introducing the DNA of the present invention into the host is not particularly limited. And introduced into a host in a state of being incorporated into a vector described in 1. The host cell is not particularly limited as long as it can express the DNA of the present invention and produce FADGDH. Specifically, prokaryotic cells such as Escherichia coli and Bacillus subtilis, eukaryotic cells such as yeast, mold, insect cells, plant culture cells, and mammalian cells can be used.

  When the host is a prokaryotic cell, examples include the genus Escherichia, the genus Bacillus, the genus Brevibacillus, the genus Corynebacterium, and the like. Examples include Escherichia coli C600, Escherichia coli HB101, Escherichia coli DH5α, Bacillus subtilis, Examples include Brevibacillus choshinensis and Corynebacterium glutamicum. Examples of the vector include pBR322, pUC19, and pBluescript.

  When the host is yeast, examples include Saccharomyces genus, Schizosaccharomyces genus, Candida genus, Pichia genus, Cryptococcus genus, etc., respectively, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Pichia pastoris, An example is Cryptococcus sp. Examples of the vector include pAUR101, pAUR224, and pYE32. When the host is a filamentous fungal cell, Aspergillus genus, Trichoderma genus, Metalidium genus and the like are mentioned as examples, and Aspergillus oryzae, Aspergillus niger, Trichoderma reesei, Metalidium sp.

  In the present invention, it is also preferable to use the Metalidium sp. F2114 strain from which FADGDH is isolated as a host. That is, in the transformant, exogenous DNA is usually present in the host cell, but a transformant obtained by so-called self-cloning using a microorganism from which the DNA is derived as a host is also a preferred embodiment.

  The transformant of the present invention is preferably the above-mentioned 7. It is prepared by transfection or transformation using the expression vector shown in 1. Transformation may be transient or stable. Transfection and transformation can be performed using calcium phosphate coprecipitation method, electroporation, lipofection, microinjection, Hanahan method, lithium acetate method, protoplast-polyethylene glycol method, and the like.

  Since the transformant of the present invention has the ability to produce the FADGDH of the present invention, the FADGDH of the present invention can be efficiently produced using the transformant.

9. Production method of flavin-binding glucose dehydrogenase The FADGDH of the present invention is typically produced by culturing a microorganism capable of producing the FADGDH of the present invention. The microorganism used for the culture is not particularly limited as long as it has the ability to produce FADGDH of the present invention. Metallicium sp. F2114 strain shown in the above and 8. The transformant shown in can be suitably used.

  The above-mentioned Metalidium SP F2114 strain is a strain possessed by Hyphagenesis Co., Ltd.

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

  The medium is not particularly limited as long as the microorganism to be used can grow. For example, carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein Nitrogen sources such as hydrolysates, bran and meat extracts, and further added with inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts and zinc salts can be used. In order to promote the growth of the microorganisms to be used, vitamins, amino acids and the like may be added to the medium.

  When culturing Metalidium sp. F2114 strain to obtain FADGDH of the present invention, culture conditions may be selected in consideration of the nutritional physiological properties of the microorganism. In many cases, it is advantageous to use liquid culture and industrially perform aeration and agitation culture. However, when productivity is considered, it may be advantageous to carry out by solid culture.

  The pH of the medium is only required to be suitable for the growth of the microorganism to be cultured. For example, it is adjusted to about 3 to 8, preferably about 5 to 7, and the culture temperature is usually about 10 to 50 ° C., preferably about 25 to 35. Culturing is carried out under aerobic conditions at about 0 ° C. for 1 to 15 days, preferably about 1 to 7 days. As the culture method, for example, a shaking culture method or an aerobic deep culture method using a jar fermenter can be used.

  After culturing under the above conditions, FADGDH is preferably recovered from the culture solution or the bacterial cells. When using microorganisms that secrete FADGDH outside the cells, for example, the culture supernatant is filtered, centrifuged to remove insolubles, concentrated by ultrafiltration membrane, salting out such as ammonium sulfate precipitation, dialysis, The present enzyme can be obtained by performing separation and purification by appropriately combining various types of chromatography. The flavin-binding glucose dehydrogenase produced by Metalidium sp. F2114 strain is basically a secreted protein.

  On the other hand, when recovering from the microbial cells, for example, the microbial cells are crushed by pressure treatment, ultrasonic treatment, mechanical method, or a method using an enzyme such as lysozyme, and if necessary, such as EDTA. This enzyme can be obtained by adding a chelating agent and a surfactant to solubilize FADGDH, separating and collecting the solution as an aqueous solution, and performing separation and purification. After the cells are collected from the culture solution in advance by filtration, centrifugation, or the like, the above series of steps (crushing, separating, and purifying the cells) may be performed.

  Purification includes, for example, concentration under reduced pressure, membrane concentration, salting-out treatment such as ammonium sulfate and sodium sulfate, or precipitation treatment by a fractional precipitation method using a hydrophilic organic solvent such as methanol, ethanol, acetone, etc., heat treatment or isoelectric point treatment. In addition, gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like can be combined as appropriate.

  When column chromatography is used, 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 (GE Healthcare Bioscience, Inc.) can be used. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

  When collecting (extracting, purifying, etc.) a protein having glucose dehydrogenase activity from the culture solution, one or more of glucose dehydrogenase activity, maltose activity, thermal stability, etc. are used as indicators. May be.

  In each purification process, in principle, fractionation is performed using GDH activity as an index, and the process proceeds to the next step. However, this does not apply when appropriate conditions can be set in advance by a preliminary test or the like.

  If this enzyme is obtained as a recombinant protein, various modifications are possible. For example, if a DNA encoding this enzyme and other appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, the peptide consists of a recombinant protein linked to any peptide or protein. This enzyme can be obtained. In addition, modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur. By the modification as described above, extraction of recombinant protein, simplification of purification, addition of biological function, and the like are possible.

10. A method for measuring glucose using glucose dehydrogenase, such as a method for measuring glucose, has already been established in the art. Therefore, according to a known method, the amount or concentration of glucose in various samples can be measured using the FADGDH of the present invention. As long as the concentration or amount of glucose can be measured using the FADGDH of the present invention, the mode thereof is not particularly limited. For example, the FADGDH of the present invention is allowed to act on glucose in a sample, and the electrons accompanying the glucose dehydrogenation reaction This can be done by measuring the structural change of a receptor (eg, DCPIP) by absorbance. More specifically, the above 1. According to the method shown in FIG. The measurement of the glucose concentration according to the present invention can be carried out by adding the FADGDH of the present invention to a sample or by adding and mixing them. The sample containing glucose is not particularly limited, and examples thereof include blood, beverages, and foods. The amount of enzyme added to the sample is not particularly limited as long as the glucose concentration or amount can be measured.

10-1. Glucose Assay Kit The glucose assay kit of the present invention comprises FADGDH according to the present invention in an amount sufficient for at least one assay. Typically, it includes the FADGDH of the present invention, buffers, mediators and other necessary reagents for measurement, a glucose standard solution for creating a calibration curve, and usage guidelines. The kit of the invention can be provided, for example, as a lyophilized reagent or as a solution in a suitable storage solution. In addition, in the reagent containing FADGDH, as a stabilizer and / or an activator, proteins such as bovine serum albumin and sericin, surfactants such as Triton X-100, Tween 20, cholate and deoxycholate, glycine Amino acids such as serine, glutamic acid, glutamine, aspartic acid, asparagine, glycylglycine, sugars such as trehalose, inositol, sorbitol, xylitol, glycerol, sucrose and / or sugar alcohols, inorganic salts such as sodium chloride, potassium chloride, Furthermore, hydrophilic polymers such as pullulan, dextran, polyethylene glycol, polyvinyl pyrrolidone, carboxymethyl cellulose, and polyglutamic acid may be added as appropriate.

10-2. Glucose sensor A glucose sensor using FADGDH according to the present invention uses a carbon electrode, a gold electrode, a platinum electrode, or the like as an electrode, and the enzyme of the present invention is immobilized on this electrode. As immobilization methods, there are a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a method of using a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc. NAD or NADP Such a coenzyme or an electron mediator typified by ferrocene or a derivative thereof may be fixed in a polymer or adsorbed and fixed on an electrode, or may be used in combination. Typically, after the FADGDH of the present invention is immobilized on a carbon electrode using glutaraldehyde, the glutaraldehyde can be blocked by treatment with a reagent having an amine group. Examples of electron mediators that can be used include those that receive electrons from FAD, which is a coenzyme of GDH, and can donate electrons to color-developing substances and electrodes, such as ferricyanide salts, phenazine etsulfate, phenazine methosulfate, and phenylenediamine. N, N, N ′, N′-tetramethylphenylenediamine, 1-methoxy-phenazine methosulfate, 2,6-dichlorophenolindophenol, 2,5-dimethyl-1,4-benzoquinone, 2,6-dimethyl Examples include, but are not limited to, -1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone, nitrosoaniline, ferrocene derivatives, osmium complexes, ruthenium complexes and the like. Further, in the GDH composition on the electrode, as a stabilizer and / or an activator, a protein such as bovine serum albumin or sericin, a surfactant such as Triton X-100, Tween 20, cholate or deoxycholate , Glycine, serine, glutamic acid, glutamine, aspartic acid, asparagine, glycylglycine and other amino acids, trehalose, inositol, sorbitol, xylitol, glycerol, sucrose and other sugars and / or sugar alcohols, sodium chloride, potassium chloride and other inorganic substances It may contain a salt or a hydrophilic polymer such as pullulan, dextran, polyethylene glycol, polyvinyl pyrrolidone, carboxymethyl cellulose, polyglutamic acid.

  Measurement of glucose concentration using a sensor can be performed 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. As a working electrode, an electrode on which FADGDH of the present invention is immobilized is used, and a counter electrode (for example, platinum electrode) and a reference electrode (for example, 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. The description of the following examples does not limit the invention in any way.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to a following example.

<Example 1>
Acquisition of Metalidium sp. F2114 strain GDH gene (1) Preparation of Metalidium sp. F2114 strain genomic DNA 50 mL YPD medium (0.5 wt% yeast extract, 1 wt% peptone, 2 wt% glucose) in 500 mL Sakaguchi flask And sterilized by autoclaving to prepare a medium for preculture. Metallicium sp. F2114 strain, which has been reconstituted in DP plate medium, is inoculated into a pre-culture medium with one platinum ear, shaken at 25 ° C and 180 rpm for 3 days, and the cells are obtained by filtering the cells with Miracloth. did.

  The obtained bacterial cells were frozen and crushed in liquid nitrogen, 5 ml of phenol: chloroform: isoamyl alcohol (25: 24: 1, pH 7.2) 5 ml was added and suspended sufficiently, and then centrifuged at 3000 rpm for 20 minutes at room temperature. About 4 ml of the aqueous layer was used as a sample. Genomic DNA was obtained from this sample using MagExtortor-PlantGenome (Toyobo) according to the attached instructions.

(2) Determination of genomic sequence of Metalidium sp. F2114 strain The genomic DNA obtained in (1) was fragmented using Nextera XT Sample prep kit (manufactured by Illumina) according to the attached instructions, and tagged.

The tagged genomic DNA fragmented was clustered on a flow cell using Miseq Reagent kit v2 300 cycles (Illumina), and data acquisition was performed using Miseq (Illumina). Based on the acquired FASTQ file, de novo assembly was performed in accordance with a regular method using CLC GENOMICS Workbench Ver7.5, and the whole genome information of Metardium sp. F2114 strain was acquired.

(3) Acquisition of GDH gene derived from Metalidium sp. F2114 strain Based on the whole genome information obtained in (2), a BLAST search was performed using the existing gonococcal FADGDH as a query, and Metallicium sp described in SEQ ID NO: 3 was obtained. -A gene sequence of GDH derived from F2114 strain was obtained.

(4) Expression of GDH gene derived from Metalidium sp. F2114 strain The gene sequence of GDH derived from Metallicium sp. F2114 strain described in SEQ ID NO: 3 was connected under the amylase promoter of pUSA vector, and Aspergillus oryzae NS4 strain was used with this vector. (Transformation System for Aspergillus oryzae with Double auxomorphic Mutations, niaD and sC (Bioscience Biotechnology Biochemistry, 1997, Vol. 13, 1997, Vol. 13). Transformation is based on the literature Transformation of Filamentous Fungi Based on Hygromycin B and Phlemycin Registration Marker (METHODS IN ENZYMOLOGY, 1992, Vol. 216-4). Conversion was carried out.

  The obtained Aspergillus oryzae NS4 strain transformant is inoculated into 50 mL of maltose medium (5 wt% yeast extract, 5 wt% maltose) in a 500 mL Sakaguchi flask and sterilized by autoclave at 30 ° C. and 180 rpm. Cultured for 72 hours. The cells were removed with Miracloth, and the culture supernatant was used as a crude enzyme solution. The GDH activity in the obtained crude enzyme solution is measured according to 2. Was confirmed according to the method for measuring FADGDH activity shown in 1., and a GDH activity of 10.71 U / ml was confirmed.

(5) Acquisition of cDNA sequence of GDH derived from Metalidium sp. F2114 strain Aspergillus oryzae NS4 strain transformant obtained in (4) was added to 50 mL of maltose medium (5 wt% yeast extract, 5 wt% maltose) in 500 mL Sakaguchi. It put into the flask, inoculated into what was sterilized by the autoclave, and it culture | cultivated at 30 degreeC180rpm for 72 hours. After filtering the cells with Miracloth, total RNA was obtained using ISOGEN (Nippon Gene) according to the attached instructions.

  From the obtained RNA, 3'RACE and 5'RACE were performed using a Smart RACE cDNA Amplification kit (Clontech) according to the attached instructions. As a result of nucleotide sequence analysis of DNA fragments contained in a plurality of plasmids obtained in accordance with the above method, the cDNA sequence of GDH derived from Metalidium sp. F2114 strain having a total chain length of 1782 bp shown in SEQ ID NO: 2 was clarified. It was. The amino acid sequence of the enzyme gene predicted from the gene sequence is shown in SEQ ID NO: 1.

  The amino acid sequence described in SEQ ID NO: 1 is 31% homologous to the amino acid sequence described in Patent Document 1 (FADGDH derived from Mucor himalis), and the amino acid sequence described in Patent Document 2 (FADGDH derived from Aspergillus oryzae). 58%, amino acid sequence described in Patent Document 3 (Botrytis tulipae, Dumontinia tuberose, and FAGDDH derived from Ovulinia azaleae) 51-54% homologous, amino acid sequence described in Patent Document 4 (Aureobasidium pulbululumNulplumNulpululNulpululNulplumNulpululNulpululNulpululNulpululNulpululNulululNululululNulululNululululNulululNululululNulbulululNulbulululNulbulululNulbululNulbulum cladosporioides, cladosporium funiculosum, Ladosporium oxysporum, Cladosporia sp. T799, Cladosporum sp. FADGDDH) has 50% homology and can be said to be a novel sequence.

<Example 2>
Acquisition of Metabolic SP sp. F2114-derived GDH 60 mL of DP medium was placed in a 500 mL Sakaguchi flask and sterilized by autoclave to prepare a medium for preculture. An Aspergillus oryzae NS4 strain transformant (previously obtained in Example 1 (4)) reconstituted in DP plate medium was inoculated into a pre-culture medium with one platinum ear, and cultured with shaking at 25 ° C. and 180 rpm for 3 days. A culture broth was obtained.

  Next, 6.0 L production medium (yeast extract 5.0% by weight, maltose weight 5.0%, pH 6.0) was placed in a 10 L jar fermenter and sterilized by autoclaving to prepare a main culture medium. . 50 mL of the seed culture solution was inoculated into the main culture medium, and cultured for 3 days under conditions of a culture temperature of 25 ° C., a stirring speed of 350 rpm, an aeration rate of 2.0 L / min, and a tube pressure of 0.2 MPa. Thereafter, the culture solution was concentrated to 3 L to obtain a crude enzyme solution.

  500 mL of phenyl sepharose FastFlow (GE Healthcare), which was gradually added to the crude enzyme solution to 40% saturation and equilibrated in advance with 50 mM potassium phosphate buffer (pH 6.0) containing 40% saturated ammonium sulfate. And eluted with a linear gradient of 50 mM phosphate buffer (pH 6.0). Superdex G25 column (manufactured by GE Healthcare) was prepared by concentrating the eluted GDH fraction with a hollow fiber membrane (manufactured by Spectrum Laboratories) having a molecular weight cut-off of 10,000 and equilibrating with 50 mM potassium phosphate buffer (pH 6.0). And desalting was carried out. Thereafter, the desalted fraction was applied to a DEAE Sepharose FastFlow (GE Healthcare) column equilibrated with 50 mM potassium phosphate buffer (pH 6.0) to adsorb only contaminating proteins. Then, the obtained fraction was applied to a Superdex S-200 (GE Healthcare) column to obtain a purified enzyme. Hereinafter, the obtained purified GDH is also referred to as “Mes-GDH”.

<Example 3>
Low-temperature reactivity Regarding Mes-GDH obtained in Example 2, the above-mentioned 2. The activity at 5 ° C., 25 ° C., and 37 ° C. was measured according to the FADGDH activity measurement method shown in FIG. The enzyme concentration was adjusted so that the final concentration was 4.8 μg / ml.

<Comparative Example 1>
In order to examine the temperature dependence of the existing FADGDH, the FADGDH amino acid sequence derived from Aspergillus oryzae described in Patent Document 2 was produced by recombination in Aspergillus oryzae, and a purified enzyme was prepared as Comparative Example 1. The activity was measured at 5 ° C., 25 ° C., and 37 ° C. according to the FADGDH activity measurement method shown in FIG. The enzyme concentration was adjusted to a final concentration of 3.8 μg / ml.

<Comparative Example 2>
In order to examine the temperature dependence of the existing FADGDH, the FADGDH amino acid sequence derived from Mucor Himalis described in Patent Document 1 was produced by recombination in yeast, and a purified enzyme was prepared as Comparative Example 2. The activity was measured at 5 ° C., 25 ° C., and 37 ° C. according to the FADGDH activity measurement method shown in FIG. The enzyme concentration was adjusted to a final concentration of 4.7 μg / ml.

Table 2 shows the relative values of the enzyme activity at 25 ° C. and 37 ° C. of the enzyme activity at 5 ° C. in Example 3, Comparative Example 1 and Comparative Example 2.

As shown in Table 2, the Mes-GDH obtained in Example 2 has an activity at 5 ° C. with an activity at 25 ° C. of 100%, and an activity at 37 ° C. of 100%. In all of the effects at 5 ° C., a higher numerical value than the existing FADGDH was shown.

<Example 4>
Reaction temperature dependence / optimum reaction temperature Using the Mes-GDH enzyme solution (2.5 μg / mL) obtained in Example 2, the optimum activity temperature was examined. Mes-GDH activity was determined at 5 ° C, 10 ° C, 25 ° C, 30 ° C, 37 ° C, 45 ° C, 50 ° C, 55 ° C, and 60 ° C. The results are shown in FIG.

As a result, Mes-GDH showed the highest activity value at 60 ° C. From the above, it was shown that the optimum activity temperature of Mes-GDH has an optimum activity at 60 ° C.

<Example 5>
Thermal stability The thermal stability of Mes-GDH obtained in Example 2 was examined. Mes-GDH was added to 50 mM potassium phosphate buffer (pH 6.0) at 2 U / ml, and each temperature (4 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C.) for 15 minutes, The GDH activity was measured using the FADGDH activity measurement method shown in Fig. 4, and the residual ratio was determined by comparison with the GDH activity before the treatment. The results are shown in FIG.

As a result, Mes-GDH had a residual rate of 90% or more when treated at a temperature in the range of 4 ° C to 50 ° C. The residual activity after treatment at 55 ° C. was 78.1%.

<Example 6>
pH dependence The optimum pH of Mes-GDH obtained in Example 2 was examined. 100 mM potassium acetate buffer (pH 5.0-5.5, plotted with ■ in FIG. 3) 100 mM MES-NaOH buffer (pH 5.5-6.4, plotted with □ in FIG. 3), 100 mM potassium phosphate A buffer solution (pH 6.3-7.8, plotted with ▲ in FIG. 3) and a 100 mM Tris-HCl buffer solution (pH 7.0-8.0, plotted with Δ in FIG. 2) were used. The activity at 37 ° C. at each pH was measured, and the relative activity at each pH was determined using the highest activity value as a reference (100%). The results are shown in FIG.

  As a result, the optimum activity pH of Mes-GDH was pH 6.3 when a potassium phosphate buffer was used. Moreover, in the range of pH 6.0-6.7, the relative activity of 80% or more was shown compared with the case where the activity in pH 6.3 was 100%. From the above, it was confirmed that the optimal activity pH of Mes-GDH was pH 6.0 to 6.7.

<Example 7>
pH stability Using the Mes-GDH enzyme solution (2 U / mL) obtained in Example 2, pH stability was examined. 100 mM Glycine-NaOH buffer (pH 2.5-3.5, plotted with ■ in FIG. 4), 100 mM potassium acetate buffer (pH 3.5-pH 5.5: plotted with □ in FIG. 4), 100 mM MES-NaOH Buffer (pH 5.5-pH 6.5: plotted with ▲ in FIG. 4), 100 mM potassium phosphate buffer (pH 6.0-pH 8.0: plotted with △ in FIG. 4), 100 mM Tris-HCl buffer (PH 7.5-pH 9.0: plotted with ● in FIG. 4), 100 mM Gricyne-NaOH buffer solution (pH 8.5-pH 10.5: plotted with ○ in FIG. 4), 25 ° C., 16 hours, The activity was measured when glucose was used as a substrate after maintaining the enzyme in each buffer solution. The activity value after the treatment was compared with the activity value in the same buffer before the treatment, and the residual activity rate was determined. The results are shown in FIG.

  As a result, the modified FADGDH had an activity remaining rate of 90% or more in the range of pH 4.5 to pH 7.0. From the above, it was confirmed that the stable pH range of the modified FADGDH is pH 4.5 to pH 7.0.

<Example 8>
Molecular weight estimation by SDS-PAGE The Mes-GDH obtained in Example 2 was denatured by heat treatment at 100 ° C. for 10 minutes, and then at 37 ° C. with 5 U of endoglycosidase (EndoH: New England Biolabs). After treatment for 1 hour, the sugar chain added to the protein was degraded. Next, SDS-polyacrylamide gel electrophoresis using Nu-PAGE 4-12% Bis-Tris Gel (manufactured by Invitrogen) was performed on the enzyme that decomposed the sugar chain. As a result, a band was mainly observed at 55 kDa. The band was cut out from the gel, digested with trypsin, and then the molecular weight of the digested fragment was measured by LC / MS / MS analysis, and the protein was identified by MASCOT analysis. . As a result, since the peptide obtained from the band of 55 kDa showed the sequence and hit obtained in Example 1, it was estimated that this band was GDH.

  The glucose dehydrogenase produced according to the present invention can be supplied as a raw material for a blood glucose level measurement reagent, a blood glucose sensor, and a glucose determination kit.

Claims (12)

A flavin-binding glucose dehydrogenase that satisfies the following (a) to (d).
(A) Polypeptide consisting of the following [1] or [2] polypeptide [1] Polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 [2] Same as the amino acid sequence shown in SEQ ID NO: 1 A polypeptide having an amino acid sequence of 99% or more and having glucose dehydrogenase activity (b) When the activity at 25 ° C is 100%, the activity at 5 ° C is 35% or more (C) Residual activity after treatment at 50 ° C. for 15 minutes is 90% or more (d) Optimal activity temperature is 60 ° C. The flavin-binding glucose dehydrogenase according to claim 1, which satisfies at least one of the following (e) and (f):
(E) pH stability: residual enzyme activity after treatment of 2 U / mL enzyme at 25 ° C. for 16 hours is at least 90% in the range of pH 4.5 to pH 7.0. (F) Optimal pH: Shows the highest activity between pH 6.0 and 6.7, and in that range shows relative activity of 80% or more compared to the case where the activity at pH 6.3 (phosphate buffer) is 100%. The glucose dehydrogenase according to claim 1 or 2, wherein the polypeptide portion has a molecular weight of about 55 kDa. The glucose dehydrogenase according to any one of claims 1 to 3, which is derived from a filamentous fungus belonging to the genus Metalidium. DNA of any of the following (A) to (F).
(A) DNA encoding the amino acid sequence represented by SEQ ID NO: 1,
(B) DNA consisting of the base sequence shown in SEQ ID NO: 2,
(C) a DNA comprising a nucleotide sequence having an identity of 99% or more with the nucleotide sequence represented by SEQ ID NO: 2 and encoding a polypeptide having glucose dehydrogenase activity;
(D) DNA consisting of the base sequence represented by SEQ ID NO: 3,
(E) a polymorphism having a glucose dehydrogenase activity, comprising a nucleotide sequence having an identity with the nucleotide sequence of SEQ ID NO: 3 of 99% or more, spliced during transcription and translation in a eukaryotic host DNA encoding the peptide A vector incorporating the DNA according to claim 5. A transformant into which the vector according to claim 6 has been introduced. The method for producing the flavin-binding glucose dehydrogenase according to claim 1, wherein the microorganism containing the DNA according to claim 5 is cultured, and the obtained culture solution is purified. The method for producing a flavin-binding glucose dehydrogenase according to claim 8, comprising a step of purifying a culture solution obtained by culturing the transformant according to claim 7. A method for measuring a glucose concentration, comprising causing the flavin-binding glucose dehydrogenase according to claim 1 to act on glucose. A glucose assay kit comprising the flavin-binding glucose dehydrogenase according to claim 1. A glucose sensor comprising the flavin-binding glucose dehydrogenase according to claim 1.