JP6465156B2 - Novel glucose dehydrogenase - Google Patents

Description

  The present invention relates to glucose dehydrogenase. More specifically, the present invention relates to a flavin-binding glucose dehydrogenase, a DNA encoding the same, a bacterium producing the enzyme, a method for producing the enzyme, a glucose measuring method using the enzyme, 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 has an electrode and an enzyme reaction layer formed on an insulating substrate.

  Examples of the enzyme used here include glucose dehydrogenase (GDH) and glucose oxidase (GO). It has been pointed out that the method using GO (EC 1.1.3.4) is easily affected by dissolved oxygen in the measurement sample, and the dissolved oxygen affects the measurement result. On the other hand, although GDH is not influenced by dissolved oxygen, for example, pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) (EC 1.1.5.2 (formerly EC 1.1.9.17)) is Since it also acts on sugars other than glucose, such as maltose and lactose, it is not suitable for accurate blood glucose measurement.

  Flavin adenine dinucleotide-dependent glucose dehydrogenase (hereinafter also referred to as “FADGDH”) is not affected by dissolved oxygen and hardly acts on maltose. Patent Documents 1 to 6 and Non-Patent Documents 1 to 6 report those derived from Aspergillus terreus or Aspergillus oryzae, or modified ones thereof. However, since these enzymes have a relatively high reactivity with xylose (Patent Document 1), there is room for improvement when measuring blood glucose in those who are undergoing a xylose tolerance test. In recent years, on the other hand, flavin-binding GDH (Patent Document 6), which has a relatively low action on xylose, and modified GDH (Patent Document 7) having the advantages of GO and GDH have been developed, but there is still room for improvement. is there

WO2004 / 058958 WO2006 / 101239 JP2007-289148 JP2008-237210A WO2008 / 059777 WO2010 / 140431 WO2011 / 0668050

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

  Under the current situation as described above, the present inventors have repeatedly studied day and night to develop a novel glucose dehydrogenase that is more suitable for use in SMBG, and in addition to excellent substrate specificity, The present inventors have found that the use of an enzyme having affinity and excellent stability shortens the measurement time and enables accurate blood glucose measurement with a small amount of enzyme. In addition, since an SMBG sensor using an enzyme usually involves a heat treatment step, it is desirable that the enzyme has excellent thermal stability that does not lose enzyme activity by heat treatment in addition to the above-described characteristics. . Therefore, an object of the present invention is to provide a new glucose dehydrogenase suitable for use in an SMBG sensor by being excellent in substrate characteristics, affinity for the substrate, thermal stability, and the like.

  In order to solve the above-mentioned problems, the present inventors have conducted day and night studies and screened many microorganisms that have not been reported to produce glucose dehydrogenase so far. It was found to have enzyme activity. As a result of isolating and purifying the enzyme and examining its properties, the present inventors have found that it is flavin-binding glucose dehydrogenation, has excellent substrate specificity, high affinity for D-glucose, and excellent heat. It was found that it has stability. Furthermore, the present inventors determined the amino acid sequence and gene sequence of the isolated enzyme, and found that these sequences are novel enzymes different from those of FADGDH reported so far.

  The present invention has been completed as a result of further research and improvement based on such knowledge, and representative examples of the present invention are as follows.

Item 1
A flavin-binding glucose dehydrogenase comprising the polypeptide of any of the following (a) to (c);
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) a polypeptide having a glucose dehydrogenase activity, comprising an amino acid sequence obtained by substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 ,
(C) A polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having glucose dehydrogenase activity.
Item 2
DNA of any of the following (A) to (E):
(A) DNA encoding the amino acid sequence represented by SEQ ID NO: 1,
(B) DNA comprising the base sequence represented by SEQ ID NO: 2,
(C) DNA comprising a nucleotide sequence having a homology with the nucleotide 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 encodes a polypeptide having glucose dehydrogenase activity;
(E) a polypeptide having glucose dehydrogenase activity, wherein one or several bases in the base sequence shown in SEQ ID NO: 2 are substituted, deleted, inserted, added and / or inverted. DNA encoding
(F) In the amino acid sequence shown in SEQ ID NO: 1, a polyamino acid sequence comprising one or several amino acid residues substituted, deleted, inserted, added, or inverted, and having glucose dehydrogenase activity DNA encoding a peptide.
Item 3
A vector incorporating the DNA of Item 2.
Item 4
A transformant comprising the vector according to Item 3.
Item 5
The method for producing a flavin-binding glucose dehydrogenase according to claim 1, comprising culturing the transformant according to item 4.
Item 6
A method for measuring a glucose concentration, comprising causing the flavin-binding glucose dehydrogenase according to Item 1 to act on glucose.
Item 8
A glucose assay kit comprising the flavin-binding glucose dehydrogenase according to Item 1.
Item 9
A glucose sensor comprising the flavin-binding glucose dehydrogenase according to Item 1.
Term A. A flavin-binding glucose dehydrogenase having the following characteristics (1) to (4).
(1) Molecular weight: The molecular weight of the polypeptide portion of the enzyme measured by SDS-polyacrylamide electrophoresis is about 69 kDa.
(2) Km value: Km value with respect to D-glucose is about 10 mM or less. (3) Temperature stability: stable at 50 ° C. or less (4) pH stability: stable term in the range of pH 3.0 to 8.0 The flavin-binding glucose dehydrogenase according to Item A, further comprising the following property (5):
(5) Substrate specificity: C. The reactivity to D-xylose is 1.8% or less when the reactivity to D-glucose is 100%. The flavin-binding glucose dehydrogenase according to Item A or B, further comprising the following property (6):
(6) Optimal activity temperature: 45 ° C to 50 ° C
Term D. The flavin-binding polymorphic glucose dehydrogenase according to any one of Items A to C, further comprising the following property (7):
(7) Optimum active pH: 8.0
Term E. The flavin-binding glucose dehydrogenase according to any one of Items A to D, further comprising the following property (8):
(8) Origin: Item F. derived from a microorganism classified into the genus Mucor. The method for producing a flavin-binding glucose dehydrogenase according to any one of Items A to E, comprising culturing a microorganism classified into the genus Mucor and collecting glucose dehydrogenase. Term G. A method for measuring a glucose concentration, comprising causing the flavin-binding glucose dehydrogenase according to any one of Items A to E to act on glucose.
Term H. A glucose assay kit comprising the flavin-binding glucose dehydrogenase according to any one of Items A to E.
Term I. A glucose sensor comprising the flavin-binding glucose dehydrogenase according to any one of Items A to E.

  The flavin-binding glucose dehydrogenase of the present invention (hereinafter sometimes referred to as “FGDH”) has glucose dehydrogenase activity and has a high affinity for D-glucose (that is, for D-glucose). (Km value is significantly lower), so that the D-glucose concentration in the sample can be measured in a shorter time with a smaller amount of enzyme. In addition, since the FGDH of the present invention has a significantly low reactivity to D-xylose, it is possible to accurately measure the amount or concentration of glucose even when D-glucose and D-xylose coexist in the sample. To. Therefore, the FGDH of the present invention is suitable for blood glucose level measurement under a xylose tolerance test. Furthermore, since the FGDH of the present invention is excellent in thermal stability, it enables efficient production of sensor strips involving heat treatment and the like. In addition, since the FGDH of the present invention is stable over a wide range of pH, it is suitable for use under a wide range of conditions. Because of these characteristics, the FGDH of the present invention makes it possible to accurately measure the glucose concentration in any sample containing D-glucose (for example, blood or food (such as seasonings or beverages)). Furthermore, since the DNA of the present invention encodes the FGDH of the present invention, it is possible to efficiently produce the FGDH of the present invention using a genetic engineering technique.

The influence of pH with respect to the activity of Mucor subtilissimus NBRC6338 origin FGDH is shown. The influence of the temperature with respect to the activity of Mucor subtilissimus NBRC6338 origin FGDH is shown. The result of having measured pH stability of Mucor subtilissimus NBRC6338 origin FGDH is shown. The result of having measured the temperature stability of Mucor subtilissimus NBRC6338 origin FGDH is shown. The relationship between the reaction rate of Mucor subtilismus NBRC6338-derived FGDH and the substrate concentration is shown.

Hereinafter, the present invention will be described in detail.
1. 1. Flavin-binding glucose dehydrogenase 1-1. Glucose dehydrogenase activity Flavin-binding glucose dehydrogenase is an enzyme having a physicochemical property 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 can bear the transfer of electrons in the reaction catalyzed by FGDH. For example, 2,6-dichlorophenolindophenol (DCPIP), phenazine methosulfate (PMS), 1-methoxy-5-methylphenadium methyl sulfate, a ferricyan compound, and the like can be used.

  The glucose dehydrogenase activity can be measured by a known method. For example, using DCPIP as an electron acceptor, the activity can be measured using the change in absorbance of the sample at a wavelength of 600 nm before and after the reaction as an index. More specifically, the activity can be measured using the following reagents and measurement conditions.

Method for measuring glucose dehydrogenase activity <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>
Prewarm 3 mL of reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 mL of FGDH solution and mixing gently, the absorbance change at 600 nm was recorded for 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control, and from the linear part (ie, the reaction rate became constant). Measure the change in absorbance per minute (ΔODTEST). In the blind test, a solvent that dissolves FGDH is added to the reagent mixture instead of the FGDH solution, and the change in absorbance per minute (ΔODBLANK) is measured in the same manner. From these values, FGDH activity is determined according to the following equation. Here, 1 unit (U) in FGDH activity is 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 ) × 3.1 × dilution ratio} / {16.3 × 0.1 × 1.0}

In the formula, 3.1 is the amount of the reaction reagent + enzyme solution (mL), 16.3 is the molar molecular extinction coefficient (cm ² / micromole) 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.

  The FGDH of the present invention is a flavin-binding GDH that requires flavin as a prosthetic group.

  The FGDH of the present invention is preferably isolated FGDH or purified FGDH. Further, the FGDH of the present invention may be present in a state dissolved in a solution suitable for the above storage or in a lyophilized state (for example, in powder form). “Isolated” when used in relation to the enzyme (FGDH) of the present invention substantially includes components other than the enzyme (for example, contaminating proteins derived from host cells, other components, culture fluid, etc.). No) states. 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 FGDH of the present invention may be present in a solution (eg, buffer) suitable for storage or measurement of enzyme activity.

1-2. Polypeptide The FGDH of the present invention is preferably composed of any of the following polypeptides (a) to (c).
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1;
(B) a polypeptide having a glucose dehydrogenase activity, comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted, added and / or inverted in the amino acid sequence shown in SEQ ID NO: 1 ;
(C) A polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having glucose dehydrogenase activity.

  As shown in Example 5, the amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence of FGDH derived from Mucor subtilismus NBRC6338 and satisfies all the following characteristics of 1-3 to 1-10.

The polypeptide of (b) above has substitution, deletion, insertion, addition and / or substitution 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. A polypeptide consisting of an inverted amino acid sequence (hereinafter sometimes collectively referred to as “mutation”). Here, “several” means that the glucose dehydrogenase activity and preferably the characteristics of 1-3 to 1-10 (particularly 1-3, 1-4, 1-7, and 1-8) described later are maintained. For example, it is a number corresponding to less than about 20% of all amino acids, preferably a number corresponding to less than about 15%, more preferably a number corresponding to less than about 10%, More preferably it is a number corresponding to less than about 5%, most preferably a number corresponding to less than about 1%. More specifically, the number of amino acid residues to be mutated is, for example, 2 to 127, preferably 2 to 96, more preferably 2 to 64, still more preferably 2 to 32, and even more. The number is preferably 2 to 20, more preferably 2 to 15, still more preferably 2 to 10, and particularly preferably 2 to 5.

  When the mutation is an amino acid substitution, the type of substitution is not particularly limited, but conservative amino acid substitution is preferable from the viewpoint of not significantly affecting the FGDH phenotype. “Conservative amino acid substitution” refers to substitution of an amino acid residue with an amino acid residue having a side chain of similar properties. Depending on the side chain of the amino acid residue, a basic side chain (eg lysine, arginine, histidine), an acidic side chain (eg aspartic acid, glutamic acid), an uncharged polar side chain (eg glycine, asparagine, glutamine, serine, threonine, tyrosine) Cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), aromatic side chains (eg tyrosine, phenylalanine, Like tryptophan and histidine). 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 (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Press, New York) and random mutation. Introduction Method (Molecular Cloning, Third Edition, Chapter)
13, Cold Spring Harbor Laboratory Press, New York) and the like by using a known method to introduce a mutation into the DNA encoding FGDH of the present invention described later. Variant FGDH can also be obtained by other methods such as ultraviolet irradiation. Variants FGDH include naturally occurring variants (for example, single nucleotide polymorphisms) such as cases based on individual differences of microorganisms holding FGDH, species or genus differences.

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

  The polypeptide of the above (c) is compared with the amino acid sequence shown in SEQ ID NO: 1 as long as it retains glucose dehydrogenase activity, preferably as long as it retains the following properties 1-3 to 1-10. A polypeptide comprising an amino acid sequence having an identity of 80% or more. Preferably, the identity between the amino acid sequence of FGDH of the present invention and the amino acid sequence shown in 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.

The identity of amino acid sequences can be calculated using analysis tools that are commercially available or available through telecommunication lines (Internet). For example, the homology algorithm BLAST (Basic local alignment) of the National Center for Biotechnology Information (NCBI) search tool) https://www.ncbi.nlm.nih.gov/BLAST/ can be calculated by using the default (initial setting) parameters.

1-3. Substrate specificity The FGDH of the present invention is excellent in substrate specificity. In particular, the FGDH of the present invention has a significantly low reactivity to at least D-xylose, based on the reactivity to D-glucose. More specifically, the FGDH of the present invention preferably has a reactivity to D-xylose of 1.8% or less when the reactivity to the same concentration of D-glucose is 100%.

  The FGDH of the present invention preferably has low reactivity to D-galactose and maltose in addition to low reactivity to D-xylose. The reactivity of FGDH of the present invention to D-galactose is usually 5% or less, preferably 3% or less, more preferably 2% or less, and the reactivity to D-glucose at the same concentration is 100%. Preferably it is 1.5% or less, Most preferably, it is 1.3% or less.

  The reactivity of FGDH of the present invention to maltose is usually 5% or less, preferably 4% or less, more preferably 3% or less, assuming that the reactivity to D-glucose of the same concentration is 100%. Preferably it is 2.3% or less.

  The lower limit of reactivity to D-xylose, D-galactose and maltose based on the reactivity of FGDH of the present invention to D-glucose is not particularly limited, but the lower limit is close to 0% or 0%. Can be a value.

  The reactivity of FGDH to each saccharide is as described in 1-1. In the method for measuring glucose dehydrogenase activity shown in FIG. 4, the determination may be made by substituting D-glucose with another sugar (for example, D-xylose, D-galactose, or maltose) and comparing the activity in the case of D-glucose. it can. However, the concentration of each saccharide in the case of comparison is based on 50 mM.

  The FGDH of the present invention having excellent substrate specificity as described above is preferable as an enzyme for accurately measuring the amount of glucose in a sample. That is, according to the FGDH of the present invention, even when impurities such as maltose, D-galactose, and D-xylose are present in the sample, it is possible to accurately measure the amount of target D-glucose. . Therefore, this enzyme is suitable for applications in which the presence of such contaminants is expected or concerned (typically, measurement of the amount of glucose in blood), and can be used in various applications including such applications. Applicable and highly versatile.

1-4. Affinity for D-glucose The FGDH of the present invention preferably has a high affinity for D-glucose, which is the original substrate. Due to the high affinity, even if the concentration of D-glucose in the sample is low, the above-described catalytic reaction can proceed, and more accurate measurement of D-glucose concentration, measurement in a shorter time, This is because it contributes to measurement with a smaller amount of enzyme. The affinity of FGDH for D-glucose is indicated by the Km value. The Km value is a value obtained from the so-called Michaelis-Menten equation, specifically, 1-1. In the activity measurement method shown in Fig. 4, the D-glucose concentration is changed, the activity at each concentration is measured, and a line weaver bark plot is created.

  Judging from the reaction kinetics of the enzyme, the lower the Km value, the higher the affinity of the enzyme for the substrate, and even when the substrate concentration is low, the enzyme can form a complex with the substrate. Can proceed. The Km value for D-glucose of FGDH of the present invention is preferably 10 mM or less, more preferably 9 mM or less, still more preferably 8 mM or less, still more preferably 7 mM or less, and particularly preferably 6.7 mM or less. is there.

1-5. Optimum active pH
As shown in Examples, the FGDH of the present invention preferably exhibits the highest activity at pH 8.0 (Tris HCl buffer). In addition, at pH 7.5 to 8.0 (TrisHCl buffer), the FGDH of the present invention preferably exhibits a relative activity of 80% or more, with the activity at pH 8.0 (Tris HCl buffer) being 100%. That is, the optimum active pH of the FGDH of the present invention is 7.5 to 8.0, preferably pH 8.0. On the other hand, FGDH of the present invention exhibits the highest activity around pH 6.5 to 7.5 in the potassium phosphate buffer and MES-NaOH buffer, as shown in the examples described later. Therefore, when these buffers are used, it is preferable that pH 6.5 to 7.5 is the optimum active pH.

1-6. Optimum activity temperature The optimum activity temperature of the FGDH of the present invention is preferably 45 ° C to 50 ° C. Here, “45 ° C. to 50 ° C.” typically means that the optimum activation temperature is around 45 ° C. to 50 ° C. and further has a certain acceptable width. In the present specification, the optimum activity temperature is determined by measuring the enzyme activity in a PIPES-NaOH buffer (pH 6.5) at an enzyme concentration of 0.1 U / mL, as shown in Examples described later.

1-7. pH stability In the present specification, when the residual enzyme activity after treating a 2 U / mL enzyme at 25 ° C. for 16 hours under a specific pH condition is 95% or more compared to the enzyme activity before the treatment In addition, the enzyme is judged to be stable at the pH condition. The FGDH of the present invention is preferably stable at least over the entire pH range of 3.0 to 8.0.

1-8. Temperature Stability In this specification, the residual enzyme activity after treating 2 U / mL of enzyme in a suitable buffer solution (for example, potassium acetate buffer (pH 5.0)) for 15 minutes under a specific temperature condition When there is no substantial decrease compared to the previous enzyme activity (that is, maintaining about 90% or more), the enzyme is judged to be stable at the temperature condition. The FGDH of the present invention is preferably stable at least in the temperature range of 0 ° C to 50 ° C.

  The FGDH of the present invention preferably has at least one or more of the characteristics shown in the above 1-3 to 1-8, more preferably two or more, and still more preferably the three characteristics. Further, more preferably four or more, more preferably five or more, still more preferably six or more, particularly preferably all of them. The FGDH of the present invention may have the characteristics of 1-3 to 1-8 in any combination, but has the characteristics of 1-3, 1-4, 1-7, and 1-8. Is preferred.

1-9. Molecular Weight The molecular weight of the polypeptide moiety constituting the FGDH of the present invention is preferably about 69 kDa as measured by SDS-PAGE. The term “about 69 kDa” means that a range in which a person skilled in the art normally determines that there is a band at a position of 69 kDa when molecular weight is measured by SDS-PAGE is included. “Polypeptide moiety” means FGDH in a state where sugar chains are not substantially bound. When the FGDH of the present invention produced by a microorganism is a glycan-linked type, the glycan is removed by treating it with heat treatment or saccharide hydrolase (that is, a “polypeptide moiety”). be able to. The state in which sugar chains are not substantially bound allows the presence of sugar chains inevitably remaining in sugar chain-bound FGDH treated by heat treatment or sugar hydrolase. Therefore, when FGDH is not inherently a sugar chain-binding type, it itself corresponds to a “polypeptide moiety”.

  The means for removing the sugar chain from the sugar chain-bound FGDH is not particularly limited. For example, as shown in the examples described later, the sugar chain-bound FGDH was denatured by heating at 100 ° C. for 10 minutes. Then, it can implement by processing for 6 hours at 37 degreeC using N-glycosidase F (made by Roche Diagnostics).

  When the FGDH of the present invention is bound to a sugar chain, its molecular weight is not particularly limited as long as it does not negatively affect glucose dehydrogenase activity, substrate specificity, affinity for D-glucose, and the like. For example, the molecular weight of the FGDH of the present invention in a state where sugar chains are bound is preferably 103,000 to 143,000 Da as measured by SDS-PAGE. Glycan-linked FGDH is preferable from the viewpoint of making the enzyme more stable and from the viewpoint of enhancing 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.

1-10. Origin The origin of the FGDH of the present invention is not particularly limited as long as it has the properties described above. The FGDH of the present invention can be derived from, for example, a microorganism belonging to the genus Mucor. Although it does not restrict | limit especially as microorganisms which belong to Mucor genus, For example, Mucor subtilismus, Mucor guilliermondi, Mucor plinii, Mucor javanicus, Mucor circinolides, and Mucor fiemis. Silvaticus etc. can be illustrated. More specifically, Mucor subtilissimus NBRC63
38 can be exemplified. Mucor subtilissimus NBRC6338 is a strain stored in NBRC (NITE Biological Resource Center) (Biotechnology Division, Biotechnology Headquarters, National Institute for Product Evaluation and Technology) and may be transferred through a prescribed procedure. it can.

  Examples of other organisms from which the FGDH of the present invention is derived include microorganisms present in water systems such as soil, rivers and lakes or in the ocean, microorganisms resident on the surface or inside various animals and plants, and the like. Microorganisms that grow in a low temperature environment, a high temperature environment such as a volcano, an oxygen-free / high-pressure / no-light environment such as the deep sea, and a special environment such as an oil field may be used as the isolation source.

  The FGDH of the present invention includes not only FGDH directly isolated from microorganisms, but also those obtained by modifying the isolated FGDH by amino acid sequence or the like by a protein engineering method or by genetic engineering techniques. . For example, microorganisms classified into the family Aceraceae, and more specifically, Mucor guilliermondii, Mucor plainii, Mucor javanicus, Mucor cilinelloides, Mucor subtilisimus, and Mucor heimalis. Those derived from microorganisms belonging to silvaticus may be modified.

2. DNA encoding flavin-binding glucose dehydrogenase
The DNA of the present invention comprises the above-mentioned 1. DNA encoding FGDH, specifically, one of the following (A) to (F).
(A) DNA encoding the amino acid sequence represented by SEQ ID NO: 1;
(B) DNA comprising the base sequence represented by SEQ ID NO: 2;
(C) DNA comprising a nucleotide sequence having a homology with the nucleotide sequence represented by SEQ ID NO: 2 of 80% or more and encoding a polypeptide having glucose dehydrogenase activity;
(D) DNA containing a DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 2 and that encodes a polypeptide having glucose dehydrogenase activity;
(E) a polypeptide having glucose dehydrogenase activity, wherein one or several bases in the base sequence shown in SEQ ID NO: 2 are substituted, deleted, inserted, added and / or inverted. DNA encoding
(F) In the amino acid sequence shown in SEQ ID NO: 1, a polyamino acid sequence comprising one or several amino acid residues substituted, deleted, inserted, added, or inverted, and having glucose dehydrogenase activity DNA encoding a peptide.

  As used herein, “DNA encoding a protein” refers to DNA from which the protein is obtained when it is expressed, that is, DNA having a base sequence corresponding to the amino acid sequence of the protein. Therefore, DNA that differs depending on codon degeneracy is also included.

In the DNA of the present invention, the protein having the amino acid sequence encoded by the DNA of the present invention has glucose dehydrogenase activity and preferably at least one of the above properties 1-2 to 1-10 (particularly, the above 1-3, 1-4). 1-7 and 1-8), the homology with the nucleotide sequence shown in SEQ ID NO: 2 is 80% or more, preferably 85% or more, more preferably 88% or more, and still more preferably 90%. More preferably, the nucleotide sequence is 93% or more, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

  The homology of the base sequence can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet), and is calculated using software such as FASTA, BLAST, PSI-BLAST, SSEARCH, etc. The Specifically, main initial conditions generally used for BLAST search are as follows. That is, in Advanced BLAST 2.1, by using blastn as a program and searching with various parameters set to default values, the homology value (%) of the nucleotide sequence can be calculated.

  In the DNA of the present invention, the protein encoded by it has glucose dehydrogenation activity, preferably the above-mentioned characteristics 1-2 to 1-10, more preferably the above 1-3, 1-4, 1-7 and 1 As long as it has at least one of the characteristics (-8), it may be DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 2. Here, “stringent conditions” 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 are described in, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Press, New York) and Current protocol Biomolecules in molecular biology. 1987).

Specific stringent conditions include, for example, a hybridization solution (50% formamide, 10 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 5 × Denhardt solution, 1% SDS, 10% Incubate at about 42 ° C. to about 50 ° C. with dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5), and then with 0.1 × SSC, 0.1% SDS And washing at about 65 ° C to about 70 ° C. More preferable stringent conditions include, for example, 50% formamide, 5 × SSC (0.15 M NaCl, 15 mM sodium as a hybridization solution).
(citrate, pH 7.0), 1 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)).

  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.

  With respect to the DNAs (E) and (F) above, “several” has the same meaning as described in 1-2 above. That is, “several” means that the glucose dehydrogenase activity and preferably the characteristics of 1-3 to 1-10 (particularly 1-3, 1-4, 1-7, and 1-8) described later are maintained. As many as, for example, a number corresponding to less than about 20% of the total DNA, preferably a number corresponding to less than about 15%, more preferably a number corresponding to less than about 10%, even more preferred. Is a number corresponding to less than about 5%, most preferably a number corresponding to less than about 1%. More specifically, the number of bases to be mutated is, for example, 2 to 382, preferably 2 to 286, more preferably 2 to 290, still more preferably 2 to 95, and still more preferably. 2 to 19, more preferably 2 to 15, even more preferably 2 to 10, and particularly preferably 2 to 5.

  In a preferred embodiment, the DNA encoding FGDH of the present invention is DNA that is 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 (F).

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). Can be easily prepared by using various genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. (Molecular Cloning 2d Ed, Cold
Spring Harbor Lab. Press (1989); secondary biochemistry experiment course "gene research method I, II, III", Japan Biochemical Society edition (1986) 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.

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 FGDH 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); Science 12
2, 778 (1983) etc.].

  The source microorganism for preparing the cDNA library is not particularly limited as long as it is a microorganism that expresses the FGDH of the present invention, but is preferably a microorganism classified into the genus Mucor. More specifically, the above 1-10. Can be mentioned.

  Isolation of total RNA from the above-mentioned microorganism, 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 [Science 130, 1350 (1985)] or a modified method of DNA or RNA can be preferably used. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method [Rapid amplification of cDNA ends; experimental medicine, 12 (6), 35
(1994)], especially the 5′-RACE method [M. A. Frohman, et al. , Proc. Natl. Acad. Sci. USA. , 8, 8998 (1988)].

  Primers used when adopting the PCR method can also 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 FGDH of the present invention can be easily and stably produced in large quantities.

3. Vector The vector of the present invention comprises the above-mentioned 2. This is a vector in which a DNA encoding FGDH of the present invention described in 1. 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 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).

4). 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 FGDH. Specifically, prokaryotic cells such as Escherichia coli and Bacillus subtilis, eukaryotic cells such as yeast, mold, insect cells, and mammalian cells can be used. When the host is Escherichia coli, Escherichia coli C600, Escherichia coli HB101, Escherichia coli DH5α and the like are used, and examples of the vector include pBR322, pUC19, pBluescript and the like. When the host is yeast, examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Pichia pastoris, and vectors include pAUR101, pAUR224, and pYE32. In the case where the host is a filamentous fungal cell, for example, Aspergillus oryzae, Aspergillus niger, Mucor himalis and the like can be exemplified. It is also preferable to use as a host a microorganism belonging to the genus Mucor from which the FGDH of the present invention has been isolated. 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 3. It is prepared by transfection or transformation using the expression vector shown in 1. Transformation may be transient or stable. Transfection and transformation consisted of calcium phosphate co-precipitation, electroporation (Potter,
H. et al. , Proc. Natl. Acad. Sci. U. S. A. 81, 7161-7165 (1984)), lipofection (Felgner,
P. L. et al. , Proc. Natl. Acad. Sci. U. S. A. 84, 7413-7417 (1984)), microinjection (Graessmann, M. & Graessmann, A., Proc. Natl.
Acad. Sci. U. S. A. 73, 366-370 (1976)), the method of Hanahan (Hanahan, D., J. Mol. Biol. 166, 557-580 (1983)), the lithium acetate method (Schiestl, R. H. et.
al. Curr. Genet. 16, 339-346 (1989)), protoplast-polyethylene glycol method (Yelton, MM et al., Proc. Natl. Acad. Sci. 81, 1470-1474 (1984)) and the like. be able to.

  Since the transformant of the present invention has the ability to produce the FGDH of the present invention, it can be used to produce the FGDH of the present invention efficiently.

5. Production method of flavin-binding glucose dehydrogenase The FGDH of the present invention is typically produced by culturing a microorganism having the ability to produce the FGDH of the present invention. The microorganism used for the culture is not particularly limited as long as it has the ability to produce the FGDH of the present invention. And a wild-type microorganism belonging to the genus Mucor shown in 4. The transformant shown in can be suitably used.

Microorganisms classified into the above genus Mucor are, for example, NBRC (NITE).
It is a strain stored in Biological Resource Center (Biological Resource Division, Biotechnology Headquarters, National Institute of Technology and Evaluation), and can be sold through a predetermined procedure.

  The culture method and culture conditions are not particularly limited as long as the FGDH of the present invention is produced. That is, on the condition that FGDH 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 microorganisms classified into the genus Mucor to obtain the FGDH of the present invention, culture conditions may be selected in consideration of the nutritional physiological properties of the microorganisms. 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 3 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, it is preferable to recover FGDH from the culture solution or the cells. When using a microorganism that secretes FGDH outside the cells, for example, the culture supernatant is filtered, centrifuged, etc. to remove insoluble matters, then concentrated with an 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. Flavin-binding glucose dehydrogenase produced by microorganisms belonging to the genus Mucor 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. The present enzyme can be obtained by adding a chelating agent and a surfactant to solubilize GDH, separating and collecting it as an aqueous solution, separating and purifying it. 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 step, in principle, fractionation is performed using FGDH 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.

  When the FGDH of the present invention is used as a purified preparation, for example, it is preferable to purify it to a specific activity of 110 to 210 (U / mg), preferably 140 to 180 (U / mg). The final form may be liquid or solid (including powder).

  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.

6). Method for Measuring Glucose A method for measuring glucose using glucose dehydrogenase 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 FGDH of the present invention. As long as the concentration or amount of glucose can be measured using the FGDH of the present invention, the mode is not particularly limited. For example, the FGDH of the present invention is allowed to act on glucose in a sample, and the electrons associated with the dehydrogenation reaction of glucose. This can be done by measuring the structural change of a receptor (eg, DCPIP) by absorbance. More specifically, the above 1-1. 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 FGDH 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.

The measurement of the glucose concentration in the form of a sensor to be described later can be performed, for example, 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 the working electrode, an electrode on which the FGDH of the present invention is immobilized is used, 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.

7). Glucose Assay Kit The glucose assay kit of the present invention contains the FGDH of the present invention in an amount sufficient for at least one assay. Typically, the kit includes the FGDH of the present invention, plus buffers necessary for the assay, mediators, glucose standard solution for creating a calibration curve, and directions for use. The FGDH of the present invention can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution.

8). Glucose Sensor The present invention also provides a glucose sensor using the FGDH of the present invention. The glucose sensor of the present invention can be produced by using a carbon electrode, a gold electrode, a platinum electrode, or the like as an electrode and immobilizing the enzyme of the present invention on this electrode. Examples of the immobilization method 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, and a redox polymer. In addition, it may be fixed in a polymer or adsorbed and fixed on an electrode together with an electron mediator represented by ferrocene or a derivative thereof, or may be used in combination. Since FGDH of the present invention is excellent in thermal stability, it can be immobilized under relatively high temperature conditions (for example, 50 ° C. and 55 ° C.). Typically, after FGDH of the present invention is immobilized on a carbon electrode using glutaraldehyde, glutaraldehyde can be blocked by treatment with a reagent having an amine group.

  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 the working electrode, an electrode on which the FGDH of the present invention is immobilized is used, 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 Restoration of Strains A strain belonging to the genus Mucor was obtained from the International Cooperation Division, National Institute of Technology and Evaluation Biotechnology Center. Since the obtained strain was an L-dried preparation, the ampoule was opened, 100 μL of reconstituted water was injected, the dry cells were suspended, and the suspension was dropped into the reconstitution medium for 3 days at 25 ° C. The strain was restored by stationary culture for 7 days. As the restoration water, sterilized water (distilled water treated at 120 ° C. for 20 minutes in an autoclave) is used, and as the restoration medium, DP medium (dextrin 2.0%, polypeptone 1.0%, KH ₂ PO ₄ . 0%, agarose 1.5%).

Example 2 Recovery of Culture Supernatant A medium containing 2 g of wheat germ and 2 mL of water was sterilized by autoclaving at 120 ° C. for 20 minutes, and each strain of the genus Mucor restored in Example 1 was inoculated with one platinum ear. The culture was stationary at 25 ° C. for 3 to 7 days. After the culture, 4 mL of 50 mM potassium phosphate buffer (pH 6.0) containing 2 mM EDTA was added, and the mixture was sufficiently suspended by vortexing. After adding a small amount of glass beads to the suspension, it was crushed under a condition of 3,000 rpm for 3 minutes × 2 times with a bead shocker (manufactured by Yasui Kikai Co., Ltd.), 4 ° C., 2,000 × g, 5 minutes. Centrifugation was performed under the above conditions, and the recovered supernatant was used as a crude enzyme solution.

Example 3 Confirmation of Glucose Dehydrogenase Activity Glucose dehydrogenase activity in the crude enzyme solution obtained in Example 2 was measured according to 1-1. It was measured using the glucose dehydrogenase activity measuring method shown in 1. The results are shown in Table 1.

  As shown in Table 1, it was confirmed that GDH activity was present in the crude enzyme solution derived from Mucor subtilismus NBRC6338.

Example 4 Purification of Mucor subtilissimus NBRC6338-derived GDH 50 mL of DP liquid medium was placed in a 500 mL Sakaguchi flask and sterilized by autoclaving to prepare a medium for preculture. Mucor subtilismus NBRC6338, which was previously reconstituted with DP plate medium, was inoculated into a preculture medium with one platinum ear, and cultured with shaking at 25 ° C. and 180 rpm for 3 days to obtain a seed culture solution.

  Next, 6.0 L of production medium (yeast extract 2.0%, glucose 1%, pH 6.0) was placed in a 10 L jar fermenter and sterilized by an autoclave to obtain a main culture medium. 50 mL of the seed culture solution was inoculated into the main culture medium and cultured for 3 days under the conditions of a culture temperature of 25 ° C., a stirring speed of 600 rpm, an aeration rate of 2.0 L / min, and a tube pressure of 0.2 MPa. Thereafter, the culture solution was filtered with a filter cloth, and the cells were collected. The obtained bacterial cells were suspended in 50 mM potassium phosphate buffer (pH 6.0).

  The suspension was fed to a French press (manufactured by Niro Soavi) at a flow rate of 160 mL / min and crushed at 1000 to 1300 bar. Subsequently, ammonium sulfate (manufactured by Sumitomo Chemical Co., Ltd.) was gradually added to the crushed solution so as to become 0.2 saturation, and after stirring at room temperature for 30 minutes, excess precipitate was removed using a filter aid. . Next, it concentrated using the UF membrane (Millipore Co., Ltd. product) of the molecular weight cut off 10,000, and desalted the concentrate using the gel of Sephadex G-25. Thereafter, ammonium sulfate was gradually added to the desalted solution to 0.5 saturation, and 400 mL of PS Sepharose FastFlow previously equilibrated with 50 mM potassium phosphate buffer (pH 6.0) containing 0.5 saturated ammonium sulfate. It was applied to a column (manufactured by GE Healthcare) and eluted with a linear gradient of 50 mM phosphate buffer (pH 6.0). The eluted GDH fraction was concentrated with a hollow fiber membrane (Spectrum Laboratories) having a molecular weight cut off of 10,000, and then applied to a DEAE Sepharose Fast Flow (GE Healthcare) column to obtain a purified enzyme. The resulting purified enzyme was subjected to SDS-polyacrylamide gel electrophoresis (Past Gel 10-15%, Phassystem GE Healthcare). In this case, phosphorylase b (97,400 dalton), bovine serum albumin (66,267 dalton), aldolase (42,400 dalton), carbonic anhydrase (30,000 dalton), trypsin inhibitor (protein molecular weight marker) 20,100 Dalton).

  As a result, since a single band was obtained, it was confirmed that GDH was sufficiently purified. From the mobility compared to molecular weight markers, the molecular weight of FGDH was found to be 109,000-143,000 daltons.

Example 5 Molecular Weight of Isolated GDH Peptide Part The GDH purified in Example 4 was denatured by heat treatment at 100 ° C. for 10 minutes, and then 5 U of N-glycosidase F (manufactured by Roche Diagnostics) At 37 ° C. for 1 hour to decompose the sugar chain added to the protein. Then, it measured by SDS-polyacrylamide gel electrophoresis by the same method as Example 4. The same molecular weight marker as in Example 4 was used. As a result, it was found that the molecular weight of the purified FGDH polypeptide part was about 69,000 daltons.

Example 6 Substrate Specificity 1-1. According to the method for measuring the activity of FGDH shown in Fig. 4, the activity of GDH purified in Example 4 was measured using D-glucose, maltose, D-galactose, and D-xylose as substrates. The activity when D-glucose was used as a substrate was defined as 100%, and the activity against other sugars compared with that was determined. The concentration of each sugar was 50 mM. The results are shown in Table 2.

  From the results of Table 2, the substrate specificity of the FGDH of the present invention is 2.5% for the apparent activity for maltose, D-galactose, and D-xylose when the activity value for D-glucose is 100%. It was as follows and it was shown that FGDH of this invention is excellent in substrate specificity.

Example 7 Optimum active pH
Using the purified FGDH enzyme solution (0.5 U / mL) obtained in Example 4, the optimum pH was examined. 100 mM potassium acetate buffer (pH 5.0-5.5, plotted with ■ in the figure), 1
00 mM MES-NaOH buffer (pH 5.5-6.5, plotted with □ in the figure), 10
Using 0 mM potassium phosphate buffer (pH 6.0-8.0, plotted with ▲ in the figure), 100 mM Tris-HCl buffer (pH 7.5-9.0, plotted with △ in the figure), each pH The enzyme reaction was performed at a temperature of 37 ° C., and the relative activities were compared. The results are shown in FIG.

  As a result, the optimum activity pH of FGDH of the present invention showed the highest activity value at pH 8.0 when the Tris-HCl buffer was used. Moreover, when potassium phosphate buffer was used, the highest activity was shown in the range of pH 6.0-7.5. Furthermore, when MES-NaOH buffer was used, the highest activity was shown at pH 6.5.

Example 8 Optimal activity temperature Using the purified FGDH enzyme solution (0.1 U / mL) obtained in Example 4, the optimal activity temperature was examined. 42 mM PIPES-NaOH buffer (pH 6.5) was used as the buffer solution, and the activity at 37 ° C., 40 ° C., 45 ° C., 50 ° C., 55 ° C., and 60 ° C. was determined. The results are shown in FIG.

As a result, the FGDH of the present invention showed the highest activity value in the range of 45 ° C. to 50 ° C., and the temperature range showing the relative activity of 80% or more with respect to the maximum activity value was 40 ° C. to 50 ° C. . From the above, it was shown that the optimal activity temperature of flavin-binding FGDH is around 40 ° C to 50 ° C.

Example 9 pH stability Using the purified FGDH enzyme solution (2 U / mL) obtained in Example 4, pH stability was examined. 100 mM glycine-HCl buffer (pH 2.5-pH 3.5: plotted with ■ in the figure) 100 mM acetate-potassium buffer (pH 3.0-pH 5.5: plotted with □ in the figure), 100 mM MES-NaOH buffer Solution (pH 5.5-pH 6.5: plotted with ▲ mark), 100 mM potassium phosphate buffer (pH 6.0-pH 8.0: plotted with △ mark), 100 mM Tris-HCl buffer (pH 7. 5-pH 9.0: plotted with a circle in the figure), 100 mM glycine-NaOH buffer solution (pH 9.0-pH 10.5: plotted with a circle in the figure) at 25 ° C. for 16 hours in each buffer. The activity was measured when the enzyme was maintained and the subsequent glucose was used as a substrate. The activity value after the treatment and the activity value before the treatment were compared to determine the residual activity rate. The results are shown in FIG.

  As a result, the activity remaining rate was 95% or more in the range of pH 3.0 to pH 8.0. From this, it was shown that the stable pH range is pH 3.0 to pH 8.0.

Example 10 Temperature Stability Using the purified FGDH enzyme solution (2 U / mL) obtained in Example 4, temperature stability was examined. The FGDH enzyme solution was treated with each temperature (4 ° C, 30 ° C, 40 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C) for 15 minutes using 100 mM potassium acetate buffer (pH 5.0). Then, GDH activity was measured and the residual rate was measured compared with the GDH activity before a process. The results are shown in FIG.

  As a result, the FGDH of the present invention had a residual rate of 96% after treatment in the temperature range of 4 ° C to 50 ° C. From this, it was shown that FGDH is stable at 50 ° C. or lower.

Example 11 Measurement of Km Value The activity of the FGDH enzyme purified in Example 4 was measured by changing the concentration of the substrate D-glucose, and a graph of the substrate concentration and the reaction rate was prepared (FIG. 5). Based on this, a Lineweaver-burk plot was created and the Km value was calculated. As a result, the Km value for D-glucose of FGDH of the present invention was 6.7 mM, and it was found that the affinity for D-glucose was high.

Example 12 Isolation of DNA encoding FGDH (1) Extraction of chromosomal DNA Mucor subtilis NBRC6338 was treated with YG medium (Yeast)
After culturing overnight at 25 ° C. using a Sakaguchi flask containing 50 ml of Extract 1%, Glucose 2%), the culture solution was filtered using a Buchner funnel and a Nutsche suction bottle to obtain bacterial cells. Among them, about 0.3 g of the microbial cells were frozen in liquid nitrogen, the mycelia were crushed using a mortar, and an extraction buffer (1% hexamethylmethyl bromide, 0.7 M NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM. It was suspended in 12 ml of EDTA, 1% mercaptoethanol). Continue rotating and stirring at room temperature for 30 minutes, add an equal amount of phenol: chloroform: isoamyl alcohol (25: 24: 1) solution, stir and centrifuge (1,500 g, 5 minutes, room temperature) to remove the supernatant. Obtained. An equal volume of chloroform: isoamyl alcohol (24: 1) solution was added to the obtained supernatant and stirred, followed by centrifugation (1,500 g, 5 minutes, room temperature). An equal volume of isopropanol was gently added to the resulting supernatant. Chromosomal DNA precipitated by this treatment was centrifuged (20,000 g, 10 minutes, 4 ° C.), and the resulting precipitate was washed with 70% ethanol and vacuum dried. The chromosomal DNA thus obtained is again 4 m
After dissolving in 1 TE and adding 200 μl of 10 mg / ml RNase A (Sigma Aldrich Japan Co., Ltd.), the mixture was incubated at 37 ° C. for 30 minutes. Next, after adding 40 μl of 20 mg / ml Proteinase K, recombinant, PCR Grade (Roche Diagnostics) solution and incubating at 37 ° C. for 30 minutes, an equal amount of phenol: chloroform: isoamyl alcohol (25: 24: 1) The solution was added. After stirring, the mixture was centrifuged (1,500 g, 5 minutes, room temperature) to obtain a supernatant. After repeating this washing operation twice, an equal volume of chloroform: isoamyl alcohol (24: 1) solution was added to the resulting supernatant and stirred, followed by centrifugation (1,500 g, 5 minutes, room temperature). It was. The supernatant obtained as a result is subjected to centrifugation (20,000 g, 20 minutes, 4 ° C.) by adding 1/10 volume of 3M NaOAc (pH 4.8) and 2.5 times volume of ethanol. It was collected by The recovered chromosomal DNA was washed with 70% ethanol, vacuum-dried, and finally dissolved in 400 μl of TE solution to obtain a chromosomal DNA solution having a concentration of about 1 mg / ml.

(2) Design of synthetic primer The amino acid sequence of Aspergillus oryzae-derived GDH shown in SEQ ID NO: 3 (amino acid sequence shown in SEQ ID NO: 4 in the sequence table of Patent 4292486) and other known amino acid sequences are relatively amino acid sequences. Based on the region that can be determined to be conserved, degenerate primers, degeF30 and degeR13 (SEQ ID NOs: 4 and 5) containing a mixed base were synthesized.

(3) Acquisition of FGDH gene partial sequence by PCR method PCR was performed under the recommended conditions using DNA polymerase KOD-Plus (manufactured by Toyobo) using the chromosomal DNA prepared in (1) above as a template. As the primer, the primer (SEQ ID NOs: 4 and 5) prepared in (2) above was used. When the PCR reaction solution was subjected to agarose gel electrophoresis, a band corresponding to a length of about 1300 bp was confirmed. The amplified DNA fragment was purified and a cloning kit Target Clone-Plus (manufactured by Toyobo) And was cloned into the vector pTA2 and transformed into Escherichia coli DH5α competent cell (manufactured by Toyobo) to obtain a transformant. The transformant was cultured in LB medium, the plasmid was extracted, and the base sequence analysis of the region corresponding to the enzyme gene was performed. The sequence reaction was performed using BigDye ™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Japan Co., Ltd.) according to the instruction manual of the product. An ABI PRISM 310 sequencer (Applied Biosystems Japan Co., Ltd.) was used for the analysis. In order to perform a base sequence analysis of the enzyme gene, the primers (SEQ ID NOs: 4 and 5) used above were used. As a result of the base sequence analysis, a partial sequence of FGDH of about 1300 bp was obtained.

(4) Acquisition of FGDH gene full-length sequence Based on the base sequence obtained as described above, two types of inverse PCR directed toward the outside of the gene in order to acquire a gene sequence in the N-terminal direction including the start codon. Primers, InvF1 and InvR1 (SEQ ID NOs: 6, 7) were newly designed. Using this PCR primer for inverse PCR, the chromosomal DNA obtained in (1) above was treated with the restriction enzyme HincII and ligated as a template under the conditions recommended using DNA polymerase KOD-Plus (manufactured by Toyobo). Inverse PCR was performed. Thereby, the sequence analysis of the amplified fragment was performed in the same manner as described above, and the upstream base sequence including the sequence presumed to be the start codon was clarified. In addition, in order to obtain a gene sequence in the C-terminal direction including a stop codon, the above-described inverse PCR primers, InvF2 and InvR2 (SEQ ID NOs: 8 and 9) were used to restrict the genomic DNA obtained in (1) above. Treated with the enzyme XbaI,
Inverse PCR was performed in the same manner as described above using the ligated template as a template. Thereby, the sequence analysis of the amplified fragment was performed in the same manner as described above, and the upstream base sequence including the sequence presumed to be a stop codon was clarified.

(5) Determination of N-terminus and C-terminus The determination of N-terminus is based on the sequence obtained in (4) from the viewpoints of amino acid sequence homology, base sequence length, etc., using known information to the maximum. A multifaceted comparison was made to determine the start codon. The determination of the C terminal was also judged in the same manner.

(6) Primer design for amplifying the full length GDH gene derived from Mucor subtilismus NBRC6338 Based on the judgment result in (5), a primer that anneals upstream of the start codon, 5UTR F1 (SEQ ID NO: 10), and downstream of the stop codon A primer for annealing, 3UTR R1 (SEQ ID NO: 11) was designed.

(7) Determination of cDNA sequence Mucor subtilismus NBRC6338 was treated with YG medium (Yeast
After culturing overnight at 25 ° C. using a Sakaguchi flask containing 50 ml of Extract 1%, Glucose 2%), the culture solution was filtered using a Buchner funnel and a Nutsche suction bottle to obtain bacterial cells. Among them, about 0.3 g of cells were frozen in liquid nitrogen, and the mycelium was pulverized using a mortar. Subsequently, mRNA was obtained from the pulverized cells using ISOGEN (manufactured by Nippon Gene) according to the protocol of this kit. Using this as a template, ReverseTra-Ace (manufactured by Toyobo Co., Ltd.) was operated according to the protocol, reverse transcription was performed, and cDNA was synthesized. As a primer for reverse transcription, 3UTR R1 (SEQ ID NO: 11) prepared in (6) was used. Subsequently, PCR was carried out under the recommended conditions using DNA polymerase KOD-Plus (manufactured by Toyobo) using the cDNA synthesized above as a template. As the primer, the primer (SEQ ID NOs: 10 and 11) prepared in (6) above was used. When the PCR reaction solution was subjected to agarose gel electrophoresis, a band corresponding to a length of about 2000 bp was confirmed. The sequence analysis of the amplified DNA fragment was performed in the same manner as described above. In this cDNA sequence analysis, nucleotide sequences derived from three different clones were analyzed. In addition, the primer described above was used for the analysis of the base sequence. Thus, the cDNA sequence of FGDH derived from Mucor subtilismus NBRC6338 shown in SEQ ID NO: 2 was determined. The amino acid sequence of the enzyme gene encoded by the cDNA sequence is shown in SEQ ID NO: 1.

(8) Amino acid sequence comparison with known GDH The identity of the amino acid sequence identified in (7) above with the known FADGDH derived from Aspergillus oryzae and FADGDH derived from Aspergillus terreus was 34% and 33%, respectively. . For amino acid sequence analysis, the default (initial setting) parameter is set in the homology algorithm BLAST (Basic local alignment search tool) https://www.ncbi.nlm.nih.gov/BLAST/ of the National Center for Biotechnology Information (NCBI). It was calculated by using.

Example 13 Expression of Mucor subtilismus NBRC6338-derived FGDH in Aspergillus oryzae and confirmation of enzyme activity (1) Construction of expression vector Recombinant plasmid containing the Mucor subtilismus NBRC6338-derived FGDH gene (SEQ ID NO: 2) After transforming a commercially available E. coli competent cell (E. coli DH5α; manufactured by Toyobo Co., Ltd.) with pMsGDH, the transformant was transformed into a liquid medium (1%) containing ampicillin (50 mg / ml; manufactured by Nacalai Tesque).
Plasmids were prepared from cells obtained by ingesting polypeptone, 0.5% yeast extract, 0.5% NaCl; pH 7.3) and shaking culture at 30 ° C overnight. The gene region was excised from the plasmid with a restriction enzyme, mixed with a vector pUSA that had been treated with the same restriction enzyme, and ligation was performed by adding an equal amount of ligation reagent (Toyobo Lagation High) to the mixture and incubating. did. In this way, the ligated DNA was transformed into Escherichia coli DH5α strain competent cells (Toyobo Competent High DH5α) according to the protocol attached to the product to obtain the transformants. The transformant was cultured in LB medium, and the plasmid was extracted. In this way, pUSAMsGDH designed to allow mass expression in Aspergillus oryzae was obtained.

(2) Transformation Subsequently, transformation into Aspergillus oryzae was performed. The method is described in Biosci. B
iotech. Biochem. 61 (8) 1367-1369.1997. In addition, this strain is Biosci. Biotech. Bioche
m. , 61 (8) 1367-1369.1997, and obtained from the Liquor Research Institute together with the pUSAR plasmid.

(3) Culture The transformant was cultured using a 10 L jar fermenter (BMS10-PI / Biot). The cells were cultured in a medium (5% yeast extract, 2% high-new AM, 5% sun malt) under the conditions of a medium volume of 7.0 L, a stirring speed of 600 rpm, a temperature of 30 ° C., and an aeration rate of 1.0 vvm. When the GDH activity was confirmed using this culture solution as a crude enzyme solution, it was confirmed that the GDH of the present invention was expressed. GDH activity was not observed in the host before transformation.

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

  The FGDH of the present invention is excellent in substrate specificity and makes it possible to measure the amount of glucose more accurately. Therefore, it can be said that the FGDH of the present invention is suitable for measurement of blood glucose level.

Claims (8)

SDS- polyacrylamide electrophoresis molecular weight measured by gel is about 69 kDa, or less Km value of about 10mM for D- glucose, flavin-binding glucose dehydrogenase consisting of the polypeptides of the lower Symbol (b);
( B) a polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having glucose dehydrogenase activity. Homology with the nucleotide sequence represented by SEQ ID NO: 2 is the nucleotide sequence is 90% or more, and has a glucose dehydrogenase activity, Km value for D- glucose is about 10mM or less, SDS- molecular weight measured by polyacrylamide electrophoresis of about 6 9 kDa, DNA encoding the polypeptide.   A vector incorporating the DNA according to claim 2.   A transformant comprising the vector according to claim 3.   The method for producing a flavin-binding glucose dehydrogenase according to claim 1, comprising culturing the transformant according to claim 4. 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.

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