JP2014180223A - Aldohexose dehydrogenase and production method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel aldohexose dehydrogenase (AHDH) with excellent character and to provide a technique using it.SOLUTION: The invention relates to a NAD-dependent aldohexose dehydrogenase which is a polypeptide of following (a) or (b): (a) a polypeptide consisting of an amino acid sequence which has an identity of not less than 60% to a specific amino acid sequence and has an AHDH activity; (b) a polypeptide consisting of an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, added and/or inverted in the amino acid sequence and has an AHDH activity.

Description

  The present invention relates to a novel aldohexose dehydrogenase, a method for producing the same, and a technique for applying the enzyme.

Aldohexose dehydrogenase (EC 1.1.1.119) (hereinafter sometimes referred to as “AHDH”) catalyzes the reversible oxidation reaction of D-aldohexose and its analogs, as shown in the following formula: .
D-aldohexose + NAD (P) → D-hexonic acid-δ-lactone + NAD (P) H + H ⁺
(Here, NAD represents oxidized nicotinamide adenine dinucleotide, NADH represents reduced nicotinamide adenine dinucleotide, NADP represents oxidized nicotinamide adenine dinucleotide phosphate, and NADPH represents reduced nicotinamide adenine dinucleotide. (Indicates nucleotide phosphate.)

  Aldohexose includes D-mannose. D-mannose is mainly used in the living body for synthesizing sugar chains such as glycoproteins and glycolipids, and plays an important role in the biosynthesis and secretion of glycoproteins and the functional expression of glycoproteins. D-mannose is present in human blood. For example, it is suggested that D-mannose is useful for diagnosis of various diseases because the blood concentration is increased in patients with diabetes or fungal infections with poor glycemic control ( Patent Documents 1 and 2).

  AHDH has been clarified in several bacteria. For example, Non-patent Document 1 describes NADP-dependent AHDH derived from Gluconobacter cerinus. Moreover, although the detailed classification of genus species is unknown, NAD-dependent AHDH derived from a microorganism called Pseudomonad MSU-1 is described in Non-Patent Document 2. It is also known that NAD-dependent AHDH exists in Thermoplasma acidophilum, which is a thermoacidophilic archaea (Non-patent Document 3).

  By the way, since NAD is several times cheaper as a reagent than NADP, NAD-dependent NAD-dependent AHDH derived from Pseudomonad MSU-1 and Thermoplasma acid phyllum, which are NAD-dependent, are more industrially available preferable. However, Pseudomonad MSU-1-derived NAD-dependent AHDH has a half-life of 40 seconds when heat-treated at 55 ° C., and is not necessarily suitable for industrial use where enzyme stability is required.

JP2004-81045A JP2004-33077A

Gad Avigad, Yair Alloy, and Sasa England J. et al. Biol. Chem. 243 (8) 1968 pp 1936-1941 A. Stephen Dahms and Richard L. Anderson J.M. Biol. Chem. 1972 247: 2222-2227. Nishiya et al. , Bioscience, Biotechnology, and Biochemistry Vol. 68 (2004) No. 12P 2451-2456

  An object of the present invention is to provide a new AHDH having more excellent characteristics in relation to the use of the AHDH described above and a technology using the same.

  As a result of repeated studies day and night to solve the above problems, the present inventors have unexpectedly succeeded in isolating completely new AHDH from Thermoplasma volcanium, which is a thermophilic archaea. And when the characteristic of this AHDH was investigated, it discovered that it had the characteristic (for example, pH stability and thermal stability) excellent in industry. Based on this knowledge and further discovery, the present inventors have completed the present invention.

A typical present invention is as follows.
Item 1.
NAD-dependent aldohexose dehydrogenase, which is a polypeptide of the following (a) or (b):
(A) a polypeptide comprising an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 1 and having an aldhexose dehydrogenase activity (b) one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1 Is a polypeptide comprising an amino acid sequence substituted, deleted, inserted, added and / or inverted, and having aldohexose dehydrogenase activity.
Item 2.
Item 2. The aldohexose dehydrogenase according to Item 1, which is stable at pH 4.5 to 11.
Item 3.
Item 3. The aldohexose dehydrogenase according to Item 1 or 2, which is stable at a temperature of 70 ° C. or lower.
Item 4.
The aldohexose dehydrogenase according to any one of Items 1 to 3, further having one or more of the following properties (A) to (C):
(A) Optimum pH: 6.9 to 9.7
(B) Km value for D-mannose: 20-50 mM
(C) The specific activity using D-mannose as a substrate is 15 U / mg or more.
Item 5.
Item 5. A polynucleotide encoding the aldohexose dehydrogenase according to any one of Items 1 to 4.
Item 6.
Item 6. The polynucleotide according to Item 5, which is any of the following (c) to (f).
(C) a polynucleotide comprising the base sequence of SEQ ID NO: 2;
(D) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1;
(E) a polynucleotide comprising a base sequence having an identity with the base sequence of SEQ ID NO: 2 of 80% or more and encoding a polypeptide having aldhexose dehydrogenase activity;
(F) A polynucleotide 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 aldhexose dehydrogenase activity.
Item 7.
Item 7. A vector comprising the polynucleotide according to Item 5 or 6.
Item 8.
Item 8. A transformant comprising the vector according to item 7.
Item 9.
Item 10. A method for producing aldhexose dehydrogenase, comprising the step of culturing the transformant according to Item 8, and the step of purifying the obtained culture broth.
Item 10.
Item 5. A method for measuring D-mannose concentration, which comprises the step of contacting the aldohexose dehydrogenase according to any one of Items 1 to 4 with a sample containing D-mannose.
Item 11.
Item 5. A product comprising the aldhexose dehydrogenase according to any one of Items 1 to 4.
Item 12.
Item 5. A sensor for measuring D-mannose concentration, comprising the aldohexose dehydrogenase according to any one of Items 1 to 4.

  Since AHDH of the present invention can use D-mannose as a main substrate, it can be used to measure the D-mannose concentration of a sample (for example, blood). In addition, since AHDH of the present invention can also use glucose and / or xylose as a substrate, it can be used to measure these concentrations. Furthermore, the AHDH of the present invention can be used in any application for dehydrogenating the sugars. The AHDH of the present invention can be excellent in pH stability. For example, high activity can be maintained in the range of pH 4.5-11. Therefore, it is suitable for use in such a wide pH environment, and is also suitable for use in combination with a mediator (for example, ferricyanide) desired to be used in the range of pH 4.5-6. Since the AHDH of the present invention can be excellent in temperature stability, it is suitable for measurement of sugar concentration in a high temperature environment and production of a sensor for measurement.

The result of SDS-PAGE of typical AHDH of the present invention is shown. The result of having measured the activity with respect to various saccharide | sugar of typical AHDH of this invention is shown. The result of having measured the optimum pH of typical AHDH of this invention is shown. The result of having measured the temperature stability of typical AHDH of this invention is shown. The result of having measured pH stability of typical AHDH of this invention is shown. The activity value measured by changing the D-mannose concentration of representative AHDH of the present invention is shown. The relationship of the reaction rate of typical AHDH of this invention and D-glucose concentration is shown. The relationship between the reaction rate of typical AHDH of the present invention and the NAD concentration is shown.

Hereinafter, the present invention will be described in detail.
1. Aldohexose dehydrogenase activity The polypeptide of the present invention is preferably the following (a) or (b):
(A) a polypeptide comprising an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 1 and having aldohexose dehydrogenase activity;
(B) A polypeptide comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, added and / or inverted in the amino acid sequence of SEQ ID NO: 1 and having an aldhexose dehydrogenase activity.

  Aldohexose dehydrogenase activity means an activity that catalyzes a reaction that oxidizes a hydroxyl group of aldohexose in the presence of an electron acceptor. In this document, unless otherwise specified, “enzyme activity”, “activity” or “AHDH activity” means aldhexose dehydrogenase activity. Aldohexose means any monosaccharide of 6 carbon atoms having four chiral centers, such as D-mannose, D-glucose, D-galactose, D-allose, D-altrose, D-talose. , D-growth and D-idose. From the viewpoint that it can be used as a diagnostic marker for diabetes and fungal infections, preferred aldohexoses are D-mannose and D-glucose. In one embodiment, the aldohexose in the present invention may be D-mannose.

  The electron acceptor (mediator) is not particularly limited as long as it is capable of transferring electrons in the dehydrogenation reaction of aldohexose with the polypeptide of the present invention. For example, 2,6-dichlorophenol indophenol (DCPIP), phenazine methosulfate (PMS), 1-methoxy-5-methylphenadium methyl sulfate, ferricyanide, and the like can be used. From the viewpoint of being relatively inexpensive and highly versatile, a preferable electron acceptor is a ferricyanide, and examples thereof include a ferricyanide salt, particularly potassium ferricyanide.

2. Method for Measuring AHDH Activity AHDH activity can be measured by various known methods. In this document, AHDH activity is performed according to the following method unless otherwise specified. In addition, although D-mannose is described here as aldohexose, other aldohexoses (for example, lactose, maltose, and sucrose) can be measured by replacing D-mannose.
<Reagent>
100 mM Tris-HCl buffer pH 8.0
80 mg / mL NAD ⁺ aqueous solution 1.5 M D-mannose aqueous solution enzyme diluted solution 20 mM potassium phosphate buffer pH 7.0 containing 0.1% Triton X and 0.3 M NaCl

<Procedure 1>
The AHDH solution is diluted to 0.8 to 1.2 U / mL with the above enzyme-diluted solution that has been ice-cooled in advance, and the solution that has been ice-cooled is used as the enzyme solution.

<Procedure 2>
2.6 mL of the above Tris-HCl buffer solution, 0.3 mL of D-mannose aqueous solution, and 0.1 mL of NAD ⁺ aqueous solution are mixed, and preheated at 37 ° C. for 5 minutes is used as a reaction mixture.
<Measurement conditions>
After adding 0.05 mL of the enzyme solution to 3.0 mL of the reaction mixture and mixing gently, the absorbance change at 340 nm was changed 2 to 3 with a spectrophotometer (optical path length 1.0 cm) controlled at 37 ° C. with water as a control. Record for minutes, and then measure the change in absorbance per minute (ΔODTEST) from the linear portion (ie, after the reaction rate is constant). In the blind test, 0.05 mL of enzyme diluted solution is added instead of the enzyme solution, and the change in absorbance per minute (ΔODBLANK) is measured in the same manner. From these values, AHDH activity is determined according to the following formula. Here, 1 unit (U) in AHDH activity is the amount of enzyme that produces NADH in 1 minute under the above measurement conditions.

AHDH activity (U / mL) = [(ΔOD _TEST− ΔOD _BLANK ) × 3.05 × dilution ratio]
/(6.22×1.0×0.05)
The numerical values described in the above formula mean the following.
3.05: Volume after mixing AHDH solution (mL)
6.22: NADH molecular extinction coefficient of NADH (cm ² / micromol)
1.0: Optical path length (cm)
0.05: Amount of AHDH solution to be added (mL)
It is.

3. Amino Acid Sequence The amino acid sequence shown in SEQ ID NO: 1 is isolated from Thermoplasma volcanium GSS1 strain (hereinafter sometimes simply referred to as “Tv strain”), which is a thermophilic archaea, as shown in the Examples described later. 2 is an amino acid sequence of a polypeptide having aldohexose dehydrogenase activity. The polypeptide of the present invention comprises an amino acid sequence having a certain identity or more with the amino acid sequence of SEQ ID NO: 1, and has an aldhexose dehydrogenase activity (and preferably at least one of the characteristics shown in the following 4 to 10). It is preferable to have. The degree of identity is, for example, 60% or more, preferably 70% or more, more preferably 80%, still more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, particularly preferably. Is 99% or more. Such a polypeptide comprising an amino acid sequence having a certain identity or more with the amino acid sequence of SEQ ID NO: 1 can be prepared, for example, using a genetic engineering technique as described later.

  As long as the polypeptide of the present invention retains aldohexose dehydrogenase activity, one or several amino acid residues are substituted, deleted, inserted, added and / or reversed in the amino acid shown in SEQ ID NO: 1. It may be a polypeptide having an amino acid sequence (hereinafter sometimes collectively referred to as “mutation”). Here, “several” is not limited as long as the aldhexose dehydrogenase activity is maintained. For example, 2 to 100, preferably 2 to 80, more preferably 2 to 60, and still more preferably 2 to 50. The number is more preferably 2 to 30, more preferably 2 to 20, 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 used from the viewpoint of not significantly affecting the phenotype of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1. Preferably there is. “Conservative amino acid substitution” refers to substitution of an amino acid residue with an amino acid residue having a side chain of similar properties. The amino acid residue is a basic side chain (for example, lysine, arginine, histidine), an acidic side chain (for example, aspartic acid, glutamic acid), an uncharged polar side chain (for example, glycine, asparagine, glutamine, serine, Threonine, tyrosine, cysteine), nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), 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. As used herein, “conservative amino acid substitution” means substitution of amino acids within the same family exemplified above.

  One or several mutations may be artificially introduced or may exist originally. In the case of artificial introduction, it can be performed by any method. For example, it can be introduced using restriction enzyme treatment, treatment with exonuclease or DNA ligase, position-directed mutagenesis, random mutagenesis, ultraviolet irradiation and the like. Variant aldohexose dehydrogenase into which one or several mutations are introduced includes naturally occurring variants (for example, single nucleotide polymorphisms) based on individual differences in organisms, species or genera. The region for introducing the mutation is not particularly limited, but it is preferably introduced in a region that does not correspond to the catalytic site and / or the substrate binding site from the viewpoint of not negatively affecting the activity.

  Amino acid sequence identity can be calculated using analysis tools that are commercially available or available through telecommunication lines (Internet), preferably the National Biotechnology Information Center (NCBI) homology algorithm BLAST. (Basic local alignment search tool) http: // www. ncbi. nlm. nih. It is possible to calculate by using default (initial setting) parameters in gov / BLAST /.

4). pH Stability The polypeptide of the present invention is preferably stable at least in the range of pH 4.5-11. In this specification, stable under a specific pH condition means that the residual enzyme activity after treatment of 0.5 mg / mL enzyme at 25 ° C. for 19 hours is 80% compared to the enzyme activity before treatment. % Or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and particularly preferably substantially 100 is maintained.

5. Temperature stability It is preferable that the polypeptide of this invention is stable in the temperature range of 70 degrees C or less (0-70 degreeC) at least. Here, being stable under a specific temperature condition means that 1 mg / mL of purified enzyme is maintained for 15 minutes in an appropriate buffer (for example, potassium phosphate buffer, pH 7.0) set at a specific temperature. This means that the residual enzyme activity after the treatment is 80% or more, preferably 90% or more, more preferably 95% or more, compared with the enzyme activity before the treatment.

6). PH optimum
The polypeptide of the present invention preferably has an optimum pH of at least 6.9 to 9.7. The optimum pH of 6.9 to 9.7 means that when the AHDH activity is measured at an interval where the range of the pH value does not exceed 1 in this range, the pH showing the highest activity value in the range is shown. The relative activity (%) when the activity is 100 (%) is 80% or more, preferably 85% or more, more preferably 95% or more. The optimum pH can be measured using any buffer, but the following buffers are preferably used: pH 4.0 to 5.7 (acetate buffer), pH 5.8 to 7.7 (phosphorus). Acid potassium buffer), pH 7.3-8.8 (Tris-HCl buffer), pH 8.3-9.7 (Glycine-NaOH buffer). For example, the polypeptide of the present invention preferably exhibits a relative activity of 80% or more at least in the range of pH 6.9 to 9.7, with the activity at pH 9.7 (glycine hydrochloride buffer) being 100%. As shown in the examples described later, AHDH derived from the Tv strain exhibits the highest activity at pH 9.7 (glycine hydrochloride buffer), and pH 6.9 to 9.7 including pH 7.3 to 8.8. It is included in the optimum pH range.

7). Km value for D-mannose When the polypeptide of the present invention uses NAD as a coenzyme, the Km value for D-mannose is preferably 100 mM or less, more preferably 50 mM or less. On the other hand, the lower limit of the Km value for D-mannose when NAD of the polypeptide of the present invention is used as a coenzyme is not particularly limited. For example, the preferable lower limit is 20 mM, more preferably 30 mM, and still more preferably. 40 mM.

8). Substrate specificity The polypeptide of the present invention is excellent in substrate specificity for D-mannose. While many conventionally known sugar dehydrogenases have a high substrate specificity for D-glucose and a relatively low substrate specificity for D-mannose, the polypeptides of the present invention are directed against D-glucose. The reactivity may be 10 times or more higher than the reactivity.

9. Specific activity The polypeptide of the present invention preferably has a high specific activity when reacted with D-mannose using nicotinamide adenine dinucleotide (NAD) as a coenzyme. The specific activity can be determined by measuring the enzyme activity when the polypeptide is in a solid form (including powder form), with the solid weight regarded as the protein weight. When the polypeptide is in a liquid form, the protein weight is measured using, for example, absorbance at A280 nm or a protein determination reagent (trade name: Bio-Rad Protein Assay Concentrated Dye Reagent) manufactured by Bio-Rad, and enzyme activity is measured. Can be obtained. If the polypeptide is solid and appears to contain components other than AHDH, the specific activity can be determined by dissolving it in an appropriate solution and handling it as a liquid preparation.

  The specific activity of the polypeptide of the present invention is preferably 5 (U / mg) or more, more preferably 10 (U / mg) or more, still more preferably 12 (U / mg) or more, and even more preferably 15 (U / Mg) or more. The upper limit of the specific activity of the polypeptide of the present invention is not particularly limited, but is preferably 50 (U / mg) or less, more preferably 40 (U / mg) or less, still more preferably 35 (U / mg) or less, and even more. Preferably it is 30 (U / mg) or less. The purified preparation of the polypeptide of the present invention may be liquid or solid (including powder).

10. Molecular Weight of Subunit The molecular weight of the monomer of the polypeptide of the present invention is preferably about 27000 to 33000 as measured by SDS-PAGE. “About 27000 to 33000” means that a range of positions that a person skilled in the art normally determines that there is a band at a position of 27000 to 33000 when the molecular weight is measured by SDS-PAGE. “Polypeptide moiety” means a polypeptide that is substantially free of sugar chains. When the polypeptide of the present invention produced by a microorganism is a sugar chain-bound type, it is treated with heat treatment or sugar hydrolase to remove the sugar chain (ie, “polypeptide moiety”). can do. The state in which the sugar chain is not substantially bound allows the presence of sugar chains inevitably remaining in the sugar chain-binding polypeptide treated by heat treatment or sugar hydrolase. Therefore, when the polypeptide is not inherently sugar chain-linked, it itself corresponds to the “polypeptide moiety”. 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.

11. Origin The origin of the polypeptide of the present invention is not particularly limited. Examples of the organism from which the polypeptide 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 in various animals and plants, and the like. Isolation sources may be those derived from 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.

  Typically, the polypeptides of the present invention are derived from microorganisms. Examples of the microorganism include, for example, hyperthermophilic protozoa, and specifically include the genus Pyrodictim, the genus Sulfolobus, the genus Desulfurococcus, and the genus Thermoproteus. , Genus Thermofilum, genus Thermoplasma and the like. A preferred microorganism is a genus Thermoplasma, more preferably Thermoplasma acidophilum, and still more preferably Thermoplasma volcanium.

  The polypeptide of the present invention includes not only those isolated directly from microorganisms but also those obtained by modifying the isolated polypeptide by a protein engineering method and those modified by a genetic engineering method. For example, it may be a modified enzyme obtained from the aforementioned microorganisms classified into the genus Thermoplasma.

  The polypeptide of the present invention preferably satisfies at least one of the above properties 4 to 10, more preferably at least 2, more preferably at least 3, even more preferably at least 4, and even more preferably at least It is preferred to satisfy 5, more preferably at least 6. When two or more of the characteristics 4 to 10 are satisfied, the combination is arbitrary, but preferably includes the pH stability of 4 and the temperature stability of 5.

12 Polynucleotide The polynucleotide of the present invention preferably encodes the above polypeptide. Examples of such a polynucleotide include any of the following (c) to (g):
(C) a polynucleotide comprising the base sequence of SEQ ID NO: 2;
(D) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1;
(E) a polynucleotide comprising a base sequence having an identity with the base sequence of SEQ ID NO: 2 of 80% or more and encoding a polypeptide having aldhexose dehydrogenase activity;
(F) A polynucleotide 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 aldhexose dehydrogenase activity.

  As used herein, “polynucleotide encoding a polypeptide” means a polynucleotide from which the polypeptide is obtained when it is expressed, that is, a polynucleotide having a base sequence corresponding to the amino acid sequence of the polypeptide. . Thus, polynucleotides that differ due to codon degeneracy are also included.

  The polynucleotide consisting of the base sequence of SEQ ID NO: 2 encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 derived from the Tv strain, as shown in the above-mentioned Examples. The polynucleotide of the present invention has the nucleotide sequence of SEQ ID NO: 2 and an arbitrary one as long as the polypeptide encoded by the polynucleotide has aldohexose dehydrogenase activity (and preferably at least one of the characteristics shown in the above 4 to 10). Preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, particularly preferably 99% or more. Have identity.

  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, it is possible to calculate a base sequence identity value (%).

  In another embodiment, the polynucleotide of the invention has an aldhexose dehydrogenase activity (and preferably at least one of the properties shown in 4-10 above), and the polypeptide that it encodes is shown in SEQ ID NO: 2. It is preferably a polynucleotide that hybridizes under stringent conditions with a base sequence complementary to the base sequence. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.

  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% dextran. Incubate at about 42 ° C. to about 50 ° C. with sulfuric acid, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5), and then with 0.1 × SSC, 0.1% SDS The conditions for washing at about 65 ° C. to about 70 ° C. can be mentioned. More preferable stringent conditions include, for example, 50% formamide, 5 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 1 × Denhardt solution, 1% SDS, 10% dextran sulfate as a hybridization solution. Examples include conditions using 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)). The stringent conditions are preferably so-called highly stringent conditions.

  Polynucleotides that hybridize 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 expression vectors. It can be easily removed by expressing it in a suitable host and measuring the enzyme activity by a known method.

  The polynucleotide of the present invention is preferably present in an isolated state. Here, the “isolated polynucleotide” means a state separated from other components such as nucleic acids and proteins that coexist in the natural state. However, 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) may be included. 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, chemicals used in the synthesis process, and the like. The polynucleotide of the present invention also includes DNA (cDNA) complementary to the above DNAs (c) to (g). Further, the polynucleotide of the present invention may be either DNA or RNA.

  The polynucleotide of the present invention can be obtained by any method based on the information in this specification or the attached sequence listing (particularly, SEQ ID NO: 2). For example, it can be easily prepared by using a chemical nucleic acid synthesis method, a genetic engineering method, a molecular biological method, a biochemical method, or the like. As a chemical nucleic acid synthesis method, a solid phase synthesis method by a phosphoramidite method can be exemplified. This synthesis method can be performed using an automatic synthesizer.

  As a genetic engineering technique, a cDNA library is prepared according to a conventional method from an appropriate source microorganism in which the polypeptide of the present invention is expressed, and the DNA sequence of the present invention (for example, the base of SEQ ID NO: 2) is prepared from the library. This can be carried out by selecting a desired clone using an appropriate probe or antibody peculiar to (sequence). The microorganism for preparing the cDNA library is not particularly limited as long as it is a microorganism that expresses the polypeptide of the present invention, and preferably includes the microorganism described in the above-mentioned “Derivation” section.

  Separation of total RNA from microorganisms, separation and purification of mRNA, acquisition and cloning of cDNA, determination of base sequence, 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 the polynucleotide, 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, it is preferable to employ the RACE method. Primers used when adopting the PCR method can also be designed and synthesized as appropriate based on the nucleotide sequence of SEQ ID NO: 2. In addition, isolation and purification of the amplified DNA or RNA fragment can be performed according to a conventional method as described above, and can be performed using, for example, gel electrophoresis, hybridization, or the like.

  By using the polynucleotide of the present invention, the polypeptide of the present invention can be produced easily, efficiently and stably.

13. Vector The vector of the present invention is a recombinant vector into which a polynucleotide encoding the polypeptide of the present invention described in the above “Polynucleotide” section 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 polynucleotide of the present invention in an appropriate host cell. As long as the expression is possible, the type and structure are not particularly limited. 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 modified product thereof, λ phage or a modified product thereof, pBR322 or a modified product 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 using standard recombinant DNA techniques (for example, Molecular Cloning, Third Edition, 1.84). , Cold Spring Harbor Laboratory Press, New York).

14 Transformant The transformant of the present invention is preferably a transformant introduced into any host cell in a state where the polynucleotide of the present invention can be expressed. The means for introducing the polynucleotide of the present invention into the host is not particularly limited. For example, the polynucleotide is introduced into the host in a state of being incorporated into the vector described in the above “Vector” section. The host cell is not particularly limited as long as it can express the polynucleotide of the present invention and produce the polypeptide of the present invention. Specifically, prokaryotic cells such as Escherichia coli and Bacillus subtilis, eukaryotic cells such as yeast, mold, insect cells, and mammalian cells can be used. Of these, Escherichia coli, Bacillus subtilis and filamentous fungi are preferable, and Escherichia coli is more preferable.

  When the host is Escherichia coli, a K-12-derived strain is particularly preferable, and BL21 (DE3), BB4, BM25.5, BMH71-18mutS, BW313, C-la, C600, CJ236, DH1, DH5, DH5α, DH10B, DP50supF, ED8654, ED8767, ER1647, HB101, HMS174, HST02, HST04dam- / dcm-, HST08 Premium, JM83, JM101, JM105, JM106, JM107, JM108, JM109, JM110, K802, K803, LE392, MC1061, 194118, M1061, 194118, M1381, 1938 Use RR1, TAP90, TG1, TG2, TH2, XL1-Blue, Χ-1776, γ-1088, γ-1089, γ-1090, etc. Can do. In this case, M13 phage or a modified form thereof, λ phage or a modified form thereof, pBR322 or a modified form thereof (pB325, pAT153, pUC8, etc.), pUC19, pUC57, pBluescript, pET22b, pUC18, pHSG398, pHSG399, pRIT2T , PUEX1 to 3, pKK223-3, pINIII1, pTTQ18, pGEMEX-1, pGH-L9, pKK233-2, and the like.

  When the host is Bacillus subtilis, examples include Bacillus subtilis, Brevibacillus brevis, Brevibacillus choshinensis, and the like. Examples of the vector in this case include pTB53 or a modified form thereof, pHY300PLK or a modified form thereof, pAL10, pAL12, pHT01, pHT08, pHT09, pHT10, pHT43, pNY326, pNCMO2, and the like.

  When the host is yeast, the genus Saccharomyces, Schizosaccharomyces, Candida, Pichia, Cryptococcus and the like can be mentioned. Specific hosts belonging to these genera include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Pichia pastoris, and Cryptococcus sp. Examples of the vector in this case include pYepSec1, pMFa, pAUR101, pAUR224, and pYE32.

  When an insect cell is used as a host, examples of the vector include pAc and pVL. When a mammalian cell is used as a host, for example, pCDM8, pMT2PC, etc. can be used as a vector.

  When the host is a filamentous fungal cell, examples include Aspergillus genus, Trichoderma genus, Coretotricum genus, and the like, and examples thereof include Aspergillus oryzae, Aspergillus niger, Trichoderma reesei, and Choletotricum hyaeris.

  In the transformant, exogenous DNA is usually present in the host cell, but a transformant obtained by so-called self-cloning using the microorganism from which the DNA is derived as a host is also a preferred embodiment.

  The transformant of the present invention is preferably prepared by transfection or transformation using the expression vector shown above. 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 polypeptide of the present invention, it can be used to efficiently produce the polypeptide of the present invention.

15. Method for Producing Polypeptide The polypeptide of the present invention is produced by culturing a microorganism having the ability to produce the polypeptide of the present invention. The microorganism to be used for the culture is not particularly limited as long as it has the ability to produce the polypeptide of the present invention. For example, the microorganism is a wild-type microorganism shown in the above-mentioned “derived” section and the above-mentioned “transformant” section. A transformant can be preferably used.

  The culture method and culture conditions are not particularly limited as long as the polypeptide of the present invention is produced. That is, on the condition that the polypeptide of the present invention 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, phosphate salts, manganese salts and iron salts can be used. Vitamins, amino acids and the like can be added to the medium to promote the growth of the microorganisms used.

  Commercially available LB medium (Luria-Bertai Medium), M9 medium (M9 Minimal Medium), NZCYM medium (NZCYM Medium), NZYM medium (NZYM Medium), NZM medium (NZM Medium, B medium) (Terrific Broth), 2XYT medium (2XYT Medium) can be used.

  When the microorganism of the present invention is cultured to produce the polypeptide of the present invention, the 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 4 to 9, preferably about 6 to 8, 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 about 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 the polypeptide of the present invention from the culture solution or the bacterial cells. When using a microorganism that secretes the polypeptide outside the cell body, for example, the culture supernatant is filtered, centrifuged, etc. to remove insolubles, then concentrated with an ultrafiltration membrane, salting out such as ammonium sulfate precipitation, dialysis, etc. The present enzyme can be obtained by separating and purifying by appropriately combining various chromatographies.

  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 enzyme can be obtained by adding a chelating agent and a surfactant to solubilize the polypeptide, 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).

  Collection (extraction, purification, etc.) of a protein having AHDH activity from the culture solution can be performed using any one or more of AHDH activity, thermal stability, and the like as an index.

  In each purification step, it is preferable to perform fractionation using AHDH activity as an index and proceed 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 obtaining the polypeptide of the present invention as a recombinant protein, various modifications are possible. For example, when a polynucleotide encoding the polypeptide of the present invention and another appropriate polynucleotide are inserted into the same vector and a recombinant protein is produced using the vector, any peptide or polypeptide is linked. The polypeptide of the present invention which is a recombinant protein 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.

16. Product The product of the present invention contains the polypeptide of the present invention. In this document, “product” means a product that constitutes a part or all of one set used by a user for the purpose of executing a certain application and includes the AHDH of the present invention. For example, the product of the present invention may be in the form of a composition or sensor comprising the polypeptide of the present invention.

The product of the present invention can be applied to various uses and is not particularly limited, but typically, one using one of the following two principles can be exemplified.
(I) Measure the amount (concentration) of a substrate such as D-mannose by AHDH.
(II) To generate an electric current by an enzymatic reaction with AHDH.

  Examples of using the principle of (I) above include uses for in-vitro diagnosis (for example, measurement of sugar used as a substrate by AHDH). For example, a method for measuring D-mannose, which is a monosaccharide, has already been established in the art. Therefore, according to a known method, the amount or concentration of D-mannose can be measured using the AHDH of the present invention.

  Hereinafter, a case where D-mannose is measured will be described as an example.

  The embodiment is not particularly limited as long as the concentration or amount of D-mannose is measured using the AHDH of the present invention. For example, D-mannose can be produced with the aid of D-mannose or any other conjugating enzyme. Various forms such as reagents, kits, and sensors for measuring biological components such as substances (for example, fructose capable of producing D-mannose by allowing D-mannose isomerase to act as a conjugate enzyme) can be exemplified.

  In the case of the composition for measuring D-mannose, NADH generated by the AHDH reaction reduces the electron acceptor such as DCPIP through AHDH and returns itself to NAD, resulting in a change in the structure of DCPIP. The concentration of D-mannose can be determined by colorimetric determination of the difference. The sample containing D-mannose is not particularly limited, and examples thereof include blood, beverages, and foods.

  The composition for measuring D-mannose may be in the form of a kit. The kit contains, for example, the polypeptide of the present invention in an amount sufficient for at least one assay, and typically includes a buffer, mediator, and D-mannose standard solution for creating a calibration curve necessary for the assay. As well as directions for use. The AHDH 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.

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

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

  Examples of using the principle of (II) include various forms such as an enzyme electrode (may be an immobilized electrode), an enzyme sensor, a fuel cell, and an electronic device having one or a plurality of fuel cells. It can be illustrated.

  Fuel cells that extract electrons by the oxidation reaction of D-mannose using AHDH have already been established in the art. Therefore, according to a known method, a fuel cell can be manufactured and operated using the AHDH of the present invention. The mode of the fuel cell is not particularly limited. For example, the fuel cell can be operated as a battery by the following means. First, AHDH of the present invention is immobilized together with an electron mediator such as AHDH or an osmium complex in a negative electrode of a biofuel cell. On the other hand, in the positive electrode, an oxidoreductase selected from bilirubin oxidase (BOD), laccase, ascorbate oxidase and the like and a mediator such as hexacyanoferrate ion are immobilized. Furthermore, a structure in which the negative electrode and the positive electrode are opposed to each other via an electrolyte layer that does not have electron conductivity and conducts only protons, and in the negative electrode, D-mannose supplied as fuel is decomposed by the polypeptide of the present invention to generate electrons. And protons (H +) are generated. In the positive electrode, water is generated by protons transported from the negative electrode through the electrolyte layer, electrons sent from the negative electrode through an external circuit, and oxygen in the air, for example. The fuel containing D-mannose is not particularly limited, and examples thereof include blood, beverages, and foods.

  The fuel cell of the present invention can be used for any application as long as it requires electric power, and the size is not limited. Specifically, this fuel cell is used for, for example, an electronic device, a moving body (automobile, motorcycle, aircraft, rocket, spacecraft, etc.), power unit, construction machine, machine tool, power generation system, cogeneration system, etc. Can do.

Hereinafter, although this invention is demonstrated with reference to an Example and a test example, this invention is not limited to these.

Example 1 Cloning of Thermoplasma volcanium-derived AHDH Thermoplasma volcanium GSS1 strain was obtained from yeast extract (Difco) 1 g / L, glucose 10 g / L, (NH ₄ ) ₂ SO ₄ 1.32 g / L, KH ₂ PO ₄ . 372 g / L, MgSO ₄ · 7H ₂ O 0.247 g / L, CaCl ₂ · 2H ₂ O 0.074 g / L, 10 mL / L trace element solution (FeCl ₃ · 6H ₂ O 1.93 g / L, MnCl ₂ 4H ₂ O 0.18 g / L, Na ₂ B ₄ O ₇ · 10H ₂ O 0.45 g / L, ZnSO ₄ · 7H ₂ O 22 mg / L, CuCl ₂ · 2H ₂ O 5 mg / L, NaMoO 4 · 2H ₂ O 3 mg / L, VOSO 4 · 2H ₂ O 3 mg / L, CoSO ₄ · 7H ₂ O 1 m g / L) was added and cultured with shaking at 58 ° C. overnight in a medium adjusted to pH 1.3 with sulfuric acid. Thereafter, the cells were collected by centrifugation (4,000 rpm, 15 minutes). The Tv strain is available from the Center for Biotechnology Technology (NBRC No. 15438).

  The culture solution remaining in the centrifuge tube was wiped off with Kimwipe, and then suspended in 15 mL of a solution containing 20% sucrose, 100 mM Tris-hydrochloric acid (pH 8.0), 50 mM EDTA, 0.1% SDS per gram of cells. 150 μL of protease K solution (10 mg / mL) and 150 μL of RNAse A (10 mg / mL) were added and maintained at 37 ° C. for 12 hours. This was treated with an equal volume of chloroform / phenol solution, and then the operation of separating the aqueous layer by centrifugation was repeated three times.

  After adding 600 μL of 5 M NaCl to the obtained aqueous layer and mixing, 0.8 times the amount of isopropyl alcohol was added, and DNA that appeared after inversion mixing was wound around a glass rod to obtain a purified DNA preparation. This purified DNA was redissolved in a 10 mM Tris-HCl (pH 8.0) solution (hereinafter abbreviated as TE) containing 5 mL of 1 mM EDTA, and 200 μL of 5 M NaCl was added and mixed, and then 0.8 times the amount of isopropyl alcohol. The DNA extracted again was washed with 70% ethanol solution, air-dried, and dissolved in 1 mL TE (Tris-HCl-EDTA) buffer.

  A sense primer consisting of 37 bases shown in SEQ ID NO: 3 and an antisense primer consisting of 34 bases shown in SEQ ID NO: 4 were synthesized. Using this primer set and Pfuturbo DNA polymerase, the DNA obtained in Example 1 was used as a template. PCR was performed in the following cycle.

Step 1: 94 ° C for 1 minute
Step 2: 50 ° C, 1 minute
Step 3: 72 ° C, 2.5 minutes (30 cycles)
When the obtained PCR product was run on a 1% agarose gel, a specific band having a size of 0.78 kb was observed. This DNA fragment was digested with restriction enzymes NdeI and XhoI, and this DNA fragment was also ligated with the ring-opened product of vector pET28 obtained by digesting with NdeI and XhoI. In this way, a recombinant vector pET / Tv was obtained. Escherichia coli XL1 / Blue strain (Stratagene) was transformed with this recombinant vector.

  pET / Tv of about 0. The nucleotide sequence of the 8 kbp inserted DNA was determined using BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and ABI3100 (Applied Biosystems). The determined open reading frame of the nucleotide sequence and the amino acid sequence corresponding thereto are shown in SEQ ID NO: 2 and SEQ ID NO: 1, respectively. The molecular weight of the protein determined from the amino acid sequence was 27000-33000.

Example 2 Production of transformant
A competent cell of Escherichia coli BL21 (DE3) RIL strain (Stratagene) was transformed with pET / Tv to obtain a transformant Escherichia coli BL21 (DE3) RIL (pET / Tv).

Example 3 Production of purified AHDH derived from Tv strain from transformant
Dispense 100 mL of LB medium into a 500 mL flask, perform autoclaving at 121 ° C. for 20 minutes, allow to cool, and add 0.1 mL of 20 mg / mL kanamycin and 34 mg / mL chloramphenicol (Nacalai Tesque), which are separately sterile filtered did. This medium was inoculated with 1 mL of a culture solution of Escherichia coli BL21 (DE3) RIL (pET / Tv) previously cultured in an LB medium with shaking at 37 ° C. for 15 hours, and cultured with aeration and stirring at 37 ° C. for 8 hours.

  Dispense 900 mL of LB medium into a 3 L flask, perform autoclaving at 121 ° C. for 20 minutes, allow to cool, and separately separate 20 mg / mL kanamycin and 34 mg / mL chloramphenicol (manufactured by Nacalai Tesque) 0.9 mL. Added. This medium was inoculated with 100 mL of a culture solution of Escherichia coli BL21 (DE3) RIL (pET / Tv) cultured for 8 hours, and cultured with aeration and stirring at 30 ° C. for 15 hours. The AHDH activity in the cells contained in 1 mL of the culture solution at the end of the culture was about 4 U / mL.

  The cells in the culture were collected by centrifugation and suspended in 50 mM Na phosphate buffer (pH 8.0) containing 300 mM NaCl, 1 mg / ml lysozyme. The cell suspension was crushed with ultrasonic waves and centrifuged to obtain a supernatant. The obtained crude enzyme solution was heat-treated at 60 ° C. for 60 minutes and centrifuged to obtain a supernatant.

  The supernatant was adsorbed on HIS-SELECT (sigma), washed with a buffer containing 50 mM Naphosphate pH 6.0, 300 mM NaCl, 10% glycerol, and then separated and purified with 0-400 mM imidazole linear gradient. went. Next, a fraction containing a large amount of AHDH was collected and dialyzed (25 mM Tris pH 7.5, 20% glycerol) to obtain a purified enzyme preparation. The AHDH preparation obtained by this method showed an almost single band by electrophoresis (SDS-PAGE). At this time, phosphorylase b (97,000 dalton), albumin (66,000 dalton), ovalbumin (45,000 dalton), carbonic anhydrase (30,000 dalton), trypsin inhibitor (20, 100 Dalton) and α-lactalbumin (14,400 Dalton) were used. SDS-PAGE of purified AHDH derived from Tv strain is shown in FIG.

Example 4 Substrate specificity of Tv strain-derived purified AHDH
Using the purified AHDH obtained in Example 3, the activity against various sugars was measured using NAD as a coenzyme. The results are shown in FIG. 2 and Table 1 below. As a result, it was found that this enzyme has high activity against D-mannose and 2-deoxy-D-glucose. Moreover, although it was lower than the activity with respect to these two substrates, it turned out that this enzyme has activity also with D-glucose and reacts also with lactose which is a disaccharide.

Example 5 Optimum pH of Tv strain-derived purified AHDH
The optimum pH for the purified AHDH (2 U / mL) obtained in Example 3 was examined. 100 mM acetate buffer (pH 4.0 to 5.7, plotted with ♦ in the figure), 100 mM potassium phosphate buffer (pH 5.8 to 7.7, plotted with ■ in the figure), 100 mM Tris-HCl buffer ( pH 7.3 to 8.8, plotted with ▲ in the figure), 100 mM glycine-NaOH buffer (8.3 to 9.7, plotted with ● in the figure) were used. At each pH, enzyme reaction was performed at a temperature of 25 ° C., and the relative activities were compared. The results are shown in FIG. The pH range in which the THD strain-derived AHDH exhibits a residual activity of 80% or more was pH 6.9 to 9.7. The pH range showing Ta-derived residual activity of 80% or more was pH 8.4 to 9.7. The pH range of the Tv strain-derived AHDH showing a residual activity of 90% or more was pH 6.9 to 9.7. The pH range for Ta-derived residual activity of 90% or more was pH 7.3 to 9.7. This indicates that Tv strain-derived AHDH has a wide optimum pH range.

Example 6 Thermostability of Tv strain-derived purified AHDH Using the AHDH enzyme solution (1 mg / mL) obtained in Example 3, temperature stability was examined. Using 20 mM potassium phosphate buffer (pH 7.0), the AHDH enzyme solution obtained in Example 3 was subjected to each temperature (4 ° C., 20 ° C., 30 ° C., 40 ° C., 50 ° C., 60 ° C., 70 ° C., After treatment at 80 ° C. and 90 ° C. for 15 minutes, the activity remaining rate was measured. The results are shown in FIG. AHDH derived from the Tv strain had a residual activity of 95.9% at 70 ° C and 23.6% at 80 ° C. From this, it was confirmed that AHDH derived from Tv strain is excellent in thermal stability.

Example 7 pH stability of purified AHDH derived from Tv strain The pH stability of the AHDH enzyme solution (0.5 mg / mL) obtained in Example 3 was examined. 100 mM acetate buffer (pH 3.5-6.0, plotted with ◆ in the figure), 100 mM potassium phosphate buffer, liquid (pH 6.0-pH 8.0: plotted with ■ in the figure), 100 mM Tris-HCl buffer Solution (pH 7.0-pH 9.0: plotted with ▲ in the figure), 100 mM glycine-NaOH buffer solution (pH 9.0-11.0, plotted with ● in the figure) and treated at 25 ° C. for 19 hours. The residual rate of activity was measured. The results are shown in FIG. As a result, it was found that the enzyme activity was maintained at 90% or more at least in the range of pH 4.5 to 11.0 and was stable.

Example 8 Measurement of Km value for D-mannose of purified AHDH derived from Tv strain In the activity measurement method described in 1-1 above, the activity was measured by changing the concentration of D-mannose as a substrate. The measurement results are shown in FIG. A Lineweaver-burk plot was created from FIG. 6, and the Km value was calculated. As a result, it was found that the Km value for D-mannose of Tv strain-derived AHDH was 44 mM.

Example 9 Measurement of Km value for D-glucose Activity of AHDH derived from Tv strain was measured by changing the concentration of D-glucose, and a Lineweaver-burk plot was prepared from the substrate concentration and reaction rate graph (FIG. 7). The Km value was calculated. As a result, it was found that the Km value for D-glucose of Tv strain-derived AHDH was 895 mM. The Km value for D-glucose of AHDH derived from the Ta strain is 208 mM as disclosed in JP-A-2004-33077.

Example 10 Measurement of Km value for NAD For AHDH derived from Tv strain, the activity was measured by changing the concentration of NAD, and a Lineweaver-burk plot was prepared from the graph of substrate concentration and reaction rate (FIG. 8). Was calculated. As a result, it was found that the Km value for NAD of Tv strain-derived AHDH was 0.15 mM.

  The aldohexose dehydrogenase produced according to the present invention can be supplied as a D-mannose measurement reagent, a sensor, a D-mannose concentration determination kit, and a fuel cell material.

Claims (12)

NAD-dependent aldohexose dehydrogenase, which is a polypeptide of the following (a) or (b):
(A) a polypeptide comprising an amino acid sequence having 60% or more identity with the amino acid sequence of SEQ ID NO: 1 and having an aldhexose dehydrogenase activity (b) one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1 Is a polypeptide comprising an amino acid sequence substituted, deleted, inserted, added and / or inverted, and having aldohexose dehydrogenase activity. The aldohexose dehydrogenase according to claim 1, which is stable at pH 4.5-11. The aldohexose dehydrogenase according to claim 1 or 2, which is stable at a temperature of 70 ° C or lower. Furthermore, the aldohexose dehydrogenase in any one of Claims 1-3 which has one or more of the following (A)-(C) characteristics:
(A) Optimum pH: 6.9 to 9.7
(B) Km value for D-mannose: 20-50 mM
(C) The specific activity using D-mannose as a substrate is 15 U / mg or more. A polynucleotide encoding the aldohexose dehydrogenase according to any one of claims 1 to 4. The polynucleotide according to claim 5, which is any of the following (c) to (f).
(C) a polynucleotide comprising the base sequence of SEQ ID NO: 2;
(D) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1;
(E) a polynucleotide comprising a base sequence having an identity with the base sequence of SEQ ID NO: 2 of 80% or more and encoding a polypeptide having aldhexose dehydrogenase activity;
(F) A polynucleotide 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 aldhexose dehydrogenase activity. A vector comprising the polynucleotide according to claim 5 or 6. A transformant comprising the vector according to claim 7. A method for producing aldohexose dehydrogenase, comprising a step of culturing the transformant according to claim 8 and a step of purifying the obtained culture broth. A method for measuring a D-mannose concentration, comprising a step of contacting the aldohoxose dehydrogenase according to any one of claims 1 to 4 with a sample containing D-mannose. A product comprising the aldohexose dehydrogenase according to any one of claims 1 to 4. The sensor for D-mannose density | concentration measurement containing the aldohexose dehydrogenase in any one of Claims 1-4.

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