throbber
PCT/JP2007/063147
`
`DESCRIPTION
`
`FAD-CONJUGATED GLUCOSE DEHYDROGENASE GENE
`
`Technical Field
`
`[0001]
`
`The present invention relates to a novel gene
`
`(polynucleotide) encoding a flavin adenine dinucleotide (FAD)
`
`conjugated glucose dehydrogenase; a process for the production
`
`of the enzyme using a transformant cell transfected with the
`
`gene; a recombinant FAD-conjugated glucose dehydrogenase; and
`
`a method for the determination of glucose, a reagent
`
`composition for use in the determination of glucose, a
`
`biosensor for use in the determination of glucose and others,
`
`each characterized by using the enzyme.
`
`Background Art
`
`[0002]
`
`The blood glucose level is an important marker for
`
`diabetes. As for an examination for diabetes, other than a
`
`clinical examination performed in a hospital laboratory or the
`
`like, a simple determination (point-of-care testing (POCT))
`
`such as a simple examination by a medical staff member or the
`
`like or a self-examination by a patient himself or herself is
`
`performed.
`
`Ikeda-PHC Exhibit 2011
`LifeScan v. Ikeda and PHC
`PGR2019-00032
`
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`[0003]
`
`This simple determination is performed using a glucose
`
`diagnostic kit or a determination device (POCT device) such
`
`as a biosensor, and in such a POCT device, conventionally a
`
`glucose oxidase has been used. However, such a glucose oxidase
`
`is affected by a dissolved oxygen concentration and an error
`
`in the measured value is caused. Therefore, it is recommended
`
`to use of a glucose dehydrogenase which is not affected by
`
`oxygen.
`
`[0004]
`
`Examples of the glucose dehydrogenase include a
`
`coenzyme-unconjugated glucose dehydrogenase which requires
`
`nicotinamide adenine dinucleotide (NAD) or nicotinamide
`
`adenine dinucleotide phosphate (NADP) as a coenzyme and a
`
`coenzyme-conjugated glucose dehydrogenase which requires
`
`pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide
`
`(FAD) or the like as a coenzyme.
`
`Among these, the
`
`coenzyme-conjugated glucose dehydrogenase has advantages that
`
`the enzyme is less likely to be affected by impurities as
`
`compared with the coenzyme-unconjugated glucose dehydrogenase,
`
`the determination sensitivity is high, and further, in
`
`principle, the POCT device can be produced at low cost.
`
`[0005]
`
`However, a conventional PQQ-conjugated glucose
`
`dehydrogenase has low stability and also has a disadvantage
`
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`
`that it reacts also with maltose and galactose. Maltose is
`
`a sugar used in an infusion, and when the PQQ-conjugated glucose
`
`dehydrogenase reacts with maltose, a blood glucose POCT device
`
`displays a higher blood glucose level than the actual value.
`
`Due to this, a patient administers an unnecessary insulin
`
`injection to the patient himself or herself, resulting in the
`
`occurrence of a hypoglycemic event such as impaired
`
`consciousness or comatose states, which has been a big problem.
`
`[0006]
`
`In particular, as for the current use of the blood glucose
`
`POCT device, not only it is used for simply determining the
`
`blood glucose, but importance as a means for self-care and
`
`self-treatment by a patient is increasing and the use of a
`
`self-monitoring of blood glucose (SMBG) device to be used for
`
`the purpose at home is expanding. Therefore, the demand for
`
`determination accuracy is considered to be very high.
`
`[0007]
`
`In fact, an official notice to draw attention about the
`
`use of a blood glucose meter using an enzyme requiring PQQ as
`
`a coenzyme was issued from the Ministry of Health, Labour and
`
`Welfare in Japan in February 2005 to patients under
`
`administration of maltose infusion or dialysate containing
`
`icodextrin (Pharmaceutical and Food Safety Bureau Notice No.
`
`0207005 issued on February 7, 2005, etc.).
`
`[0008]
`
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`On the other hand, as the coenzyme-conjugated glucose
`
`dehydrogenase which catalyzes the dehydrogenation reaction of
`
`glucose and requires FAD as a coenzyme, an Agrobacterium
`
`tumefaciens-derived enzyme (J. Biol. Chem. (1967) 242:
`
`3665-3672), a Cytophaga marinoflava-derived enzyme (Appl.
`
`Biochem. Biotechnol. (1996) 56: 301-310), a Halomonas sp.
`
`a-15-derived enzyme (Enzyme Microb. Technol. (1998) 22:
`
`269-274), an Agaricus bisporus-derived enzyme (Arch.
`
`Microbiol. (1997) 167: 119-125, Appl. Microbiol. Biotechnol.
`
`(1999) 51: 58-64), and a Macrolepiota rhacodes-derived enzyme
`
`(Arch. Microbiol. (2001) 176: 178-186) have been reported.
`
`However, these enzymes oxidize a hydroxy group at the 2- and/or
`
`3-position of glucose, have a high activity for maltose, and
`
`have a low selectivity for glucose.
`
`Further, a
`
`coenzyme-conjugated glucose dehydrogenase derived from
`
`Burkholderia cepacia having a high activity for maltose in the
`
`same manner is also known. However, an original naturally
`
`occurring enzyme is a heterooligomer enzyme comprising three
`
`kinds of subunits: a, l, and y, and is known as a membrane-bound
`
`enzyme. Therefore, there are problems that a lysis treatment
`
`is required for obtaining this enzyme, simultaneous cloning
`
`of a necessary subunit is required for exhibiting a sufficient
`
`activity by cloning, and so on.
`
`[0009]
`
`On the other hand, the present inventors have purified
`
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`
`a novel soluble coenzyme-conjugated glucose dehydrogenase
`
`which requires FAD as a coenzyme and is not a membrane-bound
`
`type from Aspergillus terreus (Patent document 1) . This
`
`coenzyme-conjugated glucose dehydrogenase described in Patent
`
`document 1 has unprecedented excellent properties that it
`
`oxidizes a hydroxy group at the 1-position of glucose, has
`
`excellent substrate (glucose) recognition performance, is not
`
`affected by dissolved oxygen, and also has a low activity for
`
`maltose (the activity for maltose is 5% or less and the activity
`
`for galactose is also 5% or less with the activity for glucose
`
`taken as 100%) .
`
`[0010]
`
`However, the coenzyme-conjugated glucose dehydrogenase
`
`described in Patent document 1 is isolated and extracted from
`
`a liquid culture of a wild-type microorganism (such as a
`
`microorganism belonging to the genus Aspergillus) , and the
`
`production amount thereof is limited. Besides the fact that
`
`the production amount of the enzyme is extremely small, a large
`
`amount of sugars are linked to the enzyme, and the enzyme is
`
`in the form covered with sugars which are different from
`
`N-linked or 0 -linked sugar chains bound to a common enzyme
`
`(which might be called "a sugar-embedded enzyme") . Therefore,
`
`the activity of the enzyme is difficult to detect (the enzymatic
`
`activity is low) , the sugar chains cannot be enzymatically or
`
`chemically removed, and as a result, in electrophoresis, almost
`
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`no staining is achieved by common protein staining (coomassie
`
`brilliant blue G-250 or the like), and also it is difficult
`
`to read amino terminal and internal amino acid sequences of
`
`the enzyme which provide information necessary for acquiring
`
`a gene from the enzyme subjected to a common purification
`
`procedure. Accordingly, it is not publicly known that the
`
`cloning of a gene of this enzyme was successful or the
`
`expression of the activity of this enzyme was confirmed.
`
`[0011]
`
`On the other hand, the existence of a coenzyme-conjugated
`
`glucose dehydrogenase derived from Aspergillus oryzae was
`
`suggested in 1967 (Non-patent document 1). However, only
`
`partial enzymatic properties were revealed, and although a
`
`property that the enzyme does not act on maltose was suggested,
`
`there has been no detailed report with respect to the
`
`coenzyme-conjugated glucose dehydrogenase derived from
`
`Aspergillus oryzae since then, and also there has been no
`
`subsequent report with respect to a coenzyme-conjugated
`
`glucose dehydrogenase derived from other microorganisms or an
`
`enzyme which oxidizes a hydroxy group at the 1-position of
`
`glucose, and also no report with respect to the amino acid
`
`sequence or gene of the coenzyme-conjugated glucose
`
`dehydrogenase has been found at all.
`
`[0012]
`
`Further, an idea of using a glucose dehydrogenase EC 1.
`
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`1. 99. 10 in glucose determination (see Patent document 2) is
`
`known, however, an FAD-conjugated glucose dehydrogenase has
`
`not been produced at a practical level, and the enzyme has not
`
`been actually used in a sensor or put into a practical use.
`
`The reason is considered that the activity of this enzyme in
`
`microbial cells was very low, and even if the enzyme was
`
`secreted to the outside of microbial cells, the amount thereof
`
`was very small, and moreover, the enzyme was covered with a
`
`large amount of sugars, and therefore the activity was low,
`
`and even the detection thereof was difficult, and thus the gene
`
`thereof could not be cloned.
`
`[0013]
`
`Patent document 1: WO 2004/058958
`
`Patent document 2: JP-A-59-25700
`
`Non-patent document 1: Biochem. Biophys. Acta., 139,
`
`277-293, 1967
`
`Disclosure of the Invention
`
`Problems that the Invention is to Solve
`
`[0014]
`
`As genetic engineering methods for modifying a
`
`PQQ-conjugated glucose dehydrogenase, a lot of techniques have
`
`already been known, and these conventional techniques mainly
`
`provide a modified PQQ-conjugated glucose dehydrogenase for
`
`improving disadvantages of the conventional PQQ-conjugated
`
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`
`glucose dehydrogenase such as low substrate specificity and
`
`low stability of the enzyme and a modified genetic material
`
`for producing the
`
`modified PQQ-conjugated glucose
`
`dehydrogenase by genetic engineering.
`
`[0015]
`
`However, in the case of the modified PQQ-conjugated
`
`glucose dehydrogenase produced using the modified genetic
`
`material, the activity for maltose is still more than about
`
`10% with the activity for glucose taken as 100%, or as a result
`
`of decreasing the reactivity to maltose, also the primary
`
`reactivity (specific activity) to glucose is decreased.
`
`Therefore, the function as a glucose sensor is not sufficient
`
`from the viewpoint of the activity determined by an
`
`electrochemical determination method using a sufficient
`
`amount of a substrate, and the current situation is that the
`
`enzyme cannot be used in a POCT device or the like. In addition,
`
`the coenzyme PQQ required for the expression of the activity
`
`of the PQQ-conjugated glucose dehydrogenase has a problem that
`
`it cannot be produced in Escherichia coli which is widely and
`
`generally used as a recombinant host and it is necessary to
`
`produce a recombinant by limiting it to a host microorganism
`
`that produces PQQ (Pseudomonas or the like).
`
`[0016]
`
`Accordingly, an object of the invention is to solve the
`
`above problems and to provide a novel gene (polynucleotide)
`
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`encoding an FAD-conjugated glucose dehydrogenase having
`
`excellent properties that it has excellent reactivity to
`
`glucose, excellent thermal stability, and excellent
`
`substrate-recognition performance and also has a low activity
`
`for maltose; a process for the production of the enzyme using
`
`a transformant cell transfected with the gene; and a method
`
`for the determination of glucose, a reagent composition for
`
`use in the determination of glucose, a biosensor for use in
`
`the determination of glucose and others, each characterized
`
`by using the obtained enzyme.
`
`Means for Solving the Problems
`
`[0017]
`
`The present inventors made intensive studies in order
`
`to achieve the above object, and as a result, they found that
`
`in order to significantly express an FAD-conjugated glucose
`
`dehydrogenase in an Aspergillus oryzae strain, it was necessary
`
`that an amino acid sequence (AGVPWV) be contained in a
`
`polypeptide encoding a gene of the enzyme, and also confirmed
`
`that the activity was substantially lost when at least one amino
`
`acid residue in the amino acid sequence was deleted, and thus,
`
`the invention was completed. That is, the invention relates
`
`to the following aspects.
`
`[0018]
`
`[Aspect 1] A polynucleotide encoding an FAD-conjugated
`
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`glucose dehydrogenase, comprising a polypeptide containing an
`
`amino acid sequence: Xl-X2-X3-X4-X5-X6 (wherein X1 and X2
`
`independently represent an aliphatic amino acid residue; X3
`
`and X6 independently represent a branched amino acid residue;
`
`and X4 and X5 independently represent a heterocyclic amino acid
`
`residue or an aromatic amino acid residue) .
`
`[Aspect 2] A polynucleotide encoding a polypeptide (a) ,
`
`(b) or (c) defined below:
`
`(a) a polypeptide which comprises an amino acid sequence
`
`represented by SEQ ID NO: 1;
`
`(b) a polypeptide which comprises an amino acid sequence
`
`having substitution, deletion, or addition of one to several
`
`amino acid residues in the amino acid sequence of the amino
`
`acid sequence (a) and has an FAD-conjugated glucose
`
`dehydrogenase activity; or
`
`(c) a polypeptide which comprises an amino acid sequence
`
`having a homology of 70% or more to the amino acid sequence
`
`(a) and has an FAD-conjugated glucose dehydrogenase activity.
`
`[Aspect 3] A polynucleotide (d) , (e) or (f) defined
`
`below:
`
`(d) a polynucleotide which comprises a base sequence
`
`represented by SEQ ID NO: 2 or 3;
`
`(e) a polynucleotide which hybridizes to a
`
`polynucleotide comprising a base sequence complementary to a
`
`polynucleotide comprising a base sequence (d) under stringent
`
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`conditions and encodes a polypeptide having an FAD-conjugated
`
`glucose dehydrogenase activity; or
`
`(f) a polynucleotide which comprises a base sequence
`
`having a homology of 70% or more to the polynucleotide
`
`comprising abase sequence (d) and encodes a polypeptide having
`
`an FAD-conjugated glucose dehydrogenase activity.
`
`[Aspect 4] A polynucleotide which has a DNA fragment
`
`amplifiable by PCR using a combination of a sense primer
`
`comprising a base sequence encoding the amino acid sequence:
`
`AGVPWV with a reverse primer comprising a base sequence on the
`
`3'-terminal side of a polynucleotide encoding an
`
`FAD-conjugated glucose dehydrogenase derived from Aspergillus
`
`oryzae or a combination of an antisense primer for a base
`
`sequence encoding the amino acid sequence: AGVPWV with a
`
`forward primer comprising a base sequence on the 5'-terminal
`
`side of a polynucleotide encoding an FAD-conjugated glucose
`
`dehydrogenase derived from Aspergillus oryzae, and encodes a
`
`polypeptide having an FAD-conjugated glucose dehydrogenase
`
`activity.
`
`[Aspect 5] A polynucleotide which hybridizes to a probe
`
`comprising a base sequence encoding the amino acid sequence:
`
`AGVPWV under stringent conditions and encodes a polypeptide
`
`having an FAD-conjugated glucose dehydrogenase activity.
`
`[Aspect 6] A polynucleotide encoding an FAD-conjugated
`
`glucose dehydrogenase derived from Aspergillus oryzae,
`
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`characterized by showing a value of enzymatic activity for
`
`maltose of 10% or less and a value of enzymatic activity for
`
`D-galactose of 5% or less with a value of enzymatic activity
`
`for D-glucose taken as 100%.
`
`[Aspect 7] A polynucleotide encoding an FAD-conjugated
`
`glucose dehydrogenase derived from Aspergillus oryzae,
`
`characterized by having an enzymatic activity of 300 U/mg or
`
`more.
`
`[Aspect 8] A recombinant vector containing the above
`
`polynucleotide.
`
`[Aspect 9] A transformant cell produced using the above
`
`recombinant vector.
`
`[Aspect 10] A process for the production of an
`
`FAD-conjugated glucose dehydrogenase, characterized by
`
`culturing the above transformant cell, and collecting an
`
`FAD-conjugated glucose dehydrogenase having an activity to
`
`dehydrogenate glucose from the resulting culture.
`
`[Aspect 11] A recombinant FAD-conjugated glucose
`
`dehydrogenase encoded by the above polynucleotide.
`
`[Aspect 12] A method for the determination of glucose,
`
`characterized by using the above FAD-conjugated glucose
`
`dehydrogenase.
`
`[Aspect 13] A reagent composition for use in the
`
`determination of glucose, characterized by comprising the
`
`above FAD-conjugated glucose dehydrogenase.
`
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`[Aspect 14] A biosensor for use in the determination of
`
`glucose, characterized by using the above FAD-conjugated
`
`glucose dehydrogenase.
`
`Advantage of the Invention
`
`[0019]
`
`By using the polynucleotide of the invention, an
`
`FAD-conjugated glucose dehydrogenase having excellent
`
`properties that it has excellent substrate (glucose)
`
`recognition performance and also has a low activity for maltose
`
`can be produced uniformly in a large amount by, for example,
`
`a recombinant DNA technique.
`
`Further, in the thus produced enzyme, the sugar amount
`
`which is a problem of the FAD-conjugated glucose dehydrogenase
`
`can be controlled according to the purpose, and therefore, by
`
`preparing the enzyme in which the sugar content has been reduced,
`
`in the determination of blood glucose or the like, it is also
`
`possible to alter the activity for sugars (such as glucose)
`
`in a sample.
`
`Brief Description of the Drawings
`
`[0020]
`
`[Fig. 1] Fig. 1 shows a calibration curve of glucose
`
`concentration using an enzyme-immobilized electrode.
`
`[Fig. 2] Fig. 2 shows the detection results of a target
`
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`gene by PCR. The symbols in the drawing are as follows. M:
`
`200 bp DNA ladder marker (manufactured by Takara Bio Inc.);
`
`1: Aspergillus oryzae NBRC4268; 2: Aspergillus oryzae NBRC
`
`5375; 3: Aspergillus oryzae NBRC 6215; 4: Aspergillus oryzae
`
`NBRC 4181; 5: Aspergillus oryzae NBRC 4220; 6: Aspergillus
`
`oryzae NBRC 100959
`
`[Fig. 3] Fig. 3 shows the detection results of a target
`
`gene by Southern hybridization. The symbols in the drawing
`
`are the same as in Fig. 2.
`
`Best Mode for Carrying Out the Invention
`
`[0021]
`
`It is one of the technically important points that an
`
`amino acid sequence: X1-X2-X3-X4-X5-X6 (wherein X1 and X2
`
`represent the same or a different aliphatic amino acid residue;
`
`X3 and X6 represent the same or a different branched amino acid
`
`residue; and X4 and X5 represent the same or a different
`
`heterocyclic amino acid residue or aromatic amino acid residue) ,
`
`in other words, a polypeptide comprising 6 amino acid residues
`
`is contained in the FAD-conjugated glucose dehydrogenase of
`
`the invention. According to this configuration, the enzyme
`
`is significantly expressed in microbial cells. Incidentally,
`
`the expressed enzyme is not necessarily secreted to the outside
`
`of microbial cells, and remains in microbial cells in some cases.
`
`In contrast, as specifically shown in Examples of this
`
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`description, even if a gene encodes an enzyme considered to
`
`be an FAD-conjugated glucose dehydrogenase based on the
`
`homology of the entire amino acid sequence or the like, when
`
`the gene does not encode the polypeptide comprising the amino
`
`acid sequence, it does not express a protein having an
`
`FAD-conjugated glucose dehydrogenase activity.
`
`[0022]
`
`The above-mentioned amino acid sequence comprising 6
`
`amino acid residues is preferably located at positions 202 to
`
`207 of a polypeptide which is an FAD-conjugated glucose
`
`dehydrogenase, or at least one of X1 to X6 is as follows: X1
`
`is alanine (A), X2 is glycine (G) , X3 is valine (V), X4 is proline
`
`(P), X5 is tryptophan (W), or X6 is valine (V). For example,
`
`as a preferred example, the amino acid sequence: AGVPWV (SEQ
`
`ID NO: 4) can be exemplified.
`
`[0023]
`
`In the invention, the "FAD-conjugated glucose
`
`dehydrogenase" refers to a soluble protein which catalyzes a
`
`reaction of the dehydrogenation (oxidation) of a hydroxy group
`
`at the 1-position of glucose in the presence of an electron
`
`acceptor and has an activity for maltose relative to the
`
`activity for glucose of 10% or less, and the enzyme is
`
`characterized by the following properties.
`
`1) Flavin adenine dinucleotide (FAD) is required as a
`
`coenzyme.
`
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`2) Oxygen is not used as an electron acceptor.
`
`3) The activity for maltose relative to the activity for
`
`glucose is 10% or less.
`
`[0024]
`
`Among the FAD-conjugated glucose dehydrogenases of the
`
`invention, as the enzyme having the amino acid sequence: AGVPWV,
`
`particularly, one derived from Aspergillus oryzae is preferred.
`
`Typical examples of a strain thereof include NBRC 5375 strain,
`
`NBRC 4079 strain, NBRC 4203 strain, NBRC 4214 strain, NBRC 4268
`
`strain, NBRC 5238 strain, NBRC 6215 strain, NBRC 30104 strain,
`
`and NBRC 30113 strain as shown in the following Table 1. The
`
`amino acid sequence: AGVPWV is contained in the amino acid
`
`sequence of the enzyme in the vicinity of positions 202 to 207
`
`(derived from NBRC 5375 strain) (in the case of an enzyme
`
`derived from other strain, positions corresponding to the
`
`positions) with the initiator amino acid residue Min a signal
`
`sequence region counted as position 1.
`
`[0025]
`
`For example, the amino acid sequence of the
`
`FAD-conjugated glucose dehydrogenase expressed by Aspergillus
`
`oryzae NBRC 5375 strain is represented by SEQ ID NO: 1
`
`(containing a signal peptide), the base sequence of a
`
`chromosomal DNA encoding the same is represented by SEQ ID NO:
`
`2, and a cDNA corresponding to the amino acid residues
`
`represented by SEQ ID NO: 1 is represented by SEQ ID NO: 3.
`
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`Incidentally, in SEQ ID NO: 2 or 3, the base sequence encoding
`
`the amino acid sequence: AGVPWV is GCTGGTGTTCCATGGGTT (SEQ ID
`
`NO: 5).
`
`[0026]
`
`Accordingly, the polynucleotide of the invention
`
`includes, in addition to those derived from Aspergillus oryzae
`
`strains described above, a polynucleotide encoding a
`
`polypeptide (a), (b) or (c) defined below:
`
`(a) a polypeptide which comprises an amino acid sequence
`
`represented by SEQ ID NO: 1;
`
`(b) a polypeptide which comprises an amino acid sequence
`
`having substitution, deletion, or addition of one to several
`
`amino acid residues in the amino acid sequence (a) and has an
`
`FAD-conjugated glucose dehydrogenase activity; or
`
`(c) a polypeptide which comprises an amino acid sequence
`
`having a homology of 70% or more to the amino acid sequence
`
`(a) and has an FAD-conjugated glucose dehydrogenase activity.
`
`[0027]
`
`Further, the polynucleotide of the invention includes
`
`a polynucleotide (d), (e) or (f) defined below:
`
`(d) a polynucleotide which comprises a base sequence
`
`represented by SEQ ID NO: 2 or 3;
`
`(e) a polynucleotide which hybridizes to a
`
`polynucleotide comprising a base sequence complementary to a
`
`polynucleotide comprising a base sequence (d) under stringent
`
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`PCT/JP2007/063147
`
`conditions and encodes a polypeptide having an FAD-conjugated
`
`glucose dehydrogenase activity; or
`
`(f) a polynucleotide which comprises a base sequence
`
`having a homology of 70% or more to the polynucleotide
`
`comprising abase sequence (d) and encodes a polypeptide having
`
`an FAD-conjugated glucose dehydrogenase activity.
`
`[0028]
`
`In particular, it is preferred that the above polypeptide
`
`(b) or (c) contains the amino acid sequence: X1-X2-X3-X4-X5-X6,
`
`or the polynucleotide (e) or (f) contains a base sequence
`
`encoding the amino acid sequence. Further, it is preferred
`
`that this amino acid sequence is AGVPWV.
`
`[0029]
`
`In this description, the amino acid sequence or base
`
`sequence having a homology of 70% or more refers to a sequence
`
`showing a homology of at least 70%, preferably 75% or more,
`
`more preferably 80% or more, further more preferably 90% or
`
`more, particularly preferably 95% or more to the full-length
`
`of a standard sequence to be compared, respectively. The
`
`homology percentage of such a sequence can be calculated using
`
`a disclosed or commercially available software with an
`
`algorithm that makes a comparison using the standard sequence
`
`as a reference sequence. For example, BLAST, FASTA, or GENETYX
`
`(manufactured by Software Development Co., Ltd.), or the like
`
`can be used. These can be used with default parameters.
`
`18
`
`Page 18 of 80
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`

`

`PCT/JP2007/063147
`
`[0030]
`
`In the invention, as specific conditions for the
`
`"hybridization
`
`under
`
`stringent
`
`conditions"
`
`when
`
`polynucleotides are hybridized, for example, incubation at
`
`42°C in 50% formamide, 5 x SSC (150 mM sodium chloride, 15 mM
`
`trisodium citrate, 10 mM sodium phosphate, 1 mM
`
`ethylenediaminetetraacetic acid, pH 7.2), 5 x Denhardt's
`
`solution, 0.1 SDS, 10% dextran sulfate, and 100 [1.g/mL modified
`
`salmon sperm DNA, followed by washing of the filter at 42°C
`
`in 0.2 x SSC can be exemplified.
`
`[0031]
`
`Further, the polynucleotide of the invention includes
`
`a polynucleotide which has a DNA fragment amplifiable by PCR
`
`using a combination of a sense primer comprising a base sequence
`
`encoding the amino acid sequence: AGVPWV with a reverse primer
`
`comprising a base sequence on the 3'-terminal side of a
`
`polynucleotide
`
`encoding
`
`an
`
`FAD-conjugated
`
`glucose
`
`dehydrogenase derived from Aspergillus oryzae or a combination
`
`of an antisense primer for a base sequence encoding the amino
`
`acid sequence: AGVPWV with a forward primer comprising a base
`
`sequence on the 5'-terminal side of a polynucleotide encoding
`
`an FAD-conjugated glucose dehydrogenase derived from
`
`Aspergillus oryzae, and encodes a polypeptide having an
`
`FAD-conjugated glucose dehydrogenase activity.
`
`[0032]
`
`19
`
`Page 19 of 80
`
`

`

`PCT/JP2007/063147
`
`Alternatively, the polynucleotide of the invention
`
`includes a polynucleotide which hybridizes to a probe
`
`comprising a base sequence encoding the amino acid sequence:
`
`AGVPWV under stringent conditions and encodes a polypeptide
`
`having an FAD-conjugated glucose dehydrogenase activity.
`
`[0033]
`
`The base sequence encoding the amino acid sequence:
`
`AGVPWV is preferably GCTGGTGTTCCATGGGTT.
`
`Further, the
`
`respective conditions for the above-mentioned PCR and
`
`hybridization under stringent conditions can be suitably
`
`selected by those skilled in the art in accordance with the
`
`description of Examples in this description.
`
`[0034]
`
`Further, the polynucleotide of the invention includes
`
`a polynucleotide encoding an FAD-conjugated glucose
`
`dehydrogenase, which shows a value of enzymatic activity for
`
`maltose of 10% or less, preferably 5% or less, more preferably
`
`3% or less, and a value of enzymatic activity for D-galactose
`
`of 5% or less, preferably
`
`or less, more preferably
`
`or
`
`less, further more preferably 1% or less with a value of
`
`enzymatic activity for D-glucose taken as 100%; or a
`
`polynucleotide
`
`encoding
`
`an
`
`FAD-conjugated
`
`glucose
`
`dehydrogenase having an enzymatic activity of a specific
`
`activity per protein of 300 U/mg or more, preferably 500 U/mg
`
`or more, more preferably 1,000 U/mg or more. Incidentally,
`
`20
`
`Page 20 of 80
`
`

`

`PCT/JP2007/063147
`
`the "specific activity per protein" as used herein is, for
`
`example, a measurement determined in a state confirmed as a
`
`single band by SDS-PAGE of a concentrated culture supernatant
`
`as described in Example 7 of this description.
`
`[0035]
`
`Incidentally, in the invention, the "polynucleotide"
`
`refers to a molecule having 100 or more phosphate esters of
`
`nucleosides in which a purine or a pyrimidine is attached to
`
`a sugar via a 13 -N-glycosidic bond (ATP (adenosine triphosphate) ,
`
`GTP (guanosine triphosphate), CTP (cytidine triphosphate), or
`
`UTP (uridine triphosphate); or dATP (deoxyadenosine
`
`triphosphate), dGTP (deoxyguanosine triphosphate), dCTP
`
`(deoxycytidine triphosphate), or dTTP (deoxythymidine
`
`triphosphate).
`
`Specific examples thereof include a
`
`chromosomal DNA encoding an FAD-conjugated glucose
`
`dehydrogenase, a mRNA transcribed from the chromosomal DNA,
`
`a cDNA synthesized from the mRNA, and a polynucleotide
`
`amplified by PCR using any of these as a template. An
`
`"oligonucleotide" refers to a molecule in which 2 to 99
`
`nucleotides are linked to one another.
`
`Further, the
`
`"polypeptide" refers to a molecule formed from. 30 or more amino
`
`acid residues which are linked to one another through an amide
`
`bond (peptide bond) or an unnatural residual linkage, and also
`
`those with the addition of a sugar chain, those with the
`
`artificial chemical modification, and the like are included.
`
`21
`
`Page 21 of 80
`
`

`

`PCT/JP2007/063147
`
`[0036]
`
`The most specific mode of the polynucleotide (gene) of
`
`the invention is a polynucleotide containing the base sequence
`
`represented by SEQ ID NO: 2 or 3. The polynucleotide which
`
`is a chromosomal DNA typified by SEQ ID NO: 2 can be obtained
`
`by, for example, preparing a chromosomal DNA library from
`
`Aspergillus oryzae NBRC 5375 strain, and screening the
`
`chromosomal DNA library by a method known to those skilled in
`
`the art using a plurality of oligonucleotide probes prepared
`
`based on amino acid sequences obtained by determining amino
`
`acid residues of N-terminal and internal sequences of an
`
`FAD-conjugated glucose dehydrogenase derived from Aspergillus
`
`terreus described in Patent document 1 by the Edman sequencing
`
`method or the like, and the genome sequence information of
`
`Aspergillus oryzae (NBRC 100959 strain) disclosed in DOGAN
`
`(Database of the Genomes Analyzed at NITE) (website
`
`http://www.bio.nite.go.jp/dogan/Top) in January, 2006 as a
`
`result of the "Aspergillus oryzae genome analysis project".
`
`[0037]
`
`The labeling of the probe can be performed by an arbitrary
`
`method known to those skilled in the art such as a radioisotope
`
`(RI) method or a non-RI method, however, a non-RI method is
`
`preferably used. Examples of the non-RI method include a
`
`fluorescence labeling method, a biotinylation method, and a
`
`chemiluminescence method, however, a fluorescence labeling
`
`22
`
`Page 22 of 80
`
`

`

`PCT/JP2007/063147
`
`method is preferably used. As a fluorescent substance, those
`
`capable of binding to a base moiety of an oligonucleotide is
`
`suitably selected and can be used, and specifically, a cyanine
`
`dye (such as Cy3 or Cy5 in Cy DyeTM series) , a rhodamine 6G reagent,
`
`N-acetoxy-N2- acetylaminofluorene (AAF),
`
`AAIF (iodine
`
`derivative of AAF), or the like can be used.
`
`[0038]
`
`Alternatively,
`
`the polynucleotide which is a cDNA
`
`typified by SEQ ID NO: 3 can be obtained by, for example, as
`
`specifically described in Examples of this specification, any
`
`of a variety of PCR methods known to those skilled in the art
`
`using the oligonucleotide primer (probe) set prepared in the
`
`above with a cDNA library as a template, or also by the RT-PCR
`
`method using the total RNA or mRNA extracted from. Aspergillus
`
`oryzae NBRC 5375 strain as a template. Incidentally, in the
`
`case where a primer is designed, the size (the number of bases)
`
`of the primers is from 15 to 40 bases, preferably from 15 to
`
`30 bases in consideration of achieving the specific annealing
`
`thereof to a template DNA. However, in the case where LA (long
`
`and accurate) PCR is performed, the size of at least 30 bases
`
`is effective. Complementary sequences between the both
`
`primers should be avoided so that a set of or a pair (2 strands)
`
`of primers comprising a sense strand (on the 5'-terminal side)
`
`and an antisense strand (on the 3f -terminal side) may not anneal
`
`to each other. Further, in order to secure stable binding to
`
`23
`
`Page 23 of 80
`
`

`

`PCT/JP2007/063147
`
`the template DNA, the GC content should be about 50% so that
`
`GC-rich or AT-rich regions should not be unevenly distributed
`
`within the primers. Since the annealing temperature depends
`
`on Tm (melting temperature), primers having a Tm value in the
`
`range from 55 to 65°C and similar to each other are selected
`
`in order to obtain a highly specific PCR product. In addition,
`
`it is necessary to note that the final concentration of the
`
`primers used in PCR should be adjusted to about 0.1 to about
`
`1 plii and the like. Further, a commercially available software
`
`for primer designing, for example, OligoTM (manufactured by
`
`National Bioscience, Inc. (U.S.A.) ) , GENETYX (manufactured by
`
`Software Development Co., Ltd.) , or the like can be also used.
`
`[0039]
`
`Incidentally, such an oligonucleotide probe or an
`
`oligonucleotide primer set can also be prepared by cleaving
`
`the cDNA which is the polynucleotide of the invention with a
`
`suitable restriction enzyme.
`
`[0040]
`
`Further, the polynucleotide of the invention can be
`
`pr

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