JP6934720B2 - Flavin-bound glucose dehydrogenase with improved substrate specificity - Google Patents

Description

The present invention relates to a flavin-linked glucose dehydrogenase having improved substrate specificity, a gene and recombinant DNA thereof, and a method for producing a flavin-linked glucose dehydrogenase having improved substrate specificity.

Blood glucose concentration (blood glucose level) is an important marker of diabetes. As a device for a diabetic patient to control his / her own blood glucose level, a self-monitoring blood glucose measuring device (SELF Monitoring of Blood Glucose: SMBG) using an electrochemical biosensor is widely used. Conventionally, enzymes using D-glucose as a substrate, such as glucose oxidase (GOD), have been used in biosensors used in SMBG devices. However, since GOD has the property of using oxygen as an electron acceptor, in the SMBG device using GOD, when the dissolved oxygen in the measurement sample affects the measured value and an accurate measured value cannot be obtained. Can occur.

On the other hand, various glucose dehydrogenases (hereinafter, GDH) are known as another enzyme that uses D-glucose as a substrate but does not use oxygen as an electron acceptor. Specifically, GDH (NAD (P) -GDH) of the type that uses nicotinamide dinucleotide (NAD) or nicotinamide dinucleotide phosphate (NADP) as a coenzyme, and pyrroloquinoline quinone (PQQ) are used as coenzymes. GDH (PQQ-GDH) has been found and is used in biosensors of SMBG equipment. However, NAD (P) -GDH has a problem that the enzyme is poorly stable and a coenzyme needs to be added, and PQQ-GDH has low substrate specificity, and D-glucose to be measured. In addition, since it acts on sugar compounds such as maltose, D-galactose and D-xylose, sugar compounds other than D-glucose in the measurement sample affect the measured value, and an accurate measured value can be obtained. There is a problem that it cannot be done.

In recent years, when measuring the blood glucose level of a diabetic patient who has been receiving an infusion solution using an SMBG device using PQQ-GDH as a biosensor, PQQ-GDH also acts on the maltose contained in the infusion solution. A measured value higher than the actual blood glucose level is obtained, and there have been reports of cases in which a patient develops hypoglycemia or the like due to treatment based on this value. It has also been found that similar events can occur in patients undergoing galactose stress testing and xylose absorption testing (see, eg, Non-Patent Document 1). In response to this, the Ministry of Health, Labor and Welfare's Pharmaceutical and Food Safety Bureau conducted a cross-reactivity test for the purpose of investigating the effect of each sugar added to the D-glucose solution on the blood glucose measurement value. When / dL of D-galactose or 200 mg / dL of D-xylose was added, the measured value of the blood glucose measurement kit using the PQQ-GDH method was 2.5 to 2.5 to the actual D-glucose concentration. It was found that the value was about 3 times higher. That is, it was found that the measured values were inaccurate due to maltose, D-galactose, and D-xylose that could be present in the measurement sample, and were not affected by the sugar compound that causes such measurement errors, and D- There is an urgent need for the development of GDH having high substrate specificity capable of specifically measuring glucose.

Against the background as described above, a type of GDH that utilizes a coenzyme other than the above has been attracting attention. For example, Non-Patent Documents 2 to 5 have reports on GDH derived from Aspergillus oryzae, and for example, Patent Documents 1 to 3 have a flavin-bound type using flavin adenine dinucleotide (FAD) derived from the genus Aspergillus as a coenzyme. Glucose dehydrogenase (FAD-GDH) is disclosed.

However, although the above enzymes have the property of being less reactive with one or several sugar compounds other than D-glucose, they are also reactive with maltose, D-galactose, and D-xylose. Does not have the property of being low enough. In response to these, the applicant found that FAD-GDH found in the genus Mucor, which is a type of mucor, is sufficiently low in reactivity with maltose, D-galactose, and D-xylose. (See, for example, Patent Document 4). In addition, using this GDH, it is possible to accurately measure the D-glucose concentration even under the presence of maltose, D-galactose, and D-xylose in a certain concentration range without being affected by those sugar compounds. It has been confirmed that it is possible (see, for example, Patent Document 4). Such excellent substrate specificity is a major feature showing the practical superiority of FAD-GDH derived from the genus Mucor.

On the other hand, in order to further improve the convenience in self-blood glucose measurement, the measurement time is shortened by further improving the measurement sensitivity, the measurement system is further reduced in scale, and the required measurement sample is continuously reduced. Being pursued. For example, as a means for improving the measurement sensitivity, it is assumed that the amount of the glucose measuring enzyme mounted on the glucose sensor is increased. However, under the condition where a large amount of enzyme is present, which is expected in such a usage, even when the above-mentioned FAD-GDH derived from the genus Mucor is used, maltose or D-xylose present at a certain concentration or higher can be obtained. Reactivity has been confirmed to some extent, and there is still room for improvement in efforts to reduce reactivity with sugar compounds other than D-glucose.

As an attempt to modify the existing FAD-GDH for the purpose of improving the substrate specificity of FAD-GDH, the activity on D-xylose is increased by introducing an amino acid substitution into FAD-GDH derived from the genus Aspergillus. A method for obtaining a reduced FAD-GDH variant is disclosed (see, for example, Patent Documents 5-6). However, FAD-GDH derived from the genus Aspergillus is considerably more reactive to D-xylose than the natural FAD-GDH derived from the genus Mucor, and has a FAD-GDH variant derived from the genus Aspergillus disclosed so far. However, it is hard to say that its substrate specificity is sufficient. Further, Patent Document 7 discloses a method for obtaining a FAD-GDH variant having reduced activity on D-xylose and maltose by introducing an amino acid substitution into FAD-GDH derived from the genus Mucor. Attempts to impart high specificity are continuously sought.
On the other hand, a glucose measuring enzyme having a high specific activity can reduce the amount of the glucose measuring enzyme mounted on the glucose sensor, so that the measurement time can be shortened by further improving the measurement sensitivity and the measurement system can be further reduced in scale. Since it is considered to be effective for reducing the amount of the required measurement sample, an enzyme having high isoisomerism while maintaining the specific activity is required.

Japanese Unexamined Patent Publication No. 2007-289148 Japanese Patent No. 4494978 International Publication No. 07/139013 Japanese Patent No. 4648993 Japanese Unexamined Patent Publication No. 2008-237210 International Publication No. 09/0846116 International Publication No. 13/06570

Safety Information No. 206 for Pharmaceuticals and Medical Devices (Pharmaceuticals and Medical Devices Safety Information No. 206), October 2004, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare Studios on the glucose dehydrogenase of Aspergillus oryzae. I. Induction of it systems by p-benzoquinone and hydroquinone, T. et al. C. Bak, and R. Sato, Biochim. Biophyss. Acta, 139, 265-276 (1967). Studios on the glucose dehydrogenase of Aspergillus oryzae. II. Pharmaceutical and physical and chemical properties, T. et al. C. Bak, Biochim. Biophyss. Acta, 139, 277-293 (1967). Studios on the glucose dehydrogenase of Aspergillus oryzae. III. General enzymatic products, T.I. C. Bak, Biochim. Biophyss. Acta, 146, 317-327 (1967). Studios on the glucose dehydrogenase of Aspergillus oryzae. IV. Histidyl reserve as an active site, T.I. C. Bak, and R. Sato, Biochim. Biophyss. Acta, 146, 328-335 (1967).

In the present invention, it has excellent substrate specificity for D-glucose, does not easily act on sugar compounds other than D-glucose, such as D-xylose and maltose, and when used for measuring D-glucose, these D- An object of the present invention is to provide a novel FAD-GDH that is not easily affected even when a sugar compound other than glucose coexists.

As a result of intensive studies to solve the above problems, the present inventors have found that the modified FAD-GDH obtained by substituting a specific amino acid residue in FAD-GDH derived from the genus Mucor is specific to D-glucose. The present invention has been completed by finding that it has excellent properties, is less likely to act on D-xylose and maltose, and is less susceptible to the coexistence of sugar compounds other than D-glucose.

That is, the present invention is as follows.
(1) It has an amino acid sequence shown by SEQ ID NO: 1, an amino acid sequence that is 70% or more identical to the amino acid sequence, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence.
The position corresponding to alanine at position 42 in the amino acid sequence shown in SEQ ID NO: 1.
Position corresponding to valine at position 83 in the amino acid sequence shown in SEQ ID NO: 1,
The position corresponding to serine at position 174 in the amino acid sequence shown in SEQ ID NO: 1.
The position corresponding to threonine at position 367 in the amino acid sequence shown in SEQ ID NO: 1.
The position corresponding to alanine at position 385 in the amino acid sequence shown in SEQ ID NO: 1.
The position corresponding to threonine at position 386 in the amino acid sequence shown in SEQ ID NO: 1.
The position corresponding to leucine at position 391 in the amino acid sequence shown in SEQ ID NO: 1.
The position corresponding to phenylalanine at position 460 in the amino acid sequence shown in SEQ ID NO: 1.
The position corresponding to threonine at position 461 in the amino acid sequence shown in SEQ ID NO: 1.
One or one at the position corresponding to the amino acid selected from the group consisting of the position corresponding to serine at position 467 in the amino acid sequence shown in SEQ ID NO: 1 and the position corresponding to glycine at position 468 in the amino acid sequence shown in SEQ ID NO: 1. Having more amino acid substitutions, the ratio of reactivity to D-xylose to reactivity to D-glucose (Xyl / Glc (%)) is reduced as compared to before the substitution. Flavin-bound glucose dehydrogenase, characterized by.
(2) The amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence 90% or more identical to the amino acid sequence, or the position corresponding to the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence. Amino acids
Whether the amino acid at the position corresponding to alanine at position 42 in the amino acid sequence shown in SEQ ID NO: 1 is glycine.
Whether the amino acid at the position corresponding to valine at position 83 in the amino acid sequence shown in SEQ ID NO: 1 is either alanine or glycine.
Whether the amino acid at the position corresponding to serine at position 174 in the amino acid sequence shown in SEQ ID NO: 1 is either proline or leucine.
Whether the amino acid at the position corresponding to threonine at position 367 in the amino acid sequence shown in SEQ ID NO: 1 is alanine, serine, or asparagine.
Whether the amino acid at the position corresponding to alanine at position 385 in the amino acid sequence shown in SEQ ID NO: 1 is threonine, aspartic acid, or arginine.
Whether the amino acid at the position corresponding to threonine at position 386 in the amino acid sequence shown in SEQ ID NO: 1 is valine, isoleucine, glycine, leucine, glutamic acid, glutamine, asparagine, or serine.
Whether the amino acid at the position corresponding to leucine at position 391 in the amino acid sequence shown in SEQ ID NO: 1 is valine.
Whether the amino acid at the position corresponding to phenylalanine at position 460 in the amino acid sequence shown in SEQ ID NO: 1 is either tyrosine or leucine.
Whether the amino acid at the position corresponding to threonine at position 461 in the amino acid sequence shown in SEQ ID NO: 1 is methionine, phenylalanine, glycine, aspartic acid, or cysteine.
Whether the amino acid at the position corresponding to serine at position 467 in the amino acid sequence shown in SEQ ID NO: 1 is leucine, methionine, isoleucine, valine, aspartic acid, or glutamic acid.
Alternatively, flavin-linked glucose dehydrogenase in which the amino acid at the position corresponding to glycine at position 468 in the amino acid sequence shown in SEQ ID NO: 1 is threonine.
(3) The ratio of the reactivity to D-xylose to the reactivity to D-glucose (Xyl / Glc (%)) is reduced by 10% or more as compared with that before the introduction of the above substitution. The flavin-bound glucose dehydrogenase according to (1) or (2).
(4) The flavin-linked glucose dehydrogenase gene encoding the flavin-linked glucose dehydrogenase according to any one of (1) to (3).
(5) A recombinant vector containing the flavin-linked glucose dehydrogenase gene according to (4).
(6) A host cell containing the recombinant vector according to (5).
(7) A method for producing a flavin-bound glucose dehydrogenase, which comprises the following steps:
(A) The step of culturing the host cell according to (6).
(B) A step of expressing a flavin-bound glucose dehydrogenase gene contained in the host cell.
(C) A step of isolating flavin-bound glucose dehydrogenase from the culture.
(8) A method for measuring glucose, which comprises using the flavin-bound glucose dehydrogenase according to (1) to (3).
(9) A glucose assay kit containing the flavin-bound glucose dehydrogenase according to (1) to (3).
(10) A glucose sensor containing the flavin-bound glucose dehydrogenase according to (1) to (3).

According to the present invention, it is possible to provide FAD-GDH having excellent substrate specificity, which maintains specific activity and has reduced reactivity with D-xylose and / or maltose.

(Principle of action and activity measurement method of FAD-GDH of the present invention)
The FAD-GDH of the present invention, like the known wild-type or mutant FAD-GDH, catalyzes the reaction of oxidizing the hydroxyl group of D-glucose to produce glucono-δ-lactone in the presence of an electron acceptor.
The activity of FAD-GDH of the present invention utilizes this principle of action, for example, using the following measurement system using phenazinemethsulfate (PMS) and 2,6-dichloroindophenol (DCIP) as electron acceptors. Can be measured.
(Reaction 1) D-glucose + PMS (oxidized type)
→ D-glucono-δ-lactone + PMS (reduced type)
(Reaction 2) PMS (reduced type) + DCIP (oxidized type)
→ PMS (oxidized type) + DCIP (reduced type)

Specifically, first, in (Reaction 1), PMS (reduced form) is produced with the oxidation of D-glucose. Then, as the PMS (reduced form) is oxidized by the subsequent progress (reaction 2), DCIP is reduced. The degree of disappearance of this "DCIP (oxidized type)" can be detected as the amount of change in absorbance at a wavelength of 600 nm, and the enzyme activity can be determined based on this amount of change.
Specifically, the activity of flavin-bound GDH can be measured according to the following procedure. Mix 2.05 mL of 50 mM phosphate buffer (pH 6.5), 0.6 mL of 1MD-glucose solution and 0.15 mL of 2 mM DCIP solution, and incubate at 37 ° C. for 5 minutes. Then 0.1 mL of 15 mM PMS solution and 0.1 mL of enzyme sample solution are added to initiate the reaction. The absorbance at the start of the reaction and over time is measured to determine the amount of decrease in absorbance (ΔA600) at 600 nm with the progress of the enzymatic reaction per minute, and the flavin-bound GDH activity is calculated according to the following formula. At this time, flavin-bound GDH activity defines 1 U as the amount of enzyme that reduces 1 μmol of DCIP per minute in the presence of D-glucose at a concentration of 200 mM at 37 ° C.

In the formula, 3.0 is the liquid volume of the reaction reagent + enzyme reagent (mL), 16.3 is the mmol molecular extinction coefficient (cm ^{2 /} μmol) under the present activity measurement conditions, and 0.1 is the liquid volume of the enzyme solution. (ML), 1.0 is the optical path length (cm) of the cell, ΔA600 _blank is the absorbance at 600 nm per minute when the reaction was started by adding 10 mM acetate buffer (pH 5.0) instead of the enzyme sample solution. The amount of decrease in, df represents the dilution multiple.

(Amino acid sequence of FAD-GDH of the present invention)
The FAD-GDH of the present invention has an amino acid sequence represented by SEQ ID NO: 1, or has a high identity with the amino acid sequence, for example, preferably 70% or more, more preferably 75% or more, more preferably 80% or more, and more. A sequence consisting of preferably 85% or more, more preferably 90% or more, most preferably 95% or more of the same amino acid sequence, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence. In the amino acid sequence described in No. 1, the position corresponding to the 42nd position, the position corresponding to the 83rd position, the position corresponding to the 174th position, the position corresponding to the 376th position, the position corresponding to the 385th position, the position corresponding to the 386th position, 391 At the position corresponding to the amino acid at the position selected from the position corresponding to the position 460, the position corresponding to the 461 position, the position corresponding to the 467 position, and the position corresponding to the 468th position. It is characterized by having one or more amino acid substitutions.

Preferably, in FAD-GDH of the present invention, the amino acid substitution at the position corresponding to the 42-position described above is a substitution in which alanine at the position corresponding to the 42-position is replaced with glycine, which corresponds to the 83-position described above. The amino acid substitution at the position corresponding to the position 83 is a substitution in which valine at the position corresponding to the 83rd position is replaced with either alanine or glycine, and the amino acid substitution at the position corresponding to the above-mentioned position 174 is the substitution at the position 174. The substitution in which the serine at the corresponding position is replaced with either proline or leucine, and the amino acid substitution at the position corresponding to the above-mentioned 367 position means that the threonine at the position corresponding to the 367 position is alanine, serine, or asparagine. The amino acid substitution at the position corresponding to the 385th position is a substitution in which the threonine at the position corresponding to the 385th position is replaced with any of alanine, aspartic acid, and arginine. The above-mentioned amino acid substitution at the position corresponding to the 386 position is a substitution in which the threonine at the position corresponding to the 386 position is replaced with any one of valine, isoleucine, glycine, leucine, glutamic acid, glutamine, asparagine and serine. The amino acid substitution at the position corresponding to the 391 position described above is a substitution in which leucine at the position corresponding to the 391 position is replaced with valine, and the amino acid substitution at the position corresponding to the 460 position described above is 460. The phenylalanine at the position corresponding to the position is replaced with either tyrosine or leucine, and the amino acid substitution at the position corresponding to the above-mentioned 461 position means that the threonine at the position corresponding to the 461 position is methionine, phenylalanine, The substitution is substituted with any of glycine, aspartic acid, and cysteine, or the amino acid substitution at the position corresponding to the above-mentioned 467 position means that the serine at the position corresponding to the 467 position is leucine, methionine, isoleucine, valine, It is a substitution substituted with either aspartic acid or glutamate, and the amino acid substitution at the position corresponding to the above-mentioned 468 position is a substitution in which the glycine at the position corresponding to the 468 position is replaced with threonine.

Among the FAD-GDH of the present invention, an example of a more preferable one is a multiple mutant having a plurality of combinations of the above-mentioned substitutions. For example, the present invention includes a double mutant having two combinations of the above substitutions, a triple mutant having three combinations, and a multiple mutant having a large number of mutations in combination. Accumulation of such mutations can produce FAD-GDH with further reduced activity on D-xylose and / or maltose.
In addition, in producing the above-mentioned multiple mutants, substitutions at positions other than the above-mentioned various substitutions can be combined. When such a substitution position is introduced alone, even if it does not exert a remarkable effect as in the above-mentioned substitution site, it can be introduced in combination with the above-mentioned substitution site. It can be synergistically effective.

In addition to the above-mentioned mutations that do not easily act on D-xylose and maltose, the FAD-GDH of the present invention includes mutations that improve thermal stability, mutations that improve specific activity, and pH. Known mutations aimed at exerting different kinds of effects, such as an effect of improving resistance to a specific substance or the like, may be arbitrarily combined. Even when such different types of mutations are combined, those FAD-GDHs are included in the present invention as long as the effects of the present invention can be exerted.

As will be described later, in the FAD-GDH of the present invention, for example, a gene encoding an amino acid sequence close to the amino acid sequence of SEQ ID NO: 1 is first obtained by an arbitrary method, and at a position equivalent to a predetermined position of SEQ ID NO: 1. It can also be obtained by introducing an amino acid substitution at any position.
Examples of the target amino acid substitution introduction method include a method of randomly introducing a mutation or a method of introducing a site-specific mutation at a assumed position. The former method includes the error prone PCR method (Techniques, 1,11-15, (1989)) and the XL1-Red competent cell (STRATAGENE), which is prone to error in plasmid replication and modification during proliferation. There is a method using (manufactured by the company). In addition, as the latter method, a three-dimensional structure is constructed by crystal structure analysis of the target protein, amino acids that are expected to give the desired effect are selected based on the information, and a commercially available Quick Change Site Directed Mutagesis Kit is used. There is a method of introducing a site-directed mutagenesis by (manufactured by STRATAGENE) or the like. Alternatively, as the latter method, there is also a method of introducing a site-specific mutation by selecting an amino acid that is expected to give the desired effect by using the three-dimensional structure of a known protein having high homology with the target protein. ..

Further, for example, "a position equivalent to the amino acid sequence of SEQ ID NO: 1" as used herein means the amino acid sequence of SEQ ID NO: 1 and homology with SEQ ID NO: 1 (preferably 80% or more, more preferably 85% or more). , More preferably 90% or more, most preferably 95% or more) means the same position in the alignment when aligned with another FAD-GDH having an amino acid sequence. The amino acid sequence homology should be calculated by a program such as Maximum Matching or Search Homology of GENETYX-Mac (manufactured by Software Development) or a program such as Maximum Matching or Multiple Alignment of DNASIS Pro (manufactured by Hitachi Soft). Can be done.

As a method for specifying the "position corresponding to the amino acid", the amino acid sequences are compared using a known algorithm such as the Lipman-Person method, and the largest conserved amino acid residue present in the amino acid sequence of FAD-GDH is used. It can be done by giving homology. By aligning the amino acid sequence of FAD-GDH in this way, it is possible to determine the position of homologous amino acid residues in each FAD-GDH sequence in the sequence regardless of insertion or deletion in the amino acid sequence. Is. Homological positions are considered to be co-located in the three-dimensional structure and can be presumed to have similar effects on the substrate specificity of the FAD-GDH of interest.

Although various variations of the FAD-GDH of the present invention are assumed within the above-mentioned range of identity, the enzymatic scientific properties of the various FAD-GDH are the same as those of the FAD-GDH of the present invention described herein. As long as they are, they can all be included in the FAG-GDH of the present invention. FAD-GDH having such an amino acid sequence has high substrate specificity and is less susceptible to the coexistence of sugar compounds other than D-glucose, such as D-xylose and maltose, and is industrially useful. Is.

Further, in the FAD-GDH of the present invention, the amino acid substitution at the position corresponding to the above-mentioned 42 position is a substitution in which the alanine at the position corresponding to the 42 position is replaced with glycine, which corresponds to the above-mentioned 83 position. The amino acid substitution at the position corresponding to the position is that the valine at the position corresponding to the 83rd position is either alanine or glycine, or the amino acid substitution at the position corresponding to the 174th position is the position corresponding to the 174th position. The fact that serine is either proline or leucine, or the amino acid substitution at the position corresponding to position 376 means that the threonine at position corresponding to position 376 is either alanin, serine, or asparagine, or The amino acid substitution at the position corresponding to the 385 position means that the threonine at the position corresponding to the 385 position is any of alanine, aspartic acid, and arginine, or the amino acid substitution at the position corresponding to the 386 position means. The threonin at the position corresponding to position 386 is any of valine, isoleucine, glycine, leucine, glutamic acid, glutamine, asparagine, and serine, or the amino acid substitution at the position corresponding to position 391 corresponds to position 391. The amino acid substitution at the position corresponding to the 460 position means that the leucine at the position corresponding to the position is valine, or the phenylalanine at the position corresponding to the 460 position is either tyrosine or leucine, or corresponds to the position 461. The amino acid substitution at the position corresponding to the position 461 is that the threonine at the position corresponding to the position 461 is any of methionine, phenylalanine, glycine, aspartic acid, and cysteine, and the amino acid substitution at the position corresponding to the position 467 is the position 467. The serine at the position corresponding to is one of leucine, methionine, isoleucine, valine, aspartic acid, and glutamic acid, or the amino acid substitution at the position corresponding to the 468th position means that the glycine at the position corresponding to the 468th position is It is important that it is threonine, not whether it is due to an artificial replacement operation. For example, if a protein whose amino acid at the above position is originally different from the residue desired in the present invention is used as a starting material, and a desired substitution is introduced therein using a known technique, these are used. The desired amino residue of is introduced by substitution. On the other hand, when a desired protein is obtained by total synthesis of a known peptide, or when a gene sequence is totally synthesized so as to encode a protein having a desired amino acid sequence and a desired protein is obtained based on the gene sequence, or The FAD-GDH of the present invention can be obtained without going through a step of artificial replacement, for example, when some of those found as natural forms originally have such a sequence.

(Improvement of Substrate Specificity in FAD-GDH of the Present Invention)
The FAD-GDH of the present invention is characterized by having high substrate specificity. Specifically, the FAD-GDH of the present invention reacts to maltose, D-galactose, and D-xylose in the same manner as the mosquito-derived FAD-GDH described in Japanese Patent No. 4648993 previously found by the present inventors. It is characterized by extremely low sex. Specifically, when the reactivity with D-glucose is 100%, the reactivity with maltose, D-galactose and D-xylose is 2% or less. Since FAD-GDH used in the present invention has such high substrate specificity, it can be used for samples of patients receiving an infusion solution containing maltose and patients undergoing a galactose loading test and a xylose absorption test. The amount of D-glucose can be accurately measured without being affected by sugar compounds such as maltose, D-galactose, and D-xylose contained in the measurement sample. Furthermore, since the FAD-GDH of the present invention has a higher substrate specificity for D-glucose as compared with the mucor-derived FAD-GDH described in Japanese Patent No. 4648993, it is further industrially useful. Sex is expected.

Further, the FAD-GDH of the present invention has a very low measured value when measured using a sugar compound such as maltose, D-galactose, D-xylose as a substrate instead of D-glucose as described above, and further. , Maltose, D-galactose, D-xylose and the like, it is preferable that D-glucose can be accurately measured even under conditions congested with sugar compounds. Specifically, when the reactivity to D-glucose in the absence of these contaminating sugar compounds is 100%, one or more selected from maltose, D-galactose, and D-xylose are selected as the contaminating sugar compounds. The measured value when present is 96% to 103%, and the measured value is 96% to 104% even when three kinds of maltose, D-galactose and D-xylose are simultaneously present as the contaminating sugar compound. preferable.
When FAD-GDH having such characteristics is used, it is possible to accurately measure the amount of D-glucose even in the presence of maltose, D-galactose, and D-xylose in the measurement sample. be.

The enzymatic chemistry of various FAD-GDHs can be investigated using known techniques for identifying the properties of the enzyme, eg, the methods described in the Examples below. Various properties of the enzyme can be investigated to some extent in the culture medium of microorganisms producing various FAD-GDH and in the middle of the purification process, and more specifically, can be investigated using the purified enzyme.

The modified FAD-GDH of the present invention is the ratio of reactivity to D-xylose at the same molar concentration to reactivity to D-glucose under reaction conditions based on the above-mentioned activity measurement method (Xyl / Glc (%)). ) And / or the ratio of reactivity to maltose at the same molar concentration to reactivity to D-glucose (Mal / Glc (%)) is reduced compared to before the introduction of amino acid substitution, preferably. Is 20% or more, more preferably 30% or more, further preferably 40% or more, and most preferably 50% or more.
In the FAD-GDH of the present invention, only one of Xyl / Glc (%) and Mal / Glc (%) may be reduced to a preferable degree as compared with FAD-GDH before introducing the amino acid substitution. Alternatively, both may be reduced to a preferable degree. It is more preferable if the reactivity to both substrates is reduced.

Further, as described above, since FAD-GDH of the present invention is originally an enzyme having excellent substrate specificity for D-glucose, it is about the same as a normal fasting blood glucose level (≦ 126 mg / dL [7 mM]). In the measurement of reactivity using molar concentrations of D-xylose and maltose, it is assumed that almost no measured values suggesting the effect of their coexistence are detected. Therefore, when measuring the reactivity of D-xylose and maltose in FAD-GDH of the present invention, for example, under the condition that D-xylose and / or maltose coexist at an excessive concentration such as 200 mM. It is preferable to perform the measurement using a large amount of enzyme solution having the same molar concentration of D-glucose reactivity of 2 U / ml or more, preferably 5 U / ml or more, more preferably 10 U / ml or more. By performing the measurement under such conditions, it is possible to examine the influence of coexisting substances assuming the same conditions as when a large amount of enzyme is mounted on the glucose sensor.

The ratio of reactivity to D-xylose at the same molar concentration to reactivity to D-glucose (Xyl / Glc (%)) and reaction to maltose at the same molar concentration to reactivity to D-glucose. Since the sex ratio (Mal / Glc (%)) differs depending on the type of transformant, its culture conditions, enzyme activity measurement conditions, etc., it is necessary to compare the respective values before and after the introduction of amino acid substitution under the same conditions. There is.

(Example of natural FAD-GDH from which FAD-GDH of the present invention is derived)
The FAD-GDH of the present invention can also be obtained by modifying a known FAD-GDH. Preferable examples of known FAD-GDH-derived microorganisms include Mucoromycotina, preferably Mucorales, more preferably Mucorales, and even more preferably Mucorales. Specific examples thereof include FAD-GDH derived from the genus Mucor, Absidia, Actinomucor, and Circinella.
Examples of specific preferred microorganisms classified into the genus Mucor include Mucor plainii, Mucor javanicus, and Mucor circinelloides f. circinelloides, Mucor guilliermondii, Mucor hiemalis f. Examples thereof include silvaticus, Mucor subtilissimus, and Mucor dimorphosporus. More specifically, Mucor plainii, Mucor javanicus, and Mucor circinelliides described in Patent Document 5 f. circinelloides, Mucor guilliermondii NBRC9403, Mucor hiemalis f. Examples thereof include silvaticus NBRC6754, Mucor subtilissimus NBRC6338, Mucor RD056860, Mucor dimorphosporus NBRC5395 and the like. Examples of specific preferable microorganisms classified into the genus Absidia include Absidia cylindrospora and Absidia hyalospora. More specifically, Absidia cylindrospora and Absidia hyalospora described in Patent Document 5 can be mentioned. Examples of specific preferred microorganisms that are classified into the genus Actinomucor include Actinomucor elegans. More specifically, Actinomucor elegans described in Patent Document 5 can be mentioned. Examples of specific preferred microorganisms classified into the genus Circinella include Circinella minor, Circinella mucoroides, Circinella muscae, Circinella rigida, and Circinella circin. More specifically, mention may be made of Circinella minor NBRC6448, Circinella mucoroides NBRC4453, Circinella muscae NBRC6410, Circinella rigida NBRC6411, Circinella simplex NBRC6412, Circinella umbellata NBRC4452, Circinella umbellata NBRC5842, Circinella RD055423 and Circinella RD055422. The NBRC strain and the RD strain are stored strains of NBRC (Biotechnology Center, Product Evaluation Technology Infrastructure Organization).

(Acquisition of a gene encoding FAD-GDH of the present invention)
In order to efficiently obtain the FAD-GDH of the present invention, it is preferable to use a genetic engineering method. In order to obtain the gene encoding FAD-GDH of the present invention (hereinafter, FAD-GDH gene), a commonly used gene cloning method may be used. For example, in order to obtain the FAD-GDH of the present invention by using a known FAD-GDH as a starting material and modifying it, a conventional method is used from known microbial cells and various cells having the ability to produce FAD-GDH. For example, chromosomal DNA or mRNA can be extracted by the method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using mRNA as a template. Using the chromosomal DNA or cDNA thus obtained, a library of chromosomal DNA or cDNA can be prepared.

Next, a method of synthesizing an appropriate probe DNA based on known FAD-GDH amino acid sequence information and using this to select a FAD-GDH gene having high substrate specificity from a library of chromosomal DNA or cDNA, or The purpose of encoding FAD-GDH with high substrate specificity by preparing an appropriate primer DNA based on the above amino acid sequence and performing an appropriate polymerase chain reaction (PCR method) such as the 5'RACE method or the 3'RACE method. It is also possible to amplify the DNA containing the gene fragment of the above gene and ligate these DNA fragments to obtain the DNA containing the full length of the target FAD-GDH gene.

Using a known FAD-GDH as a starting material, as a method for obtaining FAD-GDH having high substrate specificity of the present invention, a mutation is introduced into a gene encoding the starting material FAD-GDH and expressed from various mutant genes. It is possible to adopt a method of making a selection using the enzymatic scientific properties of FAD-GDH to be used as an index.
Mutation treatment of the FAD-GDH gene, which is a starting material, can be carried out by any known method depending on the intended mutation form. That is, a method of contacting and acting the FAD-GDH gene or the recombinant DNA in which the gene is integrated with a drug that is a mutagen; an ultraviolet irradiation method; a genetic engineering method; or a method that makes full use of a protein engineering method, etc. Can be widely used.

Examples of the mutagen drug used in the above mutation treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrite, sulfite, hydrazine, formic acid, 5-bromouracil and the like. be able to.
The conditions for contact and action can be set according to the type of drug used and the like, and are not particularly limited as long as a desired mutation can actually be induced in the Mucor-derived FAD-GDH gene. Usually, the desired mutation can be induced by contacting and acting at the above drug concentration of preferably 0.5 to 12 M at a reaction temperature of 20 to 80 ° C. for 10 minutes or longer, preferably 10 to 180 minutes. Even when irradiating with ultraviolet rays, it can be carried out according to the conventional method as described above (Hyundai Chemistry, p24-30, June 1989 issue).

As a method that makes full use of the protein engineering method, a method known as Site-Specific Mutagenesis can be generally used. For example, the Kramer method (Nucleic Acids Res., 12,9441 (1984): Materials Enzymol., 154,350 (1987): Gene, 37,73 (1985)), the Eckstein method (Nucleic Acids Res., 13,8749). 1985): Nucleic Acids Res., 13, 8765 (1985): Nucleic Acids Res, 14, 9679 (1986)), Kunkel method (Proc. Natl. Acid. Sci. USA, 82, 488 (1985). ): Methods Enzymol., 154,367 (1987)) and the like. Specific methods for converting the base sequence in DNA include, for example, a commercially available kit (Transformer Mutagenesis Kit; Clonetech, EXOIII / Mung Bean Deletion Kit; Stratagen, Quick Change Site, etc.) Can be mentioned.

It is also possible to use a technique known as the general Polymerase Chain Reaction (Technique, 1, 11 (1989)).
In addition to the above gene modification method, a modified FAD-GDH gene having high desired substrate specificity can be directly synthesized by an organic synthesis method or an enzyme synthesis method.

When determining or confirming the DNA base sequence of the FAD-GDH gene of the present invention selected by any method as described above, for example, a multicapillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter) or the like can be used. Just do it.

(Vector and host cell into which the FAD-GDH gene of the present invention has been inserted)
The FAD-GDH gene of the present invention obtained as described above is incorporated into a vector such as a bacteriophage, cosmid, or a plasmid used for transformation of prokaryotic cells or eukaryotic cells by a conventional method, and corresponds to each vector. The host cell to be transformed can be transformed or transduced by a conventional method.
Examples of prokaryotic host cells include microorganisms belonging to the genus Escherichia, such as Escherichia coli K-12 strain, Escherichia collie BL21 (DE3), Escherichia collie JM109, Escherichia collie DH5α, Escherichia collie W3110, Escherichia collie C600, etc. Both are manufactured by Takara Bio Co., Ltd.). They are transformed or transduced into them to obtain a host cell (transformant) into which DNA has been introduced. As a method for transferring the recombinant DNA into such a host cell, for example, in the case where the host cell is a microorganism belonging to Escherichia collie, a method of transferring the recombinant DNA in the presence of calcium ions can be adopted, and further. The electroporation method may be used. Further, a commercially available competent cell (for example, ECOS Compent Escherichia Collie BL21 (DE3); manufactured by Nippon Gene) may be used.

An example of a eukaryotic host cell is yeast. Examples of the yeast classified into yeast include yeasts belonging to the genus Zygosaccharomyces, the genus Saccharomyces, the genus Pichia, the genus Candida, and the like. The insert gene may contain a marker gene that allows selection of transformed cells. Examples of the marker gene include genes that complement the auxotrophy of the host, such as URA3 and TRP1. In addition, it is desirable that the inserted gene contains a promoter or other control sequence (for example, enhancer sequence, terminator sequence, polyadenylation sequence, etc.) capable of expressing the gene of the present invention in a host cell. Specific examples of the promoter include a GAL1 promoter, an ADH1 promoter and the like. Known methods for transformation into yeast include, for example, a method using lithium acetate (MethodsMol. Cell. Biol., 5, 255-269 (1995)) and electroporation (J Microbiol Methods 55 (2003) 481). -484) and the like can be preferably used, but the transformation is not limited to this, and transformation may be carried out by using various arbitrary methods including the spheroplast method and the glass bead method.

Other examples of eukaryotic host cells include mold cells such as the genus Aspergillus and the genus Trichoderma. The inserted gene includes a promoter capable of expressing the gene of the present invention in a host cell (for example, tef1 promoter) and other control sequences (for example, secretory signal sequence, enhancer sequence, terminator sequence, polyadenylation sequence, etc.). Is desirable. In addition, the inserted gene may contain a marker gene for enabling selection of transformed cells, such as niaD and pyrG. Further, the inserted gene may contain a homologous recombination region for insertion into an arbitrary chromosomal site. As a method for transformation into filamentous fungi, a known method, for example, a method using polyethylene glycol and calcium chloride after protoplastization (Mol. Gen. Genet., 218, 99-104 (1989)) is preferably used. Can be done.

(Manufacturing of FAD-GDH of the present invention)
The FAD-GDH of the present invention is obtained by culturing a host cell producing the FAD-GDH of the present invention obtained as described above, expressing the flavin-bound glucose dehydrogenase gene contained in the host cell, and then the culture. It may be produced by isolating a flavin-bound glucose dehydrogenase from a substance.
In order to culture the eukaryotic host cells, for example, a YPD (Bactopeptone 2%, Bactate yeast extract 1%, Glucose 2%) liquid medium widely used in the culture of Saccharomyces cerevisiae is preferably used. However, if there are other nutrient sources or ingredients that can improve the production amount of flavine-bound GDH used in the present invention by adding them, they can be added alone or in combination. You may.
The carbon source used in the medium may be any assimilated carbon compound, and examples thereof include glucose, starch hydrolysate, glycerin, fructose, and molasses. The nitrogen source may be any available nitrogen compound, and examples thereof include yeast extract, peptone, meat extract, corn steep liquor, soybean flour, malt extract, amino acids, ammonium sulfate, ammonium nitrate and the like. Examples of the inorganic substance include various salts such as salt, potassium chloride, magnesium sulfate, manganese chloride, ferrous sulfate, primary potassium phosphate, dipotassium phosphate, sodium carbonate, and calcium chloride. In addition, vitamins, antifoaming agents and the like may be added as needed.

As the medium for culturing the nuclear host cells, for example, sodium chloride, first phosphate, etc. are added to one or more nitrogen sources such as yeast extract, tripton, peptone, meat extract, corn steep liquor or leachate of soybean or wheat bran. Add one or more of inorganic salts such as potassium, dipotassium phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate, and if necessary, add sugar raw materials, vitamins, etc. as appropriate. Is used.

The culturing conditions may differ depending on the microorganism to be cultivated. For example, the initial pH of the medium is adjusted to pH 5 to 10, the culturing temperature is 20 to 40 ° C., and the culturing time is 15 to 25 hours, 1 to 2. It can be appropriately set for days, 10 to 50 hours, etc., and is carried out by aeration-stirring deep culture, shaking culture, static culture, or the like.
For example, as an example of the medium and culture conditions for culturing yeast of the genus Saccharomyces, a medium containing 2% bactopeptone, 1% bactoeast extract, and 2% glucose was used at 30 ° C. and 200 rpm for 24 hours. Shaking is mentioned. For example, the culture of Escherichia collie is carried out at a culture temperature of 10 to 42 ° C., preferably a culture temperature of about 25 ° C. for 4 to 24 hours, and more preferably a culture temperature of about 25 ° C. for 4 to 8 hours. , Shaking culture, static culture, etc.

After completion of the culture, in order to collect the flavin-bound GDH from the inside of the culture or the cultured cells, a usual enzyme collection means can be used.
When the enzyme is present in the cells, it is preferable to separate the cells from the culture by, for example, filtration, centrifugation, or the like, and collect the enzyme from the cells. For example, a method of destroying bacterial cells using a normal destruction means such as an ultrasonic crusher, a French press, or a dynomill, a method of lysing a bacterial cell wall using a cell wall lytic enzyme such as lysozyme, Triton X-100, etc. A method of extracting an enzyme from a cell using the above-mentioned surfactant can be adopted alone or in combination.
When the enzyme is present outside the cells, the cells may be separated by an operation such as filtration or centrifugation, and the supernatant may be collected. Then, the insoluble matter is removed by filtration or centrifugation to obtain an enzyme extract. In order to isolate and purify flavin-bound GDH from the obtained extract as necessary, nucleic acids are removed as necessary, and then ammonium sulfate, alcohol, acetone, etc. are added thereto for fractionation and precipitation. A product can be collected to obtain the crude enzyme of FAD-GDH of the present invention.

The crude enzyme of FAD-GDH of the present invention can also be further purified by any known means. To obtain a purified enzyme preparation, for example, a gel filtration method using sephadex, ultrogel, biogel, etc .; an adsorption elution method using an ion exchanger; an electrophoresis method using a polyacrylamide gel, etc.; adsorption using hydroxyapatite. Elution method; Sedimentation method such as sucrose density gradient centrifugation method; Affinity chromatography method; Fractionation method using molecular sieving membrane or hollow fiber membrane, etc., or a combination thereof was carried out for purification. The FAD-GDH enzyme preparation of the present invention can be obtained.

(D-glucose measuring method using FAD-GDH of the present invention)
The present invention also discloses a glucose assay kit containing the FAD-GDH of the present invention, for example, by using such a glucose assay kit, D-glucose (blood glucose) in blood using the FAD-GDH of the present invention. Value) can be measured.
The glucose assay kit of the present invention comprises a modified FAD-GDH according to the present invention in an amount sufficient for at least one assay. Typically, the glucose assay kits of the invention, in addition to the modified FAD-GDH of the invention, use the buffers required for the assay, mediators, D-glucose standard solution for making calibration curves, and use. Includes guidelines. Modified FAD-GDH according to the present invention can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution.

In the case of the colorimetric glucose assay kit, the D-glucose concentration can be measured, for example, as follows. The reaction layer of the glucose assay kit contains FAD-GDH, electron acceptors, and N- (2-acetamido) imide diacetic acid (ADA) and bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis) as reaction promoters. A liquid or solid composition containing one or more substances selected from the group consisting of −Tris), sodium carbonate and imidazole is retained. Here, a pH buffer and a color-developing reagent are added as needed. A sample containing D-glucose is added thereto, and the mixture is reacted for a certain period of time. During this period, the absorbance corresponding to the maximum absorption wavelength of the electron acceptor that fades due to reduction or the dye that is polymerized and produced by receiving an electron from the electron acceptor is monitored. In the case of the rate method, the rate of change in absorbance per hour, and in the case of the endpoint method, the change in absorbance up to the point when all D-glucose in the sample is oxidized, a standard concentration of D-glucose solution is used in advance. The D-glucose concentration in the sample can be calculated based on the calibration curve prepared in the above.

As mediators and color-developing reagents that can be used in this method, for example, 2,6-dichlorophenol indophenol (DCIP) can be added as an electron acceptor, and D-glucose can be quantified by monitoring the decrease in absorbance at 600 nm. be. Further, phenazinemethsulfate (PMS) is added as an electron acceptor, and nitrotetrazolium blue (NTB) is added as a coloring reagent, and the amount of diformazan produced is determined by measuring the absorbance at 570 nm to calculate the D-glucose concentration. It is possible. Needless to say, the electron acceptor and the color-developing reagent used are not limited thereto.

(Glucose sensor containing FAD-GDH of the present invention)
The present invention also discloses a glucose sensor using the FAD-GDH of the present invention. As the electrode, a carbon electrode, a gold electrode, a platinum electrode, or the like is used, and the enzyme of the present invention is immobilized on the electrode. Immobilization methods include a method using a cross-linking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, an oxidation-reduced polymer, etc., or a ferrocene or a derivative thereof. It may be fixed in a polymer or adsorbed and fixed on an electrode together with a representative electronic mediator, or these may be used in combination. Typically, the modified FAD-GDH of the present invention is immobilized on a carbon electrode with glutaraldehyde and then treated with a reagent having an amine group to block glutaraldehyde.

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

As a specific example, the 1.5 U FAD-GDH of the present invention is immobilized on a glassy carbon (GC) electrode, and the response current value with respect to the D-glucose concentration is measured. To the electrolytic cell, 1.8 ml of 50 mM potassium phosphate buffer (pH 6.0) and 0.2 ml of a 1 M potassium hexacyanoferrate (III) aqueous solution (potassium ferricyanide) are added. A GC electrode is connected to a potentiostat BAS100B / W (manufactured by BAS), the solution is stirred at 37 ° C. and + 500 mV is applied to the silver-silver chloride reference electrode. A 1MDD-glucose solution is added to these systems so that the final concentration is 5, 10, 20, 30, 40, 50 mM, and the steady-state current value is measured for each addition. This current value is plotted against a known D-glucose concentration (5, 10, 20, 30, 40, 50 mM) and a calibration curve is created. This makes it possible to quantify D-glucose with an enzyme-immobilized electrode using the FAD-bound glucose dehydrogenase of the present invention.

Since the FAD-GDH of the present invention has excellent substrate specificity even when compared with the conventional FAD-GDH derived from the genus Mucor, it has an excellent effect especially when applied to a glucose sensor as described above. Is expected to play. In the glucose sensor, it is assumed that the enzymatic reaction is carried out under special conditions in which a larger amount of enzyme is loaded, as compared with the case where it is applied to a liquid reagent kit or the like. This is because the need to reduce the impact is particularly high.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the technical scope of the present invention is not limited to those examples.

In the present invention, the substrate specificity and thermal stability of the modified FAD-GDH were evaluated according to the methods of the following test examples unless otherwise specified.

[Test example]
(1) Preparation of yeast transformants expressing various modified FAD-GDHs A set encoding the Mucor plasmid-derived FAD-GDH gene (wild-type MpGDH gene) of SEQ ID NO: 2 according to the method described in Patent Document 7. A transformant plasmid (pYES2C-Mp (wild type)) was obtained.
Using the obtained recombinant plasmid pYE2C-Mp as a template, a synthetic nucleotide for introducing each amino acid substitution, KOD-Plus- (manufactured by Toyobo Co., Ltd.), was used, and a PCR reaction was carried out under the following conditions.
That is, 5 μl of 10 × KOD-Plus-buffer solution, 5 μl of dNTPs mixed solution prepared to have dNTPs of 2 mM each, 2 μl of 25 mM _{sulfonyl 4} solution, 50 ng of pYE2C-Mp as a template, and the above synthetic oligonucleotide. To each of 15 pmol and 1 Unit of KOD-Plus- were added, and the total volume was adjusted to 50 μl with sterilized water to prepare a “reaction solution”. Incubate the prepared reaction solution at 94 ° C. for 2 minutes using a thermal cycler (manufactured by Eppendorf), followed by "94 ° C., 15 seconds"-"55 ° C., 30 seconds"-"68 ° C., 8 minutes". The cycle of was repeated 30 times.

A part of the reaction solution after the above treatment was electrophoresed on a 1.0% agarose gel, and it was confirmed that about 8 kbp of DNA was specifically amplified. The amplified DNA was treated with a restriction enzyme DpnI (manufactured by New England Biolabs) and then transformed by mixing with competent cells of Escherichia coli JM109 strain (manufactured by Nippon Gene) according to the attached protocol. Then, each of the obtained transformants was applied to LB-amp agar medium and cultured. The grown colonies were inoculated into an LB-amp liquid medium, cultured with shaking, and various plasmid DNAs containing about 8 kbp of amplified DNA were prepared using a GenElute Plasmamid Miniprep Kit (manufactured by sigma) according to the attached protocol. For example, pYE2C-Mp-V83A, pYE2C-Mp-V83G, etc. in Example 1) were isolated. Next, the nucleotide sequence of the DNA encoding the MpGDH gene in these various plasmid DNAs was determined using the multicapillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter), and in each sequence, the amino acid shown in SEQ ID NO: 1 was determined. It was confirmed that the amino acid at a predetermined position in the sequence was substituted. In this way, a yeast expression vector pYE2C-Mp (modified form, for example, pYE2C-Mp-V83A, pYE2C-Mp-V83G, etc. in Example 1) encoding a modified MpGDH in which a predetermined amino acid is substituted is obtained. bottom.
After that, S. Using a transformation kit for yeast (manufactured by Invitrogen), pYE2C-Mp (wild type) and pYES2C-Mp (modified type, for example, pYE2C-Mp-V83A, pYE2C- in Example 1) into which various mutations were introduced. By transforming Mp-V83G, etc.) into an Inv-Sc strain (manufactured by Invitrogen), a yeast transformant Sc-Mp (wild type) strain expressing wild-type MpGDH and various modified MpGDH are expressed. Yeast transformant Sc-Mp (modified type, for example, Sc-Mp-V83A, Sc-Mp-V83G, etc. in Example 1) strains were obtained, respectively.

(2) Evaluation of substrate specificity Yeast transformant Sc-Mp (wild type) and various yeast transformants Sc-Mp (modified form, for example, Sc-Mp-V83A, Sc-Mp- in Example 1). V83G, etc.) in 5 mL of pre-culture liquid medium "0.67% (w / v) amino acid-free yeast nitrogen base (BD), 0.192% (w / v) uracil-free yeast synthetic drop, respectively. In the out medium additive (manufactured by sigma), 2.0% (w / v) raffinose, the cells were cultured at 30 ° C. for 24 hours. Then, 1 mL of the preculture solution was added to 4 mL of the liquid medium for main culture "0.67% (w / v) amino acid-free yeast nitrogen base, 0.192% (w / v) uracil-free yeast synthetic dropout medium. In addition to "additives, 2.5% (w / v) D-galactose, 0.75% (w / v) raffinose", incubate at 30 ° C. for 16 hours. This culture broth was separated into bacterial cells and culture supernatant by centrifugation (10,000 × g, 4 ° C., 3 minutes), and the culture supernatant was used for evaluation of substrate specificity.
Specifically, the activity of each substrate was measured by changing the substrate of the above-mentioned activity measurement method from D-glucose to a system of D-xylose having the same molar concentration. Then, from these values, "the ratio of reactivity to D-xylose to reactivity to D-glucose (Xyl / Glc (%))" was calculated.
The (Xyl / Glc (%)) of wild-type MpGDH expressed in the Sc-Mp (wild-type) strain was 1.47%. Such substrate specificity is extremely excellent as compared with other conventionally known FAD-GDH, and it is expected that D-glucose, which is a substance to be measured, can be measured accurately.
Further, when the value of Xyl / Glc (%) in Mucor-derived FAD-GDH before the introduction of the site-specific mutation in various mutants is set to 100%, the relative value of the modified FAD-GDH after the introduction of the site-specific mutation is shown. The "Xyl / Glc ratio" representing the specificity of the substrate was calculated. "In the modified FAD-GDH having an Xyl / Glc ratio of less than 100, the reactivity to D-xylose is decreased and the substrate specificity is increased as compared with FAD-GDH before the introduction of the site-specific mutation. The smaller the value, the greater the degree.

(3) Evaluation of specific activity Yeast transformant Sc-Mp (wild type) and various yeast transformants Sc-Mp (modified type, for example, Sc-Mp-T367A, Sc-Mp-A385T in Example 2) Etc.), respectively, in 10 mL of pre-culture liquid medium "0.67% (w / v) amino acid-free yeast nitrogen base (BD), 0.192% (w / v) uracil-free yeast synthetic dropout. Incubation was carried out at 30 ° C. for 24 hours in "addition for medium (manufactured by sigma), 2.0% (w / v) raffinose". Then, 10 mL of the preculture solution was added to 40 mL of the liquid medium for main culture "0.67% (w / v) amino acid-free yeast nitrogen base, 0.192% (w / v) uracil-free yeast synthetic dropout medium. In addition to the additive, 2.5% (w / v) D-galactose, 0.75% (w / v) raffinose, the cells were cultured at 30 ° C. for 14 hours. This culture broth was separated into cells and culture supernatant by centrifugation (10,000 × g, 4 ° C., 3 minutes), and it was confirmed that the culture supernatant had GDH activity and used in the following operations. ..
The collected culture supernatant containing FAD-GDH to be evaluated was concentrated with a centrifugal filter unit (Amicon Ultra-15 30K, manufactured by MERCK MILLIPORE), and then replaced with 20 mM potassium phosphate buffer (pH 6.0). ..
Next, the recovered concentrate was placed on a column of Superdex 200 10/300 GL (manufactured by GE Healthcare Bioscience) pre-equilibrium with 20 mM potassium phosphate buffer (pH 6.0) containing 150 mM sodium chloride to activate it. Purified enzyme was obtained by collecting the fraction.
The specific activity of the purified enzyme was measured as the activity (U / A280) per absorbance (A280) at 280 nm using the above-mentioned GDH activity measurement method. Then, the "relative specific activity" in the modified MpGDH when the specific activity of the wild-type MpGDH was set to 100 was calculated. When the relative specific activity is not much less than 100, it can be seen that the modified MpGDH does not have a significantly lower specific activity.

[Example 1]
(Preparation of various modified MpGDH and evaluation of substrate specificity)
According to the method described in the above test example, a PCR reaction was carried out using pYE2C-Mp (wild type) as a template plasmid with a combination of synthetic nucleotides having the SEQ ID NOs shown in Table 1. Next, the E. coli JM109 strain was transformed with a vector containing amplified DNA, and the base sequence of the DNA encoding MpGDH in the plasmid DNA retained by the grown colony was determined to determine the basis of the DNA according to SEQ ID NO: 1. Alanine at position 83 of the amino acid sequence is alanine, alanine at position 42 is glycine, valine at position 83 is glycine, serine at position 174 is proline, serine at position 174 is leucine, and threonine at position 376 is alanine. Threonin at position 376 is for serine, threonine at position 376 is for asparagine, alanine at position 385 is for aspartic acid, alanine at position 385 is for aspartic acid, alanine at position 385 is for arginine, and threonine at position 386 is for valine. 386th threonine is isoleucine, 386th threonine is glycine, 386th threonine is leucine, 386th threonine is glutamic acid, 386th threonine is glutamine, 386th threonine is aspartic acid, 386th. Threonine to serine, leucine at position 391 to valine, phenylalanine at position 460 to tyrosine, phenylalanine at position 460 to leucine, threonine at position 461 to methionine, threonine at position 461 to phenylalanine, threonine at position 461. Is glycine, threonine at position 461 is aspartic acid, threonine at position 461 is cysteine, serine at position 467 is leucine, serine at position 467 is methionine, serine at position 467 is isoleucine, and serine at position 467. PYE2C-Mp-A42G, pYE2C-Mp-V83A, pYE2C- which are recombinant plasmids in which serine at position 467 is replaced with aspartic acid, serine at position 467 is replaced with glutamic acid, and glycine at position 468 is replaced with alanine. Mp-V83G, pYE2C-Mp-S174P, pYE2C-Mp-S174L, pYE2C-Mp-T367A, pYE2C-Mp-T367S, pYE2C-Mp-T367N, pYE2C-Mp-A385T, pYE2C-Mp-A385T, pYE2C-Mp-A385T A385R, pYE2C-Mp-T386V, pYE2C-Mp-T386I, pYE2C-Mp-T386G, pYE2C-Mp-T386L, pYE2C-Mp-T386E, pYE2C-Mp-T386Q, pYE2C-Mp-T386Q, pYE2C-M. pYE2C-Mp-L39 1V, pYE2C-Mp-F460Y, pYE2C-Mp-F460L, pYE2C-Mp-T461M, pYE2C-Mp-T461F, pYE2C-Mp-T461G, pYE2C-Mp-T461D, pYE2C-Mp, pYE2C-Mp-T461D pYE2C-Mp-S467M, pYE2C-Mp-S467I, pYE2C-Mp-S467V, pYE2C-Mp-S467D, pYE2C-Mp-S467E, and pYE2C-Mp-G468T were obtained, respectively.

Then, the recombinant plasmids pYE2C-Mp-A42G, pYE2C-Mp-V83A, pYE2C-Mp-V83G, pYE2C-Mp-S174P, pYE2C-Mp- S174L, pYE2C-Mp-T367A, pYE2C-Mp-T367S, pYE2C-Mp-T367N, pYE2C-Mp-A385T, pYE2C-Mp-A385D, pYE2C-Mp-A385R, pYE2C-Mp-A385R, pYE2C-M. pYE2C-Mp-T386G, pYE2C-Mp-T386L, pYE2C-Mp-T386E, pYE2C-Mp-T386Q, pYE2C-Mp-T386N, pYE2C-Mp-T386S, pYE2C-Mp-T386S, pYE2C-Mp-Mp-L39 Mp-F460L, pYE2C-Mp-T461M, pYE2C-Mp-T461F, pYE2C-Mp-T461G, pYE2C-Mp-T461D, pYE2C-Mp-T461C, pYE2C-Mp-S467L, pYE2C-Mp-S467L Using S467I, pYE2C-Mp-S467V, pYE2C-Mp-S467D, pYE2C-Mp-S467E, pYE2C-Mp-G468T, the Inv-Sc strain was transformed and acquired according to the item of Test Example (2). Convertible strains (Sc-Mp-A42G, Sc-Mp-V83A, Sc-Mp-V83G, Sc-Mp-S174P, Sc-Mp-S174L, Sc-Mp-T367A, Sc-Mp-T367S, Sc-Mp-T367N , Sc-Mp-A385T, Sc-Mp-A385D, Sc-Mp-A385R, Sc-Mp-T386V, Sc-Mp-T386I, Sc-Mp-T386G, Sc-Mp-T386L, Sc-Mp-T386E, Sc -Mp-T386Q, Sc-Mp-T386N, Sc-Mp-T386S, Sc-Mp-L391V, Sc-Mp-F460Y, Sc-Mp-F460L, Sc-Mp-T461M, Sc-Mp-T461F, Sc-Mp -T461G, Sc-Mp-T461D, Sc-Mp-T461C, Sc-Mp-S467L, Sc-Mp-S467M, Sc-Mp-S467I, Sc-Mp-S467V, Sc-Mp-S467D, Sc-Mp-S467E , Sc-Mp-G468T,) Was cultured, and the GDH activity in the culture supernatant was measured.

Subsequently, using the culture supernatants of the above-mentioned various variants whose GDH activity was confirmed, the reaction to D-xylose with respect to the reactivity to D-glucose based on the procedure of the item of the above-mentioned test example (2). The sex ratios "Xyl / Glc (%)" and "Xyl / Glc ratio" were measured.

For example, in Table 1, "V83A" means that V (Val) at the 83rd position is replaced with A (Ala).

As shown in Table 1, the sites of SEQ ID NO: 1 with respect to wild-type MpGDH at positions 42, 83, 174, 376, 385, 386, 391, 460, 461, 467, or 468. Specific mutagenesis, specifically A42G, V83A, V83G, S174P, S174L, T367A, T367S, T367N, A385T, A385D, A385R, T386V, T386I, T386G, T386L, T386E, T386Q, T386T Reactivity of FAD-GDH to xylose is reduced by introducing site-specific mutations in F460Y, F460L, T461M, T461F, T461G, T461D, T461C, S467L, S467M, S467I, S467V, S467D, S467E, G468T. It was confirmed that.

[Example 2]
(Evaluation of specific activity in various modified MpGDH)
The specific activities of the mutants A42G, T367A, A385T, T386S, F460Y, S467V, S467D, and S467E obtained in Example 1 were measured. According to the method described in Test Example (3), a PCR reaction was carried out using pYE2C-Mp (wild type) as a template plasmid with a combination of synthetic nucleotides having the SEQ ID NOs shown in Table 2. Next, the E. coli JM109 strain was transformed with a vector containing the amplified DNA, and the DNA encoding MpGDH in the plasmid DNA held by the grown colony was sequenced to determine the base sequence of the DNA according to SEQ ID NO: 1. Sleonine at position 367 in the amino acid sequence is alanine, alanine at position 385 is threonine, threonine at position 386 is serine, phenylalanine at position 460 is tyrosine, serine at position 467 is valine, and serine at position 467 is aspartic acid. In addition, pYE2C-Mp-A42G, pYE2C-Mp-T367A, pYE2C-Mp-A385T, pYE2C-Mp-T386S, pYE2C-Mp-F460Y, pYE2C, which are recombinant plasmids in which serine at position 467 is replaced with glutamic acid. Mp-S467V, pYE2C-Mp-S467D, and pYE2C-Mp-S467E were obtained, respectively.

Then, the recombinant plasmids pYE2C-Mp-A42G, pYE2C-Mp-T367A, pYE2C-Mp-A385T, pYE2C-Mp-T386S, pYE2C-Mp- Inv-Sc strain was transformed and obtained transformant (Sc-Mp) using F460Y, pYE2C-Mp-S467V, pYE2C-Mp-S467D, pYE2C-Mp-S467E according to the item of Test Example (3). -A42G, Sc-Mp-T367A, Sc-Mp-A385T, Sc-Mp-T386S, Sc-Mp-F460Y, Sc-Mp-S467D, Sc-Mp-S467E) were cultured, and GDH in the culture supernatant was performed. The activity was measured.

Subsequently, using the culture supernatants of the above-mentioned various variants whose GDH activity was confirmed, the reaction to D-xylose with respect to the reactivity to D-glucose based on the procedure of the item of the above-mentioned test example (2). The sex ratios "Xyl / Glc (%)" and "Xyl / Glc ratio" were measured.
Subsequently, using the culture supernatants of the above-mentioned various variants whose GDH activity was confirmed, purification was performed based on the procedure of the item of Test Example (3) above, and the specific activity of the purified enzyme was measured. Then, the "relative specific activity" was calculated.
As a comparative example, pYE2C-Mp-L121M, pYE2C-Mp-W123V, pYE2C-Mp-S612C, and pYE2C-Mp-W569Y described in Patent Document 7 were transformed and obtained from the Inv-Sc strain according to the item of Test Example (1). The transformants (Sc-Mp-L121, Sc-Mp-W123V, Sc-Mp-S612C, Sc-Mp-W569Y) were cultured, and the GDH activity of the culture supernatant was measured.
Subsequently, the ratio of the reactivity to D-xylose to the reactivity to D-glucose based on the procedure of the item of Test Example (2) using the culture supernatants of the above-mentioned various variants whose GDH activity was confirmed. "Xyl / Glc (%)" and "Xyl / Glc ratio" were measured.
Subsequently, using the culture supernatants of the above-mentioned various mutants whose GDH activity was confirmed, purification was performed based on the procedure of the item of Test Example (3) above, and then the purified enzyme had "relative specific activity". (%) ”Was measured.
In all the comparative examples, the substrate specificity was as low as 34 to 64%, but the relative specific activity was about 60%. On the other hand, the enzyme of the present invention has improved substrate specificity by 10% or more while maintaining a specific activity of 65% or more.

[Example 3]
(Preparation of various modified MrdGDH and evaluation of substrate specificity)
Japanese Patent Application Laid-Open No. 2013-176363 discloses the sequence information of GDH (hereinafter referred to as MrdGDH) derived from Mucor RD056860 of SEQ ID NO: 56, which has 73% identity with MpGDH. Therefore, it was decided to verify as follows whether or not the reactivity to xylose is similarly reduced by introducing the amino acid substitution found in Example 1 into MrdGDH. First, the nucleotide sequence of SEQ ID NO: 57, in which the amino acid of SEQ ID NO: 56 was encoded and the codon was modified for recombinant expression, was obtained by total synthesis. Using this as a template, the synthetic nucleotides of SEQ ID NOs: 58 and 59 and Prime STAR Max DNA polymerase (manufactured by TakaRa) were used, and a PCR reaction was carried out according to the attached protocol. The PCR reaction solution was electrophoresed on a 1.0% agarose gel, and about 2 kb of "DNA fragment for insert" was purified using RECOCHIP (manufactured by TakaRa). In addition, the plasmid pYES2 / CT (manufactured by Invitrogen) for expressing Saccharomyces cerevisiae was treated with the restriction enzyme KpnI (manufactured by New England Biolabs), and the reaction solution after the restriction enzyme treatment was electrophoresed on a 1.0% agarose gel and RECOCHIP. (Manufactured by TakaRa) was used to purify about 6 kb of "DNA fragment for vector". Subsequently, the purified "DNA fragment for insert" and "DNA fragment for vector" are ligated using the In-Fusion HD Cloning Kit (manufactured by Cloning) according to the attached plasmid, and MpGDH is expressed under the GAL1 promoter. Recombinant plasmid pYE2C-Mrd was prepared. Next, in the same manner as described in the above test example, a PCR reaction was carried out using pYE2C-Mrd (wild type) as a template plasmid with a combination of synthetic nucleotides having the SEQ ID NOs shown in Table 3. The position corresponding to MpGDH in MrdGDH was determined by using the multiple alignment program ClustalW (http://www.genome.jp/tools/crustalw/) on the WEB. As a result, the positions corresponding to threonine at position 386 and serine at position 467 in MpGDH were threonine at position 383 and threonine at position 464 in MrdGDH, respectively. Next, the E. coli JM109 strain was transformed with a vector containing the amplified DNA, and the DNA encoding MrdGDH in the plasmid DNA held by the grown colony was sequenced to determine the base sequence of the DNA according to SEQ ID NO: 56. PYE2C-Mrd-T383S, which is a recombinant plasmid in which threonine at position 383 of the amino acid sequence is replaced with serine, threonine at position 383 is replaced with aspartic acid, threonine at position 464 is replaced with aspartic acid, and threonine at position 464 is replaced with glutamic acid. pYE2C-Mrd-T383N, pYE2C-Mrd-T464D, and pYE2C-Mrd-T464E were obtained, respectively.

Then, the test was performed using the recombinant plasmids pYE2C-Mrd-T383S, pYE2C-Mrd-T383N, pYE2C-Mrd-T464D, and pYE2C-Mrd-T464E encoding the various modified MrdGDH described above into which site-specific mutations were introduced. According to the item of Example (2), the transformation of the Inv-Sc strain and the culture of the obtained transformants (Sc-Mrd-T383S, Sc-Mrd-T383N, Sc-Mrd-T464D, Sc-Mrd-T464E) are carried out. The GDH activity in the culture supernatant was measured.

Subsequently, using the culture supernatants of the above-mentioned various variants whose GDH activity was confirmed, the reaction to D-xylose with respect to the reactivity to D-glucose based on the procedure of the item of the above-mentioned test example (2). The sex ratios "Xyl / Glc (%)" and "Xyl / Glc ratio" were measured.

As shown in Table 3, by introducing site-specific mutations at positions 383 and 464 to wild-type MrdGDH of SEQ ID NO: 56, specifically, by introducing site-specific mutations of T383S, T383N, T464D, and T464E. It was confirmed that the reactivity to xylose was reduced as in MpGDH.

Further, using the culture supernatant of the above-mentioned MrdGDH mutants (T383S, T464D), the specific activity of the purified enzyme was measured based on the procedure of the item of the above-mentioned test example (3), and the "relative specific activity" was determined. Calculated. As a result, the relative specific activities of T383S and T464D were 100% and 123%, respectively, and these mutations are very effective for reducing the reactivity to xylose without lowering the specific activity even in MrdGDH. I understand.

As described above, the FAD-GDH of the present invention contains a large amount of sugar compounds other than D-glucose such as D-xylose because the substrate specificity for D-glucose is sufficiently high while maintaining the specific activity. The D-glucose concentration can be accurately measured even when the D-glucose in the sample is measured under the above-mentioned conditions or when the enzyme concentration is high. For example, in the application to a glucose sensor, the conventional FAD- It is expected that more accurate and highly sensitive measurement will be possible as compared with the case of using GDH.

Claims (13)

(A) In the amino acid sequence shown by SEQ ID NO: 1,
Mutation of the amino acid at position 42 corresponding to alanine to glycine,
Mutation of the amino acid at the position corresponding to valine at position 83 to alanine or glycine,
Mutation of the amino acid at the position corresponding to serine at position 174 to proline or leucine,
Mutation of the amino acid at the position corresponding to threonine at position 367 to alanine, serine or asparagine,
Mutation of the amino acid corresponding to alanine at position 385 to threonine, aspartic acid or arginine,
Mutation of the amino acid corresponding to threonine at position 386 to valine, isoleucine, glycine, leucine, glutamic acid, glutamine, asparagine or serine,
Mutation of the amino acid corresponding to leucine at position 391 to valine,
Mutation of the amino acid at position 460 corresponding to phenylalanine to tyrosine or leucine,
Mutation of the amino acid corresponding to threonine at position 461 to methionine, phenylalanine, glycine, aspartic acid or cysteine,
Selected from the group consisting of mutations of the amino acid corresponding to serine at position 467 to leucine, methionine, isoleucine, valine, aspartic acid or glutamic acid, and mutation of the amino acid corresponding to glycine at position 468 to threonine. Amino acid sequence in which at least one mutation has been introduced,
(B) An amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1 and that retains at least one mutation, or 1 or number in the amino acid sequence shown in (C) (A). A modified flavine-bound glucose dehydrogenase comprising an amino acid sequence in which an amino acid is deleted, substituted, inserted or added and retains at least one of the above mutations.
Compared with before the mutation, the ratio of reactivity to D-xylose to reactivity to D-glucose (Xyl / Glc (%)) was reduced, and the amino acid sequence shown in SEQ ID NO: 1 relative specific activity of 100% against D- glucose wild-type flavin-binding glucose dehydrogenase consisting of, specific activity against D- glucose of the modified flavin-binding glucose dehydrogenase is at least 65%, the modified flavin-binding glucose Dehydrogenase. (A') In the amino acid sequence shown by SEQ ID NO: 56,
Valine amino acid position corresponding to position 383 of threonine, isoleucine, glycine, leucine, glutamic acid, glutamine, asparagine or mutation to serine, and 464 of the threonine mutation to aspartate or glutamate of amino acid positions corresponding A modified flavin-linked glucose dehydrogenase comprising an amino acid sequence in which at least one mutation selected from the group consisting of is introduced.
The ratio of reactivity to D-xylose (Xyl / Glc (%)) to reactivity to D-glucose is reduced as compared to before the mutation, and the amino acid sequence shown in SEQ ID NO: 56. relative to a wild-type flavin-binding specific activity of 100% against D- glucose-glucose dehydrogenase consisting of specific activity against D- glucose of the modified flavin-binding glucose dehydrogenase is 100% or more, modified flavin-binding glucose Dehydrogenase. The ratio of reactivity to D-xylose to reactivity to D-glucose (Xyl / Glc (%)) is characterized by being reduced by 10% or more as compared with that before the introduction of the above-mentioned mutation. The modified flavin-bound glucose dehydrogenase according to claim 1. Modified flavin-binding glucose dehydrogenase gene encoding a modified flavin-binding glucose dehydrogenase according to any one of claims 1 to 3. Recombinant vector comprising a modified flavin-binding glucose dehydrogenase gene according to claim 4, wherein. A host cell containing the recombinant vector according to claim 5. A method for producing a modified flavin-bound glucose dehydrogenase.
Method including the following steps:
(A) The step of culturing the host cell according to claim 6.
(B) A step of expressing a modified flavin-bound glucose dehydrogenase gene contained in the host cell.
(C) A step of isolating a modified flavin-bound glucose dehydrogenase from the culture. A method for measuring glucose, which comprises using the modified flavin-bound glucose dehydrogenase according to any one of claims 1 to 3. A glucose assay kit comprising the modified flavin-bound glucose dehydrogenase according to any one of claims 1 to 3. A glucose sensor comprising the modified flavin-bound glucose dehydrogenase according to any one of claims 1 to 3. (A) In the amino acid sequence shown by SEQ ID NO: 1,
Mutation of the amino acid at position 42 corresponding to alanine to glycine,
Mutation of the amino acid at the position corresponding to valine at position 83 to alanine or glycine,
Mutation of the amino acid at the position corresponding to serine at position 174 to proline or leucine,
Mutation of the amino acid at the position corresponding to threonine at position 367 to alanine, serine or asparagine,
Mutation of the amino acid corresponding to alanine at position 385 to threonine, aspartic acid or arginine,
Mutation of the amino acid corresponding to threonine at position 386 to valine, isoleucine, glycine, leucine, glutamic acid, glutamine, asparagine or serine,
Mutation of the amino acid corresponding to leucine at position 391 to valine,
Mutation of the amino acid at position 460 corresponding to phenylalanine to tyrosine or leucine,
Mutation of the amino acid corresponding to threonine at position 461 to methionine, phenylalanine, glycine, aspartic acid or cysteine,
Selected from the group consisting of mutations of the amino acid corresponding to serine at position 467 to leucine, methionine, isoleucine, valine, aspartic acid or glutamic acid, and mutation of the amino acid corresponding to glycine at position 468 to threonine. Amino acid sequence in which at least one mutation has been introduced,
(B) An amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1 and that retains at least one mutation, or 1 or number in the amino acid sequence shown in (C) (A). A step of preparing a modified flavin-linked glucose dehydrogenase to be evaluated, which comprises an amino acid sequence in which individual amino acids are deleted, substituted, inserted or added, and which retains at least one mutation, and the modified form to be evaluated. From the flavin-bound glucose dehydrogenase, the ratio of reactivity to D-xylose (Xyl / Glc (%)) to the reactivity to D-glucose was reduced as compared with before the mutation, and SEQ ID NO: 1 A step of selecting a modified flavin-linked glucose dehydrogenase having a specific activity of 65% or more for D-glucose with respect to 100% of the specific activity of the wild-type flavin-linked glucose dehydrogenase consisting of the amino acid sequence shown in A method for producing a modified flavin-bound glucose dehydrogenase, which comprises. (A') In the amino acid sequence shown by SEQ ID NO: 56,
Valine amino acid position corresponding to position 383 of threonine, isoleucine, glycine, leucine, glutamic acid, glutamine, asparagine or mutation to serine, and 464 of the threonine mutation to aspartate or glutamate of amino acid positions corresponding From the step of preparing a modified flavin-linked glucose dehydrogenase to be evaluated, which comprises an amino acid sequence in which at least one mutation selected from the group consisting of is introduced, and the modified flavin-linked glucose dehydrogenase to be evaluated. The ratio of reactivity to D-xylose (Xyl / Glc (%)) to the reactivity to D-glucose is reduced as compared with that before the mutation, and it consists of the amino acid sequence shown by SEQ ID NO: 56. Modified flavin-bound type, which comprises the step of selecting a modified flavin-bound glucose dehydrogenase having a specific activity of 100% or more of D-glucose with respect to 100% of the specific activity of wild-type flavin-bound glucose dehydrogenase to D-glucose. Method for producing glucose dehydrogenase. A method for producing a glucose sensor, which comprises using the modified flavin-bound glucose dehydrogenase obtained by the production method according to claim 11 or 12 for a glucose sensor.