JP2018201486A - Glucose dehydrogenase with high storage stability and continuous glucose monitoring - Google Patents

Abstract

To provide FAD-GDH with improved storage stability.SOLUTION: Disclosed herein is a FAD-GDH comprising an amino acid sequence having at least 70% identity to a specific amino acid sequence or an amino acid sequence with one or several deletion, substitution or addition of amino acids in the above amino acid sequence, where the FAD-GDH has amino acid substitutions at positions corresponding to the positions 196, 228, 600, 267, 276, 55, 230, 235, 239, 435, 587, 588, and/or 591 of the specific amino acid sequence, and exhibits improved storage stability compared to the amino acid sequence not substituted.SELECTED DRAWING: None

Description

  The present invention relates to a glucose dehydrogenase excellent in storage stability, a glucose measurement method such as continuous blood glucose measurement using the same, and a method for producing glucose dehydrogenase.

  Blood glucose concentration (blood glucose level) is an important marker of diabetes. Autologous blood glucose measurement and continuous blood glucose measurement are important for the diagnosis and management of diabetes. Particularly in recent years, the importance of continuous blood glucose measurement is increasing in order to more accurately capture blood glucose fluctuations that vary among individuals. Continuous blood glucose measurement can measure blood glucose concentration for a period of several days to 1 or 2 weeks. The continuous blood glucose measurement device has a glucose sensor.

  The glucose concentration can be measured using glucose oxidase (GOD). GOD uses oxygen as an electron acceptor when it oxidizes glucose. This can be influenced by dissolved oxygen present in the measurement sample. To the best of our knowledge, all current commercial CGM (continuous glucose monitoring) devices are based on GOD. It is known that a specific GOD, for example, a GOD derived from a microorganism such as Aspergillus niger, for example, Aspergillus niger, retains activity even under severe heat conditions (Non-patent Document 1). For this reason, heat stable GOD is generally used in CGM devices. CGM is called continuous blood glucose measurement, or continuous glucose monitoring (herein these terms are interchangeable).

  Construction of a CGM device using GDH derived from Burkholderia cepacia is being studied. However, a CGM device constructed using CGM using GOD or GDH derived from Burkholderia cepacia requires calibration several times a day (for example, Non-Patent Document 2). Many commercial CGM devices also require calibration several times a day. This is thought to be because GOD and GDH are deactivated over time.

Patent Document 1 discloses GDH derived from the genus Mucor.
Non-Patent Document 3 discloses FAD-GDH derived from Mucor prainii. This document describes that GOD (EC 1.1.3.4) has been widely used as an amperometric glucose sensor because of its high thermal stability and high glucose selectivity.

  Patent Document 2 discloses GDH derived from Burkholderia cepacia. The disclosed GDH is a membrane-bound protein and has a cytochrome domain. The amino acid sequence of GDH derived from B. cepacia is not similar to FAD-dependent GDH derived from the genus Mucor.

Patent Document 3 discloses GDH derived from Mucor guilliermondii (MgGDH).
Patent Document 4 discloses GDH derived from Mucor hiemalis (MhGDH).

  Patent Document 5 discloses CsGDH derived from Circinella simplex and CRGDH derived from Circinella sp.

Patent Document 6 discloses MrdGDH derived from Mucor RD056860.
Patent Document 7 discloses MsGDH derived from Mucor subtilissimus.
Patent Document 8 and Patent Document 9 each disclose GDH derived from M. prainii.

International Publication No. 2015/099112 (US2016 / 0319246) International Publication No. 2002/036779 (US2004 / 0023330) International Publication No. 2013/051682 JP 2013-116102 International Publication No. 2013/022074 (US2014 / 0154777) JP2013-176363 JP 2013-176364 International Publication No. 2012/169512 (US2014 / 0287445) International Publication No. 2015/129475 (EP3112461) International Publication No. 2017/1957665

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 27, Issue of July 4, pp. 24324-24333, 2003 Sekimoto, et al., American Diabetes Association, 76th Scientific Sessions, 2016, 881-P-2016 Satake, et al., J Biosci Bioeng. 2015 Nov; 120 (5): 498-503

  An object of this invention is to provide GDH which can be used for CGM etc. and was excellent in long-term stability.

  CGM can theoretically be implemented by a glucose sensor having GDH or GOD. Glucose dehydrogenase uses an electron acceptor to dehydrogenate glucose. This can be advantageous for measurement because it is less susceptible to dissolved oxygen.

  However, to the best of the inventors' knowledge, all currently marketed CGM devices are based on GOD. This is probably due to the thermal stability of GOD. It is known that a specific GOD, for example, a GOD derived from a microorganism such as Aspergillus niger, for example, Aspergillus niger, retains activity even under severe heat conditions (Non-patent Document 1). GOD is known to retain higher activity under severe thermal conditions compared to GDH, especially for enzymes suitable for glucose sensors. This suggests that GOD is more stable than GDH.

  In this context, the inventors have surprisingly found that certain FAD-dependent glucose dehydrogenase mutants have higher storage stability compared to unmodified FAD-dependent glucose dehydrogenase, The present invention has been completed. Furthermore, the inventors surprisingly found that certain FAD-dependent glucose dehydrogenase mutants, when stored at ambient temperature for extended periods of time, compared to commercial glucose oxidase (GOD) under the same conditions, That is, the present inventors have found that it has higher storage stability and completed the present invention. This was particularly surprising in view of the fact that commercially available GOD retained higher enzyme activity than FAD-dependent glucose dehydrogenase under accelerated test conditions including heat treatment. That is, the present inventors have found that an enzyme having higher thermal stability is not always higher in storage stability. In addition, the present inventors have found that mutations that contribute to improving the thermal stability of an enzyme do not necessarily contribute to improving the storage stability of the enzyme, and mutations that contribute to improving the storage stability of the enzyme. It has been found that it does not necessarily contribute to the improvement of the thermal stability of the enzyme. In addition, the present inventors have found a FAD-dependent glucose dehydrogenase mutant with increased storage stability and completed the present invention.

  Based on this finding, the present invention provides a FAD-GDH mutant excellent in storage stability, a production method thereof, and a continuous glucose monitoring method using the enzyme. A continuous glucose monitoring device comprising a FAD-GDH variant is also provided.

That is, the present invention includes the following embodiments.
[1] A FAD-dependent glucose dehydrogenase (FAD-GDH) mutant,
i) the amino acid sequence represented by SEQ ID NO: 1,
ii) In the amino acid sequence shown in SEQ ID NO: 1, positions 196, 228, 600, 267, 276, 55, 230, 235, 239, 435, 587, 588, and 591 An amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than
iii) an amino acid sequence having 70% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, 3, 6 or 40, or
iv) The amino acid sequence represented by SEQ ID NO: 1, 3, or 6 has a sequence identity of 70% or more, and the amino acid sequence of the homologous region is 90 with the homologous region in the amino acid sequence represented by SEQ ID NO: 1. % Amino acid sequence having amino acid sequence identity,
Corresponds to positions 196, 228, 600, 267, 276, 55, 230, 235, 239, 435, 587, 588, and / or 591 in the amino acid sequence of SEQ ID NO: 1. A FAD-dependent glucose dehydrogenase mutant having one or more amino acid substitutions at a position, and having improved storage stability compared to an unmodified FAD-dependent glucose dehydrogenase having no such substitution.
[2] In FAD-GDH,
The amino acid at the position corresponding to position 196 in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine, phenylalanine, lysine, glutamine, asparagine, histidine, methionine, alanine, or arginine;
The amino acid at the position corresponding to position 228 in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine, threonine, histidine, serine, glutamine, or cysteine,
The amino acid at the position corresponding to position 600 in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan;
The amino acid at the position corresponding to position 267 in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine, histidine or glutamine,
The amino acid at the position corresponding to position 276 in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine, leucine, valine, methionine, cysteine, phenylalanine, tryptophan or tyrosine,
The amino acid at the position corresponding to position 55 in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine, serine, threonine, glutamine or cysteine,
The amino acid at the position corresponding to position 230 in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine, serine, threonine, asparagine, arginine, lysine, or histidine;
The amino acid at the position corresponding to position 235 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, serine or glycine;
The amino acid at the position corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, serine or glycine;
The amino acid at the position corresponding to position 435 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine, threonine, asparagine or glutamine,
The amino acid at the position corresponding to position 587 in the amino acid sequence of SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid,
The amino acid at the position corresponding to position 588 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine, threonine, asparagine or glutamine, and / or the amino acid at the position corresponding to position 591 in the amino acid sequence of SEQ ID NO: 1 is lysine, Substituted with arginine or histidine,
2. The FAD-GDH mutant according to 1, which has improved storage stability compared to an unmodified FAD-GDH having no such substitution.
[3] In FAD-GDH,
The amino acid at the position corresponding to position 196 in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine, phenylalanine, lysine, glutamine, asparagine, histidine, methionine, or alanine;
The amino acid at the position corresponding to position 228 in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine, threonine, or histidine;
The amino acid at the position corresponding to position 600 in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine,
An amino acid at a position corresponding to position 267 in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine;
The amino acid at the position corresponding to position 276 in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine,
The amino acid at the position corresponding to position 55 in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine,
The amino acid at the position corresponding to position 230 in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine,
An amino acid at a position corresponding to position 235 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine;
The amino acid at the position corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine;
The amino acid at the position corresponding to position 435 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine,
An amino acid at a position corresponding to position 587 in the amino acid sequence of SEQ ID NO: 1 is substituted with aspartic acid,
The amino acid at the position corresponding to position 588 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine, and / or the amino acid at the position corresponding to position 591 in the amino acid sequence of SEQ ID NO: 1 is replaced with lysine,
3. The FAD-GDH variant according to 1 or 2, which is a FAD-GDH variant, which has improved storage stability compared to an unmodified FAD-GDH having no such substitution.
[4] In addition,
An amino acid at a position corresponding to position 192 in the amino acid sequence of SEQ ID NO: 1 is substituted with proline;
The amino acid at the position corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine,
The amino acid at the position corresponding to position 218 in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine;
The amino acid at the position corresponding to position 226 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, and / or the amino acid at the position corresponding to position 232 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine,
FAD-GDH variant in any one of 1-3.
[5]
i) The amino acid at the position corresponding to position 196 in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine, phenylalanine, lysine or asparagine, and the amino acid at the position corresponding to position 228 in the amino acid sequence of SEQ ID NO: 1 is asparagine, threonine or A variant substituted with histidine,
ii) The amino acid at the position corresponding to position 192 in the amino acid sequence of SEQ ID NO: 1 is substituted with proline, and the amino acid at the position corresponding to position 196 in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine, phenylalanine, lysine or asparagine Mutants,
iii) The amino acid at the position corresponding to position 226 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, and the amino acid at the position corresponding to position 228 in the amino acid sequence of SEQ ID NO: 1 is replaced with asparagine, threonine or histidine. Mutant,
iv) The amino acid at the position corresponding to position 230 in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine, the amino acid at the position corresponding to position 232 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, and the amino acid sequence of SEQ ID NO: 1 Wherein the amino acid at the position corresponding to position 235 is substituted with alanine, and the amino acid at the position corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine,
v) The amino acid at the position corresponding to position 192 in the amino acid sequence of SEQ ID NO: 1 is substituted with proline, the amino acid at the position corresponding to position 196 in the amino acid sequence of SEQ ID NO: 1 is replaced with asparagine, and the amino acid sequence of SEQ ID NO: 1 The amino acid at the position corresponding to position 212 in the amino acid was substituted with leucine, the amino acid at the position corresponding to position 218 in the amino acid sequence of SEQ ID NO: 1 was replaced with histidine, and the amino acid at the position corresponding to position 226 in the amino acid sequence of SEQ ID NO: 1 A 192P / 196N / 212L / 218H / 226T / 228N mutant in which the amino acid is substituted with threonine and the amino acid at the position corresponding to position 228 in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine;
vi) In the mutant v), the amino acid at the position corresponding to position 230 in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine, and the amino acid at the position corresponding to position 232 in the amino acid sequence of SEQ ID NO: 1 is alanine 192P / 196N in which the amino acid at the position corresponding to position 235 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, and the amino acid at the position corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine / 212L / 218H / 226T / 228N / 230Q / 232A / 235A / 239A mutant, and
vii) The M3 variant having the amino acid sequence of SEQ ID NO: 40 in the variant of vi) above,
The FAD-GDH variant according to any one of 1 to 4, which is selected from the group consisting of and has improved storage stability as compared with FAD-GDH having no such substitution.
[6] Residual activity (%) of unsubstituted FAD-GDH heated at pH 7.4 at 20-40 ° C., assuming that the enzyme activity of unsubstituted FAD-GDH before heating is 100%, Compared with the residual activity (%) of the FAD-GDH mutant heated at 20-40 ° C. and pH 7.4 when the enzyme activity of the FAD-GDH mutant before warming was taken as 100%,
(a) Residual activity (%) after heating for 1 week is improved by 1% or more.
(b) Residual activity (%) after 2 weeks of warming is improved by 3% or more.
(c) Residual activity (%) after heating for 3 weeks is improved by 3% or more, or
(d) Residual activity (%) after heating for 4 days is improved by 1% or more.
FAD-GDH variant in any one of 1-5.
[7] A FAD-GDH gene encoding the FAD-GDH mutant according to any one of 1 to 6.
[8] A vector comprising the FAD-GDH gene according to 7.
[9] A host cell into which the vector according to 8 has been introduced.
[10] Method for producing FAD-GDH including the following steps:
Culturing the host cell according to 9,
Expressing the FAD-GDH gene contained in the host cell, and isolating FAD-GDH from the culture.
[11] A glucose assay kit comprising the FAD-GDH mutant according to any one of 1 to 6.
[12] A glucose sensor comprising the FAD-GDH mutant according to any one of 1 to 6.
[13] A continuous blood glucose measurement device comprising the glucose sensor according to 12.
[14] A glucose measurement method using the FAD-GDH mutant according to any one of 1 to 6, the kit according to 11, the sensor according to 12, or the device according to 13.
[15] The method according to 14, wherein continuous measurement is performed at 20 to 40 ° C. for 1 day to 2 weeks.

  This specification includes the disclosure of Japanese Patent Application No. 2017-111101, which is the basis of the priority of the present application.

  According to the present invention, FAD-GDH excellent in storage stability can be provided. This is particularly useful in continuous glucose measurement and continuous glucose monitoring applications. This is because the long-term stability of FAD-GDH allows GDH to be used over the entire lifetime of the sensor without the need for calibration by fingertip puncture. Most GOD-based CGM systems require calibration by fingertip puncture over the entire lifetime of the sensor at regular intervals, for example every 12 hours. This is thought to be due to the fact that the activity of GOD rapidly decreases at the ambient temperature of 30-40 ° C where the CGM device is used. In contrast, the FAD-GDH variants of the present invention surprisingly retain high stability (enzyme activity) at ambient temperatures of 30-40 ° C. over long periods of time, for example several weeks, without recalibration It has been found by the inventor that it can be used at a lower or less frequent calibration.

Multiple alignment of GDH from various sources is shown. MpGDH is a GDH derived from Mucor prainii (SEQ ID NO: 1), MhGDH is a GDH derived from Mucor hiemalis (SEQ ID NO: 3), MrdGDH is a GDH derived from Mucor RD056860 (SEQ ID NO: 4), and MsGDH is a GDH derived from Mucor subtilissimus (sequence) No. 5), MgGDH is Gcor derived from Mucor guilliermondii (SEQ ID NO: 6), CsGDH is derived from Circinella simplex GDH (SEQ ID NO: 7), CrGDH is derived from Circinella genus (SEQ ID NO: 8), and McGDH is This is GDH (SEQ ID NO: 9) derived from Mucor circinelloides. It is a continuation of FIG. 1-1. It is a continuation of FIG. 1-2. It is a continuation of FIGS. 1-3.

(Operational principle and activity measuring method of FAD-GDH of the present invention)
The FAD-GDH of the present invention catalyzes a reaction in which a hydroxyl group of glucose is oxidized to produce glucono-δ-lactone in the presence of an electron acceptor.

  The activity of the FAD-GDH of the present invention is measured using this principle of action, for example, using the following system using phenazine methosulfate (PMS) and 2,6-dichloroindophenol (DCIP) as electron acceptors. can do.

(Reaction 1) D-glucose + PMS (oxidized form)
→ D-glucono-δ-lactone + PMS (reduced)
(Reaction 2) PMS (reduced) + DCIP (oxidized)
→ PMS (oxidation type) + DCIP (reduction type)

  In (Reaction 1), PMS (reduced form) is produced with the oxidation of glucose. By continuing (reaction 2), DCIP is reduced as PMS (reduced form) is oxidized. The disappearance degree of this “DCIP (oxidized type)” is 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.

  The activity of FAD-GDH of the present invention is measured according to the following procedure. Mix 2.05 mL of 100 mM phosphate buffer (pH 7.0), 0.6 mL of 1M D-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 with time is measured to determine the amount of decrease in absorbance per minute (ΔA600) at 600 nm accompanying the progress of the enzyme reaction, and the GDH activity is calculated according to the following formula. At this time, the GDH activity is defined as 1 U of 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 reaction reagent + enzyme reagent solution volume (mL), 16.3 is the millimolar molecular extinction coefficient (cm ^{2 /} μmol) under the conditions of this activity measurement, 0.1 is the enzyme solution solution volume (mL), and 1.0 is Cell optical path length (cm), ΔA600 _blank is the amount of decrease in absorbance per minute at 600 nm when the reaction is started by adding the buffer used for enzyme dilution instead of the enzyme sample solution, df is the dilution factor Represent.

(Amino acid sequence of FAD-GDH mutant of the present invention)
The FAD-GDH variant of the present invention has the amino acid sequence represented by SEQ ID NO: 1, 3, 6 or 40, or has high identity with the amino acid sequence, for example, 70% or more, 71% or more, 72% or more, 73% 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% As described above, for example, 99% or more identical amino acid sequence, or one or several amino acids in the amino acid sequence (for example, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 , 5, 4, 3 or 2) amino acids Or consisting of an added amino acid sequence, and in the amino acid sequence described in SEQ ID NO: 1, positions 55, 192, 196, 212, 218, 226, 228, 230, 232, 235, 239, One or more, for example 1-18, for example 2-12, amino acids at positions corresponding to amino acids selected from positions 267, 276, 435, 587, 588, 591 and / or 600 It may have a substitution.

  In certain embodiments, the GDH of the invention is a variant of FAD-dependent GDH. In certain embodiments, native GDH is excluded from the GDH of the invention.

  In certain embodiments, a FAD-GDH variant of the invention may have an amino acid substitution at a position corresponding to position 192 of SEQ ID NO: 1. Although position 192 of SEQ ID NO: 1 is serine, the amino acid before substitution is not essential and the amino acid before substitution is not limited thereto. The amino acid after substitution can be proline. In the present specification, this amino acid substitution may be described as “S192P” or “192P”, but both are essentially synonymous.

  Hereinafter, similarly, in various embodiments, the FAD-GDH mutant of the present invention has an amino acid substitution at a position (in the GDH) corresponding to the predetermined position described in SEQ ID NO: 1 shown in the following table. sell.

  For example, although position 228 of SEQ ID NO: 1 is aspartic acid, in the mutant of the present invention, the amino acid at the position corresponding to position 228 of SEQ ID NO: 1 can be substituted with asparagine, serine, threonine, glutamine, cysteine or histidine. Other positions and substituted amino acids can be similarly read from the above table.

In certain embodiments, a variant of the invention can be D55N or have the mutation.
In certain embodiments, a variant of the invention can be or have the mutation S192P.
In certain embodiments, a variant of the invention can be or have the mutation S196N, S196Y, S196F or S196K.
In certain embodiments, a variant of the invention can be I212L or have the mutation.
In certain embodiments, a variant of the invention can be A218H or have the mutation.
In certain embodiments, a variant of the invention can be or have the mutation I226T.
In certain embodiments, a variant of the invention can be D228N, D228T, or D228H or have the mutation.
In certain embodiments, a variant of the invention can be E435S or have the mutation.
In certain embodiments, a variant of the invention can be or have the mutation S587D.
In certain embodiments, a variant of the invention can be or have the mutation P588S.
In certain embodiments, a variant of the invention may be M591K or have the mutation.
In certain embodiments, a variant of the invention may be V600I or have the mutation.
In certain embodiments, a variant of the invention can be 267R or have the mutation.
In certain embodiments, a variant of the invention can be 276I or have the mutation.
In certain embodiments, a variant of the invention may be 230Q or have the mutation.
In certain embodiments, a variant of the invention can be 232A or have the mutation.
In certain embodiments, a variant of the invention can be 235A or have the mutation.
In certain embodiments, a variant of the invention can be 239A or have the mutation.
In these cases, the position means the position in the GDH corresponding to the position of SEQ ID NO: 1.

  For GDH from other origins, amino acid substitution can be performed at a position in GDH from the other origin corresponding to the predetermined position of SEQ ID NO: 1. The corresponding positions are shown below.

  For example, in the table, the position and amino acid corresponding to position 218 of SEQ ID NO: 1 is alanine at position 219 in McGDH (SEQ ID NO: 9) derived from Mucor circinelloides. Other positions can be similarly read from the table.

  In certain embodiments, the FAD-GDH variants of the present invention may be multiple variants having multiple, eg 2-12, amino acid substitutions as described above. In the present specification, multiple mutants may be indicated using the “/” symbol. For example, in one embodiment, the FAD-GDH mutant of the present invention has an amino acid substitution at a position corresponding to position 192 of SEQ ID NO: 1 and a double mutant having an amino acid substitution at a position corresponding to position 196 of SEQ ID NO: 1. It can be. This double mutant is sometimes referred to herein as an “S192 / S196” or “192/196” mutant, but these are synonymous. For example, in one embodiment, the FAD-GDH variant of the present invention has an amino acid substitution to proline (192P) at a position corresponding to position 192 of SEQ ID NO: 1, and to an asparagine at a position corresponding to position 196 of SEQ ID NO: 1. Double mutants with the amino acid substitution of (196N). This double mutant may be described as “S192P / S196N” or “192P / 196N” in the present specification, but these are synonymous.

  Hereinafter, a part of GDH multiple mutants encompassed by the present invention will be exemplified by the same notation method. In the table, the position indicates the position of SEQ ID NO: 1 for convenience, but the position in the mutant is the position corresponding to the predetermined position of SEQ ID NO: 1 (in the mutant).

（いずれも配列番号１における位置）

(Both positions in SEQ ID NO: 1)

（いずれも配列番号１における位置）

(Both positions in SEQ ID NO: 1)

（いずれも配列番号１における位置）

(Both positions in SEQ ID NO: 1)

（いずれも配列番号１における位置）

(Both positions in SEQ ID NO: 1)

（いずれも配列番号１における位置）

(Both positions in SEQ ID NO: 1)

（いずれも配列番号１における位置）

(Both positions in SEQ ID NO: 1)

（いずれも配列番号１における位置）

(Both positions in SEQ ID NO: 1)

（いずれも配列番号１における位置）

(Both positions in SEQ ID NO: 1)

The mutant of the present invention may be a single mutant having a mutation at the position described in Table 3-1.
The mutant of the present invention may be a double mutant having a mutation at the position described in Table 3-2 or 3-6.
The mutant of the present invention may be a triple mutant having a mutation at the position described in Tables 3-3 to 3-5 or 3-7.
The mutant of the present invention may be a quadruple mutant having a mutation at the position described in Table 3-8.
The mutant of the present invention may be a quadruple to ten-fold mutant or a quadruple to 18-fold mutant in which the mutations listed in the above table are combined. For each position of these variants, the substituted amino acids can be those listed in Table 1.

When a specific mutant is exemplified, the mutant of the present invention is
196N / 228N, 196N / 228T, 196N / 228H, 196Q / 228N, 196Q / 228T, 196Q / 228H,
196A / 228N, 196A / 228T, 196A / 228H, 196F / 228N, 196F / 228T, 196F / 228H,
196H / 228N, 196H / 228T, 196H / 228H, 196K / 228N, 196K / 228T, 196K / 228H,
196M / 228N, 196M / 228T, 196M / 228H, 196Y / 228N, 196Y / 228T, 196Y / 228H,
196N / 600I, 196Q / 600I, 196A / 600I, 196F / 600I, 196H / 600I, 196K / 600I, 196M / 600I, 196Y / 600I,
196N / 267R, 196Q / 267R, 196A / 267R, 196F / 267R, 196H / 267R, 196K / 267R, 196M / 267R, 196Y / 267R,
196N / 276I, 196Q / 276I, 196A / 276I, 196F / 276I, 196H / 276I, 196K / 276I, 196M / 276I, 196Y / 276I,
196N / 55N, 196Q / 55N, 196A / 55N, 196F / 55N, 196H / 55N, 196K / 55N, 196M / 55N, 196Y / 55N,
228N / 600I, 228T / 600I, 228H / 600I,
228N / 267R, 228T / 267R, 228H / 267R,
228N / 276I, 228T / 276I, 228H / 276I,
228N / 55N, 228T / 55N, 228H / 55N,
E230Q / V232A / V235A / T239A / 55N, E230Q / V232A / V235A / T239A / S192P, E230Q / V232A / V235A / T239A / S196N, E230Q / V232A / V235A / T239A / I212L, E230Q / V232A / V235A / T239A / A218 E230Q / V232A / V235A / T239A / I226T, E230Q / V232A / V235A / T239A / D228N, E230Q / V232A / V235A / T239A / 435S, E230Q / V232A / V235A / T239A / S587D, E230Q / V232A / V235A / T239A / S588 E230Q / V232A / V235A / T239A / M591K, E230Q / V232A / V235A / T239A / V600I,
S192P / S196N,
S192P / S196N / E230Q / V232A / V235A / T239A,
I212L / A218H,
I212L / A218H / E230Q / V232A / V235A / T239A,
I226T / D228N,
I226T / D228N / E230Q / V232A / V235A / T239A,
I212L / A218H / I226T,
I212L / A218H / I226T / E230Q / V232A / V235A / T239A,
S192P / I212L / A218H / I226T,
S192P / I212L / A218H / I226T / E230Q / V232A / V235A / T239A,
S192P / S196N / I212L / A218H,
S192P / S196N / I212L / A218H / E230Q / V232A / V235A / T239A,
S192P / S196N / I226T / D228N,
S192P / S196N / I226T / D228N / E230Q / V232A / V235A / T239A,
I212L / A218H / I226T / D228N,
I212L / A218H / I226T / D228N / E230Q / V232A / V235A / T239A,
S192P / S196N / I212L / A218H / I226T / D228N,
S192P / S196N / I212L / A218H / I226T / D228N / E230Q / V232A / V235A / T239A,
D55N / S192P / S196N / I212L / A218H / I226T / D228N,
D55N / S192P / S196N / I212L / A218H / I226T / D228N / 435S,
D55N / S192P / S196N / I212L / A218H / I226T / D228N / 435S / S587D,
D55N / S192P / S196N / I212L / A218H / I226T / D228N / 435S / S587D / P588S,
D55N / S192P / S196N / I212L / A218H / I226T / D228N / 435S / S587D / P588S / M591K,
D55N / S192P / S196N / I212L / A218H / I226T / D228N / 435S / S587D / P588S / M591K / V600I, or
D55N / S192P / S196N / I212L / A218H / I226T / D228N / E230Q / V232A / V235A / T239A / 435S / S587D / P588S / M591K / V600I
May have mutations. In these cases, the position means the position in the GDH corresponding to the position of SEQ ID NO: 1.

  Moreover, when producing the above multiple mutants, substitutions at positions other than the above-mentioned various substitutions can be combined. Such substitution positions are introduced in combination with the above-described substitution sites, even when those substitutions are introduced alone, even if they do not have a significant effect as in the above-mentioned substitution sites, It can be synergistic.

  The FAD-GDH of the present invention has a mutation that improves thermal stability, a mutation that improves thermal stability, a mutation that reduces the reactivity to maltose, and temperature dependency, as described above. Mutations exhibiting other effects, such as mutations that improve resistance to pH and specific substances, for example, various known mutations that exhibit such effects may be arbitrarily combined. Even when such different types of mutations are combined, those FAD-GDH mutants are included in the present invention as long as the effects of the present invention can be exhibited.

  In certain embodiments, as the starting GDH, International Publication No. 2015/099112 (US2016 / 0319246), International Publication No. 2012/169512 (US2014 / 0287445) or International Publication No. 2015/129475 (EP3112461) or International Publication No. 2017 GDH described in / 1957665 or the GDH gene encoding it can be used. The entire contents of which are incorporated herein by reference. The obtained GDH gene can be mutated to introduce amino acid substitution into GDH. Mutations can be introduced at positions disclosed in these documents or at positions corresponding thereto. In certain embodiments, the Mucor prainii derived GDH (MpGDH, SEQ ID NO: 1) can be modified by introducing a N66Y / N68G / C88A / Q233R / T387C / E554D / L557V / S559K mutation. For convenience this is sometimes referred to as the M1 variant. In one embodiment, Mucor prainii derived GDH (MpGDH, SEQ ID NO: 1) can be modified by introducing an A175C / N214C / G466D mutation into M1. For convenience this is sometimes referred to as the M2 variant. Such GDH can be used as a starting GDH to introduce the mutation of the present invention.

  As described later, the FAD-GDH mutant of the present invention, for example, first obtains a gene encoding an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 1 by any method and corresponds to a predetermined position of SEQ ID NO: 1. It can also be obtained by introducing an amino acid substitution at any position in the position.

  Examples of the method for introducing amino acid substitution include a method of introducing mutations at random or a method of introducing site-specific mutations at assumed positions. The former includes error-prone PCR (Techniques, 1, 11-15, (1989)) and XL1-Red competent cells (manufactured by STRATAGENE) that are prone to errors in plasmid replication and prone to modification during propagation. ). Also, as the latter, a three-dimensional structure is constructed by analyzing the crystal structure 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 Mutagenesis Kit (manufactured by STRATAGENE) is selected. There is a method of introducing site-specific mutation by, for example. Alternatively, modeling can be performed using a three-dimensional structure of a known protein having high homology with the target protein, an amino acid expected to impart the target effect can be selected, and a site-specific mutation can be introduced.

  As used herein, the “position corresponding to the amino acid sequence of SEQ ID NO: 1” means the same position in the alignment when the amino acid sequence of SEQ ID NO: 1 is aligned with another amino acid sequence. . As other amino acid sequences, high amino acid sequence identity with SEQ ID NO: 1 (for example, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% As mentioned above, amino acid sequences having 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example 99% or more) can be mentioned. Amino acid sequence identity should be calculated using GENETYX Ver.11 (Genetics) maximum matching and search homology programs, or DNASIS Pro (Hitachi Soft) maximum matching and multiple alignment programs. Can do.

  As a method for specifying the “position corresponding to the amino acid sequence of SEQ ID NO: 1”, for example, the amino acid sequences are compared using a known algorithm such as the Lippmann-Person method and are present in the amino acid sequence of FAD-GDH. This can be done by giving maximum identity to conserved amino acid residues. By aligning the amino acid sequence of FAD-GDH in this way, the position of the corresponding amino acid residue in each FAD-GDH sequence can be determined regardless of insertion or deletion in the amino acid sequence. Is possible. In particular, it can be determined when comparing amino acid sequences having amino acid sequence identity of 70% or more. Corresponding positions are considered to exist at the same position in the three-dimensional structure, and it can be reasonably estimated that they have a similar effect on the storage stability of the target FAD-GDH. Depending on the GDH, the position corresponding to the predetermined position of SEQ ID NO: 1 may not exist due to the lack of a specific domain or loop as a result of the alignment. A position where the corresponding position does not exist is not limited to amino acid substitution, and amino acid substitution may be introduced at another position where the corresponding position can be determined.

(About homology region)
Amino acid sequence identity or similarity should be calculated using GENETYX Ver.11 (Genetics) maximum matching and search homology programs, or DNASIS Pro (Hitachi Solutions) maximum matching and multiple alignment programs. Can do. In order to calculate amino acid sequence identity, when two or more GDHs are aligned, the positions of amino acids that are identical in the two or more GDHs can be examined. Based on such information, the same region in the amino acid sequence can be determined.

  It is also possible to examine amino acid positions that are similar in two or more GDH. For example, CLUSTALW can be used to align a plurality of amino acid sequences. In this case, Blosum62 is used as an algorithm, and amino acids that are judged to be similar when a plurality of amino acid sequences are aligned may be referred to as similar amino acids. In the variants of the invention, amino acid substitutions may be due to substitutions between such similar amino acids. By such alignment, a position occupied by a region having the same amino acid sequence and a similar amino acid can be examined for a plurality of amino acid sequences. Based on such information, a homology region (conserved region) in an amino acid sequence can be determined.

  In the present specification, the “homology region” is a region in which when two or more GDHs are aligned, the amino acids at the corresponding positions of the reference GDH and the GDH to be compared are the same or are composed of similar amino acids. It is a region composed of 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more consecutive amino acids. For example, in FIG. 1, GDH whose sequence identity of the full length amino acid sequence is 70% or more is aligned. Among these, positions 31 to 41 with respect to the Mucor genus GDH represented by SEQ ID NO: 1 are composed of the same amino acid, and thus correspond to the homology region. Similarly, based on the Mucor genus GDH represented by SEQ ID NO: 1, positions 31 to 41, 58 to 62, 71 to 85, 106 to 116, 119 to 127, 132 to 134, 136 to 144 , 150-153, 167-171, 219-225, 253-262, 277-281, 301-303, 305-312, 314-319, 324-326, 332-337 , 339-346, 348-354, 386-394, 415-417, 454-459, 476-484, 486-491, 508-511, 518-520, 522-524 Positions 526 to 528, 564 to 579, 584 to 586, 592 to 595, 597 to 599, 607 to 617, and 625 to 630 are homologous. It can be applicable to the region.

  The GDH of the present invention is 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73 when aligned with GDH having the amino acid sequence shown in SEQ ID NO: 1, 3, 6 or 40 %, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, It has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, for example 99% or more full-length amino acid sequence identity and has glucose dehydrogenase activity. Furthermore, the amino acid sequence in the homology region of the GDH variant of the present invention is 80%, 81%, 82%, 83%, 84%, 85% with the amino acid sequence of the homology region in SEQ ID NO: 1, 3, or 6. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, eg, 99% or more sequence identity .

  The FAD-GDH of the present invention is expected to have various variations within the above-mentioned range of identity, but the enzymatic scientific properties of the various FAD-GDH are the same as the FAD-GDH of the present invention described herein. As long as they are all included in the FAD-GDH of the present invention. FAD-GDH having such an amino acid sequence is FAD-GDH with high storage stability and is industrially useful.

  In the FAD-GDH mutant of the present invention, it is important that the amino acid after substitution at the above position is as described above, for example, that the amino acid at the position corresponding to position 192 of SEQ ID NO: 1 is proline after substitution. It does not matter whether it is due to an artificial replacement operation. For example, starting from a protein in which the amino acid at the above position is originally different from the residue desired in the present invention, such as the protein described in SEQ ID NO: 1, a desired substitution is performed there using a known technique. In the case of introduction, these desired amino residues are introduced by substitution. On the other hand, when a desired protein is obtained by publicly known total peptide synthesis, or when a gene sequence is totally synthesized to encode a protein having a desired amino acid sequence, and a desired protein is obtained based on this, or In the case where some of those found as natural types originally have such a sequence, the FAD-GDH of the present invention can be obtained without going through a step of artificial substitution. However, natural products and natural sequences themselves are excluded from the FAD-GDH mutant of the present invention. For example, regarding position 192 of SEQ ID NO: 1, the amino acid at the corresponding position is proline in the natural sequence does not fall under the “192P” variant referred to in this specification.

(Storage stability of FAD-GDH)
The storage stability of conventional GOD, FAD-GDH, and further the FAD-GDH mutant of the present invention is evaluated under the conditions described in the activity measurement method and the storage stability measurement method described in this specification. By comparing the storage stability of GDH before and after amino acid substitution, the improvement in storage stability due to the substitution can be evaluated. The storage stability is determined from the initial activity in the storage stability test and the residual activity after a predetermined period. In order to calculate the ratio of initial activity to residual activity, enzyme activity is measured under the same conditions in the initial state and after warming. The activity in the initial state of the test is defined as 100%, and GDH is heated for a predetermined period of time, for example, 1 day to 3 weeks, for example, at an ambient temperature of 20-40 ° C., and then the remaining activity is measured. In certain embodiments, the pH of the storage test solution is from 6.9 to 7.4, such as 7.4. This is because the pH of blood is near neutral. The pH can be adjusted with a conventional buffer such as a phosphate buffer such as PBS, but the buffer and the buffer are not limited thereto.

In one embodiment, the FAD-GDH variant of the invention has a pH of 7.4
(a) 60% or more, for example 65% or more, 70% or more, 75% or more, for example 80% or more of initial activity 100% when heated at 20-40 ° C., for example 40 ° C., for 1 week 90% or more, such as 95%, 96% or more, 97% or more, 98% or more, such as 99% or more of the dehydrogenase activity is retained,
(b) 40% or more, for example 45% or more, 50% or more, 55% or more, for example 60% or more of the initial activity 100% when heated at 20-40 ° C., for example 40 ° C. for 2 weeks 70% or more, 75% or more, such as 80% or more, 90% or more, such as 95% or more, 96% or more, 97% or more, 98% or more, such as 99% dehydrogenase activity is retained,
(c) 30% or more, for example 35% or more, 40% or more, 45% or more, 50% or more of the initial activity 100% when heated at 20-40 ° C., for example 40 ° C. for 3 weeks, 55% or more, such as 60%, 70% or more, 75% or more, such as 80% or more, 90% or more, such as 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% dehydrogenase activity is retained Or
(d) 60%, 70% or more, 71% or more, 72% or more, 73% or more, 74% of initial activity 100% when heated at 20-40 ° C., for example, 40 ° C. for 4 days 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, for example 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more 87% or more, 88% or more, 89% or more, 90% or more, such as 95% or more, 96% or more, 97% or more, 98% or more, for example 99%, dehydrogenase activity is retained,
It may have storage stability (long-term stability).

In one embodiment, the FAD-GDH variant of the invention has a pH of 7.4
(a) 40% or more, 50% or more, 60% or more, for example, 65% or more, 70% or more of initial activity 100% when heated at 20-40 ° C., for example, 25 ° C. for 1 week, 75% or more, such as 80% or more, 90% or more, such as 95%, 96% or more, 97% or more, 98% or more, such as 99% or more dehydrogenase activity is retained,
(b) 40% or more, 50% or more, 40% or more, for example 45% or more, 50% or more of initial activity 100% when heated at 20-40 ° C., for example, 25 ° C. for 2 weeks, 55% or more, such as 60% or more, 70% or more, 75% or more, such as 80% or more, 90% or more, such as 95% or more, 96% or more, 97% or more, 98% or more, such as 99% dehydrogenase activity. Retained,
(c) 40% or more, 50% or more, 30% or more, for example, 35% or more, 40% or more of initial activity 100% when heated at 20-40 ° C., for example, 25 ° C. for 3 weeks, 45% or more, 50% or more, 55% or more, such as 60%, 70% or more, 75% or more, such as 80% or more, 90% or more, such as 95% or more, 96% or more, 97% or more, 98% or more, For example, 99% dehydrogenase activity is retained, or
(d) 40% or more, 50% or more, 60%, 70% or more, 71% or more, 72% of the initial activity 100% when heated at 20-40 ° C., for example, 25 ° C. for 4 days 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, for example 80% or more, 81% or more, 82% or more, 83% or more, 84% or more 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, for example, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% dehydrogenase activity is retained To be
It may have storage stability (long-term stability).

In one embodiment, the FAD-GDH variant of the invention has a pH of 7.4
(a) 60% or more, for example 65% or more, 70% or more, 75% or more, for example 80% or more, out of initial activity 100% when heated at 20-40 ° C., for example, 20 ° C. for 1 week 90% or more, such as 95%, 96% or more, 97% or more, 98% or more, such as 99% or more of the dehydrogenase activity is retained,
(b) 40% or more, for example 45% or more, 50% or more, 55% or more, for example 60% or more of the initial activity 100% when heated at 20-40 ° C., for example 20 ° C. for 2 weeks 70% or more, 75% or more, such as 80% or more, 90% or more, such as 95% or more, 96% or more, 97% or more, 98% or more, such as 99% dehydrogenase activity is retained,
(c) 30% or more, for example, 35% or more, 40% or more, 45% or more, 50% or more of the initial activity 100% when heated at 20-40 ° C., for example, 20 ° C. for 3 weeks, 55% or more, such as 60%, 70% or more, 75% or more, such as 80% or more, 90% or more, such as 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% dehydrogenase activity is retained Or
(d) 60%, 70% or more, 71% or more, 72% or more, 73% or more, 74% of initial activity 100% when heated at 20-40 ° C., for example, 20 ° C. for 4 days 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, for example 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more 87% or more, 88% or more, 89% or more, 90% or more, such as 95% or more, 96% or more, 97% or more, 98% or more, for example 99%, dehydrogenase activity is retained,
It may have storage stability (long-term stability).

In certain embodiments, the FAD-GDH variant of the invention comprises
When the enzyme activity of unsubstituted FAD-GDH before heating is defined as 100%, the remaining activity (%) of unsubstituted FAD-GDH heated at pH 7.4 at 20-40 ° C, and before heating In comparison with the residual activity (%) of the FAD-GDH mutant heated at 20-40 ° C. and pH 7.4 when the enzyme activity of the FAD-GDH mutant was 100%,
(a) Residual activity (%) after heating for 1 week is 1% or more, 1.5% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% For example, 55% or more,
(b) Residual activity (%) after heating for 2 weeks is 1% or more, 1.5% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, for example, 30% or more, 35% or more, 40 % Or higher, 45% or higher, 50% or higher, for example 55% or higher,
(c) Residual activity (%) after heating for 3 weeks is 1% or more, 1.5% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, for example, 30% or more, 35% or more, 40 % Or higher, 45% or higher, 50% or higher, for example 55% or higher, or
(d) Residual activity (%) after heating for 4 days is 1% or more, 1.5% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 20% or more, 25% or more, for example, 30% or more, 35% or more, 40 % Or more, 45% or more, 50% or more, for example 55% or more. In the present specification, such improvement in residual activity is sometimes referred to as improvement in storage stability (long-term stability). For example, the residual activity of unmodified FAD-GDH heated for 7 days at 40 ° C. is 63.9%, and the residual activity of the FAD-GDH mutant having the mutation of the present invention heated under the same conditions is 73.9% The FAD-GDH mutant of the present invention has a 10% improvement in storage stability. Such variants are encompassed by the present invention. Such a mutant can be used for continuous blood glucose measurement. Even if the storage stability improvement caused by a certain mutation is about 1%, GDH suitable for continuous glucose measurement is obtained as long as the storage stability is improved as compared with unmutated GDH. It can be said that this problem has been solved. Moreover, even if the improvement in storage stability caused by a certain mutation is about 1%, GDH with higher storage stability can be produced by combining a plurality of such mutations, for example. Such mutations can also be combined with mutations that alter other enzyme properties. The group of different single mutations that can be used in combination opens up protein engineering options.

(Acquisition of 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 technique. In order to obtain a gene encoding FAD-GDH of the present invention (hereinafter referred to as FAD-GDH gene), a generally 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, it is possible to obtain a conventional method from known microbial cells or 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 chromosomal DNA or cDNA library can be prepared.

  Next, based on the known amino acid sequence information of FAD-GDH, an appropriate probe DNA is synthesized, and using this, a method for selecting a FAD-GDH gene with high substrate specificity from a chromosomal DNA or cDNA library, or Based on the above amino acid sequence, an appropriate primer DNA is prepared, and FAD-GDH with high substrate specificity is encoded by an appropriate polymerase chain reaction (PCR method) such as 5'RACE method or 3'RACE method. DNAs containing the gene fragment can be amplified, and these DNA fragments can be ligated to obtain 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 excellent storage stability of the present invention, a mutation is introduced into the gene encoding the starting material FAD-GDH, and various mutant genes are used. A method of performing selection using the enzymatic scientific properties of the expressed FAD-GDH as an index may be employed.

  Mutation treatment of the FAD-GDH gene as a starting material can be performed by any known method depending on the intended mutation form. That is, a method in which a FAD-GDH gene or a recombinant DNA in which the gene is incorporated is brought into contact with and acting on a mutagen; an ultraviolet irradiation method; a genetic engineering method; or a method using a protein engineering method, etc. Can be widely used.

  Examples of the mutagen used in the mutation treatment include hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine, nitrous acid, sulfite, hydrazine, formic acid, or 5-bromouracil. be able to.

  The conditions for the contact and action can be determined according to the type of drug used, and are not particularly limited as long as a desired mutation can be actually induced in the Mucor genus-derived FAD-GDH gene. Usually, a 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 more, preferably for 10 to 180 minutes. Even when ultraviolet irradiation is performed, it can be performed according to a conventional method as described above (Hyundai Kagaku, p24-30, June 1989 issue).

  As a method of making full use of protein engineering techniques, a technique known as Site-Specific Mutagenesis can be generally used. For example, Kramer method (Nucleic Acids Res., 12, 9441 (1984): Methods Enzymol., 154, 350 (1987): Gene, 37, 73 (1985)), 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. As a specific method for converting a base sequence in DNA, for example, use of a commercially available kit (Transformer Mutagenesis Kit; Clonetech, EXOIII / Mung Bean Deletion Kit; Stratagene, Quick Change Site Directed Mutagenesis Kit; Stratagene, etc.) Is mentioned.

  In addition, a technique known as a general polymerase chain reaction can be used (Technique, 1, 11 (1989)).

  In addition to the gene modification method described above, a desired modified FAD-GDH gene having excellent storage stability 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, Inc.) or the like can be used. It ’s fine.

(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 microorganisms that are classified into the subfamily Pleurotus, preferably Pleurotus, more preferably Pleuromyceae, and more preferably Pleurotus. Specific examples include FAD-GDH derived from the genus Mucor, the genus Absidia, the genus Actinomucor, the genus Circinella, and the like.

  Specific examples of preferred microorganisms classified into the genus Mucor, such as Mucor prainii, Mucor javanicus, Mucor circinelloides f. Circinelloides, Mucor guilliermondii, Mucor hiemalis f. Silvaticus, Mucor subtilissimus, Mucor dimorphosporus, etc. It is done. More specifically, Mucor prainii, Mucor javanicus, Mucor circinelloides f. Circinelloides, Mucor guilliermondii NBRC9403, Mucor hiemalis f. Silvaticus NBRC6754, Mucor subtilissimus NBRC6338, RC60 It is done. Specific examples of preferred 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 exemplified. Actinomucor elegans can be mentioned as an example of a specific preferable microorganism that is classified into the genus Actinomucor. More specifically, Actinomucor elegans described in Patent Document 5 can be exemplified. Specific examples of preferable microorganisms classified into the genus Circinella include Circinella minor, Circinella mucoroides, Circinella muscae, Circinella rigida, Circinella simplex, and Circinella umbellata. More specifically, Circinella minor NBRC6448, Circinella mucoroides NBRC4453, Circinella muscae NBRC6410, Circinella rigida NBRC6411, Circinella simplex NBRC6412, Circinella umbellata NBRC4452, ircinella umbellata 542 irc In addition, NBRC strain and RD strain are storage strains of NBRC (National Institute of Technology and Evaluation Biotechnology Center).

(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, a 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 conventional methods.

  Examples of prokaryotic host cells include microorganisms belonging to the genus Escherichia, such as Escherichia coli K-12, Escherichia coli BL21 (DE3), Escherichia coli JM109, Escherichia coli DH5α, Escherichia coli W3110, Escherichia coli C600, etc. Both are manufactured by Takara Bio Inc.). They are transformed or transduced to obtain host cells into which DNA has been introduced (transformants). As a method for transferring a recombinant vector into such a host cell, for example, when the host cell is a microorganism belonging to Escherichia coli, a method of transferring a recombinant DNA in the presence of calcium ions can be employed. An electroporation method may be used. Furthermore, a commercially available competent cell (for example, ECOS Competent Escherichia Collie BL21 (DE3); manufactured by Nippon Gene) may be used.

  An example of a eukaryotic host cell is yeast. Examples of microorganisms classified as yeast include yeasts belonging to the genus Zygosaccharomyces, the genus Saccharomyces, the genus Pichia, the genus Candida and the like. The inserted gene may include a marker gene to enable 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, the inserted gene preferably 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 the host cell. Specific examples of the promoter include GAL1 promoter and ADH1 promoter. As a method for transformation into yeast, known methods such as a method using lithium acetate (Methods Mol. Cell. Biol., 5, 255-269 (1995)) and electroporation (J Microbiol Methods 55 (2003) 481 -484) and the like can be preferably used, but the present invention is not limited to this, and transformation may be performed using various arbitrary methods including a spheroplast method and a glass bead method.

  Other examples of eukaryotic host cells include mold cells such as the genus Aspergillus and the genus Tricoderma. The inserted gene contains a promoter capable of expressing the gene of the present invention in the host cell (eg, tef1 promoter) and other regulatory sequences (eg, 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, for example, niaD and pyrG. Furthermore, 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 do.

(High-throughput screening)
GDH can further be subjected to high-throughput screening to obtain functional GDH mutants. For example, a library of transformed or transduced strains containing the mutated GDH gene may be prepared and used for high-throughput screening based on microtiter plates, or for ultra-high-throughput screening based on droplet microfluids May be provided. As an example, a combinatorial library of mutant genes encoding variants is constructed, and then phage display (eg Chem. Rev. 105 (11): 4056-72, 2005), yeast display (eg Comb Chem High Throughput Screen. 2008; 11 (2): 127-34), bacterial display (for example, Curr Opin Struct Biol 17: 474-80, 2007) and the like, and a method of screening a large population of mutant GDH. See also Agresti et al, "Ultrahigh-throughput screening in drop-based microfluidics for directed evolution" Proceedings of the National Academy of Sciences 107 (9): 4004-4009 (Mar, 2010). The same literature description of ultra-high throughput screening techniques that can be used to screen for GDH variants is incorporated herein by reference. For example, a library can be constructed by error-prone PCR. Alternatively, using saturation mutagenesis, a library may be constructed by introducing mutations targeting the regions or positions described in the present specification or the regions or positions corresponding thereto as targets. The library can be used to transform suitable cells, such as electrically competent EBY-100 cells, to obtain approximately 10 7 mutants. Yeast cells transformed with the library can then be subjected to cell sorting. Polydimethoxylsiloxane (PDMS) microfluidic devices made using standard soft lithography methods may be used. A monodisperse droplet can be formed using a flow focus device. The formed droplets containing the individual variants can be subjected to a suitable sorting device. When cells are sorted, the presence or absence of GDH activity can be used. For example, a reaction solution having a composition that develops color when the above-described GDH acts may be used. For example, when DCIP is used, the absorbance at 600 nm may be measured using a 96-well plate, 192-well plate, 384-well plate, 9600-well plate, etc. and a plate reader. Mutation introduction and selection may be repeated multiple times.

(Method for producing FAD-GDH mutant of the present invention)
The FAD-GDH mutant of the present invention is obtained by culturing a host cell that produces the FAD-GDH of the present invention obtained as described above, expressing a FAD-GDH gene contained in the host cell, and then culturing the culture. The FAD-GDH mutant may be isolated from the product.

  Examples of the culture medium for culturing the host cells include yeast extract, tryptone, peptone, meat extract, corn steep liquor, or one or more nitrogen sources such as soybean or wheat bran leachate, sodium chloride, potassium monophosphate One or more inorganic salts such as dibasic potassium phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate were added, and saccharide raw materials, vitamins, etc. were added as necessary Things are used.

  Although the initial pH of a culture medium is not limited, For example, it can adjust to pH 6-9. The culture is performed at a culture temperature of 10 to 42 ° C., preferably at a culture temperature of around 25 ° C. for 4 to 24 hours, more preferably at a culture temperature of around 25 ° C. for 4 to 8 hours, aeration and agitation deep culture, shaking culture, and stationary. What is necessary is just to implement by culture | cultivation etc.

  After completion of the culture, the FAD-GDH of the present invention is collected from the culture. For this, an ordinary known enzyme collecting means may be used. For example, the cells are subjected to ultrasonic disruption treatment, grinding treatment, etc., or the enzyme is extracted using a lytic enzyme such as lysozyme or yatalase, or shaken or left in the presence of toluene. Thus, the enzyme can be lysed and discharged from the cell. Then, this solution is filtered, centrifuged, etc. to remove the solid part, and if necessary, nucleic acid is removed with streptomycin sulfate, protamine sulfate, manganese sulfate or the like, and then ammonium sulfate, alcohol, acetone or the like is added thereto. The fraction is collected and the precipitate is collected to obtain the FAD-GDH crude enzyme of the present invention.

  The crude enzyme of FAD-GDH of the present invention can be further purified using any known means. In order to obtain a purified enzyme preparation, for example, a gel filtration method using Sephadex, Ultrogel or biogel; an adsorption elution method using an ion exchanger; an electrophoresis method using a polyacrylamide gel; an adsorption using hydroxyapatite Elution method; Sedimentation method such as sucrose density gradient centrifugation; Affinity chromatography method; Fractionation method using molecular sieve membrane, hollow fiber membrane, etc. The FAD-GDH enzyme preparation of the present invention can be obtained.

(Glucose measurement method using FAD-GDH of the present invention)
The present invention also provides a glucose assay kit comprising the FAD-GDH mutant of the present invention. Blood glucose (blood glucose level) can be measured using the FAD-GDH of the present invention.

  The glucose assay kit of the present invention contains an amount of the FAD-GDH variant of the present invention sufficient for at least one assay. Typically, the glucose assay kit of the present invention contains, in addition to the FAD-GDH mutant of the present invention, a buffer solution necessary for the assay, a mediator, and a glucose standard solution for preparing a calibration curve. The FAD-GDH mutant used in the glucose measurement method and glucose assay kit of the present invention can be provided in various forms, for example, as a lyophilized reagent or dissolved in an appropriate storage solution.

  In the case of a colorimetric glucose assay kit, the glucose concentration can be measured, for example, as follows. The reaction layer of the glucose assay kit contains FAD-GDH, an electron acceptor, and N- (2-acetamido) imidodiacetic acid (ADA), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis) as a reaction accelerator. -Tris), a liquid or solid composition containing one or more substances selected from the group consisting of sodium carbonate and imidazole. Here, a pH buffering agent and a coloring reagent are added as necessary. A sample containing glucose is added to this and allowed to react for a certain time. During this time, the absorbance corresponding to the maximum absorption wavelength of the dye that is polymerized and formed by receiving electrons from the electron acceptor or the electron acceptor that fades upon reduction is monitored. In the case of the rate method, from the rate of change of absorbance per time, in the case of the endpoint method, the calibration was made in advance using a standard concentration glucose solution from the change in absorbance up to the point when all the glucose in the sample was oxidized. Based on the calibration curve, the glucose concentration in the sample can be calculated.

  As a mediator and coloring reagent that can be used in this method, for example, 2,6-dichlorophenolindophenol (DCPIP) is added as an electron acceptor, and glucose can be quantified by monitoring the decrease in absorbance at 600 nm. . In addition, phenazine methosulfate (PMS) is added as an electron acceptor, nitrotetrazolium blue (NTB) is further added as a coloring reagent, and the amount of diformazan produced is determined by measuring the absorbance at 570 nm to calculate the glucose concentration. Is possible. Needless to say, the electron acceptor and the coloring reagent used are not limited to these.

(Glucose sensor containing FAD-GDH mutant of the present invention)
The present invention also discloses a glucose sensor using the FAD-GDH mutant of the present invention. In certain embodiments, a glucose sensor has a working electrode comprising a FAD-GDH variant of the invention, a reference electrode and a counter electrode. As the working electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and the FAD-GDH of the present invention is immobilized on this electrode. Additionally or alternatively, an electron mediator may be immobilized on the working electrode. The counter electrode can be a conventional electrode such as a platinum electrode or Pt / C. The reference electrode can be a conventional electrode such as an Ag / AgCl electrode. Immobilization methods include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc., or ferrocene or a derivative thereof. It may be fixed in a polymer or adsorbed and fixed on an electrode together with a representative electron mediator, or a combination thereof may be used. Typically, FAD-GDH of the present invention is immobilized on a carbon electrode using glutaraldehyde and then treated with a reagent having an amine group to block glutaraldehyde. The FAD-GDH mutant of the present invention can be protected by a protective film and used for a glucose sensor. As the protective film, a known protective film or a general protective film for protecting the enzyme used in the sensor can be used.

  In some embodiments, an enzyme membrane carrying an enzyme in the membrane may be used as the protective membrane. The enzyme membrane can be formed by immobilizing a GDH enzyme and a protein such as albumin with a crosslinking agent (glutaraldehyde or the like). The enzyme membrane can be formed on the first permeable membrane (also referred to as a selectively permeable membrane). Thereafter, a second permeable membrane may be further formed on the enzyme membrane on the first permeable membrane (also referred to as a restricted permeable membrane). The restricted permeable membrane can be formed from a silicone membrane, a fluorine-based polymer membrane, a urethane polymer membrane, or a polycarbonate membrane. The first permeable membrane, the enzyme membrane, and the second permeable membrane may have a thickness of 0.1 to 10 μm. The permselective membrane may be one that allows only hydrogen peroxide to pass therethrough and prevents permeation of substances that interfere with measurement. The restricted permeation membrane may be one that prevents adsorption of contaminants and restricts glucose permeation. Each permeable membrane may have micropores, which may limit the permeation of the substance.

  In an embodiment, the protective film may be prepared by forming an enzyme layer on a substrate and immersing the substrate in a protective film solution to coat the substrate. For the enzyme layer containing GDH, additives such as sodium polyacrylate, trehalose and glucomannan can be used. The protective film solution may be a poly-4-vinylpyridine solution or the like, but is not limited thereto.

  Methods for producing a glucose sensor are known in the literature, such as Liu, et. Al., Anal. Chem. 2012, 84, 3403-3409 and Tsujimura, et. Al., J. Am. Chem. Soc. 2014, 136, 14432-14437 (all of which are incorporated herein by reference in their entirety). In a known method, GDH can be used instead of GOD.

For example, a glucose sensing reagent can be prepared by mixing a redox polymer such as a polymer containing an osmium complex with GDH present in another polymer such as polyethylene glycol in a suitable buffer such as HEPES buffer. . An osmium complex is a bipyridine molecule such as 2,2'-bipyridine, a biimidazole molecule such as 2,2'-biimidazole, a pyridine-imidazole compound such as 2- (2-pyridyl) imidazole, or a combination thereof. It can be an osmium complex complexed with one or more compounds comprising. The organic molecule forming the complex may be substituted with an alkyl group such as C1-C6 alkyl such as a methyl group or an ethyl group, if necessary. In one embodiment, the osmium complex may be as follows:
[Os Organic Molecular Complex]-[Polymer]
One of the organic molecules forming the complex may optionally be attached to the polymer via a linker. Thus, in another embodiment, the osmium complex can be as follows:
[Os Organic Molecular Complex]-[Linker]-[Polymer]
Exemplary molecules include Ohara et al., Anal Chem. 1993 Dec 1; 65 (23): 3512-7 and Antiochia et al., Materials Sciences and Applications Vol. 4 No. 7 A2 (2013) (both are fully incorporated by reference). Incorporated herein). Examples of polymers containing osmium complexes include osmium bis (2,2'-bipyridine) chloride complexed with poly (1-vinylimidazole), poly (4-vinylpyridine) complexed osmium bis (2,2 ' -Bipyridine) chloride, poly (1-vinylimidazole) _n- [osmium (4,4′-dimethyl-2,2′-bipyridyl) ₂ Cl ₂ ] ^{2 + / +} and their derivatives.

  The solution can be deposited on a carbon sensor to form an active electrode region that functions as a working electrode containing redox polymer wired GDH. A mixture of membrane polymer and crosslinker solution can be added to form a membrane on the sensor. The membrane may comprise a polymer such as poly (1-vinylimidazole), poly (4-vinylpyridine), derivatives or combinations thereof. Polymers containing the above osmium complexes can be used in combination with such membrane polymers to form hydrogel films.

  In one embodiment, the glucose sensor can include a printed electrode. In this case, an electrode can be formed on the insulating substrate. Specifically, the electrode can be formed on the substrate by a printing technique such as photolithography or screen printing, gravure printing or flexographic printing. Examples of the material constituting the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, and polyester. Materials that are highly resistant to various solvents or chemicals may be used.

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

  As a specific example, the 1.5 U FAD-GDH mutant of the present invention is immobilized on a glassy carbon (GC) electrode, and the response current value with respect to the glucose concentration is measured. In the electrolytic cell, 1.8 ml of 50 mM potassium phosphate buffer (pH 6.0) and 0.2 ml of 1M potassium hexacyanoferrate (III) aqueous solution (potassium ferricyanide) are added. Connect the GC electrode to potentiostat BAS100B / W (manufactured by BAS), stir the solution at 37 ° C, and apply +500 mV to the silver-silver chloride (saturated KCl) reference electrode. A 1M D-glucose solution is added to these systems to a final concentration of 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 mM, and a steady-state current value is measured for each addition. This current value is plotted against a known glucose concentration (1, 2, 3, 4, 5, 10, 20, 30, 40, 50 mM) to create a calibration curve. Thus, glucose can be quantified with the enzyme-immobilized electrode using the FAD-bound glucose dehydrogenase of the present invention.

  The FAD-GDH mutant of the present invention shows good storage stability. Such FAD-GDH mutant can continuously measure the glucose concentration of the sample at 20 to 40 ° C. The continuous measurement can be from 1 day to 3 weeks, such as from 1 day to 2 weeks. It is also advantageous when transporting liquid reagents for a long time at 40 ° C. or lower. In particular, it can be used for continuous blood glucose measurement in which blood glucose is continuously measured near body temperature. For example, when the FAD-GDH mutant having storage stability of the present invention is used as an enzyme electrode, a stable electrode can be obtained. The electrode having such stability can be used for continuous blood glucose measurement.

  In one embodiment, a continuous blood glucose measurement device having the glucose sensor of the present invention is provided. The continuous blood glucose measurement device may include a CGM (continuous glucose monitoring) device or an FGM (Flash glucose monitoring) device that can grasp a blood glucose level change for 24 hours to 2 weeks. In one embodiment, a method for measuring continuous blood glucose using the FAD-GDH mutant of the present invention is provided.

  In certain embodiments, continuous blood glucose measurements can be made with or without recalibration. In some embodiments, the recalibration frequency can be set lower than the conventional method, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 Can be done daily.

  In certain embodiments, the CGM device may have a glucose sensor for placement under the skin. The sensor can be worn for a certain period, for example 1 day to 3 weeks. The sensor may be disposable or reusable. The sensor can have a sensor membrane layer to prevent direct contact between the tissue and the enzyme. The sensor can be designed to be inserted into the abdomen using an applicator. In certain embodiments, the present invention provides a CGM device further comprising a transmitter and a sensor to transmitter connection. The transmitter may not be implanted. Preferably, the transmitter is communicable with the radio receiver. The CGM device may further have an electrical receiver that continuously displays the glucose level. The CGM device may optionally have a calibration fingerstick (fingertip piercing member). The CGM device can have instructions for use.

  The enzyme activity of GOD can be measured by a known method. For example, the enzyme activity of GOD can be measured by a method of measuring GDH activity, for example, under conditions where an excess of mediators that accept electrons are present. Alternatively, the enzyme activity of GOD can be measured by an assay system using oxygen as an acceptor. Also, the enzyme activity of GOD can be determined according to the supplier's instructions. In order to calculate the residual activity ratio, the initial activity and the residual activity are measured in the same system.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.

Example 1. Introduction of Mucor gene-derived GDH gene into host and confirmation of GDH activity Roughly explained, first of all, each of N66Y / N68G / C88A / Q233R / T387C / E554D / L557V / S559K was added to Mucor gene-derived GDH (MpGDH, SEQ ID NO: 1). A gene encoding a modified GDH (MpGDH-M1) into which a mutation was introduced was obtained. The amino acid sequence of MpGDH-M1 is shown in SEQ ID NO: 10, and the base sequence of the gene is shown in SEQ ID NO: 11. A DNA construct was prepared by inserting the target gene MpGDH-M1 gene into the multicloning site of the plasmid yeast expression plasmid pYES2 / CT by a conventional method. At the In-Fusion Cloning Site at the multiple cloning site of pYES2 / CT, the MpGDH-M1 gene is ligated according to the protocol attached to the kit using the In-Fusion HD Cloning Kit described above, and the construct plasmid (pYES2 / CT-MpGDH-M1) was obtained.

  Using the obtained recombinant plasmid pYES2 / CT-MpGDH-M1 as a template, PCR was performed using the synthetic oligonucleotide of SEQ ID NO: 14-19, KOD-Plus- (manufactured by Toyobo) under the following conditions.

That is, 5 μl of 10 × KOD-Plus-buffer, 5 μl of dNTPs mixed solution prepared so that each dNTP is 2 mM, 2 μl of 25 mM MgSO ₄ solution, and a DNA construct in which MpGDH-M1 gene as a template is linked. Was added in an amount of 15 ng, and 1 unit of KOD-Plus- was added, and the total volume was adjusted to 50 μl with sterilized water. The prepared reaction solution was incubated at 94 ° C. for 2 minutes using a thermal cycler (Eppendorf), followed by “94 ° C., 15 seconds” — “50 ° C., 30 seconds” — “68 ° C., 8 minutes” This cycle was repeated 30 times.

  A part of the reaction solution was electrophoresed on a 1.0% agarose gel, and it was confirmed that about 8,000 bp of DNA was specifically amplified. The DNA thus obtained was treated with a restriction enzyme DpnI (manufactured by NEW ENGLAND BIOLABS), and the remaining template DNA was cleaved. Then, Escherichia coli JM109 was transformed and developed on an LB-amp agar medium. The grown colonies were inoculated into 2.5 ml of LB-amp medium [1% (W / V) bactotryptone, 0.5% (W / V) peptone, 0.5% (W / V) NaCl, 50 μg / ml Ampicillin] The culture was obtained by shaking at 37 ° C. for 20 hours. The culture was collected by centrifuging at 7,000 rpm for 5 minutes to obtain bacterial cells. Next, the recombinant plasmid was extracted from the cells using QIAGEN tip-100 (Qiagen) and purified to obtain 2.5 μg of DNA. The base sequence of DNA encoding MpGDH-M1 in the plasmid was determined using a multicapillary DNA analysis system Applied Biosystems 3130xl Genetic Analyzer (manufactured by Life Technologies). As a result, 175 of the amino acid sequence described in SEQ ID NO: 1 was determined. A DNA construct encoding MpGDH-M1 / A175C / N214C / G466D (SEQ ID NO: 12), a mutant in which the alanine at position is replaced with cysteine, the asparagine at position 214 with cysteine, and the glycine at position 466 with aspartic acid Obtained (SEQ ID NO: 13). This modified GDH was designated as MpGDH-M2.

Example 2 Preparation of FAD-GDH mutant Next, PCR was performed under the same conditions as described above using the synthetic oligonucleotides of SEQ ID NOs: 20-37 and 42-49, KOD-Plus- (manufactured by Toyobo Co., Ltd.), and site-specific mutation Was introduced.

  In the table, for example, “S192P” means that serine at position 192 in SEQ ID NO: 1 is substituted with proline. “S192P / S196N” means that serine at position 192 in SEQ ID NO: 1 is substituted with proline and serine at position 196 is substituted with asparagine, and the symbol “/” has both substitutions.

  Then, using the transformation kit for S. cerevisiae (manufactured by Invitrogen), INVSc1 strain using pYES2 / CT-M2 and a modified type into which various mutations were introduced (for example, pYES2 / CT-M2-S192P / S196N) By transforming (produced by Invitrogen), a yeast transformant Sc-M2 strain that expresses GDH, and a yeast transformant that expresses various modified MpGDH, such as the Sc-M2-S192P / S196N strain, etc. Respectively.

(Evaluation of storage stability of yeast-expressed FAD-GDH)
Each of yeast transformant Sc-M2, various yeast transformants Sc-M2-S192P / S196N, etc., was added to each 5 mL of a liquid medium for preculture [0.67% (w / v) amino acid-free yeast nitrogen base (BD), 0.192% (w / v) uracil-free yeast synthetic dropout medium additive (manufactured by sigma), 2.0% (w / v) raffinose] was cultured at 30 ° C. for 24 hours. Thereafter, 1 mL of the preculture solution was added to 4 mL of a liquid medium for main culture [0.67% (w / v) amino acid-free yeast nitrogen base, 0.192% (w / v) uracil-free yeast synthesis dropout medium additive, 2.5 % (w / v) D-galactose, 0.75% (w / v) raffinose], and cultured at 30 ° C. for 16 hours. This culture solution was separated into cells and culture supernatant by centrifugation (10,000 × g, 4 ° C., 3 minutes), and the culture supernatant was collected. 3 ml of this culture supernatant was concentrated to 50 μl with 0.5 ml of Amicon Ultra and replaced with 1 × PBS solution. These were used as test samples. Furthermore, GDH-M2 expressed and purified by Aspergillus sojae using the same procedure as described in International Publication No. 2010/140431, and the stable enzyme GDH-M2 after dialysis expressed in yeast are stable. It was confirmed that the sex did not change.

Example 3 Storage stability test of FAD-GDH mutant Next, the prepared mutant was heated in a PBS solution (2 ml) at pH 7.4 at 40 ° C. for a predetermined period. Subsequently, the residual activity after a predetermined period was measured.

GDH activity is measured when the FAD-GDH activity of each sample is measured based on the GDH activity measurement method described above after a predetermined period, and the enzyme activity immediately before storage at 40 ° C. is taken as 100%. The activity value after a predetermined period of time at 40 ° C. was calculated as “activity remaining rate (%)”.
The results of the storage stability test are shown in the table.

  As shown in the table, the mutant of the present invention has improved storage stability compared to the unsubstituted one. The mutants MpGDH-M2 / S196N and MpGDH-M2 / D228N were subjected to a heat treatment at 60 ° C. for 15 minutes, and the thermal stability was also evaluated. There was no improvement in stability. Similarly, in D55N, E435S, K267R, T276I, S587D, P588S, M591K, and V600I, heat treatment was performed at 55 ° C. for 15 minutes, and the thermal stability was also evaluated, but compared with the unmodified enzyme, it was significant. No improvement in thermal stability was observed. Therefore, it was confirmed that the mutation that contributes to the improvement of storage stability of GDH does not necessarily contribute to the improvement of thermal stability. International Publication No. 2017/195765 describes that MpGDH-M1 (SEQ ID NO: 10) had a residual activity rate of 56% after heat treatment at 50 ° C. for 15 minutes. According to the investigation by the present inventors, when the wild type MhGDH (SEQ ID NO: 3) was similarly heat-treated at 50 ° C. for 15 minutes, the residual activity rate was 60%. That is, MpGDH-M1 and MhGDH have almost the same thermal stability. However, when these were tested for storage stability in the same procedure as above, MpGDH-M1 had a residual activity rate of 15.5% after storage at 40 ° C. for 1 day, and a residual activity rate of 0% after storage for 5 days. In contrast, MhGDH had a residual activity rate of 32.6% after storage at 40 ° C. for 5 days. Therefore, the mutation contributing to the thermal stability of GDH does not necessarily correlate with the mutation contributing to storage stability.

  Subsequently, multiple mutations were constructed by combining mutations with improved storage stability. Using the synthetic oligonucleotides of SEQ ID NOS: 22-25 and 50-53 described in the following table, KOD-Plus- (manufactured by Toyobo Co., Ltd.), PCR reaction was carried out under the same conditions as described above to introduce site-specific mutations. .

  Next, the mutant prepared as described above was heated in a PBS solution (2 ml) at pH 7.4 at 40 ° C. for a predetermined period. Subsequently, the residual activity after a predetermined period was measured. As a result of the storage stability test, the obtained multiple mutant M2-6T is more than 95.3% active even when stored at 40 ° C for 7 days compared to 100% at 0 ° C Was found to remain. Furthermore, the mutant M2-10T combined with E230Q / V232A / V235A / T239A has 96.6% activity remaining compared to the case where the residual activity rate at 40 ° C. and 0 day is 100%, and further storage stability Was found to improve.

  Subsequently, using MpGDH-M3 having S192P / S196N / I212L / A218H / I226T / D228N / E230Q / V232A / V235A / T239A and having 93% identity with MpGDH and having the amino acid sequence of SEQ ID NO: 40, Similarly, it was verified whether the storage stability was good. The amino acid sequence of MpGDH-M3 is shown in SEQ ID NO: 40, and the base sequence of the gene is shown in SEQ ID NO: 41. In the same manner as described above, a DNA construct was prepared by inserting the target gene MpGDH-M3 gene into the multicloning site of the plasmid yeast expression plasmid pYES2 / CT by a conventional method. Next, a yeast transformant was prepared, and a culture supernatant containing MpGDH-M3 was collected. Test samples substituted with PBS were prepared in the same manner as described above, and the storage stability at 40 ° C. was evaluated.

  As a result, when the residual activity rate of MpGDH-M3 at 40 ° C. and 0 day was defined as 100%, it was found that 95.3% or more of MpGDH-M3 remained even after storage at 40 ° C. for 7 days.

  Next, an enzyme electrode was prepared using MpGDH-M3, and the stability of the electrode was evaluated. As a mediator, an Os polymer in which peroxidase was deactivated by heat treatment of Refill kit peroxidase (Os Polymer / Peroxidase) (manufactured by BAS, Cat No.002096) at 98 ° C. for 1 hour was used. 0.5 μL of Os polymer was applied to a glassy carbon electrode (manufactured by BAS) and air-dried. Subsequently, 4 U of MpGDH-M3 was applied and air-dried, and then 1 μL of 2% polyethylene glycol glycidyl ether (average molecular weight 500, manufactured by Sigma) was applied and allowed to stand at 4 ° C. for 16 hours. For comparison, electrodes were similarly prepared using MLD and GLD3 which are FAD-GDH purchased from Funakoshi. GLD1 and GLD3 were used for electrode preparation after purification using gel filtration chromatography.

  The obtained FAD-GDH-immobilized electrode was washed with ultrapure water, then placed in a 15 ml falcon tube and stored at 40 ° C. Cyclic voltammetry was used for electrode evaluation. Specifically, FAD-GDH immobilized electrode as working electrode, silver-silver chloride reference electrode (manufactured by BAS) as reference electrode, platinum electrode (manufactured by BAS) as counter electrode, ALS electrochemical analyzer 814D (manufactured by BAS) Connected to. Three poles were placed in 10 mL of 100 mM potassium phosphate buffer (pH 7.0), and cyclic voltammetry was performed by sweeping the voltage in the range of 0 mV to +600 mV (vs. Ag / AgCl) while stirring at 650 rpm with a stirrer. . The sweep speed was 10 mV / sec. Subsequently, a 2M glucose solution was added to a final concentration of 50 mM glucose, and cyclic voltammetry was performed in the same manner. The value obtained by subtracting the oxidation current value when glucose was not added from the oxidation current value when glucose was added at +500 mV (vs. Ag / AgCl) was recorded. The recorded value before storage at 40 ° C. was taken as 100%, and the stability after one week storage (residual activity rate (%)) was compared to evaluate the stability of the electrode.

  As a result, MpGDH-M3 was able to construct a very stable electrode as compared to MpGDH before mutagenesis. Furthermore, it was shown that MpGDH-M3 is more stable than commercially available GLD1 and GLD3. Note that, in continuous glucose monitoring and the like, since a protective film or the like is generally provided on the electrode, the conditions of this example are considered to be severer conditions than actual monitoring.

  As described above, the FAD-GDH mutant of the present invention has excellent storage stability and retains activity for a long period of time. This is excellent in stability and can measure glucose while maintaining the initial activity in continuous glucose monitoring as compared with a conventional GOD used for a sensor.

  Next, mutation was introduced into wild-type MpGDH instead of MpGDH-M2, and a storage stability test was performed. First, using a synthetic oligonucleotide of SEQ ID NOs: 54-66, KOD-Plus- (manufactured by Toyobo Co., Ltd.), a PCR reaction was performed under the same conditions as described above to introduce site-specific mutations.

  Subsequently, a yeast transformant was prepared in the same manner as described above, and a culture supernatant containing MpGDH and MpGDH mutant was collected. Test samples substituted with PBS were prepared in the same manner as described above, and the storage stability at 20 ° C. was evaluated.

  As a result, the storage stability of MpGDH / E230Q was improved compared to that before the mutation introduction. That is, unmodified MpGDH had a residual activity rate of 81.9% after 20 ° C. and 7 days compared to a residual activity rate of 100% at 20 ° C. and 0 day storage, whereas the MpGDH / E230Q mutant was The residual activity rate after 20 days at 20 ° C. was 83.4%, compared with 100% residual activity rate after storage at 20 ° C. for 0 days. The MpGDH / E230Q mutant was subjected to a heat treatment at 40 ° C. for 15 minutes, and its thermal stability was also evaluated. However, no significant improvement in thermal stability was observed compared to the unmodified enzyme.

  Subsequently, the storage stability at 25 ° C. was evaluated. As a result, the unmodified MpGDH had a residual activity rate of 40.5% after 25 days at 25 ° C compared to 100% of the residual activity rate stored at 25 ° C for 0 days, whereas the MpGDH-S196A mutant The residual activity rate after 7 days at 25 ° C. was 42.4%, compared with 100% residual activity rate after storage at 25 ° C. for 0 day. Various mutants were also evaluated.

  As a result, the storage stability of the mutant of the present invention was improved compared to that before the introduction of the mutation. At positions 196 and 228, it was found that even if the amino acid was replaced with other amino acids other than asparagine in the storage stability-enhancing mutation found in MpGDH-M2, it contributed to the improvement of storage stability.

(Preparation and storage stability evaluation of various modified MgGDH)
In Japanese Patent Laid-Open No. 2017-000137, sequence information of GDH derived from Mucor guillimonmondii (hereinafter referred to as MgGDH) having an identity of 78% with MpGDH is disclosed. Therefore, it was decided to verify whether the storage stability was improved by introducing the amino acid substitution found in Example 3 into MgGDH as follows.

  The amino acid sequence of MgGDH is shown in SEQ ID NO: 6, and the base sequence of the gene is shown in SEQ ID NO: 39. In the same manner as in Example 1, PCR reaction was performed using the synthetic oligonucleotides of SEQ ID NOs: 67 to 72 to introduce site-specific mutations.

  Subsequently, a yeast transformant was prepared in the same manner as described above, and a culture supernatant containing MgGDH and the MgGDH mutant was collected. Test samples substituted with PBS were prepared in the same manner as described above, and the storage stability at 25 ° C. was evaluated.

  As a result, it was found that also in MgGDH, a mutant into which a mutation whose storage stability was improved with MpGDH was improved in storage stability compared with that before the mutation was introduced. That is, unmodified MgGDH had a residual activity rate of 55.1% after 25 days at 25 ° C. compared to 100% of the residual activity rate stored at 0 ° C., whereas the MgGDH / H191N mutant The residual activity rate after 25 days at 25 ° C. and 5 days storage is 59.0% compared to 100% residual activity at 25 ° C. and 0 days storage, and the MgGDH / D227A mutant has a residual activity rate of 100% storage at 25 ° C. and 0 days. Compared to%, the residual activity rate after 7 days at 25 ° C. was 79.7%.

  In addition, the unmodified MgGDH had a remaining activity rate of 69.9% at 25 ° C. after 4 days compared to the remaining activity rate of 100% at 25 ° C. and 0 day storage, whereas the MgGDH-Q50N mutant was The residual activity rate after 4 days at 25 ° C. was 72.0% compared to 100% residual activity rate after storage at 25 ° C. for 0 day.

(Preparation and storage stability evaluation of various modified MhGDH)
In Japanese Patent Laid-Open No. 2013-116102, sequence information of GDH (hereinafter referred to as MhGDH) derived from Mucor himalis of SEQ ID NO: 3 having 78% identity with MpGDH is disclosed. Therefore, it was decided to verify whether the storage stability was similarly improved by introducing the amino acid substitution found in Example 3 into MhGDH as follows.

  The amino acid sequence of MhGDH is shown in SEQ ID NO: 3, and the base sequence of the gene is shown in SEQ ID NO: 38. PCR reaction was performed using the synthetic oligonucleotides of SEQ ID NOs: 73 to 78 in the same manner as in Example 3 to introduce site-specific mutations.

  Subsequently, a yeast transformant was prepared in the same manner as described above, and a culture supernatant containing MhGDH and MhGDH mutant was collected. Test samples substituted with PBS were prepared in the same manner as described above, and the storage stability at 40 ° C. was evaluated.

  As a result, it was found that also in MhGDH, a mutant introduced with a mutation whose storage stability was improved by MpGDH was improved in storage stability as compared with that before the mutation was introduced. Therefore, it is considered that the mutation of the present invention similarly improves the storage stability of GDH derived from other sources.

  The entire contents of WO 2017/1957665 are incorporated herein by reference. Although the invention has been described by way of example, various modifications can be made without departing from the spirit of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 Mucor prainii GDH (MpGDH) aa
SEQ ID NO: 2 MpGDH gene DNA
Sequence number 3 Mucor hiemalis GDH (MhGDH) aa
SEQ ID NO: 4 Mucor RD056860 GDH (MrdGDH) aa
Sequence number 5 Mucor subtilissimus GDH (MsGDH) aa
Sequence number 6 Mucor guilliermondii GDH (MgGDH) aa
Sequence number 7 Circinella simplex GDH (CsGDH) aa
Sequence number 8 Circinella genus GDH (CrGDH) aa
SEQ ID NO: 9 Mucor circinelloides GDH (McGDH) aa
SEQ ID NO: 10 MpGDH-M1 GDH aa
SEQ ID NO: 11 MpGDH-M1 gene DNA
Sequence number 12 MpGDH-M1 / A175C / N214C / G466D aa (M2)
SEQ ID NO: 13 MpGDH-M2 gene DNA
SEQ ID NO: 14-37 Primer SEQ ID NO: 38 MhGDH gene DNA
SEQ ID NO: 39 MgGDH gene DNA
SEQ ID NO: 40 MpGDH-M3 aa
SEQ ID NO: 41 MpGDH-M3 gene DNA
SEQ ID NOs: 42-78 primers

Claims (15)

A FAD-dependent glucose dehydrogenase (FAD-GDH) variant comprising:
i) the amino acid sequence represented by SEQ ID NO: 1,
ii) In the amino acid sequence shown in SEQ ID NO: 1, positions 196, 228, 600, 267, 276, 55, 230, 235, 239, 435, 587, 588, and 591 An amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than
iii) an amino acid sequence having 70% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, 3, 6 or 40, or
iv) The amino acid sequence represented by SEQ ID NO: 1, 3, or 6 has a sequence identity of 70% or more, and the amino acid sequence of the homologous region is 90 with the homologous region in the amino acid sequence represented by SEQ ID NO: 1. % Amino acid sequence having amino acid sequence identity,
Corresponds to positions 196, 228, 600, 267, 276, 55, 230, 235, 239, 435, 587, 588, and / or 591 in the amino acid sequence of SEQ ID NO: 1. A FAD-dependent glucose dehydrogenase mutant having one or more amino acid substitutions at a position, and having improved storage stability compared to an unmodified FAD-dependent glucose dehydrogenase having no such substitution. In FAD-GDH,
The amino acid at the position corresponding to position 196 in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine, phenylalanine, lysine, glutamine, asparagine, histidine, methionine, alanine, or arginine;
The amino acid at the position corresponding to position 228 in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine, threonine, histidine, serine, glutamine, or cysteine,
The amino acid at the position corresponding to position 600 in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan;
The amino acid at the position corresponding to position 267 in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine, histidine or glutamine;
The amino acid at the position corresponding to position 276 in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine, leucine, valine, methionine, cysteine, phenylalanine, tryptophan or tyrosine,
The amino acid at the position corresponding to position 55 in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine, serine, threonine, glutamine or cysteine,
The amino acid at the position corresponding to position 230 in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine, serine, threonine, asparagine, arginine, lysine, or histidine;
The amino acid at the position corresponding to position 235 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, serine or glycine;
The amino acid at the position corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, serine or glycine;
The amino acid at the position corresponding to position 435 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine, threonine, asparagine or glutamine,
The amino acid at the position corresponding to position 587 in the amino acid sequence of SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid,
The amino acid at the position corresponding to position 588 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine, threonine, asparagine or glutamine, and / or the amino acid at the position corresponding to position 591 in the amino acid sequence of SEQ ID NO: 1 is lysine, Substituted with arginine or histidine,
The FAD-GDH variant according to claim 1, wherein the FAD-GDH variant has improved storage stability compared to an unmodified FAD-GDH that does not have the substitution. In FAD-GDH,
The amino acid at the position corresponding to position 196 in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine, phenylalanine, lysine, glutamine, asparagine, histidine, methionine, or alanine;
The amino acid at the position corresponding to position 228 in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine, threonine, or histidine;
The amino acid at the position corresponding to position 600 in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine,
An amino acid at a position corresponding to position 267 in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine;
The amino acid at the position corresponding to position 276 in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine,
The amino acid at the position corresponding to position 55 in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine,
The amino acid at the position corresponding to position 230 in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine,
An amino acid at a position corresponding to position 235 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine;
The amino acid at the position corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine;
The amino acid at the position corresponding to position 435 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine,
An amino acid at a position corresponding to position 587 in the amino acid sequence of SEQ ID NO: 1 is substituted with aspartic acid,
The amino acid at the position corresponding to position 588 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine, and / or the amino acid at the position corresponding to position 591 in the amino acid sequence of SEQ ID NO: 1 is replaced with lysine,
The FAD-GDH mutant according to claim 1 or 2, wherein the FAD-GDH mutant has improved storage stability compared to an unmodified FAD-GDH having no such substitution. further,
An amino acid at a position corresponding to position 192 in the amino acid sequence of SEQ ID NO: 1 is substituted with proline;
The amino acid at the position corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine,
The amino acid at the position corresponding to position 218 in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine;
The amino acid at the position corresponding to position 226 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, and / or the amino acid at the position corresponding to position 232 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine,
The FAD-GDH mutant according to any one of claims 1 to 3. i) The amino acid at the position corresponding to position 196 in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine, phenylalanine, lysine or asparagine, and the amino acid at the position corresponding to position 228 in the amino acid sequence of SEQ ID NO: 1 is asparagine, threonine or A variant substituted with histidine,
ii) The amino acid at the position corresponding to position 192 in the amino acid sequence of SEQ ID NO: 1 is substituted with proline, and the amino acid at the position corresponding to position 196 in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine, phenylalanine, lysine or asparagine Mutants,
iii) The amino acid at the position corresponding to position 226 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, and the amino acid at the position corresponding to position 228 in the amino acid sequence of SEQ ID NO: 1 is replaced with asparagine, threonine or histidine. Mutant,
iv) The amino acid at the position corresponding to position 230 in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine, the amino acid at the position corresponding to position 232 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, and the amino acid sequence of SEQ ID NO: 1 Wherein the amino acid at the position corresponding to position 235 is substituted with alanine, and the amino acid at the position corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine,
v) The amino acid at the position corresponding to position 192 in the amino acid sequence of SEQ ID NO: 1 is substituted with proline, the amino acid at the position corresponding to position 196 in the amino acid sequence of SEQ ID NO: 1 is replaced with asparagine, and the amino acid sequence of SEQ ID NO: 1 The amino acid at the position corresponding to position 212 in the amino acid was substituted with leucine, the amino acid at the position corresponding to position 218 in the amino acid sequence of SEQ ID NO: 1 was replaced with histidine, and the amino acid at the position corresponding to position 226 in the amino acid sequence of SEQ ID NO: 1 A 192P / 196N / 212L / 218H / 226T / 228N mutant in which the amino acid is substituted with threonine and the amino acid at the position corresponding to position 228 in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine;
vi) In the mutant v), the amino acid at the position corresponding to position 230 in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine, and the amino acid at the position corresponding to position 232 in the amino acid sequence of SEQ ID NO: 1 is alanine 192P / 196N in which the amino acid at the position corresponding to position 235 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, and the amino acid at the position corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine / 212L / 218H / 226T / 228N / 230Q / 232A / 235A / 239A mutant, and
vii) The M3 variant having the amino acid sequence of SEQ ID NO: 40 in the variant of vi) above,
The FAD-GDH variant according to any one of claims 1 to 4, which is selected from the group consisting of and has improved storage stability compared to FAD-GDH having no such substitution. When the enzyme activity of unsubstituted FAD-GDH before heating is defined as 100%, the remaining activity (%) of unsubstituted FAD-GDH heated at pH 7.4 at 20-40 ° C, and before heating In comparison with the residual activity (%) of the FAD-GDH mutant heated at 20-40 ° C. and pH 7.4 when the enzyme activity of the FAD-GDH mutant was 100%,
(a) Residual activity (%) after heating for 1 week is improved by 1% or more.
(b) Residual activity (%) after 2 weeks of warming is improved by 3% or more.
(c) Residual activity (%) after heating for 3 weeks is improved by 3% or more, or
(d) Residual activity (%) after heating for 4 days is improved by 1% or more.
The FAD-GDH mutant according to any one of claims 1 to 5.   The FAD-GDH gene which codes the FAD-GDH variant of any one of Claims 1-6.   A vector comprising the FAD-GDH gene according to claim 7.   A host cell into which the vector according to claim 8 has been introduced. Method for producing FAD-GDH including the following steps:
Culturing the host cell of claim 9;
Expressing the FAD-GDH gene contained in the host cell, and isolating FAD-GDH from the culture.   A glucose assay kit comprising the FAD-GDH mutant according to any one of claims 1 to 6.   The glucose sensor containing the FAD-GDH variant of any one of Claims 1-6.   A continuous blood glucose measurement device comprising the glucose sensor according to claim 12.   A glucose measurement method using the FAD-GDH mutant according to any one of claims 1 to 6, the kit according to claim 11, the sensor according to claim 12, or the device according to claim 13.   The method according to claim 14, wherein continuous measurement is performed at 20 to 40 ° C. for 1 day to 2 weeks.

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