JP2011115156A - Method for improving temperature dependency of flavin-adenine-dinucleotide-dependent glucose dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a modified FAD-dependent glucose dehydrogenase free from defects of known FAD-dependent glucose dehydrogenase, and having sufficient reactivity in a wide temperature range and excellent substrate specificity. <P>SOLUTION: There is provided the modified FAD-dependent glucose dehydrogenase having improved temperature dependency and lowered action to xylose compared with unmodified FAD-dependent glucose dehydrogenase and produced by modifying the FAD-dependent glucose dehydrogenase on a genetic level. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for improving temperature dependency of flavin adenine dinucleotide-dependent glucose dehydrogenase (hereinafter also referred to as FADGDH) and improving substrate specificity, and encodes the modified FADGDH The present invention relates to a gene, a method for producing the modified FADGDH, and various applications of the modified FADGDH to a glucose measuring reagent.

  In recent years, the incidence of diabetes has been increasing year by year, and it is estimated that the total number of people who are said to be diabetic reserves will be more than 10 million in Japan alone. In addition, interest in lifestyle-related diseases has increased greatly, and opportunities for self-management of blood glucose levels are increasing. Against this backdrop, development of technology for blood glucose self-monitoring is an important technology for diabetics to manage their daily blood glucose levels. With regard to blood glucose measurement technology, practical use is progressing in many ways, and electrochemical sensing is advantageous in terms of reducing the amount of specimen, shortening measurement time, and downsizing the apparatus.

  As a sensing technique in blood glucose measurement technology, an enzyme using glucose in blood as a substrate is used. An example of such an enzyme is glucose oxidase (EC 1.1.3.4). Glucose oxidase has the advantage of high specificity to glucose and high stability to heat. The blood glucose sensor using glucose oxidase is measured by the electron generated in the process of oxidizing glucose and converting it to D-glucono-δ-lactone through the mediator. The glucose oxidase is a proton generated by the reaction. There is a problem in that the measured value is affected because it is easy to pass to the dissolved oxygen.

  In order to avoid such a problem, pyrroloquinoline quinone-dependent glucose dehydrogenase (hereinafter also referred to as PQQGDH) (EC 1.1.5.2 (formerly EC 1.1.9.17)) is used as an enzyme for blood glucose sensors. It is used. PQQGDH is advantageous in that it is not affected by dissolved oxygen, but it has a disadvantage that it has poor substrate specificity, and acts on saccharides other than glucose such as maltose and lactose, thereby impairing the accuracy of measurement values.

Therefore, a flavin adenine dinucleotide-dependent glucose dehydrogenase (hereinafter referred to as FAD-dependent glucose dehydrogenase) is described as FADGDH as a glucose dehydrogenase (hereinafter also referred to as GDH) that is not affected by dissolved oxygen and has excellent substrate specificity. ) Is attracting attention. FADGDH is described in Non-Patent Documents 1 to 6, and has been known for a long time.
Patent document 1 describes the gene sequence and amino acid sequence of FADGDH derived from Aspergillus terreus.
Patent Document 2 describes Aspergillus oryzae-derived FADGDH, and Patent Document 3 describes modified FADGDH with improved thermal stability by modifying Aspergillus oryzae-derived FADGDH. Patent Document 4 describes modified FADGDH in which FADGDH derived from Aspergillus oryzae and FADGDH derived from Aspergillus terreus are modified to improve thermal stability and xylose activity.

  On the other hand, Non-Patent Document 7 states that, as a caution point of the SMBG (Self Monitoring Blood Glucose) device, when the environmental temperature condition is a low temperature range and a high temperature range, the measured value is out of the allowable range of ISO15197. There are many devices that come off, and it is pointed out that if the measured value shows extremely low or high values, it can cause medical accidents.

WO2004 / 058958 JP2007-289148 JP2008-237210A WO2008 / 059777

Biochim Biophys Acta. 1967 Jul 11; 139 (2): 265-76. Biochim Biophys Acta. 1967 Jul 11; 139 (2): 277-93. Biochim Biophys Acta. 146 (2): 317-27 Biochim Biophys Acta. 146 (2): 328-35 J Biol Chem (1967) 242: 3665-3672. Appl Biochem Biotechnol (1996) 56: 301-310 Equipment / Reagents 32 (6), 2009: 707-713

  An object of the present invention is to provide an enzyme that can be used as a reagent for measuring blood glucose level, which is further advantageous in practical use as compared with the known enzymes for blood glucose sensor as described above.

  The inventors of the present invention have focused on the variation and abnormality of measured values according to changes in the environmental temperature at which the blood glucose concentration is measured, and have found that the following problems may exist.

In the development stage, the enzyme reaction conditions are mainly examined at a specific temperature (for example, 37 ° C.). In contrast, the temperature at which a diabetic patient actually measures glucose using a glucose sensor is at room temperature. If the enzyme reactivity varies due to temperature changes, the measured values will vary.
Some glucose sensors have a built-in correction function in anticipation of such temperature fluctuations, but the function is not perfect.
Some known FADGDHs act on xylose. Xylose is a monosaccharide used for carbohydrate digestion and absorption tests. Therefore, when a patient performing a xylose absorption test uses a blood glucose sensor, it reacts not only with glucose in blood but also with xylose, so that the measured value is higher than the original glucose concentration.

Based on the above consideration, the present inventors examined the temperature dependence of the known FADGDH disclosed in Patent Document 2 or Patent Document 3. As a result, it was found that FADGDH disclosed in Patent Document 3 has an activity value at 25 ° C. of 63% and an activity value at 5 ° C. of 40% when the activity value at 37 ° C. is 100%. That is, it was found that the measurement accuracy of this FADGDH decreases depending on the environmental temperature when measuring the glucose concentration. That is, the 37% activity decreases between the activity value at 37 ° C. and the activity value at 25 ° C. Such fluctuations in activity are factors that hinder accurate measurement of glucose concentration using such enzymes. This is because, for example, in some glucose sensors, the glucose concentration is estimated based on the amount of product resulting from the dehydrogenation reaction catalyzed by GDH within a certain time.
In the present invention, the “temperature-dependent value” means that the activity value of FADGDH before modification is divided by the activity value at 25 ° C. by the activity value at 37 ° C. The value obtained by performing the same calculation is further divided by the value of FADGDH before modification.
If the temperature dependence value is 1.1, the difference between 37 ° C. and 25 ° C. is calculated to be about 30%, and the difference between 25 ° C. and 30 ° C. is estimated to be reduced to about 12-13%. The When this is considered as the difference between the maximum value and the minimum value of the variation in data, it is preferable that it is within about 6 to 7%.
Furthermore, if the temperature dependency is 1.2, the difference between 37 ° C. and 25 ° C. is estimated to be about 24%, and the difference between 25 ° C. and 30 ° C. is estimated to be reduced to about 10%. If this is considered as the difference between the maximum value and the minimum value of the variation in data, it will be within about ± 5%, which is more preferable.

  If the change in reactivity due to the environmental temperature can be reduced, it can be expected to improve the measurement accuracy and accuracy. In the present application, smoothing the peak of the optimum temperature is expressed as improvement of temperature dependency.

As a result of intensive research, the present inventors have found a modified FADGDH having a temperature dependency improved over that of the wild type FADGDH before modification by substituting a specific amino acid of the known FADGDH with another amino acid. The present invention was completed by assembling a glucose measurement system using
In addition, the present inventors examined the xylose activity of FADGDH described in Patent Document 1 (see Patent Document 4), and the xylose activity of FADGDH is 10% when the reactivity to glucose is 100%. It was found to react to some extent. That is, it has been found that when measuring the glucose concentration using this FADGDH, the accuracy of the measured value is impaired.
As a result of intensive studies, the present inventors have replaced temperature-specific FADGDH specific amino acids with other amino acids to improve temperature dependence and reduce xylose activity as compared with FADGDH before modification. The modified FADGDH was found and the present invention was completed.

Hereinafter, representative examples of the present invention will be exemplified.
Item 1.
The following protein (A1) or (A2).
(A1) A protein comprising an amino acid sequence containing any of the amino acid substitutions shown in the following (a) in the amino acid sequence of FAD-dependent glucose dehydrogenase (FADGDH) described in SEQ ID NO: 2:
(A) G59A, G59C, G59D, G59F, G59H, G59K, G59L, G59M, G59N, G59P, G59Q, G59S, G59T, G59W, G59Y, R82A, R82C, R82H, R82K, R82Q, R82V, R82Y, V108A, V108D , V108E, V108F, V108H, V108L, V108M, V108Q, V108R, V108S, V108T, F505D, F505L, F505M, F505N, F505Q, F505R, F505Y;
(A2) a protein satisfying the following (i) to (iii):
(I) an amino acid sequence having the following amino acid (b) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(B) 59A, 59C, 59D, 59F, 59H, 59K, 59L, 59M, 59N, 59P, 59Q, 59S, 59T, 59W, 59Y, 82A, 82C, 82H, 82K, 82Q, 82V, 82Y, 108A, 108D 108E, 108F, 108H, 108L, 108M, 108Q, 108R, 108S, 108T, 505D, 505L, 505M, 505N, 505Q, 505R, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has improved temperature dependence over the protein having the amino acid sequence of SEQ ID NO: 2.
Item 2.
The following protein (B1) or (B2).
(B1) A protein comprising an amino acid sequence containing any of the amino acid substitutions shown in the following (c) in the amino acid sequence of FAD-dependent glucose dehydrogenase (FADGDH) described in SEQ ID NO: 2:
(C) G59C, G59D, G59K, G59M, G59Q, G59S, G59T, R82H, R82Q, V108M, V108T, F505Y;
(B2) a protein satisfying the following (i) to (iii):
(I) an amino acid sequence having the following amino acid (d) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(D) 59C, 59D, 59K, 59M, 59Q, 59S, 59T, 82H, 82Q, 108M, 108T, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has a reduced xylose activity compared to a protein having the amino acid sequence of SEQ ID NO: 2.
Item 3.
The following protein (C1) or (C2).
(C1) A protein comprising an amino acid sequence containing any of the amino acid substitutions shown in (e) below in the amino acid sequence of FAD-dependent glucose dehydrogenase (FADGDH) described in SEQ ID NO: 2:
(E) G59C, G59D, G59K, G59M, G59Q, G59S, G59T, R82H, R82Q, V108M, V108T, F505Y;
(C2) a protein satisfying the following (i) to (iii):
(I) an amino acid sequence having the following amino acid (f) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(F) 59C, 59D, 59K, 59M, 59Q, 59S, 59T, 82H, 82Q, 108M, 108T, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has an improved temperature dependency over the protein having the amino acid sequence of SEQ ID NO: 2, and has a reduced xylose activity.
Item 4.
Item 4. A gene encoding the protein according to any one of Items 1 to 3.
Item 5.
Item 5. A vector comprising the gene according to item 4.
Item 6.
Item 6. A transformant transformed with the vector according to Item 5.
Item 7.
A method for producing a protein having glucose dehydrogenase activity, comprising culturing the transformant according to item 6 and collecting a protein having glucose dehydrogenase activity.
Item 8.
Item 4. A glucose assay kit comprising the protein according to any one of Items 1 to 3.
Item 9.
Item 4. A glucose sensor comprising the protein according to any one of Items 1 to 3.
Item 10.
The glucose measuring method containing the protein in any one of claim | item 1-3.

The protein of the present invention is glucose dehydrogenase excellent in temperature dependency. That is, the protein of the present invention is glucose dehydrogenase in which the influence on the activity due to the temperature change of the use environment is reduced. Therefore, by using the protein of the present invention, it is possible to measure the glucose concentration in the sample more accurately without being influenced by the temperature change of the use environment. Since the protein of the present invention has a reduced influence on the enzyme activity due to temperature changes in the environment of use, for example, the glucose concentration in the sample can be more accurately even at temperatures lower or higher than normal room temperature. Makes it possible to measure. Therefore, the protein of the present invention is useful as an enzyme for clinical tests for measuring blood glucose concentration.
The protein of the present invention is not only temperature-dependent but also has reduced action on xylose. Therefore, from the viewpoint of substrate specificity, the protein of the present invention is excellent in measuring blood glucose concentration.
By the method of the present invention, it is possible to efficiently produce a novel protein having the excellent characteristics as described above.

FIG. 1 shows a change in the activity of FADGDH containing the amino acid sequence represented by SEQ ID NO: 2 due to a change in temperature. FIG. 2 is a diagram comparing the amino acid sequence of Aspergillus oryzae-derived wild-type FADGDH (SEQ ID NO: 1) with the amino acid sequence of Aspergillus oryzae-derived wild-type FADGDH (SEQ ID NO: 3).

The present invention is described in detail below.
In this specification, the amino acid which comprises an amino acid sequence is described by the alphabet one letter or three letters. For example, glycine is written as Gly or G. The position of the amino acid on the amino acid sequence is specified using a number. For example, when the 59th glycine is substituted with alanine, it is expressed as “G59A”. “59A” indicates that the amino acid at position 59 (or position 59) is Ala (A) regardless of the presence or absence of mutation. In SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, the amino acid notation is numbered with methionine as 1.

  In the present specification, SEQ ID NO: 1 shows the amino acid sequence of FADGDH derived from wild-type Aspergillus oryzae. SEQ ID NO: 2 shows an amino acid sequence in which glycine at position 163 in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine and valine at position 551 is substituted with cysteine. The protein consisting of the amino acid sequence represented by SEQ ID NO: 2 retains glucose dehydrogenase activity. Hereinafter, the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is appropriately referred to as FADGDH of SEQ ID NO: 2. The amino acid sequences of SEQ ID NOs: 1 and 2 are disclosed in, for example, Patent Document 2 or Patent Document 3.

Modified FADGDH with improved temperature dependence
One embodiment of the present invention is the protein described below.
In the amino acid sequence of FADGDH described in SEQ ID NO: 1 or SEQ ID NO: 2, it has an amino acid substitution at any of positions 59, 82, 108, and 505, or an equivalent position, and is more temperature-dependent than before modification Modified FADGDH with improved properties.

Specifically, the following protein (A1) or (A2) is exemplified.
(A1) A protein comprising an amino acid sequence containing any of the amino acid substitutions shown in the following (a) in the amino acid sequence of FAD-dependent glucose dehydrogenase (FADGDH) described in SEQ ID NO: 2:
(A) G59A, G59C, G59D, G59F, G59H, G59K, G59L, G59M, G59N, G59P, G59Q, G59S, G59T, G59W, G59Y, R82A, R82C, R82H, R82K, R82Q, R82V, R82Y, V108A, V108D , V108E, V108F, V108H, V108L, V108M, V108Q, V108R, V108S, V108T, F505D, F505L, F505M, F505N, F505Q, F505R, F505Y;
(A2) a protein satisfying the following (i) to (iii):
(I) an amino acid sequence having the following amino acid (b) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(B) 59A, 59C, 59D, 59F, 59H, 59K, 59L, 59M, 59N, 59P, 59Q, 59S, 59T, 59W, 59Y, 82A, 82C, 82H, 82K, 82Q, 82V, 82Y, 108A, 108D 108E, 108F, 108H, 108L, 108M, 108Q, 108R, 108S, 108T, 505D, 505L, 505M, 505N, 505Q, 505R, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has improved temperature dependence over the protein having the amino acid sequence of SEQ ID NO: 2.

Modified FADGDH with reduced xylose activity
In the amino acid sequence of FADGDH described in SEQ ID NO: 1 or SEQ ID NO: 2, it has an amino acid substitution at any one of positions 59, 82, 108, and 505, or an equivalent position thereof, which is more xylose than before modification Modified FADGDH having reduced properties is also an embodiment of the present invention.

Specifically, the following protein (B1) or (B2) is exemplified.
(B1) A protein comprising an amino acid sequence containing any of the amino acid substitutions shown in the following (c) in the amino acid sequence of FAD-dependent glucose dehydrogenase (FADGDH) described in SEQ ID NO: 2:
(C) G59C, G59D, G59K, G59M, G59Q, G59S, G59T, R82H, R82Q, V108M, V108T, F505Y;
(B2) a protein satisfying the following (i) to (iii):
(I) an amino acid sequence having the following amino acid (d) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(D) 59C, 59D, 59K, 59M, 59Q, 59S, 59T, 82H, 82Q, 108M, 108T, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has a reduced xylose activity compared to a protein having the amino acid sequence of SEQ ID NO: 2.

Modified FADGDH with improved temperature dependence and reduced xylose activity
In the amino acid sequence of FADGDH described in SEQ ID NO: 1 or SEQ ID NO: 2, it has an amino acid substitution at any of positions 59, 82, 108, and 505, or an equivalent position, and is more temperature-dependent than before modification Modified FADGDH with improved properties and reduced xylose activity is also an embodiment of the present invention.

Specifically, the following protein (C1) or (C2) is exemplified.
(C1) A protein comprising an amino acid sequence containing any of the amino acid substitutions shown in (e) below in the amino acid sequence of FAD-dependent glucose dehydrogenase (FADGDH) described in SEQ ID NO: 2:
(E) G59C, G59D, G59K, G59M, G59Q, G59S, G59T, R82H, R82Q, V108M, V108T, F505Y;
(C2) a protein satisfying the following (i) to (iii):
(I) an amino acid sequence having the following amino acid (f) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(F) 59C, 59D, 59K, 59M, 59Q, 59S, 59T, 82H, 82Q, 108M, 108T, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has an improved temperature dependency over the protein having the amino acid sequence of SEQ ID NO: 2, and has a reduced xylose activity.

Temperature dependency In this application, temperature dependency means the property that the glucose dehydrogenase activity which a protein has changes with the temperature change of the environment where protein exists. “Improved temperature dependency” means that a change in enzyme activity accompanying a change in environmental temperature is small, and a more constant enzyme activity is maintained under a wide range of temperature conditions.
Whether the temperature dependency is improved is determined based on the temperature dependency of FADGDH consisting of the amino acid sequence represented by SEQ ID NO: 2, and specifically, determined according to the following (1) to (5).
(1) The activity value (U / ml) after treatment at 37 ° C. for 24 hours is measured, and this is designated as A. (2) The activity value (U / ml) after treatment at 25 ° C. for 24 hours is measured, and this is designated as B. (3) The relative value (%) of B when A is 100% is calculated, and this is defined as C.
(4) The C value of FADGDH of SEQ ID NO: 2 is set to 1, and (A1) the relative value of C of a modified protein such as a protein is determined. This is D. (D is also called the temperature dependence ratio.)
(5) As shown in Example 1 below, the enzyme activity at 25 ° C. of FADGDH of SEQ ID NO: 2 is 63%, assuming that the enzyme activity at 37 ° C. is 100%. Therefore, when the C value of the modified protein is greater than 63%, the value of D is greater than 1. That is, the value of D of the modified FADGDH is improved as it approaches 1.59 starting from 1 and has a lower temperature dependency.
The proteins of the present invention have a value of D greater than at least 1 because they have improved temperature dependence. Preferably, the value of D is 1.02 or more, more preferably 1.05 or more, still more preferably 1.1 or more, still more preferably 1.15 or more, still more preferably 1.2 or more, More preferably, it is 1.25 or more, More preferably, it is 1.3 or more.

(A1) The amino acid substitution contained in the amino acid sequence of the protein may be any of the substitutions listed as (a) above.
Preferred amino acid substitutions from the viewpoint of improving temperature dependence are G59A, G59C, G59D, G59F, G59H, G59K, G59L, G59M, G59N, G59P, G59Q, G59S, G59T, G59W, G59Y, R82A, R82C, R82H, R82K. , R82Q, R82V, R82Y, V108A, V108F, V108H, V108M, V108Q, V108R, V108S, V108T, F505D, F505L, F505M, F505N, F505Q, F505R, and F505Y. Modified FADGDH having these amino acid substitutions has a value of D of 1.02 or more.
More preferred amino acid substitutions are G59A, G59C, G59F, G59H, G59K, G59L, G59M, G59N, G59P, G59Q, G59S, G59T, G59W, G59Y, R82A, R82C, R82H, R82K, R82Q, R82V, R82Y, V108R, Any of F505L, F505Q, F505R, and F505Y. Modified FADGDH having these amino acid substitutions has a value of D of 1.05 or more.
More preferred amino acid substitution is any of G59C, G59F, G59H, G59K, G59L, G59M, G59N, G59Q, G59T, G59W, G59Y, R82C, R82H, R82Q, R82V, R82Y, F505Q, and F505R. Modified FADGDH having these amino acid substitutions has a value of D of 1.1 or more.
Further preferred amino acid substitutions are any of G59C, G59F, G59L, G59M, G59T, G59Y, R82H, R82Y, F505Q and F505R. Modified FADGDH having these amino acid substitutions has a value of D of 1.15 or more.
More preferred amino acid substitutions are G59F, G59M, G59T, G59Y and R82Y. The modified FADGDH having this amino acid substitution has a value of D of 1.2 or more.
As long as the temperature dependency which (A1) protein has is improved, two or more specific amino acids illustrated above may exist in a different position in an amino acid sequence.

On the other hand, the action on xylose is reduced along with the improvement of temperature dependency.
In the present invention, the xylose activity is expressed as a relative ratio% between the reaction rate when glucose is used as a substrate and the reaction rate when xylose is used as a substrate (glucose is defined as 100%). The value of the xylos activity of FADGDH consisting of the amino acid sequence shown in SEQ ID NO: 2 is 1, and the relative ratio% of each modified enzyme is calculated. Therefore, it is determined that the smaller the value, the lower the xylose activity.

  The modified FADGDH of the present invention is preferably a modified FADGDH in which the xylose activity ratio is reduced to 0.9 or less compared to that before modification. More preferably, the modified FADGDH is reduced to 0.8 or less compared to before modification.

  The method for measuring temperature dependence and xylose activity is determined using the FADGDH activity measurement method described below.

(B1) The amino acid substitution contained in the amino acid sequence of the protein may be any of the substitutions listed as (c) above.
A preferable amino acid substitution from the viewpoint of reduced activity on xylose is any one selected from the group consisting of G59C, G59D, G59K, G59M, G59Q, G59S, G59T, R82H, R82Q, V108M, V108T, and F505Y. It is. The modified FADGDH having these amino acid substitutions has a xylose activity ratio of 0.97 or less compared to that before modification.
More preferred amino acid substitution is any of G59M, G59Q, R82H, R82Q, and F505Y. The modified FADGDH having these amino acid substitutions has a xylose activity ratio of 0.9 or less compared to that before modification.
A more preferred amino acid substitution is G59M. The modified FADGDH having these amino acid substitutions has a xylose activity ratio of 0.8 or less compared to that before modification.
A plurality of the specific amino acids exemplified above may be present at different positions in the amino acid sequence as long as the action of (B1) protein on xylose is reduced.

(C1) The amino acid substitution contained in the amino acid sequence of the protein may be any of the substitutions listed as (e) above.
As long as the specific amino acids exemplified above have improved temperature dependency of (C1) protein and (C1) the activity of the protein on xylose has been reduced, multiple amino acids exist at different positions in the amino acid sequence. May be.

  Two or more amino acid substitutions in the above (A1), (B1) and (C1) may be present as long as the substituted amino acid positions are different, and any combination thereof may be used.

(A1) The protein may have further mutation (amino acid substitution, addition, deletion, insertion) in its amino acid sequence as long as it has glucose dehydrogenase activity and temperature dependency is improved. For example, you may have two or more amino acid substitution selected from said (a) in a different site | part.
In the amino acid sequence of SEQ ID NO: 2, the amino acid sequence in which arginine at position 163 is replaced with glycine and cysteine at position 551 is replaced with valine is the amino acid sequence of wild-type FADGDH (SEQ ID NO: 1). Therefore, in the protein (A1), in addition to the above specific amino acid substitution, the 163rd amino acid may be substituted with glycine, and the 551st amino acid may be substituted with valine.

(A2) The protein satisfies the following requirements (i) to (iii).
(I) comprising an amino acid sequence containing any of the amino acid substitutions shown in the following (b) in the amino acid sequence containing at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(B) 59A, 59C, 59D, 59F, 59H, 59K, 59L, 59M, 59N, 59P, 59Q, 59S, 59T, 59W, 59Y, 82A, 82C, 82H, 82K, 82Q, 82V, 82Y, 108A, 108D 108E, 108F, 108H, 108L, 108M, 108Q, 108R, 108S, 108T, 505D, 505L, 505M, 505N, 505Q, 505R, 505Y;
(Ii) having glucose dehydrogenase activity;
(Iii) It has improved temperature dependence over the protein having the amino acid sequence of SEQ ID NO: 2.
That is, the (A2) protein contains an amino acid sequence having at least 60% amino acid homology with the amino acid sequence shown in SEQ ID NO: 2 and contains at least one amino acid shown in (b) above. Furthermore, the (A2) protein retains glucose dehydrogenase activity and has an improved temperature dependence compared to FADGDH of SEQ ID NO: 2.

(B2) The protein satisfies the following requirements (i) to (iii).
(I) an amino acid sequence having the following amino acid (d) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(D) 59C, 59D, 59K, 59M, 59Q, 59S, 59T, 82H, 82Q, 108M, 108T, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has a reduced xylose activity compared to a protein having the amino acid sequence of SEQ ID NO: 2.
That is, the (B2) protein contains an amino acid sequence having at least 60% amino acid homology with the amino acid sequence shown in SEQ ID NO: 2 and contains at least one amino acid shown in (d) above. Furthermore, the (B2) protein retains glucose dehydrogenase activity and has a reduced xylose activity as compared to the FADGDH of SEQ ID NO: 2.

(C2) The protein satisfies the following requirements (i) to (iii).
(I) an amino acid sequence having the following amino acid (f) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(F) 59C, 59D, 59K, 59M, 59Q, 59S, 59T, 82H, 82Q, 108M, 108T, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has an improved temperature dependency over the protein having the amino acid sequence of SEQ ID NO: 2, and has a reduced xylose activity.
That is, the (C2) protein contains an amino acid sequence having at least 60% amino acid homology with the amino acid sequence shown in SEQ ID NO: 2, and contains at least one amino acid shown in (f) above. Furthermore, the (C2) protein retains glucose dehydrogenase activity, has improved temperature dependence compared to the FADGDH of SEQ ID NO: 2, and has reduced xylose activity.

  An amino acid sequence having an amino acid sequence having 60% or more homology with at least one of SEQ ID NO: 2 and encoding a protein having glucose dehydrogenase activity is derived from, for example, Aspergillus terreus shown in SEQ ID NO: 3. The amino acid sequence of wild type FADGDH is mentioned. The amino acid sequence is disclosed in Patent Document 5.

  In the present specification, the homology of amino acid sequences means a value compared with GENETYX software. For example, when the amino acid sequence of Aspergillus oryzae-derived wild-type FADGDH (SEQ ID NO: 1) and the amino acid sequence of Aspergillus oryzae-derived wild-type FADGDH (SEQ ID NO: 3) were compared and analyzed with GENETYX software, 63.5 % Homology (see FIG. 2). The homology between the amino acid sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 3 is 63.4%.

  A protein containing 60% homology with the amino acid sequence of SEQ ID NO: 2 that retains glucose dehydrogenase activity and improved temperature dependence is, for example, aligned for SEQ ID NO: 1 or 2 and SEQ ID NO: 3. It can be produced by adding mutations to regions other than the conservative region.

  A region that is assumed to have no effect on enzyme activity and temperature dependency even when a mutation is added to the amino acid sequence is a region located on the protein surface from the X-ray crystal structure. For example, 1st to 40th, 111th to 190th, 213th to 310th, 340th to 397th, 420th to 435th, 455th to 492th, 514th to 538th, 552th to 572th is there.

  The amino acid sequences contained in the (A2) protein, (B2) protein, and (C2) protein are not particularly limited as long as they satisfy the requirements (i) to (iii) above, but preferably the amino acid sequence represented by SEQ ID NO: 2 The homology is 70% or more, more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more. The amino acid homology in the present specification means amino acid identity.

By having any of the specific amino acids listed in (b) above, the (A2) protein has a temperature dependency superior to that of FADGDH of SEQ ID NO: 2. Among the specific amino acids listed in (b) above, preferred amino acids are 59A, 59C, 59D, 59F, 59H, 59K, 59L, 59M, 59N, 59P, 59Q, 59S, 59T, 59W, 59Y, 82A, Any selected from the group consisting of 82C, 82H, 82K, 82Q, 82V, 82Y, 108A, 108F, 108H, 108M, 108Q, 108R, 108S, 108T, 505D, 505L, 505M, 505N, 505Q, 505R, and 505Y It is.
More preferred specific amino acids are 59A, 59C, 59F, 59H, 59K, 59L, 59M, 59N, 59P, 59Q, 59S, 59T, 59W, 59Y, 82A, 82C, 82H, 82K, 82Q, 82V, 82Y, 108R. , 505L, 505Q, 505R, and 505Y.
More preferred specific amino acids are selected from the group consisting of 59C, 59F, 59H, 59K, 59L, 59M, 59N, 59Q, 59T, 59W, 59Y, 82C, 82H, 82Q, 82V, 82Y, 505Q, and 505R. Either.
Further preferred specific amino acids are any selected from the group consisting of 59C, 59F, 59L, 59M, 59T, 59Y, 82H, 82Y, 505Q and 505R.
Further preferred specific amino acids are any selected from the group consisting of 59F, 59M, 59T, 59Y and 82Y.
As long as the temperature dependency which (A2) protein has is improved, two or more specific amino acids illustrated above may exist in a different position in an amino acid sequence.

By having any of the specific amino acids listed in (d) above, the (B2) protein has a reduced activity on xylose than FADGDH of SEQ ID NO: 2.
Among the specific amino acids listed in the above (d), preferred amino acids are any selected from the group consisting of 59C, 59D, 59K, 59M, 59Q, 59S, 59T, 82H, 82Q, 108M, 108T, and 505Y. It is.
Further preferred specific amino acids are any of 59M, 59Q, 82H, 82Q, and 505Y.
A more preferred amino acid substitution is 59M.
As long as the temperature dependency which (B2) protein has is improved, two or more specific amino acids illustrated above may exist in a different position in an amino acid sequence.

By having any of the specific amino acids listed in (f) above, the (C2) protein has a reduced activity on xylose than FADGDH of SEQ ID NO: 2.
The specific amino acids exemplified above may be present at different positions in the amino acid sequence as long as the (C2) protein has improved temperature dependence and reduced xylose activity.

The above conversion position is a protein having FADGDH activity derived from an origin other than that derived from Aspergillus oryzae (SEQ ID NO: 1) or a modified version thereof (SEQ ID NO: 2) (for example, Aspergillus terreus) It may be an equivalent position in the amino acid sequence. This equivalent position can be determined based on the knowledge of the primary structure (for example, alignment) and the three-dimensional structure of the amino acid sequence. The alignment can be compared using GENETYX WIN (manufactured by GENETICS CORPORATION).

In the present specification, the homology of amino acid sequences means a value compared with GENETYX software.
Further, in the present specification, a position equivalent to position 54 of SEQ ID NO. 1 in a certain amino acid sequence is determined to be equivalent to a position corresponding to position 54 of SEQ ID NO. 1 when the sequences are compared with GENETYX software. Similarly, the position equivalent to position 58 of SEQ ID NO: 1 in the amino acid sequence is determined to be equivalent to the position corresponding to position 58 of SEQ ID NO: 1 when comparing the primary structure (for example, alignment) of the sequence with GENETYX software. You may refer to the knowledge of a three-dimensional structure etc. further as needed.
Specifically, from the comparison data of the alignment of the amino acid sequence of FADGDH derived from the wild strain of Aspergillus oryzae and the amino acid sequence of FADGDH derived from the wild strain of Aspergillus terreus, the position equivalent to the 59th position of the present invention is aspergillus It corresponds to glycine at position 55 of FADGDH derived from wild Teleus strain. The position equivalent to the 82nd position of the present invention corresponds to the 78th position arginine of FADGDH derived from the wild Aspergillus terreus strain. The position equivalent to position 108 of the present invention corresponds to the 104th alanine of FADGDH derived from Aspergillus terreus wild strain. The position equivalent to position 505 of the present invention corresponds to phenylalanine at position 501 of FADGDH derived from Aspergillus terreus wild strain.
In the above (c), the homology of the amino acid sequence with at least either SEQ ID NO: 1 or SEQ ID NO: 2 is preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95%. That's it.
In the protein represented by any one of the above F to H (a) to (c), one or several amino acids are further deleted, substituted or added (inserted) at a position other than the position where the amino acid is substituted. A protein having an amino acid sequence and having glucose dehydrogenase activity is also an embodiment of the present invention.
The GENETYX software used was version 6.1 sold by GENETYX CORPORATION.

The FADGDH variant of the present invention can be prepared by various known means.
Below, the manufacturing method of the FADGDH modification which modified | denatured FADGDH derived from the wild type Aspergillus oryzae shown by sequence number 1 is illustrated. Although a manufacturing method is not specifically limited, It can manufacture in the procedure as shown below.
As a method for modifying the amino acid sequence constituting the FAD-dependent glucose dehydrogenase, a commonly used technique for modifying genetic information is used. That is, DNA having genetic information of a modified protein is created by converting a specific base of DNA having genetic information of the protein, or by inserting or deleting a specific base. As a specific method for converting a base sequence in DNA, for example, a commercially available kit (Transformer Mutagenesis Kit; Clonetech, EXOIII / Mung Bean Selection Kit; manufactured by Stratagene, Quick
Use of Change Site Directed Mutagenesis Kit (manufactured by Stratagene, etc.) or use of polymerase chain reaction (PCR).

  The prepared DNA having the genetic information of the FAD-dependent glucose dehydrogenase variant is transferred to a host microorganism in a state of being linked to a plasmid, and becomes a transformant that produces the FAD-dependent glucose dehydrogenase variant. As the plasmid in this case, for example, Escherichia coli JM109, Escherichia coli DH5α, Escherichia coli W3110, Escherichia coli C600 and the like can be used. As a method for transferring the recombinant vector to the host microorganism, for example, when the host microorganism is a microorganism belonging to Escherichia coli, a method of transferring the recombinant DNA in the presence of calcium ions can be employed. A poration method may be used. Furthermore, a commercially available competent cell (for example, competent high JM109; manufactured by Toyobo) may be used.

  In addition, the gene encoding the FADGDH variant, the vector containing the gene, and the transformant transformed with the vector obtained in these processes are also included as embodiments of the present invention.

The gene that is the basis for modification of the modified FADGDH of the present invention is not particularly limited, but it is desirable to use FADGDH derived from the genus Aspergillus. More preferably, it is FADGDH derived from Aspergillus oryzae or Aspergillus terreus.
Examples of FADGDH derived from Aspergillus oryzae include the protein represented by SEQ ID NO: 1 or SEQ ID NO: 2.
As the FADGDH derived from Aspergillus terreus, the protein represented by SEQ ID NO: 3 can be exemplified.

One embodiment of the present invention is a gene encoding the protein described in any of the above.

Examples of the gene encoding the protein before modification include the base sequence represented by SEQ ID NO: 4 as the gene encoding the protein represented by SEQ ID NO: 1 above. Moreover, as a gene which codes the protein represented by said sequence number 2, the base sequence represented by sequence number 5 can be illustrated. Moreover, as a gene encoding the protein represented by SEQ ID NO: 3 (FADGDH derived from Aspergillus terreus), the base sequence represented by SEQ ID NO: 6 can be exemplified.
In the gene of the present invention, the portion encoding the amino acid at any of the 59th, 82nd, 108th, and 505 positions in the gene sequence encoding the protein before modification, or an amino acid at an equivalent position thereof, Substituted to encode other amino acids.

  The gene encoding the FADGDH variant of the present invention is a protein that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the base sequence described in SEQ ID NO: 4, 5 or 6 and has FADGDH activity DNA encoding Here, the stringent condition refers to a condition in which nucleic acids having high homology, for example, hybridize at a temperature ranging from Tm of a perfectly matched hybrid to a temperature 15 ° C., preferably 10 ° C. lower than the Tm. Specifically, for example, it refers to conditions for hybridization in a general hybridization buffer solution at 68 ° C. for 20 hours. In the present invention, the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4, 5 or 6 has 50% or more homology, preferably 80% or more, more preferably 90% or more, more preferably Is considered to correspond to stringent conditions for a base sequence encoding an amino acid sequence having a homology of 95% or more.

  The gene of the present invention may include a modified codon usage for the purpose of improving GDH expression, for example.

  In one embodiment of the present invention, a vector having the above gene, a transformant transformed with the vector, culturing the transformant, and collecting a protein having glucose dehydrogenase activity are obtained. It is a production method.

  For example, the above GDH gene is inserted into an expression vector (many things such as plasmids are known in the art), and an appropriate host (many things such as E. coli are known in the art). Transform. After culturing the obtained transformant and collecting the bacterial cells from the culture solution by centrifugation or the like, the bacterial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, such as EDTA A water-soluble fraction containing GDH can be obtained by adding a chelating agent or a surfactant and solubilizing. Alternatively, the expressed GDH can be secreted directly into the culture medium by using an appropriate host vector system.

  The GDH-containing solution obtained as described above is precipitated by, for example, vacuum concentration, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. You just have to let them know. Heat treatment and isoelectric point treatment are also effective purification means. Further, purified GDH can be obtained by performing gel filtration using an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

These can proceed according to the following literature, for example.
(A) Protein Experiment Protocol Volume 1 Functional Analysis, Volume 2 Structural Analysis (Syujunsha) Supervised by Yoshifumi Nishimura and Shigeo Ohno (b) Revised Protein Experiment Notes Extraction and separation and purification (Yodosha) Masato Okada, Kaoru Miyazaki Editing It can also be carried out by the method exemplified below.

  The prepared DNA having the genetic information of the protein is transferred into the host microorganism in a state of being linked to the vector.

Suitable vectors are those constructed for gene recombination from phages or plasmids that can grow independently in a host microorganism. Examples of the phage include Lambda gt10 and Lambda gt11 when Escherichia coli is used as the host microorganism. Examples of plasmids include pBR322, pUC19, pKK223-3, and pBluescript when Escherichia coli is used as a host microorganism. Of these, pBluescript or the like that retains a promoter that can be recognized in Escherichia coli upstream of the cloning site is preferable.
In addition, a suitable host microorganism is not particularly limited as long as the recombinant vector is stable, can self-propagate and can express a foreign gene. In Escherichia coli, Escherichia coli W3110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH5α, and the like can be used.

  As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, a method for transferring the 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, competent high DH5α; manufactured by Toyobo) may be used. When yeast is used as the host, the lithium method or electroporation method is used. When filamentous fungi are used, the protoplast method or the like is used.

  In the present invention, methods for obtaining a gene encoding GDH include the following methods. Using Aspergillus oryzae genome sequence information, a predicted GDH gene can be found. Next, mRNA is prepared from Aspergillus oryzae cells and cDNA is synthesized. Using the cDNA thus obtained as a template, the GLD gene is amplified by the PCR method, and this gene is bound to the vector at the blunt end or sticky end of both DNAs with DNA ligase or the like to construct a recombinant vector. After transferring the recombinant vector to a replicable host microorganism, a recombinant microorganism containing a gene encoding GDH is obtained using the marker of the vector.

  The microorganism which is a transformant obtained as described above can stably produce a large amount of GDH by being cultured in a nutrient medium. The selection of the recombinant may be carried out by searching for a microorganism that simultaneously expresses the marker of the vector and the GDH activity. For example, a microorganism that grows in a selective medium based on a drug resistance marker and generates GDH may be selected.

  The base sequence of the GDH gene was decoded by the dideoxy method described in Science, Vol. 214, 1205 (1981). The amino acid sequence of GDH was estimated from the base sequence determined as described above.

As described above, transfer from a recombinant vector carrying a GDH gene once selected to a recombinant vector that can be replicated in another microorganism can be carried out by using a restriction enzyme or PCR method from the recombinant vector carrying the GDH gene. It can be easily carried out by collecting the DNA that is the GDH gene and binding it to other vector fragments. Further, for transformation of other microorganisms with these vectors, a competent cell method using calcium treatment, an electroporation method, a protoplast method, or the like can be used.

  The GDH gene of the present invention may have a DNA sequence in which a part of each amino acid residue of the translated amino acid sequence of the gene is deleted or substituted as long as it has glucose dehydrogenase activity. It may have a DNA sequence in which other amino acid residues are added or substituted.

  As a method for modifying the gene encoding wild-type GDH, a commonly performed technique for modifying genetic information is used. That is, a DNA having genetic information of a modified protein is created by converting a specific base of DNA having genetic information of the protein, or by inserting or deleting a specific base. Specific methods for converting bases in DNA include, for example, commercially available kits (Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Deletion Kit; manufactured by Stratagene, QuickChange SiteDirectedMetStainedMistageStained Kit, etc.) Use of the chain reaction method (PCR) is mentioned.

  The culture form of the host microorganism as a transformant may be selected in consideration of the nutritional physiological properties of the host. In many cases, it is advantageous to use liquid culture and industrially perform aeration and agitation culture. However, in consideration of productivity, it may be advantageous to use a filamentous fungus as a host and perform the solid culture.

  As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, lactose, molasses, pyruvic acid and the like are used. The nitrogen source may be any available nitrogen compound. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

  The culture temperature can be appropriately changed within the range in which the bacteria grow and produce GDH, but is preferably about 20 to 37 ° C. Although the culture time varies slightly depending on the conditions, the culture may be completed at an appropriate time in consideration of the time when GDH reaches the maximum yield, and is usually about 6 to 48 hours. The pH of the medium can be appropriately changed within the range in which the bacteria grow and produce GDH, but is preferably in the range of about pH 6.0 to 9.0.

  Although the culture solution containing the cells producing GDH in the culture can be collected and used as it is, generally, when GDH is present in the culture solution according to a conventional method, filtration, centrifugation, etc. It is used after separating the GDH-containing solution and the microbial cells. When GDH is present in the microbial cells, the microbial cells are collected from the obtained culture by means of filtration or centrifugation, and then the microbial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme. Further, if necessary, a chelating agent such as EDTA and a surfactant are added to solubilize GDH and separated and collected as an aqueous solution.

The GDH-containing solution obtained as described above is precipitated by, for example, vacuum concentration, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. You just have to let them know. Heat treatment and isoelectric point treatment are also effective purification means. Thereafter, purified GDH can be obtained by performing gel filtration using an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

  For example, gel filtration using Sephadex gel (manufactured by GE Healthcare Bioscience), DEAE Sepharose CL-6B (manufactured by GE Healthcare Bioscience), octyl Sepharose CL-6B (manufactured by GE Healthcare Bioscience) And purified by column chromatography such as) to obtain a purified enzyme preparation. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

In the present invention, FADGDH activity is measured under the following conditions.
[Test example]
<Reagent>
50 mM PIPES buffer pH 6.5 (including 0.1% Triton X-100)
24 mM PMS solution 2.0 mM 2,6-dichlorophenolindophenol (DCPIP) solution 1M D-glucose solution


The above PIPES buffer 21.9 ml, DCPIP solution 1.0 ml, PMS solution 2.0 ml, and D-glucose solution 4.5 ml are mixed to obtain a reaction reagent.

<Measurement conditions>
Pre-warm 3 ml of reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 ml of GDH solution and mixing gently, the absorbance change at 600 nm was recorded for 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control, and the absorbance change per minute (ΔOD _TEST) ). In the blind test, a change in absorbance per minute (ΔOD _BLANK ) is similarly measured by adding a solvent that dissolves GDH to the reagent mixture instead of the GDH solution. From these values, the GDH activity is determined according to the following formula. Here, 1 unit (U) in GDH activity is defined as the amount of enzyme that reduces 1 micromole of DCPIP per minute in the presence of 200 mM D-glucose. Moreover, when measuring the activity value in 25 degreeC, it changes by changing the operation described above 37 degreeC into 25 degreeC. When measuring the reactivity to xylose, 1M D-xylose may be used instead of the above 1M D-glucose solution.

Activity (U / ml) =
{− (ΔOD _TEST −ΔOD _BLANK ) × 3.0 × dilution ratio} / {16.3 × 0.1 × 1.0}

In the formula, 3.0 is the amount of the reaction reagent + enzyme solution (ml), 16.3 is the molar molecular extinction coefficient (cm ² / micromole) under the conditions for this activity measurement, and 0.1 is the solution of the enzyme solution. Amount (ml), 1.0 indicates the optical path length (cm) of the cell.

Glucose Assay Kit The present invention also features a glucose assay kit comprising a modified FADGDH according to the present invention. The glucose assay kit of the present invention comprises a modified FADGDH according to the present invention in an amount sufficient for at least one assay. Typically, the kit contains, in addition to the modified FADGDH of the present invention, buffers necessary for the assay, mediators, a glucose standard solution for creating a calibration curve, and directions for use. The modified FADGDH according to the invention can be provided in various forms, for example as a lyophilized reagent or as a solution in a suitable storage solution.

Glucose Sensor The present invention also features a glucose sensor that uses a modified FADGDH according to the present invention. As the electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and the enzyme of the present invention is immobilized on this electrode. Immobilization methods include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc., or ferrocene or a derivative thereof. It may be fixed in a polymer or adsorbed and fixed on an electrode together with a representative electron mediator, or a combination thereof may be used. Typically, the modified FADGDH 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 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 the modified FADGDH of the present invention is immobilized is used as a working electrode, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCl electrode) are used. After a constant voltage is applied to the carbon electrode and the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.

  Hereinafter, the present invention will be specifically described by way of examples.

Example 1 Evaluation of temperature dependence of FADGDH FADGDH of SEQ ID NO: 2 was used. A FADGDH purified sample was prepared by the method described above, and activity measurement was performed at a predetermined temperature condition in 38 mM PIPES buffer (pH 6.5). FIG. 1 shows a temperature dependence curve of the enzyme activity. The vertical axis shows the relative activity value at each temperature, with the activity value at 37 ° C. being 100%. When the activity value at 37 ° C. is 100%, the activity value at 25 ° C. is about 63%, and the activity value at 5 ° C. is about 40%.

Example 2 Production of Modified FADGDH Gene A commercially available Escherichia coli competent cell (E. coli DH5a; manufactured by TOYOBO) was transformed with a recombinant plasmid pAOGDH-M76 containing the gene (SEQ ID NO: 2) encoding FADGDH, and ampicillin. And agar medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar; pH 7.3), and then cultured at 30 ° C. overnight. The obtained transformant was fed with a liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl; pH 7.3) containing ampicillin (50 mg / ml; manufactured by Nacalai Tesque), and 30 Cultured with shaking at 0 ° C. overnight. A plasmid was prepared from the obtained cells by a conventional method.
Using the plasmid as a template, the synthetic oligonucleotide of SEQ ID NO: 7 designed to substitute the 59th glycine with alanine and the synthetic oligonucleotide complementary thereto, and the 82nd arginine of SEQ ID NO: 8 designed to substitute the alanine Synthetic oligonucleotide and its complementary synthetic oligonucleotide, SEQ ID NO: 9 designed to replace 108th valine with alanine and its complementary synthetic oligonucleotide, 505th phenylalanine to be substituted with alanine The designed synthetic oligonucleotide of SEQ ID NO: 10 and a synthetic oligonucleotide complementary thereto are modified using QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE). To prepare a H.

Example 3 Preparation of Alanine-Modified FADGDH Plasmid A commercially available competent cell (E. coli DH5a; manufactured by TOYOBO) was transformed with the obtained modified FADGDH, and 37 ° C. on an LB agar medium containing ampicillin. Cultured for 16 hours. Then, the modified FADGDH single colony was ingested into an LB liquid medium containing ampicillin and cultured overnight at 30 ° C. with shaking. A plasmid was extracted from the obtained cells by a conventional method. The site of the extracted plasmid was identified using a DNA sequencer (ABI PRISM ™ 3700 DNA Analyzer; manufactured by Perkin-Elmer), and modified FADGDH substituted with alanine was obtained. Thereafter, a plasmid in which the 59th glycine was replaced with alanine was pAOGDH-M76-G59A, a plasmid in which the 82nd arginine was replaced with alanine was pAOGDH-M76-R82A, and a plasmid in which the 108th valine was replaced with alanine was pAOGDH-M76. -V108A, A plasmid in which the 505th phenylalanine was substituted with alanine was named pAOGDH-M76-F505A.

Example 4 Preparation of Crude Enzyme Solution Containing Modified FADGDH and Comparison of Temperature Dependence and Xylose Activity pAOGDH-M76-G59A, pAOGDH-M76-R82A, pAOGDH-M76-V108A and pAOGDH-M76 obtained in Example 3 -Commercially available E. coli competent cells (E. coli DH5a; manufactured by TOYOBO) were transformed with F505A, and cultured on an LB agar medium containing ampicillin at 37 ° C for 16 hours. Thereafter, a single colony of alanine-modified FADGDH was ingested into an LB liquid medium containing ampicillin and cultured overnight at 30 ° C. with shaking. Bacteria obtained by centrifugation are collected from a part of the culture solution, and a crude enzyme solution is prepared by crushing the cells using glass beads in 50 mM phosphate buffer (pH 6.0). did.

Using the prepared crude enzyme solution, GDH activity at 25 ° C. and 37 ° C. and xylose activity at 37 ° C. were measured by the activity measuring method described above. Table 1 shows the results.
Table 1 shows the results of comparing the temperature dependence of G59A, R82A, V108A and F505A when cultured in 5 ml LB medium / test tube at 30 ° C. for 24 hours.
Compared with the modified site and the temperature dependence, it was confirmed that the substitution of 59, 82, and 108 with alanine improved the temperature dependence over FADGDH before modification. 108 sites were candidates. Further, the 505 site was inactivated by substituting with alanine, but it was selected as a candidate because there was a possibility that the temperature dependence could be improved by substituting with other amino acids other than alanine.

Example 5 Optimization of 59-site amino acid Using the pAOGDH-M76 used in Example 2 as a template, the synthetic oligonucleotide of SEQ ID NO: 11 designed to replace the 59th tyrosine with the other 19 amino acids, and A modified FADGDH was prepared using QuickChange ™ Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) as a complementary synthetic oligonucleotide. E. coli competent cells (E. coli DH5a; manufactured by TOYOBO) were transformed with the modified FADGDH, and cultured on an LB agar medium containing ampicillin at 37 ° C. for 16 hours. Thereafter, the single colony having the modified FADGDH was ingested into an LB liquid medium containing ampicillin and cultured overnight at 30 ° C. with shaking. Thereafter, 1 ml of the culture solution was ingested, and the plasmid was extracted by a conventional method. The site of the extracted plasmid was identified using a DNA sequencer (ABI PRISM ™ 3700 DNA Analyzer; manufactured by Perkin-Elmer). PAOGDH-M76-G59A is a plasmid having a modified FADGLD with 59 sites replaced with A, pAOGDH-M76-G59C is a plasmid having a modified FADGLD with 59 sites replaced with C, and a modified FADGLD with 59 sites replaced with D PAOGDH-M76-G59D, a plasmid having modified FADGLD with 59 sites replaced with E, pAOGDH-M76-G59E, a plasmid having modified FADGLD with 59 sites replaced with G, pAOGDH-M76-G59F, PAOGDH-M76-G59H was substituted for the plasmid having the modified FADGLD with 59 sites replaced with H, and pAOGDH-M76-G59I was replaced with the plasmid having the modified FADGLD with 59 sites replaced with I. PAOGDH-M76-G59K is a plasmid having a modified FADGLD, pAOGDH-M76-G59L is a plasmid having a modified FADGLD in which the 59 site is replaced with L, and pAOGDH-M76- is a plasmid having a modified FADGLD in which the 59 site is replaced with M. G59M, pAOGDH-M76-G59N is a plasmid having a modified FADGLD with 59 sites replaced with N, pAOGDH-M76-G59P is a plasmid having a modified FADGLD with 59 sites replaced with P, and 59 is replaced with Q PAOGDH-M76-G59Q is a plasmid having type FADGLD, pAOGDH-M76-G59R is a plasmid having modified FADGLD in which 59 site is substituted with R, and modified FADGLD in which 59 site is substituted with S PAOGDH-M76-G59S, pAOGDH-M76-G59T having a modified FADGLD with 59 sites replaced with T, and pAOGDH-M76-G59V, 59 having a modified FADGLD with 59 sites replaced with V The plasmid having the modified FADGLD with the site replaced with W was named pAOGDH-M76-G59W, and the plasmid with the modified FADGLD with the 59 site replaced with Y was named pAOGDH-M76-G59Y.

Example 6 Comparison of temperature dependence and xylose activity of modified FADGDH in which amino acid at 59 sites was optimized Temperature dependence of modified FADGDH in which 59 sites were substituted with 19 amino acids in the same manner as in Example 5 And xylose activity was measured. Table 2 shows the temperature dependence and xylose activity of the modified FADGDH.
Table 2 shows the results of comparing the temperature dependence and xylose activity of the modified FADGDH in which the G59 site is modified when cultured in 5 ml LB medium / test tube at 30 ° C. for 24 hours.
Modified FADGDH with 59 replaced with A, Modified FADGDH with 59 replaced with C, Modified FADGDH with 59 replaced with D, Modified FADGDH with 59 replaced with F, 59 Replaced with H Modified FADGDH, modified FADGDH with 59 sites replaced with K, modified FADGDH with 59 sites replaced with L, modified FADGDH with 59 sites replaced with M, modified FADGDH with 59 sites replaced with N, 59 Modified FADGDH with the site replaced with P, Modified FADGDH with 59 replaced with Q, Modified FADGDH with 59 replaced with S, Modified FADGDH with 59 replaced with T, 59 Replaced with W Modified FADGDH, modified FADGDH with 59 sites replaced with Y, is more temperature dependent than FADGDH before modification It was good. Further, modified FADGDH with 59 sites replaced with C, modified FADGDH with 59 sites replaced with D, modified FADGDH with 59 sites replaced with K, modified FADGDH with 59 sites replaced with M, and 59 sites with Q The modified FADGDH in which the 59 is replaced with S, the modified FADGDH in which the 59 site is replaced with S, and the modified FADGDH in which the 59 site is replaced with T not only have improved temperature dependence, but also have a lower xylose activity than the FADGDH before the modification. .

Example 7 Optimization of amino acid at 82 sites A synthetic oligonucleotide of SEQ ID NO: 12 designed to substitute 82 other amino acids in the same manner as in Example 5 and a complementary oligonucleotide complementary thereto The amino acids used were optimized. PFAOGDH-M76-R82A is a plasmid having a modified FADGLD with 82 sites replaced with A, pAOGDH-M76-R82C is a plasmid having a modified FADGLD with 82 sites replaced with C, and a modified FADGLD with 82 sites replaced with D PAOGDH-M76-R82D, a plasmid having modified FADGLD with 82 sites replaced with E, pAOGDH-M76-R82E, a plasmid having modified FADGLD with 82 sites replaced with G, pAOGDH-M76-R82G, PAOGDH-M76-R82H was substituted for the plasmid having the modified FADGLD with 82 sites replaced with H, and pAOGDH-M76-R82I was substituted for the plasmid having the modified FADGLD with 82 sites replaced with I. PAOGDH-M76-R82K is a plasmid having a modified FADGLD, pAOGDH-M76-R82L is a plasmid having a modified FADGLD in which 82 sites are substituted with L, and pAOGDH-M76- is a plasmid having a modified FADGLD in which 82 sites are substituted with M. R82M, pFAOGDH-M76-R82N with a modified FADGLD plasmid with 82 sites replaced with N, pAOGDH-M76-R82P with 82 sites replaced with P, pAOGDH-M76-R82P with 82 sites replaced with Q PAOGDH-M76-R82Q for a plasmid having type FADGLD, pAOGDH-M76-R82F for a plasmid having modified FADGLD with 82 sites substituted with F, and a modified FADGLD with 82 sites substituted for S PAOGDH-M76-R82S, pFAOGDH-M76-R82T having a modified FADGLD with 82 sites replaced with T, and pAOGDH-M76-R82V, 82 having a modified FADGLD with 82 sites replaced with V The plasmid having the modified FADGLD with the site replaced with W was named pAOGDH-M76-R82W, and the plasmid with the modified FADGLD with the 82 site replaced with Y was named pAOGDH-M76-R82Y.

Example 8 Comparison of temperature dependence and xylose activity of modified FADGDH with optimized amino acid at 82 sites Temperature dependence of modified FADGDH in which 82 amino acids were substituted with 19 amino acids in the same manner as in Example 6 And xylose activity was measured. Table 3 shows the results of temperature dependence and xylose activity of the modified FADGDH.
Table 3 shows the results of comparing the temperature dependence and xylose activity of the modified FADGDH in which the R82 site is modified when cultured in 5 ml LB medium / test tube at 30 ° C. for 24 hours.
Modified FADGDH with 82 sites replaced with A, Modified FADGDH with 82 sites replaced with C, Modified FADGDH with 82 sites replaced with H, Modified FADGDH with 82 sites replaced with K, Replaced 82 sites with Q The modified FADGDH, the modified FADGDH in which the 82 site was substituted with V, and the modified FADGDH in which the 82 site was substituted with Y improved the temperature dependency over FADGDH before the modification. Further, the modified FADGDH in which the 82 site was substituted with H and the modified FADGDH in which the 82 site was substituted with Q not only improved the temperature dependence, but also reduced the xylose activity compared to FADGDH before the modification.

Example 9 Optimization of amino acid at 108 sites A synthetic oligonucleotide of SEQ ID NO: 13 designed to substitute 108 other amino acids with the other 19 amino acids in the same manner as in Example 5 and a synthetic oligonucleotide complementary thereto The amino acids used were optimized. PFAOGDH-M76-V108A is a plasmid having a modified FADGLD with 108 sites replaced with A, pAOGDH-M76-V108C is a plasmid having a modified FADGLD with 108 sites replaced with C, and a modified FADGLD with 108 sites replaced with D PAOGDH-M76-V108D, a plasmid having modified FADGLD with 108 sites replaced with E, pAOGDH-M76-V108E, a plasmid having modified FADGLD with 108 sites replaced with G, pAOGDH-M76-V108G, PAOGDH-M76-V108H is a plasmid having a modified FADGLD in which 108 sites are replaced with H, and pAOGDH-M76-V10 is a plasmid having a modified FADGLD in which 108 sites are replaced with I. I, pAOGDH-M76-V108K is a plasmid having a modified FADGLD in which 108 sites are replaced with K, and pAOGDH-M76-V108L is a plasmid having a modified FADGLD in which 108 sites are replaced with L, and 108 is replaced with M. PAOGDH-M76-V108M is a plasmid having type FADGLD, pAOGDH-M76-V108N is a plasmid having modified FADGLD in which 108 sites are substituted with N, and pAOGDH-M76- is a plasmid having modified FADGLD in which 108 sites are substituted with P A plasmid having a modified FADGLD in which V108P and 108 sites are substituted with Q is pAOGDH-M76-V108Q, and a plasmid having a modified FADGLD in which 108 sites are substituted with R is pAOGDH-M76- A plasmid having modified FADGLD in which 108R and 108 sites are replaced with S is pAOGDH-M76-V108S, and a plasmid having modified FADGLD in which 108 sites are replaced with T is modified with pAOGDH-M76-V108T and 108 sites are replaced with F. PAOGDH-M76-V108F is a plasmid having type FADGLD, pAOGDH-M76-V108W is a plasmid having modified FADGLD in which 108 sites are replaced with W, and pAOGDH-M76- is a plasmid having modified FADGLD in which 108 sites are replaced with Y. It was named V108Y.

Example 10 Comparison of temperature dependence and xylose activity of modified FADGDH in which 108 amino acids were optimized Temperature dependence of modified FADGDH in which 108 amino acids were substituted with 19 amino acids in the same manner as in Example 6 And xylose activity was measured. Table 4 shows the results of temperature dependence and xylose activity of the modified FADGDH.
Table 4 shows the results of comparing the temperature dependence and xylose activity of the modified FADGDH in which the V108 site is modified when cultured in 5 ml LB medium / test tube at 30 ° C. for 24 hours.
Modified FADGDH with 108 sites replaced with A, Modified FADGDH with 108 sites replaced with D, Modified FADGDH with 108 sites replaced with E, Modified FADGDH with 108 sites replaced with F, 108 Sites replaced with F Modified FADGDH, modified FADGDH with 108 sites replaced with H, modified FADGDH with 108 sites replaced with L, modified FADGDH with 108 sites replaced with M, modified FADGDH with 108 sites replaced with Q, 108 The temperature dependence of the modified FADGDH in which the site was replaced with R, the modified FADGDH in which the 108 site was replaced with S, and the modified FADGDH in which the 108 site was replaced with T were improved as compared to the FADGDH before the modification. In addition, the modified FADGDH in which 108 sites were replaced with M and the modified FADGDH in which 108 sites were replaced with T not only improved the temperature dependency, but also decreased the xylose activity compared to FADGDH before the modification.

Example 11 Optimization of amino acid at 505 site In the same manner as in Example 5, a synthetic oligonucleotide of SEQ ID NO: 14 designed to replace 505 site with 19 other amino acids and a synthetic oligonucleotide complementary thereto were prepared. The amino acids used were optimized. PAOGDH-M76-F505A is a plasmid having a modified FADGLD in which the 505 site is replaced with A, pAOGDH-M76-F505C is a plasmid having the modified FADGLD in which the 505 site is replaced with C, and a modified FADGLD in which the 505 site is replaced with D PAOGDH-M76-F505D, a plasmid having a modified FADGLD in which the 505 site is replaced by E, pAOGDH-M76-F505E, a plasmid having a modified FADGLD in which the 505 site is replaced by G, pAOGDH-M76-F505G, PAOGDH-M76-F505H is a plasmid having a modified FADGLD in which the 505 site is replaced with H, and pAOGDH-M76-F50 is a plasmid having a modified FADGLD in which the 505 site is replaced with I. I, pAOGDH-M76-F505K is a plasmid having a modified FADGLD in which the 505 site is replaced by K, and pAOGDH-M76-F505L is a plasmid having a modified FADGLD in which the 505 site is replaced by L, and M is replaced by a 505 site. PAOGDH-M76-F505M is a plasmid having type FADGLD, pAOGDH-M76-F505N is a plasmid having modified FADGLD in which 505 site is substituted with N, and pAOGDH-M76- is a plasmid having modified FADGLD in which 505 site is substituted with P PAOGDH-M76-F505Q, a plasmid having the modified FADGLD in which the 505 site is replaced with R, and pAOGDH-M76- PAOGDH-M76-F505S, a plasmid having a modified FADGLD in which the 505R and 505 sites are replaced with S, and pAOGDH-M76-F505T, a plasmid having the modified FADGLD in which the 505 site is replaced with T, and a modification in which the 505 site is replaced with V PAOGDH-M76-F505V for a plasmid having type FADGLD, pAOGDH-M76-F505W for a plasmid having modified FADGLD in which the 505 site is replaced with W, and pAOGDH-M76- for a plasmid having modified FADGLD in which the 505 site is replaced with Y It was named F505Y.

Example 12 Comparison of temperature dependence and xylose activity of modified FADGDH in which amino acid at 505 site was optimized Temperature dependence of modified FADGDH in which 505 site was substituted with 19 amino acids in the same manner as in Example 6 And xylose activity was measured. Table 5 shows the results of temperature dependency and xylose activity of the modified FADGDH.
Table 5 shows the results of comparing the temperature dependence and xylose activity of the modified FADGDH in which the F505 site has been modified when cultured in 5 ml LB medium / test tube at 30 ° C. for 24 hours.
Modified FADGDH with 505 replaced with D, Modified FADGDH with 505 replaced with L, Modified FADGDH with 505 replaced with M, Modified FADGDH with 505 replaced with N, Replaced 505 with Q The modified FADGDH, the modified FADGDH in which the 505 site is replaced with R, and the modified FADGDH in which the 505 site is replaced with Y have improved temperature dependency over FADGDH before modification. Moreover, the modified FADGDH in which the 505 site was replaced with Y not only improved the temperature dependency, but also reduced the xylose activity compared to FADGDH before the modification.

  The use of FAD-dependent glucose dehydrogenase with improved temperature dependency and improved substrate specificity according to the present invention makes it possible to improve the accuracy of glucose measurement and contribute to industries such as medical fields. is there.

Claims (10)

The following protein (A1) or (A2).
(A1) A protein comprising an amino acid sequence containing any of the amino acid substitutions shown in the following (a) in the amino acid sequence of FAD-dependent glucose dehydrogenase (FADGDH) described in SEQ ID NO: 2:
(A) G59A, G59C, G59D, G59F, G59H, G59K, G59L, G59M, G59N, G59P, G59Q, G59S, G59T, G59W, G59Y, R82A, R82C, R82H, R82K, R82Q, R82V, R82Y, V108A, V108D , V108E, V108F, V108H, V108L, V108M, V108Q, V108R, V108S, V108T, F505D, F505L, F505M, F505N, F505Q, F505R, F505Y;
(A2) a protein satisfying the following (i) to (iii):
(I) an amino acid sequence having the following amino acid (b) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(B) 59A, 59C, 59D, 59F, 59H, 59K, 59L, 59M, 59N, 59P, 59Q, 59S, 59T, 59W, 59Y, 82A, 82C, 82H, 82K, 82Q, 82V, 82Y, 108A, 108D 108E, 108F, 108H, 108L, 108M, 108Q, 108R, 108S, 108T, 505D, 505L, 505M, 505N, 505Q, 505R, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has improved temperature dependence over the protein having the amino acid sequence of SEQ ID NO: 2. The following protein (B1) or (B2).
(B1) A protein comprising an amino acid sequence containing any of the amino acid substitutions shown in the following (c) in the amino acid sequence of FAD-dependent glucose dehydrogenase (FADGDH) described in SEQ ID NO: 2:
(C) G59C, G59D, G59K, G59M, G59Q, G59S, G59T, R82H, R82Q, V108M, V108T, F505Y;
(B2) a protein satisfying the following (i) to (iii):
(I) an amino acid sequence having the following amino acid (d) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(D) 59C, 59D, 59K, 59M, 59Q, 59S, 59T, 82H, 82Q, 108M, 108T, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has a reduced xylose activity compared to a protein having the amino acid sequence of SEQ ID NO: 2. The following protein (C1) or (C2).
(C1) A protein comprising an amino acid sequence containing any of the amino acid substitutions shown in (e) below in the amino acid sequence of FAD-dependent glucose dehydrogenase (FADGDH) described in SEQ ID NO: 2:
(E) G59C, G59D, G59K, G59M, G59Q, G59S, G59T, R82H, R82Q, V108M, V108T, F505Y;
(C2) a protein satisfying the following (i) to (iii):
(I) an amino acid sequence having the following amino acid (f) in an amino acid sequence having at least 60% homology with the amino acid sequence shown in SEQ ID NO: 2;
(F) 59C, 59D, 59K, 59M, 59Q, 59S, 59T, 82H, 82Q, 108M, 108T, 505Y
(Ii) having glucose dehydrogenase activity;
(Iii) It has an improved temperature dependency over the protein having the amino acid sequence of SEQ ID NO: 2, and has a reduced xylose activity.   The gene which codes the protein in any one of Claims 1-3.   A vector comprising the gene according to claim 4.   A transformant transformed with the vector according to claim 5.   A method for producing a protein having glucose dehydrogenase activity, comprising culturing the transformant according to claim 6 and collecting a protein having glucose dehydrogenase activity.   A glucose assay kit comprising the protein according to claim 1.   The glucose sensor containing the protein in any one of Claims 1-3. The glucose measuring method containing the protein in any one of Claims 1-3.