WO2010137489A1

WO2010137489A1 - Glucose dehydrogenase having altered properties

Info

Publication number: WO2010137489A1
Application number: PCT/JP2010/058332
Authority: WO
Inventors: 洋志相場; 雅夫北林; 芳昭西矢
Original assignee: 東洋紡績株式会社
Priority date: 2009-05-28
Filing date: 2010-05-18
Publication date: 2010-12-02
Also published as: JP5846908B2; JPWO2010137489A1

Abstract

Disclosed is a glucose dehydrogenase (GDH) which has high thermal stability and high substrate specificity and can exhibit a high affinity for a substrate when nicotinamide adenine dinucleotide (NAD) is used as a coenzyme. A GDH having high thermal stability and strict substrate specificity can be obtained from hyperthermophilic archaea Thermoproteus sp. strain GDH1. Further, a modified GDH which can exhibit an improved affinity for glucose when NAD is used as a coenzyme can be successfully produced by substituting an amino acid residue located at a specific position in the aforementioned GDH by a different amino acid residue.

Description

Glucose dehydrogenase with modified properties

The present invention relates to a glucose dehydrogenase that can be used in a reagent for measuring a glucose concentration and a glucose sensor. Moreover, it is related with the manufacturing method of this enzyme, the composition for glucose determination using this enzyme, and a glucose sensor.

NAD (P) -dependent glucose dehydrogenase (EC 1.1.1.47, hereinafter referred to as GDH, NAD) which acts as a coenzyme with nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) (P) -dependent glucose dehydrogenase (also referred to as NAD (P) -GDH) is an enzyme mainly used for blood glucose concentration measurement and catalyzes the following reaction.

D-glucose + NAD (P) → D-glucono-δ-lactone + NAD (P) H

Glucose oxidase is another known enzyme for quantifying blood glucose. However, since this enzyme can use molecular oxygen as an electron acceptor, the effect of dissolved oxygen concentration on the measurement of glucose concentration. The problem of receiving is pointed out. Since glucose dehydrogenase is not affected by such dissolved oxygen, it has become a mainstream enzyme for glucose sensors in recent years. GDH includes a pyrroloquinoline quinone (PQQ) -dependent type and a flavin-dependent type in addition to the NAD (P) -dependent type. PQQ-dependent GDH, typified by those derived from Acinetobacter baumannii, has a problem in substrate specificity, such as having reactivity equivalent to glucose with respect to maltose. Further, as flavin-dependent GDH, for example, those derived from Aspergillus terreus are known, and the substrate specificity is more strict than PQQ-dependent GDH, however, the ratio of glucose to xylose is about 9%. Therefore, the substrate specificity is not necessarily sufficient. Further, the temperature stability is limited to about 50 ° C., which is not sufficient.

Known NAD (P) -GDH is well known from bacteria belonging to the genus Bacillus, for example, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, etc. Have been reported as GDH producing bacteria. These bacteria-derived NAD (P) -GDH still have problems such as having activity against xylose and the like, or poor thermal stability.

Patent Document 1 describes four types of B.I. The properties of megaterium-derived GDH isozymes are described, and their reactivity with respect to 100 mM xylose is converted to glucose ratio (%) by 12 for GDH (I), 3.5 for GDH (II), and GDH (III), respectively. Is 1.8 and GDH (IV) is 7.1. GDH (III), which has the lowest reactivity to xylose, loses its activity completely by heating at 40 ° C. for 20 minutes in the absence of NaCl, and has poor thermal stability. Although this GDH (III) improves thermal stability under the limited condition that a very high concentration NaCl of 2M exists, even in that case, the activity is completely lost by heat treatment at 70 ° C. for 20 minutes. , Not necessarily enough.

Regarding the stability problem, for example, Patent Document 2 describes B.I. Although it is described that the thermal stability has been improved by substituting a specific amino acid residue of Megathelium-derived GDH, it is necessary to add a very high concentration of inorganic salt in order to exhibit sufficient thermal stability, Even GDH, which shows the highest thermal stability, loses its activity completely by heat treatment at about 75 ° C. for 20 minutes in the absence of sodium chloride. Since it is known that the presence of salts such as sodium chloride and sodium maleate accelerates the decomposition of NAD, it is practical to add a high concentration of sodium chloride to stabilize GDH. Preference is given to GDH, which has sufficient thermal stability in the absence of sodium chloride.

Also, Patent Document 3 describes B.I. Although NAD (P) -GDH with improved thermal stability in the absence of sodium chloride has been described by producing chimeras of multiple types of GDH including megathelium-derived GDH, it solves the problem of substrate specificity There is no intention, and the chimeric GDH has another problem that it is excellent in alkali resistance, but is not sufficiently stable in the neutral and acidic pH regions below pH 7.5. NAD (P), which is an oxidized coenzyme, is known to be extremely unstable in the alkaline region. In order to ensure the stability of these oxidized coenzymes, the pH is usually near neutral or lower. Considering the production of a glucose quantitative composition, it is extremely disadvantageous in practice.

In 1986, it was derived from Sulforobus solfataricus (Non-patent Document 1), in 1989, from Thermoplasma acidophilum (Non-patent document 2), and in 1997, Thermoproteus tenax. GDH derived from (Thermoproteus tenax) (Non-patent Document 3) has been reported.

In using such a hyperthermophile-derived GDH, it is advantageous in that it is not necessary to carry out functional modification to increase heat resistance like the GDH exemplified above. However, while these enzymes are excellent in thermal stability, they have a problem that the substrate specificity is inferior to those derived from bacteria. S. For GDH derived from Solfataricus, when NADP is used as a coenzyme, the substrate specificity is broad, and the activity against galactose and xylose is higher than that at glucose at a substrate concentration of 40 mmol / L. When NAD is used as a coenzyme, the specificity for glucose is relatively high, but the activity for xylose is still as high as about 26% of glucose. T. Acidophilum-derived GDH exhibits activity against galactose when NADP is used as a coenzyme, and the ratio to glucose is as high as 70%. In addition, T.W. Tenax-derived GDH also shows high reactivity with xylose. In measuring the blood glucose concentration, the fact that the substrate specificity of GDH used is low and that it reacts with substances other than glucose results in a loss of the accuracy of blood glucose level measurement, which is extremely inconvenient.

As described above, known GDH has problems in thermal stability and / or substrate specificity, and practical GDH that can simultaneously satisfy both characteristics of thermal stability and substrate specificity has been reported so far. There was no example.

In addition, when quantifying glucose, measurement at 37 ° C. is assumed for a liquid reagent for clinical tests, and measurement at 10 ° C. to 40 ° C. is generally assumed for a simple glucose sensor. Of course, it is more preferable that the enzyme used for glucose determination has a higher specific activity in these temperature ranges.

In addition, when quantifying glucose using NAD (P) -dependent GDH, it is necessary to add NAD or NADP to the composition for quantification containing GDH, but NADP is more expensive than NAD. For economic reasons, it is desirable that the GDH has a higher affinity for NAD.

In addition, it is assumed that the glucose sensor is used in a temperature range of 10 ° C. to 40 ° C. for quantitative determination of glucose. In general, a chemical reaction via an enzyme catalyzed by an enzyme shows a behavior in which the reaction rate increases as the temperature increases, and the reaction rate rapidly decreases as the enzyme is deactivated at a temperature exceeding a certain temperature. . Such a temperature-dependent change in enzyme activity can be a factor that causes the quantitative value of glucose to change depending on the outside temperature at the time of measurement. In order to avoid this, for example, a glucose sensor is provided with a thermometer, and a measure for correcting the measured value from the temperature at the time of measurement is made. However, it is pointed out in Non-Patent Document 4 that such a temperature correction function cannot sufficiently eliminate the influence of the environmental temperature on the quantitative value. Therefore, in order to reduce the influence of the environmental temperature on the quantitative value of glucose, it is more desirable that the enzyme activity has a small fluctuation in the temperature range of 10 ° C. to 40 ° C. as a characteristic of the enzyme itself.

JP-A-4-258289 Japanese Patent No. 3220471 JP2003-310274

The thermal stability of wild-type GDH (GDH consisting of the same amino acid sequence as SEQ ID NO: 2) and mutant GDH of R202S + W334G, D58V + R202S + Y328D, and R202S + W334R is shown. The vertical axis represents the activity remaining rate (relative activity after 30 minutes of heating treatment under each temperature condition, where the GDH activity before the heating treatment is 100;%), and the horizontal axis represents the temperature during the heating treatment. . The pH stability of R202S + W334G mutant type GDH is shown. The vertical axis represents the activity remaining rate (relative activity after heating at 25 ° C. for 16 hours under each pH condition, where the GDH activity before the heating treatment is 100;%), and the horizontal axis represents the pH of the reaction solution. However, pH 3.6 to 6.0 is 50 mM acetate buffer, pH 6.1 to 8.0 is 50 mM potassium phosphate buffer, pH 7.0 to 8.9 is 50 mM Tris-HCl buffer, pH 9.0 to 10. 7 is data using 50 mM glycine-NaOH buffer. The pH stability of D58V + R202S + Y328D mutant GDH is shown. The vertical axis represents the activity remaining rate (relative activity after heating at 25 ° C. for 16 hours under each pH condition, where the GDH activity before the heating treatment is 100;%), and the horizontal axis represents the pH of the reaction solution. However, pH 3.6 to 6.0 is 50 mM acetate buffer, pH 6.1 to 8.0 is 50 mM potassium phosphate buffer, pH 7.0 to 8.9 is 50 mM Tris-HCl buffer, pH 9.0 to 10. 7 is data using 50 mM glycine-NaOH buffer. The pH stability of R202S + W334R mutant GDH is shown. The vertical axis represents the activity remaining rate (relative activity after heating at 25 ° C. for 16 hours under each pH condition, where the GDH activity before the heating treatment is 100;%), and the horizontal axis represents the pH of the reaction solution. However, pH 3.5 to 6.0 is 50 mM acetate buffer, pH 6.1 to 8.0 is 50 mM potassium phosphate buffer, pH 7.0 to 9.0 is 50 mM Tris-HCl buffer, pH 9.0 to 10. 7 is data using 50 mM glycine-NaOH buffer. The pH dependence of R202S + W334G mutant GDH activity is shown. The vertical axis represents relative activity (relative activity under each pH condition when the activity under the condition that maximizes the activity value is 100), and the horizontal axis represents the reaction pH. However, pH 3.6 to 6.0 is 50 mM acetate buffer, pH 5.9 to 7.8 is 50 mM potassium phosphate buffer, pH 8.0 to 8.9 is 50 mM Tris-HCl buffer, pH 8.8 to 10. 4 is data using 50 mM glycine-NaOH buffer. The pH dependency of D58V + R202S + Y328D mutant GDH activity is shown. The vertical axis represents relative activity (relative activity under each pH condition when the activity under the condition that maximizes the activity value is 100), and the horizontal axis represents the reaction pH. However, pH 3.6 to 6.1 is 50 mM acetate buffer, pH 5.9 to 7.8 is 50 mM potassium phosphate buffer, pH 8.0 to 8.9 is 50 mM Tris-HCl buffer, pH 8.8 to 10. 4 is data using 50 mM glycine-NaOH buffer. The pH dependency of R202S + W334R mutant GDH activity is shown. The vertical axis represents relative activity (relative activity under each pH condition when the activity under the condition that maximizes the activity value is 100), and the horizontal axis represents the reaction pH. However, pH 3.6 to 6.1 is 50 mM acetate buffer, pH 5.9 to 7.8 is 50 mM potassium phosphate buffer, pH 8.0 to 8.9 is 50 mM Tris-HCl buffer, pH 8.8 to 10. 4 is data using 50 mM glycine-NaOH buffer. The temperature-dependent variation of the activity of wild-type GDH (GDH consisting of the same amino acid sequence as SEQ ID NO: 2) and R202S + W334R mutant GDH from 10 ° C. to 37 ° C. is shown. The vertical axis represents relative activity (relative activity under each temperature condition when the activity at a reaction temperature of 37 ° C. is 100), and the horizontal axis represents the reaction temperature.

An object of the present invention is to provide GDH having high heat stability and high affinity for glucose as a substrate.

Also, an object of the present invention is to provide GDH having high thermal stability and high affinity for NAD.

Also, an object of the present invention is to provide GDH having high thermal stability and high specific activity in a temperature range of 37 ° C. or lower.

Also, an object of the present invention is to provide a GDH having high thermal stability and further reduced temperature-dependent activity fluctuation.

Another object of the present invention is GDH having high thermostability and substrate specificity, high specific activity in a temperature range of 37 ° C. or lower, and affinity for NAD as a coenzyme and glucose as a substrate. It is to provide a GDH that is higher and has reduced temperature-dependent activity fluctuations.

As a result of diligent research to achieve the above object, the present inventors have succeeded in replacing GDH with high thermal stability and high affinity for NAD with Thermoproteus sp. GDH1 which is a hyperthermophilic archaeon. Strain (Thermoproteus sp. GDH1).
The NAD (P) -dependent GDH obtained from this strain shows substantial activity against saccharides other than glucose, that is, xylose, galactose, maltose, lactose, sorbitol, sucrose, and mannose when NAD is used as a coenzyme. It was superior to the known hyperthermophilic archaeon-derived enzyme in that it was not.

(1)
However, the enzyme can use both NAD and NADP as coenzymes, but its affinity for NADP is overwhelmingly high, and the Michaelis constant (Km) at a reaction temperature of 60 ° C. is 10. While it is 3 mM, it is 0.075 mM for NADP, and there is a difference of 100 times or more. From this, it is speculated that this enzyme originally uses NADP as a coenzyme in the metabolic system.
However, considering industrial use, NADP is more expensive than NAD, and the use of NADP results in an increase in cost in the production of glucose sensors and glucose quantification reagents. In addition, since NADP is more unstable than NAD, it is more advantageous to use NAD as a coenzyme.
Although it is possible for GDH to use NAD as a coenzyme, the Michaelis constant (Km) for glucose, which is a substrate when NAD is used as a coenzyme, is as high as about 64 mM. For example, the normal value of blood glucose concentration is less than 110 mg / dl, that is, less than about 6 mM in terms of molar concentration, which is 1/10 or less of the Michaelis constant for glucose when the GDH uses NAD as a coenzyme. It is. The lower the substrate concentration than the Michaelis constant for the enzyme substrate, the lower the activity of the enzyme. This means that a signal intensity for quantifying glucose cannot be sufficiently obtained in a glucose sensor using GDH.
In addition, when performing glucose determination by the end point method, an enzyme having a high Michaelis constant for the substrate has a drawback that it takes a long time to completely oxidize glucose in the sample. In order to make this enzyme more suitable for practical use, it was necessary to reduce the Michaelis constant for the substrate glucose and to modify it to GDH having sufficient activity even for low-concentration glucose.
Therefore, the present inventors next tried to modify the GDH protein obtained from Thermoproteus sp. GDH1 strain, and replaced NAD as a coenzyme by substituting a specific amino acid residue in the GDH protein. In this case, the present inventors have succeeded in further increasing the affinity for glucose and completed the present invention.

(2)
Alternatively, since the enzyme has a low affinity for NAD as described above, a large amount of NAD is required in producing a glucose sensor and a glucose quantification reagent, and the cost is increased accordingly. Was included.
Moreover, it was reported that the well-known hyperthermophilic archaeon-derived GDH described in Non-Patent Documents 1 to 3 described above also has a high affinity for NADP.
Therefore, the present inventors next attempted to modify the GDH protein obtained from Thermoproteus sp. GDH1 strain, and further substituted the specific amino acid residue in the GDH protein to further increase the affinity for NAD. It succeeded in raising and came to complete this invention.

(3)
Alternatively, this enzyme has a problem that it has a low activity in a low temperature region of 37 ° C. or lower, like other hyperthermophile-derived enzymes. The specific activity of the enzyme at 60 ° C. is 1670 U / mg when NAD is used as a coenzyme, but the specific activity decreases with a decrease in the reaction temperature, and the Vmax at 37 ° C. is approximately About 290 U / mg, and the specific activity at 37 ° C. calculated according to the activity measurement method and protein quantification method described later is 172 U / mg. The temperature condition for quantifying glucose is usually 37 ° C. for a liquid glucose quantification reagent, and 10 to 40 ° C. for a simple colorimetric or electrochemical glucose sensor. As expected, higher activity at 37 ° C. or lower is advantageous in practice.
Therefore, the present inventors next attempted to modify the GDH protein obtained from Thermoproteus sp. GDH1 strain, and substituted specific amino acid residues in the GDH protein to thereby obtain a specific activity at 37 ° C. or lower. The present invention has been completed successfully.

(4)
Alternatively, when the activity at 37 ° C. is 100, the relative activity of the enzyme is 33 at 25 ° C. and 4 at 10 ° C., and the temperature-dependent activity fluctuation is large.
Then, the present inventors next tried to modify the GDH protein obtained from Thermoproteus sp. The present invention has been completed by successfully reducing temperature-dependent fluctuations in activity values.

(5)
In addition, the present inventors attempted to modify the GDH protein obtained from Thermoproteus sp. GDH1 strain, and substituted specific amino acid residues in the GDH protein, thereby allowing specific activity in a temperature range of 37 ° C. or lower. The present inventors have succeeded in simultaneously modifying the properties of affinity for NAD and glucose, and temperature-dependent activity value fluctuations in the temperature range of 37 ° C. or lower, thereby completing the present invention.

That is, the present invention has the following configuration.
Item 1-1.
A mutant glucose dehydrogenase obtained by substituting the amino acid residue corresponding to the 202nd arginine in the amino acid sequence shown in SEQ ID NO: 2 with any of glycine, alanine, leucine, isoleucine, serine, threonine, asparagine, and lysine .
Item 1-2.
Item 1. The mutant glucose dehydrogenase according to Item 1-1, wherein 1 to several amino acid residues other than the 202nd arginine are deleted, substituted, inserted and / or added.
Item 1-3.
1 selected from the group consisting of 58th aspartic acid, 286th valine, 328th tyrosine, 334th tryptophan, 339th isoleucine, 340th lysine, 341th threonine and 345th leucine in the amino acid sequence set forth in SEQ ID NO: 2 Item 1. The mutant glucose dehydrogenase according to Item 1-1, further comprising substitution of several amino acid residues with other residues.
Item 1-4.
In the amino acid sequence described in SEQ ID NO: 2 (D58V + Y328D), selected from V286A, Y328T, Y328E, W334G, W334R, W334H, W334A, W334K, I339P, K340R, T341R, T341G, T341P, T341M, and T341Q Item 12. The mutant glucose dehydrogenase according to Item 1-1, further comprising one or several amino acid substitutions.
Item 1-5.
The amino acid residue corresponding to the 202nd arginine in the amino acid sequence shown in SEQ ID NO: 2 is substituted with serine, and (D58V + Y328D), V286A, Y328T, Y328E, W334G, W334R, W334H, W334A, W334K, I339P, K340R, Item 1. The mutant glucose dehydrogenase according to Item 1-1, further comprising one or several amino acid substitutions selected from the group consisting of T341R, T341G, T341P, T341M, T341S, and L345Q.
Item 2-1.
A mutant glucose dehydrogenase having high affinity for glucose, wherein the amino acid residue corresponding to the 202nd arginine in the amino acid sequence described in SEQ ID NO: 2 is substituted with serine.
Item 2-2.
Item 2. The mutant glucose dehydrogenase according to Item 2-1, wherein one or several amino acid residues other than the 202nd arginine are deleted, substituted, inserted and / or added.
Item 2-3.
In the amino acid sequence shown in SEQ ID NO: 2, the 3rd alanine, 46th isoleucine, 49th lysine, 58th aspartic acid, 74th glycine, 80th valine, 85th leucine, 89th threonine, 121st alanine, 124 Serine, 168th lysine, 173th alanine, 206th serine, 208th lysine, 294th leucine, 298th glutamic acid, 303th histidine, 306th glutamic acid, 311th leucine, 323rd alanine, 328th tyrosine, 330th glutamic acid 1 to several amino acids selected from the group consisting of 334th tryptophan, 335th threonine, 338th aspartic acid, 341th threonine, 343th leucine and 345th leucine Comprising further combined substitution of another amino acid residue of residue, mutant glucose dehydrogenase according to Item 2-1.
Item 2-4.
In the amino acid sequence shown in SEQ ID NO: 2, A3V, I46V, K49R, D58V, G74D, V80A, L85H, T89K, A121T, S124L, S124P, K168E, A173T, S206N, K208R, L294W, E298G, H303R, E306G, L311P, A323T, Y328D, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, E330G, W334G, W334R, W334H, W334A, W334K, W334S, T335N, D338G, D338V, T341R, P345L, P345T, L345P Item 2. The mutant glucose protein according to Item 2-1, which is further combined with one or several amino acid substitutions selected from Rogenaze.
Item 2-5.
In the amino acid sequence shown in SEQ ID NO: 2, the amino acid residue corresponding to the 202nd arginine is substituted with serine, and A3V, (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), V80A, L85H, T89K, A121T, S124L, (K168E + E330G), A173T, S206N, K208R, L294W, E298G, H303R, L311P, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334R, W334T, W334T, W334T, W334T, W334T, W334T (I339P + T341G), (I339P + T341R), (I339P + T341L), (I339P + T 41K), T341R, T341G, T341M, L343P, comprising further combining one of the amino acid substitutions selected from the group consisting of L345Q and L345P, mutant glucose dehydrogenase according to Item 2-1.
Item 3-1.
A mutant glucose dehydrogenase having an improved specific activity at 37 ° C. or lower, wherein the amino acid residue corresponding to the 202nd arginine in the amino acid sequence shown in SEQ ID NO: 2 is substituted with serine.
Item 3-2.
Item 3. The mutant glucose dehydrogenase according to Item 3-1, wherein one or several amino acid residues other than the 202nd arginine are deleted, substituted, inserted and / or added.
Item 3-3.
In the amino acid sequence shown in SEQ ID NO: 2, the 3rd alanine, 46th isoleucine, 49th lysine, 58th aspartic acid, 74th glycine, 80th valine, 85th leucine, 89th threonine, 121st alanine, 124 Serine, 168th lysine, 173th alanine, 206th serine, 208th lysine, 294th leucine, 298th glutamic acid, 303th histidine, 306th glutamic acid, 311th leucine, 323rd alanine, 328th tyrosine, 330th glutamic acid 1 to several amino acids selected from the group consisting of 334th tryptophan, 335th threonine, 338th aspartic acid, 341th threonine, 343th leucine and 345th leucine Comprising further combined substitution of another amino acid residue of residue, mutant glucose dehydrogenase according to Item 3-1.
Item 3-4.
In the amino acid sequence shown in SEQ ID NO: 2, A3V, I46V, K49R, D58V, G74D, V80A, L85H, T89K, A121T, S124L, S124P, K168E, A173T, S206N, K208R, L294W, E298G, H303R, E306G, L311P, A323T, Y328D, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, E330G, W334G, W334R, W334H, W334A, W334K, W334S, T335N, D338G, D338V, T341R, P345L, P345T, L345P Item 3. The mutant glucose protein according to Item 3-1, further combining one or several amino acid substitutions selected from Rogenaze.
Item 3-5.
In the amino acid sequence shown in SEQ ID NO: 2, the amino acid residue corresponding to the 202nd arginine is substituted with serine, and A3V, (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), V80A, L85H, T89K, A121T, S124L, (K168E + E330G), A173T, S206N, K208R, L294W, E298G, H303R, L311P, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334R, W334T, W334T, W334T, W334T, W334T, W334T (I339P + T341G), (I339P + T341R), (I339P + T341L), (I339P + T 41K), T341R, T341G, T341M, L343P, comprising further combining one of the amino acid substitutions selected from the group consisting of L345Q and L345P, mutant glucose dehydrogenase according to Item 3-1.
Item 4-1.
In the amino acid sequence shown in SEQ ID NO: 2, the 202nd arginine is substituted with serine, and the 46th isoleucine, 49th lysine, 58th aspartic acid, 74th glycine, 89th threonine, 168th lysine, 124th serine, 206 Serine, 208th lysine, 294th leucine, 303th histidine, 306th glutamic acid, 311th leucine, 323rd alanine, 328th tyrosine, 330th glutamic acid, 334th tryptophan, 335th threonine, 338th aspartic acid and 345th A mutant glucose dehydrogenase obtained by substituting one or several amino acids selected from the group consisting of leucine with other amino acids.
Item 4-2.
Arginine at position 202 is substituted with serine in the amino acid sequence shown in SEQ ID NO: 2, and (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), T89K, S124L, (K168E + E330G), S206N, K208R, L294W, H303R, L311P, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334R, W334H, W334A, W334K, (W334S + T335N), a combination of amino acids selected from the group consisting of a combination of D338V, D338G, L345Q and L345P. Item 4. The mutant glucose dehydrogenase according to Item 4-1.
Item 4-3.
Arginine at position 202 is substituted with serine in the amino acid sequence shown in SEQ ID NO: 2, and (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), T89K, S124L, (K168E + E330G), S206N, K208R, L294W, H303R, L311P, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334R, W334H, W334A, W334K, (W334S + T335N), a combination selected from the group consisting of D338V, D338G, L345Q and L345P. The mutant glucose dehydrogenase according to Item 4-1.
Item 5-1.
A mutant glucose dehydrogenase in which the 202nd arginine is substituted with serine and the 334th tryptophan is substituted with glycine in the amino acid sequence shown in SEQ ID NO: 2.
Item 5-2.
A mutant glucose dehydrogenase in which the 202nd arginine is substituted with serine and the 334th tryptophan is substituted with arginine in the amino acid sequence shown in SEQ ID NO: 2.
Item 5-3.
A mutant glucose dehydrogenase in which the 58th aspartic acid is substituted with valine, the 202nd arginine is substituted with serine, and the 328th tyrosine is substituted with aspartic acid in the amino acid sequence shown in SEQ ID NO: 2.
Item 6.
Item 4. A gene encoding a mutant glucose dehydrogenase according to any one of Items 1-1 to 5-3.
Item 7.
Item 7. A vector comprising the gene according to item 6.
Item 8.
Item 8. A transformant transformed with the vector according to Item 7.
Item 9.
A method for producing a mutant glucose dehydrogenase, comprising culturing the transformant according to Item 8.
Item 10.
A glucose assay kit comprising the mutant glucose dehydrogenase according to any one of Items 1-1 to 5-3.
Item 11.
A glucose sensor comprising the mutant glucose dehydrogenase according to any one of Items 1-1 to 5-3.
Item 12.
Item 4. A glucose measurement method comprising the mutant glucose dehydrogenase according to any one of Items 1-1 to 5-3.

According to the present invention, it is possible to obtain GDH having excellent thermal stability and enhanced affinity for glucose. The GDH is useful as a raw material for glucose sensors and glucose determination reagents.

According to the present invention, GDH having excellent thermal stability and enhanced affinity for NAD can be obtained. Moreover, the amount of NAD used can be reduced when producing a glucose sensor and a glucose quantitative reagent using the GDH.

According to the present invention, GDH excellent in thermal stability and having improved specific activity at 37 ° C. or lower can be obtained. The GDH is useful as a raw material for glucose sensors and glucose determination reagents.

According to the present invention, it is possible to obtain GDH having excellent thermal stability and reduced temperature-dependent activity value fluctuation in a temperature range of 37 ° C. or lower. Moreover, the amount of NAD used can be reduced when producing a glucose sensor and a glucose quantitative reagent using the GDH.

According to the present invention, it has excellent thermal stability and substrate specificity, has high specific activity in a temperature range of 37 ° C. or lower, has high affinity for NAD and glucose, and is temperature-dependent in a temperature range of 37 ° C. or lower. GDH with reduced variation in activity value can be obtained. Moreover, the amount of NAD used can be reduced when producing a glucose sensor and a glucose quantitative reagent using the GDH. At the same time, the influence of the environmental temperature on the quantitative value of glucose can be reduced.

[1] Mutant glucose dehydrogenase of the present invention [1-1]
One embodiment of the present invention is a mutant glucose dehydrogenase having high thermostability and high affinity for glucose.

Specifically, the 202th arginine residue in the amino acid sequence shown in SEQ ID NO: 2 is replaced with one amino acid residue selected from the group consisting of glycine, alanine, leucine, isoleucine, serine, methionine, asparagine, and lysine. Examples thereof include glucose dehydrogenase substituted with a group.
Here, SEQ ID NO: 2 is an amino acid sequence of glucose dehydrogenase obtained by the present inventors from Thermoproteus sp. GDH1 strain (Thermoproteus sp. GDH1). The acquisition method is described in Japanese Patent Application No. 2008-60032, which will be described later in Examples.

As a GDH serving as a base for introducing a mutation, a GDH comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is a preferred example, but at least 50% or more compared to the amino acid sequence shown in SEQ ID NO: It is estimated that GDH having a homology of 60% or more, more preferably 70% or more, and most preferably 80% or more is also available.
Such GDH preferably includes NAD (P) -GDH derived from a hyperthermophilic archaeon, preferably GDH derived from Thermoproteus.
This explanation is similarly applied to the mutant glucose dehydrogenase of the present invention described in [1-2], [1-3] and [1-4] described later.

When such a GDH is used as a base for mutagenesis, use a sequence analysis software such as GENETYX to identify an arginine residue that is considered to be present at the position equivalent to position 202 in the amino acid sequence described in SEQ ID NO: 2. Can do. Mutant GDH obtained by substituting this with other amino acid residues is also included in the equivalent scope of the present invention.
This explanation is similarly applied to the mutant glucose dehydrogenase of the present invention described in [1-2], [1-3] and [1-4] described later.

In addition to substitution of serine for an arginine residue present at position 202 or equivalent thereto in SEQ ID NO: 2, the present invention provides 58th aspartic acid, 74th glycine, 80th valine, 85th leucine, 89th threonine. 121th alanine, 124th serine, 168th lysine, 173th alanine, 206th serine, 208th lysine, 294th leucine, 298th glutamic acid, 303th histidine, 306th glutamic acid, 311th leucine, 323rd alanine, 328 Further substitution of one to several amino acid residues selected from the group consisting of th-tyrosine, 334th tryptophan, 338th aspartic acid, 341th threonine, 343th leucine and 345th leucine with other residues Is a mutant glucose dehydrogenase to be together.
The substitution residue to be introduced into each site may be any one that improves the specific activity of the glucose dehydrogenase at 37 ° C. as an effect after amino acid substitution. Valine, 46th valine, 49th arginine, 58th valine, 74th aspartic acid, 80th alanine, 85th histidine, 89th lysine, 121th threonine, 124th leucine, 168 Is glutamic acid, 173 is threonine, 206 is asparagine, 208 is arginine, 294 is tryptophan, 298 is glycine, 303 is arginine, 306 is glycine, 311 is proline, 323 is threonine, 328 The second is serine and threonine One of valine, glycine, glutamic acid and leucine, 330th is glycine, 334th is glycine, arginine, histidine, alanine, lysine and serine, 338th is valine or glycine 341th is arginine, leucine, lysine and glycine , Any one of methionine and proline, 343 is preferably substituted with th-proline and 345-th with glutamine or prosin, respectively.
Substitution of 46th isoleucine and 124th serine, substitution of 58th aspartic acid and 328th tyrosine, substitution of 74th glycine, 306th glutamic acid and 323rd alanine, substitution of 168th lysine and 330th glutamic acid, respectively, as a set It is preferable to do this.

As a more preferable example of the amino acid substitution to be introduced, in addition to substitution of the amino acid residue corresponding to the 202nd arginine with serine, A3V, (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), V80A, L85H, T89K , A121T, S124L, (K168E + E330G), A173T, S206N, K208R, L294W, E298G, H303R, L311P, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334W, T334W, T334W, T334W, T334W , D338V, (I339P + T341G), (I339P + T341R), (I339P + T341L), (I339 + T341K), T341R, T341G, T341M, L343P, double or multiple mutations enzymes were also added one amino acid substitution selected from the group consisting of L345Q and L345P are preferred.

[1-2]
One of the embodiments of the present invention is a mutant glucose dehydrogenase having high thermostability and high affinity for NAD. More preferably, it is a mutant glucose dehydrogenase having excellent substrate specificity in addition to the above properties.

Specifically, the 202th arginine residue in the amino acid sequence shown in SEQ ID NO: 2 is replaced with one amino acid residue selected from the group consisting of glycine, alanine, leucine, isoleucine, serine, threonine, asparagine, and lysine. Examples thereof include glucose dehydrogenase substituted with a group.

Furthermore, the present invention relates to the substitution of arginine residue present at position 202 or equivalent thereto in SEQ ID NO: 2 with any of glycine, alanine, leucine, isoleucine, serine, threonine, asparagine, and lysine. A further combination of substitution of any one of the amino acid residues of any of the first aspartic acid, 286th valine, 328th tyrosine, 334th tryptophan, 336th histidine, 340th arginine, 341th threonine and 345th leucine with another amino acid residue Is a mutant glucose dehydrogenase.
The substitution residue introduced into each site may be any residue that reduces the Michaelis constant for NAD of glucose dehydrogenase as an effect after amino acid substitution. Examples of such substitution residues include, for example, the 58th asparagine. Acid is valine, 286th valine is alanine, 328th tyrosine is aspartic acid, 334th tryptophan is glycine or arginine, 340th arginine is lysine, 341th threonine is arginine, glycine, proline, methionine and serine In any case, the 345th leucine is preferably substituted with glutamine.
The substitution of the 58th aspartic acid and the substitution of the 328th tyrosine are preferably introduced as a set.

As a more preferable example of the amino acid substitution to be introduced, the mutant GDH of R202S is added to (D58V + Y328D), V286A, Y328T, Y328E, W334G, W334R, W334H, W334A, W334K, I339P, K340R, T341R, T341T, P341M, A double or triple mutant enzyme to which any one of the amino acid substitutions of T341S and L345Q is further added is preferable.

[1-3]
One embodiment of the present invention is a mutant glucose dehydrogenase having high thermostability and high specific activity at a temperature of 37 ° C. or lower. Also preferred is glucose dehydrogenase which is further excellent in substrate specificity.

Specifically, glucose dehydrogenase formed by substituting serine for the arginine residue present at position 202 in the amino acid sequence shown in SEQ ID NO: 2 can be exemplified.

In addition to substitution of serine for an arginine residue present at position 202 or equivalent thereto in SEQ ID NO: 2, the present invention provides the third alanine, 46th isoleucine, 49th lysine, 58th aspartic acid, 74th glycine. 80th valine, 85th leucine, 89th threonine, 121st alanine, 124th serine, 168th lysine, 173th alanine, 206th serine, 208th lysine, 294th leucine, 298th glutamic acid, 303th histidine, 306 1st selected from the group consisting of glutamic acid, 311th leucine, 323rd alanine, 328th tyrosine, 334th tryptophan, 338th aspartic acid, 341th threonine, 343th leucine and 345th leucine A mutant glucose dehydrogenase comprising further combining substitution of other residues of the amino acid residues of the stone several places.
The substitution residue to be introduced into each site may be any one that improves the specific activity of the glucose dehydrogenase at 37 ° C. as an effect after amino acid substitution. Valine, 46th valine, 49th arginine, 58th valine, 74th aspartic acid, 80th alanine, 85th histidine, 89th lysine, 121th threonine, 124th leucine, 168 Is glutamic acid, 173 is threonine, 206 is asparagine, 208 is arginine, 294 is tryptophan, 298 is glycine, 303 is arginine, 306 is glycine, 311 is proline, 323 is threonine, 328 The second is serine and threonine One of valine, glycine, glutamic acid and leucine, 330th is glycine, 334th is glycine, arginine, histidine, alanine, lysine and serine, 338th is valine or glycine 341th is arginine, leucine, lysine and glycine , Any one of methionine and proline, 343 is preferably substituted with th-proline and 345-th with glutamine or prosin, respectively.
Substitution of 46th isoleucine and 124th serine, substitution of 58th aspartic acid and 328th tyrosine, substitution of 74th glycine, 306th glutamic acid and 323rd alanine, substitution of 168th lysine and 330th glutamic acid, respectively, as a set It is preferable to do this.

As a more preferable example of the amino acid substitution to be introduced, in addition to substitution of the amino acid residue corresponding to the 202nd arginine with serine, A3V, (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), V80A, L85H, T89K , A121T, S124L, (K168E + E330G), A173T, S206N, K208R, L294W, E298G, H303R, L311P, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334W, T334W, T334W, T334W, T334W , D338V, (I339P + T341G), (I339P + T341R), (I339P + T341L), (I339 + T341K), T341R, T341G, T341M, L343P, 1 kind of further double or multiple mutations enzyme plus amino acid substitutions selected from the group consisting of L345Q and L345P are preferred.

[1-4]
One embodiment of the present invention is a mutant glucose dehydrogenase having high thermostability and reduced temperature-dependent activity value fluctuation in a temperature range of 37 ° C. or lower. More preferably, it is a mutant glucose dehydrogenase having excellent substrate specificity in addition to the above properties. Even more preferably, the glucose dehydrogenase is stable in the pH range near neutrality in addition to the above properties.

Specifically, the arginine residue present at the 202nd position in the amino acid sequence shown in SEQ ID NO: 2 is substituted with serine, and 1 to several amino acid residues other than the 202nd serine are substituted with other amino acid residues. Is a mutant glucose dehydrogenase.

The position at which amino acid substitution is introduced in addition to substitution of serine at 202nd arginine is preferably 46th isoleucine, 49th lysine, 58th aspartic acid, 74th glycine, 89th threonine, 168th lysine, 124th serine. 206th serine, 208th lysine, 294th leucine, 303th histidine, 306th glutamic acid, 311th leucine, 323rd alanine, 328th tyrosine, 330th glutamic acid, 334th tryptophan, 335th threonine, 338th aspartic acid, The 345th leucine is mentioned.
The substituted residue introduced into each site may be any residue that reduces temperature-dependent activity fluctuation in a temperature range of 37 ° C. or lower as an effect after amino acid substitution. For example, 46th isoleucine is valine, 49th lysine is arginine, 58th aspartate valine, 74th glycine aspartate, 89th threonine is lysine, 168th lysine glutamate, 124th serine is proline or Lysine, 206th serine asparagine, 208th lysine, arginine, 294th leucine, tryptophan, 303th histidine arginine, 306th glutamic acid glycine, 311th leucine proline, 323th alanine threonine, 328th tyrosine Is aspartic acid, serine, threonine, valine, glycine, glutamic acid and leucine, 330th glutamic acid is glycine, 334th tryptophan is glycine, arginine, histidine, alanine, serine and lysine 335th It is preferable to substitute threonine for asparagine, 338th aspartic acid for valine or glycine, and 345th leucine for glutamine or proline, respectively.
Preferred examples of the combination of two or more substitutions among the above amino acid residues include 46th isoleucine and 124th serine, 58th aspartic acid and 328th tyrosine, 74th glycine, 306th glutamic acid and 323rd alanine. Examples include, but are not limited to, combinations of amino acid substitutions of the 168th lysine, 330th glutamic acid, 334th tryptophan, and 335th threonine residues.

As a more preferable example of the amino acid substitution to be introduced, the mutant GDH of R202S includes (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), T89K, S124L, (K168E + E330G), S206N, K208R, R294L, H Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334R, W334H, W334A, W334K, (W334S + T335N), D338V, D338G, L345Q, and L345P. Double to quadruple mutant enzymes are preferred.

[1-5]
One embodiment of the present invention is a mutant glucose dehydrogenase having high thermostability and increased specific activity in a temperature range of 37 ° C. or lower. Furthermore, it is a mutant glucose dehydrogenase having an increased affinity for NAD as a coenzyme and at the same time an increased affinity for glucose as a substrate when NAD is used as a coenzyme. Furthermore, in addition to the above characteristics, it is a mutant glucose dehydrogenase that has reduced temperature-dependent activity value fluctuations in a temperature range of 37 ° C. or lower. And in addition to the said characteristic, it is a mutant | variant glucose dehydrogenase which is further excellent in substrate specificity. Furthermore, in addition to the above properties, it is a glucose dehydrogenase that is stable in a pH range near neutrality.

Specifically, it is a mutant glucose dehydrogenase in which the 202th arginine residue in the amino acid sequence shown in SEQ ID NO: 2 is substituted with serine, and the 334th tryptophan residue is substituted with glycine. .
Alternatively, it is a mutant glucose dehydrogenase in which the 202nd arginine residue in the amino acid sequence shown in SEQ ID NO: 2 is substituted with serine and the 334th tryptophan residue is substituted with arginine.
Alternatively, the 58th aspartic acid residue in the amino acid sequence shown in SEQ ID NO: 2 is substituted with valine, the 202th arginine residue is substituted with serine, and the 328th tyrosine residue is replaced with an aspartic acid residue. It is a mutant glucose dehydrogenase obtained by substitution.

As a GDH serving as a base for introducing a mutation, a GDH comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is a preferred example, but at least 50% or more compared to the amino acid sequence shown in SEQ ID NO: It is estimated that GDH having a homology of 60% or more, more preferably 70% or more, and most preferably 80% or more is also available.
Such GDH preferably includes NAD (P) -GDH derived from a hyperthermophilic archaeon, preferably GDH derived from Thermoproteus.

When such GDH is used as a base for mutagenesis, an aspartic acid residue considered to be present at the position equivalent to the 58th position in the amino acid sequence described in SEQ ID NO: 2 using the sequence analysis software such as GENETYX, the 202nd position Can be identified as arginine residues considered to be present at positions equivalent to tyrosine, tyrosine residues considered to be present at positions equivalent to position 328, and tryptophan residues present at positions equivalent to position 334. Mutant GDH obtained by substituting these with other amino acid residues is also included in the equivalent scope of the present invention.

[2] Definition of terms and the like in this specification [2-1] Notation of amino acid substitution In this specification, the notation of amino acid substitution is the original amino acid residue, the position from the N-terminal, the amino acid residue after substitution, For example, V286A means that the 286th valine in SEQ ID NO: 2 is substituted with alanine. Alphabet notation is R for arginine, S for serine, V for valine, A for alanine, W for tryptophan, G for glycine, P for proline, T for threonine, M for methionine, Q for glutamine, K for lysine, D for Aspartic acid, L is leucine, I is isoleucine, and Y is tyrosine.
Here, (D58V + Y328D) means having the amino acid substitution represented by D58V and Y328D at the same time, and the enzyme obtained by further adding (D58V + Y328D) amino acid substitution to the mutant GDH of R202S is represented by D58V + R202S + Y328D. It can be called triple mutant GDH.

[2-2] Affinity for substrate In the present invention, affinity for a substrate is evaluated by the Michaelis constant (Km) for glucose.
The Michaelis constant (Km) for glucose described in the present invention is a value obtained by measurement and calculation according to a measurement example described later.
The Michaelis constant for glucose in the mutant GDH of the present invention is preferably 50 mM or less, more preferably 35 mM or less, still more preferably 20 mM or less, still more preferably 10 mM or less, and most preferably 5 mM. It is as follows.
From another point of view, the Michaelis constant (Km) for glucose after introduction of mutation is preferably 78% or less, more preferably 55% or less, still more preferably 30% or less, still more preferably 16% relative to the wild type. Hereinafter, it is most preferably 8% or less.

In the present invention, the affinity for NAD is evaluated by the Michaelis constant (Km) for NAD.
The Michaelis constant (Km) for NAD described in the present invention is a value obtained by measurement and calculation according to the method described in “Example of calculation of Michaelis constant (Km) for nicotinamide adenine dinucleotide (NAD)” described later. .
The Michaelis constant for NAD in the mutant GDH of the present invention is preferably 5 mM or less, more preferably 2 mM or less, still more preferably 1.25 mM or less, even more preferably 1 mM or less, and most preferably Is 0.5 mM or less.
From another viewpoint, the degree of reduction of the Michaelis constant (Km) relative to NAD due to the introduction of mutation is preferably 60% or less, more preferably 25% or less, still more preferably 15% or less, and still more preferably, compared to the wild type. 12% or less, most preferably 6% or less.

[2-3] Thermal Stability The GDH of the present invention preferably has the necessary thermal stability in addition to the above characteristics. Thermal stability is evaluated by the activity maintained even after 30 minutes of heating in a 0.1 M potassium phosphate buffer (pH 8.0) containing 5 U / ml GDH. Is done.
The necessary thermal stability means that the activity remaining rate when heated at 80 ° C. is preferably 50% or more, more preferably 80% or more, and most preferably 90% or more, compared to the pre-heating treatment. .
From another point of view, the maximum limit value of the temperature condition where the activity remaining rate after the heating treatment for 30 minutes is 90% or more is preferably 70 ° C. or more and 90 ° C. or less, more preferably 75 ° C. or more and 85 ° C. Glucose dehydrogenase which is:

[2-4] Substrate specificity In addition to the above properties, the GDH of the present invention preferably has the necessary substrate specificity.
Substrate specificity is evaluated according to “Substrate specificity calculation example” described later. The substrate specificity of glucose dehydrogenase described in the present invention is preferably 5% or less, more preferably 3% or less, and still more preferably 2% or less, as a function of maltose, galactose, and xylose as a glucose ratio. And most preferably less than 1%.

[2-5] Specific activity The specific activity described in the present invention is measured and calculated by the method described in “Examples of protein quantification and calculation of specific activity” described later.
The specific activity of the mutant GDH of the present invention is preferably 220 U / mg or more, more preferably 400 U / mg or more, still more preferably 500 U / mg or more, and most preferably 600 U / mg or more. .
From another viewpoint, the degree of improvement in the specific activity of the mutant GDH according to the present invention is preferably 1.3 times or more, more preferably 2.3 times or more, still more preferably 2.9 times or more as a wild type ratio, Most preferably, it is 3.5 times or more.

[2-6] Temperature-Dependent Activity Variation In the present invention, the temperature-dependent activity variation is evaluated as a 25 ° C./37° C. activity temperature ratio, and the calculation method is described later in “25 ° C./37° C. activity”. It is a method described in “Example of calculating temperature ratio”.
An increase in this value means that the temperature dependent variation of activity between 25 ° C. and 37 ° C. is reduced.
In light of biochemical knowledge, the enzyme activity at 25 ° C cannot be significantly higher or lower than the surrounding temperature region, and the relative activity at 25 ° C (the activity at 37 ° C is defined as 100). High means that the relative activity is high in a wide temperature range of less than 37 ° C., at least in the temperature range of 10 ° C. or more and less than 37 ° C., and thus the temperature dependence of the activity in the temperature range of at least 10 ° C. or more and 37 ° C. or less. It is thought that this suggests that there is a reduction in general fluctuations.
The 25 ° C./37° C. active temperature ratio in the mutant GDH of the present invention is preferably 0.40 or more, more preferably 0.45 or more, and most preferably 0.5 or more.
From another point of view, the degree of increase in the 25 ° C / 37 ° C activity temperature ratio due to the introduction of mutation is preferably 1.2 times or more, more preferably 1.4 times or more, and most preferably 1.5 times the wild type ratio. It is more than double.

[2-7] pH stability In addition to the above characteristics, the GDH of the present invention is preferably stable in a wide pH range centering on a neutral range.
The pH stability described in the present invention is the activity remaining ratio after incubation with respect to GDH activity before incubation by incubating at 25 ° C. for 16 hours in a 50 mM buffer solution containing a GDH concentration of 10 U / ml. Evaluate as
Under these conditions, the pH range showing an activity remaining rate of 80% or more is at least 5.5 to 9.5, more preferably 5.0 to 9.9, and still more preferably 5.0 to 10. 7.

[3] A gene encoding the mutant glucose dehydrogenase of the present invention, a vector containing the gene, a transformant transformed with the vector, and a mutant glucose dehydrogenase characterized by culturing the transformant Manufacturing method [3-1]
One embodiment of the present invention is a gene encoding a mutant glucose dehydrogenase having high thermostability and high affinity for NAD.
One of the embodiments of the present invention is a gene encoding a mutant glucose dehydrogenase having high thermal stability and high specific activity at a temperature of 37 ° C. or lower.
[3-2]
In addition, a vector containing the gene, a transformant transformed with the vector, and a method for producing a mutant glucose dehydrogenase characterized by culturing the transformant are also included as embodiments of the present invention.
As a method for producing the GDH of the present invention, a method of producing a polynucleotide encoding the amino acid sequence of the GDH and transforming it into a host cell and culturing it is preferable.

As a method for producing a polynucleotide encoding GDH of the present invention and a plasmid capable of expressing a gene comprising the polynucleotide, for example, a DNA encoding the amino acid sequence set forth in SEQ ID NO: 1 or a plasmid comprising the DNA And a method for introducing a desired mutation into this, and a method for artificially chemically synthesizing the entire polynucleotide having the base sequence encoding GDH of the present invention and inserting it into a plasmid by restriction enzyme treatment and ligation. However, it is not limited to these.
Here, SEQ ID NO: 1 is a gene encoding the amino acid sequence of glucose dehydrogenase obtained by the present inventors from Thermoproteus sp. GDH1 strain (Thermoproteus sp. GDH1). A method for obtaining DNA encoding the amino acid sequence described in SEQ ID NO: 2 (SEQ ID NO: 1) and a plasmid into which the DNA has been inserted is described in Japanese Patent Application No. 2008-60032, and will be described later in the Examples.

And, as a method for introducing a mutation into DNA for producing GDH of the present invention, for example, a sequence in which a portion corresponding to a codon encoding an amino acid residue to be substituted is replaced with a codon encoding an amino acid after substitution And a method for extending a DNA having a sequence into which a mutation has been introduced using a DNA encoding SEQ ID NO: 2 (typified by SEQ ID NO: 1) as a template using this primer and DNA polymerase. Used. When performing site-specific modification of such genes, it is also possible to use various commercially available site direct mutagenesis kits such as Clontech's Transformer Mutagenesis Kit or Stratagene's QuickChange Site Direct Mutagenesis Kit. Although it is possible, it is not limited to these.

In addition, as a method for obtaining DNA used for GDH production of the present invention, a DNA strand is chemically synthesized or a synthesized partially overlapping oligo DNA short chain is connected by using a PCR method. It is also possible to construct a DNA encoding the full length of the GDH of the present invention.
The advantage of constructing full-length DNA in combination with chemical synthesis or PCR is that the codons used can be designed over the entire length of the gene in accordance with the host into which the gene is introduced. A plurality of codons encoding the same amino acid are not used uniformly, and the frequency of use varies depending on the species. In general, codons contained in genes that are highly expressed in a given species contain many codons that are frequently used in that species, and conversely, the presence of codons that are used infrequently becomes a bottleneck for genes with low expression levels. There are many examples that prevent high expression. In the expression of heterologous genes, many examples have been reported so far in which the expression level of the heterologous protein has been increased by replacing the gene sequence with a codon frequently used in the host organism. It is expected to be effective in increasing the expression level of heterologous genes.

For the above reasons, it is desirable to modify the DNA encoding the GDH of the present invention into a codon more suitable for the host cell into which it is introduced (ie, a codon frequently used in the host). The codon usage frequency of each host is defined by the ratio of each codon used in all genes existing on the genome sequence of the host organism, and is expressed, for example, by the number of times used per 1000 codons. In addition, the codon usage frequency can be approximately calculated from a sequence of a plurality of representative genes in an organism whose whole genome sequence has not been elucidated.
For the data of codon usage in the host organism to be recombined, for example, the genetic code usage database published on the website of Kazusa DNA Research Institute (http://www.kazusa.or.jp) is used. Or refer to the literature describing the codon usage in each organism, or determine the codon usage data of the host organism used. Refer to the obtained data and the gene sequence to be introduced, and replace the less frequently used codons in the host organism with the most frequently used codons that encode the same amino acid. Good.
As such a frequently used codon, for example, when the host is E. coli K12, Gly is GGT or GGC, Glu is GAA, Asp is GAT, Val is GTG, and Ala is Gly. GCG and Arg are CGT or CGC, Ser is AGC, Lys is AAA, Ile is ATT or ATC, Thr is ACC, Leu is CTG, Gln is CAG, Pro is CCG, and the like.

The DNA encoding the GDH of the present invention is transformed in a state of being connected to a recombinant vector. The recombinant vector of the present invention is not particularly limited as long as it can be replicated and maintained or autonomously propagated in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors and viral vectors.
The recombinant vector is simply a known cloning vector or expression vector available in the art, and the DNA encoding the above GDH is converted to an appropriate restriction enzyme and ligase, or, if necessary, a linker or adapter DNA. Can be prepared by ligation using
In addition, a gene fragment amplified using a DNA polymerase that adds a single base to the amplification end, such as Taq polymerase, can be connected to a vector by TA cloning.

Examples of vectors include Escherichia coli-derived plasmids such as pBR322, pBR325, pUC18 and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillus subtilis-derived plasmids such as pUB110, pTP5 and pC194. Moreover, bacteriophage such as λ phage, papovirus such as SV40, bovine papilloma virus (BPV), retrovirus such as Moloney murine leukemia virus (MoMuLV), adenovirus (AdV), adeno-associated virus (AAV), Illustrative are animal and insect viruses such as vaccinia virus, baculovirus.

In particular, the present invention provides a GDH expression vector in which DNA encoding GDH is placed under the control of a promoter functional in the intended host cell.
As a vector to be used, a promoter region that functions in various prokaryotic and / or eukaryotic host cells and can control transcription of a gene located downstream thereof (for example, trp promoter when the host is E. coli, When the host is Bacillus subtilis, such as lac promoter, lectA promoter, etc. When the host is a yeast, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. When the host is a mammalian cell SV40-derived early promoter, MoMuLV-derived long terminal repeat, adenovirus-derived early promoter, and the like, and a transcription termination signal of the gene, that is, a terminator region, The terminator region is not particularly limited as long as it is linked via a sequence containing at least one restriction enzyme recognition site, preferably a unique restriction site that cleaves the vector only at that site. It further contains a selection marker gene for selection of transformants (a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, a gene that complements auxotrophic mutations, etc.) It is preferable. Furthermore, when the DNA encoding GDH to be inserted does not contain a start codon and a stop codon, the start codon (ATG or GTG) and the stop codon (TAG, TGA, TAA) are respectively located downstream of the promoter region and the terminator region. A vector contained upstream of is preferably used.
When a bacterium is used as a host cell, the expression vector generally needs to contain a replicable unit capable of autonomous replication in the host cell in addition to the promoter region and terminator region described above. The promoter region includes an operator and a Shine-Dalgarno (SD) sequence in the vicinity of the promoter.
When yeast, animal cells or insect cells are used as the host, the expression vector preferably further contains an enhancer sequence, 5 ′ and 3 ′ untranslated regions of GDH mRNA, a polyadenylation site, and the like.

The GDH of the present invention can be produced by culturing a transformant containing the GDH expression vector prepared as described above in a medium and recovering GDH from the resulting culture.

The medium to be used preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large Examples include soybean cake and potato extract. Further, other nutrients [for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.)] may be included as desired. .

Culturing is performed by methods known in the art. Specific media and culture conditions used according to the host cells are exemplified below, but the culture conditions in the present invention are not limited to these.
When the host is a bacterium, actinomycetes, yeast, filamentous fungus or the like, for example, a liquid medium containing the above nutrient source is suitable. A medium having a pH of 5 to 9 is preferred. When the host is Escherichia coli, LB medium and M9 medium [Miller. J. et al. , Exp. Mol. Genet, p. 431, Cold Spring Harbor Laboratory, New York (1972)]. Culturing can be performed usually at 14 to 43 ° C. for about 3 to 72 hours with aeration and agitation, if necessary. When the host is Bacillus subtilis, it can be performed usually at 30 to 40 ° C. for about 16 to 96 hours with aeration and stirring as necessary. When the host is yeast, as a medium, for example, Burkholder minimal medium [Bostian. K. L. et al, Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)], and the pH is preferably 5-8. The culture is usually carried out at about 20 to 35 ° C. for about 14 to 144 hours, and if necessary, aeration or stirring can be performed.
When the host is an animal cell, the medium is, for example, minimal essential medium (MEM) containing about 5 to 20% fetal calf serum [Science, 122, 501 (1952)], Dulbecco's modified Eagle medium (DMEM) [Virology, 8 , 396 (1959)], RPMI 1640 medium [J. Am. Med. Assoc. , 199, 519 (1967)], 199 medium [Proc. Soc. Exp. Biol. Med. , 73, 1 (1950)] or the like. The pH of the medium is preferably about 6 to 8, and the culture is usually carried out at about 30 to 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed.
When the host is an insect cell, for example, Grace's medium containing fetal calf serum [Proc. Natl. Acad. Sci. USA, 82, 8404 (1985)], etc., and the pH is preferably about 5-8. Cultivation is usually carried out at about 20 to 40 ° C. for 15 to 100 hours, and if necessary, aeration or stirring can be performed.

Purification of GDH can be performed by appropriately combining various commonly used separation techniques depending on the fraction in which GDH activity is present.
GDH present in the culture medium is obtained by centrifuging or filtering the culture to obtain a culture supernatant (filtrate), for example, salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration. By appropriately selecting a known separation method such as non-denaturing PAGE, SDS-PAGE, ion exchange chromatography, hydroxylapatite chromatography, affinity chromatography, reverse phase high performance liquid chromatography, isoelectric focusing, etc. Obtainable.

On the other hand, GDH present in the cytoplasm collects cells by centrifuging or filtering the culture and suspending it in an appropriate buffer, such as sonication, lysozyme treatment, freeze-thawing, osmotic shock, and / or triton. After crushing (dissolving) the cells and the organelle membrane by treatment with a surfactant such as -X100, the debris is removed by centrifugation or filtration to obtain a soluble fraction. It can be isolated and purified by treating with a method.

As a means for obtaining recombinant GDH quickly and conveniently, an amino acid sequence (for example, a base such as histidine, arginine, lysine, etc.) that can be adsorbed to a metal ion chelate at a portion (preferably N or C terminal) of the coding sequence of GDH. A DNA sequence encoding a sequence consisting of a natural amino acid, preferably a sequence consisting of histidine (so-called “tag”), is added by genetic manipulation and expressed in a host cell, and from the GDH activity fraction of the culture of the cell, A method of separating and recovering GDH by affinity with a carrier on which a metal ion chelate is immobilized is preferably exemplified.
The DNA sequence encoding the amino acid sequence that can be adsorbed to the metal ion chelate is obtained by, for example, using a hybrid primer in which the DNA sequence is linked to the base sequence encoding the C-terminal amino acid sequence of GDH in the process of cloning the DNA encoding GDH. The DNA can be introduced into the GDH coding sequence by performing PCR amplification or inserting the DNA encoding GDH in frame into an expression vector containing the DNA sequence before the stop codon. The metal ion chelate adsorbent used for purification contains transition metals such as cobalt, copper, nickel, iron divalent ions, or iron, aluminum trivalent ions, etc., preferably cobalt or nickel divalent ions. The solution is prepared by contacting a ligand, such as an iminodiacetic acid (IDA) group, a nitrilotriacetic acid (NTA) group, a tris (carboxymethyl) ethylenediamine (TED) group, etc., in contact with the ligand. The matrix part of the chelate adsorbent is not particularly limited as long as it is a normal insoluble carrier.
Alternatively, affinity purification can be performed using glutathione-S-transferase (GST), maltose binding protein (MBP), HA, FLAG peptide or the like as a tag.

In the purification step, membrane concentration, reduced pressure concentration, activator and stabilizer addition may be performed as necessary. In particular, since the present GDH is excellent in heat resistance, a heating treatment within a range in which other host cell-derived contaminating proteins can be heat denatured and GDH activity can be maintained is effective in greatly improving GDH purity. The solvent used in these steps is not particularly limited, but various buffer solutions represented by K-phosphate buffer solution, Tris-HCl buffer solution, GOOD buffer solution and the like having a buffer capacity in the range of about pH 6-9 are preferable. .

When the GDH thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein is obtained as a salt, a method known per se Alternatively, the salt can be converted to a free form or other salt by a method analogous thereto.
Further, as a stabilizer and / or activator for a solution or composition containing the GDH, a protein such as bovine serum albumin or sericin, a surface activity such as Triton X-100, Tween 20, cholate or deoxycholate Agents, amino acids such as glycine, serine, glutamic acid, glutamine, aspartic acid, asparagine, glycylglycine, sugars and / or sugar alcohols such as trehalose, inositol, sorbitol, xylitol, glycerol, sucrose, sodium chloride, potassium chloride, etc. Inorganic salts may be added as appropriate.

The purified enzyme is liquid and can be used for industrial use, but can also be pulverized or further granulated. The liquid enzyme is pulverized by lyophilization by a conventional method.

Further, the GDH of the present invention can also be synthesized by in vitro translation using a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc., using RNA corresponding to the DNA encoding it as a template. can do.
Whether the RNA encoding the GDH of the present invention is purified from the host cell expressing the RNA using the conventional method for the mRNA encoding the GDH of the present invention described above in the method for obtaining the cDNA encoding the GDH of the present invention. Alternatively, it can be obtained by preparing cRNA using DNA encoding GDH as a template and using a cell-free transcription system containing RNA polymerase. As the cell-free protein transcription / translation system, a commercially available one can be used. M.M. et al. "Transscription and Translation", Hames B .; D. and Higgins S. J. et al. eds. , IRL Press, Oxford 179-209 (1984).
Commercially available cell lysates include those derived from E. coli. Examples include E. coli S30 extract system (Promega) and RTS 500 Rapid Translation System (Roche), and those derived from rabbit reticulocytes are those of Rabbit Reticulocyte Lysate System (Promega) PROTEIOSTM (made by TOYOBO) etc. are mentioned. Of these, those using wheat germ lysate are preferred. As a method for producing wheat germ lysate, for example, Johnston F. et al. B. et al. , Nature, 179: 160-161 (1957) or Erickson A. et al. H. et al. , Meth. Enzymol. 96: 38-50 (1996).

The production of GDH by chemical synthesis is, for example, synthesized by using a peptide synthesizer based on the amino acid sequence obtained by introducing a desired mutation into SEQ ID NO: 1, that is, the amino acid sequence of GDH of the present invention. This can be done.
The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. When the partial peptide or amino acid capable of constituting the GDH of the present invention is condensed with the remaining part, and the product contains a protecting group, the protecting group is eliminated, whereby the target protein can be produced. Here, the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the method described in the following (1) and (2).
(1) M.M. Bodanszkyand M.M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

The GDH of the present invention thus obtained can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
When the GDH obtained by the above method is a free form, the free form can be converted into an appropriate salt by a known method or a method according thereto, and conversely, when a protein is obtained as a salt. The salt can be converted into a free form or other salt by a known method or a method analogous thereto.

[4] Reagent for measuring glucose The reagent for measuring glucose of the present invention typically comprises the GDH of the present invention, a coenzyme, a buffer, a glucose standard solution for preparing a calibration curve, and a usage guideline. Further, it preferably contains a reagent necessary for measurement such as a mediator. Further, in the reagent containing GDH, as a stabilizer and / or an activator, a protein such as bovine serum albumin or sericin, a surfactant such as Triton X-100, Tween 20, cholate or deoxycholate, glycine Amino acids such as serine, glutamic acid, glutamine, aspartic acid, asparagine, glycylglycine, sugars such as trehalose, inositol, sorbitol, xylitol, glycerol, sucrose and / or sugar alcohols, and inorganic salts such as sodium chloride and potassium chloride You may add suitably.

[5] Glucose assay kit The glucose assay kit of the present invention typically comprises the GDH of the present invention, a coenzyme, a buffer, a mediator and other reagents necessary for measurement, a glucose standard solution for preparing a calibration curve, and Includes usage guidelines.
The kit of the invention can be provided, for example, as a lyophilized reagent or as a solution in a suitable storage solution. Further, in the reagent containing GDH, as a stabilizer and / or an activator, a protein such as bovine serum albumin or sericin, a surfactant such as Triton X-100, Tween 20, cholate or deoxycholate, glycine Amino acids such as serine, glutamic acid, glutamine, aspartic acid, asparagine, glycylglycine, sugars such as trehalose, inositol, sorbitol, xylitol, glycerol, sucrose and / or sugar alcohols, and inorganic salts such as sodium chloride and potassium chloride You may add suitably.

[6] Glucose sensor In the glucose sensor of the present invention, a carbon electrode, a gold electrode, a platinum electrode, or the like is used as an electrode, and GDH is immobilized on the electrode. As immobilization methods, there are a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a method of using a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc. NAD or NADP Such a coenzyme or an electron mediator may be fixed in a polymer or adsorbed and fixed on an electrode, or a combination thereof may be used.
The GDH of the present invention is immobilized on the electrode in the form of coexisting with the coenzyme NAD or NADP, but may be immobilized in the absence of the coenzyme and supplied as a separate layer or in solution. Is possible. Typically, the 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.
Examples of the electron mediator to be used include those that receive electrons from NAD or NADP, which are GDH coenzymes, and can donate electrons to the color-developing substances and electrodes. Phenylenediamine, N, N, N ′, N′-tetramethylphenylenediamine, 1-methoxy-phenazine methosulfate, 2,6-dichlorophenolindophenol, 2,5-dimethyl-1,4-benzoquinone, 2,6 Examples include, but are not limited to, dimethyl-1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone, nitrosoaniline, ferrocene derivatives, osmium complexes, ruthenium complexes and the like.
In addition, in the GDH composition on the electrode, surfactants such as proteins such as bovine serum albumin and sericin, Triton X-100, Tween 20, cholate and deoxycholate are used as stabilizers and / or activators. , Glycine, serine, glutamic acid, glutamine, aspartic acid, asparagine, glycylglycine and other amino acids, trehalose, inositol, sorbitol, xylitol, glycerol, sucrose and other sugars and / or sugar alcohols, sodium chloride, potassium chloride and other inorganic substances Salts may be added as appropriate, or a hydrophilic polymer represented by pullulan, dextran, polyethylene glycol, carboxymethyl cellulose and the like may be included as an excipient.

The glucose concentration can be measured as follows. A reaction solution containing a buffer solution, GDH, and NAD or NADP as a coenzyme is placed in a constant temperature cell and maintained at a constant temperature. A sample containing glucose is added thereto and reacted at a constant temperature for a fixed time. During this time, the absorbance at 340 nm is monitored. From the rate of increase in absorbance per hour for the rate method, and the absorbance increase up to the point when all glucose in the sample is oxidized for the endpoint method, a calibration curve prepared in advance with a standard concentration glucose solution is used. Originally, the glucose concentration in the sample can be calculated. In addition, in the case of performing quantification by colorimetry in the visible light region, an appropriate mediator and coloring reagent may be added.
For example, glucose can be quantified by adding 2,6-dichlorophenolindophenol (DCPIP) and monitoring the decrease in absorbance at 600 nm. Further, it is possible to determine the amount of diformazan produced by adding phenazine method (PMS) as a mediator and nitrotetrazorium blue (NTB) as a coloring reagent and measuring the absorbance at 570 nm to calculate the glucose concentration. Needless to say, the mediator and coloring reagent used are not limited to these.

The glucose concentration can also be measured as follows. Put the buffer in the thermostat cell and add coenzyme and mediator as needed to maintain constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate, or the like can be used. An electrode on which the GDH 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.

Activity measurement examples In the present invention, GDH activity is carried out according to the following method unless otherwise specified.
Place 2.9 mL of the reaction solution (0.1 mol / L Tris, 10 mmol / L β-NAD ⁺ , 150 mmol / L D-glucose, pH 8.0) in a quartz cell, and preheat at 37 ° C. for 5 minutes. And 0.1 mL of GDH solution is added and mixed, it is made to react at 37 degreeC for 5 minutes, 340 nm light absorbency is measured in the meantime. The degree of increase in absorbance per minute (ΔOD _TEST ) is calculated from the linear portion of the absorbance change. In the blind test, a buffer solution is added in place of the GDH solution and mixed, and similarly, incubated at 37 ° C. for 5 minutes, the absorbance at 340 nm is recorded, and the absorbance change per minute (ΔOD _BLANK ) is calculated. The activity value (U / mL) is calculated by applying these values to the following equation. Here, the amount of enzyme that reduces 1 micromole of coenzyme per minute in the presence of a substrate is defined as 1 U.

GDH activity (U / mL) = [(ΔOD _TEST −ΔOD _BLANK ) × 3.0 × dilution ratio] / (6.22 × 1.0 × 0.1)

Here, 3.0: Volume after mixing with GDH solution (mL)
6.22: millimolar molecular extinction coefficient of NADH (cm ² / micromolar)
1.0: Optical path length (cm)
0.1: Volume of GDH solution to be added (mL)
It is.

Example of Calculation of Protein Quantification and Specific Activity The amount of protein described in the present invention is measured by measuring absorbance at 280 nm.
That is, the enzyme solution was diluted with distilled water so that the absorbance at 280 nm was in the range of 0.1 to 1.0, and the absorbance (Abs) at 280 nm was measured using an absorptiometer corrected with zero point using distilled water. Measure. The protein concentration described in the present invention approximates 1 Abs≈1 mg / ml, and is expressed by a value obtained by multiplying the absorbance measurement and the measured solution dilution rate.
The specific activity described in the present invention is the activity (U / mg) of GDH per mg as the amount of protein by this measurement method, and the GDH activity at this time is obtained by measuring according to the above activity measurement example. Value.

Calculation Example of Michaelis Constant (Km) for Nicotinamide Adenine Dinucleotide (NAD) The method for calculating the Michaelis constant (Km) for NAD described in the present invention is performed by the following measurement method.
That is, the concentration of β-NAD + in the reaction solution composition described in the above activity measurement example as a measurement solution is 20 mmol / L, 10 mmol / L, 5 mmol / L, 2.5 mmol / L, 1 mmol / L, 0.5 mmol / L. 6 types of reaction solutions were prepared, and GDH solutions (solutions adjusted to have an activity value of 0.8 U / ml in the above-mentioned activity measurement example) using the respective measurement solutions according to the method of the above-described activity measurement example. ΔOD (ΔODTEST−ΔODBLANK) is measured. Based on these measured values, the Michaelis constant (Km) is calculated according to the Lineweaver-Burk plot method (both reciprocal plot method).

Calculation Example of Michaelis Constant (Km) for Glucose The method for calculating the Michaelis constant (Km) for the substrate described in the present invention is performed by the following measurement method.
That is, six types of D-glucose concentrations in the reaction liquid composition described in the above activity measurement examples as measurement solutions were 1000 mmol / L, 200 mmol / L, 100 mmol / L, 50 mmol / L, 20 mmol / L, 10 mmol / L. And using each measurement solution according to the method of the above activity measurement example, ΔOD (ΔODTEST) of the GDH solution (solution adjusted so that the activity value in the above activity measurement example is 0.8 U / ml) -ΔODBLANK). Based on these measured values, the Michaelis constant (Km) is calculated according to the Lineweaver-Burk plot method (both reciprocal plot method).

Calculation Example of Substrate Specificity The substrate specificity evaluation method described in the present invention is performed by the following measurement method. That is, reaction solutions containing 150 mmol / L of maltose, galactose, and xylose were prepared in place of D-glucose in the reaction solution composition described in the above-mentioned activity measurement example as a measurement solution, and these were used for activity according to the activity measurement example. Measure the value. A value obtained by dividing the activity value using these reaction solutions by the activity value when glucose is used as a substrate is calculated as the reactivity to each substrate (vs. glucose).

Calculation Example of 25 ° C./37° C. Active Temperature Ratio The calculation method of the 25 ° C./37° C. active temperature ratio described in the present invention is performed as follows.
That is, the 25 ° C / 37 ° C activity temperature ratio is calculated by dividing the activity value obtained by carrying out the activity measurement by the above activity measurement example at the reaction temperature of 25 ° C by the activity value measured at 37 ° C. .

Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.

First, the gene (SEQ ID NO: 1) encoding the amino acid sequence (amino acid sequence described in SEQ ID NO: 2) of glucose dehydrogenase obtained by the present inventors from Thermoproteus sp. GDH1 strain (Thermoproteus sp. GDH1) and the gene Methods for obtaining the plasmid into which is inserted are shown in Test Examples 1 to 4 below. (This method is also described in Japanese Patent Application No. 2008-60032.)

<Test Example 1>
Culture of hyperthermophilic archaeon and purification of GDH The inventors isolated the hyperthermophilic archaeon from hot spring water in Kohojima, Kagoshima Prefecture. This strain was estimated to be a bacterium belonging to the genus Thermoproteus from the base sequence of 16S rRNA, and further had the following characteristics (A) to (G). (A) The base sequence shown in SEQ ID NO: 3 is included as the base sequence on the genomic DNA encoding 16S rRNA. (B) It can grow at a temperature of 80 ° C. or higher, and the optimum growth temperature is about 90 ° C. (C) The GC content of the genomic DNA is 58 to 62 mol%. (D) Absolute anaerobic bacteria. (E) Good growth when thiosulfate is added as an electron acceptor. (F) It can grow at a NaCl concentration of 1% or less. (G) The shape is a long koji mold having a length of 10 to 30 μm and a width of about 5 μm. This strain having the above characteristics was named Thermoproteus sp. GDH1 strain (Thermoproteus sp. GDH1).
In culturing GDH1 strain, 0.5% tryptone, 0.5% yeast extract, 0.5% sodium thiosulfate, 0.5% sodium chloride, 0.005% sodium sulfide, and 5 mg / A medium containing L resazurin as a component was placed in an anaerobic glove box, and nitrogen substitution was repeated to remove oxygen in the medium. Here, the above isolate was planted and statically cultured at 85 ° C. for 3 days. Furthermore, the proliferating cells were inoculated into a medium in which glucose having a final concentration of 0.5% was added to the medium composition, and anaerobic culture was performed at 85 ° C. for 3 days. 7 L of the culture solution was centrifuged using a high-speed cooling centrifuge, and the cells were collected by removing the supernatant. This microbial cell was suspended in 20 mL of 50 mM potassium phosphate buffer (pH 7.0) and placed on ice. Using an ultrasonic crusher (Tomy Seiko Co., Ltd., UD-201), the output was 3 at a driving rate of 40%. The cells were divided and the cells were crushed. The crushing liquid was further centrifuged to remove solid residues, and a GDH crude extract was obtained. In this crude extract, ammonium sulfate was dissolved to a final concentration of 30% and stirred at room temperature for 20 minutes to precipitate contaminating proteins. The precipitate was removed by centrifugation, and ammonium sulfate was added and dissolved to a final concentration of 48%, followed by stirring at room temperature for 20 minutes to precipitate a fraction containing GDH. The supernatant was removed by centrifugation, and the obtained GDH fraction was dissolved in 20 mL of 50 mM potassium phosphate buffer (pH 7.0). This liquid was applied to ResourceQ (manufactured by GE Healthcare) having a column volume of 6 mL to adsorb contaminating proteins on the column, and GDH was permeated. Ammonium sulfate was dissolved in this permeate so as to have a final concentration of 22.8%, and applied to a resource ISO column (manufactured by GE Healthcare, volume 6 mL) as a hydrophobic column and adsorbed. The adsorbed protein was eluted with a gradient of ammonium sulfate concentration of 22.8% to 0% to collect fractions having GDH activity. Furthermore, gel filtration was performed using Superdex 200 as a separation column and a buffer solution of pH 7.0 containing 50 mM Tris and 0.15 mM sodium chloride as an elution buffer. The obtained GDH fraction was used as a purified solution.

<Test Example 2>
Cloning of GDH gene In 10 μL of the GDH solution obtained in Example 1, an equal volume of 2 × SDS sample buffer (10 mM Tris-HCl, 10% glycerol, 2% SDS, 0.1% bromophenol blue, 2% (v / v) 2-mercaptoethanol, pH 6.8) was added and boiled at 100 ° C. for 10 minutes. This was applied to a 12.5% acrylamide gel, and after electrophoresis at 40 mA, the gel was subjected to CBB staining using CBB Stain One (manufactured by Nacalai Tesque). The main band of the sample was cut out from the stained gel, and the peptide sequence was analyzed with a mass spectrometer. Based on the obtained deduced amino acid sequence, a degenerate PCR primer containing a mixed base was prepared, and a PCR reaction was performed using genomic DNA as a template. This PCR reaction solution was applied to a 1% agarose gel, electrophoresed, stained with ethidium bromide, and then a band of an internal partial fragment of the GDH gene amplified under UV irradiation was cut out. Then, DNA was extracted and purified from the cut gel piece using Wizard SV Gel and PCR Clean-up System (Promega). The obtained DNA fragment was ligated to the cloning vector pTA2 attached to this kit in the manner of TA cloning using Toyobo's TARGET Clone Plus. The ligation product was added to E. coli JM109 strain competent cells (Toyobo Competent High JM109), transformed by heat shock, coated on LB agarose plate containing 100 μg / mL ampicillin, and cultured at 37 ° C. overnight. Transformant colonies were formed. Multiple colonies are inoculated into 5 mL of LB medium (containing 100 μg / mL of ampicillin) and cultured overnight, and the manual of this kit is used from the culture solution using Quantum Prep Miniprep Kit (BioRad). The plasmid was extracted according to By analyzing the base sequence of the extracted plasmid insert, the partial base sequence of the target GDH gene was determined. Further, based on the determined sequence, a primer directed to the outside of the internal partial sequence was prepared, and using this primer and LA PCR in vitro Cloning Kit (manufactured by Takara Bio Inc.), the 5 ′ and 3 ′ end regions of the GDH gene The entire nucleotide sequence of the gene was determined by performing amplification and sequencing. The determined base sequence is shown in SEQ ID NO: 1, and the deduced amino acid sequence is shown in SEQ ID NO: 2.

<Test Example 3>
Construction of GDH expression vector A PCR reaction was carried out using primers designed to have a sequence in which NdeI was added to the start codon of the GDH gene and a BamHI site was added immediately after the stop codon using the hyperthermophilic genomic DNA as a template. The reaction solution was applied to a 1% agarose gel, electrophoresed, stained with ethidium bromide, and then the GDH gene band amplified under UV irradiation was excised. Then, DNA was extracted and purified from the cut gel piece, and the obtained DNA fragment was inserted into the cloning vector pTA2 attached to this kit (pTA2TGDH1) using TARGET Clone Plus. In order to replace the NdeI site (CATATG) present in the inserted GDH gene with another base sequence without changing the encoded amino acid, the following operation was performed. PTA2TGD obtained above using as a primer an oligo DNA having a base sequence consisting of 5′-AGCACGGGCATTTGGGGGCCTCC-3 ′ (SEQ ID NO: 4) and 5′-GGAGCCCCCAAATGCCGTGCT-3 ′ (SEQ ID NO: 5)
A reaction similar to PCR was performed using H1 as a template using a thermal cycler. Subsequently, 2% DpnI to the reaction solution was added to the reaction solution, and the template (pTA2TGDH1) was digested by treatment at 37 ° C. for 1 hour. This DpnI treatment solution was added to E. coli JM109 strain competent cells (Toyobo Competent High JM109), transformed by heat shock, coated on LB agarose plate containing 100 μg / mL ampicillin, and cultured at 37 ° C. overnight. Thus, a transformant colony was formed. A plurality of colonies were each inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and cultured overnight, and a plasmid was extracted from the culture solution using a Quantum Prep plasmid miniprep kit. The base sequence of the obtained plasmid was analyzed, and the codon encoding the 113th isoleucine in the GDH amino acid sequence was converted from ATA to ATT, that is, the 339th A in the GDH gene base sequence was replaced with T. It was confirmed that the plasmid pTA2TGDH2 was sequence-corrected. This pTA2TGDH2 was treated with restriction enzymes NdeI and BamHI, electrophoresed on a 1% agarose gel to cut out a gel piece containing the GDH gene (having NdeI and BamHI cleavage ends at 5 ′ and 3 ′ ends, respectively), Wizard DNA was extracted and purified using SV Gel and PCR Clean-up System. This was mixed with the expression vector pET21a treated with the same restriction enzyme, and this mixture was mixed with an equal amount of Ligation High (manufactured by Toyobo) and incubated at 16 ° C. for 30 minutes for ligation. This ligation solution is added to E. coli JM109 strain competent cells, transformed by heat shock, spread on LB agarose plates containing 100 μg / mL ampicillin, and cultured overnight at 37 ° C. to form transformant colonies. It was. Among the transformant colonies, colonies whose insertion was confirmed by colony direct PCR were inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and cultured overnight. From the cells obtained by centrifuging the culture solution, the plasmid was recovered using a plasmid extraction kit. By analyzing the sequence of the insert of this plasmid, it was confirmed that it had the correct gene sequence and used as an expression vector (pET21aTGDH2).

<Test Example 4>
Expression and purification of GDH gene pET21aTGDH2 obtained in Example 3 was introduced into Escherichia coli BL21 (DE3) competent cells (manufactured by Stratagene) according to the attached manual, and a transformant was obtained. The transformed colony was suspended in 8 mL of LB medium (containing 100 μg / mL ampicillin) in a test tube and cultured with shaking at 37 ° C. overnight. 8 mL each of the obtained culture solution was inoculated into 4 800 mL LB medium (containing 100 μg / mL ampicillin) in a 2 L Sakaguchi flask. The flask was shaken at 37 ° C. and 120 rpm for 3 hours, and when the turbidity at 660 nm reached about 0.6, IPTG was added to a final concentration of 0.1 mM, and further shaken at 37 ° C. and 120 rpm for 4 hours. The culture was continued. The culture solution was centrifuged with a high-speed cooling centrifuge, the supernatant was removed by decantation, and the obtained bacterial cells were suspended in 70 mL of 50 mM Tris-hydrochloric acid buffer + 0.1 M NaCl (pH 8.0). The suspension was treated for 20 minutes at an output of 4 and a driving rate of 40% using an ultrasonic crusher (UD-201, manufactured by Tommy Seiko Co., Ltd.) to crush the cells. The crushed liquid was centrifuged to remove the residue, and a GDH crude extract was obtained. This crude extract was treated at 85 ° C. for 30 minutes to denature contaminating proteins, and the denatured proteins were removed by centrifugation. The supernatant fraction was passed through a resourceQ column buffered with 50 mM Tris-HCl / 0.1 M NaCl (pH 8.0), and 21.3% ammonium sulfate was dissolved in the permeate. This solution was adsorbed on a resource ISO column buffered with 50 mM Tris-HCl · 22.8% ammonium sulfate (pH 8.0), and gradient elution was performed up to an ammonium sulfate concentration of 0% to collect GDH fractions. This fraction was further subjected to gel filtration using a superdex 200 column, and the obtained GDH fraction was used as a purified recombinant GDH solution. This purified solution was confirmed to be a pure product showing a single band by CBB staining by SDS-PAGE.

<Example 1>
PET21aTGDH2 obtained in the same manner as in Test Example 3 for site-directed mutagenesis of the residue corresponding to the 202nd arginine was digested with the restriction enzymes NdeI and BamHI, applied to a gel containing 1% agarose, electrophoresed, and ethidium bromide A DNA fragment encoding wild-type GDH is obtained by excising a band of about 1 kb under ultraviolet irradiation and purifying and extracting the excised gel using Toyobo's nucleic acid purification kit (MagExtrantor-Gel & PCR Clean Up-). Got.
This purified DNA is mixed with NdeI / BamHI restriction enzyme treatment expression vector pBluescriptKSN + (pBluescriptKS + β-galactosidase gene translation start codon position into which NdeI site is introduced by base substitution) and ligated for expression plasmid pBSTGDH2 was prepared.
In substituting the 202nd arginine with another arbitrary amino acid residue, a mismatch primer was designed. The primer sequences are R202X-F: 5'-CGTGGCCCAGNNSCCGCCGGAT-3 '(SEQ ID NO: 6), R202X-R: 5'-ATCCGGCGSNNCGTGGCCACCG-3' (SEQ ID NO: 7). However, N in the sequence represents a mixed base containing A, T, G and C, and S represents a mixed base containing G and C.
Using this mismatch primer, a DNA polymerase expression plasmid library in which an arbitrary mutation was introduced into the 202nd Arg was prepared through extension reaction with DNA polymerase and digestion of template DNA by treatment with restriction enzyme DpnI. This library solution was added to Escherichia coli JM109 strain competent cell (Toyobo Competent High JM109), and transformation was performed by heat shock according to the manual attached to this product.
50 of the resulting transformed colonies were picked up with toothpicks, each inoculated into 5 ml of LB medium (containing 100 μg / ml ampicillin) in a test tube, and cultured with shaking at 37 ° C. for 24 hours. 1 ml of the culture solution was transferred to a 1.5 ml Eppendorf tube, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed to obtain bacterial cells. The cells were suspended in 1 ml of 20 mM potassium phosphate buffer (pH 8.0), and the cells were disrupted by ultrasonic treatment.
First, measure the activity of this disrupted solution as described in the activity measurement example, extract and purify the plasmid from the culture solution with a miniprep for the activity confirmed, and analyze the sequence to the site corresponding to the 202nd arginine. Introduced amino acid substitutions were examined.
In addition, for these mutant GDH, 50 ml GDH production medium (2.4% yeast extract, 2.4% peptone, 1.25% monohydrogen phosphate) in an E. coli JM109 strain transformed with the expression plasmid was placed in a 500 ml Sakaguchi flask. 2 potassium, 0.23% monopotassium dihydrogen phosphate, 0.4% glycerol, pH 7.0), and cultured by shaking at 37 ° C. and 180 rpm for 24 hours for expression.
After centrifuging the obtained culture solution, the supernatant was discarded to obtain bacterial cells, which were suspended in 10 ml of 20 mM potassium phosphate buffer (pH 8.0) and disrupted by ultrasonic waves. Further, 1.52 g of ammonium sulfate was added to this crushed solution to dissolve it, and the mixture was heated at 60 ° C. for 1 hour, and further the precipitate was removed by centrifugation. This solution was applied to Octyl-Sepharose resin (manufactured by GE Healthcare) buffered with 20 mM potassium phosphate buffer (pH 8.0) containing 15.2% ammonium sulfate to adsorb GDH to the resin. Then, GDH was eluted by passing the buffer through a gradient while changing the ammonium sulfate concentration from 15.2% to 0% and simultaneously the ethylene glycol concentration from 0% to 0.1%. Finally, G-25 buffered with 50 mM potassium phosphate buffer (pH 7.0)
Desalting was performed using Sepharose resin to obtain purified GDH.

With respect to the purified GDH thus obtained, the calculation of the Michaelis constant for NAD and the calculation of the Michaelis constant for glucose were respectively measured and calculated according to the above calculation examples.
In addition, the wild-type GDH that was similarly expressed and purified using pBSTGDH2 as an expression vector was similarly calculated for the Michaelis constant for NAD and the Michaelis constant for glucose.
The results are shown in Table 1. From this result, in the mutant enzyme in which the amino acid residue corresponding to the 202nd arginine is substituted with any of glycine, alanine, leucine, isoleucine, serine, threonine, asparagine, and lysine, the Michaelis constant for NAD compared to the wild type Was found to be reduced. Further, in the mutant enzyme in which the amino acid residue corresponding to the 202nd arginine is substituted with any of glycine, alanine, serine, threonine, and asparagine, the Michaelis constant for the substrate (glucose) is lower than that of the wild type. It was found.

Furthermore, the specific activities at 37 ° C. of the wild-type GDH and R202S mutant enzyme obtained in Test Example 4 were calculated according to the methods described above. As a result, wild type GDH was 172 U / mg, while R202S was 229 U / mg. The purity of R202S mutant GDH purified according to the purification method described above is about 85-90% of the total protein according to SDS-PAGE, that is, the specific activity of R202S mutant GDH itself exceeds at least 229 U / mg. It can be said that it has. From the above, it was confirmed that the specific activity of R202S mutant GDH was increased as compared to wild type GDH.

<Example 2>
Site-directed mutagenesis to the 340th lysine and its vicinity The expression plasmid of the mutant enzyme (R202S) obtained by substituting the 202nd arginine with serine obtained in Example 1 was used as a template, and further 339 in the same manner as in Example 1. Examination of the introduction of amino acid substitutions to the 341 th to 341 th was carried out, selection of mutant enzymes presumed to be modified by special products, and confirmation of the introduced amino acid substitutions. For each of the purified enzymes prepared as in Example 1, specific activity, Michaelis constant for NAD, and Michaelis constant for glucose were calculated according to the methods described above.
Table 2 shows the introduced amino acid substitutions and the characteristics of each mutant enzyme. In addition to R202S, the specific activity at 37 ° C. was further improved in the mutant GDH in which any mutation of T341R, T341G, and T341M was further introduced. Further, in addition to R202S, in the mutant GDH in which any mutation of I339P, K340R, T341R, T341P, T341M, T341S and T341G was further introduced, the Michaelis constant for NAD was further reduced. At the same time, the combination of K202R with R202K and R202N was also examined. As with the combination with R202S, the Michaelis constant for NAD was significantly reduced. Furthermore, in the mutant GDH in which any mutation of T341R, T341M, and T341G was further introduced in addition to R202S, a significant decrease in the Michaelis constant for glucose was found.

Subsequently, the introduction of mutation into 341 threonine was examined based on R202S + I339P. This R202S + I339P was obtained as a mutant enzyme having a reduced Michaelis constant for the coenzyme NAD, and its specific activity at 37 ° C. is 132 U / mg, which is rather lower than that of the wild type. However, a surprisingly dramatic improvement in specific activity at 37 ° C. was recognized by combining this mutant enzyme with the 341st amino acid substitution.
The effective combinations were R202S + I339P + T341G, R202S + I339P + T341R, R202S + I339P + T341L and R202S + I339P + T341K shown in Table 2. R202S + I339P has a higher Michaelis constant for glucose than wild type, but is higher than R202S alone mutation.
However, in addition to this R202S + I339P, by further adding any mutation of T341G, T341R, T341L and T341K, a significant reduction in the Michaelis constant for glucose was observed.

<Example 3>
Modification by random mutagenesis By carrying out error-prone PCR using the expression plasmid of the mutant enzyme (R202S) obtained by substituting serine for the 202nd arginine obtained in Example 1, the full length of the GDH gene and its upstream / downstream The region containing the part was amplified.
Error-prone PCR was performed using Diversify ™ PCR Random Mutagenesis Kit manufactured by Clontech, according to the manual attached to this kit, and mutations were randomly introduced into the GDH gene.
The amplified DNA was treated with restriction enzymes NdeI and BamHI, mixed with an expression vector pBluescript KSN + solution treated with the same restriction enzyme, and ligated to prepare a random mutant GDH expression plasmid library.
This library solution was transformed into E. coli JM109 competent cells, and the resulting transformed colonies were inoculated into 400 μL of LB medium in a 96-well microplate and cultured with shaking at 37 ° C. for 24 hours using a microplate shaker. .
The obtained culture broth was crushed by repeatedly freezing with liquid nitrogen and thawing with a 60 ° C. heat bath water bath, and 10 μL of the disrupted solution was added to 200 μL of an assay solution (50 mM D-glucose, 2 mM β-NAD ⁺ , 0 .Suspension in 2 mM 2,6-dichlorophenolindophenol (DCPIP), 0.1 M Tris-HCl, pH 8.0) and leave at room temperature to determine the presence or absence of activity by visually observing the blue fading of DCPIP did.
The activity of the liquid with recognized activity was measured according to the above activity measurement example. Moreover, in order to evaluate the decrease in Michaelis constant with respect to NAD, among the reaction solution compositions described in the activity measurement examples, two types of reaction solutions with NAD concentrations of 0.2 mM and 5 mM were prepared and the activity was measured. The ratio of 0.2 mM / 5 mM was calculated by dividing the activity value measured using 0.2 mM NAD by the activity value measured using 5 mM NAD.
Further, in order to evaluate the decrease in Michaelis constant with respect to glucose, among the reaction solution compositions described in the activity measurement examples, two types of reaction solutions having glucose concentrations of 15 mM and 150 mM were prepared and the activity was measured. The ratio of 15 mM / 150 mM was calculated by dividing the activity value measured using, by the activity value measured using 150 mM glucose.
Those whose values were increased compared to the R202S mutant enzyme produced by culturing and disrupting were selected. About 10,000 colonies were assayed, 25 strains were selected and the introduced mutation was identified by sequence analysis of the plasmid, and each mutant enzyme was cultured and expressed and purified according to the procedure of Example 1, and various enzymes The characteristics were investigated. Furthermore, according to the method described in “Example of calculating the temperature ratio of 25 ° C./37° C.” above, the temperature dependency of each mutant enzyme was also evaluated.

Table 3 shows the introduced mutations and the characteristics of each mutant enzyme. Mutations were observed with the modified effective from the viewpoint of specific activity increase, A3V + R202S, I46V + S124P + R202S, K49R + R202S, D58V + R202S + Y328D, G74D + R202S + E306G + A323T, V80A + R202S, L85H + R202S, T89K + R202S, A121T + R202S, S124L + R202S, K168E + R202S + E330G, A173T + R202S, R202S + S206N, R202S + K208R, R202S + L294W, R202S + E298G, R202S + H303R, R202S + L311P , R202S + Y328S, R202S + W334G, R202S + D338V, R202S + D338G, R202S + L343P, R202S + L3 5Q, include the R202S + L345P.

Also, mutations that have been altered from the viewpoint of reducing the Michaelis constant for NAD include D58V + R202S + Y328D, R202S + V286A, R202S + W334G, R202S + H336P, and R202S + L345Q.

In addition, mutations having a modification effect from the viewpoint of reducing the Michaelis constant for the substrate glucose include I46V + S124P + R202S, D58V + R202S + Y.
328D, G74D + R202S + E306G + A323T, T89K + R202S, A121T + R202S, S124L + R202S, R202S + V286A, R202S + L294W, R202S + W334G, R202S + D338V, R202S + L345Q, and R202S + L345.

Furthermore, mutations that have been altered only from the viewpoint of reducing the temperature-dependent activity fluctuation range, that is, increasing the temperature ratio of 25 ° C./37° C. include I46V + S124P + R202S, K49R + R202S, D58V + R202S + Y328D, G74D + R202S + E306G + A323T, T89K + R202S + S202S + S202S + S202S + S202S + R202S + K208R, R202S + L294W, R202S + E298G, R202S + H303R, R202S + L311P, R202S + Y328S, R202S + W334G, R202S + D338V, R202S + D338G, R202S + L345Q, R202S + L345.

In particular, as a mutant GDH that has a modification effect in all these aspects and maintains low reactivity that does not cause a practical problem with respect to maltose, galactose, and xylose as substrate specificity, D58V + R202S + Y328D and R202S + W334G 2 There are types.

<Example 4>
Examination of introduced residues at sites with high alteration effect by amino acid substitution Details of alteration effects due to differences in amino acids substituted for 328th tyrosine and 334th tryptophan presumed to be sites with high alteration effect from the results of Example 3 investigated.
According to the method described in Example 1, mismatch primers of a sequence in which a mixed base was introduced so that an arbitrary amino acid residue was introduced into a codon site encoding each site were prepared, and an R202S mutant GDH expression plasmid was used as a template. Site-directed mutagenesis was performed.
A mutant GDH expression library in which an arbitrary codon substitution was introduced at each site was subjected to the same screening as in Example 3. The obtained modified GDH was cultured and expressed and purified according to the procedure of Example 1, and Example 3 was obtained. The characteristics of each enzyme were examined in the same manner. Moreover, the plasmid was extracted and purified from the culture solution for each, and the introduced amino acid residue was identified by analyzing the sequence.

The results are shown in Table 4. For the 328th tyrosine, Y328T, Y328V, Y328G, Y328E, Y328L and for the 334th tryptophan, W334R, W334H, W334A, W334K were confirmed to contribute to further increase in specific activity in combination with R202S. It was. In addition, a mutant enzyme having an amino acid substitution introduced at the 335th position, ie, R202S + W334S + T335N, was also obtained in spite of the mutation introduction operation at the 334th position. This also showed a higher specific activity than the R202S single mutation.

Further, mutations that contribute to the reduction of the Michaelis constant (Km) for NAD include Y328T, Y328E, and Y328R for the 328th tyrosine, and W334R, W334H, W334A, and W334K for the 334th tryptophan, respectively, with R202S. It was confirmed that the combination was effective.

In addition, the combination of any one of the mutations Y328T, Y328V, Y328G, Y328E, Y328L, W334R, W334H, W334A, W334K, W334S + T335N and R202S further reduces the Michaelis constant for glucose compared to the mutation of R202S alone. It was confirmed that

Further, from the viewpoint of reducing the temperature-dependent activity value fluctuation range, that is, increasing the 25 ° C./37° C. active temperature ratio, each mutation of Y328T, Y328V, Y328G, Y328E, Y328L, W334R, W334H, W334A, W334K, W334S + T335N It was confirmed that the combination of any of the above and R202S was effective.

<Example 5>
Scale-up production of mutant GDH The D58V + R202S + Y328D mutant GDH expression plasmid obtained in Example 3, the R202S + W334G mutant GDH expression plasmid, and the R202S + W334R expression plasmid obtained in Example 4 were each transformed into competent high JM109. Recombinant E. coli was obtained. Each recombinant Escherichia coli was placed in a 200 ml Sakaguchi flask in a 200 ml preculture medium (0.5% yeast extract, 0.25% peptone, 0.5% sodium chloride, 0.5% glucose, 100 μg / ml ampicillin sodium). , PH 7.4), and cultured with shaking at 30 ° C. and 180 rpm for 16 hours. The obtained precultured solution was mixed with 6 L of GDH production medium (2.4% yeast extract, 2.4% peptone, 1.25% dihydrogen phosphate, 0.23% phosphate in 10 L jar fermenter. 2 hydrogen 1 potassium, 0.4% glycerol, 0.1% antifoaming agent, 100 μg / ml ampicillin sodium, pH 7.0), the whole amount was charged, aeration rate 2 L / min, stirring speed 310 rpm, tank pressure 0.02 MPa, The culture was aerated and stirred at a temperature of 37 ° C. for 24 hours. The resulting culture is centrifuged to obtain bacterial cells, which are suspended in 1 L of 20 mM potassium phosphate buffer (pH 8.0), and the bacterial cells are removed at an average pressure of 80 MPa using a French press bacterial cell crusher. It was crushed. Further, 152 g of ammonium sulfate per liter was added to this crushed solution for dissolution, followed by heating at 60 ° C. for 1 hour, and further removing the precipitate by centrifugation. This solution was applied to Octyl-Sepharose resin (manufactured by GE Healthcare) buffered with 20 mM potassium phosphate buffer (pH 8.0) containing 15.2% ammonium sulfate to adsorb GDH to the resin. The resin was washed with 20 mM potassium phosphate buffer (pH 8.0) containing 6% ammonium sulfate. Then, the GDH was eluted by passing the buffer through a gradient while changing the ammonium sulfate concentration from 7.6% to 0% and simultaneously the ethylene glycol concentration from 0% to 0.2%. Next, G-25 buffered with 50 mM potassium phosphate buffer (pH 7.0).
Desalting and buffer replacement were performed using Sepharose resin. Finally, the GDH solution was passed through DEAE-Sepharose buffered with 50 mM potassium phosphate buffer (pH 7.0) to adsorb the contaminating protein to the resin, and the permeate was used as purified GDH.
As a result of SDS-PAGE, each mutant GDH thus obtained was confirmed to have a high purity that forms a single band by CBB staining. The specific activity of each GDH in this state was 857 U / mg for R202S + W334G. D58V + R202S + Y328D was 889 U / mg, and R202S + W334R was 923 U / mg.

<Example 6>
Thermal stability of mutant GDH For each GDH obtained in Example 5 and wild-type GDH obtained in Test Example 4, a GDH concentration of 5 U / ml was obtained using 50 mM potassium phosphate buffer (pH 8.0). After dilution, each solution was heated at 50 ° C., 60 ° C., 70 ° C., 80 ° C., and 90 ° C. for 30 minutes, and the ratio of the GDH activity after heating to the GDH activity before heating (activity) (Residual rate) was examined. The results are shown in FIG. In the treatment at 70 ° C. for 30 minutes, the activity of the wild type and the three mutant GDHs were not reduced. The activity remaining rate after 30 minutes of treatment at 80 ° C. was about 90% for R202S + W334R, D58V + R202S + Y328D, and R202S + W334G, and the wild type was 99%. In addition, the activity remaining rate after treatment at 90 ° C. for 30 minutes was 75% for R202S + W334G, 34% for D58V + R202S + Y328D, 76% for R202S + W334R, and 91% for the wild type. All mutant GDHs maintain at least 85% activity at 80 ° C. for 30 minutes and at least 30% activity at 90 ° C. for 30 minutes, and maintain a high level of thermal stability after mutation introduction. It was confirmed.
The other mutant GDH in the present invention is presumed to have the same stability, and the maximum limit point of the temperature condition in which 90% or more of the activity remains after each mutant GDH is heated for 30 minutes, It is thought that it exists in the range of 70 degreeC or more and 90 degrees C or less.

<Example 7>
PH stability of mutant GDH For each GDH obtained in Example 5, the stable pH region of GDH was examined as follows. First, a buffer was prepared in the range of pH 3.5 to 11.0. The buffer species used were sodium acetate (pH 3.5-6.0), potassium phosphate (pH 6.0-8.0), Tris-HCl (pH 7.0-9.0), glycine NaOH (pH 9.0- 11.0) and all buffer concentrations are 50 mM. Each buffer was diluted to a GDH concentration of 10 U / ml, the pH of each diluted solution was measured, and each solution was incubated at 25 ° C. for 16 hours. The ratio of GDH activity after incubation to GDH activity before incubation (activity remaining rate) was examined. The pH stability of each mutant GDH activity of R202S + W334G, D58V + R202S + Y328D, and R202S + W334R is shown in FIG. 2, FIG. 3, and FIG. 4, respectively. The pH range where the activity remaining rate is 80% or more is 5.0 to 10.7 for R202S + W334R, 5.0 to 9.9 for D58V + R202S + Y328D, and 5.0 to 10.7 for R202S + W334G, all of which have a wide pH range. It was confirmed to be stable in the region.
In addition, it is estimated that other mutant GDHs have the same pH stability, and an activity remaining rate of 80% or more is exhibited even after incubation at 25 ° C. for 16 hours at least in the range of pH 6.0 to 9.0. It is considered a thing.

<Example 8>
PH Dependency of Mutant GDH Activity Value For each GDH obtained in Example 5, the pH dependency of GDH activity value was examined as follows. Among the compositions shown in the activity measurement examples, various buffers were used in place of 0.1 mol / L Tris, and various pH measurement solutions were prepared in the pH range of 3.5 to 11.0. The buffer species used were sodium acetate (pH 3.5-6.0), potassium phosphate (pH 6.0-8.0), Tris-HCl (pH 8.0-9.0), glycine NaOH (pH 9.0- 11.0), and the buffer concentration in the measurement solution is all 50 mM. Using each measurement solution, the GDH activity at each pH was measured according to the procedure of the activity measurement example described above. The relative activity value at each pH was calculated with the activity value under the condition showing the highest activity as 100. The pH dependence of each mutant GDH activity of R202S + W334G, D58V + R202S + Y328D, and R202S + W334R is shown in FIG. 5, FIG. 6, and FIG. 7, respectively. The optimum reaction pH was about 9.0 in all cases, but the relative activities at pH 7.0 were 87 for R202S + W334R, 66 for D58V + R202S + Y328D, and 74 for R202S + W334G, all showing sufficient activity in the neutral region. confirmed.

<Example 9>
Detailed substrate specificity of mutant GDH The substrate specificity of each GDH obtained in Example 5 was examined in more detail. The method follows the above-mentioned evaluation example of substrate specificity, but in addition to maltose, galactose, and xylose as substrates to be used, 2-deoxyglucose, sorbose, mannose, fructose, lactose, sorbitol, mannitol, saccharose, inositol, maltitol The reactivity (vs. glucose) was similarly measured for lactitol. The results are shown in Table 5. Each GDH was confirmed to maintain good substrate specificity.

<Example 10>
Temperature-dependent activity variation of mutant GDH The wild-type GDH obtained in Test Example 4 and the R202S + W334R mutant GDH obtained in Example 5 were reacted at 10 ° C., 20 ° C., 25 ° C., 30 ° C., and 37 ° C. The activity was measured, and the relative activity at each temperature when the activity at 37 ° C. was taken as 100 was calculated. The measurement was performed according to the above activity measurement example, and the activity was measured by setting the preheating and the temperature during the reaction to the temperature of each temperature condition. The results are shown in FIG. The mutant GDH was shown to have little temperature-dependent activity fluctuation in a temperature range of at least 10 ° C. and 37 ° C. Similarly, the mutant GDH having a higher temperature ratio of 25 ° C / 37 ° C compared to the wild type is presumed to have less temperature-dependent activity fluctuation than the wild type in the temperature range of at least 10 ° C and 37 ° C. Is done.

The glucose dehydrogenase produced according to the present invention can be supplied as a raw material for a reagent for measuring blood glucose level, a blood glucose sensor, and a glucose determination kit.

Claims

A mutant glucose dehydrogenase obtained by substituting the amino acid residue corresponding to the 202nd arginine in the amino acid sequence shown in SEQ ID NO: 2 with any of glycine, alanine, leucine, isoleucine, serine, threonine, asparagine, and lysine .
The mutant glucose dehydrogenase according to claim 1, wherein one or several amino acid residues other than the 202nd arginine are deleted, substituted, inserted and / or added.
1 selected from the group consisting of 58th aspartic acid, 286th valine, 328th tyrosine, 334th tryptophan, 339th isoleucine, 340th lysine, 341th threonine and 345th leucine in the amino acid sequence described in SEQ ID NO: 2 The mutant glucose dehydrogenase according to claim 1, further comprising a combination of substitution of several amino acid residues with other residues.
In the amino acid sequence described in SEQ ID NO: 2 (D58V + Y328D), selected from V286A, Y328T, Y328E, W334G, W334R, W334H, W334A, W334K, I339P, K340R, T341R, T341G, T341P, T341M, and T341Q The mutant glucose dehydrogenase according to claim 1, further comprising one or several amino acid substitutions.
The amino acid residue corresponding to the 202nd arginine in the amino acid sequence shown in SEQ ID NO: 2 is substituted with serine, and (D58V + Y328D), V286A, Y328T, Y328E, W334G, W334R, W334H, W334A, W334K, I339P, K340R, The mutant glucose dehydrogenase according to claim 1, further comprising one or several amino acid substitutions selected from the group consisting of T341R, T341G, T341P, T341M, T341S and L345Q.
A mutant glucose dehydrogenase having high affinity for glucose, wherein the amino acid residue corresponding to the 202nd arginine in the amino acid sequence described in SEQ ID NO: 2 is substituted with serine.
The mutant glucose dehydrogenase according to claim 6, wherein one or several amino acid residues other than the 202nd arginine are deleted, substituted, inserted and / or added.
In the amino acid sequence shown in SEQ ID NO: 2, the 3rd alanine, 46th isoleucine, 49th lysine, 58th aspartic acid, 74th glycine, 80th valine, 85th leucine, 89th threonine, 121st alanine, 124 Serine, 168th lysine, 173th alanine, 206th serine, 208th lysine, 294th leucine, 298th glutamic acid, 303th histidine, 306th glutamic acid, 311th leucine, 323rd alanine, 328th tyrosine, 330th glutamic acid 1 to several amino acids selected from the group consisting of 334th tryptophan, 335th threonine, 338th aspartic acid, 341th threonine, 343th leucine and 345th leucine Comprising further combined substitution of another amino acid residue of residue, mutant glucose dehydrogenase according to claim 6.
In the amino acid sequence shown in SEQ ID NO: 2, A3V, I46V, K49R, D58V, G74D, V80A, L85H, T89K, A121T, S124L, S124P, K168E, A173T, S206N, K208R, L294W, E298G, H303R, E306G, L311P, A323T, Y328D, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, E330G, W334G, W334R, W334H, W334A, W334K, W334S, T335N, D338G, D338V, T341R, P345L, P345T, L345P The mutant glucose protein according to claim 6, further comprising one or more amino acid substitutions selected from Rogenaze.
In the amino acid sequence shown in SEQ ID NO: 2, the amino acid residue corresponding to the 202nd arginine is substituted with serine, and A3V, (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), V80A, L85H, T89K, A121T, S124L, (K168E + E330G), A173T, S206N, K208R, L294W, E298G, H303R, L311P, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334R, W334T, W334T, W334T, W334T, W334T, W334T (I339P + T341G), (I339P + T341R), (I339P + T341L), (I339P + T 41K), T341R, T341G, T341M, L343P, comprising further combining one of the amino acid substitutions selected from the group consisting of L345Q and L345P, mutant glucose dehydrogenase according to claim 6.
A mutant glucose dehydrogenase having an improved specific activity at 37 ° C. or lower, wherein the amino acid residue corresponding to the 202nd arginine in the amino acid sequence shown in SEQ ID NO: 2 is substituted with serine.
The mutant glucose dehydrogenase according to claim 11, wherein one or several amino acid residues other than the 202nd arginine are deleted, substituted, inserted and / or added.
In the amino acid sequence shown in SEQ ID NO: 2, the 3rd alanine, 46th isoleucine, 49th lysine, 58th aspartic acid, 74th glycine, 80th valine, 85th leucine, 89th threonine, 121st alanine, 124 Serine, 168th lysine, 173th alanine, 206th serine, 208th lysine, 294th leucine, 298th glutamic acid, 303th histidine, 306th glutamic acid, 311th leucine, 323rd alanine, 328th tyrosine, 330th glutamic acid 1 to several amino acids selected from the group consisting of 334th tryptophan, 335th threonine, 338th aspartic acid, 341th threonine, 343th leucine and 345th leucine Comprising further combined substitution of another amino acid residue of residue, mutant glucose dehydrogenase according to claim 11.
In the amino acid sequence shown in SEQ ID NO: 2, A3V, I46V, K49R, D58V, G74D, V80A, L85H, T89K, A121T, S124L, S124P, K168E, A173T, S206N, K208R, L294W, E298G, H303R, E306G, L311P, A323T, Y328D, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, E330G, W334G, W334R, W334H, W334A, W334K, W334S, T335N, D338G, D338V, T341R, P345L, P345T, L345P The mutant glucose deoxypeptide according to claim 11, further comprising one or several amino acid substitutions selected from Dehydrogenase.
In the amino acid sequence shown in SEQ ID NO: 2, the amino acid residue corresponding to the 202nd arginine is substituted with serine, and A3V, (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), V80A, L85H, T89K, A121T, S124L, (K168E + E330G), A173T, S206N, K208R, L294W, E298G, H303R, L311P, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334R, W334T, W334T, W334T, W334T, W334T, W334T (I339P + T341G), (I339P + T341R), (I339P + T341L), (I339P + T 41K), T341R, T341G, T341M, L343P, comprising further combining one of the amino acid substitutions selected from the group consisting of L345Q and L345P, mutant glucose dehydrogenase according to claim 11.
In the amino acid sequence shown in SEQ ID NO: 2, the 202nd arginine is substituted with serine, and the 46th isoleucine, 49th lysine, 58th aspartic acid, 74th glycine, 89th threonine, 168th lysine, 124th serine, 206 Serine, 208th lysine, 294th leucine, 303th histidine, 306th glutamic acid, 311th leucine, 323rd alanine, 328th tyrosine, 330th glutamic acid, 334th tryptophan, 335th threonine, 338th aspartic acid and 345th A mutant glucose dehydrogenase obtained by substituting one or several amino acids selected from the group consisting of leucine with other amino acids.
Arginine at position 202 is substituted with serine in the amino acid sequence shown in SEQ ID NO: 2, and (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), T89K, S124L, (K168E + E330G), S206N, K208R, L294W, H303R, L311P, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334R, W334H, W334A, W334K, (W334S + T335N), a combination of amino acids selected from the group consisting of a combination of D338V, D338G, L345Q and L345P. The mutant glucose dehydrogenase according to claim 16.
Arginine at position 202 is substituted with serine in the amino acid sequence shown in SEQ ID NO: 2, and (I46V + S124P), K49R, (D58V + Y328D), (G74D + E306G + A323T), T89K, S124L, (K168E + E330G), S206N, K208R, L294W, H303R, L311P, Y328S, Y328T, Y328V, Y328G, Y328E, Y328L, W334G, W334R, W334H, W334A, W334K, (W334S + T335N), a combination selected from the group consisting of D338V, D338G, L345Q and L345P. The mutant glucose dehydrogenase according to claim 16.
A mutant glucose dehydrogenase in which the 202nd arginine is substituted with serine and the 334th tryptophan is substituted with glycine in the amino acid sequence shown in SEQ ID NO: 2.
A mutant glucose dehydrogenase in which the 202nd arginine is substituted with serine and the 334th tryptophan is substituted with arginine in the amino acid sequence shown in SEQ ID NO: 2.
A mutant glucose dehydrogenase in which the 58th aspartic acid is substituted with valine, the 202nd arginine is substituted with serine, and the 328th tyrosine is substituted with aspartic acid in the amino acid sequence shown in SEQ ID NO: 2.
A gene encoding the mutant glucose dehydrogenase according to any one of claims 1 to 21.
A vector comprising the gene according to claim 22.
A transformant transformed with the vector of claim 23.
A method for producing a mutant glucose dehydrogenase, comprising culturing the transformant according to claim 24.
A glucose assay kit comprising the mutant glucose dehydrogenase according to any one of claims 1 to 21.
A glucose sensor comprising the mutant glucose dehydrogenase according to any one of claims 1 to 21.
A glucose measuring method comprising the mutant glucose dehydrogenase according to any one of claims 1 to 21.