Abstract:
A mutant protein having diaphorase activity is provided. A mutant protein includes an amino acid sequence obtained by deletion, replacement, addition, or insertion of at least one amino acid residue of a native-form amino acid sequence of SEQ. ID. No. 1, wherein the mutant protein has diaphorase activity with an enzyme activity of 245 or more.

Description:
CROSS REFERENCES TO RELATED APPLICATIONS  
       [0001]     The present application claims priority to Japanese Patent Application JP 2005-343605 filed in the Japanese Patent Office on Nov. 29, 2005, and Japanese Patent Application JP 2006-231228 filed in the Japanese Patent Office on Aug. 28, 2006, the entire contents of which are incorporated herein by reference.  
       BACKGROUND  
       [0002]     The present application relates to a mutant protein having diaphorase activity. More specifically, the present application relates to a mutant protein having diaphorase activity and having predetermined levels or more of enzyme activity and heat resistance.  
         [0003]     Enzymes are biocatalysts for allowing many reactions for the maintenance of life to smoothly proceed under mild conditions in vivo. Enzymes turn over in vivo, are produced in vivo according to need, and express their catalytic functions.  
         [0004]     Techniques for making use of enzymes in vitro have already been used practically or studied to achieve practical use. For example, technology for using enzymes has been developed in various technical fields, such as the production of useful materials, the production of energy-related materials, measurement or analysis, environmental conservation, and medical care. In relatively recent years, technologies, such as an enzyme cell (for example, see Japanese Unexamined Patent Application Publication No. 2004-71559), which is a type of fuel cell, an enzyme electrode, and an enzyme sensor (sensor for measurement of a chemical substance using an enzymatic reaction), have been developed.  
         [0005]     In general, enzymes are denatured by degrees of heat and pH. Hence, enzymes have low stability in vitro compared with other chemical catalysts such as metal catalysts. Accordingly, when enzymes are used in vitro, it is important to allow the enzymes to more stably work in response to environmental changes and to allow the activity of the enzymes to be maintained.  
         [0006]     When an enzyme is used in vitro, a method for artificially modifying the nature and function of the enzyme and a method for producing the environment of the site in which the enzyme functions are employed. With respect to the former method, it is common that the base sequence of a gene encoding a protein is artificially modified, and the modified gene is expressed in an organism such as  Escherichia coli  to form an artificially mutated protein, and then the mutant protein having the target function and nature is separated by screening (for example, see Japanese Unexamined Patent Application Publication No. 2004-298185).  
       SUMMARY  
       [0007]     In consideration of the wide availability of diaphorase in vitro, it is desirable to provide a mutant protein having predetermined levels of diaphorase activity and heat resistance.  
         [0008]     According to an embodiment, there is provided a mutant protein including an amino acid sequence obtained by deletion, replacement, addition, or insertion of at least one amino acid residue of the native-form amino acid sequence of SEQ. ID. No. 1 (211 amino acid residues), wherein the mutant protein has diaphorase activity with an enzyme activity of 245 or more. Furthermore, there is provided a mutant protein having diaphorase activity in which enzyme activity is 245 or more, and residual activity is 27% or more and more preferably 41% or more after heating.  
         [0009]     Furthermore, there is provided a mutant protein including an amino acid sequence obtained by deletion, replacement, addition, or insertion of at least one amino acid residue of a native-form amino acid sequence of SEQ. ID. No. 1, wherein the mutant protein has diaphorase activity with an enzyme activity of 170 or more and with residual activity of 41% or more after heating.  
         [0010]     These mutant proteins are variants of proteins which have diaphorase activity and which are derived from, for example, thermophilic  Bacillus  bacteria, in particular,  Bacillus stearothermophilus.  More specifically, examples of the mutant proteins include mutant proteins having amino acid sequences of SEQ. ID. Nos. 2 to 56.  
         [0011]     In the amino acid sequence of SEQ. ID. No. 2 (hereinafter, referred to as “K139N/A187E”), lysine at the 139th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with asparagine, and alanine at the 187th position from the N-terminus is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 3 (hereinafter, referred to as “F105L”), phenylalanine at the 105th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with leucine. In the amino acid sequence of SEQ. ID. No. 4 (hereinafter, referred to as “G122D”), glycine at the 122th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with asparatic acid. In the amino acid sequence of SEQ. ID. No. 5 (hereinafter, referred to as “G131E”), glycine at the 131th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 6 (hereinafter, referred to as “A146G”), alanine at the 146th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glycine. In the amino acid sequence of SEQ. ID. No. 7 (hereinafter, referred to as “R147H”), arginine at the 147th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with histidine.  
         [0012]     In the amino acid sequence of SEQ. ID. No. 8 (hereinafter, referred to as “H34Q”), histidine at the 34th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glutamine. In the amino acid sequence of SEQ. ID. No. 9 (hereinafter, referred to as “F105H”), phenylalanine at the 105th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with histidine. In the amino acid sequence of SEQ. ID. No. 10 (hereinafter, referred to as “A113E”), alanine at the 113th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glutamic acid.  
         [0013]     In the amino acid sequence of SEQ. ID. No. 11 (hereinafter, referred to as “K123E”), lysine at the 123th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 12 (hereinafter, referred to as “K139N”), lysine at the 139th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with asparagine. In the amino acid sequence of SEQ. ID. No. 13 (hereinafter, referred to as “R147S”), arginine at the 147th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with serine. In the amino acid sequence of SEQ. ID. No. 14 (hereinafter, referred to as “G149D”), lysine at the 149th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid. In the amino acid sequence of SEQ. ID. No. 15 (hereinafter, referred to as “G154D”), glycine at the 154th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid. In the amino acid sequence of SEQ. ID. No. 16 (hereinafter, referred to as “A156E”), alanine at the 156th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 17 (hereinafter, referred to as “M159T”), methionine at the 159th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with threonine. In the amino acid sequence of SEQ. ID. No. 18 (hereinafter, referred to as “A187E”), alanine at the 187th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 19 (hereinafter, referred to as “A187T”), alanine at the 187th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with threonine. In the amino acid sequence of SEQ. ID. No. 20 (hereinafter, referred to as “A187V”), alanine at the 187th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with valine.  
         [0014]     In the amino acid sequence of SEQ. ID. No. 21 (hereinafter, referred to as “R64H/A146T”), arginine at the 64th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with histidine, and alanine at the 146th position is replaced with threonine. In the amino acid sequence of SEQ. ID. No. 22 (hereinafter, referred to as “E85D/R147H”), glutamic acid at the 85th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, and arginine at the 147th position is replaced with histidine. In the amino acid sequence of SEQ. ID. No. 23 (hereinafter, referred to as “F105L/A187E”), phenylalanine at the 105th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with leucine, and alanine at the 187th position is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 24 (hereinafter, referred to as “A113E/K126N”), alanine at the 113th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glutamic acid, and lysine at the 126th position is replaced with asparagine. In the amino acid sequence of SEQ. ID. No. 25 (hereinafter, referred to as “Y151H/A187E”), tyrosine at the 151th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with histidine, and alanine at the 187th position is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 26 (hereinafter, referred to as “G122D/A187E”), glycine at the 122th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, and alanine at the 187th position is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 27 (hereinafter, referred to as “G149D/A187E”), glycine at the 149th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, and alanine at the 187th position is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 28 (hereinafter, referred to as “G149S/A187E/L207W”), glycine at the 149th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with serine, alanine at the 187th position is replaced with glutamic acid, and leucine at the 207th position is replaced with tryptophan. In the amino acid sequence of SEQ. ID. No. 29 (hereinafter, referred to as “F105L/A187E/L207W”), phenylalanine at the 105th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with leucine, alanine at the 187th position is replaced with glutamic acid, and leucine at the 207th position is replaced with tryptophan. In the amino acid sequence of SEQ. ID. No. 30 (hereinafter, referred to as “G66R/F105L/A187E/K192R”), glycine at the 66th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with arginine, phenylalanine at the 105th position is replaced with leucine, alanine at the 187th position is replaced with glutamic acid, and lysine at the 192th position is replaced with arginine.  
         [0015]     In the amino acid sequence of SEQ. ID. No. 31 (hereinafter, referred to as “A146G/L207W”), alanine at the 146th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glycine, and leucine at the 207th position is replaced with tryptophan. In the amino acid sequence of SEQ. ID. No. 32 (hereinafter, referred to as “F105L/A187E/Q171P”), phenylalanine at the 105th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with leucine, alanine at the 187th position is replaced with glutamic acid, and glutamine at the 171th position is replaced with proline. In the amino acid sequence of SEQ. ID. No. 33 (hereinafter, referred to as “A78E/F105L/A187E”), alanine at the 78th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glutamic acid, phenylalanine at the 105th position is replaced with leucine, and alanine at the 187th position is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 34 (hereinafter, referred to as “F105L/K149N/V168L/A187E”), phenylalanine at the 105th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with leucine, lysine at the 149th position is replaced with asparagine, valine at the 168th position is replaced with leucine, and alanine at the 187th position is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 35 (hereinafter, referred to as “G154D/G180R”), glycine at the 154th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, and glycine at the 180th position is replaced with arginine. In the amino acid sequence of SEQ. ID. No. 36 (hereinafter, referred to as “F107I”), phenylalanine at the 107th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with isoleucine. In the amino acid sequence of SEQ. ID. No. 37 (hereinafter, referred to as “G185R”), glycine at the 185th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with arginine. In the amino acid sequence of SEQ. ID. No. 38 (hereinafter, referred to as “Y151H/G185R”), tyrosine at the 151th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with histidine, and glycine at the 185th position is replaced with arginine. In the amino acid sequence of SEQ. ID. No. 39 (hereinafter, referred to as “Y151H/G185R”), glycine at the 122th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, and glycine at the 185th position is replaced with arginine. In the amino acid sequence of SEQ. ID. No. 40 (hereinafter, referred to as “G149D/G185R”), glycine at the 149th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, and glycine at the 185th position is replaced with arginine.  
         [0016]     In the amino acid sequence of SEQ. ID. No. 41 (hereinafter, referred to as “G149D/G185R/A208V”), glycine at the 149th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, glycine at the 185th position is replaced with arginine, and alanine at the 208th position is replaced with valine. In the amino acid sequence of SEQ. ID. No. 42 (hereinafter, referred to as “F107I/G185R”), phenylalanine at the 107th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with isoleucine, and glycine at the 185th position is replaced with arginine. In the amino acid sequence of SEQ. ID. No. 43 (hereinafter, referred to as “F107I/G185R/A208V”), phenylalanine at the 107th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with isoleucine, glycine at the 185th position is replaced with arginine, and alanine at the 208th position is replaced with valine. In the amino acid sequence of SEQ. ID. No. 44 (hereinafter, referred to as “F107I/G185R/Q171P”), phenylalanine at the 107th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with isoleucine, glycine at the 185th position is replaced with arginine, and glutamine at the 171th position is replaced with proline. In the amino acid sequence of SEQ. ID. No. 45 (hereinafter, referred to as “V80D/F107I/G185R”), valine at the 80th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, phenylalanine at the 107th position is replaced with isoleucine, and glycine at the 185th position is replaced with arginine. In the amino acid sequence of SEQ. ID. No. 46 (hereinafter, referred to as “F107I/K139N/V168L/G185R”), phenylalanine at the 107th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with isoleucine, lysine at the 139th position is replaced with asparagine, valine at the 168th position is replaced with leucine, and glycine at the 185th position is replaced with arginine. In the amino acid sequence of SEQ. ID. No. 47 (hereinafter, referred to as “F150V”), phenylalanine at the 150th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with valine. In the amino acid sequence of SEQ. ID. No. 48 (hereinafter, referred to as “A193E”), alanine at the 193th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 49 (hereinafter, referred to as “F150V/A193E”), phenylalanine at the 150th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with valine, and alanine at the 193th position is replaced with arginine. In the amino acid sequence of SEQ. ID. No. 50 (hereinafter, referred to as “Y151H/A193E”), tyrosine at the 151th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with histidine, and alanine at the 193th position is replaced with glutamic acid.  
         [0017]     In the amino acid sequence of SEQ. ID. No. 51 (hereinafter, referred to as “G122D/A193E”), glycine at the 122th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, and alanine at the 193th position is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 52 (hereinafter, referred to as “G149D/A193E/A208V”), glycine at the 149th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, alanine at the 193th position is replaced with glutamic acid, and alanine at the 208th position is replaced with valine. In the amino acid sequence of SEQ. ID. No. 53 (hereinafter, referred to as “F150V/A193E/A208V”), phenylalanine at the 150th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with valine, alanine at the 193th position is replaced with glutamic acid, and alanine at the 208th position is replaced with valine. In the amino acid sequence of SEQ. ID. No. 54 (hereinafter, referred to as “F150V/A193E/Q171P”), phenylalanine at the 150th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with valine, alanine at the 193th position is replaced with glutamic acid, and glutamine at the 171th position is replaced with proline. In the amino acid sequence of SEQ. ID. No. 55 (hereinafter, referred to as “V80D/F150V/A193E”), valine at the 80th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with aspartic acid, phenylalanine at the 150th position is replaced with valine, and alanine at the 193th position is replaced with glutamic acid. In the amino acid sequence of SEQ. ID. No. 56 (hereinafter, referred to as “K139N/F150V/V168L/A193E”), lysine at the 139th position from the N-terminus of the native-form amino acid sequence of SEQ. ID. No. 1 is replaced with asparagine, phenylalanine at the 150th position is replaced with valine, valine at the 168th position is replaced with leucine, and alanine at the 193th position is replaced with glutamic acid.  
         [0018]     The mutant proteins include a protein having an amino acid sequence obtained by deletion, replacement, addition, or insertion of at least one amino acid residue of any one of the amino acid sequences of SEQ. ID. Nos. 2 to 56 as well as the variant proteins having the amino acid sequences of SEQ. ID. Nos. 2 to 56.  
         [0019]     Key technical terms related to the present invention will be described.  
         [0020]     The term “diaphorase” means an enzyme having activity in which the enzyme catalyzes the oxidation of NADH or NADPH with dye, such as potassium ferricyanide, methylene blue, 2,6-dichloroindophenol, or a tetrazolium salt, i.e., the term “diaphorase” means an enzyme having diaphorase activity. The diaphorase is widely distributed in the range from microorganisms, such as bacteria and yeast, to mammals. The diaphorase plays an important part in an electron transport system in vivo. NADH or NADPH formed by dehydrogenation of a substrate caused by an NAD+- or NADP+-dependent dehydrogenase is oxidized by an electron acceptor in the presence of the diaphorase, resulting in a reduced form of the electron acceptor.  
         [0021]     The term “mutant protein” means a protein expressed from a gene obtained by artificially modifying the base sequence in a DNA encoding an amino acid sequence constituting a protein.  
         [0022]     The term “enzyme activity” generally means the catalytic rate of a reaction under a predetermined condition. In the present invention, the term “enzyme activity” means the catalytic rate of a reaction in which reduced nicotinamide dinucleotide (NADH) reduces 2-amino-1,4-naphthoquinone (ANQ) to yield oxidized nicotinamide dinucleotide (NAD+) and 2-amino-1,4-dihydroxynaphthalene. Specifically, the term “enzyme activity” is defined as the number of moles of a product resulting from a reaction catalyzed by one mole of an enzyme per unit time in a 0.1 M potassium phosphate buffer at 25° C. under an argon atmosphere or a nitrogen atmosphere in the presence of 0.3 mM ANQ and 40 mM NADH. Accordingly, the unit is sec −1 . Note that an enzyme activity of 245 or more corresponds to about 1.5 times or more that of a native protein having diaphorase activity and derived from  Bacillus stearothermophilus.    
         [0023]     The term “residual activity after heating” may also be referred to as “residual enzyme activity” or “retention of enzyme activity”. The term “residual activity after heating” means a value representing a change in activity before and after an enzyme is subjected to predetermined heating. That is, enzyme activity is measured under the same condition before and after heating. The term “residual activity” means the percentage of activity after heating to activity before heating. In the present invention, the term “heating” means stationary treatment in a buffer solution at 80° C. for 10 minutes. The ratio of the enzyme activity after the heating to the enzyme activity before the heating is represented by percentage. Note that a residual activity of 41% or more corresponds to about 1.5 times or more that of a native protein having diaphorase activity and derived from  Bacillus stearothermophilus.    
         [0024]     A mutant protein according to an embodiment of the present invention has diaphorase activity and a predetermined level or more of enzyme activity and/or heat resistance.  
         [0025]     Additional features and advantages are described herein, and will be apparent from, the following Detailed Description and the figures. 
     
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0026]      FIG. 1  is a diagrammatic illustration showing the flow of an experiment according to Example 2 (library preparation by random mutation and screening).  
         [0027]      FIG. 2  is part of a photograph, as an alternative to a drawing, of a well plate when a total of about 8,000 colonies are screened.  
         [0028]      FIG. 3  is part of a photograph of a well plate resulting from a double check experiment.  
         [0029]      FIG. 4  shows tables, as an alternative to drawings, summarizing factors that reflect ease of binding of substrates to active sites of wild-type diaphorase and G122D mutant diaphorase.  
         [0030]      FIG. 5  shows a conformation observed by simulation of wild-type diaphorase.  
         [0031]      FIG. 6A  is a front structural view illustrating the vicinity of FAD in the conformation obtained by simulation of R147H mutant diaphorase.  
         [0032]      FIG. 6B  is a top structural view illustrating the vicinity of FAD in the conformation obtained by simulation of R147H mutant diaphorase.  
         [0033]      FIG. 7A  is a front structural view illustrating the vicinity of FAD in the conformation obtained by simulation of G122D mutant diaphorase.  
         [0034]      FIG. 7B  is a top structural view illustrating the vicinity of FAD in the conformation obtained by simulation of G122D mutant diaphorase.  
         [0035]      FIG. 8  shows the electrostatic potential surface and the conformation obtained by simulation of wild-type diaphorase.  
         [0036]      FIG. 9  shows the electrostatic potential surface and the conformation obtained by simulation of R147H mutant diaphorase.  
         [0037]      FIG. 10  shows the electrostatic potential surface and the conformation obtained by simulation of G122D mutant diaphorase.  
         [0038]      FIG. 11  shows conformational diagrams illustrating hydrogen bonds obtained by simulation of wild-type diaphorase and R147H mutant diaphorase.  
         [0039]      FIG. 12  shows conformational diagrams illustrating hydrogen bonds obtained by simulation of wild-type diaphorase and G122D mutant diaphorase. 
     
    
     DETAILED DESCRIPTION  
       [0040]     A description in further detail is provided below according to an embodiment.  
       EXAMPLE 1  
       [0041]     Cloning, Expression, and Purification of Diaphorase Derived From  Bacillus Stearothermophilus    
         [0042]     (1-1) Isolation and Purification of Genomic DNA from  Bacillus Stearothermophilus    
         [0043]      Bacillus stearothermophilus  was purchased from Japan Collection of Microorganisms (JCM), Riken, (JCM No. 2501, NCBI accession number of a diaphorase gene: AF112858). A lyophilizate of  Bacillus stearothermophilus  was cultured on agar medium A overnight at 55° C.  
         [0044]     The resulting colony was similarly cultured on fresh agar medium A to form a pure colony. The colony was partially picked up, cultured in liquid medium A overnight at 55° C., and centrifuged to collect the bacterium. Genomic DNA was isolated with a Wizard Genomic DNA Purification Kit (Promega Corporation) (Details on a process were given in an instruction manual included with a product). The composition of medium A was described in Table 1 (in 1 L, pH 7.0 to 7.2).  
                       TABLE 1                           Meat Extract (Merck)   10   g       Bacto Peptone (DIFCO)   10   g       NaCl (Wako)   5   g       Agar, guranulated (DIFCO)   20   g (when an agar medium was made)                  
 
         [0045]     (1-2) Cloning of Diaphorase Gene  
         [0046]     A diaphorase gene was cloned by PCR from the genomic DNA obtained in item (1-1). Pfu DNA polymerase (Stratagene) was used as a DNA polymerase. A primer having sequences showing in Table 2 was used.  
                           TABLE 2                           sense_DI   ggaattccat atgatgacaa   SEQ. ID. No. 57               acgtattgtac at               Antisense_DI   cgggatcctt aaaacgtgtg   SEQ. ID. No. 58           cgccaagt                  
 
         [0047]     The resulting diaphorase gene, which was a PCR product, was purified with PCR Cleanup Kit (Qiagen) and verified by agarose gel electrophoresis. Furthermore, a base sequence was verified with a DNA sequencer.  
         [0048]     (1-3) Introduction of Diaphorase Gene into Vector  
         [0049]     Cloned fragments of the diaphorase gene were treated with BamH I and Nde I and purified with PCR Cleanup Kit (Qiagen). Vector pET12a (Novagen) was treated with BamH I and Nde I and purified similarly. These two types of fragments were ligated with T4 ligase. XL1-blue electrocompetent cells (Stratagene) were transformed with the resulting products and cultured in LB-amp medium to increase production.  
         [0050]     The resulting plasmid was treated with Bss I. Insertion of the diaphorase gene was verified by agarose gel electrophoresis. The base sequence was measured and analyzed. The results showed a slight difference between the resulting base sequence and the base sequence stored in a database (NCBI). This was probably because the strain purchased from Japan Collection of Microorganisms (JCM), Riken, was slightly different from a strain described in the database, so that the base sequence of the cloned DI was inconsistent with the base sequence of the DI stored in the database. In the genotype (base sequence), there were inconsistencies at 11 points. Among these, in the phenotype (amino acid sequence), there were inconsistencies at two residues (see Table 3).  
                       TABLE 3                       Amino acid residue No.   Resulting gene   Database                   28   Glutamic acid   Aspartic acid       61   Aspartic acid   Glycine                  
 
         [0051]     A gene was formed with Quick Change Site-Directed Mutagenesis Kit (Stratagene) so that the two amino acid residues were modified to be identical to those described in the database. This gene was named as “pET12a-BsDI”.  
         [0052]     (1-4) Transformation of  E. Coli    
         [0053]     The pET12a-BsDI was inserted and transformed into  E. coli  BL21 (DE3) (Novagen) by heat shock. After preculture in SOC for 1 hr at 37° C., the resulting transformant was developed on LB-amp agar medium. Part of the colonies was cultured in a liquid medium. The expression of diaphorase was verified by SDS-PAGE.  
         [0054]     (1-5) Cryopreservation Sample of Transformant  
         [0055]     First, 3 mL of a medium containing the transformant was centrifuged.  E. coli  pellets were suspended in 2XYT medium, and the resulting mixture was mixed and then stored at −80° C.  
         [0056]     (1-6) Mass Culture and Purification of Protein  
         [0057]     The frozen sample of BL21 (DE3)/pET12a-BsDI was developed on LB-amp agar medium. The resulting colony was picked up and precultured in 100 mL of LB-amp medium until OD600 reached about 1. The resulting preculture was developed in 18 L of LB-amp medium and cultured with shaking at 37° C. until OD600 was saturated about 2. The culture was centrifuged at 5 kG to harvest a bacterium (yield: 20 g, wet weight). The bacterial pellets were frozen at −80° C. and then melted. The bacterial pellets were treated by sonication at 0° C. in 200 mL of a solution containing 50 mM Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM DTT, and 1 mM PMSF to cause bacteriolysis. The resulting lysate was centrifuged at 9.5 kG to recover a solution fraction.  
         [0058]     Purification was performed by an ammonium sulfate precipitation method. Powdery ammonium sulfate was gradually added under stirring to form a 35% saturated solution. The solution was left standing overnight. Precipitates were removed by centrifugation at 9.5 kG. The solution was desalted with a dialysis membrane tube (final solution: 5 mM Tris-HCl, pH 7.8). Next, 50 mL of a sample concentrated by ultrafiltration was passed through an anion-exchange column (Sepharose Q FastFlow, Amersham Bioscience) to harvest a diaphorase-containing fraction. The resulting fraction was concentrated by ultrafiltration (the amount of the solution: 20 mL, Centriplus Centrifugal Filter Unit YM-30, Millipore). The resulting sample was passed through a gel filtration column (Sephacryl S-200, Amersham Bioscience) to collect a diaphorase-containing fraction.  
       EXAMPLE 2  
       [0059]     Preparation of Mutant Library by Random Mutation of Diaphorase Derived from  Bacillus Stearothermophilus  and Screening of Thermostable Mutant  
         [0060]      FIG. 1  shows the flow of an experiment conducted in EXAMPLE 2. A gene library of a diaphorase mutant was constructed by error-prone PCR. The gene was introduced into vector DNA and expressed in  E. coli.  The resulting library was subjected to thermostability screening to extract a target thermostable diaphorase mutant.  
         [0061]     (2-1) Error-Prone PCR with GeneMorph (Registered Trademark)  
         [0062]     This is a method for randomly mutating cloned DNA fragments using misreading by a polymerase of a base sequence. A variety of methods have been reported. GeneMorph (registered trademark, Stratagene) was selected here among commercially available methods. The pET12a-BsDI integrated with the diaphorase gene of  Bacillus stearothermophilus  was used as a template DNA. A primer that was used for cloning this gene was also used.  
         [0063]     Table 4 shows sequences of the primer. The primer has the sequence of Nde I at the 5′ terminus of a coding strand and the sequence of BamH I at the 5′ terminus of a complementary strand (underlined portion). Thus, error-prone PCR products can be inserted into a multicloning site of pET12a by treatment with these restriction enzymes (similar to the cloning of the native diaphorase).  
                           TABLE 4                           sense_DI   ggaattccatatgatgacaaa   SEQ. ID. No. 59               cgtattgtacat               Antisense_DI   cgggatccttaaaagtgtgcg   SEQ. ID. No. 60           ccaagt                  
 
         [0064]     PCR was performed according to the manual of GeneMorph (registered trademark). Table 5 shows the composition of a reaction mixture. Table 6 shows a temperature profile.  
                             TABLE 5                       &lt;Composition of reaction mixture&gt;                                41.5   μL   water       5.0   μL   10× Mutazyme reaction buffer       1.0   μL   40 mM dNTP mix (200 μM each final)       0.5   μL   primer mix (250 ng/μL of each primer)       1.0   μL   Mutazyme DNA polymerase (2.5 U/μL)       1.0   μL   template (10 pg/μL - 10 ng/μL)       50.0   μL   in total                  
 
         [0065]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                   
               
               
                 &lt;Temperature profile&gt; 
               
             
          
           
               
                   
                   
                 Number of 
                   
                   
               
               
                   
                 Segment 
                 cycle 
                 Temperature 
                 duration 
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 1 
                 96° C. 
                 30 
                 sec 
               
               
                   
                 2 
                 30 
                 96° C. 
                 30 
                 sec 
               
               
                   
                   
                   
                 53° C. * 1   
                 30 
                 sec 
               
               
                   
                   
                   
                 72° C. 
                 1 
                 min * 2   
               
               
                   
                 3 
                 1 
                 72° C. 
                 10 
                 min 
               
               
                   
                   
               
               
                   
                   * 1  Primer Tm −5° C.    
               
               
                   
                   * 2  1 min for ≦1 kb target    
               
             
          
         
       
     
         [0066]     (2-2) Introduction of Diaphorase Gene into Vector  
         [0067]     The total amount of the error-prone PCR products other than the amount of the products used for agarose gel electrophoresis was used for the treatment with the restriction enzymes, Nde I and BamH I. After the reaction was performed at 37° C. for 2 hours, the resulting reaction product was purified with Qiaquick PCR purification Kit (Qiagen). The vector pET12a was treated with the restriction enzymes, Nde I and BamH I, in the same way as the PCR products (at 37° C. for 2 hours).  
         [0068]     The reaction products resulting from the treatment with the restriction enzymes were separated by low-melting-point agarose gel electrophoresis. The target open-circular vector DNA was purified with Qiaquick Gel Extraction Kit (Qiagen). The products purified by treatment of the vector with the restriction enzymes were treated with an alkaline phosphatase to dephosphorylate the 5′ terminus. The reaction products were purified with Qiaquick PCR purification Kit (Qiagen). The resulting error-prone PCR products, i.e., diaphorase mutant gene library, were ligated into the vector that was treated with the restriction enzymes and dephosphorylated. A ligation reaction was performed with Ligation Kit Mighty Mix (Takara Bio Inc). The reaction product was purified by ethanol precipitation.  
         [0069]     (2-3) Preparation of Competent Cell and Transformation  
         [0070]     Electrocompetent cells (competency: about 10 8 /ng) of in-house prepared BL21 (DE3) were used as competent cells. Next, 40 μL of the competent cell frozen sample was melted on ice, and 0.5 μL of the DNA sample having a concentration of about 1 ng/μL was mixed thereto. The total mixture was charged into an electroporation cuvette with a gap of 0.1 cm. Transformation was performed by applying 1,800 kV. Then, 960 μL of an SOC medium was added thereto. The resulting mixture was precultured by shaking for 1 hour at 37° C. The resulting culture was inoculated on 5 to 50 μL of LB-amp agar medium and incubated at 37° C. overnight.  
         [0071]     (2-4) Screening Method  
         [0072]     Each colony on the agar medium obtained in item (2-3) was inoculated using a toothpick into LB-amp liquid medium (150 μL) on 96-well plate. Two wells were occupied by a strain of  E. coli  that produces a wild type. The top of the well plate was sealed with a gas-permeable adhesive sheet (ABgene) and covered with an accompanying lid. The cultures were cultured with shaking at 37° C. overnight (about 14 hours). Next, 25 μL each of the resulting cultures was placed into 25 μL of a 0.2 N NaOH aqueous solution that has been aliquoted in another well plate. After the mixture was well mixed by pipetting, the plate was covered with a lid and incubated at 37° C. for 15 minutes with an incubator to cause bacteriolysis.  
         [0073]     Next, 100 μL of 0.1 M K-pi (pH 6.8) was added thereto at room temperature to neutralize the mixture. One of the two wild-type samples was separated, charged into a microtube, and stored at room temperature, the sample being used as an unheated control sample. The plate was sealed with a commercially available OPP tape, heated at 80° C. for 75 minutes with an incubator, and left standing to cool to room temperature. The separated wild-type sample was returned to the plate. Then, 10 μL of a 20 mM anthraquinone sulfonic acid (AQS) in a 20% DMSO solution and 50 μL of a 80 mM NADH aqueous solution prepared just before using it were added to each sample. The plate was sealed with the OPP tape and stirred for 5 seconds with a vortex mixer. Revelation was recorded with a camera. Samples having strong coloration due to reduction of AQS compared with the coloring of the wild-type sample were selected as candidates for thermostable mutants.  
         [0074]     (2-5) Preservation of Sample  
         [0075]     In the samples selected from the screening, part of each culture remaining in the 96-well plate was inoculated into 4.5 mL of LB medium and cultured overnight. The plasmid was purified and stored in a freezer. Furthermore, each culture was separately inoculated into 4 mL of LB medium and cultured until O.D. 600 reached about 0.4 and centrifuged to collect a bacterium. The resulting bacterium was suspended in 2 mL of 2×YT medium, frozen with liquid nitrogen, and preserved at −80° C.  
         [0076]     (2-6) Abundant Expression and Purification of Diaphorase Mutant  
         [0077]     Abundant expression and purification of a diaphorase mutant were performed by a method described above. In the abundant expression,  E. coli  was cultured in 1 L of LB-amp medium. The volume and the like in the following purification steps were adjusted according to a culture scale.  
         [0078]     (2-7) Activity Evaluation Test  
         [0079]     Activity evaluation of a diaphorase mutant was performed under the conditions described below. A reaction solution contained 100 mM K-pi (pH 8.0), [ANQ]=0.3 mM, [NADH]=40 mM, and [diaphorase]=48 nM. Deoxygenation was sufficiently performed by argon bubbling before measurement. The reaction was performed under argon atmosphere. The addition of diaphorase initiated the reaction. The extent of reaction was monitored by means of a reduction in the absorbance of ANQ at 520 nm (molar absorption coefficient: 680 M −1 cm −1 ) to calculate the reaction rate.  
         [0080]     (2-8) Heat Resistance Test  
         [0081]     A purified diaphorase mutant sample solution in 50 mM Tris-HCl (pH 7.8) and a 300 mM NaCl solution was concentrated by ultrafiltration, and the buffer was replaced to prepare a 0.1 M K-pi (pH 8.0) solution. This solution was appropriately diluted in such a manner that the absorbance of diaphorase at 460 nm was 0.1 (the solution with an enzyme concentration of 8.3 μM). This solution was incubated at 80° C. for 10 minutes with an aluminum block heater or the like and immediately cooled on ice. Activity was measured after sufficiently cooled. A control experiment was made with a sample that was not incubated.  
         [0082]     (2-9) Result  
         [0083]     A total of about 8,000 colonies were screened according to the above-described method.  FIG. 2  is part of a photograph of a well plate used in this Example.  FIG. 2  shows an example of the detection of diaphorase maintaining activity during screening. Arrows A and A indicate samples of candidates of thermostable mutants detected in this plate. Arrow B indicates a wild-type sample as a control. Arrow C indicates a wild-type sample that is not subjected to heat treatment.  
         [0084]     In consideration of possible errors, such as the variation between plates and difference in level of expression between strains, selected samples were screened again. That is, cryopreserved  E. coli  samples were streak-cultured on LB agar medium. The resulting colonies were inoculated on a 96-well plate and heated similarly. However, in order to minimize the error, 8 colonies per sample were screened.  
         [0085]      FIG. 3  is part of a photograph of a well plate resulting from a double check experiment. In  FIG. 3 , the same mutant sample was disposed along a column. As a control, the wild-type sample after heat treatment was disposed at column  11  and  4  wells located at the upper side of column  24 , and the wild-type sample not subjected to heat treatment was disposed at column  12  and  4  wells located at the lower side of column  24 . In this example of the photograph shown in  FIG. 3 , samples disposed columns  7 ,  14 ,  17 ,  19 ,  20 , and  21  were positive. The samples were selected as candidates for thermostable mutants.  
         [0086]     Tables 7 to 12 show results of the heat resistance test of the candidates for the thermostable diaphorase mutants.  
                                             TABLE 7                       SEQ. ID.       Activity   Residual activity       No.   Type of mutant   (S −1 )   (%)                                1   WT (wild type, control)   168   23       2   K139N/A187E   367   53       3   F105L   246   48       4   G122D   362   28       5   G131E   250   28       6   A146G   263   9       7   R147H   315   4       8   H34Q   228   8       9   F105H   283   33       10   A113E   143   46                  
 
         [0087]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                   
               
               
                 SEQ. ID. 
                   
                 Activity 
                 Residual activity 
               
               
                 No. 
                 Type of mutant 
                 (S −1 ) 
                 (%) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 11 
                 K123E 
                 155 
                 34 
               
               
                 12 
                 K139N 
                 263 
                 25 
               
               
                 13 
                 R147S 
                 226 
                 17 
               
               
                 14 
                 G149D 
                 168 
                 22 
               
               
                 15 
                 G154D 
                 247 
                 24 
               
               
                 16 
                 A156E 
                 318 
                 27 
               
               
                 17 
                 M159T 
                 196 
                 28 
               
               
                 18 
                 A187E 
                 407 
                 52 
               
               
                 19 
                 A187T 
                 328 
                 17 
               
               
                 20 
                 A187V 
                 214 
                 33 
               
               
                   
               
             
          
         
       
     
         [0088]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                   
               
               
                 SEQ. ID. 
                   
                 Activity 
                 Residual activity 
               
               
                 No. 
                 Type of mutant 
                 (S −1 ) 
                 (%) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 21 
                 R64H/A146T 
                 169 
                 20 
               
               
                 22 
                 E85D/R147H 
                 321 
                 37 
               
               
                 23 
                 F105L/A187E 
                 241 
                 46 
               
               
                 24 
                 A113E/K126N 
                 211 
                 31 
               
               
                 25 
                 Y151H/A187E 
                 284 
                 20 
               
               
                 26 
                 G122D/A187E 
                 356 
                 61 
               
               
                 27 
                 G149D/A187E 
                 215 
                 53 
               
               
                 28 
                 G149S/A187E/L207W 
                 212 
                 38 
               
               
                 29 
                 F105L/A187E/L207W 
                 524 
                 15 
               
               
                 30 
                 G66R/F105L/A187E/K192R 
                 428 
                 24 
               
               
                   
               
             
          
         
       
     
         [0089]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                   
               
               
                 SEQ. ID. 
                   
                 Activity 
                 Residual activity 
               
               
                 No. 
                 Type of mutant 
                 (S −1 ) 
                 (%) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 31 
                 A146G/L207W 
                 213 
                 15 
               
               
                 32 
                 F105L/A187E/Q171P 
                 283 
                 68 
               
               
                 33 
                 A78E/F105L/A187E 
                 284 
                 68 
               
               
                 34 
                 F105L/K149N/V168L/A187E 
                 270 
                 55 
               
               
                 35 
                 G154D/G180R 
                 297 
                 33 
               
               
                 36 
                 F107I 
                 283 
                 53 
               
               
                 37 
                 G185R 
                 446 
                 58 
               
               
                 38 
                 Y151H/G185R 
                 315 
                 15 
               
               
                 39 
                 G122D/G185R 
                 387 
                 63 
               
               
                 40 
                 G149D/G185R 
                 264 
                 54 
               
               
                   
               
             
          
         
       
     
         [0090]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                   
               
               
                 SEQ. ID. 
                   
                 Activity 
                 Residual activity 
               
               
                 No. 
                 Type of mutant 
                 (S −1 ) 
                 (%) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 41 
                 G149D/G185R/A208V 
                 223 
                 26 
               
               
                 42 
                 F107I/G185R 
                 305 
                 48 
               
               
                 43 
                 F107I/G185R/A208V 
                 545 
                 73 
               
               
                 44 
                 F107I/G185R/Q171P 
                 410 
                 21 
               
               
                 45 
                 V80D/F107I/G185R 
                 437 
                 72 
               
               
                 46 
                 F107I/K139N/V168L/G185R 
                 283 
                 47 
               
               
                 47 
                 F150V 
                 380 
                 58 
               
               
                 48 
                 A193E 
                 497 
                 63 
               
               
                 49 
                 F150V/A193E 
                 412 
                 51 
               
               
                 50 
                 Y151H/A193E 
                 358 
                 18 
               
               
                   
               
             
          
         
       
     
         [0091]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                   
               
               
                 SEQ. ID. 
                   
                 Activity 
                 Residual activity 
               
               
                 No. 
                 Type of mutant 
                 (S −1 ) 
                 (%) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 51 
                 G122D/A193E 
                 418 
                 68 
               
               
                 52 
                 G149D/A193E/A208V 
                 275 
                 30 
               
               
                 53 
                 F150V/A193E/A208V 
                 572 
                 78 
               
               
                 54 
                 F150V/A193E/Q171P 
                 458 
                 43 
               
               
                 55 
                 V80D/F150V/A193E 
                 512 
                 82 
               
               
                 56 
                 K139N/F150V/V168L/A193E 
                 294 
                 37 
               
               
                   
               
             
          
         
       
     
         [0092]     For example, in mutant proteins having the amino acid sequence of SEQ. ID. Nos. 2 to 7, 9, 12, 15, 16, 18, 19, 22, 25, 26, 29, 30, 32 to 40, and 42 to 56, enzyme activity (reaction rate) was significantly improved compared with the wild type (WT). Furthermore, for example, in mutant proteins having the amino acid sequence of SEQ. ID. Nos. 2, 3, 18, 23, 26, 27, 32 to 34, 36, 37, 39, 40, 42, 43, 45 to 49, 51, and 53 to 55, residual activity after heat treatment was particularly satisfactory compared with the wild type (WT).  
         [0093]     Furthermore, in this experimental system, a target diaphorase mutant having improved thermostability was successfully prepared. Accordingly, the method for constructing a mutant library by random mutation using error-prone PCR and the method of screening by heat treatment were practically useful.  
       EXAMPLE 3  
       [0094]     Detailed Study of Thermostable Diaphorase Mutant  
         [0095]     Among the mutants obtained by the above-described study, with respect to G122D in which enzyme activity was improved compared with the wild type, a study based on enzyme kinetics was conducted.  
         [0096]     The enzymatic reaction rate was plotted against the concentration of ANQ in the presence of NADH 40 mM. Furthermore, the enzymatic reaction rate was plotted against the concentration of NADH in the presence of 2.2 mM ANQ. The results were well consistent with the Michaelis-Menten equation. Then, kcat, KM (NADH), and KM (ANQ) were determined on the basis of the equation.  
         [0097]     Kcat, KM (NADH), and KM (ANQ) of wild-type (native) diaphorase (DI (DH “Amano” 3)) are shown for comparison. Diaphorase shows a ping-pong reaction mechanism. The term “kcat” means a turnover number per unit time in a catalytic reaction. The terms “KM (NADH)” and “KM (ANQ)” refer to Michaelis constants for substrates and are factors that reflect ease of binding of substrates to active sites of the enzymes.  FIG. 4 , as an alternative to tables, summarizes the results.  
         [0098]     The results demonstrates that the mediator ANQ binding site of the mutant has a property of ease of bonding compared with the wild type (native) (see ANQ association table in  FIG. 4 ). On the other hand, with respect to the NADH binding site, factors were not different from those of the wild type (native) or may be reduced (see NADH association table in  FIG. 4 ). Thus, it can be predicted that the higher catalytic ability of the mutant (mutant protein) is attributed to the acquisition of affinity for the ANQ substrate.  
         [0099]     This means that the mutant does not exhibit higher catalytic activity at higher concentrations but exhibits an advantage at low concentrations. For example, this may lead to an advantage that the concentration of the mediator ANQ in an enzyme battery can be suppressed.  
       EXAMPLE 4  
     Study by Molecular Dynamics Simulation  
       [0100]     In this Example, the conformation of each mutant protein R147H and G122D was estimated by molecular dynamics simulation. The conformation of the wild-type protein was compared with that of the mutant protein. The relationship among the conformation of the mutant diaphorase, enzyme activity thereof, and thermostability was studied.  
         [0101]     General information on the computation and computational model of the molecular dynamics simulation is described below.  
         [0102]     In this simulation, commercially available protein modeling software, “Discovery Studio Modeling” (hereinafter, referred to as “DS Modeling”) was used. In the simulation, “DS Modeling 1.1” was used for initial modeling of the conformation of a protein, and “DS Modeling 1.5” was used for calculation and analysis using a force field.  
         [0103]     With respect to the wild-type diaphorase, similarity search was performed by position specific iterative BLAST (PST-BLAST) to make a search for a protein having high similarity. Then, a protein having the highest score in the search was used as a template, and the initial modeling of the wild-type diaphorase and mutant diaphorase was performed by 3-D Alignment. Initial modeling of a coenzyme (FAD) was also performed.  
         [0104]     With respect to the initial modeling, the chemistry at harvard macromolecular mechanics (CHARMm) force field was assigned to each atom, and the structure was optimized by molecular mechanics calculation. The structure was optimized by 1,000 steps of calculation using the steepest-descent method and then 5,000 steps of calculation using the adapted basis Newton-Raphson method.  
         [0105]     In order to consider thermodynamic conditions, the set condition was changed from 50 K to 300 K through 2,000 steps (1 step was 1 femtosecond). Then, the structure at 300 K was calculated.  
         [0106]     The number of particles n, a volume V, and a temperature T were set at a constant (NVT ensemble). Equilibration calculation at 300 K was performed for 1 nanosecond (1 step is 1 femtosecond).  
         [0107]     Molecular dynamics (MD) calculation was performed for 1 nanosecond (1 step was 1 femtosecond). The motion of each atom was tracked to perform energetic analysis.  
         [0108]     Thereby, the conformation of each of the wild-type diaphorase and the mutant diaphorase was simulated through the above-described steps. FIGS.  5  to  11  show the results.  
         [0109]      FIG. 5  shows a conformation observed by simulation of wild-type diaphorase. This conformation is a final structure after MD calculation. In  FIG. 5 , “FAD” indicates the site of FAD (coenzyme). “R147” and “G122” each indicate the site of an amino acid residue replaced in the mutant protein.  
         [0110]     As shown in  FIG. 5 , “R147” is located in the vicinity of FAD, i.e., “R147” is located in the vicinity of an active site for an enzymatic reaction. “G122” is located at a position remote from the active site for the enzymatic reaction. However, “G122” is present at a position at which three α-helices gather and is present at an important position in the conformation of the protein.  
         [0111]      FIGS. 6A  to  7 B are each a photograph, as an alternative to a drawing, showing the results of structure analysis in the vicinity of the active site of the enzymatic reaction.  
         [0112]      FIGS. 6A and 6B  each show the vicinity of FAD in the conformation of R147H mutant diaphorase obtained by simulation.  FIGS. 7A and 7B  each show the vicinity of FAD in the conformation of G122D mutant diaphorase.  FIGS. 6A and 7A  are each a front structural view.  FIGS. 6B and 7B  are each a top structural view.  
         [0113]     In the above-described enzyme activity analysis, the simulated mutant protein had high enzyme activity (reaction rate) compared with the wild-type diaphorase. On the other hand, as shown in  FIGS. 6A  to  7 B, mutant diaphorase is different in the position of Trp103 from wild-type diaphorase (see  FIG. 5 ).  
         [0114]     That is, Trp103 in the wild type is remote from flavin in FAD, whereas Trp103 in the mutant is located below flavin. As a result, Asn104 in the wild type is located above the plane of flavin and is thus close to the active center (N atom disposed at the middle) of flavin, whereas Asn104 in the mutant is located below the plane of flavin and is thus remote from the active center of flavin.  
         [0115]     The results suggest that in the conformation of diaphorase, mutation such that the amino acid residue (Asn104) located at the 104th position of the amino acid sequence is remote from the active center of flavin of coenzyme FAD improves the enzyme activity of diaphorase. That is, the results suggest that in the conformation of diaphorase, the enzyme activity of the mutant protein having a modified structure in which the amino acid residue located at the 104th position of the amino acid sequence is remote from the active center of flavin of coenzyme in the conformation is higher than that of the wild-type diaphorase.  
         [0116]     FIGS.  8  to  10  are each a photograph, as an alternative to the drawing, showing the results of structure analysis on the basis of an electrostatic potential surface.  
         [0117]      FIG. 8  shows the electrostatic potential surface and the conformation obtained by simulation of wild-type diaphorase.  FIG. 9  shows the electrostatic potential surface and the conformation obtained by simulation of R147H mutant diaphorase.  FIG. 10  shows the electrostatic potential surface and the conformation obtained by simulation of G122D mutant diaphorase. In each of FIGS.  8  to  10 , the left figure is a conformational view showing the electrostatic potential surface. Furthermore, in each of the figures, the term “HYDROPHILIC” indicates a hydrophilic portion, and “DISAPPEAR HYDROPHILICITY” indicates a hydrophobic portion.  
         [0118]     As shown in  FIG. 8 , the vicinity of the flavin-binding site in the wild-type diaphorase is substantially hydrophobic. However, a hydrophilic portion is partly present. In contrast, the hydrophilic portion disappears in the mutant diaphorase.  
         [0119]     ANQ, which is a substrate of diaphorase, is a hydrophobic agent. Thus, the above-described results suggest that in the conformation of diaphorase, since the structure in the vicinity of the coenzyme-binding site is changed to be hydrophobic, the interaction with ANQ increases to improve the enzyme activity of diaphorase. That is, the results suggest that the enzyme activity of the mutant protein having a modified structure in which hydrophobicity in the vicinity of the coenzyme-binding site in the conformation is higher than that of the wild-type protein is higher than that of the wild-type diaphorase.  
         [0120]      FIGS. 11 and 12  are each a photograph, as an alternative to the drawing, showing hydrogen bonds.  
         [0121]      FIG. 11  shows conformational diagrams illustrating hydrogen bonds obtained by simulation of wild-type diaphorase and R147H mutant diaphorase.  FIG. 12  shows conformational diagrams illustrating hydrogen bonds obtained by simulation of wild-type diaphorase and G122D mutant diaphorase. In each of the figures, the left conformational diagram represents the hydrogen bonds in the wild-type diaphorase, and the right conformational diagram represents the hydrogen bonds in the mutant diaphorase.  
         [0122]     As shown in  FIGS. 11 and 12 , the results of the simulation demonstrated that the number of hydrogen bonds in R147H mutant diaphorase decreased, and the number of hydrogen bonds in G122D mutant diaphorase increased.  
         [0123]     The results of the above-described thermostability analysis demonstrated that the thermostability of R147H mutant diaphorase decreased, whereas the thermostability of G122D mutant slightly increased.  
         [0124]     The results suggest findings about the thermostability of diaphorase described below.  
         [0125]     In the case of R147H mutant diaphorase, the vicinity of the FAD-binding site is modified. The modification reduces the number of hydrogen bonds in the modified region, thus degrading thermostability.  
         [0126]     In the case of G122D mutant diaphorase, although the number of hydrogen bonds increases, the modification is not close to the FAD-binding site; hence, thermostability is not so improved. However, G122 is located at a position at which three α-helices in the conformation of diaphorase gather; hence, the increase in the number of hydrogen bonds affects the vicinity of the FAD-binding site via the helices, thereby thermostability increases to some extent.  
         [0127]     In summary, the results suggest that the modification for preventing a change in conformation due to the coenzyme (FAD) can improve the thermostability of diaphorase. That is, the results suggest that the thermostability of the mutant protein having a modified structure capable of preventing a change in conformation due to the coenzyme is higher than that of the wild-type diaphorase.  
         [0128]     Specifically, with respect to the conformation of diaphorase, the results suggest that the modification such that the number of hydrogen bonds increases in the vicinity of the FAD-binding site; or the modification such that a change in conformation in the vicinity of the FAD-binding site is prevented improves the thermostability of diaphorase.  
         [0129]     It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.