Patent Publication Number: US-9410184-B2

Title: Glucose dehydrogenase

Description:
TECHNICAL FIELD 
     The present invention relates to a pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQGDH) and to its preparation and use in the glucose assay. 
     BACKGROUND ART 
     The blood glucose level is an important marker for diabetes. The use of PQQGDH has already been commercialized as one method for measuring the glucose concentration. 
     PQQGDH is a glucose dehydrogenase that employs pyrroloquinoline quinone as a coenzyme, and it catalyzes the oxidation of glucose with the production of gluconolactone. PQQGDH is known to occur as a membrane-bound enzyme and as a water-soluble enzyme. Membrane-bound PQQGDHs are single peptide proteins with molecular weights of approximately 87 kDa and are widely encountered in various Gram-negative bacteria. Water-soluble PQQGDH, on the other hand, has been identified in several strains of  Acinetobacter calcoaceticus , and its structural gene was cloned and its amino acid sequence was determined (GenBank accession number X15871 ; Mol. Gen. Genet . (1989), 217: 430-436). The results of X-ray crystal structural analysis of water-soluble PQQGDH from  Acinetobacter calcoaceticus  have been reported and the higher order structure of the enzyme, and most importantly the active center, has been elucidated. (A. Oubrie et al.,  J. Mol. Biol.,  289, 319-333 (1999); A. Oubrie et al.,  The EMBO Journal,  18(19), 5187-5194 (1999); A. Oubrie et al.,  PNAS,  96(21), 11787-11791 (1999)). Water-soluble PQQGDH from  Acinetobacter baumannii  has also been identified (GenBank accession number E28183). 
     PQQGDHs have a high oxidation activity for glucose and do not require oxygen as an electron acceptor because they are coenzyme-linked enzymes. As a result they are expected to find application in glucose assays and particularly as the recognition element of glucose sensors. A problem with PQQGDHs, however, is their low selectivity for glucose. In particular, PQQGDH also has a high activity for maltose, and thus accurate assay is difficult in patients receiving a maltose-containing infusion solution. In this case, the apparent blood sugar level will be higher than the actual blood sugar level, which could lead to a risk of hypoglycemia caused by administering insulin to the patient based on the measured level. Accordingly, a PQQGDH that exhibits a higher selectivity for glucose versus maltose is desired for the enzyme used for measurement of the blood sugar level. 
     The present inventor has already reported several modified PQQGDHs that exhibit an increased selectivity for glucose (for example, WO 00/66744, Japanese Patent Application Laid-open No. 2001-346587, and WO 2004/005499), but a modified PQQGDH that exhibits an even higher selectivity and/or an even higher enzymatic activity is still required. 
     The reference documents cited herein are listed below. The contents of these documents are hereby incorporated by reference in its entirety. None of these documents are admitted to constitute a prior art of the present invention.
     Patent Document 1: WO 00/66744   Patent Document 2: Japanese Patent Application Laid-open No. 2001-346587   Patent Document 3: WO 2004/005499   Nonpatent Document 1 : Mol. Gen. Genet . (1989), 217:430-436   Nonpatent Document 2: A. Oubrie et al. (1999)  J. Mol. Biol.,  289, 319-333   Nonpatent Document 3: A. Oubrie et al. (1999)  The EMBO Journal,  18(19), 5187-5194   Nonpatent Document 4: A. Oubrie et al. (1999)  PNAS,  96(21), 11787-11791   

     DISCLOSURE OF THE INVENTION 
     An object of the present invention is to provide a modified pyrroloquinoline quinone glucose dehydrogenase that exhibits a high selectivity for glucose. 
     The present inventor discovered that the selectivity for glucose is increased by the insertion, in a particular position in water-soluble PQQGDH, of a peptide fragment comprising 4 or 5 amino acids having a predetermined sequence. 
     The present invention provides a modified pyrroloquinoline quinone glucose dehydrogenase in which the amino acid residue G at Position 99 of a pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) represented by SEQ ID NO: 1 or the amino acid residue G at Position 100 of the PQQGDH represented by SEQ ID NO: 3 is substituted by the amino acid sequence TGZN (where Z is SX, S, or N and X is any amino acid residue), and wherein from 1 to 10 of amino acid residues at Positions 1 to 98 and amino acid residues at Positions 100 to 478 of SEQ ID NO: 1, or amino acid residues at Positions 1 to 99 and amino acid residues at Positions 101 to 480 of SEQ ID NO: 3, may be substituted by any other amino acid residue(s). Z in the modified pyrroloquinoline quinone glucose dehydrogenase of the present invention is preferably SX and is particularly preferably SN. 
     Preferably, the modified pyrroloquinoline quinone glucose dehydrogenase of the present invention further comprises one or more mutations selected from the group consisting of the following amino acid substitutions: 
     Q192G, Q192A, or Q192S; 
     L193X (where X is any amino acid residue); 
     E277X (where X is any amino acid residue); 
     A318X (where X is any amino acid residue); 
     Y367A, Y367F, or Y367W; 
     G451C; and 
     N452X (where X is any amino acid residue). 
     In another aspect the present invention provides a gene coding for the modified pyrroloquinoline quinone glucose dehydrogenase according to the present invention, a recombinant vector comprising the gene, and a transformant or transfectant that has been transformed with the recombinant vector. The present invention further provides a method of preparing the modified pyrroloquinoline quinone glucose dehydrogenase, comprising culturing a transformant that was transformed by a recombinant vector comprising a gene coding for the modified pyrroloquinoline quinone glucose dehydrogenase according to the present invention; and recovering the modified pyrroloquinoline quinone glucose dehydrogenase from the culture. 
     In an additional aspect the present invention provides a glucose assay kit comprising the modified pyrroloquinoline quinone glucose dehydrogenase according to the present invention. The present invention additionally provides an enzyme electrode comprising the modified pyrroloquinoline quinone glucose dehydrogenase according to the present invention, as well as a glucose sensor comprising this enzyme electrode as a working electrode. 
     The modified pyrroloquinoline quinone glucose dehydrogenase of the present invention exhibits a high selectivity for glucose as well as a high glucose oxidation activity, and can therefore be used for the highly selective and highly sensitive assay of glucose. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the enzymatic activity of the modified PQQGDH of the present invention for glucose and maltose. 
     
    
    
     PREFERRED EMBODIMENT OF THE INVENTION 
     Structure of the Modified PQQGDH 
     The modified pyrroloquinoline quinone glucose dehydrogenase of the present invention is characterized in that the amino acid residue G at Position 99 of the water-soluble PQQGDH shown by SEQ ID NO: 1 or the amino acid residue G at Position 100 of the PQQGDH shown by SEQ ID NO: 3 is substituted by the amino acid sequence TGZN (where Z is SX, S, or N and X is any amino acid residue). As used herein, the position of an amino acid in the amino acid sequence of the water-soluble PQQGDHs is numbered by assigning 1 to the initiation methionine. 
     The PQQGDH represented by SEQ ID NO: 1 is a PQQGDH from  Acinetobacter calcoaceticus  (GenBank accession number X15871) and the PQQGDH represented by SEQ ID NO: 3 is a PQQGDH from  Acinetobacter baumannii  (GenBank accession number E28183). These PQQGDHs have an approximately 92% homology at the level of the amino acid sequence. The alignment of the two sequences is shown below. 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
             
            
               
                 _aln.pos 
                          10        20        30        40        50        60 
                   
               
               
                 
                   Calcoace 
                 
                  MNKHLLAKIALLSAVQLVTL-SAFADVPLTPSQFAKAKSENFDKKVILSNLNKPHALLWGPDNQIWLT 
                 (SEQ ID NO: 1) 
               
               
                 
                   Baumannii 
                 
                  MNKHLLAKITLLGAAQLFTFHTAFADIPLTPAQFAKAKTENFDKKVILSNLNKPHALLWGPDNQIWLT 
                 (SEQ ID NO: 3) 
               
               
                 _consrvd 
                  ************ * ** *   * **** ****** ***************************** 
                   
               
               
                   
               
               
                 _aln.p 
                  70        80        90       100       110       120       130 
                   
               
               
                 
                   calocoace 
                 
                  ERATGKILRVNPESGSVKTVFQVPEIVNDADGQNGLLGFAFHPDFKNNPYIYISGTFKNPKSTD 
                   
               
               
                 
                   Baumannii 
                 
                  ERATGKILRVNPVSGSAKTVFQVPEIVSDADGQNGLLGFAFHPDFKHNPYIYISGTFKNPKSTD 
                   
               
               
                 _consrvd 
                  ************ *** ********** ****************** ***************** 
                   
               
               
                   
               
               
                 _aln.pos 
                   140       150       160       170       180       190       200 
                   
               
               
                 
                   calcoace 
                 
                  KELPNQTIIRRYTYNKSTDTLEKPVDLLAGLPSSKDHQSGRLVIGPDQKIYYTIGDQGRNQLAYLFLP 
                   
               
               
                 
                   Baumannii 
                 
                  KELPNQTIIRRYTYNKTTDTFEKPIDLIAGLPSSKDHQSGRLVIGPDQKIYYTIGDQGRNQLAYLFLS 
                   
               
               
                 _consrvd 
                  **************** *** *** ** *************************************** 
                   
               
               
                   
               
               
                 _aln.pos 
                     210       220       230      240        250       260       270 
                   
               
               
                 
                   calcoace 
                 
                  NQAQHTPTQQELNGKDYHTYMGKVLRLNLDGSIPKDNPSFNGVVSHIYTLGHRNPQGLAFTPNGKLLQ 
                   
               
               
                 
                   Baumannii 
                 
                  NQAQHTPTQQELNSKDYHTYMGKVLRLNLDGSIPKDNPSFNGVVSHIYTLGHRNPQGLAFAPNGKLLQ 
                   
               
               
                 _consrvd 
                  ************* ********************************************** ******* 
                   
               
               
                   
               
               
                 _aln.pos 
                      280        290       300       310       320       330       340 
                   
               
               
                 
                   calcoace 
                 
                  SENPNSDDE INLIVKGGNYGWPNVAGYKDDSGYAYANYSAAANK-SIKDLAQNGVKVAAGVPVTKES 
                   
               
               
                 
                   Baumannii 
                 
                  SENPNSDDE INLVLKGGNYGWPNVAGYKDDSGYAYANYSAATNKSQIKDLAQNGIKVATGVPVTKES 
                   
               
               
                 _consrvd 
                  *************  *************************** **  ******** *** ******** 
                   
               
               
                   
               
               
                 _aln.pos 
                         350       360       370       380       390       400 
                   
               
               
                 
                   calcoace 
                 
                  EWTGKNFVPPLKTLYTVQDTYNYNDPTCGEMTYICWPTVAPSSAYVYKGGKKAITGWENTLLVPSLKR 
                   
               
               
                 
                   Baumannii 
                 
                  EWTGKNFVPPLKTLYTVQCTYNYNDPTCGEMAYICWPTVAPSSAYVYTGGKKAIPGWENTLLVPSLKR 
                   
               
               
                 _consrvd 
                  ******************************* *************** ****** ************* 
                   
               
               
                   
               
               
                 _aln.p 
                 410       420       430       440       450       460       470 
                   
               
               
                 
                   calcoace 
                 
                  GVIFRIKLDPTYSTTYDDAVPMFKSNNRYRDVIASPDGNVLYVLTDTAGNVQKDDGSVTNTLENPGSL 
                   
               
               
                 
                   Baumannii 
                 
                  GVIFRIKLDPTYSTTLDDAIPMFKSNNRYRDVIASPEGNTLYVLTDTAGNVQKDDGSVTHTLENPGSL 
                   
               
               
                 _consrvd 
                  *************** *** **************** ** ******************* ******** 
                   
               
               
                   
               
               
                 _aln.pos 
                  480 
                   
               
               
                 
                   calcoace 
                 
                  IKFTYKAK 
                   
               
               
                 
                   Baumannii 
                 
                  IKFTYNGK 
                   
               
               
                 _consrvd 
                  *****  * 
               
               
                   
               
            
           
         
       
     
     These two PQQGDHs have similar secondary structures, and it is known that the properties of enzymes, for example, the thermal stability and substrate specificity, are similarly changed by the substitution of corresponding amino acid residues. 
     Z in the modified pyrroloquinoline quinone glucose dehydrogenase of the present invention is preferably SX and particularly preferably is SN. Thus, the amino acid sequence TGSXN, preferably TGSNN, and particularly preferably TGSKN, TGSRN, or TGSWN is inserted in place of the amino acid residue G at Position 99 in SEQ ID NO: 1 or in place of the amino acid residue G at Position 100 in SEQ ID NO: 3. TGSN or TGNN is also preferably inserted in place of this amino acid residue G. 
     The modified PQQGDH of the present invention has a higher selectivity for glucose than naturally occurring water-soluble PQQGDH. The modified PQQGDH of the present invention preferably has a lower reactivity for maltose versus reactivity for glucose than does the wild type. Given the reactivity for glucose is 100%, preferably the activity for maltose is not more than 50%, more preferably not more than 30%, even more preferably not more than 20%, and most preferably not more than 10%. 
     The modified PQQGDH of the present invention may have other mutations in addition to the mutation in the amino acid residue at Position 99 of SEQ ID NO: 1 or at Position 100 of SEQ ID NO: 3. For example, one or more, for example, from 1 to 10, of the amino acid residues at Positions 1 to 98 or at Positions 100 to 478 of SEQ ID NO: 1, or the amino acid residues at Positions 1 to 99 or at Positions 101 to 480 of SEQ ID NO: 3, may be substituted by any other amino acid residue. 
     In a preferred embodiment of the present invention, the modified PQQGDH of the present invention has one or more mutations, preferably from 1 to 10 mutations, for example, 1, 2, 3, 4, 5, or 6 mutations, selected from the group consisting of 
     Q192G, Q192A, or Q192S; 
     L193X (where X is any amino acid residue); 
     E277X (where X is any amino acid residue); 
     A318X (where X is any amino acid residue); 
     Y367A, Y367F, or Y367W; 
     G451C; and 
     N452X (where X is any amino acid residue). 
     Wherein, the “Q192G” designation, for example, indicates that the glutamine at Position 192 in SEQ ID NO: 1, or the corresponding glutamine at Position 193 in SEQ ID NO: 3, is substituted by glycine. The other substitutional mutations are also indicated in the same manner. 
     A particularly preferred embodiment of the present invention is a modified pyrroloquinoline quinone glucose dehydrogenase comprising any of the following combinations of amino acid mutations: 
     G99(TGSXN)+Q192G+L193E; 
     G99(TGSXN)+Q192S+N452P; 
     G99(TGSXN)+Q192G+L193E+N452P; 
     G99(TGSXN)+Q192S+N452P; 
     G99(TGSNN)+N452P; 
     G99(TGSNN)+Q192G+L193E+N452P; 
     G99(TGSNN)+Q192S+N452P; 
     G99(TGSNN)+Q192G+N452P; 
     G99(TGSNN)+L193E+N452P; 
     G99(TGSNN)+Q192S+L193M+N452P; 
     G99(TGSNN)+A318Y+N452P; 
     G99(TGSNN)+Q192G; 
     G99(TGSNN)+Q192S; 
     G99(TGSNN)+Q192A; 
     G99(TGSNN)+Q192G+L193E; 
     G99(TGSNN)+Q192S+L193X; 
     G99(TGSNN)+Q192S+L193M; 
     G99(TGSNN)+Q192S+L193T; 
     G99(TGSNN)+Q192S+E277X; 
     G99(TGSNN)+Q192S+N452X; 
     G99(TGSNN)+Q192S+L193X+A318Y+N452P; 
     G99(TGSNN)+Q192S+A318X+N452P; 
     G99(TGSNN)+Q192G+L193E+A318X+N452P; 
     G99(TGSKN)+Q192S+N452P; 
     G99(TGSRN)+Q192S+N452P; 
     G99(TGSWN)+Q192S+N452P; 
     G99(TGSN)+Q192S+N452P; 
     G99(TGSN)+Q192G+L193E; 
     G99(TGSN)+Q192S+L193M; 
     G99(TGNN)+Q192S+N452P; 
     G99(TGNN)+Q192G+L193E; or 
     G99(TGNN)+Q192S+L193M. 
     Wherein X is any amino acid reside, for example, G99(TGSXN) indicates that the G at Position 99 in the PQQGDH represented by SEQ ID NO: 1 is substituted by TGSXN. 
     Another preferred embodiment of the present invention is a modified pyrroloquinoline quinone glucose dehydrogenase comprising any of the following amino acid mutations: 
     G99(TGSXN)+Q192G+L193E; 
     G99(TGSXN)+Q192S+N452P; 
     G99(TGSNN)+Q192G+L193E+N452P; 
     G99(TGSNN)+Q192S+L193M+N452P; 
     G99(TGSNN)+Q192G+L193E+A318K+N452P; or 
     G99(TGSNN)+Q192G+L193E+A318Q+N452P. 
     WO 04/005499 discloses that the glutamine residue at Position 192, the leucine residue at Position 193, and the asparagine residue at Position 452 are involved in substrate recognition and binding by PQQGDH. In general, however, it is completely unpredictable how the substrate specificity and enzymatic activity will change when mutations are simultaneously introduced in amino acid residues present in different domains. A complete loss of enzymatic activity may even occur in some cases. Accordingly, it was totally unpredictable if the selectivity for glucose would be further enhanced by the simultaneous introduction of the above-indicated substitutions and an insertion mutation at the amino acid residue at Position 99 in SEQ ID NO: 1 or the amino acid residue at Position 100 in SEQ ID NO: 3. 
     Method of Producing Modified PQQGDH 
     The sequence of the gene coding for naturally occurring water-soluble PQQGDH from  Acinetobacter calcoaceticus  is shown in SEQ ID NO: 2, while the sequence of the gene coding for naturally occurring water-soluble PQQGDH from  Acinetobacter baumannii  is shown in SEQ ID NO:4. A gene coding for the modified PQQGDH of the present invention can be constructed by replacing the nucleotide sequence coding for the amino acid residue targeted for substitution in the gene coding for naturally occurring water-soluble PQQGDH with a nucleotide sequence coding for the desired amino acid residue. Methods for such a site-specific sequence substitution are well known in the art; for example, by PCR using suitably designed primers, as is described in the examples provided below. 
     The thus obtained mutant gene is inserted in an expression vector (for example, a plasmid), which is then transformed into a suitable host (for example,  E. coli ). A large number of vector/host systems for the expression of foreign protein are known in the art. A variety of hosts, for example, bacteria, yeast, cultured cells, and so forth are available. 
     In addition, a portion of other amino acid residues in the modified PQQGDH of the present invention may also be deleted or substituted, and other amino acid residues may be added, insofar as it has a desired glucose dehydrogenase activity. Various methods for site-specific nucleotide sequence substitution are well known in the art. 
     The modified PQQGDH-expressing transformant obtained as described above is then cultured, and the cells are recovered from the culture fluid by, for example, centrifugation. The recombinant protein present in vhe periplasmic compartment is subsequently released into culture medium by grinding the cells, for example, with a French press, or by osmotic shock. Ultracentrifugation is thereafter carried out to obtain a water-soluble fraction containing the modified PQQGDH. Alternatively, through the use of a suitable host-vector system, the expressed modified PQQGDH can be secreted into the culture fluid. The modified PQQGDH of the present invention can be isolated by purifying the water-soluble fraction by, for example, ion-exchange chromatography, affinity chromatography, HPLC, and so forth. 
     Method of Measuring the Enzymatic Activity 
     The modified PQQGDH of the present invention has the ability to catalyze the oxidation of glucose with PQQ as its coenzyme, with the production of gluconolactone. To measure the enzymatic activity, the quantity of PQQ that is reduced accompanying the PQQGDH-mediated glucose oxidation can be quantitated by the color reaction of a redox dye. For example, phenazine methosulfate (PMS), 2,6-dichlorophenolindophenol (DCIP), potassium ferricyanide, ferrocene, and so forth, can be used as the chromogenic reagent. 
     Selectivity for Glucose 
     The selectivity for glucose exhibited by the modified PQQGDH of the present invention can be evaluated by measuring the enzymatic activity in the manner described above using various sugars as substrates, e.g., 2-deoxy-D-glucose, mannose, allose, 3-o-methyl-D-glucose, galactose, xylose, lactose, maltose, and so forth, and determining the relative activity with reference to the activity using glucose as the substrate. 
     The modified PQQGDH of the present invention provides a higher selectivity for glucose than the wild-type enzyme and in particular has a higher reactivity for glucose than for maltose. Accordingly, an assay kit or enzyme sensor constructed using the modified PQQGDH of the present invention will exhibit a high selectivity with regard to glucose measurement and offers the advantage of high-sensitivity glucose detection even when a maltose-containing sample is used. 
     Glucose Assay Kit 
     Another aspect of the present invention is a glucose assay kit comprising a modified PQQGDH according to the present invention. The glucose assay kit of the present invention comprises the modified PQQGDH according to the present invention in a quantity sufficient for conducting at least one assay. In addition to the modified PQQGDH of the present invention, the kit will typically comprise a buffer required for the assay, a mediator, a glucose reference solution for constructing a calibration curve, and instructions for use. The modified PQQGDH according to the present invention can be provided in various forms, for example, as a freeze-dried reagent or as a solution in a suitable storage solution. The modified PQQGDH of the present invention is preferably provided in a form of holoenzyme, but can also be provided as the apoenzyme and then converted into the holoenzyme before use. 
     Glucose Sensor 
     Additional aspect of the present invention is an enzyme electrode that carries a modified PQQGDH according to the present invention, and a glucose sensor comprising the enzyme electrode. For example, a carbon electrode, gold electrode, or platinum electrode can be used as the electrode, and the enzyme according to the present invention is immobilized on the electrode. The immobilization method can be exemplified by the use of a crosslinking reagent; enclosure in a polymer matrix; coating with a dialysis film; use of a photocrosslinking polymer, electroconductive polymer, or redox polymer; immobilization in a polymer or adsorptive immobilization on the electrode together with an electron mediator such as ferrocene and its derivatives; and the use of combinations of the preceding. The modified PQQGDH of the present invention is preferably immobilized on the electrode in a holoenzyme form, but can also be immobilized in an apoenzyme form with the PQQ being provided in a separate layer or in a solution. Typically, the modified PQQGDH of the present invention is immobilized on a carbon electrode via glutaraldehyde followed by treatment with an amine group-containing reagent to block the aldehyde groups of glutaraldehyde. 
     Measurement of the glucose concentration can be carried out as follows. Buffer solution is introduced into a thermostatted cell; PQQ, CaCl 2 , and a mediator are added; and the cell is held at a constant temperature. Potassium ferricyanide, phenazine methosulfate, and so forth, may be used as the mediator. An electrode bearing the immobilized modified PQQGDH of the present invention is used as the working electrode, in combination with a counterelectrode (for example, a platinum electrode) and a reference electrode (for example, the Ag/AgCl electrode). A constant voltage is applied to the carbon electrode, and after the current becomes constant, a glucose-containing sample is added and the increase in the current is measured. The glucose concentration in the sample can be determined using a calibration curve constructed using glucose solutions of standard concentrations. 
     The contents of all the patents and reference documents explicitly cited herein are hereby incorporated by reference in its entirety. 
     The present invention is described in detail based on the following examples, but is not limited to these examples. 
     Example 1 
     Construction of Modified PQQGDH-Encoding Genes 
     Mutation was introduced into the structural gene for water-soluble PQQGDH from  Acinetobacter calcoaceticus , which is represented by SEQ ID NO: 2. In brief, PCR was carried out using a full length forward primer for the wild-type water-soluble PQQGDH and a mutagenic reverse primer, and another PCR was carried out using a full length reverse primer and a mutagenic forward primer. These PCR products were mixed and PCR was run using a full length forward primer and reverse primer to obtain a gene coding for the mutated full length PQQGDH. The product was sequenced to conform that the desired mutation was correctly introduced. 
     The sequence of the primers for full length amplification were as follows. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 forward: AACAGACCATGGATAAACATTTATTGGC 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
                 reverse: ACAGCCAAGCTTTTACTTAGCCTTATAGG 
               
            
           
         
       
     
     The sequences of the mutagenic primers (F: forward, R: reverse) are shown in the following table. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                   
                 SEQ  
               
               
                 Muta- 
                   
                   
                 ID 
               
               
                 tion 
                 Primer Sequence 
                   
                 NO 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 TGNN 
                 ACTGGAAATAATCAGAATGGTTTATTAGGTTTT 
                 F 
                 7 
               
               
                   
                 CTGATTATTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 8 
               
               
                   
               
               
                 TGQN 
                 ACTGGACAGAATCAGAATGGTTTATTAGGTTTT 
                 F 
                 9 
               
               
                   
                 CTGATTCTGTCCAGTATCAGCATCATTGACAAT 
                 R 
                 10 
               
               
                   
               
               
                 TGSN 
                 ACTGGAAGCAATCAGAATGGTTTATTAGGTTTT 
                 F 
                 11 
               
               
                   
                 CTGATTGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 12 
               
               
                   
               
               
                 TGGN 
                 ACTGGAGGTAATCAGAATGGTTTATTAGGTTTT 
                 F 
                 13 
               
               
                   
                 CTGATTACCTCCAGTATCAGCATCATTGACAAT 
                 R 
                 14 
               
               
                   
               
               
                 TGSSN 
                 GATACTGGAAGCAGCAATCAGAATGGTTTATTA 
                 F 
                 15 
               
               
                   
                 ATTGCTGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 16 
               
               
                   
               
               
                 TGWN 
                 ACTGGATGGAATCAGAATGGTTTATTAGGTTTT 
                 F 
                 17 
               
               
                   
                 CTGATTCCATCCAGTATCAGCATCATTGACAAT 
                 R 
                 18 
               
               
                   
               
               
                 TGFN 
                 ACTGGATTTAATCAGAATGGTTTATTAGGTTTT 
                 F 
                 19 
               
               
                   
                 CTGATTAAATCCAGTATCAGCATCATTGACAAT 
                 R 
                 20 
               
               
                   
               
               
                 TGDN 
                 ACTGGAGATAATCAGAATGGTTTATTAGGTTTT 
                 F 
                 21 
               
               
                   
                 CTGATTATCTCCAGTATCAGCATCATTGACAAT 
                 R 
                 22 
               
               
                   
               
               
                 TGSHN 
                 GATACTGGAAGCCATAATCAGAATGGTTTATTA 
                 F 
                 23 
               
               
                   
                 ATTATGGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 24 
               
               
                   
               
               
                 TGSLN 
                 GATACTGGAAGCTTAAATCAGAATGGTTTATTA 
                 F 
                 25 
               
               
                   
                 ATTTAAGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 26 
               
               
                   
               
               
                 TGSVN 
                 GATACTGGAAGCGTCAATCAGAATGGTTTATTA 
                 F 
                 27 
               
               
                   
                 ATTGACGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 28 
               
               
                   
               
               
                 TGSQN 
                 GATACTGGAAGCCAAAATCAGAATGGTTTATTA 
                 F 
                 29 
               
               
                   
                 ATTTTGGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 30 
               
               
                   
               
               
                 TGSEN 
                 GATACTGGAAGCGAAAATCAGAATGGTTTATTA 
                 F 
                 31 
               
               
                   
                 ATTTTCGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 32 
               
               
                   
               
               
                 TGSDN 
                 GATACTGGAAGCGATAATCAGAATGGTTTATTA 
                 F 
                 33 
               
               
                   
                 ATTATCGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 34 
               
               
                   
               
               
                 TGSPN 
                 GATACTGGAAGCCCTAATCAGAATGGTTTATTA 
                 F 
                 35 
               
               
                   
                 ATTAGGGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 36 
               
               
                   
               
               
                 TGSTN 
                 GATACTGGAAGCACAAATCAGAATGGTTTATTA 
                 F 
                 37 
               
               
                   
                 ATTTGTGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 38 
               
               
                   
               
               
                 TGSIN 
                 GATACTGGAAGCATTAATCAGAATGGTTTATTA 
                 F 
                 39 
               
               
                   
                 ATTAATGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 40 
               
               
                   
               
               
                 TGSAN 
                 GATACTGGAAGCGCTAATCAGAATGGTTTATTA 
                 F 
                 41 
               
               
                   
                 ATTAGCGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 42 
               
               
                   
               
               
                 TGSWN 
                 GATACTGGAAGCTGGAATCAGAATGGTTTATTA 
                 F 
                 43 
               
               
                   
                 ATTCCAGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 44 
               
               
                   
               
               
                 TGSGN 
                 GATACTGGAAGCGGTAATCAGAATGGTTTATTA 
                 F 
                 45 
               
               
                   
                 ATTACCGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 46 
               
               
                   
               
               
                 TGSFN 
                 GATACTGGAAGCTTTAATCAGAATGGTTTATTA 
                 F 
                 47 
               
               
                   
                 ATTAAAGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 48 
               
               
                   
               
               
                 TGSYN 
                 GATACTGGAAGCTATAATCAGAATGGTTTATTA 
                 F 
                 49 
               
               
                   
                 ATTATAGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 50 
               
               
                   
               
               
                 TGSCN 
                 GATACTGGAAGCTGCAATCAGAATGGTTTATTA 
                 F 
                 51 
               
               
                   
                 ATTGCAGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 52 
               
               
                   
               
               
                 TGSMN 
                 GATACTGGAAGCATGAATCAGAATGGTTTATTA 
                 F 
                 53 
               
               
                   
                 ATTCATGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 54 
               
               
                   
               
               
                 TGSKN 
                 GATACTGGAAGCAAAAATCAGAATGGTTTATTA 
                 F 
                 55 
               
               
                   
                 ATTTTTGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 56 
               
               
                   
               
               
                 TGSRN 
                 GATACTGGAAGCCGTAATCAGAATGGTTTATTA 
                 F 
                 57 
               
               
                   
                 ATTACGGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 58 
               
               
                   
               
               
                 TGSNN 
                 GATACTGGAAGCAATAATCAGAATGGTTTATTA 
                 F 
                 59 
               
               
                   
                 ATTATTGCTTCCAGTATCAGCATCATTGACAAT 
                 R 
                 60 
               
               
                   
               
            
           
         
       
     
     Other substitutional mutations, e.g., mutations such as Q192G and L193E were introduced using conventional site-specific mutagenesis methods as described in WO 00/66744. In addition, modified PQQGDHs that incorporated a combination of mutations at a plurality of sites were prepared by site-specific mutagenesis using a plurality of primers that corresponded to the individual mutations or were prepared by a recombinant method using restriction enzymes. 
     Example 2 
     Production of Modified PQQGDH 
     The gene coding for wild-type PQQGDH or the gene coding for the modified PQQGDH constructed as described above was inserted in the multicloning site of an  E. coli  expression vector pTrc99A (Pharmacia), and the constructed plasmid was transformed into  E. coli . The transformant was cultured with shaking overnight at 37° C. in 450 mL L-broth containing 50 μg/mL ampicillin and 30 μg/mL chloramphenicol. The culture was inoculated into 7 L of L-broth containing 1 mM CaCl 2  and 500 μM PQQ. At approximately 3 hours after the start of cultivation, isopropylthiogalactoside was added at a final concentration of 0.3 mM and continued cultivation for 1.5 hours. The cells were recovered from the culture medium by centrifugation (5000×g, 10 minutes, 4° C.) and suspended in 150 μL 10 mM MOPS (pH 7.0). Glass beads (0.105 to 0.125 mm for bacteria) were added in an amount that was ½ to ⅔ of the volume and vortexed for 20 minutes at 4° C. 1 mL 10 mM MOPS (pH 7.0) was added and centrifuged (15,000 rpm, 20 minutes, 4° C.). The supernatant was collected and used as a crude enzyme preparation in the following examples. 
     Example 3 
     Measurement of the Enzymatic Activity 
     900 μL of a mixture of 10 mM MOPS (pH 7.0)+1 mM PQQ+1 mM CaCl 2  holoenzyme conversion solution was added to 100 μL of the crude enzyme preparation of the wild-type PQQGDH or the modified PQQGDH obtained in Example 2 and left stand (room temperature, in the dark, at least 30 minutes) to allow for conversion to the holoenzyme. After completion of conversion to the holoenzyme, the solution was diluted 10× to prepare an enzyme test solution. To 150 μL of the enzyme test solution was added 50 μL of a given concentration of the substrate (glucose or maltose), 0.6 mM phenazine methosulfate (PMS), and 0.3 mM 2,6-dichlorophenolindophenol (DCIP, final concentrations in each case), and incubated at room temperature in the total volume of 200 μL. Glucose and maltose were used as the substrates at final concentrations of 0, 1, 2, 4, 10, 20, and 40 mM. The change in the DCIP absorbance at 600 nm was monitored with a spectrophotometer. The rate of decline in the absorbance was measured to determine the reaction rate of the enzyme. In the experiments described herein, 1 unit was designated as the enzymatic activity that reduces 1 μmol of DCIP in 1 minute with the molar absorption coefficient of 16.3 mM −1  of DCIP at pH 7.0. The protein concentration was measured using a commercially available protein assay kit (BioRad). 
     Typical results are given in the following tables indicated by the ratio (%) of the activity for 4 mM maltose with reference to the activity for 4 mM glucose. 
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 TGSXN 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Wild Type 
                 79 
               
               
                   
                 Q192G + 193E 
                 9.0 
               
               
                   
                 TGSAN 
                 54 
               
               
                   
                 TGSCN 
                 47 
               
               
                   
                 TGSDN 
                 60 
               
               
                   
                 TGSEN 
                 51 
               
               
                   
                 TGSGN 
                 52 
               
               
                   
                 TGSHN 
                 46 
               
               
                   
                 TGSIN 
                 42 
               
               
                   
                 TGSKN 
                 43 
               
               
                   
                 TGSMN 
                 43 
               
               
                   
                 TGSNN 
                 42 
               
               
                   
                 TGSPN 
                 47 
               
               
                   
                 TGSQN 
                 42 
               
               
                   
                 TGSRN 
                 43 
               
               
                   
                 TGSSN 
                 57 
               
               
                   
                 TGSTN 
                 47 
               
               
                   
                 TGSVN 
                 39 
               
               
                   
                 TGSWN 
                 33 
               
               
                   
                 TGSFN 
                 58 
               
               
                   
                 TGSLN 
                 49 
               
               
                   
                 TGSYN 
                 63 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 TGSXN + Q192G + L193E 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Wild Type 
                 79 
               
               
                   
                 Q192G + L193E 
                 9.0 
               
               
                   
                 TGSGN + Q192G + L193E 
                 6.7 
               
               
                   
                 TGSMN + Q192G + L193E 
                 5.7 
               
               
                   
                 TGSNN + Q192G + L193E 
                 4.2 
               
               
                   
                 TGSPN + Q192G + L193E 
                 6.6 
               
               
                   
                 TGSQN + Q192G + L193E 
                 5.2 
               
               
                   
                 TGSRN + Q192G + L193E 
                 6.0 
               
               
                   
                 TGSSN + Q192G + L193E 
                 9.3 
               
               
                   
                 TGSTN + Q192G + L193E 
                 7.4 
               
               
                   
                 TGSVN + Q192G + L193E 
                 6.9 
               
               
                   
                 TGSAN + Q192G + L193E 
                 15 
               
               
                   
                 TGSDN + Q192G + L193E 
                 16 
               
               
                   
                 TGSEN + Q192G + L193E 
                 20 
               
               
                   
                 TGSHN + Q192G + L193E 
                 9.8 
               
               
                   
                 TGSIN + Q192G + L193E 
                 20 
               
               
                   
                 TGSKN + Q192G + L193E 
                 5.7 
               
               
                   
                 TGSCN + Q192G + L193E 
                 17 
               
               
                   
                 TGSWN + Q192G + L193E 
                 7.6 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 TGSNN + Q192S + E277X 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSNN + Q192S 
                 12 
               
               
                   
                 TGSNN + Q192S + E277A 
                 20 
               
               
                   
                 TGSNN + Q192S + E277D 
                 26 
               
               
                   
                 TGSNN + Q192S + E277I 
                 15 
               
               
                   
                 TGSNN + Q192S + E277T 
                 22 
               
               
                   
                 TGSNN + Q192S + E277Q 
                 16 
               
               
                   
                 TGSNN + Q192S + E277V 
                 23 
               
               
                   
                 TGSNN + Q192S + E277W 
                 38 
               
               
                   
                 TGSNN + Q192S + E277Y 
                 27 
               
               
                   
                 TGSNN + Q192S + E277C 
                 12 
               
               
                   
                 TGSNN + Q192S + E277F 
                 66 
               
               
                   
                 TGSNN + Q192S + E277G 
                 34 
               
               
                   
                 TGSNN + Q192S + E277H 
                 39 
               
               
                   
                 TGSNN + Q192S + E277K 
                 29 
               
               
                   
                 TGSNN + Q192S + E277L 
                 76 
               
               
                   
                 TGSNN + Q192S + E277M 
                 64 
               
               
                   
                 TGSNN + Q192S + E277N 
                 56 
               
               
                   
                 TGSNN + Q192S + E277S 
                 13 
               
               
                   
                 TGSNN + Q192S + E277R 
                 24 
               
               
                   
                 TGSNN + Q192S + E277P 
                 39 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 TGSNN + Q192S + N452X 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSNN + Q192S + N452A 
                 8.9 
               
               
                   
                 TGSNN + Q192S + N452C 
                 12 
               
               
                   
                 TGSNN + Q192S + N452D 
                 20 
               
               
                   
                 TGSNN + Q192S + N452E 
                 nd 
               
               
                   
                 TGSNN + Q192S + N452F 
                 10 
               
               
                   
                 TGSNN + Q192S + N452G 
                 14 
               
               
                   
                 TGSNN + Q192S + N452H 
                 13 
               
               
                   
                 TGSNN + Q192S + FN452I 
                 14 
               
               
                   
                 TGSNN + Q192S + N452K 
                 18 
               
               
                   
                 TGSNN + Q192S + N452L 
                 7.8 
               
               
                   
                 TGSNN + Q192S + N452M 
                 11 
               
               
                   
                 TGSNN + Q192S + N452P 
                 1.9 
               
               
                   
                 TGSNN + Q192S + N452Q 
                 nd 
               
               
                   
                 TGSNN + Q192S + N452R 
                 27 
               
               
                   
                 TGSNN + Q192S + N452S 
                 11 
               
               
                   
                 TGSNN + Q192S + N452T 
                 14 
               
               
                   
                 TGSNN + Q192S + N452V 
                 8.8 
               
               
                   
                 TGSNN + Q192S + N452W 
                 79 
               
               
                   
                 TGSNN + Q192S + N452Y 
                 9.3 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 TGSNN + Q192G/S/A 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Wild Type 
                 79 
               
               
                   
                 Q192G + L193E 
                 9.0 
               
               
                   
                 TGSNN + Q192G + L193E 
                 4.2 
               
               
                   
                 Q192G 
                 20 
               
               
                   
                 TGSNN + Q192G 
                 9.1 
               
               
                   
                 Q192A 
                 15 
               
               
                   
                 TGSNN + Q192A 
                 7.8 
               
               
                   
                 Q192S 
                 28 
               
               
                   
                 TGSNN + Q192S 
                 12 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 TGSNN + Q192S + L193X 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSNN + Q192S 
                 12 
               
               
                   
                 TGSNN + Q192G + L193E 
                 4.2 
               
               
                   
                 TGSNN + Q192S + L193G 
                 6.8 
               
               
                   
                 TGSNN + Q192S + L193A 
                 7.3 
               
               
                   
                 TGSNN + Q192S + L193K 
                 3.7 
               
               
                   
                 TGSNN + Q192S + L193R 
                 7.7 
               
               
                   
                 TGSNN + Q192S + L193H 
                 8.3 
               
               
                   
                 TGSNN + Q192S + L193D 
                 6.8 
               
               
                   
                 TGSNN + Q192S + L193E 
                 4.3 
               
               
                   
                 TGSNN + Q192S + L193N 
                 8.9 
               
               
                   
                 TGSNN + Q192S + L193Q 
                 11 
               
               
                   
                 TGSNN + Q192S + L193S 
                 5.7 
               
               
                   
                 TGSNN + Q192S + L193T 
                 6.3 
               
               
                   
                 TGSNN + Q192S + L193Y 
                 7.4 
               
               
                   
                 TGSNN + Q192S + L193C 
                 9.3 
               
               
                   
                 TGSNN + Q192S + L193M 
                 4.0 
               
               
                   
                 TGSNN + Q192S + L193F 
                 7.8 
               
               
                   
                 TGSNN + Q192S + L193W 
                 8.2 
               
               
                   
                 TGSNN + Q192S + L193V 
                 7.0 
               
               
                   
                 TGSNN + Q192S + L193I 
                 6.9 
               
               
                   
                 TGSNN + Q192S + L193P 
                 6.1 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
             
            
               
                   
                 Wild Type 
                 79 
               
               
                   
                 TGSNN 
                 42 
               
               
                   
                 TGSNN + Q192S 
                 12 
               
               
                   
                 TGSNN + N452P 
                 14 
               
               
                   
                 TGSNN + Q192G + L193E 
                 4.2 
               
               
                   
                 TGSNN + Q192S + L193M 
                 4.0 
               
               
                   
                 TGSNN + Q192S + L193T 
                 5.8 
               
               
                   
                 TGSNN + Q192S + N452P 
                 1.9 
               
               
                   
                 TGSNN + Q192G + N452P 
                 1.6 
               
               
                   
                 TGSNN + Y367A + N452P 
                 50 
               
               
                   
                 TGSNN + Y367F + N452P 
                 12 
               
               
                   
                 TGSNN + Y367W + N452P 
                 15 
               
               
                   
                 TGSNN + Q192S + Y367A + N452P 
                 nd 
               
               
                   
                 TGSNN + Q192S + Y367F + N452P 
                 2.7 
               
               
                   
                 TGSNN + Q192S + Y367W + N452P 
                 2.0 
               
               
                   
                 TGSNN + L193E + N452P 
                 1.0 
               
               
                   
                 TGSNN + Q192G + L193E + N452P 
                 0.6 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 10 
               
               
                   
                   
               
             
            
               
                   
                 Wild Type 
                 79 
               
               
                   
                 G451C 
                 74 
               
               
                   
                 TCSRN 
                 33 
               
               
                   
                 TCSRN + G451C 
                 44 
               
               
                   
                 N452P 
                 30 
               
               
                   
                 TGSNN + Q192S + L193M 
                 4.0 
               
               
                   
                 TGSNN + Q192S + L193M + N452P 
                 0.8 
               
               
                   
                 TGSNN + Q192G + L193E + E277K 
                 2.7 
               
               
                   
                 TGSNN + Q192G + L193E + N452P 
                 0.6 
               
               
                   
                 TGSNN + N452P 
                 14 
               
               
                   
                 TGSNN + A318Y + N452P 
                 7.8 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 TGSXN + Q192S + N452P 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSAN + Q192S + N452P 
                 7.6 
               
               
                   
                 TGSCN + Q192S + N452P 
                 3.9 
               
               
                   
                 TGSDN + Q192S + N452P 
                 3.6 
               
               
                   
                 TGSFN + Q192S + N452P 
                 26 
               
               
                   
                 TGSGN + Q192S + N452P 
                 3.6 
               
               
                   
                 TGSLN + Q192S + N452P 
                 2.2 
               
               
                   
                 TGSMN + Q192S + N452P 
                 2.5 
               
               
                   
                 TGSNN + Q192S + N452P 
                 1.9 
               
               
                   
                 TGSPN + Q192S + N452P 
                 19 
               
               
                   
                 TGSSN + Q192S + N452P 
                 7.4 
               
               
                   
                 TGSTN + Q192S + N452P 
                 3.8 
               
               
                   
                 TGSVN + Q192S + N452P 
                 3.7 
               
               
                   
                 TGSIN + Q192S + N452P 
                 3.4 
               
               
                   
                 TGSWN + Q192S + N452P 
                 1.2 
               
               
                   
                 TGSYN + Q192S + N452P 
                 1.4 
               
               
                   
                 TGSEN + Q192S + N452P 
                 5.8 
               
               
                   
                 TGSHN + Q192S + N452P 
                 4.0 
               
               
                   
                 TGSQN + Q192S + N452P 
                 5.1 
               
               
                   
                 TGSRN + Q192S + N452P 
                 1.5 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
               
                   
               
               
                 TGSXN + Q192G + L193E + N452P 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSAN + Q192G + L193E + N452P 
                 4.1 
               
               
                   
                 TGSFN + Q192G + L193E + N452P 
                 nd 
               
               
                   
                 TGSHN + Q192G + L193E + N452P 
                 2.3 
               
               
                   
                 TGSIN + Q192G + L193E + N452P 
                 1.6 
               
               
                   
                 TGSLN + Q192G + L193E + N452P 
                 1.4 
               
               
                   
                 TGSNN + Q192G + L193E + N452P 
                 0.6 
               
               
                   
                 TGSQN + Q192G + L193E + N452P 
                 8.4 
               
               
                   
                 TGSTN + Q192G + L193E + N452P 
                 nd 
               
               
                   
                 TGSVN + Q192G + L193E + N452P 
                 2.3 
               
               
                   
                 TGSCN + Q192G + L193E + N452P 
                 44 
               
               
                   
                 TGSDN + Q192G + L193E + N452P 
                 7.2 
               
               
                   
                 TGSMN + Q192G + L193E + N452P 
                 7.3 
               
               
                   
                 TGSKN + Q192G + L193E + N452P 
                 2.4 
               
               
                   
                 TGSPN + Q192G + L193E + N452P 
                 5.4 
               
               
                   
                 TGSRN + Q192G + L193E + N452P 
                 3.1 
               
               
                   
                 TGSSN + Q192G + L193E + N452P 
                 5.2 
               
               
                   
                 TGSWN + Q192G + L193E + N452P 
                 2.7 
               
               
                   
                 TGSYN + Q192G + L193E + N452P 
                 3.1 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
               
                   
               
               
                 TGSXN + Q192S + N452P 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSNN 
                 42 
               
               
                   
                 TGSKN 
                 43 
               
               
                   
                 TGSRN 
                 43 
               
               
                   
                 TGSWN 
                 33 
               
               
                   
                 TGSNN + Q192S 
                 12 
               
               
                   
                 TGSKN + Q192S 
                 14 
               
               
                   
                 TGSRN + Q192S 
                 8.1 
               
               
                   
                 TGSWN + Q192S 
                 7.2 
               
               
                   
                 TGSNN + N452P 
                 14 
               
               
                   
                 TGSKN + N452P 
                 16 
               
               
                   
                 TGSRN + N452P 
                 15 
               
               
                   
                 TGSWN + N452P 
                 10 
               
               
                   
                 TGSNN + Q192S + N452P 
                 1.9 
               
               
                   
                 TGSKN + Q192S + N452P 
                 1.8 
               
               
                   
                 TGSRN + Q192S + N452P 
                 1.5 
               
               
                   
                 TGSWN + Q192S + N452P 
                 1.2 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
               
                   
               
               
                 TGSNN + Q192S + L193X + A318Y + N452P 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSNN + Q192S + L193M + N452P 
                 0.8 
               
               
                   
                 TGSNN + Q192S + A318Y + N452P 
                 1.5 
               
               
                   
                 TGSNN + Q192S + L193G + A318Y + N452P 
                 1.0 
               
               
                   
                 TGSNN + Q192S + L193A + A318Y + N452P 
                 8.8 
               
               
                   
                 TGSNN + Q192S + L193K + A318Y + N452P 
                 0.5 
               
               
                   
                 TGSNN + Q192S + L193R + A318Y + N452P 
                 8.8 
               
               
                   
                 TGSNN + Q192S + L193H + A318Y + N452P 
                 9.0 
               
               
                   
                 TGSNN + Q192S + L193D + A318Y + N452P 
                 7.8 
               
               
                   
                 TGSNN + Q192S + L193E + A318Y + N452P 
                 6.6 
               
               
                   
                 TGSNN + Q192S + L193N + A318Y + N452P 
                 1.4 
               
               
                   
                 TGSNN + Q192S + L193Q + A318Y + N452P 
                 2.2 
               
               
                   
                 TGSNN + Q192S + L193S + A318Y + N452P 
                 1.0 
               
               
                   
                 TGSNN + Q192S + L193T + A318Y + N452P 
                 8.5 
               
               
                   
                 TGSNN + Q192S + L193Y + A318Y + N452P 
                 8.1 
               
               
                   
                 TGSNN + Q192S + L193C + A318Y + N452P 
                 8.9 
               
               
                   
                 TGSNN + Q192S + L193M + A318Y + N452P 
                 2.4 
               
               
                   
                 TGSNN + Q192S + L193F + A318Y + N452P 
                 8.4 
               
               
                   
                 TGSNN + Q192S + L193W + A318Y + N452P 
                 9.0 
               
               
                   
                 TGSNN + Q192S + L193V + A318Y + N452P 
                 0.9 
               
               
                   
                 TGSNN + Q192S + L193I + A318Y + N452P 
                 7.7 
               
               
                   
                 TGSNN + Q192S + L193P + A318Y + N452P 
                 8.2 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
               
                   
               
               
                 TGSNN + Q192S + A318X + N452P 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSNN + Q192S + N452P 
                 1.9 
               
               
                   
                 TGSNN + Q192S + A318G + N452P 
                 2.6 
               
               
                   
                 TGSNN + Q192S + A318K + N452P 
                 0.0 
               
               
                   
                 TGSNN + Q192S + A318R + N452P 
                 3.0 
               
               
                   
                 TGSNN + Q192S + A318H + N452P 
                 2.0 
               
               
                   
                 TGSNN + Q192S + A318D + N452P 
                 2.1 
               
               
                   
                 TGSNN + Q192S + A318E + N452P 
                 2.3 
               
               
                   
                 TGSNN + Q192S + A318N + N452P 
                 2.4 
               
               
                   
                 TGSNN + Q192S + A318Q + N452P 
                 2.8 
               
               
                   
                 TGSNN + Q192S + A318S + N452P 
                 nd 
               
               
                   
                 TGSNN + Q192S + A318T + N452P 
                 5.1 
               
               
                   
                 TGSNN + Q192S + A318Y + N452P 
                 2.3 
               
               
                   
                 TGSNN + Q192S + A318C + N452P 
                 nd 
               
               
                   
                 TGSNN + Q192S + A318M + N452P 
                 3.0 
               
               
                   
                 TGSNN + Q192S + A318F + N452P 
                 3.0 
               
               
                   
                 TGSNN + Q192S + A318W + N452P 
                 0.1 
               
               
                   
                 TGSNN + Q192S + A318V + N452P 
                 0.4 
               
               
                   
                 TGSNN + Q192S + A318L + N452P 
                 3.0 
               
               
                   
                 TGSNN + Q192S + A318I + N452P 
                 4.5 
               
               
                   
                 TGSNN + Q192S + A318P + N452P 
                 nd 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
               
                   
               
               
                 TGSNN + Q192G + L193E + A318X + N452P 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSNN + Q192G + L193E + N452P 
                 0.6 
               
               
                   
                 TGSNN + Q192G + L193E + A318G + N452P 
                 0.7 
               
               
                   
                 TGSNN + Q192G + L193E + A318K + N452P 
                 0.7 
               
               
                   
                 TGSNN + Q192G + L193E + A318R + N452P 
                 0.7 
               
               
                   
                 TGSNN + Q192G + L193E + A318H + N452P 
                 0.8 
               
               
                   
                 TGSNN + Q192G + L193E + A318D + N452P 
                 1.0 
               
               
                   
                 TGSNN + Q192G + L193E + A318E + N452P 
                 0.9 
               
               
                   
                 TGSNN + Q192G + L193E + A318N + N452P 
                 0.6 
               
               
                   
                 TGSNN + Q192G + L193E + A318Q + N452P 
                 0.5 
               
               
                   
                 TGSNN + Q192G + L193E + A318S + N452P 
                 0.6 
               
               
                   
                 TGSNN + Q192G + L193E + A318T + N452P 
                 nd 
               
               
                   
                 TGSNN + Q192G + L193E + A318Y + N452P 
                 1.0 
               
               
                   
                 TGSNN + Q192G + L193E + A318C + N452P 
                 0.7 
               
               
                   
                 TGSNN + Q192G + L193E + A318M + N452P 
                 0.5 
               
               
                   
                 TGSNN + Q192G + L193E + A318F + N452P 
                 0.8 
               
               
                   
                 TGSNN + Q192G + L193E + A318W + N452P 
                 1.0 
               
               
                   
                 TGSNN + Q192G + L193E + A318V + N452P 
                 nd 
               
               
                   
                 TGSNN + Q192G + L193E + A318L + N452P 
                 0.6 
               
               
                   
                 TGSNN + Q192G + L193E + A318I + N452P 
                 1.9 
               
               
                   
                 TGSNN + Q192G + L193E + A318P + N452P 
                 nd 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
               
                   
               
               
                 TGSI(K, R, W)N 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSNN 
                 42 
               
               
                   
                 TGSKN 
                 43 
               
               
                   
                 TGSRN 
                 43 
               
               
                   
                 TGSWN 
                 33 
               
               
                   
                 TGSNN + Q192S 
                 12 
               
               
                   
                 TGSKN + Q192S 
                 14 
               
               
                   
                 TGSRN + Q192S 
                 8.1 
               
               
                   
                 TGSWN + Q192S 
                 7.2 
               
               
                   
                 TGSNN + N452P 
                 14 
               
               
                   
                 TGSKN + N452P 
                 16 
               
               
                   
                 TGSRN + N452P 
                 15 
               
               
                   
                 TGSWN + N452P 
                 10 
               
               
                   
                 TGSNN + Q192S + N452P 
                 1.9 
               
               
                   
                 TGSKN + Q192S + N452P 
                 1.8 
               
               
                   
                 TGSRN + Q192S + N452P 
                 1.5 
               
               
                   
                 TGSWN + Q192S + N452P 
                 1.2 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
               
                   
               
               
                 TGSN 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGSN 
                 nd 
               
               
                   
                 TGSN + N452P 
                 38 
               
               
                   
                 TGSN + Q192S + N452P 
                 9.5 
               
               
                   
                 TGSN + Q192G + L193E 
                 11 
               
               
                   
                 TGSN + Q192G + L193E + N452P 
                 2.5 
               
               
                   
                 TGSN + Q192S + L193M 
                 16 
               
               
                   
                 TGSN + Q192S + L193M + N452P 
                 5.5 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
               
                   
               
               
                 TGNN 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 TGNN 
                 38 
               
               
                   
                 TGNN + N452P 
                 22 
               
               
                   
                 TGNN + Q192S + N452P 
                 5.3 
               
               
                   
                 TGNN + Q192G + L193E 
                 11 
               
               
                   
                 TGNN + Q192G + L193E + N452P 
                 2.1 
               
               
                   
                 TGNN + Q192S + L193M 
                 17 
               
               
                   
                 TGNN + Q192S + L193M + N452P 
                 2.3 
               
               
                   
                   
               
            
           
         
       
     
     As is clear from the tables, the modified PQQGDH of the present invention in all cases exhibited a reactivity for glucose that was higher than that for maltose. 
     Example 4 
     Fabrication and Evaluation of an Enzyme Sensor 
     20 mg carbon paste was added to 5 units of the modified PQQGDH of the present invention and freeze-dried. After thorough mixing, it was filled onto the surface of a carbon paste electrode that carries approximately 40 mg carbon paste, and polished on filter paper. The electrode was treated for 30 minutes at room temperature in 10 mM MOPS buffer (pH 7.0) containing 1% glutaraldehyde, and then treated for 20 minutes at room temperature in 10 mM MOPS buffer (pH 7.0) containing 20 mM lysine in order to block the glutaraldehyde. The electrode was equilibrated for at least one hour at room temperature in 10 mM MOPS buffer (pH 7.0). The electrode was stored at 4° C. 
     The glucose concentration was measured using the enzyme sensor prepared above. Glucose was quantitatively measured in the range from 0.1 mM to 5 mM using the enzyme sensor having immobilized the modified PQQGDH of the present invention. 
     Example 5 
     Measurement of the Enzymatic Activity Using a Purified Enzyme Preparation 
     A cation-exchange chromatography column packed with TSKgel CM-TOYOPEARL 650M (Tosoh Corporation) was equilibrated with 10 mM phosphate buffer at pH 7.0 and the wild-type crude enzyme or the modified PQQGDH crude enzymes obtained in Example 2 was adsorbed on the column. The column was washed with 750 mL 10 mM phosphate buffer (pH 7.0) and the enzyme was then eluted with 10 mM phosphate buffer (pH 7.0) containing from 0 to 0.2 M NaCl. The flow rate was 5 mL/minute. The fraction exhibiting GDH activity was collected and was dialyzed overnight against 10 mM MOPS-NaOH buffer (pH 7.0) to obtain an electrophoretically-homogeneous modified PQQGDH protein. The enzymatic activity of the purified enzyme preparations for glucose and maltose was measured as in Example 4. Typical results are shown in the following table. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 20 
               
             
            
               
                   
                   
               
               
                   
                 ENZYMATIC 
                   
               
               
                   
                 ACTIVITY 
                 Mal:Glu 
               
               
                   
                 (Glu; 4 mM) 
                 (%) 
               
            
           
           
               
               
               
               
            
               
                 MODIFIED PQQGDH 
                 (U/mg) 
                 4:4 
                 40:4 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 TGSNN + Q192G + L193E 
                 357 
                 4.1 
                 28* 
               
               
                 TGSNN + Q192S + N452P 
                 723 
                 3.5 
                 19* 
               
               
                 TGSNN + Q192G + L193E + N452P 
                 213 
                 0.56 
                   5.7 
               
               
                 TGSNN + Q192S + L193M + N452P 
                 285 
                 2.0 
                 16* 
               
               
                 TGSNN + Q192G + L193E + A318K + 
                 381 
                 0.99 
                   8.92 
               
               
                 N452P 
               
               
                 TGSNN + Q192G + L193E + A318Q + 
                 469 
                 0.55 
                   6.74 
               
               
                 N452P 
               
               
                   
               
               
                 *measured at Mal:Glu = 50:5 
               
            
           
         
       
     
     In addition, the enzymatic activity for glucose and maltose of the modified PQQGDH having the TGSNN+Q192G+L193E+N452P mutations is shown in  FIG. 1 . 
     Example 6 
     Comparison with Prior-Art Modified PQQGDHs 
     The enzymatic activity for glucose and maltose was measured as in Example 4 using the wild-type crude enzyme obtained in Example 2, the modified PQQGDH crude enzymes obtained in Example 2, and, for comparison, prior-art modified PQQGDHs lacking the mutation at amino acid residue G at Position 99 of PQQGDH but having the 1 or 2 or more amino acid substitutions at other positions. 
     The following table shows typical results for the enzymatic activity of the modified PQQGDH of the present invention having various mutations and substitution of 99G with TGSNN, a comparative PQQGDH having the same mutation(s) but not substitution at 99G and wild-type PQQGDH. The results are expressed by the activity for 4 mM glucose or 10 mM glucose and for the ratio of the activity for maltose to the activity for glucose (%, 4 mM maltose: 4 mM glucose or 10 mM: 10 mM). 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 21 
               
             
            
               
                   
                   
               
               
                   
                 ACTIVITY FOR 
                 Mal/Glu 
               
               
                   
                 GLUCOSE (U/mg) 
                 (%) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 4 mM 
                 10 mM 
                 4:4 
                 10:10 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 WILD TYPE 
                 47.69 
                 86.96 
                 73.5 
                 75.5 
               
               
                 TGSNN + Q192G + L193E 
                 10.63 
                 20.20 
                 4.2 
                 5.1 
               
               
                 Q192G + L193E 
                 6.11 
                 11.62 
                 9.0 
                 10.5 
               
               
                 TGSNN + Q192G 
                 15.90 
                 26.36 
                 9.1 
                 11.9 
               
               
                 Q192G 
                 19.30 
                 31.79 
                 20.2 
                 26.1 
               
               
                 TGSNN + Q192A 
                 26.26 
                 48.56 
                 7.8 
                 9.9 
               
               
                 Q192A 
                 21.44 
                 41.32 
                 15.2 
                 18.2 
               
               
                 TGSNN + Q192S 
                 35.83 
                 60.94 
                 12.9 
                 17.0 
               
               
                 Q192S 
                 26.66 
                 44.91 
                 28.0 
                 37.8 
               
               
                 TGSNN + N452P 
                 58.71 
                 110.01 
                 16.3 
                 18.7 
               
               
                 N452P 
                 56.53 
                 103.43 
                 26.4 
                 33.1 
               
               
                 TGSNN + Q192S + N452P 
                 37.98 
                 72.23 
                 1.5 
                 2.2 
               
               
                 Q192S + N452P 
                 28.93 
                 56.12 
                 5.0 
                 6.6 
               
               
                   
               
            
           
         
       
     
     As shown in the table, the enzymatic activity of the modified enzymes according to the present invention with the insertion of the TGSNN sequence for glucose is either maintained or increased compared to the corresponding modified enzymes having the same mutations but lacking the insertion of TGSNN. In addition, the maltose-versus-glucose activity ratio is reduced to about one-half to one-fifth, and the selectivity for glucose is thus improved. For example, it has already been reported that the substrate specificity is increased in the modified PQQGDH having a double mutation Q192G/L193E. The activity of the Q192G/L193E modified PQQGDH for 4 mM glucose was 6.1 U/mg and the 4 mM maltose-versus-4 mM glucose activity ratio was 9%. In contrast, the additional insertion of TGSNN provided an approximately 1.7-fold increase in the activity for 4 mM glucose to 10.63 U/mg, and a 2.14-fold increase in substrate specificity, with 4.2% for the 4 mM maltose-versus-4 mM glucose activity ratio. 
     As demonstrated by these results, the modified PQQGDH of the present invention has a high enzymatic activity for glucose and a high substrate specificity for glucose over maltose. 
     INDUSTRIAL APPLICABILITY 
     The present invention is useful for assaying glucose and particularly for measuring blood sugar level.