# Patent

## Title
Mutants of pyrroloquinoline quinine-dependent soluble glucose dehydrogenase

## Abstract

The present invention relates to novel mutants of PQQ s-GDH containing an amino acid substitution in position 428 of the protein sequence of the wild type PQQ s-GDH of Acinetobacter calcoaceticus (SEQ.ID. NO:2). The invention also relates to the use of said PQQ s-GDH mutants for the development of glucose electrodes of interest in the assay of glucose, in particular of blood glucose in diabetic subjects, and for implementing biofuel cells that utilize glucose as fuel.

## Background
The present invention relates to the field of the development of glucose electrodes that are of interest in glucose assay, in particular of blood glucose of diabetic subjects, and for the application of biofuel cells using glucose as fuel.
The present invention relates more particularly to mutants of the enzyme pyrroloquinoline-quinone soluble glucose dehydrogenase (also called PQQ s-GDH) which have advantageous properties relative to the wild-type enzyme.
Type 2 diabetes affects nearly two million people in France, to which we must add 600 000 who are unaware of their disease. In the United States the situation is even more critical. In the developed countries, diabetes is the main cause of blindness in the age range 20-65 years.
Monitoring and management of the disease are based on, among other things, daily determination of blood glucose and injection of insulin. Various companies offer glucose sensors that enable patients to measure their blood sugar level themselves. These sensors can be amperometric, potentiometric or coulometric; they are all based on the use of an enzyme capable of oxidizing glucose; the two principal enzymes being glucose oxidase and PQQ s-GDH.
The pyrroloquinoline quinone glucose dehydrogenases belong to the family of quinoproteins, which have been identified in Acinetobacter calcoaceticus; there are two types: one is a membrane enzyme and the other is a soluble enzyme. They catalyze the oxidation of glucose to D-gluconolactone and can be used as a supplier of electrons.
The PQQ s-GDH of Acinetobacter calcoaceticus is composed of a homodimer consisting of two subunits of about 50 kDa (apoenzyme) and a pyrroloquinoline quinone cofactor (PQQ) (Oubrié et al., J. Mol. Biol; 289, 319-333 (1999)). This enzyme has an active site, within which the oxidation of glucose is catalyzed in the presence of PQQ; this active site is composed of the amino acids located at positions 76, 144, 169, 343, 346 and 428.
PQQ s-GDH is of considerable industrial interest as it is easily obtainable in large amounts; it has thus become the main enzyme used in glucose sensors for self-monitoring of blood sugar level. The advantage of the quinoproteins is that they are not dependent on oxygen, in contrast to glucose oxidase which utilizes O2 as electron acceptor.
Based on its physicochemical properties, it is also conceivable to use PQQ s-GDH for making biofuel cells, the principle of which is based on the oxidation of glucose to produce an electric current.
The drawback of PQQ s-GDH is its low thermal stability, its low stability at physiological pH and its moderate activity.
Several authors have tried to develop variants of PQQ s-GDH in order to improve its properties.
“Mutant” or “variant” means a PQQ s-GDH whose protein sequence comprises the insertion, deletion and/or substitution of at least one amino acid relative to the protein sequence of the wild-type PQQ s-GDH; hereinafter, the reference nucleotide and protein sequences of PQQ s-GDH are those of the wild-type PQQ s-GDH of Acinetobacter calcoaceticus (respectively SEQ. ID. No. 1 and 2).
Mutations Aiming to Improve the Stability of PQQ s-GDH
U.S. Pat. No. 7,244,600 describes a PQQ s-GDH mutated in such a way that the two subunits are joined together by a disulfide bridge. The mutation consists of replacing at least one amino acid at positions 415 and 414 and/or simultaneously the two amino acids located at positions 340 and 418 with a cysteine residue. These modifications endow the enzyme with better thermal stability.
Mutation Aiming to Make PQQ s-GDH Less Sensitive to Inhibition by its Substrate
U.S. Pat. No. 7,244,581 describes a PQQ s-GDH in which at least one amino acid in the region 349-377 is replaced with a different amino acid. This modification leads to an enzyme which is less sensitive to inhibition by the substrate and can thus be used in the presence of high concentrations of glucose.
Mutations Aiming to Make PQQ s-GDH More Specific to its Natural Substrate (Glucose)
It is also possible to modify the wild-type PQQ s-GDH to make it more substrate-specific. An enzyme that is specific to a substrate will only catalyze the reaction involving said substrate, and conversely, an enzyme with low substrate specificity is able to catalyze reactions based on substrates structurally similar to the natural substrate. The natural substrate of PQQ s-GDH is glucose but the wild-type PQQ s-GDH has low specificity and is also able to catalyze the oxidation of other monosaccharides, and disaccharides.
Several authors have proposed modifying the wild-type PQQ s-GDH in order to make it more specific to glucose:

    
    
        thus, patent application US 2007/0105173 proposes modifying PQQ s-GDH by substituting at least one of the amino acids located at positions 49, 67 to 69, 76, 89, 129 to 131, 167 to 170, 174, 188, 189, 207, 215, 245, 249, 300, 341 to 343, 349, 351 and 429 and/or by introducing an amino acid between positions 428 and 429;
        patent application EP 1 367 120 proposes modifying PQQ s-GDH by inserting a leucine, a lysine or an alanine between positions 428 and 429;
        U.S. Pat. No. 7,037,698 describes a substitution of the amino acid at one of the positions 75, 326 to 354, 278 to 320 and 162 to 197 of PQQ s-GDH;
        patent application US 2007/0243566 recommends inserting an amino acid between positions 428 and 429 and, optionally, substituting the amino acid at position 428 with a leucine, proline or valine of PQQ s-GDH;
        international application WO 2006/085509 describes mutants of the PQQ s-GDH of Acinetobacter having one or more substitutions at positions 125, 128, 142, 168, 169, 170, 224, 230, 236, 345, 351, 416 or 428; this document then lists specific mutants having numerous mutated sites;
        patent application EP 1 666 586 cites mutants of the PQQ s-GDH of Acinetobacter baumanii bearing the insertion of a leucine, an alanine or a lysine between positions 428 and 429 or mutants for which the amino acid of position 429 is substituted with a phenylalanine, a proline, a leucine or a tyrosine;
        U.S. Pat. No. 7,132,270 proposes substituting the amino acids of position 348 with an alanine, a glycine or a serine and of position 428 with a leucine, a proline or a valine.
    
    


For use in glucose sensors, it is necessary to have PQQ s-GDH mutants that are more active, i.e. which permit a faster reaction of transformation of glucose to D-gluconolactone, which is not possible with the existing mutants.
It is thus still necessary to develop a PQQ s-GDH that would display better activity than the wild-type PQQ s-GDH while retaining satisfactory or even improved thermal and pH stability.
To meet this need, the inventors have developed novel mutants of the wild-type PQQ s-GDH of Acinetobacter calcoaceticus; these mutants are such that the asparagine located at position 428 is substituted with a cysteine, a tyrosine, an alanine, an aspartate or a glutamate.
Thus, a first object of the invention relates to a PQQ s-GDH mutant having a percentage identity of at least 80% relative to the wild-type PQQ s-GDH of Acinetobacter calcoaceticus, characterized in that its amino acid located at position 428, referring to the protein sequence of the wild-type PQQ s-GDH of Acinetobacter calcoaceticus (SEQ. ID. No. 2), is substituted with an amino acid selected from the group comprising a cysteine, a tyrosine, an alanine, an aspartate or a glutamate.
During the research that led to the present invention, the inventors also prepared a PQQ s-GDH mutant characterized in that its amino acid located at position 428, referring to the protein sequence of the wild-type PQQ s-GDH of Acinetobacter calcoaceticus (SEQ. ID. No. 2), is substituted with a lysine ∘ (see examples, part 4); this mutant has the protein sequence SEQ. ID. No. 10 and is encoded by the nucleic acid molecule of SEQ. ID. No. 9. This mutant can be prepared with the oligonucleotides of SEQ. ID. No. 17 and 18 presented in Table I below.
The numbering of the amino acids refers to the sequence of the wild-type PQQ s-GDH of Acinetobacter calcoaceticus; the PQQ s-GDH mutants according to the present invention are not, however, limited to the mutants of the wild-type PQQ s-GDH of Acinetobacter calcoaceticus, the present invention also relates to the mutants of PQQ s-GDH having a percentage identity of at least 90% relative to the wild-type PQQ s-GDH of Acinetobacter calcoaceticus. 
The identity of a sequence relative to the sequence of the wild-type PQQ s-GDH of Acinetobacter calcoaceticus (SEQ. ID. No. 2) as reference sequence is evaluated as a function of the percentage of amino acid residues that are identical, when the two sequences are aligned, so as to obtain maximum correspondence between them.
The percentage identity can be calculated by a person skilled in the art using a computer program for comparing sequences, such as for example that of the BLAST suite (Altschul et al., NAR, 25, 3389-3402). The BLAST programs are employed on the comparison window consisting of the whole of SEQ. ID. No. 2 indicated as reference sequence.
A peptide having an amino acid sequence having at least X % of identity with a reference sequence is defined, in the present invention, as a peptide whose sequence can include up to 100-X alterations for 100 amino acids of the reference sequence, while preserving the functional properties of said reference peptide, in the present case its enzyme activity of oxidation of glucose. In the sense of the present invention, the term alteration includes deletions, substitutions or consecutive or dispersed insertions of amino acids in the reference sequence.
The amino acid corresponding to the amino acid located at position 428 of the PQQ s-GDH of Acinetobacter calcoaceticus is identified by alignment of the sequence of said homologous enzyme with the PQQ s-GDH of Acinetobacter calcoaceticus. 
The invention relates in particular to a PQQ s-GDH mutant having an amino acid sequence selected from SEQ. ID. No. 4, 6, 8, 12 and 14, corresponding respectively to the amino acid sequences of cysteine, tyrosine, alanine, aspartate and glutamate mutants of PQQ s-GDH; these mutated enzymes are encoded by nucleotide fragments obtained by mutation of the gene of the PQQ s-GDH of Acinetobacter calcoaceticus with suitable pairs of oligonucleotides.
These novel PQQ s-GDH mutants according to the invention display improved performance relative to the wild-type enzyme of Acinetobacter calcoaceticus, which is the enzyme used in the commercially available glucose sensors:

    
    
        their activity is greater than that of the wild-type enzyme, especially at physiological concentrations of glucose, i.e. between 1 and 10 mM;
    
    


The activity of the enzyme can be quantified by monitoring the coloration of redox reagents occurring during the reaction of oxidation of glucose to gluconolactone by PQQ s-GDH; the redox reagents are for example phenazine methosulfate (PMS) in combination with 2,6-dichlorophenolindophenol (DCIP), potassium ferricyanide, and ferrocene.

    
    
        they are less sensitive to the inhibitory effect of glucose at high concentration.
    
    


The specificity of a PQQ s-GDH mutant for a substrate can be evaluated by comparing the enzyme activity of said mutant on glucose and on several other sugars such as disaccharides as substrate.
The advantageous properties of the PQQ s-GDH mutants according to the invention make their use particularly suitable for bioelectric systems such as biofuel cells using glucose as the source of energy and glucose biosensors.
The present invention also relates to a nucleic acid molecule coding for a PQQ s-GDH mutant according to the invention; said nucleic acid molecule being obtained by modifying a wild-type PQQ s-GDH, such as that of Acinetobacter calcoaceticus, with a pair of oligonucleotides selected from the group consisting of the pairs of oligonucleotides shown in Table I.







TABLE I







list and sequence of the oligonucleotides used


for preparing the PQQ s-GDH mutants according


to the invention.








Oligonucleotides
sequences










Pair of oligonucleotides used for preparing


the alanine mutant (N428A)








N428A_Sense
gAT ACT gCC ggA gCT gTC CAA AAA



gAT



(SEQ. ID. No. 15)


N428A_Antisense
ATC TTT TTg gAC AgC TCC ggC AgT



ATC



(SEQ. ID. No. 16)


 







Pair of oligonucleotides used for preparing the


lysine mutant (N428K)








N428K_Sense
gAT ACT gCC ggA AAG gTC CAA AAA



gAT



(SEQ. ID. No. 17)


N428K_Anitsense
ATC TTT TTg gAC CTT TCC ggC AgT



ATC



(SEQ. ID. No. 18)


 







Pair of oligonucleotides used for preparing the


tyrosine mutant (N428Y)








N428Y_Sense
gAT ACT gCC ggA TAT gTC CAA AAA



gAT



(SEQ. ID. No. 19)


N428Y_Antisense
ATC TTT TTg gAC ATA TCC ggC AgT



ATC



(SEQ. ID. No. 20)


 







Pair of oligonucleotides used for preparing the


aspartate mutant (N428D)








N428D_Sense
gAT ACT gCC ggA gAC gTC CAA AAA



gAT



(SEQ. ID. No. 21)


N482D_Antisense
ATC TTT TTg gAC gTC TCC ggC AgT



ATC



(SEQ. ID. No. 22)


 







Pair of oligonucleotides used for preparing the


glutamate mutant (N428E)








N428E_Sense
gAT ACT gCC ggA gAA gTC CAA AAA



gAT



(SEQ. ID. No. 23)


N428E_Antisense
ATC TTT TTg gAC TTC TCC ggC AgT



ATC



(SEQ. ID. No. 24)


 







Pair of oligonucleotides used for preparing the


cysteine mutant (N428C)








N428C_Sense
gAT ACT gCC ggA TgT gTC CAA AAA



gAT



(SEQ. ID. No. 25)


N428C_Antisense
ATC TTT TTg gAC ACA TCC ggC AgT



ATC



(SEQ. ID. No. 26)









The nucleic acid molecules coding for the PQQ s-GDH mutants according to the invention can notably be prepared by modifying the nucleotide sequence of the gene coding for the wild-type enzyme of sequence SEQ. ID. No. 1 produced by Acinetobacter calcoaceticus. Several techniques for modifying the sequence of the gene are known by a person skilled in the art (see the review by Igarashi et al., Archives of Biochemistry and Biophysics 428 (2004) 52-63). According to a particular manner of preparation, the nucleic acid molecules coding for the PQQ s-GDH mutants according to the invention are prepared by mutagenesis by PCR in the presence of an oligonucleotide bearing the mutation to be introduced (see the example given below).
According to a particular embodiment, the present invention relates to a nucleic acid molecule coding for a PQQ s-GDH mutant according to the invention whose sequence is selected from the group consisting of the sequences SEQ. ID. No. 3, 5, 7, 11 and 13. The nucleic acid molecules coding for the PQQ s-GDH mutants according to the invention can then be cloned into an expression vector such as a plasmid, then transformed in a suitable host such as a bacterium, a yeast or a cell culture.
Expression vector means a vector possessing a region permitting the insertion of a coding nucleotide sequence between the signals that are indispensable for its expression, notably, a promoter (constitutive or inducible), a ribosome attachment site, a transcription termination signal and, optionally, a selection marker such as an antibiotic resistance gene.
The present invention further relates to an expression vector comprising said nucleic acid molecule and to a host cell transformed with said expression vector and expressing a PQQ s-GDH mutant according to the invention.
The expression vector can be introduced into the host cell by any method known by a person skilled in the art, in particular, by altering the membrane permeability of the host cell, for example in the presence of calcium ions, or by electroporation.
After culture of the host cells transformed for expressing a PQQ s-GDH mutant according to the invention, said cells can be recovered by centrifugation, lysed in order to release the enzymes including said PQQ s-GDH mutant according to the invention.
If Escherichia coli is the host microorganism, the plasmids that can be are used are notably the plasmids pBluescript, pUC18 or similar.
As examples, the host cells that can be used comprise Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, Escherichia coli JM101, Escherichia coli DH5α, etc.
Preferably, the PQQ s-GDH mutants according to the invention are produced in a strain of Escherichia coli JM101; the nucleic acid molecule that encodes them is obtained by modifying the gene of the PQQ s-GDH of Acinetobacter calcoaceticus and cloned into the vector pUC18 (Cleton-Jansen et al., Mol. Gen. Genet. 217 (1989) 430-436). The mutants thus produced are exported into the periplasm of the bacterium owing to the signal sequence of PQQ s-GDH. The mutants are then purified after disrupting the bacteria by sonication.
The invention also relates to the use of a PQQ s-GDH mutant according to the invention for determining glucose in solution, i.e. for measuring the concentration of glucose in a sample, notably a biological sample, in particular in blood.
Determination of glucose in solution in a given biological sample can be performed by introducing a redox reagent and a PQQ s-GDH mutant according to the invention into said sample and then comparing the intensity of coloration obtained with standard solutions with a known glucose content.
The present invention also relates to a kit for determination of glucose in solution, characterized in that it comprises a PQQ s-GDH mutant according to the invention.
Typically, said assay kit additionally contains the necessary reagents for carrying out the glucose assay, in particular buffers, any buffer can be used in the kit according to the invention, we may mention non-limitatively phosphate and acetate buffers, buffer with trishydroxymethylaminomethane (TRIS), with N-morpholino-3-propanesulfonic acid (MPOS), with 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), buffer comprising a mixture of buffers such as TRIS-acetate etc., the redox reagents can be any reagent for oxidizing the PQQ s-GDH mutant, they can be selected from the group comprising phenazine methosulfate (PMS) in combination with 2,6-dichlorophenolindophenol (DCIP); potassium ferricyanide; ferrocene and complexes derived from ferrocene such as ferrocenemethanol, ferrocene carboxylic acid; and complexes of osmium and ruthenium, standard glucose solutions for constructing calibration curves, and the instructions for use necessary for carrying out the assay.
The present invention further relates to glucose electrodes comprising a conductive material such as a conductive metal, notably platinum, copper, silver, aluminum, gold or steel or carbon, such as vitreous carbon, carbon fibers, carbon nanotube fibers or of diamond etc., said conductive material is covered with a deposit comprising at least one PQQ s-GDH mutant according to the invention; said deposit can further comprise a redox polymer for improving the conductive properties of the conductive material.
The redox polymer is selected from polymers based on ferrocene, osmium and ruthenium and conductive polymers such as polypyrrole and polyananilline.
The methods of immobilization of the PQQ s-GDH mutant on said conductive material can be selected from the conventional methods that are available to a person skilled in the art, which notably comprise inclusion of the PQQ s-GDH mutant in a polymer matrix, adsorption of the PQQ s-GDH mutant on the surface of the polymer membrane, fixation by covalent bonding, or electrodeposition (Gao et al., Chem. Int. ED. 2002, 41, No. 5, 810-813).
Preferably, the immobilized PQQ s-GDH mutant consists of the apoenzyme assembled with the cofactor PQQ; but it is also possible to immobilize the apoenzyme alone and supply the cofactor PQQ additionally, for example in solution in the reaction mixture.
Such electrodes are used advantageously in bioelectric systems such as glucose-based biofuel cells or glucose biosensors.
The present invention thus also relates to a glucose biosensor comprising an electrode according to the invention.
A glucose biosensor consists of an electrode, on which a bioreceptor is immobilized that is capable of recognizing a biological target; binding of the biological target to the bioreceptor leads to physicochemical changes of the membrane and the production of an electrical signal by an electrochemical transducer (amperometric, potentiometric, conductometric, etc.) attached to the electrode; in the present case the bioreceptor is a PQQ s-GDH mutant according to the invention and the biological target is its substrate: glucose.
According to one embodiment, the electrode on which the PQQ s-GDH mutant is immobilized is also covered with a membrane which prevents detachment of said mutant from the electrode. Said membrane can consist of Nafion, cellulose or any biocompatible material, i.e. compatible with a physiological environment.
According to one embodiment of the invention, the glucose biosensor is implanted under the skin and makes it possible to record the blood glucose concentration.
The present invention also relates to biofuel cells using glucose as the source of energy and comprising a first electrode according to the invention as anode and a second electrode as cathode. The cathode can be, for example, an enzymatic electrode that makes it possible to reduce oxygen bearing an enzyme selected from the class of enzymes based on copper (multi copper oxidases) and particularly bilirubin oxidase and laccase. It can also be a metallic electrode, for example of platinum, of gold or of a platinum or gold alloy.
The invention further relates to a method of solution assay of the glucose in a sample comprising the following steps:
a) introducing, into said sample, a redox reagent, reduction of which leads to a change of color, and a PQQ s-GDH mutant, according to the invention;
b) measuring the intensity of coloration of the sample after enzymatic reaction;
c) comparing the intensity of coloration measured in step b) with the intensity measured for standard solutions having a known glucose content;
d) determining the glucose concentration in said sample.
The redox reagent whose reduction leads to a change of color is selected from phenazine methosulfate (PMS) in combination with 2,6-dichlorophenolindophenol (DCIP), potassium ferricyanide, and ferrocene.
The invention also relates to a method of glucose assay of a sample, characterized in that it comprises the following steps:
a) introducing a glucose electrode according to the invention into said sample;
b) measuring the intensity of the current in the sample;
c) comparing the intensity of the current measured in step b) with the intensity measured for standard solutions having a known glucose content;
d) determining the glucose concentration in said sample.
Besides the arrangements presented above, the invention further comprises other arrangements which will become clear from the description given below, referring to examples of application of the present invention, as well as to the appended drawings in which:



FIGURES
FIGS. 1A and 1B are schematics showing the active site of the PQQ s-GDH of Acinetobacter calcoaceticus, wild-type (FIG. 1A) or mutated at position Asn428Cys (FIG. 1B). The figure was created with the PyMol software (published by DeLano Scientific LLC, version of 2006) according to the coordinates pdb lcql described by Oubrie et al. (1999). “G” denotes glucose and “PQQ” denotes the coenzyme PQQ. The numbering of the amino acid residues of the active site corresponds to SEQ. ID. No. 2.
FIGS. 2A to 2F are graphs showing the steady-state kinetic parameters in the presence of glucose or maltose:

    
    
        of the PQQ s-GDH of Acinetobacter calcoaceticus, wild-type (WT) and mutated at position 428 by a cysteine (N428C, FIG. 2A);
        of the PQQ s-GDH of Acinetobacter calcoaceticus mutated at position 428 by a tyrosine (N428Y, FIG. 2B);
        of the PQQ s-GDH of Acinetobacter calcoaceticus mutated at position 428 by an alanine (N428A, FIG. 2C);
        of the PQQ s-GDH of Acinetobacter calcoaceticus mutated at position 428 by a lysine (N428K, FIG. 2D);
        of the PQQ s-GDH of Acinetobacter calcoaceticus mutated at position 428 by an aspartate (N428D, FIG. 2E);
        of the PQQ s-GDH of Acinetobacter calcoaceticus mutated at position 428 by a glutamate (N428E, FIG. 2F).
    
    


FIGS. 3A and 3B show the activity as a function of pH of the PQQ s-GDH of Acinetobacter calcoaceticus, wild-type (WT) and mutated at position 428 by a cysteine (N428C).
FIG. 4 shows the activity curve of the wild-type (WT) PQQ s-GDH and mutated by a cysteine at position 428 (N428C) as a function of temperature.
FIG. 5 is a graph showing the activity of PQQ s-GDH mutated at position 428 by a cysteine (N428C) as a function of different substrates.



EXAMPLES
1. Materials
1.1. Bacterial Strains of Escherichia coli 
DH5α: supE44, ΔlacU169, (Φ80 lacZDM15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1 (Hanahan, 1983). This strain is used for plasmid preparation and directed mutagenesis.
JM101: F′traD36 proA+B+ lacIq Δ(lacZ)M15/Δ(lac-proAB) glnV thi. This strain is used for the production of PQQ glucose dehydrogenase of Acinetobacter calcoaceticus in conical flasks. This strain carries the plasmid F-pro-lacI which inhibits expression of the lac promoter.
1.2. Plasmid
pgp492: donated by Nora Goosen, recombinant plasmid obtained by cloning the coding sequence of the gene of the soluble PQQ glucose dehydrogenase of Acinetobacter calcoaceticus (Cleton-Jansen et al., Mol. Gen. Genet. 217 (1989) 430-436).
1.3. Culture Medium
LB rich medium: tryptone 10 g/L; yeast extract 5 g/L; NaCl 5 g/L; distilled H2O q.s. 1 L; pH not adjusted, autoclaved for 50 min at 1 bar.
2. Genetic Engineering Techniques
2.1. Transformation of Supercompetent Bacteria
The supercompetent bacteria DH5α are prepared by Inoue's method (Sambrook and Russell (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor Laboratory Press).
2.2. Preparation of the DNA
A plasmid DNA purification kit (Quiagen) is used for the DNA preparations in small and large amounts.
2.3. Sequencing of Double-Stranded DNA
Double-stranded DNA is sequenced with the sequencing kit BigDye Terminator v1.1 or v3.1. The reagent contains the 4 ddNTPs with different fluorescent markers (BigDyeTerminators), AmpliTaq DNA Polymerase, and all other components required for the reaction. The extension products must be purified before being passed through the sequencer ABI 3130xl, to remove the unincorporated markers, salts and other contaminants.
2.4. PCR-Directed Mutagenesis
In the mutagenesis protocol, PCR is performed with the DNA polymerase Pfu of Pyrococcus furiosus, six times more faithful than the polymerase Taq for replicating the two strands of the plasmid. The oligodeoxyribonucleotides listed in Table I, each complementary to a strand of the plasmid, will serve as primers for DNA synthesis by the DNA polymerase Pfu, which leads to a plasmid mutated at the non-religated ends.
The amplified product is then treated with the endonuclease Dpn I (target sequence 5′Gm6ATC-3′), specific to the parent DNA and thus makes it possible to select the neosynthesized DNA containing the mutation. The DNA isolated from most strains of E. coli is “methylated DAM” (DAM for DNA adenine methylase; indicates the presence of a methyl on the adenines of the GATC sequences) and can therefore be digested by Dpn I. A fraction of the digestion mixture is used for transforming supercompetent DH5α bacteria, which will religate the ends of the mutated plasmid.
3. Production, Purification and Characterization of the PQQ s-GDH of Acinetobacter calcoaceticus 
3.1. Production of the Wild-Type and Mutated PQQ s-GDH
The apoenzyme s-GDH is produced in the E. coli strain JM101 by the recombinant plasmid pgp492 bearing the sequence coding for the wild-type or mutated s-GDH. A preculture of 50 mL of LB medium supplemented with ampicillin (200 mg/L) (LBA) is seeded by a clone isolated on an LB agar dish supplemented with ampicillin (100 mg/mL) and is stirred at 220 rpm overnight at 37° C. The cultures are seeded at 1/100th in LBA medium. They are incubated at 37° C. with stirring (220 rpm) until OD600nm is between 0.4 and 1 OD600nm/mL. The cultures are then induced by 400 μM of IPTG and then stirred (180-220 rpm) at 25° C. for 20 h. The cells, collected by centrifugation (5285 g, 4° C.), are washed in a buffer of Tris 20 mM, CaCl2 3 mM pH 7.5 and stored at −20° C. or at −80° C. without additive.
3.2. Purification of Wild-Type and Mutated Enzymes PQQ s-GDH
3.2.1. Cell Disruption and Treatment with DNase I
The cellular pellet, obtained from a liter of culture, is taken up in 20 mL of buffer Tris 20 mM, CaCl2 3 mM, pH 7.5 and sonicated at a sonication power of 40 W for 3 minutes with cycling of one second of ultrasound and stopped for one second. The sample obtained, called raw extract, is supplemented with final 2 mM MgCl2 and treated for 30 minutes at room temperature with DNase I (1 U/mL of raw extract). The insoluble cellular debris is then removed from the raw extract by centrifugation at 20000 g for 45 minutes.
3.2.2. Cation Exchange Chromatography
The sonication supernatant, filtered on a filter with a cutoff threshold of 0.22 μm (Millex-GS 0.22 μm, Millipore) and diluted to an OD280nm of 10, is injected onto a cation exchange column source 30S (GE Healthcare®), coupled to the system AKTA purifier (GE Healthcare®), equilibrated in buffer Tris 20 mM, CaCl2 3 mM, pH 7.5. Elution is performed with a gradient of NaCl from 0 to 1 M in the same buffer at a flow rate of 5 mL/min. The fractions containing the s-GDH protein are combined and concentrated by filtration by centrifugation on Amicon YM10 membrane. At this stage, the s-GDH protein is pure and can be stored at −20° C. in the precipitated form in the presence of ammonium sulfate (90% saturation).
3.2.3 Characterization of the Wild-Type and Mutated s-GDH Enzymes
3.2.3.1. Determination of Molecular Weight
The molecular weight of the monomer of s-GDH after purification is determined by MALDI mass spectrometry. The protein is desalted on a PD10 column (GE Healthcare) and eluted in a buffer of ammonium bicarbonate 50 mM pH 7.5. The lyophilized sample is then analyzed by MALDI-TOF and a molecular weight of 50234.83 Da is found; this molecular weight corresponds to the theoretical molecular weight of the PQQ s-GDH without the signal peptide.
3.2.3.2. Reconstitution of s-GDH with Cofactor PQQ
At the end of purification (buffer Tris 20 mM, CaCl2 3 mM pH 7.5), the enzyme solution is preincubated for 15 minutes with PQQ at room temperature. The PQQ added corresponds to a final concentration equivalent to twice the molarity of the concentration of enzyme. The excess PQQ is then removed by desalting on a PD10 column (GE Healthcare) equilibrated in a buffer Pipes 20 mM, CaCl2 3 mM pH 7.
3.2.3.3. Measurement of Concentration
The concentration of enzyme in a solution is calculated from the OD measured at 280 nm, using for the enzyme (dimeric or monomeric), with or without cofactor PQQ, an extinction coefficient of 1.28 or 1.67 l·g−1·cm−1 respectively (Olsthoorn et al., 1997).
3.2.3.4. Enzymatic Test
The enzymatic tests are carried out using a Varian spectrophotometer in a buffer of sodium phosphate 20 mM pH 7 at 37° C. in a volume of 3 mL, monitoring disappearance by reduction of DCIP by means of PMS, which acts as electron acceptor, at 600 nm as a function of time. The specific activity of the enzyme is expressed in μmol of DCIP disappeared per minute per mg of protein. The concentrations of PMS and DCIP are 0.6 and 0.06 mM respectively. The enzyme is diluted so as to measure a slope between −0.05 and −0.2 OD600nm/min.
4. Techniques for Studying the Enzymatic Properties of the Wild-Type and Mutated PQQ s-GDH
4.1. Determination of the Kinetic Constant (kcat) and Michaelis Constant (KM) in the Steady State
The experiments are conducted at 37° C. on a Varian spectrophotometer in a buffer of sodium phosphate 20 mM pH 7. The concentration of substrate (glucose and maltose) in the test varies between 0 and 800 mM.
PMS is used as first electron acceptor and the enzyme activity is monitored at 600 nm from the reduction of the DCIP used as second electron acceptor. The initial concentrations of PMS and DCIP are respectively 0.6 and 0.06 mM. The test is started by adding enzyme. The experimental points are analyzed by nonlinear regression with equations 1, 2 or 3 by means of the Sigma-plot 6.0 software according to the following equations:

kss=kcat*[S]/(KM+[S])  (1) Michaelis-Menten model

kss=kcat1*[S]/(KM1+[S])+kcat2*[S]/KM2+[S])  (2) Double hyperbole

v/vmax=[S]/Ks*(1+[S]/KS+[I]/KI)  (3) Michaelis-Menten model with competitive inhibition

Results
The graphs in FIGS. 2A, 2B, 2C, 2D, 2E and 2F show the steady-state kinetic parameters of the soluble PQQ glucose dehydrogenase of Acinetobacter calcoaceticus, wild-type (WT) and mutated at position 428 (N428C, N428Y, N428A, N428K, N428D and N428E) in the presence of glucose or maltose.
These results show that, for the two substrates maltose and glucose, the mutated enzymes according to the invention display greater activity than that of the wild-type enzyme; some of these mutants are twice as active as the wild-type enzyme.
4.2. Investigation as a Function of pH
4.2.1. Activity as a Function of pH
Investigation of the variation of the reaction rate constant as a function of pH is performed over a range of pH from 5 to 9 by incubation of the wild-type and mutated enzymes either in a mixed buffer composed of Tris 120 mM, imidazole 30 mM, acetic acid 30 mM (TIA), the ionic strength of which is adjusted to 190 mM with NaCl, or in a buffer of sodium phosphate 20 mM in the pH range from 6 to 8 (NaPi). The experiments are conducted at 37° C. using a Varian spectrophotometer. PMS is used as first electron acceptor at a concentration of 0.6 mM. The activity is monitored by the disappearance of DCIP at 600 nm used as second electron acceptor at a concentration of 0.06 mM. The test is started by adding the enzyme. The optimal activity for the wild-type or mutated enzyme corresponds to 100% and the activity relating to each pH is shown.
Results
FIGS. 3A and 3B are graphs showing activity as a function of pH, of the PQQ s-GDH of Acinetobacter calcoaceticus, wild-type (WT) and mutated at position 428 by a cysteine (N428C). These results show that the cysteine mutant of PQQ s-GDH (N428C) displays a relative activity comparable to that of the wild-type enzyme, demonstrating that the mutation does not lead to a loss of relative activity whatever the pH.
4.2.2. Stability as a Function of pH
The stability as a function of pH of the wild-type or mutated PQQ s-GDH is determined by dilution of the enzyme purified to homogeneity in a mixed buffer in the pH range from 5 to 9. This mixed buffer is composed of Tris 120 mM, imidazole 30 mM, acetic acid 30 mM, the ionic strength of which is adjusted to 190 mM with NaCl. The diluted enzyme solution, between 1 and 6 μg/ml, is preincubated at 37° C. Various samples are taken as a function of time. The residual activity is measured at 37° C. using a Varian spectrophotometer in a buffer of sodium phosphate 20 mM pH 7 in the presence of 0.06 mM DCIP, 0.6 mM PMS. The glucose concentration is 75 mM for testing the wild-type enzyme and 150 mM for testing the mutant N428C.
Results
The mutated enzyme according to the invention displays better stability than the wild-type enzyme whatever the pH and whether this stability is constant over the range of pH tested, in particular, the mutated enzyme remains stable even at pH above 7.
4.3. Investigation as a Function of Temperature
4.3.1. Activity as a Function of Temperature
The variation of the reaction rate constant is investigated as a function of pH in a buffer of sodium phosphate 20 mM pH 7, in the presence of 0.06 mM of DCIP and 0.6 mM of PMS. The glucose concentration is 75 mM for testing the wild-type enzyme and 150 mM for testing the mutant N428C. The temperature varies from 10 to 60° C. The activity is monitored on a temperature-controlled Varian spectrophotometer CARY UV Biomelt. The test is started by adding the enzyme.
Results
FIG. 4 shows the curve of activity of the wild-type (WT) and mutated (N428) PQQ s-GDH as a function of temperature.
This curve shows that between 30 and 50° C., the mutated enzyme displays better activity than the wild-type enzyme.
4.3.2. Stability of the Enzyme as a Function of Temperature
The study is conducted in a buffer of sodium phosphate 20 mM pH 7, in the presence of 0.06 mM of DCIP and 0.6 mM of PMS. The glucose concentration is 75 mM for testing the wild-type enzyme and 150 mM for testing the mutant N428C. The temperature varies from 10 to 60° C. The activity is monitored at 37° C. on a temperature-controlled Varian spectrophotometer CARY UV Biomelt. The test is started by adding the enzyme.
Results
It can be seen that in an incubation time of less than 200 minutes, the mutated enzyme N428C displays better activity at 40 and 50° C. than the wild-type enzyme, thus demonstrating its advantage for blood glucose assay kits.
4.4. Investigation as a Function of the Substrate
The specificity of the cysteine mutant (N428C) was evaluated with respect to various substrates.
The protocol employed for this measurement of specificity is identical to that described in section 4.1., using different substrates.
The results obtained are presented in Table II below and in FIG. 5.







TABLE II







Comparison of the substrate specificity of the PQQ GDH of


Acinetobacter calcoaceticus, wild-type and mutated comprising a


cysteine at position 428 (mutant N428C).










WT
N428C














%

%
% Relative



kss s−1 at
Relative
kss s−1 at
Relative
activity/



4 mM
activity/WT
4 mM
activity/
N428C


Substrate
substrate
Glucose
substrate
WT
Glucose















Glucose
806
100
1396
173
100


Maltose
660
81
861
130
61


Ribose
86
10
67
77
4


Xylose
148
18
159
107
11


Galactose


357
26
25









Results
Even if the raw values of kss of the mutant N428C are higher for most substrates than those of the wild-type enzyme; the relative activity, which characterizes the specificity of an enzyme for one substrate relative to another substrate, shows that the mutant N428C is more specific to glucose. Thus, at 4 mM of substrate, with maltose as substrate, a relative activity with respect to glucose of 81% is measured for the wild-type enzyme whereas the mutant N428C displays a relative activity of maltose relative to glucose of 61%.
This same protocol was employed for evaluating the specificity for glucose and for maltose of the aspartate (N428D), glutamate (N428E), alanine (N428A), tyrosine (N428Y) and lysine (N428K) mutants.
The results are presented in Table III below:
















N428D
N428E














kss (s−1)
relative activity/
relative
kss (s−1)
relative activity/
relative



at 5 mM
N428D glucose
activity/
at 5 mM
N428E glucose
activity/


substrate
substrate
(%)
WT (%)
substrate
(%)
WT (%)





glucose
1214
100
150
1380
100
171


maltose
1103
 91
167
1182
 85
179













N428A
N428Y














kss (s−1)
relative activity/
relative
kss (s−1)
relative activity/
relative



at 5 mM
N428A glucose
activity/
at 5 mM
N428Y glucose
activity/


substrate
substrate
(%)
WT (%)
substrate
(%)
WT (%)





glucose
1058
100
131
1353
100
167


maltose
 638
 60
 97
 716
 52
108















N426K

















kss (s−1)
relative activity/
relative






at 5 mM
N428Y glucose
activity/


substrate
substrate
(%)
WT (%)





glucose
 940
100
110


maltose
 625
 66
 94

## Claims

1. An isolated mutant of pyrroloquinoline-quinone soluble glucose dehydrogenase (PQQ s-GDH) having a percentage identity of at least 90% relative to the PQQ s-GDH of Acinetobacter calcoaceticus of SEQ. ID. No. 2, wherein the amino acid located at position 428, referring to SEQ. ID. No. 2, is substituted with an amino acid selected from the group consisting of cysteine, tyrosine, alanine and glutamate, wherein the mutant has an amino acid sequence selected from the group consisting of SEQ. ID. No. 4, 6, 8 and 14.

2. An isolated nucleic acid molecule, coding for a mutant PQQ s-GDH as claimed in claim 1.

3. The nucleic acid molecule as claimed in claim 2, having a sequence selected from the group consisting of SEQ. ID. No. 3, 5, 7 and 13.

4. A recombinant expression vector, characterized in that it comprises a nucleic acid molecule as claimed in claim 2.

5. A recombinant E. coli expressing an isolated mutant as claimed in claim 1, characterized in that it is transformed with an expression vector which comprises a nucleic acid molecule, which codes for the mutant PQQ s-GDH.

6. A glucose assay kit, characterized in that it comprises a mutant of PQQ s-GDH as claimed in claim 1.

7. A glucose electrode, characterized in that it comprises a conductive material covered with a deposit comprising at least one mutant of PQQ s-GDH as claimed in claim 1.

8. A glucose sensor, characterized in that it consists of an electrode as claimed in claim 7.

9. A glucose biofuel cell, characterized in that it comprises a first electrode as claimed in claim 7 as anode and a second electrode as cathode.

10. A method of determining glucose in solution in a sample, characterized in that it comprises the following steps:
a) introducing a redox reagent, reduction of which leads to a change of color, and a PQQ s-GDH mutant as claimed in claim 1, into said sample;
b) measuring the intensity of coloration of the sample after enzymatic reaction;
c) comparing the intensity of coloration measured in step b) with the intensity measured for standard solutions having a known glucose content;
d) determining the glucose concentration in said sample.

11. A method of glucose assay of a sample, characterized in that it comprises the following steps:
a) introducing a glucose electrode as claimed in claim 7 into said sample;
b) measuring the intensity of the current in the sample;
c) comparing the intensity of the current measured in step b) with the intensity measured for standard solutions having a known glucose content;
determining the glucose concentration in said sample.