Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions.
GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k(cat)/K(M)(glucose)=93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k(cat)/K(M)(glucose)=27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM(ox)) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependent FM(ox) reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k(cat)/K(M)(FM(ox))=27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.
Centre de Recherche Paul Pascal, CRPP-UPR 8641-CNRS, 115 Avenue Albert Schweitzer, 33600 Pessac, France.
Olivier
O
Courjean
Nicolas
N
Mano
D001674:Bioelectric Energy Sources; D001709:Biotechnology; D001786:Blood Glucose; D055664:Electrochemical Techniques / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005947:Glucose / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010407:Penicillium / Q000201:enzymology*; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D001786:Blood Glucose; D011994:Recombinant Proteins; D005949:Glucose Oxidase; D005947:Glucose; D010100:Oxygen