Extended-Range Glucose Sensor Employing Engineered Glucose Dehydrogenases

Abstract

An enzyme glucose sensor with an expanded dynamic range was constructed using a novel strategy. This strategy was based on a new concept of utilizing protein-engineered enzymes with a different Michaelis constant, which allows for the expanded dynamic range. We used the engineered Escherichia coli pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) of which His775 was substituted for Asp which showed an increased Km value (25-fold). We first constructed the composite colorimetric analytical system employing the wild-type PQQGDH and His775Asp and evaluated its dynamic range. The composite colorimetric analytical system was constructed and showed a wide dynamic range of 0.5-30 mM with less than +/-5% error. The composite colorimetric analytical system, an extended-range colorimetric analytical system, enabled the determination of the concentration of glucose over a 30-fold range that could not have been achieved using the single colorimetric analytical system. Furthermore, we have demonstrated the composite amperometric glucose sensor employing the combination of His775Asn and His775Asp. The extended-range glucose sensor acquired not only the expanded dynamic range (3-70 mM) that covered both dynamic ranges of the single enzyme sensors but also the narrower substrate specificity of glucose due to the inherited property of engineered enzymes.