IMPROVED SUBSTRATE-SPECIFICITY AND DYNAMIC-RANGE FOR GLUCOSE MEASUREMENT OF ESCHERICHIA-COLI PQQ GLUCOSE-DEHYDROGENASE BY SITE-DIRECTED MUTAGENESIS

Site directed mutagenesis study was carried out with Escherichia coli pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) by substitutio n of His775 with either Asn (H775N) or Asp (H775D). The mutated PQQGDH s had different substrate specificity and catalytic activity from the wild type PQQGDH. The K-m values of H775N for 2-deoxy-D-glucose and fo r D-allose increased for 10-fold. The K-m values for both D-mannose an d D-galactose were estimated much higher than 100 mM. H775D also showe d the increase in K-m values toward saccharides. As a result, these mu tants possessed narrower substrate specificity than wild type E. coli PQQGDH. H775D showed the increase in K-m value for glucose versus wild type PQQGDH (25-fold), therefore H775D is suitable for the direct mea surement of blood glucose. The role of His775 in E. coli. PQQGDH is al so discussed.