Based on the characterization of a PCR mutation of water-soluble glucose dehydrogenase possessing pyrroloquinoline quinone (pQQ), PQQGDH-B, Ser231Cys, we have constructed a series of Ser231 variants. The replacement of Ser231 to Cys, Met, Leu, Asp, Asn, His, or Lys resulted in an increase in thermal stability. Among these variants, Ser231Lys showed the highest level of thermal stability and also showed high catalytic activity. Considering that Ser231Lys showed more than an 8-fold increase in its half-life during the thermal inactivation at 55 degrees C compared with the wild-type enzyme, and also retained catalytic activity similar to a wild-type enzyme, the application of this mutant enzyme as a glucose sensor constituent may develop into a stable glucose sensor construction.