Creation of catalytic activity-improved hyperthermophilic PQQ-dependent aldose sugar dehydrogenase and its efficient use for high performance electro-device

Abstract

PQQ-dependent aldose sugar dehydrogenase (PQQ-ASD) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PaeASD) has great potential as an element for durable bioelectrodevices owing to its exceptional stability against high temperatures and across a broad pH spectrum. However, its application is constrained by low electric current output of the enzyme-immobilized electrodes, which is attributable to its low catalytic activity. A directed evolutionary approach was performed on PaeASD to improve enzyme activity, resulting in the identification of a PaeASD s24 mutant containing six amino acid substitutions, which exhibited a 16-fold higher specific activity than that of wild type. Although each single amino acid mutant among these substitutions exhibited lower enzyme activity than PaeASD s24, the double mutant R64Q/D350N showed enzyme activity comparable to that of PaeASD s24. These amino acids located in the vicinity of coenzyme PQQ within the PaeASD molecule are also highly conserved with those of PQQ-ASDs reported to date. Thus, these amino acids play crucial roles in the catalytic activity of PQQ-ASD. Furthermore, the K_m value for d-glucose of PaeASD s24-immobilized electrode decreased to approximately 1/3 that of the wild-type-immobilized electrode. These results indicate that the PaeASD s24 mutant is an excellent catalyst for potential bioelectrodevice applications.