RESUMO
Glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was purified about 5240-fold, using an aqueous two-phase system and triazine-dye affinity chromatography. The specific activity of the purified preparation was about 460 units/mg of protein with a final recovery of enzyme activity of about 75%. The affinity column could be regenerated and reused again several times. The purified enzyme appeared to be homogeneous when analyzed both on SDS-PAGE and native PAGE. The protein band on native PAGE coincided with the activity stain. ATP acts apparently as a competitive inhibitor for this enzyme with respect to NAD and protects the enzyme from dissociation into partially inactive dimers. In the absence of either glycerol or ATP, the enzyme dissociates into partially inactive dimers.
