Thermal and operational stabilities of Hansenula polymorpha alcohol oxidase

The thermal stability of Hansenula polymorpha alcohol oxidase (AOX) was evaluated at 50 °C. The stability of free AOX was dependent of pH, buffer and additives but independent of protein concentration. More than 80% of the initial activity was retained after 9 h in the presence of additives, such as lactose, dextran sulphate and PEG 400 and combinations thereof. In the specific case of 0.01% dextran sulphate and 50 mM lactose no activity was lost for 9 h. Salts (ammonium sulphate and chloride) had a strong destabilising effect on the enzyme. mobilisation of AOX onto controlled-pore glass (CPG) beads allowed the use of mini packed-bed bioreactors (31 mm3) to monitor ethanol concentration. The conversion decrease (80% after 4 h) during continuous oxidation of ethanol at 32 °C in phosphate buffer was attributed to inactivation by hydrogen peroxide rather than thermal deactivation. Accordingly, an in situ stabilisation strategy was devised, which consisted in promoting the instantaneous consumption of H2O2, by horseradish peroxidase (HRP) and its reducing substrates, phenol-4-sulfonic acid and 4-aminoantipyrine. This strategy led to high operational stabilities (more than 10 h with no loss in conversion degree) and was successfully applied in a flow injection analysis (FIA) system for ethanol analysis.