Thermal and operational stabilities of Hansenula polymorpha alcohol oxidase

Abstract

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. The immobilisation of AOX onto controlled-pore glass (CPG) beads allowed the use of mini packed-bed bioreactors (31 mm 3) 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 H 2O 2, 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.