Files
Abstract
Enzymes have been widely used in the food industries as biocatalysts and enzyme biosensors. However, many industrial applications of enzymes are hindered by their low stability or activity. Immobilization is a common method to stabilize enzymes. High hydrostatic pressure (HHP), normally used to kill microorganisms and inactivate enzymes in food processing, is reported to be able to stabilize and activate enzymes as well. Previously, our laboratory reported the stabilizing effect of HHP on the stability of glucose oxidase (GOx) and alcohol oxidase (AOx). We also reported the activating effect of HHP on GOx. In this study, the effect of HHP combined with immobilization in electrochemically generated poly-o-phenylenediamine (PoPD) nano-films on the stability of GOx or AOx biosensors is reported. The effect of HHP on the activity of AOx is also reported. Immobilized GOx inactivated at 70 °C and 180 MPa was 87.6 times more stable than GOx in solution inactivated at 70 °C and atmospheric pressure. Alcohol oxidase from P. pastoris has two enzyme fractions, the labile (L) fraction and the resistant (R) fraction. The R fraction is of more interest because it is ~150 times more stable than the L fraction. The reaction rate of the R fraction AOx at 50 °C was 17.9 ± 3.6 or 17.7 ± 0.8 µM min-1 at 80 or 160 MPa, respectively. It was approximately 6 times relative to the reaction rate of 3.2 ± 0.2 µM min-1 at 25 °C and atmospheric pressure. The increase in the activity of the R fraction AOx attributed to the stabilizing effect of HHP. The effect of HHP on the stability of the AOx biosensors in terms of the stability of AOx could not be determined. The pseudo-first-order rate constant of response loss of the immobilized AOx was higher than the pseudo-first-order rate constant of inactivation of the R fraction AOx in solution, suggesting the decrease in the effective amperometric response was caused by enzyme leaching out of the immobilization matrix. Indeed, the thickness of PoPD is similar to the diameter of AOx. Therefore, electrochemically generated PoPD is not an adequate immobilization material for AOx.