Application of Mixture Rule to Determine Arrhenius Activation Energy of Time Temperature Integrator Using Mixture of Laccase from Pleurotus ostreatus and PEGylated Laccase from Trametes versicolor

Abstract

Two isoenzyme mixture system was developed to freely adjust the Arrhenius activation energy (Ea), which is one of the most important parameters to design a reliable time temperature integrators (TTIs). We hypothesized that the apparent Ea of a mixture of two isozymes would be expressed in a simple linear relationship with the ratio of the mixture, although it could be expressed with a rather complicate equation. To prove our hypothesis, laccase from Pleurotus ostreatus (Ea=27.06 kJ/mol) and PEGylated laccase from Trametes versicolor (Ea=50.35 kJ/mol) were used to prepare enzyme mixtures with ratios of 0, 0.25,0.5, 0.75, and 1.0. The enzyme activity was determined by the increase of absorbance of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) incubated at 5-30 degrees C, pH 5.0, and Ea for each enzyme mixture was determined to be in the range of 27.06-50.35 kJ/mol. Relationship between Ea and a ratio of the enzyme mixture not only turned out to be linear, but also was well fitted to the linear mixture rule. This newly found linear dependency is much simpler than kinetically derived one, presumably because microscopic reaction paths and thermodynamic parameters were combined and cancelled out, resulting in linearity. This finding is important in that a mixture of two enzymes with a proper ratio determined from the simple linear mixture rule can customize Ea of an enzymatic TTI. This easier and convenient method can offer a practical and reliable way to adjust Ea of an enzymatic TTI on demand.