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-6sulphonic acid) incubated at 5−30 o 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.

Laccase and Biomass Production via Submerged Cultivation of Pleurotus ostreatus Using Wine Lees

“…The activation energy of the purified laccase was calculated as 20.00 ± 0.17 kJ/mol using ABTS as a substrate; this value is close to one obtained for laccase produced by P. ostreatus ATCC 56270 (16.3 kJ/mol), whereas another study for P. ostreatus laccase revealed a higher activation energy of 27.06 kJ/mol, which also used ABTS as its substrate [53,54]. A much lower activation energy was calculated for Pleurotus florida laccase using guaiacol as a substrate, 3.9 kJ/mol, whereas 12 kJ/mol was reported for P. sajor-caju laccase using ABTS as its substrate [55,56].…”

Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity

“…While laccase from C. gallica conjugated with mPEG had a higher catalytic activity on syringaldazine than the native enzyme. In the case of laccase of T. hirsuta , the PEGylation resulted in an enhanced enzyme stability while with increasing molecular weight of attached mPEG (5, 2 and 1.10 kDa) the substrate affinity for the laccase conjugated decreased (Vandertol‐Vanier et al ., ; Lopez‐Cruz et al ., ; Schroeder et al ., ; Kim et al ., ). The PEGylation reaction used in the above mentioned reports is usually designated as random and results in complex mixtures of conjugates with different numbers and distributions of bound mPEG chains (Gaberc‐Porekar et al ., ).…”

Preliminary Study on Biosensor-Type Time-Temperature Integrator for Intelligent Food Packaging

“…However, in our previous work using a laccase-based TTI, the E a of another redox enzyme that was also very low was successfully increased. In a previous study, the E a was increased and regulated by adding sodium azide, a competitive inhibitor of laccase, or by making a mixture of isoenzymes with a different activation energy [ 32 , 33 ]. Therefore, the low E a color of the GOx-based TTI could be increased.…”