Peroxidase time-temperature indicator

The second time-temperature indicator is based on the use of horseradish peroxidase in liquid and solid paraffin (126). The enzyme is deposited onto non-porous glass beads md mixed with melted paraffin containing the substrate. The suspension is mixed well and quickly cooled in a dry ice/acetone bath. When the enzyme is stored in solid paraffin, the activity is extremely slow. But when the temperature increases, the paraffin may melt and thereby make the enzyme reaction a millionfold faster than in the solid hydrocarbon phase. This time-temperature integrator is based on the same concept as the I-point, but here the... 

Culture media partially optimized for P. chrysosporium (50) unless otherwise indicated, nmol/min. measured at room temperature per mg dry mycelial weight for vanillylacetoneas a Mn -dependent peroxidase substrate and veratiyl alcohol as a lignin peroxidase substrate 0 denotes activity below detectable limit while — indicates assay not performed. Time, at 39 C for P. chrysosporium or at 30 C for L edodes, after inoculation when maximum activity appears (d ). Interval over which C02 evolution from dehydrogenative polymerizate (DHP) of [ring- /- C]conifeiyl alcohol is measured (days after inoculation). Culture media partially optimized for L. edodes at 22 C (83). 

Fig. 25. Linear-sweep voltammograms of HjOj (56 pM) in 0.1 M KCl, 5 mM neomycin, 5 mM HEPES, pH 7.0, initiated at various times following addition of cytochrome c peroxidase to a concentration of 0.2 pM. Scan rate 8 mVs temperature = 0°C i) 30 s ii) 178 s in) 325 s iv) 750 s v) 1080 s. The position of E for cytoehrome C, the natural reductant of CcP is indicated.

Enzymes typically function at normal body temperature, 37°C. Their activities at higher temperatures are desirable for rate acceleration, but free enzymes denature at elevated temperatures. Upon intercalation in a-ZrRP (R = OH), both HRP and Hb showed peroxidase activities at over 85°C, observed for the first time, (258) while the free enzyme/protein deactivated rapidly at these temperatures with no activity. The maximum rate of the reaction (Vmax) increased 3.6-fold, while the concentration needed to achieve half the maximum rate (K ) decreased by 20% at these higher temperatures. Such high-temperature activities of enzymes/ proteins are unusual, and they indicate the promise of a-ZrRPs for enzyme stabilization in high-temperature applications. This strategy of enzyme stabilization in a-ZrRP may provide alternatives to thermophilic enzymes obtained from thermo-philes, and these may supplement thermostable enzymes obtained by protein engineering. 

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