Kinetic study of a purified anionic peroxidase isolated from Eupatorium odoratum and its novel application as time temperature indicator for food materials

Abstract

An anionic peroxidase isoenzyme having a pI of 3.5 was purified from Eupatorium odoratum, commonly called, 'siam weed', belonging to family, Asteraceae. The molecular weight of the enzyme was identified as 55 kD. The specific activity of the crude extract was increased to 647 U/mg from 62 U/mg by ammonium sulfate precipitation. The enzyme was 114-fold purified by ion exchange chromatography and had a specific activity of 7094 IU/mg. The specificity constant (k(cat)/K-m) of the isozyme was 8.75 x 10(5) s(-1) M-1 with ABTS and 6.9 x 10(5) s(-1) M-1 With H2O2 as substrates. The enzyme was found to be very stable at room temperature (30 +/- 2 degrees C) and retained more than 90% activity even after a period of 2 months and was stable for more than 6 months at 4 +/- 1 degrees C without any additive, stabilizer or preservative. The activation energy for inactivation (Ea) of the isozyme was 120.14 kJ mol(-1) and the half-life was found to be around 34 h at 50 degrees C. The purified Eupatorium peroxidase has an optimum pH of 4.5 and optimum temperature of 55 degrees C. This isozyme was stable in metal ionic solutions and showed increased activity in presence of Hg2+, K+ and Ca2+. The enzyme can also be used as a low cost time-temperature indicator strip for which the preliminary works have already been carried out satisfactorily.