Abstract

Thermal inactivation of a new recombinant Phlebia radiata manganese peroxidase (rPr-MnP3) in the presence and absence of additives (CaCl₂ and EDTA) is described for the first time. The influence of temperature and melting points (T_m) on the stability of rPr-MnP3 and its mutant (E40H/E44H) were determined. There was no significant inactivation at 25 – 40°C. However, we observed rapid inactivation of rPr-MnP3 at 50°C and above. Addition of CaCl₂ to the enzyme mixture resulted in a marked increase in the half-life (533 min) of the wild-type enzyme compared to E40H/E44H with the half-life of 92 min. Ethylenediaminetetraacetic acid (EDTA) increased the rate of rPr-MnP3 thermal inactivation as shown by the decay constant (k_d) of 0.070 ± 0.007 min^-1 and half-life of 10 min. The decay constant (k_d) 0.029 ± 0.002 min^-1 and half-life of 24 min were obtained for the control (untreated sample). Calcium ion had protective effect on the inactivation of the wild-type enzyme but not for mutant. The mutant (E40H/E44H) was observed to be more stable with a higher melting point of 58°C than the wild-type (Tm =54°C).The inactivation effect of EDTA on the E40H/E44H was lower than that of the wild-type. Calcium ions were found to be important structural elements responsible for the enzyme stability. Our findings showed that rPr-MnP3 is a highly stable enzyme and may be of significant industrial applications.   

Key words: Peroxidase, Phlebia radiata, thermal stability, thermal inactivation, reactivation, melting point, additives.