Purification and Characterization of An Extracellular Peroxidase of a Bacterial Isolate Bacillus sp. F31

Neelam      Bansal; Shamsher   S.   Kanwar

doi:10.2174/22115501113029990009

Abstract

An extracellular peroxidase produced by a bacterial isolate Bacillus sp. F31 was purified by successive (NH₄)₂SO₄ fractionation, dialysis and DEAE-cellulose anion-exchange chromatography to 14.5-fold with a yield of ~7.0%. The molecular mass of purified peroxidase was determined to be ~37 kDa by SDS-PAGE. The optimum temperature and pH for purified peroxidase were 37°C and 5.5, respectively. However, the bacterial peroxidase was active at an alkaline pH too, unlike previously reported microbial peroxidases. The K_m(H2O2) and V_max(H2O2) of the bacterial peroxidase by using H₂O₂ as a substrate were found to be 0.133 mM and 33.3 U/mg/min, respectively representing a K_cat(H2O2) of 20.53/sec. The Bacillus sp. F31 peroxidase exhibited good affinity towards OPD as observed from measured K_m(OPD) and V_max(OPD) of 5.138 mM and 29.41 U/mg/min, respectively (Fig. 11). The half-life of the purified peroxidase was approximately 155 min at 37°C. The peroxidase activity was found to be stimulated only in the presence of Mg⁺² (1, 3 and 5 mM) ions added to the reaction cocktail whereas other salt-ions (Li₂CO₃, ZnSO₄, KH₂PO₄, NaCl, HgCl₂, CaCl₂, CuSO₄, MnSO₄ and FeSO₄) inhibited the enzyme activity. All the selected detergents (SDS, Triton X-100, Tween 80 and Tween 20) also had an inhibitory effect on the enzyme activity.