Analysis of inducible protein factor degrading apo-catalase in Enterococcus faecalis

Abstract: Catalase is a heme-containing homo-tetrameric enzyme that catalyzes the degradation of hydrogen peroxide. Apo-catalase is in the bacterium Enterococcus faecalis produced also in the absence of heme. In this work I have enriched and studied proteolytic activity causing apo-catalase degradation in stationary growth phase E. faecalis cells. The activity was enriched from soluble cell free extract of a catalase null mutant strain through fractionation by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. I purified hexahistidyl- tagged holo- and apo-catalase from E. faecalis cells and identified the N-terminal amino acid sequence of the catalase polypeptide. The rate of degradation of purified apo-catalase was determined using two methods - Western blot and an in this work developed procedure called Loss of catalase activity assay. Several features of the apo-catalase degrading activity were characterized such as protease inhibitor sensitivity, optimal pH and temperature, and molecular size. A polypeptide of about 25 kDa dominated in highly enriched protease preparation and was tentatively identified as a peptidyl-prolyl cis-trans isomerase.