title: Peroxidase activity in the sulfate-reducing bacterium Desulfotomaculum acetoxidans DSM 771.
abstract: Earlier research demonstrated the secretion of benzoate, which must be oxygenated to its 4-hydroxy derivative in order to be included in further sulfate uptake processes. The present study on Desulfotomaculum acetoxidans DSM 771 was designed to determine the activity and catalytic specificity of the enzyme (most probably peroxidase) catalyzing the hydroxylation of secreted benzoate. Peroxidase activity measured with ABTS (2,2'-azino-bis (3-ethylbenzathiazoline-6-sulfonic acid) during cultivation indicated the greatest activity on the third and thirteen days (3.4 and 2.3 nkat per ml sample respectively). The highest (0.7979) correlation coefficient was calculated between peroxidase activity and hydrogen peroxide levels. The cell walls from 3- and 13-day cultures were subjected to an isolation procedure, PIPES (piperazine-N,N'-bis (2-ethane-sulfonic acid) extract followed by preparative electrophoresis. The extracts of a approximately 30 kDa band on the gel were analyzed by Western blotting and the membrane was stained with TMB (3,3',5,5'-tetramethylbenzidine-specific for the presence of peroxidase). This same protein was incubated for 6 h with benzoate, H2O2, Na2SO4. The product formed a complex with Fe3+, whose maximum absorption spectra (501.7 nm) corresponded with a ferric complex of synthetic 4-hydroxy-3-sulfo-benzoate. The H2S level during the cultivation was higher in culture grown with 15.5 mM 4-hydroxy-3-sulfo-benzoate than in culture with lactate supplemented with 15.5 mM sulfate. The role of peroxidase in oxygen utilization and sulfate uptake is discussed.
authors.affiliation: Institute of Biology, Pedagogical University of Cracow, Kraków, Poland. Ipc@ap.krakow.pl
authors.forename: Lucyna
authors.initials: L
authors.lastname: Pawłowska-Cwiek
mesh_terms: D001565:Benzoates / Q000032:analysis / Q000378:metabolism; D002473:Cell Wall / Q000201:enzymology; D045859:Desulfotomaculum / Q000201:enzymology* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism; D006862:Hydrogen Sulfide / Q000032:analysis / Q000378:metabolism; D010100:Oxygen / Q000032:analysis / Q000378:metabolism; D010544:Peroxidases / Q000302:isolation & purification / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism; D013997:Time Factors
chemical_list: D001565:Benzoates; D013431:Sulfates; D006861:Hydrogen Peroxide; D010544:Peroxidases; D010100:Oxygen; D006862:Hydrogen Sulfide