Showing papers by "Alfons J. M. Stams published in 1989"

Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii.

Abstract: In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii. Images

Purification and characterization of an oxygen-stable carbon monoxide dehydrogenase of Methanothrix soehngenii.

Abstract: Carbon monoxide dehydrogenase was purified to apparent homogeneity from Methanothrix soehngenii. In contrast with the carbon monoxide dehydrogenases from most other anaerobic bacteria, the purified enzyme of Methanothrix soehngenii was remarkably stable towards oxygen and it was only slightly inhibited by cyanide. The native molecular mass of the carbon monoxide dehydrogenase of Methanothrix soehngenii determined by gel filtration was 190 kDa. The enzyme is composed of subunits with molecular mass of 79.4 kDa and 19.4 kDa in an α2β2 oligomeric structure. The enzyme contains 1.9 ± 0.2 (n= 3) mol Ni/mol and 19 ± 3 (n= 3) mol Fe/mol and it constitutes 4% of the soluble cell protein. Analysis of enzyme kinetic properties revealed a Km of 0.7 mM for CO and of 65 μM for methyl viologen. At the optimum pH of 9.0 the Vmax was 140 μmol of CO oxidized min−1 mg protein−1. The enzyme showed a high degree of thermostability.

Sulphur and sulphate reduction with acetate and propionate in an aerobic process for sulphide removal.

Abstract: Sulphide production rates of sulphur-and sulphate-reducing bacteria up to 50 mg per biomass support particle per day were observed in an aerated sulphide-removal reactor with polyurethane (PUR) foam as carrier material. The optimal pH and temperature for the sulphide-producing bacteria were 8.0 and 30°C respectively. Raschig rings and four types of cube-shaped PUR particles were tested as carrier materials. When using PUR particles, the sulphide production rate was always between 3% and 4% of the sulphide removal rate, dependent on the dimensions and pore size of the polyurethane support particles. With the Raschig rings this ratio was only 2% and for reactors in which no carrier materials were present it was even lower (0.6%). Media containing different mixtures of acetate, propionate, sulphur and sulphate inoculated under anoxic conditions with sludge from the aerated reactor showed the presence of acetate-degrading sulphur-reducing, but not of acetate-degrading sulphate-reducing, bacteria. With propionate as sole electron donor no degradation occurred in the presence of sulphur within 2 weeks, whereas sulphate-dependent propionate oxidation started after 5–6 days incubation. Bacteria were isolated and resembled Desulfuromonas acetoxidans and Desulfobulbus propionicus morphologically and physiologically.