Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C1-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 μM, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol-1 at a dilution rate of 0.05 h-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C1-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.@en

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 μM pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol−1 and 0.10 mmol · g−1 · h−1, respectively. Neither the Y max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.@en

Gluconobacter oxydans, an organism used for the industrial production of gluconic acid, contains two glucose dehydrogenases (GDHs) catalysing the direct oxidation of glucose to gluconic acid. In addition to a quinoprotein (PQQ-dependent), membrane-bound GDH (EC 1.1.99.17), an NADP-dependent, cytoplasmic GDH is present. From three types of experiments, evidence is presented that the quinoprotein GDH is the enzyme responsible for gluconic acid production by G. oxydans. In cell-free extracts, the activity of quinoprotein GDH was 30-fold higher than the activity of NADP-dependent GDH. A kinetic analysis of glucose-dependent oxygen uptake showed that a system with an affinity constant similar to the Km of quinoprotein GDH is involved in the process of gluconic acid production. The conclusion that gluconic acid production is essentially an extracytoplasmic process catalysed by quinoprotein GDH was confirmed in uptake experiments, which demonstrated that inhibition of glucose transport does not result in inhibition of gluconic acid production.@en

The enzyme β-galactosidase was first mentioned in the literature by Beijerinck exactly a hundred years ago. The Department of Microbiology and Enzymology of the Delft University of Technology keeps the memory of Beijerinck, its first professor, alive by maintaining a ‘Beijerinck-room’ in the attic of the building. In addition to manuscripts and laboratory notebooks, this room contains some of his chemicals and biological preparations, and it was here that we recently found a 90-year old lactase preparation. Even after storage under suboptimal conditions, the preparation still exhibited measurable enzyme activities.@en

A simple method is described for the preparation of d-xylulose. It consists of the isomerization of d-xylose with xylose isomerase (EC 5.3.1.5), yielding an equilibrium mixture of d-xylulose and d-xylose. This is followed by the quantitative oxidation of residual d-xylose to d-xylonic acid with immobilized A. calcoaceticus cells. A combination of methanol precipitation and ion exchange is used for the removal of xylonic acid. This procedure offers many advantages over existing methods for the preparation of d-xylulose. The purity of the final product compares favorably to that of a commercial d-xylulose preparation.@en

Quinoprotein glucose dehydrogenase (GDH; EC 1.1.99.17) was partially purified from cell-free extracts of Acinetobacter calcoaceticus LMD79.41. The enzyme oxidized monosaccharides (d-glucose, d-allose, 2-deoxy-d-glucose, d-galactose, d-mannose, d-xylose, d-ribose and l-arabinose) as well as disaccharides (d-lactose, d-maltose and d-cellobiose). Intact cells of A. calcoaceticus LMD79.41 also oxidized these monosaccharides, but not the disaccharides. The difference in substrate specificity can not be explained by impermeability of the outer membrane for disaccharides, since right-side-out membrane vesicles did not oxidize disaccharides either. Destruction of the cytoplasmic membrane strongly affected the catalytic properties of GDH. Not only did the affinity towards some monosaccharides change substantially, but disaccharides also became good substrates upon solubilization of the enzyme. Thus, at least in A. calcoaceticus LMD79.41, the oxidation of disaccharides by GDH can be considered as an in vitro ‘artefact’ caused by the removal of the enzyme from its natural environment.@en