Ein ungewöhnlicher Stärkeabbauweg im hyperthermophilen archaeellen Sulfatreduzierer Archaeoglobus fulgidus Stamm 7324 und Charakterisierung Stärke-abbauender Enzyme aus geothermalen Habitaten
Comparative analyses of sugar catabolism in archaea revealed that degradation of glucose and hexose polymers proceeds via modified glycolytic pathways, mainly via modified Embden-Meyerhof pathways. The utilization of sugars by Archaeoglobus fulgidus species, which represent the first isolated archaeal sulfat reducer, has not unequivocally been demonstrated so far. Thus, in this thesis it was found that Archaeoglobus fulgidus strain 7324, rather than the type strain VC16, was able to grow on starch and sulfate as energy and carbon source. Starch was degraded to acetate via an unusual starch degradation pathway. Some key enzymes were purified and characterized. Furthermore, starch degrading and modifying enzymes from geothermal habitats were characterized to find properties useful for biotechnological applications. 2.1 Starch degradation in A. fulgidus strain 7324 A. fulgidus strain 7324 was found to grow on starch (1 g/l) and sulfate (30 mM) in presence of yeast extract (0.5 g/l) as energy and carbon source. During exponential growth on starch, 1 mol of glucose-equivalent was incompletely oxidized with sulfate to approximately 2 mol acetate, 2 mol CO2 and 1 mol H2S. Beside starch, amylose and β-cyclodextrin were used as substrates. Other sugars, e.g. glucose, fructose and maltose were not utilized. This is the first report of growth of a sulfate reducer on starch, i.e. on a polymeric sugar. Extracts of starch-grown cells of A. fulgidus Stamm 7324 contained all activites of an unusual starch degradation pathway to acetate, which includes the conversion of starch to glucose 6-phosphate as well as the conversion of glucose to pyruvate via a modified Embden-Meyerhof pathway. Specific activities of the key enzymes of the starch conversion to acetate were significantly higher in starch-grown cells than in lactate-grown cells, indicating induction of these enzymes during starch catabolism. In A. fulgidus strain VC16 neither enzyme activities of starch degradation nor the corresponding homologous genes were detected. Starch degradation to glucose 6-phosphate. Extracts of starch-grown A. fulgidus strain 7324 contained all enzymes of a starch degradation pathway so far only described for Klebsiella oxytoca and Thermococcus sp. B1001. In this pathway starch is converted to cyclodextrins by means of a cyclodextrin glucanotransferase. After transport into the cell cyclodextrins are linearised via a cyclodextrinase. By means of a maltodextrin phosphorylase the resulting maltooligodextrins are cleaved by phosporylation. The resulting glucose 1-phosphat is converted via phosphoglucomutase to glucose 6-phosphat, an intermediate of glycolysis. These four enzymes were purified and characterized. The N-terminal amino acid sequences and the molecular and catalytic properties of the enzymes were very similar to their homologs of the Thermococcales. The classical enzymes of starch degradation, α-amylase and pullulanase, could not be detected in A. fulgidus strain 7324 Glucose degradation to pyruvate via a modified Embden-Meyerhof pathway. In extracts of starch-grown cells the following enzymes of a modified Embden-Meyerhof pathway were detected: ADP-dependent glucokinase, phosphoglucose isomerase, ADP-dependent 6-phosphofructokinase, fructose-1,6-phosphate aldolase, glyceraldehyde-3-phosphate: ferredoxin oxidoreductase, phosphoglycerate mutase, enolase, and pyruvate kinase. So far, this pathway was only described for Thermococcales. The first enzyme of this modified Embden-Meyerhof pathway, ADP-dependent glucokinase, was purified and characterized. The properties of the glucokinase were similar to the glucokinases from Thermococcales. So far, formation of glucose as substrate of the ADP-dependent glucokinase can not be explained by the described starch degradation pathway. Pyruvate conversion to acetate. Pyruvate conversion to acetate involved pyruvate: ferredoxin oxidoreductase and ADP-forming acetyl-CoA synthetase. Activities of phosphotransacetylase and acetate kinase could not be detected. The findings indicate that the archaeal sulfate reducer A. fulgidus strain 7324 converts starch to acetate via an unsual starch degradation pathway, a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming). This is the first report of a starch degradation pathway in a sulfate reducer. 2.2 Characterization of starch degrading enzymes from geothermal habitats Furthermore, starch degrading and modifying enzymes from geothermal habitats were characterized within the EC project ï¿½Thermogenesï¿½. The genes of these enzymes were isolated from environmental DNA and organisms, respectively, originating from geothermal habitats in Iceland. The intention of these experiments was the characterization of enzymes from the glycosylhydrolase family 13 in respect to their substrate specificity, cation dependence and stability in order to find properties of biotechnological interest. Starting from seven different isolated, sequenced and cloned. Seven different genes of starch degrading enzymes were expressed in E. coli, purified and characterized. One protein showed pullulanase activity and one neopullulanase activity, respectively; five proteins exhibited cyclodextrinase activity. The characterized proteins were highly stable at moderate thermophilic conditions and showed a broad substrate spectrum, useful properties for biotechnological applications. Differences in substrate specificity can be explained by differences in amino acid sequences, e.g. the existence of a N-terminal domain correlated with cyclodextrinase activity.
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