Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OWTU.10012/3951 |
| Date | 31 August 2008 |
| Creators | Salma, Fariha |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation |
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