Characterization of a Thermostable Xylanase from the Extremely Thermophilic Bacterium Thermotoga hypogea

Abstract

Thermotoga hypogea is an extremely thermophilic bacterium that can utilize both peptides and carbohydrates including xylan as growth substrates at 90 ºC. It was found that its xylanase activity was increased up to 9-fold in the presence of either xylan or xylose in growth media, which was independent of growth phases. More than 95% of xylanase activities were associated with cells of T. hypogea. Ultracentrifugation of the cell-free extract of the organism showed that most xylanase activities were in the soluble fraction. A xylanase was purified from the cell-free extract to homogeneity using an FPLC system. The purified enzyme had a molecular mass of 38 kDa as revealed by SDS-PAGE. Its native molecular mass was determined using gel-filtration to be 29 kDa, indicating it was a monomeric enzyme. The purified xylanase was stable with a half-life of 270 min at 90°C in the presence of xylan and BSA. Its optimal pH and temperature were determined to be 5.5 and 90°C, respectively. An apparent Km-value was determined to be 0.07% (w/v) of xylan. Hydrolytic products from xylan catalyzed by the purified enzyme were xylobiose and xylooligosaccharides (n>2). No cellulase activity was detected for the purified xylanase.