An Alkaline Active Endo-Xylanase from Bacillus halodurans S7:Molecular and Structural Aspects

Abstract: A strain of an alkaliphilic bacterium Bacillus halodurans was isolated from a water sample in Lake Shalla, a soda lake in the Ethiopian Rift Valley. From the culture of this isolate, B. halodurans S7, a xylanase which is active in a wide range of pH, with an optimum at about 9-9.5 was purified. When assayed at pH 10, the enzyme was optimally active at 70 ?C. The xylanase was free from any associated cellulase activity. The gene encoding this protein was cloned, sequenced and expressed in Escherichia coli. Based on amino acid sequence similarity, the enzyme belongs to the glycoside hydrolase family 10. Multiple sequence alignment revealed the presence of eight distinct conserved regions in the sequences of family 10 xylanases of different origins. Moreover, insert amino acids were identified in sequences of xylanases, which exhibit significant activity at or above pH 9. The crystal structure of the xylanase was determined and was found to be an elliptical eight-fold ?/?-barrel. Unlike most xylanase structures determined so far, the long axis of the barrel is longer in this xylanase. Sequence composition and structural analysis provided insights on possible mechanisms that could render the enzyme active and stable under high pH conditions. Compared to non-alkaline active xylanases, B. halodurans S7 xylanase exhibited higher % composition of arginine, glutamine and histidine. Moreover, the enzyme was found to have excess acidic residues, in particular glutamic acid, on its surface. The excess negative charge on the protein might form a water shield that offers protection in the extreme environment. Compared to other family 10 xylanases for which the structures have been determined, this xylanase has a deeper active site cleft, which might explain its high substrate specificity. The degradation of insoluble xylan, in particular from oat spelts, by the xylanase was not efficient. The hydrolytic efficiency towards this substrate was improved by the fusion of the B. halodurans S7 xylanase with N-terminal carbohydrate binding domains from Thermotoga neapolitana xylanase A. However, the chimeric enzyme was found to be heat labile compared to the native xylanase. The adaptation role of the insert amino acids found in xylanases that exhibit significant activities at elevated pH was investigated by constructing deletion mutants. From the characterization studies of the deletion variant xylanases, it seems that there is no significant shift in the pH and temperature optima of the mutant enzymes. One of the mutants was as active as the wild type xylanase whereas the activity of the other two mutants was low and the fourth mutant had no detectable activity.