Cloning, sequencing, and characterization of the bifunctional xylosidase-arabinosidase from the anaerobic thermophile thermoanaerobacter ethanolicus.

The gene for the bifunctional xylosidase-arabinosidase (xarB) from the thermophilic anaerobe Thermoanaerobacter ethanolicus JW200 was cloned, sequenced, and expressed in Escherichia coli (Genebank Accession No. AF135015). Analysis of the recombinant enzyme revealed activity against multiple substrates with the highest affinity towards p-nitrophenyl beta-D-xylopyranoside (pNPX) and highest activity against p-nitrophenyl alpha-L-arabinopyranoside (pNPAP), respectively. Thus, we classify this enzyme as a bifunctional xylosidase-arabinosidase. Even though both sequences are 96% identical on the amino acid level, excluding the amino-terminal end, a frame-shift mutation in the 5' region of the gene in T. brockii ATCC 33075 and a deletion in a downstream open reading frame in T. ethanolicus seem to have occurred through evolutionary divergence of these two species. This represents an interesting phenomenon of molecular evolution of bacterial species, as PCR analysis of the region around the deletion indicates that the deletion is not present in T. brockii ssp. finnii and T. brockii ssp. brockii type strain HTD4.