CLONING, SEQUENCING, AND EXPRESSION OF THE GENE ENCODING A LARGE S-LAYER-ASSOCIATED ENDOXYLANASE FROM THERMOANAEROBACTERIUM SP STRAIN JW SL-YS-485 IN ESCHERICHIA-COLI/

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxyl anase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned an d expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria, This PCR p roduct was used as a probe in Southern hybridizations to identify a 4. 6-kb EcoRI fragment containing the complete xynA gene. This fragment w as cloned into E. coli, and recombinant clones expressed significant l evels of xylanase activity, The purified recombinant protein had an es timated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485, The en tire insert was sequenced and analysis revealed a 4,044-bp open readin g frame encoding a protein containing 1,348 amino acid residues (estim ated molecular mass of 148 kDa). xynA was preceded by a putative promo ter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding s ite (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 38 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identi fied, and the C-terminal end of the protein contained three repeating sequences (59, 64, and 57 amino acids) that showed 46 to 68% similarit y to repeating sequences at the N-terminal end of S-layer and S-layer- associated proteins from other gram-positive bacteria, These repeats c ould permit an interaction of the enzyme with the S-layer and tether i t to the cell surface.