Molecular cloning and the nucleotide sequence of a Bacillus sp. KK-1 β-Xylosidase gene

Abstract

A gene coding for β-xylosidase from thermophilic xylanolytic Bacillus sp. KK-1 was cloned into Escherichia coli using plasmid pBR322. Recombinant plasmid DNAs were isolated from E. coli clones which were capable of hydrolyzing 4-methylumbelliferyl-β-D-xylopyranoside. Restriction analysis showed the DNAs to share a common insert DNA. Xylo-oligosaccharides, including xylotriose, xylotetraose, xylopentaose, and xylobiose were hydrolyzed to form xylose as an end product by cell-free extracts of the E. coli clones, confirming that the cloned gene from strain KK-1 is a β- xylosidase gene. The β-xylosidase gene of strain KK-1 designated as xylB was completely sequenced. The xylB gene consisted of an open reading frame of 1,602 nucleotides encoding a polypeptide of 533 amino acid residues, and a TGA stop codon. The 3' flanking region contained one stem-loop structure which may be involved in transcriptional termination. The deduced amino acid sequence of the KK-1 β-xylosidase was highly homologous to the β- xylosidases of Bacillus subtilis and Bacillus pumilus, but it showed no similarity to a thermostable β-xylosidase from Bacillus stearothermophilus.