CLONING AND DNA-SEQUENCE OF THE GENE CODING FOR BACILLUS-STEAROTHERMOPHILUS T-6 XYLANASE

Bacillus stearothermophilus T-6 produces an extracellular thermostable xylanase. Affinity-purified polyclonal serum raised against the enzym e was used to screen a genomic library of B. stearothermophilus T-6 co nstructed in lambda-EMBL3. Two positive phages were isolated, both con taining similar 13-kb inserts, and their lysates exhibited xylanase ac tivity. A 3,696-bp SalI-BamHI fragment containing the xylanase gene wa s subcloned in Escherichia coli and subsequently sequenced. The open r eading frame of xylanase T-6 consists of 1,236 bp. On the basis of seq uence similarity, two possible -10 and -35 regions, a ribosome-binding site at the 5' end of the gene and a potential transcriptional termin ation motif at the 3' end of the gene, were identified. From the previ ously known N-terminal amino acid sequence of xylanase T-6 and the pos sible ribosome-binding site, a putative 28-amino-acid signal peptide w as deduced. The mature xylanase T-6 consists of 379 amino acids with a calculated molecular weight and pI of 43,808 and 6.88, respectively. Multiple alignment of beta-glycanase amino acid sequences revealed hig hly conserved regions. Northern (RNA) blot analysis indicated that the xylanase T-6 transcript is about 1.4 kb and that the induction of thi s enzyme synthesis by xylose is on the transcriptional level.