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 enzyme was used to screen a genomic library of B. stearothermophilus T-6 constructed in lambda-EMBL3. Two positive phages were isolated, both containing similar 13-kb inserts, and their lysates exhibited xylanase activity. A 3,696-bp SalI-BamHI fragment containing the xylanase gene was subcloned in Escherichia coli and subsequently sequenced. The open reading frame of xylanase T-6 consists of 1,236 bp. On the basis of sequence similarity, two possible -10 and -35 regions, a ribosome-binding site at the 5' end of the gene and a potential transcriptional termination motif at the 3' end of the gene, were identified. From the previously known N-terminal amino acid sequence of xylanase T-6 and the possible ribosome-binding site, a putative 28-amino-acid signal peptide was 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 highly conserved regions. Northern (RNA) blot analysis indicated that the xylanase T-6 transcript is about 1.4 kb and that the induction of this enzyme synthesis by xylose is on the transcriptional level.
Gat
O
O
Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa.
Lapidot
A
A
Alchanati
I
I
Regueros
C
C
Shoham
Y
Y
D000595:Amino Acid Sequence; D001435:Bacteriophages; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004790:Enzyme Induction / Q000235:genetics; D004926:Escherichia coli; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D043325:Xylan Endo-1,3-beta-Xylosidase
D004269:DNA, Bacterial; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase