Identification and characterization of clustered genes for thermostable xylan-degrading enzymes, β-xylosidase and xylanase, of Bacillus stearothermophilus 21

Abstract

Bacillus stearothermophilus 21 is a gram-positive, facultative thermophilic aerobe that can utilize xylan as a sole source of carbon. We isolated this strain from soil, purified its extracellular xylanase and β- xylosidase, and analyzed the two-step degradation of xylan by these enzymes (T. Nanmori, T. Watanabe, R. Shinke, A. Kohno, and Y. Kawamura, J. Bacteriol. 172:6669-6672, 1990). An Escherichia coli transformant carrying a 4.2-kbp chromosomal segment of this bacterium as a recombinant plasmid was isolated. It excreted active β-xylosidase and xylanase into the culture medium. The plasmid was introduced into UV-sensitive E. coli CSR603, and its protein products were analyzed by the maxicell method. Proteins harboring β- xylosidase and xylanase activities were identified, and their molecular masses were estimated by sodium dodecyl sulfate-polyarylamide gel electrophoresis to be 75 and 40 kDa, respectively. The values were identical to those of proteins prepared from cells of B. stearothermophilus 21. The genes for both enzymes were encoded in a 3.4-kbp Pst1 fragment derived from the 4.2-kbp chromosomal segment. The nucleotide sequence of the 4.2-kbp segment was accordingly determined. The β-xylosidase gene (xylA) is located upstream of the xylanase gene (xynA) with a possible promoter and a Shine- Dalgarno sequence. The latter gene is preceded by two possible promoters and a Shine-Dalgarno sequence that are located within the 3'-terminal coding region of the former. The two genes thus appear to be, at least partly, expressed independently, which was experimentally confirmed in E. coli by deletion analysis. The xynA gene was found to belong to cellulase-xylanase family F, possessing six regions that are highly conserved in this family. However, the β-xylosidase has no detectable similarity to any other proteins in the PIR data base (release 38.0).