Isolation of two β‐xylosidase genes ofBacillus pumilusand comparison of their gene products

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Abstract
The chromosomal DNA fragments of B. pumilus IPO, a potent xylan-hydrolyzing bacterium, were ligated to a vector plasmid, pBR322, and used to transform Escherichia coli C600 cells. Two hybrid plasmids, pOXD28 and 2.6-MDa [megadalton] Bg/II fragment and the latter, a 7.7-MDa PstI fragment, both encoding .beta.-xylosidase, but xylanase is coded only on the latter hybrid plasmid. The DNA inserted in both plasmids originated from the B. pumilus chromosome, but from different regions, as shown by Southern hybridization and the analysis of restriction fragments. .beta.-Xylosidases I and II, coded on pOXN29 and pOXD28, respectively, were purified to homogeneous preparations and compared. Both were dimer enzymes consisting of 65,000-70,000-Da subunits. Specific activity and the Km value of .beta.-xylosidase I to p-nitrophenyl .beta.-D-xyloside as substrate were, respectively, 100 and 1/40 times those of .beta.-xylosidase II. The mobilities of .beta.-xylosidases I and II on polyacrylamide gel electrophoresis were also different. .beta.-Xylosidase I, the gene of which is located near the xylanase gene on pOXN29, can convert xylooligosaccharides to xylose, but .beta.-xylosidase II had little activity on xylobiose. .beta.-Xylosidase I is apparently the main enzyme for xylan hydrolysis in B. pumilus.