Sequence and properties of β‐xylosidase from Bacillus pumilus IPO

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Abstract
The nucleotide sequence of the β‐xylosidase (xynB) gene from Bacillus pamilus has been reported previously [Moriyama, H., Fukusaki, E., Crespo, J. C., Shinmyo, A. & Okada, H. (1987) Eur. J. Biochem. 166, 539–545]. However, the sequence indentified in the present study is quite different from the previously reported one. The total lenght of the PstI‐EcoRI fragment of a plasmid pOXN295 containing the xnyB gene is 2201 bp from our sequencing, while the length of the fragment in the previous data was 2466 bp. The sequences are similar in the N‐terminal (500 bp) and C‐terminal (260 bp) regions, but those in the central region are completely different. From the following observations, the previous sequence seems to have no reliable experimental basis. First, the restriction sites observed for pOXN295 are quite different from the sites deduced from the sequence. Second, the amino acid composition deduced from the sequence and the composition identified by amino acid analysis of the purified β‐xylosidase are very different. It is confirmed, on the other hand, that our new sequence agrees well with these experimental data. The enzyme was purified to homogeneity from Bacillus pumilus and Escherichia coli harboring a hybrid plasmid which highly expresses the xynB gene. The molecular mass of the enzyme was estimated to be 190 kDa by high performance gel filtration chromatography using TSK‐G3000SW and 56 kDa by SDS/polyacrylamide gel electrophoresis. The pH optimum was 7.0, and the optimum temperature was 40°C. The Vm value was estimated to be 1.23±0.14 μkat/mg (for p‐nitrophenyl β‐d‐xylosied) and 0.14±0.011 μkat/mg (for xylobiose), while Km was estimated to be 3.9±0.59 mM (for p‐nitrophenyl β‐d‐xylosied) and 8.9±1.19 mM (for xylobiose).