Sequence and properties of beta-xylosidase from Bacillus pumilus IPO. Contradiction of the previous nucleotide sequence.
The nucleotide sequence of the beta-xylosidase (xynB) gene from Bacillus pumilus 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 identified in the present study is quite different from the previously reported one. The total length of the PstI--EcoRI fragment of a plasmid pOXN295 containing the xynB 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 beta-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 degrees C. The Vm value was estimated to be 1.23 +/- 0.14 mukat/mg (or p-nitrophenyl beta-D-xyloside) and 0.14 +/- 0.011 mukat/mg (for xylobiose), while Km was estimated to be 3.9 +/- 0.59 mM (for p-nitrophenyl beta-D-xyloside) and 8.9 +/- 1.19 mM (for xylobiose).
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Department of Biotechnology, Osaka University, Japan.
Shima
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Negoro
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Urabe
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D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014995:Xylosidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase