Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520.
The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.
Tsujibo
H
H
Osaka University of Pharmaceutical Sciences, Takatsuki, Japan. tsujibo@oysun01.oups.ac.jp
Takada
C
C
Tsuji
A
A
Kosaka
M
M
Miyamoto
K
K
Inamori
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000201:enzymology; D003001:Cloning, Molecular; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D014995:Xylosidases / Q000096:biosynthesis / Q000235:genetics*
D003470:Culture Media; D011994:Recombinant Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase