Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities.
A single gene (xylB) encoding both beta-D-xylosidase (EC 3.2.1.37) and alpha-L-arabinofuranosidase (EC 3.2.1.55) activities was identified and sequenced from the ruminal bacterium Butyrivibrio fibrisolvens. The xylB gene consists of a 1.551-bp open reading frame (ORF) encoding 517 amino acids. A subclone containing a 1.843-bp DNA fragment retained both enzymatic activities. Insertion of a 10-bp NotI linker into the EcoRV site within the central region of this ORF abolished both activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytoplasmic proteins from recombinant Escherichia coli confirmed the presence of a 60,000-molecular-weight protein in active subclones and the absence of this protein in subclones lacking activity. With p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinofuranoside as substrates, the specific activity of arabinosidase was found to be approximately 1.6-fold higher than that of xylosidase. The deduced amino acid sequence of the xylB gene product did not exhibit a high degree of identity with other xylan-degrading enzymes or glycosidases. The xylB gene was located between two incomplete ORFs within the 4,200-bp region which was sequenced. No sequences resembling terminators were found within this region, and these three genes are proposed to be part of a single operon. Based on comparison with other glycosidases, a conserved region was identified in the carboxyl end of the translated xylB gene which is similar to that of glucoamylase from Aspergillus niger.
Utt
E A
EA
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611.
Eddy
C K
CK
Keshav
K F
KF
Ingram
L O
LO
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012417:Rumen / Q000382:microbiology*; D016415:Sequence Alignment; D014995:Xylosidases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D003062:Codon; D004269:DNA, Bacterial; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase