Genetic organization, sequence and biochemical characterization of recombinant beta-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI.
Endoxylanase (xynA) and beta-xylosidase (xynB) genes from Thermoanerobacterium saccharolyticum were subcloned from a cosmid clone (pXDM1) to generate pXPH3. The nucleotide sequence of a PstI-HindIII fragment in pXPH3 that contained xynB revealed an open reading frame (ORF) of 1500 bp encoding a 55 kDa protein. Another open reading frame (ORF1) of unknown function was found 21 bp downstream from the first stop codon of xynB. xynB, ORF1 and xynA had the same direction of transcription. xynB from T. saccharolyticum strain B6A-RI exhibited 45% amino acid similarity, with 18% amino acid identity to xynA of T. saccharolyticum strain B6A-RI, and 61% similarity and 37% identity with the beta-xylosidase gene from Caldocellum saccharolyticum. Recombinant beta-xylosidase was purified from E. coli (pXPH3) cells. The enzyme was a monomer with a molecular mass of 55 kDa. The specific activity and pH and temperature optima for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside (pNPX) were 5.53 U mg-1, 5.5 and 70 degrees C, respectively. The beta-xylosidase was stable at 65 degrees C, but lost activity at 85 degrees C. The purified enzyme had hydrolytic activity towards xylopentose, xylotriose, xylobiose and pNPX, but had no activity toward xylan.
Lee
Y E
YE
Department of Microbiology and Public Health, Michigan State University, East Lansing 48824.
Zeikus
J G
JG
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase