GENETIC ORGANIZATION, SEQUENCE AND BIOCHEMICAL-CHARACTERIZATION OF RECOMBINANT BETA-XYLOSIDASE FROM THERMOANAEROBACTERIUM-SACCHAROLYTICUM STRAIN B6A-RI

Endoxylanase (xynA) and beta-xylosidase (xynB) genes from Thermoanaero bacterium saccharolyticum were subcloned from a cosmid clone (pXDM1) t o generate pXPH3. The nucleotide sequence of a PstI-HindIII fragment i n pXPH3 that contained xynB revealed an open reading frame (ORF) of 15 00 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 s imilarity, with 18 % amino acid identity to xynA of T. saccharolyticum strain B6A-RI, and 61 % similarity and 37 % identity with the beta-xy losidase gene from Caldocellum saccharolyticum. Recombinant beta-xylos idase was purified from E. coli (pXPH3) cells. The enzyme was a monome r with a molecular mass of 55 kDa. The specific activity and pH and te mperature 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-degree s-C. The purified enzyme had hydrolytic activity towards xylopentose, xylotriose, xylobiose and pNPX, but had no activity toward xylan.