CHARACTERIZATION AND SEQUENCE OF A THERMOMONOSPORA-FUSCA XYLANASE

TfxA is a thermostable xylanase produced by the thermophilic soil bact erium Thermomonospora fusca. The enzyme was purified to homogeneity fr om the culture supernatant of Streptomyces lividans transformed by pla smid pGG92, which carries the gene for TfxA, xynA. The molecular mass of TfxA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis i s 32 kDa. TfxA is extremely stable, retaining 96% of its activity afte r 18 h at 75 degrees C. It has a broad pH optimum around pH 7 and reta ins 80% of its maximum activity between pH 5 and 9. The native enzyme binds strongly to both cellulose and insoluble xylan even though it ha s no activity on cellulose. Treatment of TfxA with a T. fusca protease produced a 24-kDa catalytically active fragment that had the same N-t erminal sequence as TfxA. The fragment does not bind to cellulose and binds weakly to xylan. The V-max values for TfxA and the fragment are 600 and 540 mu mol/min/mg, respectively, while the K(m)s, are 1.1 and 2.3 mg of xylan per ml, respectively. The DNA sequence of the xynA gen e was determined, and it contains an open reading frame that codes for a 42-amino-acid (42-aa) actinomycete signal peptide followed by the 3 2-kDa mature protein. There is a 21-aa Gly-Pro-rich region that separa tes the catalytic domain from an 86-aa C-terminal binding domain. The amino acid sequence of the catalytic domain of TfxA has from 40 to 72% identity with the sequences of 12 other xylanases from seven differen t organisms and belongs to family G. Two glutamic acid residues, previ ously identified as essential for catalytic activity in Bacillus pumil us XynA, are conserved in all 13 proteins. TfxA is the only thermophil ic xylanase in family G as well as one of only two family-G xylanases to contain a binding domain.