The thermophilic actinomycete Thermomonospora fusca produced endoxylanase, ae-arabinofuranosidase, ,-xylosidase, and acetyl esterase activities maximally during growth on xylan. Growth yields on glucose, xylose, or arabinose were comparable, but production of endoxylanase and I-xylosidase was not induced on these substrates. The crude xylanase activity was thermostable and relatively resistant to end product inhibition by xylobiose and xylan hydrolysis products. Six proteins with xylanase activity were identified by zymogram analysis of isoelectric focusing gels, but only a 32-kDa protein exhibiting three isomeric forms could be purified by fast protein liquid chromatography. Endoglucanases were also identified in carboxymethylcellulose-grown cultures, and their distinction from endoxylanases was confirmed. oa-Arabinofuranosidase activity was due to a single dimeric protein of 92 kDa, which was particularly resistant to end product inhibition by arabinose. Three bands of acetyl esterase activity were detected by zymogram analysis, and there was evidence that these mainly consisted of an intracellular 80-kDa protein secreted to yield active 40-kDa subunits in the culture supernatant. The acetyl esterases were found to be responsible for acetyl xylan esterase activity in T. fusca, in contrast to the distinction proposed in some other systems. The addition of purified j-xylosidase to endoxylanase increased the hydrolysis of xylan, probably by relieving end product inhibition. The enhanced saccharification of wheat straw caused by the addition of purified a-arabinofuranosidase to T. fusca endoxylanase suggested a truly synergistic relationship, in agreement with proposals that arabinose side groups on the xylan chain participate in cross-linking within the plant cell wall structure.

Xylan-degrading enzymes, including 8-xylosidase (EC 3.2.1 .37), were induced when Thermomonospora fusca was grown at 50 "C in liquid medium containing 0.2% xylan. The intracellular P-xylosidase activity was concentrated and characterized by fast protein liquid chromatography and gel electrophoresis. A zymogram technique was developed to identify 8-xylosidase directly on polyacrylamide gels. A single enzyme (168 kDa; PI 4.37) was identified and purified to homogeneity. The consistent detection of a single band on denaturing SDS gels suggested that the enzyme was composed of identical subunits; since the subunit molecular mass was 56 kDa, a trimeric structure is suggested. High activity against p-nitrophenyl P-Dxylopyranoside (pNPX) occurred in the pH range 5.0-9-0 and temperature range 40-60 "C. The enzyme was stable at room temperature at pH 6.0-8-0; it had a half-life of 8 h at 65 "C, and of 1.5 h at 70 "C. The purified enzyme did not exhibit any detectable activity against arabinoxylan, carboxymethylcellulose orp-nitrophenyl P-D-glucopyranoside. The enzyme had a K, of 0.89 mM (pNPX) and was inhibited by D-XylOSe (Ki 19 mM) but not D-glucose. The size of the T. fusca enzyme is in the range reported for the few other bacterial P-xylosidases described, but the acidic nature of the protein and its affinity for the substrate have more in common with some of the monomeric P-xylosidases described in fungi.