Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces beta-glucosidase (EC 3.2.1.21) extracellularly. The beta-glucosidase was purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With p-nitrophenyl beta-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the Km was 1.72 mM and Vmax was 326 &mgr;mol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0 at temperatures up to 50°C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl beta-D-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca2+, Mg2+, Mn2+, Cd2+ and beta-mercaptoethanol, and inhibited by Ag+, Hg2+, SDS, and p-chloromercuribenzoate (PCMB).

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of β-glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. β-Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.

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