Purification and biochemical characteristics of beta-D-glucosidase from a thermophilic fungus, Thermomyces lanuginosus-SSBP.

An extracellular xylanase was purified to homogeneity from the culture filtrate of a thermophilic fungus, Thermomyces lanuginosus-SSBP, and its biochemical characteristics were studied. A yield of 70-80% was achieved through the procedures of 80%-satd. ammonium sulphate precipitation, DEAE-Sephadex A25 and quaternary aminoethyl (QAE)-Sephadex A25 column chromatography. The molecular mass of the purified xylanase was 23.6 kDa, as analysed by SDS/PAGE, with a pI value of 3.8. The molar absorption coefficient of the absorbance at 280 nm was 6.8x10(4) M(-1).cm(-1). The specific activity, calculated using the dinitrosalicylic acid (DNS) method, was 3500 units/mg. The enzyme reactions followed Michaelis-Menten kinetics with K app m and V(max) values of 3.26 mg/ml and 6300 units/ml per mg of protein respectively, as obtained from a Lineweaver-Burk plot. The xylanase contained no other enzyme activity (cellulase, beta-glucosidase, beta-mannosidase, alpha-arabinofuranosidase, or beta-xylosidase) except for the hydrolysis of xylan substrate. The optimal temperature of the enzyme assay was 70-75 degrees C. The enzyme retained full activity after a 60 degrees C incubation for 3 h. The optimal pH of xylanase activity was 6.5 and the enzyme appeared to be stable over a broad pH range (pH 5-12) under the assay conditions. The majority of the metal ions tested had no effect on the enzyme activity, with the exception of Pb(2+) (modest inhibitor) and Hg(2+) (strong inhibitor). The results showed that one or two tryptophan residues oxidized by N-bromosuccinamide per enzyme molecule was sufficient to inhibit the enzyme activity completely, thus indicating that the tryptophan residues play an important role in the catalytical processes of the enzyme reaction. Because of the outstanding properties of the purified xylanase from the SSBP strain, this xylanase has a potential use in biopulping processes and other industrial applications.