PURIFICATION AND CHARACTERIZATION OF A COMPLEX-BOUND AND A FREE BETA-1,4-ENDOXYLANASE FROM THE CULTURE FLUID OF THE ANAEROBIC FUNGUS PIROMYCES SP STRAIN E2

Two extracellular xylanases were purified to homogeneity from the culture filtrate of the anaerobic fungus Piromyces sp. strain E2 and their properties were studied. The enzymes are present in a High Molecular Mass complex (HMM-complex) and as free protein in nearly equal amounts. Both enzymes are most likely identical as all biochemical characteristics were identical. The molecular masses of the enzymes are 12.5 kDa, as estimated by gel chromatography and electrophoretic mobility. The activities of both enzymes are optimal at pH 6.0 and 50-degrees-C and the enzymes are stable up to 72 h at 40-degrees-C. The enzymes have a pl of 9.1. The K(m) and V(max), determined with xylan from oat spelts, were 3 mg . ml-1 and 2600 IU . mg-1 protein. The enzymes are active both on soluble and insoluble oat spelt xylan. The purified xylanases are inactive against Avicel, carboxymethylcellulose, p-nitrophenyl-beta-D-glucoside, and p-nitrophenyl-beta-D-xyloside. The products of the pure enzymes are predominantly xylo-oligosaccharides, indicating that the enzymes act as endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8).