Purification and Properties of a Cellulase fromAspergillus niger

Abstract

A cellulolytic enzyme was extensively purified from a commercial crude cellulase preparation from Aspergillus niger by consecutive column chromatography. The purified enzyme was homogeneous on polyacrylamide gel as well as ampholine electrophoresis. The enzyme was an acidic protein with an isoelectric point at pH 3.67. The molecular weight of the enzyme was estimated to be 31,000 by SDS-polyacrylamide gel electrophoresis. No carbohydrate moiety seemed to be associated with the enzyme protein. The optimum pH was 4.0, and the optimum temperature of the enzyme was 45~50°C. The enzyme was completely stable over the range of pH 5.0~8.0 at 4°C for 24 hr, and retained about 50% of its original activity after heating at 70°C for 10 min. The enzyme was partly inactivated by 1 mm Ag+, Hg2+, and Fe2+. The enzyme was characterized as an endocellulase on the basis of its action on carboxymethyl cellulose and cellooligosaccharides. The enzyme split cellopentaose, retaining the β-configuration of the anomeric carbon atoms in the hydrolysis products. The Km value of the enzyme for carboxymethyl cellulose was 0.086%. It was active on carboxymethyl cellulose and cellooligosaccharides (cellotriose to cellohexaose), but not on either cellobiose or p-nitrophenyl β-d-glucoside under the assay conditions used.