Purification and properties of three cellobiases from Aspergillus niger A20

Three cellobiases, here called cellobiase A, B, and C, from the culture fil trate of Aspergillus niger A20, were purified by precipitation with ammoniu m sulphate, gel filtration through Sephadex G-75, and column chromatography of DEAE-cellulose. The purified enzymes were homogeneous on polyacrylamide disk electrophoresis. The mol wt of the purified enzymes were estimated by SDS-gel electrophoresis to be 88,000, 80,000, and 71,000 for cellobiases A , B, and C, respectively. The enzymes were active at pH 4.5 and 55-60 degre es C. The pattern of their amino acid compositions showed high contents of aspartic acid, glutamic acid, threonine, serine, and glycine. The apparent K-m values for cellobiose were 0.9, 1.63, and 1.0 mM for cellobiases A, B, and C, respectively. Calcium ions stimulated cellobiases B and C, and Co2and Mg2+ ions stimulated cellobiase A. The purified enzymes hydrolyzed cell obiose and aryl-beta-D-glucosides, but they had no action on sucrose, malto se, and cellulose. The three cellobiases catalyzed transglycosylase reactio n, and the major product formed from cellobiose was tetramer of glucose.