Chemical Properties of the Polysaccharides Associated with Acid Protease of Aspergillus oryzae Grown on Solid Bran Media

Abstract

The chemical and physical properties of the carbohydrate moiety of acid protease Al [EC 2. 4. 23. 6], one of the two acid proteases produced by Aspergillus oryzae grown on solid bran media, have been studied. The enzyme was separated into two active components (A_1a and A_1b) by isoelectric focusing; both contained approximately 50% carbohydrate. The carbohydrate portion of the major component Al. was composed of galactose, glucose, and mannose, but the minor component A_1b contained glucose as the predominant carbohydrate, with smaller amounts of galactose, fructose, xylose and mannose.
The absence of a covalent linkage between the polysaccharide and the protein in acid protease A₁ was indicated, although these two moieties were tightly associated with each other. This conclusion was based on the following observations: (a) the polysaccharides obtained after proteolytic digestion contained no detectable amino acids; (b) no hexosamines were detected in acid protease A₁; (c) the polysaccharides were not separable from the protein by a variety of procedures, including treatment with cold 15% trichloroacetic acid, 8M urea, 6M guanidine hydrochloride, and 0.1 N NaOH (at 25°C for 18 h). However, when treated with 50% acetic acid at 37°C for 48 h, the polysaccharides were completely dissociated from the protein; and (d) of several glycoproteins tested, containing an N-glycosyl or O-glycosyl linkage between the protein and carbohydrate moieties, none liberated carbohydrate residues upon treatment with 50% acetic acid.