Extracellular acid protease of Aspergillus oryzae grown on liquid media: multiple forms due to association with heterogeneous polysaccharides

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Abstract
The acid protease (EC 2.4.23.6) that is produced extracellularly when A. oryzae is grown on liquid media was isolated and characterized. The enzyme was purified by precipitation with tannic acid, chromatography on Duolite A-2, and gel filtration on Sephadex G-100. The last step yielded 4 active components, with varying MW ranging from 42,000-60,000. Two of them, designated E1 and E1a, with MW of 60,000 and 55,000, respectively, were heterogeneous on isoelectric focusing, giving at least 3 enzyme species with different isoelectric points, whereas the other two, E1b and E2, with MW of 49,000 and 42,000, respectively, were homogeneous. These 4 enzymes activated bovine pancreatic trypsinogen and had the same pH optima in the acid pH range. They had essentially the same amino acid composition and immunologically cross-reacted with each other. These catalytic, chemical, and immunological properties are similar to those of acid protease A1 and A2 from A. oryzae grown on solid bran media. Unlike acid protease from solid bran culture, which contains both carbohydrate-containing and the carbohydrate-free species, of the 4 enzymes, E1, E1a, E1b, and E2, contained carbohydrate, ranging from 18.9-43% and comprising 3 hexoses, glucose, galactose and mannose. The carbohydrate portions were polysaccharide in nature and heterogeneous with respect to both MW and sugar composition, and a part of the carbohydrate was present in the form of homopolysaccharides such as galactan and mannan. Probably polysaccharide chains with different MW weights and with different chemical compositions are apparently responsible for the microheterogeneity of acid protease.