Isolation of alkaline proteinases from Aspergillus oryzae by one-step affinity chromatography on ovoinhibitor-sepharose column

Abstract

1. 1. Alkaline proteinases from a crude preparation of Aspergillus oryzae were isolated by one-step affinity chromatography on a chicken ovoinhibitor-Sepharose column. The yield of the activity was 55% and the purified fraction had about 300-fold higher specific activity than the starting material. 2. 2. The purified fraction was found to be homogeneous in size but consisted of at least four electrophoretically distinct bands. The amino acid composition was estimated and found to be very similar to other known alkaline proteinases from Aspergillus species, thus indicating a strong genetic relationship. 3. 3. The esterolytic activity of the purified fraction was tested on specific esteratic substrates of pancreatic serine proteases. The purified fraction was active on all 3 substrates, thus indicating a rather broad specificity and the kinetic parameters were almost identical to those of pure alkaline proteinase from Aspergillus sojae. Like the latter, the purified fraction had an extremely high k cat on acetyl tri- l-alanine methyl ester.