Isolation and characterization of alkaline proteinase of Aspergillus flavus

Abstract

1. 1. A simple method was developed to isolate an alkaline proteinase from a crude enzymatic product prepared from the culture medium of Aspergillus flavus consisting of (io) adsorption of the crude product on CM-Sephadex at pH 4.5 (ii) desorption of proteinase by 0.5 M sodium acetate at pH 7, and (iii) chromatogrpahy on DEAE-Sephadex in phosphate buffers at pH 6.0. This purification results in a 120-fold increase of proteolytic activity. 2. 2. The proteinase obtained is homogeneous when subjected to disc electrophoresis and sedimentation analysis in the ultracentrifuge. The molecular weight was determined, and the following values were obtained: (i) 19 500 to 22 000 by gel filtration, (ii) 19 000 ± 1000 by light-scattering technique, and (iii) 17 800 when the molecular weight was calculated from the amino acid composition: Lys 11, His 3−4, Arg 2, Asp 21, Thr 11, Ser 20, Glu 12−13, Pro 4−5, Gly 20, Ala 23, Val 15, He 9−10, Leu 9, Tyr 5, Phe 3, Trp 2, amide-NH 3, 17. The enzyme does not contain either disulphide bonds or cysteine; the N-terminal end-group residue is glycine; the C-terminal amino acid is alanine. The enzyme contains 1 mole of a sugar component not yet identified. Treatment of the proteinase with diisopropylphosphorofluoridate provided evidence of one active serine residue. 3. 3. Similarities existing between analytical data on alkaline proteinase of Aspergillus flavus and other extracellular enzymes from Aspergilli are discussed.