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A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.


N Yoshida, S Tsuruyama, K Nagata, K Hirayama, K Noda, S Makisumi. Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase). Journal of biochemistry. 1988 Sep;104(3):451-6

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PMID: 3149277

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