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J. Biochem, 1988, Vol. 104, No. 3 451-456
© 1988 Japanese Biochemical Society

research-article

Purification and Characterization of an Acidic Amino Acid Specific Endopeptidase of Streptomyces griseus Obtained from a Commercial Preparation (Pronase)

Norio Yoshida*,1, Shigeru Tsuruyama*, Kyoko Nagata*, Kouichirou Hirayama**, Kousaku Noda*** and Satoru Makisumi*

*Department of Chemistry, Faculty of Science, Kyushu University 33 Higashi-ku, Fukuoka, Fukuoka 812
**The Third Research Department, Protein Engineering Research Institute Midori-ku, Yokohama, Kanagawa 227
***Department of Human Life Science, Fukuoka Women's University Higashi-ku, Fukuoka, Fukuoka 813

1To whom correspondence should be addressed

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 inactivat ed by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.


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