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Intracellular Protease from Streptococcus durans¹

Department of Food Science, Michigan State University, East Lansing 48823

ABSTRACT

An intracellular protease from Streptococcus durans was isolated and partially purified and characterized. A 67-fold purification was obtained with retention of 53% of the original activity. Purification was complicated by the presence of nucleic acids which could not be removed without loss of proteolytic activity. Ribonuclease inactivated the protease, but attempts to reactivate the protease by adding ribonucleic acid extracted from S. durans were unsuccessful. Disc electrophoresis of the enzyme in polyacrylamide gel showed one protein zone with an adjoining nucleic acid zone.

The protease exhibited normal reaction kinetics with respect to substrate concentrations, enzyme concentration, and reaction rate. The proteolytic activity was affected by the ionic strength of the assay reaction mixture, and an ionic strength of 0.1 was optimal. Proteolytic activity was optimal at pH 7.5, with secondary activity occurring at pH 5.5 to 6.

Divalent Ca, Co, Cu, Mg, Mn, Ni, Zn, and di- and trivalent Fe had no effect on the activity of the protease purified 67-fold, but Hg inhibited the protease about 15%. Treatment of the assay reaction mixture could be partially restored by the addition of the aforementioned ions, except Fe, Cu, and Hg.

The protease withstood heating at 97 to 99 C for 60 minutes at pH 6 and 7.5, but lost 21% of activity at pH 8.5. Casein and ß-lactoglobulin were acted upon by the enzyme, but there was no proteolytic activity detected on bovine serum albumin and hemoglobin, or in the milk-clot test.

p-Chloromercuribenzoate was the most effective inhibitor of sulfhydryl groups, followed by N-ethylmaleimide and iodoacetamide. Reducing agents and diisopropyl fluorophosphate had no effect on the proteolytic activity.

¹ Michigan Agricultural Experiment Station Journal Article Number 4883.

² Present address: Fitzsimons General Hospital, Denver, Colorado 80240.