Isolation and Properties of a Staphylococcal Protease, Preferentially Cleaving Glutamoyl‐Peptide Bonds

An extracellular protease of a mutant of strain 8325N of Staphylococcus aureus, was isolated by a three‐step procedure involving stepwise chromatography on DEAE‐cellulose, gradient chromatography on hydroxyapatite and gel filtration on Sephadex G‐150. The enzyme was purified 250 times to homogeneity with a recovery of 20–30%. The protease has a molecular weight of about 29000 and consists of a single polypeptide chain as shown by gel filtration of native enzyme on calibrated columns of Sephadex G‐150 as well as gel filtration of denaturated enzyme on Sepharose 6‐B in 6 M guanidine‐HCl and dodecylsulfate‐polyacrylamide gel electrophoresis. Peptide maps also suggest a molecular weight around 30000. It is an acidic protein and is rich in aspartic acid and asparagine, but no cysteine or cystine can be detected. The N‐terminal sequence is Val‐Ile‐Leu‐Pro‐Asn‐Asn‐Asp. The enzyme contains no carbohydrate. The protease preferentially hydrolyzes peptide bonds on the carboxyl‐terminal side of glutamic acid. In a few cases aspartoyl bonds were also cleaved. It has no activity towards dipeptides with N‐terminal glutamic acid. The activity was not affected by divalent cations or EDTA, but was completely abolished after incubation with diisopropylfluorophosphate suggesting that the enzyme is a serine protease. The purified enzyme is stable between pH 3 and 10. It has maximum activity between pH 7.5 and 7.8 with casein as substrate and between pH 4.5 and 5.0 with hemoglobin as substrate.