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Purification and characterization of alkaline protease from Alcaligenes faecalis
Author(s) -
Thangam E. Berla,
Rajkumar G. Suseela
Publication year - 2002
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20010013
Subject(s) - pmsf , alcaligenes faecalis , enzyme , casein , serine protease , protease , chemistry , zymography , alcaligenes , biochemistry , enzyme assay , substrate (aquarium) , molecular mass , chromatography , bacteria , biology , pseudomonas , ecology , genetics
Extracellular alkaline protease from the alkalophilic bacterium Alcaligenes faecalis was purified by a combination of ion‐exchange and size‐exclusion chromatographic methods, and its properties were examined. The purified enzyme had a specific activity of 563.8 μ mol of tyrosine/min per mg of protein and gave a single band on native PAGE and SDS/PAGE with a molecular mass of 67 kDa. Gelatin zymogram also revealed one clear zone of proteolytic activity which corresponded to the band obtained with native PAGE and SDS/PAGE. The enzyme had an optimal pH of 9.0 and exhibited its highest activity at 55 °C. The enzyme activity was inhibited by PMSF, suggesting the presence of serine residues at the active site. The enzyme had a K m of 1.66 mg/ml and a V max of 526 units/min per mg of protein with casein as the substrate.