Purification of Alkaline Serine Protease From Local Bacillus subtilis M33 by Two Steps: a Novel Organic Solvent and Detergent Tolerant Enzyme

Sonuc Karaboga M. N., Logoglu E.

GAZI UNIVERSITY JOURNAL OF SCIENCE, vol.32, no.1, pp.116-129, 2019 (ESCI) identifier identifier

  • Publication Type: Article / Article
  • Volume: 32 Issue: 1
  • Publication Date: 2019
  • Journal Indexes: Emerging Sources Citation Index (ESCI), Scopus, TR DİZİN (ULAKBİM)
  • Page Numbers: pp.116-129
  • Keywords: Alkaline protease, Bacillus subtilis, Enzyme purification, DEAE, STABLE PROTEASE, OXIDANT
  • Gazi University Affiliated: Yes


Alkaline proteases are important from an industrial perspective due to their wide scale applications and obtained from different sources. In this study, an alkaline protease from a newly isolated Bacillus subtilis M33 was purified by ammonium sulfate precipitation and DEAE cellulose anion exchange chromatography with 38.66% yield and 15.50 fold. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and gel chromatography indicate that the molecular weight of the purified enzyme is 39 kDa. The enzyme present pH and temperature optimum of 10.0 and 55 degrees C The purified enzyme has been found to maintain stability over a wide range (pH 8.0-11.0) for 7 days.. Phenylmethyl sulfonyl fluoride (PMSF) which is a specific inhibitor completely inhibited the enzyme activity. However, the increased activity of the enzyme in the presence of 2-mercaptoethanol and dithiothreitol indicates that the enzyme is a thiol-dependent serine protease. The enzyme retained its stability with laboratory bleaches (H2O2), surfactants (Tween 80, Triton X-100, SDS) and organic solvents such as ethanol, toluene, propanol. The enzymatic behavior of the purified enzyme in the presence of some commercial detergents was also evaluated. The two important Michaelis Menten parameters Km and Vm were calculated 0.706 mg/ml, 3000 mu M.min-1 respectively.