Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

  • Dilek Kazan
  • Aziz Akın Denizci
  • Mine N. Kerimak Öner
  • Altan Erarslan
Original Paper

Abstract

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH4)2SO4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca2+ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol−1 (44.30 kJ mol−1). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K m and k cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×103 min−1, respectively.

Keywords

Bacillus clausii Serine alkaline protease Enzyme purification Kinetic properties Enzyme characterization 

Notes

Acknowledgement

This research was supported by The Scientific and Technical Research Council of Turkey (TUBITAK) by the Project No. TBAG 2129-102T003.

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Copyright information

© Society for Industrial Microbiology 2005

Authors and Affiliations

  • Dilek Kazan
    • 1
    • 2
  • Aziz Akın Denizci
    • 1
  • Mine N. Kerimak Öner
    • 3
  • Altan Erarslan
    • 1
    • 4
  1. 1.The Scientific and Technical Research Council of TurkeyResearch Institute for Genetic Engineering and Biotechnology, Marmara Research Center CampusKocaeliTurkey
  2. 2.Department of Chemical Engineering, Faculty of EngineeringMarmara University, Göztepe CampusIstanbulTurkey
  3. 3.Department of Fermentation, Köseköy Technical CollegeKocaeli UniversityIzmit-KocaeliTurkey
  4. 4.Department of Chemistry, Section of Biochemistry, Faculty of Arts and SciencesKocaeli UniversityIzmit-KocaeliTurkey

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