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

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 degrees 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 (NH(4))(2)SO(4) 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 degrees C; however, it is shifted to 70 degrees C after addition of 5 mM Ca(2+) ions. The enzyme was stable between 30 and 40 degrees C for 2 h at pH 10.5; only 14% activity loss was observed at 50 degrees 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 degrees 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 degrees 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 microM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21 x 10(3) min(-1), respectively.

Effects of Cultural Conditions on the Production of Extracellular Protease by Streptomyces albolongus and Streptomyces aburaviensis

“…Shaking at 100 rpm markedly increase proteases production by S. aburaviensis. This finding was also reflected in other studies [24,26,28,29].…”

“…S. albolongus preferred medium with neutral pH for maximum enzyme production, but maximum protease activities S. aburaviensis was obtained in medium having initial p H 8.0. Production of microbial proteases in neutral and alkaline medium p H was also reported by other authors [25][26][27]. Temperature is an important environmental factor for growth and enzyme production.…”

Secreted Compounds of the Probiotic Bacillus clausii Strain O/C Inhibit the Cytotoxic Effects Induced by Clostridium difficile and Bacillus cereus Toxins

“…In addition, Kazan et al (55) have already characterized a serine alkaline protease from B. clausii GMBAE 42 with activity and specificity very close to those of our protease.…”

Production of moderately halotolerant, SDS stable alkaline protease from Bacillus cereus MTCC 6840 isolated from lake Nainital, Uttaranchal state, India

“…The use of alkaline proteases in detergents, food, leather and silk industries has remarkably increased in recent years (12). Of all proteases, alkaline proteases produced by Bacillus species are of utmost importance due to the stability of their enzymes under different environmental conditions (14). While several proteases from alkalophiles have been studied comprehensively, proteases from halophiles and moderate halophiles have not been investigated in detail (12).…”