Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2.
A protease-producing strain CT2 isolated from Tunisian potatoes, exhibiting a potent protease activity (prot CT2), was identified as Bacillus halotolerans according to 16S ribosomal DNA sequence analysis. Maximum prot CT2 production was obtained in medium supplemented with bean seed proteins. Proteolytic activity was purified by ammonium sulphate precipitation, Sephacryl S-200 gel filtration and SP-sepharose cation-exchange chromatography. Optimal enzyme activity was reached at pH 9 and temperature of 50 °C. Proteolytic activity was enhanced by Ca2+ and Mn2+ ions, completely inhibited by PMSF suggesting a serine protease nature and exhibited high stability in the presence of commercial detergents. Prot CT2 showed broad substrate specificity towards both synthetic and natural substrates, with a high capacity to hydrolyze legume seed proteins. Using electrophoretic analysis, its molecular weight was around 250 kDa with two major subunit showing important homologies with serine proteases belonging to the subtilisin-like serine proteases. Based on the Lineweaver-Burk plots Km and Vmax values were 10 mg/ml and 50,000 U/mg respectively. This newly described prot CT2 displays relevant properties which highlight its potential use in various industrial and biotechnological applications.
['Dorra|Gharbi|G|', 'Ines|Karkouch|K|', 'Imen|Ben Slimene|BS|', 'Laurent|Coquet|C|', 'Sana|Azaiez|A|', 'Olfa|Tabbene|T|', 'Pascal|Cosette|C|', 'Thierry|Jouenne|J|', 'Ferid|Limam|L|']
D001407:Bacillus D001426:Bacterial Proteins D002118:Calcium D010450:Endopeptidases D060026:Endophytes D004795:Enzyme Stability D006868:Hydrolysis D007477:Ions D007700:Kinetics D008345:Manganese D008970:Molecular Weight D011480:Protease Inhibitors D012336:RNA, Ribosomal, 16S D013379:Substrate Specificity D013696:Temperature
High molecular weight Protease Detergent compatible Protein hydrolysis