Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11.
The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 gel permeation chromatography. The enzyme was shown to have a relative low molecular weight of 15 kd by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and was purified 21-fold with a yield of 7.5%. It was most active at 60 degrees C, pH 10, with casein as substrate. It was stable between pH 8 and 10. This enzyme was almost 100% stable at 60 degrees C even after 350 minutes of incubation. It was strongly activated by metal ions such as Ca+2, Mg+2, and Mn+2. Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP) but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with iodoacetate, p-chloromercuric benzoate (pCMB), and beta-mercaptoethanol (beta-ME). The compatibility of the enzyme was studied with commercial and local detergents in the presence of 10mM CaCl2 and 1M glycine. The addition of 10mM CaCl2 and 1M glycine, individually and in combination, was found to be very effective in improving the enzyme stability where it retained 52% activity even after 3 hours. This enzyme improved the cleansing power of various detergents. It removed blood stains completely when used with detergents in the presence of 10mM CaCl2 and 1M glycine.
Adinarayana
Kunamneni
K
Pharmaceutical Biotechnology Division, Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India. adi
Ellaiah
Poluri
P
Prasad
Davuluri Siva
DS
D001412:Bacillus subtilis / Q000201:enzymology*; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008670:Metals / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013696:Temperature
D003902:Detergents; D008670:Metals; D012697:Serine Endopeptidases; C032923:microbial serine proteinases