Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus species HSRB08 from hotspring.
The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus species HSRB08, which was isolated from hotspring. The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 chromatography. The enzyme was shown to have molecular weight of 66 kD by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Gelatin Zymogram and was purified 15.3-fold with a yield of 7.5%. It was most active at 45 degrees C, pH 9.0, with casein as substrate. It was strongly activated by metal ions such as Ca2+, Mg2+, and Mn2+. Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with beta-mercaptoethanol (beta-ME). The compatibility of the enzyme was studied with commercial and local detergents in the presence of 10 mM CaCl2. The addition of 10 mM CaCl2 individually and in combination, was found to be very effective in improving the enzyme stability. This enzyme improved the cleansing power of various detergents. It removed blood stains completely when used with detergents in the presence of 10 mM CaCl2.
Pharmaceutical Chemistry Department, Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune-18. badheravi@rediffmail.com
Ravindra V
RV
Badhe
Sunil L
SL
Harer
Rabindra K
RK
Nanda
Sonali R
SR
Badhe
Sumitra J
SJ
Jangam
Avinash D
AD
Deshpande
D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D045482:Hot Springs / Q000382:microbiology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D057057:Serine Proteases / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D010450:Endopeptidases; D057057:Serine Proteases; C087822:alkaline protease