Purification and characterization of an extracellular protease from Bacillus subtilis EAG-2 strain isolated from ornamental plant nursery.
Bacillus subtilis EAG-2 strain isolated from an ornamental plant nursery produced a highly active extracellular protease. It was purified to apparent homogeneity by successive purification steps. The SDS-gel of purified protease revealed a single band of 27 KDa on 10% polyacrylamide gel. Proteolytic activity was confirmed by using two different zymographic methods. Interestingly, the enzyme showed two clear activity bands in both cases. The optimum proteolysis for this protease was observed at pH 8.5 and 65 degrees C. The enzyme was highly stable up to 80% after 30-50 degrees C for 60 minutes. It also remained stable at pH 6.5-9.0 after 4 hours of incubation at 37 degrees C. Its activity was reduced to 16% and 25% by PMSF and APMSF which indicates its relation to serine proteases. An increase in activity was noticed in the presence of Ca(+2), Zn(+2) and Ba(+2). On the other hand, it worked effectively with different natural substrates. Hence EAG-2 protease might be a useful contribution to the enzyme industry in Pakistan based upon its distinctive properties.
Ghafoor
Afia
A
Department of Microbiology and Molecular Genetics, Quaid-e-Azam Campus University of the Punjab, Lahore, Pakistan. afia_ghafoor@yahoo.com
Hasnain
Shahida
S
D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010944:Plants / Q000382:microbiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011480:Protease Inhibitors; D010447:Peptide Hydrolases