title: Effect of pH and temperature on stability and kinetics of novel extracellular serine alkaline protease (70 kDa).
abstract: A novel extracellular serine protease (70 kDa by SDS-PAGE) was purified and characterized. This enzyme retained more than 93% of its initial activity after preincubation for 30 min at 37 °C in the presence of 25% (v/v) tested organic solvents and showed feather degradation activity. The purified enzyme was deactivated at various combinations of pH and temperature to examine the interactive effect of them on enzyme activity. The deactivation process was modeled as first-order kinetics and the deactivation rate constant (k(d)) was found to be minimum at pH 9 and 37 °C. The kinetic analysis of enzyme over a range of pH values indicated two pK values at 6.21 and at 10.92. The lower pK value was likely due to the catalytic histidine in the free enzyme and higher pK value likely reflected deprotonation of the proline moiety of the substrate but ionization of the active site serine is another possibility. Inhibition kinetic showed that enzyme is serine protease because enzyme was competitively inhibited by antipain and aprotinin as these compounds are known to be competitive inhibitors of serine protease. The organic solvent, thermal and pH tolerances of enzyme suggested that it may have potential for use as a biocatalyst in industry.
authors.affiliation: Department of Biotechnology, National Institute of Technology, Mahatma Gandhi Avenue, Durgapur 713209, India.
authors.forename: Biswanath
authors.initials: B
authors.lastname: Bhunia
authors.forename: Bikram
authors.initials: B
authors.lastname: Basak
authors.forename: Tamal
authors.initials: T
authors.lastname: Mandal
authors.forename: Pinaki
authors.initials: P
authors.lastname: Bhattacharya
authors.forename: Apurba
authors.initials: A
authors.lastname: Dey
mesh_terms: D000818:Animals; D000976:Antipain / Q000494:pharmacology; D007611:Aprotinin / Q000494:pharmacology; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002645:Chickens; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology*; D005241:Feathers / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D057057:Serine Proteases / Q000302:isolation & purification / Q000378:metabolism*; D012997:Solvents; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D008670:Metals; D011480:Protease Inhibitors; D012997:Solvents; D000976:Antipain; D007611:Aprotinin; D010450:Endopeptidases; D057057:Serine Proteases; C087822:alkaline protease