title: Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology.
abstract: The kinetic constants for an alkaline protease from Bacillus mojavensis were determined using a central composite circumscribed design (CCCD) where concentration of substrate (casein) and the assay temperature were varied around their center point. The K(m),V(max), K(cat), activation energy (E(a)) and temperature coefficient (q(10)) were determined and the values of these kinetic constants obtained were found comparable to that obtained with conventional methods. The Michaelis-Menten constant (K(m)) for casein decreased with corresponding increase in V(max), as reaction temperature was raised from 45-60 degrees C. The protease exhibited K(m) of 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml, and 0.0250 mg/ml at 45, 50, 55, and 60 degrees C, respectively, whereas V(max) values at these temperatures were 74.07, 99.01, 116.28, and 120.48 microg/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45 degrees C until 60-65 degrees C followed by thermal inactivation. Likewise, the energy of activation (E(a)) was more between 45-55 degrees C (9747 cal/mol) compared to E(a) between 50-60 degrees C (4162 cal/mol).
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Marg, New Delhi 110 021, India. microzyme@123india.com
authors.forename: Qasim Khalil
authors.initials: QK
authors.lastname: Beg
authors.forename: R K
authors.initials: RK
authors.lastname: Saxena
authors.forename: Rani
authors.initials: R
authors.lastname: Gupta
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D002364:Caseins / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D015233:Models, Statistical; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002364:Caseins; D012697:Serine Endopeptidases; C032923:microbial serine proteinases