Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology

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 Michael-is-Menten constant (K-m) for casein decreased with corresponding increase in V-max, as reaction temperature was raised from 45-60degreesC. 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 60degreesC, respectively, whereas V-max values at these temperatures were 74.07, 99.01, 116.28, and 120.48 mug/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45degreesC until 60-65degreesC followed by thermal inactivation. Likewise, the energy of activation (E-a) was more between 45-55degreesC (9747 cal/mol compared to E-a between 50-60degreesC (4162 cal/mol). (C) 2002 Wiley Periodicals, Inc.