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Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology
Author(s) -
Beg Qasim Khalil,
Saxena R. K.,
Gupta Rani
Publication year - 2002
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10203
Subject(s) - activation energy , chemistry , arrhenius plot , analytical chemistry (journal) , michaelis–menten kinetics , response surface methodology , kinetic energy , protease , substrate (aquarium) , kinetics , casein , reaction rate constant , arrhenius equation , bacillus subtilis , chromatography , stereochemistry , nuclear chemistry , thermodynamics , enzyme , enzyme assay , biochemistry , physics , oceanography , quantum mechanics , geology , biology , bacteria , genetics
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°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°C, respectively, whereas V max values at these temperatures were 74.07, 99.01, 116.28, and 120.48 μg/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45°C until 60–65°C followed by thermal inactivation. Likewise, the energy of activation ( E a ) was more between 45–55°C (9747 cal/mol) compared to E a between 50–60°C (4162 cal/mol). © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 289–295, 2002.