A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification |
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Authors: | Khawar Sohail Siddiqui Don M. Parkin Paul M. G. Curmi Davide De Francisci Anne Poljak Kevin Barrow Malcolm H. Noble Jill Trewhella Ricardo Cavicchioli |
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Affiliation: | 1. School of Biotechnology and Biomolecular Sciences (BABS), The University of New South Wales (UNSW), Sydney, NSW 2052, Australia;2. telephone: 61‐2‐93853516;3. fax: 61‐2‐93852742;4. School of Molecular and Microbial Biosciences, The University of Sydney, NSW, Australia;5. School of Physics, UNSW, Sydney, NSW 2052, Australia and Centre for Immunology, St Vincent's Hospital, Sydney, NSW, Australia;6. Bioanalytical Mass Spectrometry Facility (BMSF), UNSW, Sydney, NSW, Australia |
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Abstract: | The alkaline protease, savinase was chemically modified to enhance the productivity of the enzyme at low temperatures on a complex polymeric protein (azocasein) substrate. At 5 and 15°C, savinase modified with ficol or dextran hydrolyzed fivefold more azocasein than the unmodified savinase. Kinetic studies showed that the catalytic improvements are associated with changes in uncompetitive substrate inhibition with Ki values of modified savinases sixfold higher than the unmodified savinase. Modeling of small‐angle scattering data indicates that two substrate molecules bind on opposing sides of the enzyme. The combined kinetic and structural data indicate that the polysaccharide modifier sterically blocks the allosteric site and reduces substrate inhibition. In contrast to the properties of cold‐active enzymes that generally manifest as low activation enthalpy and high flexibility, this study shows that increased activity and productivity at low temperature can be achieved by reducing uncompetitive substrate inhibition, and that this can be achieved using chemical modification with an enzyme in a commercial enzyme‐formulation. Biotechnol. Bioeng. 2009;103: 676–686. © 2009 Wiley Periodicals, Inc. |
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Keywords: | allostery cold‐adapted enzyme structure function and stability enzyme kinetics Savinase SAXS |
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