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A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification
Author(s) -
Siddiqui Khawar Sohail,
Parkin Don M.,
Curmi Paul M. G.,
Francisci Davide De,
Poljak Anne,
Barrow Kevin,
Noble Malcolm H.,
Trewhella Jill,
Cavicchioli Ricardo
Publication year - 2009
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.22300
Subject(s) - substrate (aquarium) , chemistry , uncompetitive inhibitor , enzyme , steric effects , protease , stereochemistry , combinatorial chemistry , organic chemistry , non competitive inhibition , biology , ecology
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 K i 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.