A novel approach for enhancing the catalytic efficiency of a protease at low temperature: reduction in substrate inhibition by chemical modification.
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 degrees 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.
Siddiqui
Khawar Sohail
KS
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.
Parkin
Don M
DM
Curmi
Paul M G
PM
De Francisci
Davide
D
Poljak
Anne
A
Barrow
Kevin
K
Noble
Malcolm H
MH
Trewhella
Jill
J
Cavicchioli
Ricardo
R
D002364:Caseins / Q000378:metabolism; D002942:Circular Dichroism; D003911:Dextrans / Q000494:pharmacology; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D005362:Ficoll / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature
D002364:Caseins; D003911:Dextrans; D020536:Enzyme Activators; C026018:azocasein; D005362:Ficoll; D012697:Serine Endopeptidases; C032923:microbial serine proteinases