A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification

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 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.

Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation

“…Conformational flexibility plays an important role in enzyme stability (24,28). High flexibility, particularly around the active site, tends to result in an enzyme with high specific activity and low activation energy (28).…”

“…High flexibility, particularly around the active site, tends to result in an enzyme with high specific activity and low activation energy (28). Chemical modification has been reported to increase the conformational flexibility of a protease, thereby enhancing the enzyme's catalytic efficiency (28).…”

Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

“…The endogenous proteases from the detergents were inactivated by incubating the detergents at 65°C for 1 h before addition. When enzyme activity was determined, the detergents were diluted to give a final concentration of 7 mg/ml for solid detergents and 1% for liquid detergents to simulate washing conditions (9). The starting concentration of the added enzyme was 16 U/ml.…”

“…High flexibility, particularly around the active site, tends to result in an enzyme with high specific activity and low activation energy (10). For example, the conformational flexibility in a protease function became higher after chemical modification, and this enhanced the catalytic efficiency of the enzyme (9). The flexibility in the active site of AmyK was shown by the loop containing residues 337 to 347 (Fig.…”

“…The commonly used protein engineering strategies include site-directed mutagenesis and directed evolution (e.g., errorprone PCR) (4)(5)(6)(7)(8)(9)(10)(11). However, there are some disadvantages for their applications for improving the catalytic efficiency.…”