Browsing by Author "Siddiqui, KS"

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    A chemically modified alpha-amylase with a molten-globule state has entropically driven enhanced thermal stability
    (Oxford University Press, 2010-10-01) Siddiqui, KS; Poljak, A; Francisci, D; Guerriero, G; Pilak, O; Burg, D; Raftery, MJ; Parkin, DM; Trewhella, J; Cavicchioli, R
    The thermostability properties of TAA were investigated by chemically modifying carboxyl groups on the surface of the enzyme with AMEs. The TAA(MOD) exhibited a 200% improvement in starch-hydrolyzing productivity at 60 degrees C. By studying the kinetic, thermodynamic and biophysical properties, we found that TAAMOD had formed a thermostable, MG state, in which the unfolding of the tertiary structure preceded that of the secondary structure by at least 20 degrees C. The X-ray crystal structure of TAAMOD revealed no new permanent interactions (electrostatic or other) resulting from the modification. By deriving thermodynamic activation parameters of TAAMOD, we rationalised that thermostabilisation have been caused by a decrease in the entropy of the transition state, rather than being enthalpically driven. Far-UV CD shows that the origin of decreased entropy may have arisen from a higher helical content of TAAMOD. This study provides new insight into the intriguing properties of an MG state resulting from the chemical modification of TAA.© 2010, Oxford University Press (OUP)
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    Novel approach for enhancing the catalytic efficiency of a protease at low temperature: reduction in substrate inhibition by chemical modification.
    (Wiley-Blackwell, 2009-07-01) Siddiqui, KS; Parkin, DM; Curmi, PMG; De Francisci, D; Poljak, A; Barrow, KD; Noble, MH; Trewhella, J; Cavicchioli, R
    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. Biotechnol. Bioeng. 2009;103: 676-686. © 2009, Wiley-Blackwell.