Glucoamylase (GA) plays an important role in the industrial conversion of raw starch to glucose syrups which are utilized in the production of ethanol and fructose sweeteners. Since the native structure is thermally unstable above temperatures of 60 C, GA digestion must proceed at low temperature where it is the rate limiting step in the industrial production of glucose syrups. To improve the overall thermal stability of GA, we have engineered the following mutants based on computer modeling methods/techniques: S30P, G137A, N20C/A27C, S30P/G137A, N20C/A27C/S30P, N20C/A27C/G137A, N20C/A27C/G137A/S30P. Our primary objective is to characterize the thermal stability of these mutants and assess the extent to which combined mutations alter the resultant differential scanning calorimetric (DSC) profiles. Our investigation has initially focused on establishing experimental conditions for DSC studies as a function of scan rate, solution pH, protein concentration, and ionic strength. Once appropriate conditions have been established to ensure reversibility of folding/unfolding, we hope to obtain Hcal, Tm and Cp of all the mutants and the wild type enzyme.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
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Johns Hopkins University
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