Glucoamylase Wild Type & Mutant Struct Stability Thermodynamic Study

Kahn, Saber

Abstract

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.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Center for Research Resources (NCRR)
• Type: Biotechnology Resource Grants (P41)
• Project #: 5P41RR004328-10
• Application #: 6122037
• Project Start: 1997-08-05
• Project End: 1998-08-04
• Support Year: 10
• Fiscal Year: 1997

Institution

• Name: Johns Hopkins University
• DUNS #: 045911138
• City: Baltimore
• State: MD
• Country: United States
• Zip Code: 21218

Publications

Jaganaman, Sunil; Pinto, Alex; Tarasev, Michael et al. (2007) High levels of expression of the iron-sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli. Protein Expr Purif 52:273-9

Todd, M J; Gomez, J (2001) Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal Biochem 296:179-87

Karantza, V; Freire, E; Moudrianakis, E N (2001) Thermodynamic studies of the core histones: stability of the octamer subunits is not altered by removal of their terminal domains. Biochemistry 40:13114-23

Griko, Y V; Remeta, D P (1999) Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin. Protein Sci 8:554-61

Chu, V; Freitag, S; Le Trong, I et al. (1998) Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin-biotin system. Protein Sci 7:848-59

Luque, I; Freire, E (1998) Structure-based prediction of binding affinities and molecular design of peptide ligands. Methods Enzymol 295:100-27

Luque, I; Gomez, J; Semo, N et al. (1998) Structure-based thermodynamic design of peptide ligands: application to peptide inhibitors of the aspartic protease endothiapepsin. Proteins 30:74-85

Gomez, J; Semo, N; Freire, E (1998) Structural thermodynamic study of the binding of renin inhibitors to endothiapepsin. Adv Exp Med Biol 436:325-8

Koder, R L; Miller, A F (1998) Overexpression, isotopic labeling, and spectral characterization of Enterobacter cloacae nitroreductase. Protein Expr Purif 13:53-60

Freire, E (1998) Statistical thermodynamic linkage between conformational and binding equilibria. Adv Protein Chem 51:255-79