title: A chemically modified alpha-amylase with a molten-globule state has entropically driven enhanced thermal stability.
abstract: 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 TAA(MOD) 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 TAA(MOD) revealed no new permanent interactions (electrostatic or other) resulting from the modification. By deriving thermodynamic activation parameters of TAA(MOD), 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 TAA(MOD). This study provides new insight into the intriguing properties of an MG state resulting from the chemical modification of TAA.
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia. k.siddiqui@unsw.edu.au
authors.forename: Khawar Sohail
authors.initials: KS
authors.lastname: Siddiqui
authors.forename: Anne
authors.initials: A
authors.lastname: Poljak
authors.forename: Davide
authors.initials: D
authors.lastname: De Francisci
authors.forename: Gea
authors.initials: G
authors.lastname: Guerriero
authors.forename: Oliver
authors.initials: O
authors.lastname: Pilak
authors.forename: Dominic
authors.initials: D
authors.lastname: Burg
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Raftery
authors.forename: Don M
authors.initials: DM
authors.lastname: Parkin
authors.forename: Jill
authors.initials: J
authors.lastname: Trewhella
authors.forename: Ricardo
authors.initials: R
authors.lastname: Cavicchioli
mesh_terms: D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019277:Entropy; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry*
chemical_list: D000516:alpha-Amylases