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. Study holds ProTherm entries: 25787, 25788, 25789, 25790, 25791, 25792, 25793, 25794 Extra Details: Unfolding at a scan rate of 60 degreesC/h activation thermodynamics; calorimetry; entropy/ enzyme kinetics; protein X-ray crystallographic structure
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:56 p.m.
|Number of data points||8|
|Proteins||Alpha-amylase A type-1/2 ; Alpha-amylase A type-1/2|
|Assays/Quantities/Protocols||Experimental Assay: Cm prot_conc:0.20 microM ; Experimental Assay: Cm prot_conc:0.21 microM ; Experimental Assay: Tm prot_conc:3.6 microM, buffers:Sodium acetate: 0.05 mM ; Experimental Assay: Tm buffers:Sodium acetate: 0.05 mM, prot_conc:3.8 microM ; Experimental Assay: Tm prot_conc:18 microM, buffers:Sodium acetate: 50 mM ; Experimental Assay: Tm prot_conc:19 microM, buffers:Sodium acetate: 50 mM|
|Libraries||Mutations for sequence ATPADWRSQSIYFLLTDRFARTDGSTTATCNTADQKYCGGTWQGIIDKLDYIQGMGFTAIWITPVTAQLPQTTAYGDAYHGYWQQDIYSLNENYGTADDLKALSSALHERGMYLMVDVVANHMGYDGAGSSVDYSVFKPFSSQDYFHPFCFIQNYEDQTQVEDCWLGDNTVSLPDLDTTKDVVKNEWYDWVGSLVSNYSIDGLRIDTVKHVQKDFWPGYNKAAGVYCIGEVLDGDPAYTCPYQNVMDGVLNYPIYYPLLNAFKSTSGSMDDLYNMINTVKSDCPDSTLLGTFVENHDNPRFASYTNDIALAKNVAAFIILNDGIPIIYAGQEQHYAGGNDPANREATWLSGYPTDSELYKLIASANAIRNYAISKDTGFVTYKNWPIYKDDTTIAMRKGTDGSQIVTILSNKGASGDSYTLSLSGAGYTAGQQLTEVIGCTTVTVGSDGNVPVPMAGGLPRVLYPTEKLAGSKICSSS|