Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties.
Submitter: Shu-Ching Ou
Submission Date: Dec. 5, 2018, 3:39 p.m.
|Number of data points||38|
|Proteins||Carbonic anhydrase 2|
|Assays/Quantities/Protocols||Experimental Assay: Thermal Stability: Tm ; Experimental Assay: T_inact: Temperature of Thermal Inactivation ; Experimental Assay: Kinetic Constant: kcat_ex/Keff_S ; Experimental Assay: kB: Rate Constant for Protein Transfer ; Derived Quantity: SD of Thermal Stability: Tm|
|Libraries||Variants for HCA2 ; Variants for HCA2_CO2_HEPES|