Characterization and engineering of a thermophilic chaperone to improve enzyme stability in denaturing co-solvents

Many enzymatic reactions proceed better in organic co-solvents due to the increased solubility of nonpolar substrates as well as the driving of reactions towards alternative products. However, the use of enzymes in the presence of organic solvents can be limited by enzyme stability, particularly in solutions containing a high concentration of a water miscible organic co-solvent. Thus, there is a need for general methods of stabilizing enzymes in aqueous-organic co-solvent mixtures. We demonstrate the use of an organic solvent-stable hyperthermophilic chaperonin subunit to prevent enzyme aggregation and preserve enzyme activity in the presence of water/co-solvent mixtures.;We have produced and characterized the recombinant thermosome (rTHS) from Methanocaldococcus jannaschii. rTHS prevents protein aggregation and maintains/restores enzyme activity in the direct presence of co-solvents. Given the unfolding pathways of the enzyme, we can predict how well rTHS inhibits enzyme deactivation and, when appropriate, can modify the unfolding environment to assist rTHS function. Specific chaperoning regions were identified within rTHS that are responsible for protein substrate refolding. In addition, we have designed, engineered, and characterized chimera proteins in which enzyme is genetically fused to rTHS to create a self-renaturing enzyme. These chimeras were immobilized to function in continuous flow reactions. This unprecedented approach of using the natural function of chaperones to assist in enzyme stabilization in co-solvents provides a means of expanding the applications of semi-aqueous enzymology.