Stabilization and activation of {alpha}-chymotrypsin in water-organic solvent systems by complex formation with oligoamines

Formation of enzyme–oligoamine complexes was suggestedas an approach to obtain biocatalysts with enhanced resistancetowards inactivation in water–organic media. Complex formationresults in broadening (by 20–40% v/v ethanol) of the rangeof cosolvent concentrations where the enzyme retains its catalyticactivity (stabilization effect). At moderate cosolvent concentrations(20–40% v/v) complex formation activates the enzyme (by3–6 times). The magnitude of activation and stabilizationeffects increases with the number of possible electrostaticcontacts between the protein surface and the molecules of oligoamines(OA). Circular dichroism spectra in the far-UV region show thatcomplex formation stabilizes protein conformation and preventsaggregation in water–organic solvent mixtures. Two populationsof the complexes with different thermodynamic stabilities werefound in -chymotrypsin (CT)–OA systems depending on theCT/OA ratio. The average dissociation constants and stoichiometriesof both low- and high-affinity populations of the complexeswere estimated. It appears that it is the low-affinity siteson the CT surface that are responsible for the activation effect.