STABILITY OF NATIVE AND COVALENTLY MODIFIED PAPAIN

Covalent modification of enzymes with large polymers can produce modif ied enzymes which retain considerable biological activity and at the s ame time display resistance to denaturation by high temperatures and c haotropic agents. The cysteine protease, papain, with potential applic ations in industry, was covalently coupled to polymeric sucrose (mol. wt 400 kDa) at different ratios, The derivatives retained >80% intrins ic catalytic activity with no change in pH optima and kinetic constant s, indicating that the gross tertiary structure was not altered by mod ification, However, they displayed better thermotolerance than native papain, as indicated by their higher T-50 values (6-10 degrees C) and their temperature optima being shifted by 10 degrees C, The half-life of modified papain, calculated from the rate of thermoinactivation, wa s prolonged by 2- to 30-fold over the native depending on the temperat ure and proportion of polymeric sucrose in the adducts, The increases in activation free energy of inactivation (1-10 kJ/mol) and activation enthalpy (4-78 kJ/mol) indicate stabilization of the protein and less er inactivation due to spontaneous unfolding, In the presence of urea, modified papain showed activation, which may be due to a loosening of the 'rigid' structure, reminiscent of the property of thermophilic en zymes.