title: Stability of native and covalently modified papain.
abstract: Covalent modification of enzymes with large polymers can produce modified enzymes which retain considerable biological activity and at the same time display resistance to denaturation by high temperatures and chaotropic agents. The cysteine protease, papain, with potential applications in industry, was covalently coupled to polymeric sucrose (mol. wt 400 kDa) at different ratios. The derivatives retained > 80% intrinsic catalytic activity with no change in pH optima and kinetic constants, indicating that the gross tertiary structure was not altered by modification. However, they displayed better thermotolerance than native papain, as indicated by their higher T50 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, was prolonged by 2- to 30-fold over the native depending on the temperature 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 lesser 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 enzymes.
authors.affiliation: Centre for Protein Engineering and Biomedical Research, Voluntary Health Services, Madras, India.
authors.forename: N
authors.initials: N
authors.lastname: Rajalakshmi
authors.forename: P V
authors.initials: PV
authors.lastname: Sundaram
mesh_terms: D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D010206:Papain / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013395:Sucrose / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: D011134:Polysaccharides; D013395:Sucrose; D014508:Urea; D010206:Papain