REDUCTION OF NEGATIVE CHARGE IN THE ASPARTYL PROTEINASE FROM THE FUNGUS MUCOR-MIEHEI BY CHEMICAL MODIFICATION OF CARBOXYL GROUPS - EFFECT ON STRUCTURE-FUNCTION

Decreased negative charge in the aspartyl proteinase from Mucor miehei (MMP) by modification of carboxyl groups with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and different nucleophiles (methyl esters of glycine, leucine, arginine, and tryptophan) reduced proteolytic and milk-clotting activity an average of 24 and 93%, respectively. A shift in the pH-activity optimum from pH 5.0 to pH 3.0 or 3.5 (depending on nucleophile), increased pH-stability, and generally lower temperature-activity optimum and range were also consequences of modification. Relative to native enzyme, enthalpy of denaturation values and peak denaturation temperatures, determined by differential scanning calorimetry, were lower only for arginine and tryptophan methyl ester-modified MMP; the kinetic and thermodynamic parameters activation energy of denaturation and change in free energy, respectively, indicated compromised stability of all carboxyl-modified derivatives. Changes in functional properties of modified MMP were associated with changes in tertiary structure as evidenced by decreased near-UV CD spectral intensity. No change in the proportions of secondary structure fractions was observed. Results from this study indicated that the reduction of negative charge via carboxyl modification was not a viable means for increasing the cheese-making potential of MMP.