The effects of chemical modification of s-amino groups (using the water soluble polyanionic copolymer ethylene/maleic anhydride) on the structure and function of various aspartyl proteinases (the proteinase from the fungus Mucor miehei (MMP), and the mammalian proteinases porcine pepsin and chymosin) were examined. Modification of 100% of the lysyl residues in the proteinases resulted in an increased milk-clotting to proteolytic activity ratio (MC/PA) for pepsin and chymosin, and a lower MC/PAfor MMP.Ammo-modified MMPwas insensitive to the proteinase inhibitor pepstatin, but modified pepsin and chymosin were inactivated. Modification shifted the pH-activity optimum of the mammalianenzymes by 0.5 pH units to a more alkaline pH and to a more acidic optimumfor MMP. Amino modification also decreased the thermostability of the fungal enzyme. Changes in tertiary structure and significant differences in the proportions of the secondary structure fractions a-helix and /?-sheet were evident only for modified MMP.Results from this study suggested that MMP was more susceptible to destabilization by charge alteration than aspartyl proteinases of mammalian origin.

Decreased negative charge in the aspartyl proteinase from Mucor miehei (MMP) by modification of carboxyl groups with 1-ethyl-3-[3-(dimethylamino)propylIcarbodiimide 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.

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