Barostability of milk plasmin activity

The influence of high pressure (HP) on the stability and activity of the mi lk alkaline proteinase plasmin was examined. Assays of enzyme activity foll owing HP treatment of plasmin in phosphate buffer (pH 6.7) indicated that t he enzyme was extremely pressure stable, retaining almost all activity even after treatment at 600 MPa for 20 min at 20 degreesC. Plasmin was also ext remely stable when HP treated in buffer containing 25 mg.mL(-1) sodium case inate, and in cheese. However, HP treatment in buffer containing 5 mg.mL(-1 ) beta -lactoglobulin resulted in enzyme inactivation at pressures > 400 MP a, indicating that the presence of beta -lactoglobulin greatly destabilises the enzyme under high pressure, which is analogous to the effect of this p rotein on the heat stability of plasmin. In separate experiments, hydrolysi s of beta -casein by plasmin at 20 degreesC for 30 min at various pressures (300-800 MPa) was studied. Parallel control incubations were performed at atmospheric pressure. Urea-PAGE analysis of digests showed that primary pro teolysis of beta -casein was decreased at P > 400 MPa. As judged from RP-HP LC analysis, production of 2%-TCA soluble peptides by plasmin appeared unaf fected at P < 700 MPa. above which pressure the rates of peptide production decreased. Overall, plasmin is relatively pressure stable in most systems and can hydrolyse its preferred substrate (<beta>-casein) at pressures up t o 700 MPa, but is sensitive to destabilisation by denatured beta -lactoglob ulin.