Barostability of milk plasmin activity

Abstract

The influence of high pressure (HP) on the stability and activity of the milk alkaline proteinase plasmin was examined. Assays of enzyme activity following HP treatment of plasmin in phosphate buffer (pH 6.7) indicated that the enzyme was extremely pressure stable, retaining almost all activity even after treatment at 600 MPa for 20 min at 20 °C. Plasmin was also extremely stable when HP treated in buffer containing 25 mg·mL-1 sodium caseinate, and in cheese. However, HP treatment in buffer containing 5 mg·mL-1 β-lactoglobulin resulted in enzyme inactivation at pressures > 400 MPa, indicating that the presence of β-lactoglobulin greatly destabilises the enzyme under high pressure, which is analogous to the effect of this protein on the heat stability of plasmin. In separate experiments, hydrolysis of β-casein by plasmin at 20 °C 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 proteolysis of β-casein was decreased at P > 400 MPa. As judged from RP-HPLC analysis, production of 2%-TCA soluble peptides by plasmin appeared unaffected 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 (β-casein) at pressures up to 700 MPa, but is sensitive to destabilisation by denatured β-lactoglobulin.