Biophysical evaluation of milk-clotting enzymes processed by high pressure

- HPP is able to promote changes in milk-clotting enzymes structure.
- HPP increased the intrinsic fluorescence under activation conditions.
- HPP promoted a drastic reduction of the fluorescence under inactivation conditions.
- HPP resulted greater changes to the coagulants secondary structures at higher pressures.
- Porcine pepsin was the most sensitive enzyme to the HPP.

High pressure processing (HPP) is able to promote changes in enzymes structure. This study evaluated the effect of HP on the structural changes in milk-clotting enzymes processed under activation conditions for recombinant camel chymosin (212 MPa/5 min/10 °C), calf rennet (280 MPa/20 min/25 °C), bovine rennet (222 MPa/5 min/23 °C), and porcine pepsin (50 MPa/5 min/20 °C) and under inactivation conditions for all enzymes (600 MPa/10 min/25 °C) including the protease from Rhizomucor miehei. In general, it was found that the HPP at activation conditions was able to increase the intrinsic fluorescence of samples with high pepsin concentration (porcine pepsin and bovine rennet), increase significantly the surface hydrophobicity and induce changes in secondary structure of all enzymes. Under inactivation conditions, increases in surface hydrophobicity and a reduction of intrinsic fluorescence were observed, suggesting a higher exposure of hydrophobic sites followed by water quenching of Trp residues. Moreover, changes in secondary structure were observed (with minor changes seen in Rhizomucor miehei protease). In conclusion, HPP was able to unfold milk-clotting enzymes even under activation conditions, and the porcine pepsin and bovine rennet were more sensitive to HPP.

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