Summary

Objectives: High pressure UV-Vis absorbance spectroscopy (HPS) was used to characterize the influence of pressure and temperature on the rates at which the proteases trypsin, chymotrypsin, and lysyl endopeptidase (Lys-C) digest their respective substrates.

Methods: Using a high pressure optical cell coupled to the HUB880 high pressure generator, real-time kinetics based on the conversion of chromogenic substrates were measured at pressure ranging from atmospheric to 420 MPa at temperatures between 23°C and 55°C.Results: The synergy between elevated pressure and temperature was most pronounced with Lys-C where the rate of substrate conversion was increased 21.2-fold at 55°C at 420 MPa relative to room temperature at atmospheric pressure, and11.2-fold compared to 37°C at atmospheric pressure. Similarly, chymotrypsin reaction rate increased 13.3-fold at 55°C at 310 MPa relative to37°C at atmospheric pressure, with an apparent inflection point near 290 MPa. In contrast, trypsin digestion rates increased only 2.1 and 1.7 fold at55°C at 0 and 280 MPa, respectively.

Conclusions: HPS facilitated the rapid optimization of digestion conditions for three proteases which considers the synergistic effects of both elevated pressure and temperature specific foreach enzyme.: High pressure UV-Vis absorbance spectroscopy (HPS) was used to characterize the influence of pressure and temperature on the rates at which the proteases trypsin, chymotrypsin, and lysyl endopeptidase (Lys-C) digest their respective substrates.