Influence of high hydrostatic pressure on the proteolysis of β-lactoglobulin A by trypsin

摘要

This work describes the effect of the hydrolysis time and pressure (0.1-400 MPa) on the proteolysis of β-lactoglobulin A (β-lg A) with trypsin, either conducting hydrolysis of β-lg under pressure or hydrolysing β-lg that was previously pressure treated. Pressurisation, before or during enzyme treatments, enhanced tryptic hydrolysis of β-lg. Trypsin degraded pressure-modified β-lg and pressure-induced β-lg aggregates, favouring proteolysis to the intermediate degradation products: (Val_(15)-Arg_(40))/ (Val_(41)-Lys_(69))S-S(Leu_(149)-Ile_(162)) and (Val_(41)-Lys_(70))S-S(Leu_(149)-Ile_(162)). These were further cleaved at the later stages of proteolysis to yield: (Val_(15)-Tyr_(20)), (Ser_(21)-Arg_(40)), (Val_(41)-Tyr_(60)), (Trp_(61)-Lys_(69))S-S(Leu_(149)-Ile_(162)) and (Trp_(61)-LyS_(70))S-S(Leu_(149)-Ile_(162)). Particularly, in the tryptic hydrolysates of pre-pressurized β-lg, two other fragments linked by disulphide bonds: (Lys_(101)-Arg_(124))S-S(Leu_(149)-Ile_(162)) and (Tyr_(102)-Arg_(124))S-S(Leu_(149)-Ile_(162)), were found. These corresponded to rearrangement products induced by SH/SS exchange between the free thiol group of Cys_(121) and Cys_(160), that normally forms the disulphide bond Cys_(66)-Cys_(160). In the light of these results, structural modifications of β-lg under high pressure are discussed.