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

Abstract

This work describes the effect of the hydrolysis time and pressure (0.1-400 MPa) on the proteolysis of beta-lactoglobulin A (beta-lg A) with trypsin, either conducting hydrolysis of beta-lg under pressure or hydrolysing beta-lg that was previously pressure treated. Pressurisation, before or during enzyme treatments, enhanced tryptic hydrolysis of beta-lg. Trypsin degraded pressure-modified beta-lg and pressure-induced beta-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 beta-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 beta-lg under high pressure are discussed.

Hydrolysis of β-lactoglobulin by trypsin under acidic pH and analysis of the hydrolysates with MALDI–TOF–MS/MS

“…The most interesting influences are attributed to temperature and pressure. Through high temperature and high pressure the protein structure is changed, leading to the exposure to the enzyme of more cleavage sites (Chicon, Belloque, Alonso, Martin-Alvarez, & Lopez-Fandino, 2008;Chicon, Belloque, Recio, & Lopez-Fandino, 2006;Iametti et al, 2002). Thus hydrolysis may be quicker although cases of enzyme inhibition were reported with protein heat denaturing (Cheison, Wang, & Xu, 2007) over time.…”

Immunoreactivity and digestibility of high-pressure-treated whey proteins

“…On the other hand, HP-induced changes in the conformation of the proteins may make enzymatic digestion easier, and HP treatment has been reported to enhance the susceptibility of b-Lg to subsequent proteolysis at atmospheric pressure (Bonomi et al, 2003;Chico´n, Belloque, Recio, & Lo´pez-Fandin˜o, 2006a;Chico´n, Lo´pez-Fandin˜o, Quiro´s, & Belloque, 2006b;Knudsen, Otte, Olsen, & Skibsted, 2002;Otte, Zakora, Qvist, Olsen, & Barkholt, 1997).…”

Pressure Cycling Technology-assisted Protein Digestion for Efficient Proteomic Analysis

“…10 High pressure is another alternative energy that has been used to increase enzymatic activity. 15 High pressure is thought to change the protein conformation and force the penetration of water molecules into the protein interior, especially into cavities, leading to denaturing of the protein. 16 In this study, we used PCT, which uses alternating cycles of atmospheric and high pressure, up to tens of kpsi (1 kpsi = 6.895 MPa).…”