Effect of molecular patch modification on the stability of dynamic high‐pressure microfluidization treated trypsin

Previous research indicated that the unfolding of trypsin induced by dynamic high–pressure microfluidization (DHPM) treatment had enhanced its hydrolytic stability. However, the unfolding trypsin is in a metastable state and will subsequently aggregate to be in the lowest energy state. In this study, monomethoxy polyethylene glycol-succinimidyl carbonates (mPEG–SC) was chosen to modify trypsin acted as a molecular patch. The results indicated mPEG–SC showed a significant effect on storage stability and thermal stability of unfolded trypsin. Compared to trypsin DHPM–treated under pressure of 100 MPa, further mPEG–SC modification increased the activity from 50% to 74% and from 70% to 87% after 4 °C for 8 days and 55 °C for 10 min, respectively. Fluorescence quenching demonstrated the conjugation of mPEG–SC and trypsin, and indicated molecular patch modification could inhibit the refolding of unfolded trypsin.Industrial relevanceDynamic high–pressure microfluidization (DHPM) is a promising technology that has been widely applied for innovative foods with modified functional properties or structure. Trypsin has been widely used in modern food industry and biological field. Either DHPM treatment or mPEG–SC modification enhanced the thermal stability of trypsin, while, the combination of the two enhanced the thermal and storage stability of trypsin more significantly. It is expected that this study can provide a new viewpoint and technical scheme for basic research on theory and application of enzyme modification.

► DHPM treatment and mPEG–SC modification showed no significance on the activity of trypsin.
► The thermal stability of trypsin was enhanced by either DHPM treatment or mPEG–SC modification.
► mPEG–SC modification of unfolded trypsin has higher thermal and storage stability than that of modified native trypsin.
► Fluorescence quenching indicated the conjugation of lysine residues of trypsin and mPEG–SC.