Directing enzymatic cross-linking activity to the air–water interface by a fusion protein approach

Enzymatic cross-linking of proteins is of great interest due to an effective and controlled way of modifying the structures of protein networks. Enzyme-aided structural engineering aims at enhanced stabilisation of foams, emulsions and dispersions by enzymatically inducing intra- and intermolecular cross-links between proteins in continuous phase and/or at interfaces. Formation of stronger interfacial structures protects air bubbles or oil droplets against coalescence, and in some applications, may even be preferable to bulk reactions. In this work we studied direction of enzymatic cross-linking reactions to the air–water interface by enhancing the functionality of enzymes as fusion proteins. We used a hydrophobin–laccase fusion protein, HFBI–MaL, where the surface active hydrophobin component facilitates the access for laccase to cross-link β-casein film directly at the air–water interface. As a result, enhanced cross-linking was shown by means of surface dilatational rheology, where increased G moduli indicated formation of a stronger film with the fusion protein compared to native laccase.