Boosting the performance of a protease with polymers and surfactants

The main objective of this PhD thesis was to gain a deep molecular understanding of interactions which govern an enzymatic activity boost on a subtilisin alkaline serine protease (PDB ID: 1ST3) in the presence of anionic surfactants and polymers. To this end, individual interactions between the protease and anionic surfactants (SLES and MES) and polymers (PAA and γ-PGA) that lead to the boosting effect were analyzed systematically in water and in a complex laundry detergent under application conditions. Different approaches comprising directed protease evolution, computational analysis, molecular modeling as well as biophysical characterization were applied to gain insights into the underlying molecular interactions. The key findings underlying mechanism of interaction of proteases with surfactants and polymers is concluded as follow. Interactions between the protease and the anionic surfactants SLES and MES micelles mainly driven by electrostatic forces were observed and common interaction sites for both surfactants close to the Ca2+ binding sites and substrate binding pockets of the protease were identified. Saturation mutagenesis on identified positions governing protease-surfactant interaction revealed protease variants (with substitutions close to Ca2+ binding sites) with up to 190% improved performance on a fabric stain in the presence of SLES or MES. Interaction studies on the protease in the presence of the anionic polyelectrolytes PAA and γ-PGA disclosed an electrostatic driven formation of enyzme-polyelectrolyte complexes (EPCs) with common interaction sites close to the Ca2+ binding sites of the protease. Binding of the polyelectrolytes on the protease enhanced proteolytic performance up to 200% and subsequent protease engineering revealed protease variants with up to 300% enhanced protease performance. In addition, complexation between PAA and the surfactants SLES or MES is bridged by electrostatic interaction via divalent cations such as Ca2+/Mg2+ yielding polyelectrolyte and surfactant complexes (PESCs) with high solubilization efficiency. Finally, the binding of the protease on these PESCs, mainly via electrostatic interaction with the polyelectrolyte or the surfactant, leads to an enhanced performance of the protease in water. The protease benefits highly likely from the properties of the formed PESCs which increase the substrate accessibility of the protease on the cotton surface. The gained molecular understanding on fundamental principles causing enhanced protease performance is likely applicable for other detergent enzymes such as amylases, lipases and cellulases and can be further applied for reengineering of enzyme, polymer and surfactant compositions in modern laundry detergents