S5: Altering Enzyme Surfaces to Dramatically Improve Performance

The properties of enzymes functioning outside of their natural milieu are often suboptimal for industrial biotechnology applications.  Historically, protein engineers have optimized enzymes through mutations at or near the active site that affect specific interactions with the substrate.  They tend to overlook the contribution of both short- and long-range non-specific interactions arising from intermolecular colloidal and surface forces that govern association and dissociation with the substrate.  In this paper we demonstrate that mutations outside of the active site improve performance in various commercial applications, for instance detergent cleaning by proteases or starch liquefaction by amylases.  We modulate the electrostatic forces between enzyme and substrate through systematic variation of the enzyme net charge by accumulation of charges mutations on its surface, i.e. charge ladders.  We have built several enzyme charge ladders that exhibit equal solution activity and stability, and vary in surface charge from as much as -12 to +12 elementary charges.  Working at a defined pH against a charged substrate, such as protein soils on cloth, there is an optimum surface charge for performance.  The figure shows protease wash performance against Blood/Milk/Ink stains in buffer and in laundry detergent as a function of enzyme surface charge. .  When viewed as charged colloids, the performance of enzymes on a charged substrate reduces to a common scale described by their zeta potential. In fact, knowing the charge of the substrate and the reaction conditions allows us to calculate the optimal enzyme surface properties. This approach enables rapid enzyme optimization for application conditions