Interfacial biocatalysis: Electrostatic interactions elucidated using an enzyme charge ladder

Surface biocatalysis is a complex multistep process that requires enzyme adsorption to facilitate catalysis, which may be assisted by enzyme diffusion at the interface. In this study, we examined the effect of charge modification and the optimization of solution conditions to enhance our understanding of interfacial interactions and to provide strategies for improving biocatalysis at the solid-liquid interface.;A protease charge ladder consisting of cumulative single amino acid charge mutations ranging from -4 to +4 with respective to the parent enzyme was utilized. An immobilized bovine serum albumin (BSA) multilayer was prepared using a layer-by-layer (LbL) self-assembly technique for the model substrate. To characterize enzyme adsorption and reaction, surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) were used. Because of the complex response of fluorescence under surface plasmon excitation, an appropriate method for quantification of the fluorescence signal was developed and applied to the protease-immobilized protein substrate system.;First, we examined the influence of electrostatic interactions on enzyme surface diffusion and the contribution of both enzyme diffusion and electrostatic interactions in interfacial biocatalysis. The interfacial reaction rate exhibited a clear maximum at intermediate enzyme adsorption for each charge variant, which increased for more positive variants. Fluorescence recovery after photobleaching measurements indicated that strong electrostatic interactions decreased the enzyme surface mobility at constant surface concentrations. We proposed that the maximum reaction rate is achieved by a balance between adsorption and surface diffusion, which is inhibited by high enzyme surface concentrations and strong electrostatic interactions. Further, an enhancement of enzyme specific activity at low surface concentrations could be directly correlated with a sharp increase in surface mobility at high ionic strength.;We also studied the effects of substrate surface charge on interfacial catalysis, which was controlled by solution pH or chemical modification of the BSA substrate. Enzyme and substrate charges were characterized at each pH value by measuring the zeta potential. Adsorption and reaction studies of the enzyme charge ladder were performed at each pH for various ionic strength values. At low substrate charge, the maximum rate increased rapidly and leveled off at low ionic strength. At higher substrate charge, the increase was gradual, occurring over a wide range of ionic strengths. The ionic strength dependence of the chemically modified substrate also fits the original trend, confirming our original conclusion.;Finally, we investigated surface catalysis of a single enzyme variant as a function of enzyme solution concentration at fixed ionic strength conditions. Again, the reaction rate showed a maximum at intermediate surface concentrations, suggesting that diffusion hindered by molecular crowding also reduces the enzyme's specific activity. At a fixed surface concentration, the higher ionic strength value displayed the larger reactivity but required a higher solution concentration to achieve. Brownian dynamics simulations were performed to further investigate enzyme diffusion and reaction. The reaction rate measured by simulations also displayed a maximum in reactivity at surface coverage values consistent with experimental data.