Biocatalysis at the Solid-Liquid Interface: Subtilisin on Surface-Bound Polypeptides and Amylase Variants on Starch Granules.

Biocatalysis at surfaces involves the use of enzymes and biological molecules to promote chemical modifications of materials at an interface. It is a growing field of study, with applications in research, medicine, and industry. With surface biocatalysis, the presence of an interface introduces the processes of adsorption and surface diffusion that are not present in bulk reactions. In addition, issues of structure and organization of insoluble substrates are of importance. Protein engineering is commonly employed to develop enzymes that perform optimally for specific surface-bound substrates or environs. However, a detailed understanding of the surface processes involved with surface biocatalysis has been elusive; hence, a coherent approach to the engineering of such enzymes is largely absent. In order to allow rational engineering of enzymes that are used with interfacial systems, a better understanding of the mechanisms that drive surface biocatalysis is required. It is to this end that this research was undertaken. In this work, we present evidence supporting that enzymatic activity on a surface-bound substrate is optimized based on an apparent competition between adsorption and surface diffusion, where adsorption that is too strong negates the benefit of surface diffusion, and binding that is too weak results in too little enzyme bound at the surface. Previous studies by others have been helpful in furthering our understanding of the behavior of enzymes at surfaces. However, most of these have employed fluorescent labels that can interfere with enzyme-substrate interactions involved with surface processes. As such, all work in this thesis was performed under label-free conditions, using an array of complementary techniques to interrogate surface processes. Two types of enzyme-substrate systems involving surface biocatalysis were investigated. The first enzyme system studied was that of subtilisin, a serine protease that breaks down proteins and polypeptides. A synthetically produced polypeptide of specified composition and secondary structure was used to form surface films of varying coverage, by mixing the polypeptide substrate with a diluent molecule and forming mixed films. By interrogating these mixed films with subtilisin, we tested the hypothesis that as films became less dense in the polypeptide, they would become more readily digested due to the enzyme having greater accessibility to the polypeptide chain. The second system used variants of a single alpha-amylase enzyme (AmyS) that digests insoluble starch granules. Substitutions were performed on outer residues of AmyS that were distant from the active site. A sequence of substitutions generated a spectrum of variants such that the surface charge of the AmyS was incrementally changed to be more negative or positive with respect to the wild-type AmyS. To establish relationships between the enzyme charge variants, adsorption, and reactivity, we measured adsorption and reaction for the AmyS variants when introduced to native starch granules. We then used several adsorption models to evaluate the strength of adsorption as well as the surface porosity and enzyme capacity of the starch granules, and related the model results to the reaction profiles of the AmyS variants. Interestingly, the results for both systems were found to be largely influenced by the properties of the substrate. For the subtilisin-polypeptide system, a pure film of the polypeptide was resistant to enzymatic digestion while mixed films showed rapid digestion, even for films that were dominated by polypeptide coverage. Critical aspects proved to be that the binding cleft of Subtilisin engages with a...