Enzymes which regulate the metabolic reactions for sustaining all living things, are the engines of life. The discovery of molecules that are able to control enzyme activity is of great interest for therapeutics and the biocatalysis industry.Peptides are promising enzyme modulators due to their large chemical diversity and the existence of well-established methods for library synthesis.Microarrays represent a powerful tool for screening thousands of molecules, on a small chip, for candidates that interact with enzymes and modulate their functions. In this work, a method is presented for screening highdensity arrays to discover peptides that bind and modulate enzyme activity. A viscous polyvinyl alcohol (PVA) solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide feature. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase (HRP), alkaline phosphatase (APase) and β-galactosidase (β-Gal) and substantially alter their activities by comparing the peptide-enzyme binding levels and bound enzyme activity on microarrays.Several peptides, selected from microarrays, were able to inhibit β-Gal in solution, which demonstrates that behaviors selected from surfaces often transfer to solution. A mechanistic study of inhibition revealed that some of the selected peptides inhibited enzyme activity by binding to enzymes and inducing aggregation.PVA-coated peptide slides can be rapidly analyzed, given an appropriate enzyme assay, and they may also be assayed under various conditions (such as temperature, pH and solvent). I have developed a general method to discover ii molecules that modulate enzyme activity at desired conditions. As demonstrations, some peptides were able to promote the thermal stability of bound enzyme, which were selected by performing the microarray-based enzyme assay at high temperature. For broad applications, selected peptide ligands were used to immobilize enzymes on solid surfaces. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activities and stabilities. Peptide-modified surfaces may prove useful for immobilizing enzymes on surfaces with optimized orientation, location and performance, which are of great interest to the biocatalysis industry.

A study with H(2)-based membrane biofilm reactors (MBfRs) was undertaken to examine the effectiveness of direct H(2) delivery in ex-situ reductive dechlorination of chlorinated ethenes. Trichloroethene (TCE) could be reductively dechlorinated to ethene with up to 95% efficiency as long as the pH-increase effects of methanogens and homoacetogens were managed and dechlorinators were selected for during start-up by creating H(2) limitation. Based on quantitative PCR, the dominant bacterial groups in the biofilm at the end of reactor operation were Dehalococcoides, Geobacter, and homoacetogens. Pyrosequencing confirmed the dominance of the dechlorinators and identified Acetobacterium as the key homoacetogen. Homoacetogens outcompeted methanogens for bicarbonate, based on the effluent concentration of acetate, by suppressing methanogens during batch start-up. This was corroborated by the methanogenesis functional gene mcrA, which was 1-2 orders of magnitude lower than the FTHFS functional gene for homoacetogens. Imaging of the MBfR fibers using scanning electron microscopy showed a distinct Dehalococcoides-like morphology in the fiber biofilm. These results support that direct addition of H(2) can allow for efficient and complete reductive dechlorination, and they shed light into how H(2)-fed biofilms, when operated to manage methanogenic and homoacetogenic activity, can be used for ex-situ bioremediation of chlorinated ethenes.

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