Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Chemical Engineering

Committee Chair/Advisor

Dr. Mark A. Blenner

Committee Member

Dr. Marc R. Birtwistle

Committee Member

Dr. Scott M. Husson

Committee Member

Dr. Alexey A. Vertegel


Soluble enzymes have the unique ability to assist the conversion of complex substrates in fewer steps compared to conventional chemical synthesis; however, when immobilized to a surface, the insoluble enzyme's activity and stability can suffer in its new environment. To improve upon future immobilized enzyme efficacy, a fundamental understanding of the interactions across the enzyme, linker, and surface during covalent, site-specific immobilization that can be detrimental to its activity is required. More specifically, using a well-characterized and model enzyme, T4 lysozyme (T4L), 28 different cysteine attachment points' effects on site-specifically immobilized activity are examined. Different combinations of heterobifunctional amine-to-sulfhydryl crosslinkers and hydrophobic and hydrophilic aminated polymer surfaces are used to immobilize T4L site-specifically via amine-to-sulfhydryl conjugation. The cysteine mutations availability and accessibility result in heuristics that minimize nonspecific adsorption while maximizing site-specific conjugation. Based on the findings, an improved method for site-specifically immobilizing biotinylated T4L on streptavidin-coated polymer beads is developed. Innate structural characteristics such as cysteine accessibility, amino acid consensus, and enzyme surface concentration enhance immobilized enzyme’s specific catalytic efficiency regardless of the attachment site and orientation.



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