Doctor of Philosophy (PhD)
The process of immobilizing enzymes onto solid supports for bioreactions has some compelling advantages compared to their solution-based counterpart including the facile separation of enzyme from products, elimination of enzyme autodigestion, and increased enzyme stability and activity. We report in this work, the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micro- and nano-pillars populated within a fluidic devices for the micro and nanoscale on-chip digestion of double-stranded DNA. Enzyme immobilization in both studies was successfully accomplished using 3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling to carboxylic acid functionalized PMMA micropillars. Our micro-scale results suggest that the reaction efficiency for the catalysis of dsDNA digestion using λ-exonuclease, including its processivity and reaction rate, were higher when the enzyme was attached to a solid support compared to the free solution digestion. We obtained a clipping rate of 1.0 x 10^3 nucleotides s^-1 for the digestion of λ-DNA (48.5 kbp) by λ-exonuclease. We suggest that the kinetic behavior of this solid-phase reactor could be described by a fractal Michaelis-Menten. Preliminary nano-scale λ-Exo immobilization experiments reveal potential enzymatic activity changes as observed in reduced digestion rates (~303 nucleotides s-1). Further studies will deduce reasoning for these observed differences. Simulation of the nanofluidic reactors reveal kinetic behavior to be mass transport limited, a result not expected due to the reduction in reactor dimensions. Nonetheless, the results from these studies work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification. As a step towards this goal, an investigation of the dynamics of DNA in these irregularly shaped structures has been performed.
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Calixte, Nyote J., "From Micro- to Nano-Scale: Applications of Solid-Phase Enzymatic Reactors for Biopolymer Disassembly" (2014). LSU Doctoral Dissertations. 924.
Soper, Steven A