Identifier

etd-08272014-211241

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

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.

Date

2014

Document Availability at the Time of Submission

Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.

Committee Chair

Soper, Steven A

Included in

Chemistry Commons

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