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      저자 : Oliver-Calixte, Nyoté (검색결과 1 건)
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      • Immobilization of Lambda Exonuclease onto Polymer Micropillar Arrays for the Solid-Phase Digestion of dsDNAs

        Oliver-Calixte, Nyoté,J.,Uba, Franklin I.,Battle, Katrina N.,Weerakoon-Ratnayake, Kumuditha M.,Soper, Steven A. American Chemical Society 2014 ANALYTICAL CHEMISTRY - Vol.86 No.9

        <P>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 the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micropillars populated within a microfluidic device for the on-chip digestion of double-stranded DNA. Enzyme immobilization was successfully accomplished using 3-(3-dimethylaminopropyl) carbodiimide/<I>N</I>-hydroxysuccinimide (EDC/NHS) coupling to carboxylic acid functionalized PMMA micropillars. Our results suggest that the 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 × 10<SUP>3</SUP> nucleotides s<SUP>–1</SUP> for the digestion of λ-DNA (48.5 kbp) by λ-exonuclease. The kinetic behavior of the solid-phase reactor could be described by a fractal Michaelis–Menten model with a catalytic efficiency nearly 17% better than the homogeneous solution-phase reaction. The results from this work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2014/ancham.2014.86.issue-9/ac5002965/production/images/medium/ac-2014-002965_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac5002965'>ACS Electronic Supporting Info</A></P>

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