| Autor: |
Oliver-Calixte NJ; Department of Chemistry, Louisiana State University , Baton Rouge, Louisiana 70803, United States., Uba FI, Battle KN, Weerakoon-Ratnayake KM, Soper SA |
| Jazyk: |
angličtina |
| Zdroj: |
Analytical chemistry [Anal Chem] 2014 May 06; Vol. 86 (9), pp. 4447-54. Date of Electronic Publication: 2014 Apr 08. |
| DOI: |
10.1021/ac5002965 |
| Abstrakt: |
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/N-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(3) nucleotides s(-1) 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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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