Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs
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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.
- ENZYME IMMOBILIZATION; CAPILLARY-ELECTROPHORESIS; MICROANALYTICAL DEVICES; COVALENT IMMOBILIZATION; PROTEIN IMMOBILIZATION; MICROFLUIDIC SYSTEMS; SURFACE MODIFICATION; REACTION-KINETICS; POROUS SILICON; DNA-MOLECULES
- Issue Date
- AMER CHEMICAL SOC
- ANALYTICAL CHEMISTRY, v.86, no.9, pp.4447 - 4454
- 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 × 103 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.
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