title: Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs.
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 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.
authors.affiliation: Department of Chemistry, Louisiana State University , Baton Rouge, Louisiana 70803, United States.
authors.forename: Nyoté J
authors.initials: NJ
authors.lastname: Oliver-Calixte
authors.forename: Franklin I
authors.initials: FI
authors.lastname: Uba
authors.forename: Katrina N
authors.initials: KN
authors.lastname: Battle
authors.forename: Kumuditha M
authors.initials: KM
authors.lastname: Weerakoon-Ratnayake
authors.forename: Steven A
authors.initials: SA
authors.lastname: Soper
mesh_terms: D004247:DNA / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005092:Exonucleases / Q000737:chemistry*; D007700:Kinetics; D011108:Polymers / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D011108:Polymers; D004247:DNA; D005092:Exonucleases