Enhancing Multistep DNA Processing by Solid-Phase Enzyme Catalysis on Polyethylene Glycol Coated Beads

Bioconjug Chem. 2018 Jul 18;29(7):2316-2324. doi: 10.1021/acs.bioconjchem.8b00299. Epub 2018 Jun 21.

Abstract

Covalent immobilization of enzymes on solid supports provides an alternative approach to homogeneous biocatalysis by adding the benefits of simple enzyme removal, improved stability, and adaptability to automation and high-throughput applications. Nevertheless, immobilized (IM) enzymes generally suffer from reduced activity compared to their soluble counterparts. The nature and hydrophobicity of the supporting material surface can introduce enzyme conformational change, spatial confinement, and limited substrate accessibility, all of which will result in loss of the immobilized enzyme activity. In this work, we demonstrate through kinetic studies that flexible polyethylene glycol (PEG) moieties modifying the surface of magnetic beads improve the activity of covalently immobilized DNA replication enzymes. PEG-modified immobilized enzymes were utilized in library construction for Illumina next-generation sequencing (NGS) increasing the read coverage across AT-rich regions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biocatalysis
  • DNA Replication
  • Enzymes, Immobilized / chemistry*
  • High-Throughput Nucleotide Sequencing / methods
  • Kinetics
  • Magnetics
  • Microspheres*
  • Polyethylene Glycols / chemistry*
  • Surface Properties

Substances

  • Enzymes, Immobilized
  • Polyethylene Glycols