title: Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase.
abstract: Directed evolution has become an important enabling technology for the development of new enzymes in the chemical and pharmaceutical industries. Some of the most interesting substrates for these enzymes, such as polymers, have poor solubility or form highly viscous solutions and are therefore refractory to traditional high-throughput screens used in directed evolution. We combined digital imaging spectroscopy and a new solid-phase screening method to screen enzyme variants on problematic substrates highly efficiently and show here that the specific activity of the enzyme galactose oxidase can be improved using this technology. One of the variants we isolated, containing the mutation C383S, showed a 16-fold increase in activity, due in part to a 3-fold improvement in K(m). The present methodology should be applicable to the evolution of numerous other enzymes, including polysaccharide-modifying enzymes that could be used for the large-scale synthesis of modified polymers with novel chemical properties.
authors.affiliation: Hercules Incorporated, Corporate Research, 500 Hercules Road, Wilmington, DE 19808, USA.
authors.forename: S
authors.initials: S
authors.lastname: Delagrave
authors.forename: D J
authors.initials: DJ
authors.lastname: Murphy
authors.forename: J L
authors.initials: JL
authors.lastname: Pruss
authors.forename: A M
authors.initials: AM
authors.lastname: Maffia
authors.forename: B L
authors.initials: BL
authors.lastname: Marrs
authors.forename: E J
authors.initials: EJ
authors.lastname: Bylina
authors.forename: W J
authors.initials: WJ
authors.lastname: Coleman
authors.forename: C L
authors.initials: CL
authors.lastname: Grek
authors.forename: M R
authors.initials: MR
authors.lastname: Dilworth
authors.forename: M M
authors.initials: MM
authors.lastname: Yang
authors.forename: D C
authors.initials: DC
authors.lastname: Youvan
mesh_terms: D019020:Directed Molecular Evolution / Q000379:methods*; D005692:Galactose Oxidase / Q000235:genetics* / Q000378:metabolism; D015698:Genomic Library; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008756:Methylgalactosides / Q000378:metabolism; D009154:Mutation
chemical_list: D008756:Methylgalactosides; D005692:Galactose Oxidase