Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase
| Autor: | Christina L. Grek, Simon Delagrave, Michael R. Dilworth, William J. Coleman, Dennis J. Murphy, Anthony M. Maffia, Douglas C. Youvan, Mary M. Yang, Edward J. Bylina, Jennifer L. Rittenhouse Pruss, Barry L. Marrs |
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| Rok vydání: | 2001 |
| Předmět: |
chemistry.chemical_classification
Genomic Library Mutation Bioengineering Polymer Biology Methylgalactosides medicine.disease_cause Directed evolution Galactose Oxidase Biochemistry Kinetics Enzyme chemistry Galactose oxidase Image Processing Computer-Assisted medicine Screening method Specific activity Directed Molecular Evolution Molecular Biology Throughput (business) Biotechnology |
| Zdroj: | Protein Engineering, Design and Selection. 14:261-267 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/14.4.261 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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