Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase

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 refrac...

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Bibliographic Details
Published in:	Protein engineering Vol. 14; no. 4; pp. 261 - 267
Main Authors:	Delagrave, Simon, Murphy, Dennis J., Pruss, Jennifer L. Rittenhouse, Maffia, Anthony M., Marrs, Barry L., Bylina, Edward J., Coleman, William J., Grek, Christina L., Dilworth, Michael R., Yang, Mary M., Youvan, Douglas C.
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 01-04-2001 Oxford Publishing Limited (England)
Subjects:	2′-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid 4-chloronaphthol 4CN ABTS alcohol oxidation digital imaging spectroscopy directed evolution Directed Molecular Evolution - methods EPP error-prone PCR galactose oxidase Galactose Oxidase - genetics Galactose Oxidase - metabolism Genomic Library high-throughput screening Image Processing, Computer-Assisted Kinetics Methylgalactosides - metabolism Mutation alcohol oxidation EPP, error-prone PCR 4CN, 4-chloronaphthol GO, galactose oxidase high-throughput screening ABTS, 2,2′-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid digital imaging spectroscopy galactose oxidase directed evolution
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Summary:	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 Km. 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.
Bibliography:	local:0140261 istex:E9A548BB03556FB3E41A12A26048F5AC94E8B1B5 ark:/67375/HXZ-0W4MJW1L-P PII:1460-213X ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0269-2139 1741-0126 1460-213X 1741-0134
DOI:	10.1093/protein/14.4.261