title: Micro-colony array based high throughput platform for enzyme library screening.
abstract: Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%.
authors.affiliation: Applied Biocatalysis Research Centre, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Pohn
authors.forename: Jochen
authors.initials: J
authors.lastname: Gerlach
authors.forename: Marcel
authors.initials: M
authors.lastname: Scheideler
authors.forename: Hermann
authors.initials: H
authors.lastname: Katz
authors.forename: Martina
authors.initials: M
authors.lastname: Uray
authors.forename: Horst
authors.initials: H
authors.lastname: Bischof
authors.forename: Ingo
authors.initials: I
authors.lastname: Klimant
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
mesh_terms: D002138:Calibration; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism*; D004950:Esterases / Q000235:genetics / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D015202:Protein Engineering; D015203:Reproducibility of Results
chemical_list: D004798:Enzymes; D050505:Mutant Proteins; D004950:Esterases