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High Throughput Screening and Directed Evolution of Tyrosine Phenol-Lyase  

Choi Su-Lim (Laboratory of Microbial Function, KRIBB)
Rha Eu-Gene (Laboratory of Microbial Function, KRIBB)
Kim Do-Young (Laboratory of Microbial Function, KRIBB)
Song Jae-Jun (Laboratory of Microbial Function, KRIBB)
Hong Seung-Pyo (Bioleaders Corporation)
Sung Moon-Hee (Bioleaders Corporation, Kookmin University)
Lee Seung-Goo (Laboratory of Microbial Function, KRIBB)
Publication Information
Microbiology and Biotechnology Letters / v.34, no.1, 2006 , pp. 58-62 More about this Journal
Abstract
Rapid assay of enzyme is a primary requirement for successful application of directed evolution technology. Halo generation on a turbid plate would be a method of choice for high throughput screening of enzymes in this context. Here we report a new approach to prepare turbid plates, by controlling the crystallization of tyrosine to form needle-like particles. In the presence of tyrosine phenol-lyase (TPL), the needle-like tyrosine crystals were converted to soluble phenol rapidly than the usual rectangular tyrosine crystals. When an error-prone PCR library of Citrobacter freundii TPL was spread on the turbid plate, approximately 10% of the colonies displayed recognizable halos after 24 hours of incubation at $37^{\circ}C$. Representative positives from the turbid plates were transferred to LB-medium in 96-wellplates, cultivated overnight, and assayed for the enzyme activity with L-tyrosine as the substrate. The assay results were approximated to be proportional to the halo size on turbid plates, suggesting the screening system is directly applicable to the directed evolution of TPL. Actually, two best mutants on the turbid plates were identified to be $2{\sim}2.5$ and 1.5-fold improved in the activity.
Keywords
Tyrosine phenol-lyase; needle-like tyrosine crystal; turbid plate; error-prone PCR; high through-put screening;
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