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Title 

High throughput screening and directed evolution of tyrosine phenol-lyase

 

Tyrosine phenol-lyase의 고속탐색기술 개발 및 방향성 분자진화

Authors 

Su-Lim ChoiEugene RhaDo Young KimJae Jun SongS P HongM H SungSeung Goo Lee

Publisher 

The Korean Society for Applied Microbiology and Biotechnology

Issue Date 

2006

Citation 

Korean Journal of Microbiology & Biotechnology, vol. 34, no. 1, pp. 58-62

Keywords 

error-prone PCRhigh throughput screeningneedle-like tyrosine crystalturbid platetyrosine phenol-lyasetyrosine phenol lyasescreening

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 phenollyase (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°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 2-2.5 and 1.5-fold improved in the activity.

ISSN 

0257-2389

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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