High Throughput Screening and Directed Evolution of Tyrosine Phenol-Lyase

Tyrosine Phenol-Lyase의 고속탐색기술 개발 및 방향성 분자진화

  • 최수림 (한국생명공학연구원 시스템미생물연구센터) ;
  • 나유진 (한국생명공학연구원 시스템미생물연구센터) ;
  • 김도영 (한국생명공학연구원 시스템미생물연구센터) ;
  • 송재준 (한국생명공학연구원 시스템미생물연구센터) ;
  • 홍승표 (바이오리더스) ;
  • 성문희 (바이오리더스, 국민대학교 생명나노화학과) ;
  • 이승구 (한국생명공학연구원 시스템미생물연구센터)
  • Published : 2006.03.01

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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