Tyrosine Phenol-Lyase의 고속탐색기술 개발 및 방향성 분자진화
High Throughput Screening and Directed Evolution of Tyrosine Phenol-Lyase
0
조회
0
다운로드
1
인용
0
참고문헌
키워드
초록
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 37oC. 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.
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 37oC. 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.
참고문헌 (8)
- Barbolina, M. V, "Citrobacter freundii tyrosinephenol-lyase", 13, pp.207~215 (2000)
- Faleev, N. G.,, "The catalytic mechanism of tyrosinephenol-lyase from Erwinia herbicola", 51, pp.363~370 (1996)
- -I, "Structure of tyrosine phenol-lyase genes from Citro-bacter freundii and structural comparison with tryptophanasefrom Escherichia coli", yohi, pp.147~151 (1991)
- Phillips, "Mechanistic deduc-tions from kinetic isotope effects and pH studies of pyridoxalphosphate dependent carbon-carbon lyases", pp.7333~7338 (1988)
- Kuchner, O., "Directed evolution ofenzyme catalysts", 15, pp.523~530 (1997)
- Lee, S. G, "Thermostable tyrosine phenol-lyase of Symbio-bacterium sp. SC-1", 11, pp.263~270 (1997)
- Lee, S. G.,, "Characterization of thermostable tyrosinephenol-lyase from an obligatory symbiotic thermophile,Symbiobacterium sp. SC-1", 32, pp.480~485 (1999)
- Olsen, M, "High-through-" (2000)