title: An improved flurogenic probe for high-throughput screening of 2-deoxyribose aldolases.
abstract: 2-Deoxyribose aldolase-catalyzed C-C bond-forming reactions have become 1 more and more important in synthesis of statins and other drug intermediates. Many methods have focused on improving the aldolase properties and harvesting new aldolases, but a good outcome depends on the efficiency of the high-throughput screening system. We have developed a visible green fluorescence probe based on a coumarin derivative, which can be reversibly modulated by a retro-aldol reaction catalyzed by 2-deoxyribose aldolase for selecting aldolase mutants with high activity. This assay system provides a convenient and effective way for high-throughput screening aldolases as the green fluorescence is sensitively detected and daylight-viewable without the need for specialist equipment. We used our probe to successfully harvest aldolase mutants with higher activities than the parent from a random mutagenesis library.
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Fei
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Xu
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, China. Electronic address: 10928041@zju.edu.cn.
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Wu
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, China.
authors.forename: Li-Rong
authors.initials: LR
authors.lastname: Yang
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry*; D017931:DNA Primers; D003855:Deoxyribose / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D019906:Nuclear Magnetic Resonance, Biomolecular; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D003855:Deoxyribose; D000446:Aldehyde-Lyases
keywords: 2-deoxyribose aldolase; Daylight-viewable fluorescence; High-throughput screen