High-throughput screening of activity and enantioselectivity of esterases
High-throughput screening of activity and enantioselectivity of esterases
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DOI：
10.1038/nprot.2006.391
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Bornscheuer, Uwe T.
中科院分区：
文献类型：
--
作者：
Boettcher, Dominique;Bornscheuer, Uwe T.
描述了用于高通量筛选酯酶的程序。这包括在微量滴定板中的酶表达和使用仲醇的乙酸酯作为模型底物测量酯酶变体的活性和对映选择性（E）。释放的乙酸在酶级联中转化，导致化学计量的NADH形成，其在分光光度计中定量。该方法允许每天筛选数千个突变体，并且已经成功地应用于鉴定E > 100的酯酶突变体，该酯酶突变体是有机合成的重要构件。该方案也可用于在常规大肠杆菌菌株中以可溶形式表达的脂肪酶和可能的其他水解酶。该方案可在3 - 4天内完成。
A procedure for the high-throughput screening of esterases is described. This includes enzyme expression in microtiter plates and the measurement of activity and enantioselectivity (E) of the esterase variants using acetates of secondary alcohols as model substrates. Acetic acid released is converted in an enzyme cascade leading to the stoichiometric formation of NADH, which is quantified in a spectrophotometer. The method allows screening of several thousand mutants per day and has already been successfully applied to identify an esterase mutant with an E > 100 toward an important building block for organic synthesis. This protocol can also be used for lipases and possibly other hydrolases that are expressed in soluble form in conventional Escherichia coli strains. This protocol can be completed in 3 - 4 days.