Böttcher Dominique, Zägel Patrick, Schmidt Marlen, Bornscheuer Uwe T
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany.
Methods Mol Biol. 2017;1539:197-204. doi: 10.1007/978-1-4939-6691-2_11.
A procedure for the high-throughput screening (HTS) of esterases is described. This includes a pretest for discrimination of active and inactive clones using an agar plate overlay assay, the 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 towards 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 E. coli strains.
本文描述了一种用于酯酶高通量筛选（HTS）的方法。该方法包括使用琼脂平板覆盖测定法对活性和非活性克隆进行区分的预测试、在微量滴定板中进行酶表达，以及使用仲醇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反应中转化，导致化学计量地形成NADH，然后在分光光度计中对其进行定量。该方法每天可筛选数千个突变体，并且已经成功应用于鉴定一种对有机合成重要结构单元的对映选择性（E）>100的酯酶突变体。该方案也可用于脂肪酶以及可能在常规大肠杆菌菌株中以可溶形式表达的其他水解酶。
