Fulton Alexander, Hayes Marc R, Schwaneberg Ulrich, Pietruszka Jörg, Jaeger Karl-Erich
Institute of Molecular Enzyme Technology, Heinrich-Heine - Universität Düsseldorf, Forschungszentrum Jülich, 52426, Jülich, Germany.
Novozymes A/S, Krogshoejvej 36, 2880, Bagsvaerd, Denmark.
Methods Mol Biol. 2018;1685:209-231. doi: 10.1007/978-1-4939-7366-8_12.
Screening is defined as the identification of hits within a large library of variants of an enzyme or protein with a predefined property. In theory, each variant present in the respective library needs to be assayed; however, to save time and consumables, many screening regimes involve a primary round to identify clones producing active enzymes. Such primary or prescreenings for lipolytic enzyme activity are often carried out on agar plates containing pH indicators or substrates as triolein or tributyrin. Subsequently, high-throughput screening assays are usually performed in microtiter plate (MTP) format using chromogenic or fluorogenic substrates and, if available, automated liquid handling robotics. Here, we describe different assay systems to determine the activity and enantioselectivity of lipases and esterases as well as the synthesis of several substrates. We also report on the construction of a complete site saturation library derived from lipase A of Bacillus subtilis and its testing for detergent tolerance. This approach allows for the identification of amino acids affecting sensitivity or resistance against different detergents.
筛选被定义为在具有预定义特性的酶或蛋白质的大量变体文库中鉴定出命中物。理论上，需要对各个文库中存在的每个变体进行检测；然而，为了节省时间和耗材，许多筛选方案都包括一轮初步筛选，以鉴定产生活性酶的克隆。这种针对脂肪分解酶活性的初步或预筛选通常在含有pH指示剂或底物（如三油酸甘油酯或三丁酸甘油酯）的琼脂平板上进行。随后，高通量筛选测定通常以微孔板（MTP）形式进行，使用显色或荧光底物，并且如果可行的话，使用自动化液体处理机器人技术。在这里，我们描述了不同的测定系统，以确定脂肪酶和酯酶的活性和对映选择性以及几种底物的合成。我们还报告了源自枯草芽孢杆菌脂肪酶A的完整位点饱和文库的构建及其对洗涤剂耐受性的测试。这种方法允许鉴定影响对不同洗涤剂的敏感性或抗性的氨基酸。
