Chapter title |
A Screening Method for P450 BM3 Mutant Libraries Using Multiplexed Capillary Electrophoresis for Detection of Enzymatically Converted Compounds.
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Chapter number | 13 |
Book title |
Directed Evolution
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Published in |
Methods in molecular biology, January 2022
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DOI | 10.1007/978-1-0716-2152-3_13 |
Pubmed ID | |
Book ISBNs |
978-1-07-162151-6, 978-1-07-162152-3
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Authors |
Gärtner, Anna, de Almeida Santos, Gustavo, Ruff, Anna Joëlle, Schwaneberg, Ulrich, Almeida Santos, Gustavo |
Abstract |
Capillary electrophoresis (CE) is an analytical method in which charged species are separated by attraction or repulsion performed in submillimeter diameter capillaries or micro- and nanofluidic channels through the application of a high voltage electric field. When capillary electrophoresis is assembled in a multicapillary instrument such as 96-well format (multiplexed), it becomes a powerful high-throughput system with the ability to simultaneously screen several types of samples like genetic mutations, metabolomes, kinase inhibitors, or enzymatic activities to name a few. The usage of a 96-multiplexed capillary electrophoresis system (96-MP-CE) represents a new platform for product-specific high-throughput screening of enzyme mutant libraries from directed evolution campaigns providing a comprehensive view on enzyme activity through the detection of all products formed. We describe the application of 96-MP-CE to screen mutant libraries of P450 BM3. MP-CE was used in directed evolution campaigns toward benzo-1,4-dioxane and α-isophorone. |
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