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. 2019 Oct 29;9:15479. doi: 10.1038/s41598-019-52077-w

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Flow chart of screening approach and variant characterization. Multiplexed capillary electrophoresis (MP-CE) was used as platform for screening of two P450 BM3 site-saturation mutagenesis libraries at positions F87 and A328 and compared to the frequently used NADPH depletion assay. A MP-CE and a NADPH based selection of variants was made and selected variants rescreened in multiplets in the corresponding platform. Variants with ≥1.3-fold increase in performance were selected and product formation of all identified substitutions compared to the WT in substrate conversion experiments. For a holistic view, rescreening was as well performed with the opposite platform. The two variants performing “best” in the respective platform (MP-CE: F87V, NADPH: A328N) were purified and their coupling efficiencies and initial oxidation rates determined.