l-amino acid oxidases (LAAOs, EC 1.4.3.2) are flavin adenine dinucleotide (FAD)-containing enzymes that catalyze the stereospecific oxidative deamination of l-amino acids to give α-keto acids and ammonia. The limited biocatalytic applications for LAAOs have been attributed to various challenges, such as low expression levels in recombinant systems (Geueke and Hummel, 2003, Nuutinen et al., 2012) or constrained substrate tolerance (Cheng et al., 2011, Liu et al., 2013). As breakthrough, following the evolutionary track of natural LAAOs and using ancestral sequence reconstruction (ASR), five ancestral LAAOs were designed (Nakano et al., 2020), from which AncLAAO-N1 was expressed with high yield and displayed a broad substrate scope, including 13 natural l-amino acids and several non-natural l-phenylalanine and l-tryptophan analogues (Nakano et al., 2019). Recently, by extending the ASR method, other AncLAAOs of increased stability have also been reported (Ishida et al., 2021, Nakano et al., 2020).

While the industrial-scale applications of AncLAAOs remain challenging due to the generation of hydrogen peroxide, one significant and direct utility of LAAOs lies in their use in coupled enzyme systems (Walton et al., 2018, Zhang et al., 2021, Zhu et al., 2019). d-Amino acid oxidases (DAAOs) were successfully employed in high-throughput screenings of directed evolution libraries of phenylalanine ammonia-lyases (PALs) engineered for d-selectivity (Parmeggiani et al., 2015). Protein engineering within the range of the natural l-selectivity of PALs, one of the most attractive biocatalysts for the production of phenylalanines (Parmeggiani et al., 2018), has been highly successful (Bencze et al., 2017, Filip et al., 2018, Hardegger et al., 2020, Nagy et al., 2019, Rowles et al., 2016, Tork et al., 2022) and requires assays amenable for the activity screens of extensive mutant libraries. Currently, the PAL assays designed for screening within the l-selectivity encompass diverse methodologies. HPLC methods (Filip et al., 2018, Nagy et al., 2019), have proven effective for smaller, rational design-based variant libraries. Conversely, other approaches, such as inductively coupled plasma–mass spectrometry (ICP-MS) (Yan et al., 2017) and desorption electrospray ionization (DESI) coupled with direct infusion of biotransformations to the mass spectrometer (DiBT-MS) (Kempa et al., 2021), require high instrumentational facilities. The fluorescent PAL-assay, involving the ferulic acid decarboxylase–mediated decarboxylation of cinnamic acid, might be hampered by the use of volatile styrenes (Moisa et al., 2020). The use of transcriptional biosensors in fluorescence activated cell sorting (FACS) (Flachbart et al., 2021, Flachbart et al., 2019), is challenging to adapt to different substrates targeted by the engineered PALs. The high-throughput applicability of the classical UV-spectroscopy based PAL-assay (Jendresen et al., 2015, Poppe et al., 2012, Tomoiaga et al., 2020) is also hindered by the high UV-background of the cells/cell lysates. Accordingly, a solid phase assay providing easy handling and facile visual detection of PALs of improved activity is still highly required among the high-throughput PAL-assay toolbox.

Herein, leveraging the availability of AncLAAO of high stability, we tested its substrate scope from the perspective of its compatibility with the substrate scope of PALs and developed a solid phase, high-throughput AncLAAO-based PAL-assay, suitable for activity screens of protein engineering-derived libraries operating within the range of l-selectivity of PALs.