How do new enzymatic motifs emerge in evolution? Charting new mechanistic solutions for biocatalytic challenges is a core obstacle in the constant evolutionary adaptation of living organisms. If understood and harnessed, these processes hold the potential to revolutionise industrial processes in the chemical and pharmaceutical industry. The recent massive release of new, man-made substance classes into the environment in the form of pesticides provides a dynamic testing ground to observe protein evolution in fast-forward mode. In this global experiment, we can observe the emergence of new enzymatic activities and compare the adaptability of different catalytic motifs. Among the xenobiotic substances used, phosphotriester pesticides range among the most toxic. Phosphotriester pesticides were designed to irreversibly inhibit the catalytic triad of synaptic acetylcholinesterase. Numerous enzymes from different evolutionary origins have rapidly evolved to hydrolyse phosphotriesters since. Interestingly, all known enzymes have convergently reached the same mechanistic solution by recruiting divalent cations as a cofactor for metal-ion catalysis. The starting point of this study is the α/β hydrolase P91. This enzyme was recently identified in a functional metagenomic screening and displays weak promiscuous phosphotriesterase (PTE) activity. P91’s PTE activity is remarkable because, in contrast to other currently known PTEs, it utilises a fundamentally different, metal-independent mechanism. At the same time, it has the same fold and a very similar active site configuration as acetylcholinesterase, the biochemical target of most organophosphate pesticides and nerve gases. In this dissertation, I subject P91 to directed evolution in order to explore the evolvability of its catalytic motif to this new enzymatic activity (chapter 2). To be able to cover otherwise inaccessible sequence space, I use droplet microfluidics to screen large libraries of enzyme variants in high throughput. Within only two rounds of evolution, P91’s phosphotriesterase activity could be increased by 400-fold. The improved enzyme displays a catalytic efficiency close to 10^6 M^(−1) s^(−1) and a turnover rate of > 10 s^(−1). This activity is comparable to the catalytic efficiencies of many metal-dependent ‘conventional’ phosphotriesterases. These result highlight that enzymes can be evolved to hydrolyse phosphotriester pesticides using a catalytic Cys–His–Asp triad, independent of metal cofactors. Detailed mechanistic analysis reveals that P91 is pre-disposed for the turnover of organophosphates due to the intrinsic suitability of its cysteine nucleophile (chapter 3). This residue is able to quickly break down the otherwise unresolvable intermediate providing the initial foothold for rounds of directed evolution to improve upon the rate-limiting initial formation of the covalent intermediate. Further phylogenetic exploration of P91 homologues reveals that this promiscuous activity is widespread within the dienelactone hydrolase protein family, a cluster of proteins in the α/β hydrolase superfamily, which possess a Cys–His–Asp catalytic triad (chapter 4). By detailed structural analysis and targeted engineering I show that the determinant for the level of displayed PTE activity is not a specific arrangement of specificity-determining residues in the active site (chapter 5), but rather the length and flexibility of loops surrounding the active site (chapter 6). In summary, these results highlight the enormous potential for synergy by combining focussed, combinatorial libraries with high-throughput droplet microfluidics. In combination, these techniques can establish new mechanistic tracks and find efficient biocatalytic solutions that – despite strong natural selective pressure – were previously unseen in Nature.