The most promising variant to emerge following three rounds of evolution (CAT3) contains 7 mutations clustered around the active site. As intended CAT3 operates as an efficient aminotransferase, promoting the conversion of 2 to 4 even at low NH2OH concentrations and high substrate loadings. For example, at 500 mM 2 and 10% NH2OH (pH 7) CAT3 produces an 8:1 ratio of 4:3. In contrast, the wild-type enzyme produces a 1:6 ratio of 4:3 under identical conditions at equilibrium. With a promising biocatalyst in hand, our attention turned to reaction intensification and product isolation. We observed that N-hydroxy-cytidine is substantially less soluble than either cytidine or uridine in aqueous solutions, presenting an opportunity for dynamic in situ crystallization of 4 to allow facile product isolation and further favour the distribution of 4:3. By increasing the substrate loading (750 mM) and reducing the reaction temperature (4 °C) in situ crystallization of 4 was observed. Following optimisation of reaction conditions, 4 was isolated in 91% yield and >98% purity in only 3 hours using 0.001 mol% CAT3.