Controllably crosslinked dual enzymes enabled by genetic-encoded non-standard amino acid for efficiently enantioselective hydrogenation

In traditional method for preparing crosslinked enzymes aggregates using glutaraldehyde, random linkage is inevitable, which often destroys the enzyme active sites and severely decreases the activity. To address this issue, using genetic encode expanding, nonstandard amino acids (NSAAs) were inserted into enzyme proteins at the preselected sites for crosslinking. When aldehyde ketone reductase (AKR), alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) were utilized as model enzymes, their mutants containing p-azido-L-phenylalanine were bio-orthogonally crosslinked with diyne to form crosslinked dual enzymes (CLDEs) acting as a cascade biological oxidation and reduction system. Then, the resultant self-purified CLDEs were characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM), etc. In the asymmetric synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol using CLDEs, high product yield (76.08%), ee value (99.99%) and reuse stability were achieved. The yield and ee value were 12.05 times and 1.39 times higher than those using traditional crosslinked enzyme aggregates, respectively. Thus, controllable insertion NSAAs in number and location can engender reasonable linkage and metal-free self-purification for target enzyme proteins. This facile and sustainable method could be further expanded to other dual and multienzyme systems for cascade biocatalysis.