HaloTag™: Evaluation of a covalent one-step immobilization for biocatalysis

Abstract Easy, fast and gentle immobilization for the efficient reuse of important biocatalysts is highly demanded. We used the commercially available HaloTag™ technology (Promega), so far relatively unknown in the context of biocatalysis, to immobilize the benzaldehyde lyase from P. fluorescence ( Pf BAL). Immobilization mediated by this fusion tag proceeds rapidly within minutes from crude extracts yielding covalently attached enzymes in high purity, making expensive and laborious previous chromatographic purification steps obsolete, which strongly reduces the costs for biocatalyst immobilization. Further, we introduce a novel design of HaloTag fusions and demonstrate the positive effect of the tag on soluble expression and activity of Pf BAL. The immobilized biocatalyst was stable at 4 °C for months and was successfully reused in several repetitive batches for the carboligation of aggressive aldehydes.