Closing the gap for efficient immobilization of biocatalysts in continuous processes: HaloTag™ fusion enzymes for a continuous enzymatic cascade towards a vicinal chiral diol

Compartmentalization of biocatalysts is an effective tool to integrate biocatalytic steps in continuous (chemo)enzymatic cascades. Therefore, efficient covalent immobilization techniques are of utmost importance, which enable a fast and selective immobilization of the enzyme directly from crude cell extracts. Here we demonstrate that the HaloTag (TM) mediates the covalent immobilization of such fusion enzymes in only a few minutes contact time with the respective modified carrier in a packed-bed reactor, thereby enabling enzyme immobilization directly in the flow setup. In this study, we evaluated this concept for a continuous enzymatic cascade towards a chiral vicinal diol by combining a variant of the benzoylformate decarboxylase from Pseudomonas putida (PpBFD) and the alcohol dehydrogenase from Lactobacillus brevis (LbADH). Limitations in PpBFD stability were overcome by optimization of buffer salt, cofactor concentration and choice of a different substrate. For optimal LbADH activity, excess acetaldehyde was removed in-line. This optimization lead to a high operational stability of the individual cascade steps up to several weeks and resulting in the efficient stereoselective production of (1S, 2S)-1-phenylpropane-1,2-diol with high conversion up to 99%, high stereoselectivities (ee/ic) up to 96% and space-time yields up to 1850 g L-1 d(-1).