초록
<P>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™ 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 <I>Pseudomonas putida</I> (<I>Pp</I>BFD) and the alcohol dehydrogenase from <I>Lactobacillus brevis</I> (<I>Lb</I>ADH). Limitations in <I>Pp</I>BFD stability were overcome by optimization of buffer salt, cofactor concentration and choice of a different substrate. For optimal <I>Lb</I>ADH 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 (1<I>S</I>,2<I>S</I>)-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<SUP>−1</SUP> d<SUP>−1</SUP>.</P><P>Graphic Abstract</P><P>Compartmentalization of biocatalysts is an effective tool to integrate biocatalytic steps in continuous (chemo)enzymatic cascades.<BR/><IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c7gc03225k'/><BR/></P>