Tailor-made catalytically active inclusion bodies for different applications in biocatalysis

Abstract

We have recently demonstrated that fusions of different target enzymes to the coiled-coil domain TDoT induced the formation of catalytically active inclusion bodies (CatIBs) in E. coli (doi:10.1016/j.jbiotec.2017.04.033). Here we show that the CatIB properties can be tailored to the requirements of different reaction systems using two different coiled-coil domains as fusion tags: TDoT and 3HAMP. As an example the benzaldehyde lyase from Pseudomonas fluorescens (PfBAL) was chosen, which catalyzes the formation of 2-hydroxy ketones and benzoins from aromatic donor aldehydes and aromatic or aliphatic acceptor aldehydes. Using these fusion tags two different kinds of PfBAL-CatIBs were successfully produced that differ in morphology, solubility during washing steps, initial rate activity, protein and lipid content. TDoT-PfBAL and 3HAMP-PfBAL CatIBs were studied relative to the soluble enzyme concerning their application in biocatalysis in continuous reaction mode and in batch using a mono- and biphasic solvent system. Both CatIBs outperformed the soluble enzyme with respect to stability under reaction conditions. In the buffer/DMSO system used for the continuous synthesis of (R)-2-hydroxy-1-phenylpropanone, TDoT-PfBAL was superior to the soluble enzyme. Besides, 3HAMP-PfBAL was more suitable in a biphasic reaction system for the synthesis of (R)-benzoins and revealed an up to 3-fold higher activity compared to the soluble enzyme.