BackgroundImmobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically.ResultsWe here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein.ConclusionWe here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1081-5) contains supplementary material, which is available to authorized users.
Optimal performance of multi-step enzymatic one-pot cascades requires a facile balance between enzymatic activity and stability of multiple enzymes under the employed reaction conditions. We here describe the optimization of an exemplary two-step one-pot recycling cascade utilizing the thiamine diphosphate (ThDP)-dependent benzaldehyde lyase from Pseudomonas fluorescens (PfBAL) and the alcohol dehydrogenase from Ralstonia sp. (RADH) for the production of the vicinal 1,2-diol (1R,2R)-1-phenylpropane-1,2-diol (PPD) using both enzymes as catalytically active inclusion bodies (CatIBs). PfBAL is hereby used to convert benzaldehyde and acetalydehyde to (R)-2-hydroxy-1-phenylpropanone (HPP), which is subsequently converted to PPD. For recycling of the nicotinamide cofactor of the RADH, benzyl alcohol is employed as co-substrate, which is oxidized by RADH to benzaldehyde, establishing a recycling cascade. In particular the application of the RADH, required for both the reduction of HPP and the oxidation of benzyl alcohol in the recycling cascade is challenging, since the enzyme shows deviating pH optima for reduction (pH 6-10) and oxidation (pH 10.5), while both enzymes show only low stability at pH > 8. This inherent stability problem hampers the application of soluble enzymes and was here successfully addressed by employing CatIBs of PfBAL and RADH, either as single, independently mixed CatIBs, or as co-immobilizates (Co-CatIBs). Single CatIBs, as well as the Co-CatIBs showed improved stability compared to the soluble, purified enzymes. After optimization of the reaction pH, the RADH/PfBAL ratio and the co-solvent content, we could demonstrate that almost full conversion (> 90%) was possible with CatIBs, while under the same conditions the soluble enzymes yielded at most > 50% conversion. Our study thus provides convincing evidence that (Co-)CatIB-immobilizates can be used efficiently for the realization of cascade reactions, i. e. under conditions where enzyme stability is a limiting issue.
Fe(II)/α-ketoglutarate-dependent dioxygenases (KDOs) catalyze a broad range of selective C–H oxidation reactions. However, the difficult production of KDOs in recombinant E. coli strains and their instability in purified form have so far limited their application in preparative biotransformations. Here, we investigated the immobilization of three KDOs (CaKDO, CpKDO, FjKDO) that catalyze the stereoselective hydroxylation of the L-lysine side chain using two one-step immobilization techniques (HaloTag®, EziG™). The HaloTag®-based immobilisates reached the best results with respect to residual activity and stability. In preparative lab-scale experiments, we achieved product titers of 16 g L−1 (3S)-hydroxy-L-lysine (CaKDO) and (4R)-hydroxy-L-lysine (FjKDO), respectively, starting from 100 mM L-lysine. Using a HaloTag®-immobilized lysine decarboxylase from Selenomonas ruminantium (SrLDC), the (3S)-hydroxy-L-lysine from the CaKDO-catalyzed reaction was successfully converted to (2S)-hydroxy-cadaverine without intermediate product purification, yielding a product titer of 11.6 g L−1 in a 15 mL consecutive batch reaction. We propose that covalent in situ immobilization is an appropriate tool to access the preparative potential of many other KDOs.
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