Bacterial inclusion bodies (IBs) have long been considered as inactive, unfolded waste material produced by heterologous overexpression of recombinant genes. In industrial applications, they are occasionally used as an alternative in cases where a protein cannot be expressed in soluble form and in high enough amounts. Then, however, refolding approaches are needed to transform inactive IBs into active soluble protein. While anecdotal reports about IBs themselves showing catalytic functionality/ activity (CatIB) are found throughout literature, only recently, the use of protein engineering methods has facilitated the ondemand production of CatIBs. CatIB formation is induced usually by fusing short peptide tags or aggregation-inducing protein domains to a target protein. The resulting proteinaceous particles formed by heterologous expression of the respective genes can be regarded as a biologically produced bionanomaterial or, if enzymes are used as target protein, carrier-free enzyme immobilizates. In the present contribution, we review general concepts important for CatIB production, processing, and application. Key points • Catalytically active inclusion bodies (CatIBs) are promising bionanomaterials. • Potential applications in biocatalysis, synthetic chemistry, and biotechnology. • CatIB formation represents a generic approach for enzyme immobilization. • CatIB formation efficiency depends on construct design and expression conditions.

Background In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their high stability, easy and cost efficient production, and recyclability and thus provide an interesting alternative to conventionally immobilized enzymes. Results Here, we present the construction and characterization of a CatIB set of the lysine decarboxylase from Escherichia coli (EcLDCc), constructed via Golden Gate Assembly. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with the artificial peptides (18AWT, L6KD and GFIL8) or the coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. The linkers were fused to the C-terminus of the EcLDCc to form a linkage between the enzyme and the aggregation inducing tags. Comprehensive morphology and enzymatic activity analyses were performed for the ten EcLDCc-CatIB variants and a wild type EcLDCc control to identify the CatIB variant with the highest activity for the decarboxylation of l-lysine to 1,5-diaminopentane. Interestingly, all of the CatIB variants possessed at least some activity, whilst most of the combinations with the rigid PT-Linker showed the highest conversion rates. EcLDCc-PT-L6KD was identified as the best of all variants allowing a volumetric productivity of 457 g L− 1 d− 1 and a specific volumetric productivity of 256 g L− 1 d− 1 gCatIB−1. Noteworthy, wild type EcLDCc, without specific aggregation inducing tags, also partially formed CatIBs, which, however showed lower activity compared to most of the newly constructed CatIB variants (volumetric productivity: 219 g L− 1 d− 1, specific volumetric activity: 106 g L− 1 d− 1 gCatIB− 1). Furthermore, we demonstrate that microscopic analysis can serve as a tool to find CatIB producing strains and thus allow for prescreening at an early stage to save time and resources. Conclusions Our results clearly show that the choice of linker and aggregation inducing tag has a strong influence on the morphology and the enzymatic activity of the CatIBs. Strikingly, the linker had the most pronounced influence on these characteristics.

In industry, enzymes are often immobilized to generate more stable enzyme preparations that are easier to store, handle, and recycle for repetitive use. Traditionally, enzymes are bound to inorganic carrier materials, which requires caseto-case optimization and incurs additional labor and costs. Therefore, with the advent of rational protein design strategies as part of bottom-up synthetic biology approaches, numerous immobilization methods have been developed that enable the one-step production and immobilization of enzymes onto biogenic carrier materials often directly within the production host, which we here refer to as in vivo immobilization. As a result, nano-to micro-meter-sized functionalized biomaterials, or biologically produced enzyme immobilizates, are obtained that can directly be used for synthetic purposes. In this Perspective, we provide an overview over established and recently emerging in vivo enzyme immobilization methods, with special emphasis on their applicability for (industrial) biocatalysis. For each approach, we present fundamental working principles as well as advantages and limitations guiding future research avenues toward sustainable applications in the bioindustry.

In industries, enzymes are often immobilized to obtain stable preparations that can be utilized in batch and flow processes. In contrast to traditional immobilization methods that rely on carrier binding, various immobilization strategies have been recently presented that enable the simultaneous production and in vivo immobilization of enzymes. Catalytically active inclusion bodies (CatIBs) are a promising example for such in vivo enzyme immobilizates. CatIB formation is commonly induced by fusion of aggregation-inducing tags, and numerous tags, ranging from small synthetic peptides to protein domains or whole proteins, have been successfully used. However, since these systems have been characterized by different groups employing different methods, a direct comparison remains difficult, which prompted us to benchmark different CatIB-formation-inducing tags and fusion strategies. Our study highlights that important CatIB properties like yield, activity, and stability are strongly influenced by tag selection and fusion strategy. Optimization enabled us to obtain alcohol dehydrogenase CatIBs with superior activity and stability, which were subsequently applied for the first time in a flow synthesis approach. Our study highlights the potential of CatIB-based immobilizates, while at the same time demonstrating the robust use of CatIBs in flow chemistry.

For the development of efficient and green industrial processes, the combination of biocatalysis and flow chemistry holds great promises. Flow chemical utilization of biocatalysts, essentially made possible by the immobilization (or retention) of enzymes in flow reactors, has attracted increased academic attention during recent years. In the present review we present an overview of immobilization strategies suitable for flow chemistry, particularly focusing on recently developed carrier‐free immobilization methods, highlighting advances in the field and presenting future trends.

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