Amine dehydrogenases (AmDHs) catalyze the enzymatic reduction of ketones to amines, serving as a suitable biocatalytic route for amine synthesis. A limited number of experimentally validated native AmDHs (nat‐AmDHs) have been reported recently, expanding the sequences with this function to complement the small set of engineered enzymes. Since researchers can now probe into the vast diversity of enzymes within niche environments by a metagenomics approach, a tandem metagenomic and bioinformatic approach is a powerful tool to identify new members of limited enzyme families to access new features in an iterative fashion. The previously untapped biocatalytic reservoirs of the ocean environment and human microbiome were screened for potential AmDHs using a hidden Markov model. Among the hundreds of hits, a subset of 18 enzymes was selected for further characterization and were confirmed to display AmDH activity. Additional analysis on six enzymes confirmed altered cofactor specificities and variation in substrate scopes, catalytic efficiencies, and active site residues compared to the reference nat‐AmDHs previously described. Particularly, MATOUAmDH2 from an eukaryotic organism demonstrated specific activity of 11.07 and 0.88 U mg−1 toward isobutyraldehyde and 1,2‐cyclohexadione respectively. Their abundance among the screened environments was also described. The protein sequence diversity of validated AmDHs reached by this metagenomics mining strategy highlights the success of such an approach. Metagenomically mined proteins, including eukaryotic ones, stand to increase the reach of biocatalysis towards enviromentally benign processes.

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Small optically active molecules, and more particularly short-chain chiral amines, are key compounds in the chemical industry and precursors of various pharmaceuticals. Their chemo-biocatalytic production on a commercial scale is already established, mainly through lipase-catalyzed resolutions leading to ChiPros™ products among others. Nevertheless, their biocatalytic synthesis remains challenging for very short-chain C4 to C5 amines due to low enantiomeric excess. To complement the possibilities recently offered by transaminases, this work describes alternative biocatalytic access using amine dehydrogenases (AmDHs). Without any protein engineering, some of the already described wild-type AmDHs (CfusAmDH, MsmeAmDH, MicroAmDH, and MATOUAmDH2) were shown to be efficient for the synthesis of hydroxylated or unfunctionalized small 2-aminoalkanes. Conversions up to 97.1% were reached at 50 mM, and moderate to high enantioselectivities were obtained, especially for (S)-1-methoxypropan-2-amine (98.1%), (S)-3-aminobutan-1-ol (99.5%), (3S)-3-aminobutan-2-ol (99.4%), and the small (S)-butan-2-amine (93.6%) with MsmeAmDH. Semi-preparative scale-up experiments were successfully performed at 150 mM substrate concentrations for the synthesis of (S)-butan-2-amine and (S)-1-methoxypropan-2-amine, the latter known as “(S)-MOIPA”. Modeling studies provided some preliminary results explaining the basis for the challenging discrimination between similarly sized substituents in the active sites of these enzymes.

Linkers are critical components of fusion proteins, as they physically separate individual domains to enable each to fold and retain function. The role of peptide linker properties was investigated for fusions of a leucine zipper immobilization domain (Z E ) to a chimeric amine dehydrogenase (AmDH) or a formate dehydrogenase (cbFDH). A linker library was developed, which varied in length, orientation, and proline content, as a way to vary stiffness. Fusion proteins were characterized by melting temperature, immobilization ability, cofactor binding, and kinetic activity. The best linker candidate for each enzyme was tested in a dual-functionality assay, where enzymatic activity of fusions immobilized in protein-inorganic supraparticles was greater than 80% after washing. The best linker for AmDH was completely different than that for cbFDH. This work highlights the need to experimentally assess linker properties in the design of new fusion proteins and provides a linker library for this purpose.

Enzyme immobilization is an essential technology for commercializing biocatalysis. It imparts stability, recoverability, and other valuable features that improve the effectiveness of biocatalysts. While many avenues to join an enzyme to solid phases exist, protein‐mediated immobilization is rapidly developing and has many advantages. Protein‐mediated immobilization allows for the binding interaction to be genetically coded, can be used to create artificial multienzyme cascades, and enables modular designs that expand the variety of enzymes immobilized. By designing around binding interactions between protein domains, they can be integrated into functional materials for protein immobilization. These materials are framed within the context of biocatalytic performance, immobilization efficiency, and stability of the materials. In this review, supports composed entirely of protein are discussed first, with systems such as cellulosomes and protein cages being discussed alongside newer technologies like spore‐based biocatalysts and forizymes. Protein‐composite materials such as polymersomes and protein–inorganic supraparticles are then discussed to demonstrate how protein‐mediated strategies are applied to many classes of solid materials. Critical analysis and future directions of protein‐based immobilization are then discussed, with a particular focus on both computational and design strategies to advance this area of research and make it more broadly applicable to many classes of enzymes.