Biocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transaminases from different species. A cysteine in a consensus sequence was converted to a single aldehyde by the formylglycine-generating enzyme for directed single-point attachment to amine beads. This immobilization was compared to cross-linked enzyme aggregates (CLEAs) and multipoint attachments to glutaraldehyde-functionalized amine- and epoxy-beads. Subsequently, the reactivity and stability (i.e., thermal, storage, and solvent stability) of all soluble and immobilized transaminases were analyzed and compared under different conditions. The effect of immobilization was highly dependent on the type of enzyme, the immobilization strategy, and the application itself, with no superior immobilization technique identified. Immobilization of HAGA-beads often resulted in the highest activities of up to 62 U/g beads, and amine beads were best for the hexameric transaminase from Luminiphilus syltensis. Furthermore, the immobilization of transaminases enabled its reusability for at least 10 cycles, while maintaining full or high activity. Upscaled kinetic resolutions (partially performed in a SpinChemTM reactor) resulted in a high conversion, maintained enantioselectivity, and high product yields, demonstrating their applicability.

Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co-substrate. A dual-enzyme recycling system overcomes this limitation: l-amino acid oxidases (LAAO) recycle the accumulating co-product of (S)-selective transaminases in the kinetic resolution of racemic amines to produce pure (R)-amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H 2 O 2 as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using substoichiometric amounts of 1 mol% of the transaminase cosubstrate as well as the initial application of l-amino acids instead of α-keto acids. With an optimized protocol, the synthetic potential of this kinetic resolution cascade was proven at the preparative scale (> 90 mg) by the synthesis of highly enantiomerically pure (R)-methylbenzylamine (> 99 %ee) at complete conversion (50 %).

The aldehyde tag is appropriate to selectively label proteins, prepare antibody-drug conjugates or to immobilize enzymes or antibodies for biotechnological and medical applications. The cysteine within the consensus sequence CxPxR of the aldehyde tag is specifically oxidized by the formylglycine-generating enzyme (FGE) to the non-canonical and electrophilic amino acid C α -formylglycine (FGly). Subsequent reductive amination is a common method for site-directed immobilization, which usually results in poor immobilization efficiency due to the reaction conditions. Here, we introduce a new solid support like agarose modified with an aryl substituted pyrazolone (Knoevenagel reagent) that was obtained in a facile and efficient 2-step synthesis. The modified agarose allowed the site-selective and efficient immobilization of aldehyde-containing small molecules, peptides and proteins -in particular enzymes -at physiological pH (6.2-8.2) without any additive or catalyst needed. In comparison to reductive amination, higher loadings and activities were achieved in various buffers at different concentrations and temperatures.

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