A modern concept of producing nitriles involves aldoxime dehydratases, which are capable of dehydrating aldoximes to the corresponding nitriles without need of a cofactor. Aldoximes as the starting material for nitrile synthesis are easily prepared by condensation of hydroxylamine with the corresponding aldehydes. In this contribution we present chemoenzymatic cascade reactions toward nitriles consisting of an initial nitroxyl radical-catalyzed oxidation of aliphatic alcohols to aldehydes using sodium hypochlorite as the oxidation agent, subsequent condensation of the aldehyde with hydroxylamine, and a biocatalytic dehydration using aldoxime dehydratases to the corresponding nitriles without isolation of intermediates and using the product nitrile as a solvent. These formal “solvent-free” cascades open up the possibility to use biorenewable sources, namely fatty acids, as the starting material for a chemoenzymatic nitrile synthesis. We were also able to apply this cascade concept for the synthesis of aliphatic dinitriles, which are used as, e.g., a precursor for polymer building blocks. Overall yields without isolation of intermediates of up to 70% with very simple product isolation were achieved.

The TEMPO-catalyzed oxidation of alcohols to aldehydes has emerged to one of the most widely applied methodologies for such transformations. Advantages are the utilization of sodium hypochlorite, a component of household bleach, as an oxidation agent and the use of water as a co-solvent. However, a major drawback of this method is the often occurring strict limitation to use dichloromethane as an organic solvent in a biphasic reaction medium with water. Previous studies show that dichloromethane cannot easily be substituted because a decrease of selectivity or inhibition of the reaction is observed by using alternative organic solvents. Thus, up to now, only a few examples are known in which after a tedious optimi- [a]

Immobilization of biocatalysts is a current topic in research enabling the easy recovery of catalysts from the reaction medium after the reaction, and it is often accompanied by a stabilization of the catalysts, which enables recycling. Within our ongoing research on the utilization of aldoxime dehydratases in the cyanide-free synthesis of nitriles through dehydration of readily available aldoximes, a screening of different immobilization methods for free enzymes was performed. The applied immobilization methods are based on covalent binding and hydrophobic interactions of the enzyme with the carrier material and whole-cell immobilization in calcium alginate beads with and without subsequent coating. In our study, we found that the immobilization with purified free aldoxime dehydratases from OxdRE (Rhodococcus erythropolis) and OxdB (Bacillus sp. strain OxB-1) leads to high immobilization efficiencies, but also to a strong loss of activity with a residual activity of <20%, regardless of the carrier material used. However, when using whole cells for immobilization instead of purified enzymes, we could increase the residual activity significantly. Escherichia coli BL21(DE3)-CodonPlus-RIL OxdRE and OxdB whole cells were entrapped in calcium alginate beads and coated with silica using tetraethylorthosilicate (TEOS), leading to immobilized catalysts with up to 75% residual activity and a higher stability compared to the free whole cells. Even after three rounds of recycling, which corresponds to a 3 d reaction time, the immobilized OxdB whole cells showed a residual activity of 85%.

Alcohol dehydrogenases catalyse the conversion of a large variety of ketone substrates to the corresponding chiral products. Due to their high regio- and stereospecificity, they are key components in a wide range of industrial applications. A novel alcohol dehydrogenase from Comamonas testosteroni (CtADH) was identified in silico, recombinantly expressed and purified, enzymatically and biochemically investigated as well as structurally characterized. These studies revealed a broad pH-profile and an extended substrate spectrum with the highest activity for compounds containing halogens as substituents and a moderate activity for bulky–bulky ketones. Biotransformations with selected ketones—performed with a coupled regeneration system for the co-substrate NADPH – resulted in conversions of more than 99% with all tested substrates and with excellent enantioselectivity for the corresponding S-alcohol products. CtADH/NADPH/substrate complexes modelled based on crystal structures of CtADH and its closest homologue suggested preliminary hints to rationalize the enzyme’s substrate preferences

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