Hydroxynitrile lyases (HNLs) catalyse the reversible cleavage of cyanohydrins to carbonyl compounds and HCN. The recent discovery of bacterial HNLs with a cupin fold gave rise to a new promising class of these enzymes. They are interesting candidates for the synthesis of cyanohydrins on an industrial scale owing to their high expression levels in Escherichia coli. The activity and enantioselectivity of the manganese‐dependent HNL from Granulicella tundricola (GtHNL) were significantly improved by site‐saturation mutagenesis of active site amino acids. The combination of beneficial mutations resulted in a variant with 490‐fold higher specific activity in comparison to the wild‐type enzyme. More importantly, GtHNL‐A40H/V42T/Q110H is a highly competitive alternative for the synthesis of chiral cyanohydrins, such as 2‐chlorobenzaldehyde cyanohydrin, (R)‐2‐hydroxy‐4‐phenylbutyronitrile, and (R)‐2‐hydroxy‐4‐phenyl‐3‐butene nitrile, which serve as intermediates for the synthesis of pharmaceuticals.

The nitroaldol (Henry) reaction is a valuable C−C bond‐forming reaction resulting in β‐nitro alcohols, which are important building blocks for the synthesis of bioactive compounds. Metal‐dependent bacterial hydroxynitrile lyases with a cupin fold couple nitromethane or nitroethane and various aldehydes to yield (R)‐β‐nitro alcohols with up to 90 % conversion and up to ≥99 % enantiomeric excess.

Hydroxynitrile lyases (HNLs) are powerful carbon–carbon bond forming enzymes. The reverse of their natural reaction – the stereoselective addition of hydrogen cyanide (HCN) to carbonyls – yields chiral cyanohydrins, versatile building blocks for the pharmaceutical and chemical industry. Recently, bacterial HNLs have been discovered, which represent a completely new type: HNLs with a cupin fold. Due to various benefits of cupins (e.g. high yield recombinant expression in Escherichia coli), the class of cupin HNLs provides a new source for interesting, powerful hydroxynitrile lyases in the ongoing search for HNLs with improved activity, enantioselectivity, stability and substrate scope. In this study, database mining revealed a novel cupin HNL from Acidobacterium capsulatum ATCC 51196 (AcHNL), which was able to catalyse the (R)-selective synthesis of mandelonitrile with significantly better conversion (97%) and enantioselectivity (96.7%) than other cupin HNLs.

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity in the cleavage and the synthesis reaction of (R)-mandelonitrile with up to 74% conversion of benzaldehyde (enantiopreference ee 89%). Both showed high similarity to proteins of the cupin superfamily which so far were not known to exhibit HNL activity. Hydroxynitrile lyases (HNLs) catalyze the reversible cleavage of cyanohydrins, yielding the respective carbonyl compound and HCN. They are of great relevance in organic synthesis, in which the ability of the enzymes to catalyze the reverse reaction forming C-C bonds in a stereoselective manner is of substantial industrial relevance (9,11,19). Hydroxynitrile lyases are quite heterogeneous exhibiting remarkable diversity with respect to their substrate specificity, mass, glycosylation, and amino acid sequence, and their similarity to oxidoreductases, ␣/␤-hydrolases, carboxypeptidases, or Zn 2ϩ -containing alcohol dehydrogenases has been reported (3,4,8,17,18,19).Hydroxynitrile lyases are widespread in plants playing a major role in disease suppression, and only recently a bacterial protein with HNL activity in the cyanohydrin cleavage reaction was reported (16). Usually plants are colonized by a range of different bacteria (14). Endophytes in particular live in close association with their host and promote plant growth and health by various mechanisms, including the production of substances with phytohormonal activity or antimicrobial substances such as antibiotics (13,15,21). The production of HCN in pseudomonads has been reported (5), for example, but it has also been reported in other bacteria. In contrast to plants, cyanogenesis in bacteria usually follows a completely different biosynthetic pathway involving a HCN synthase (2,20). In this work, we present the discovery of a new bacterial enzyme class with hydroxynitrile lyase activity initially identified by function-based screening of a gene library of an isolated bacterial endophyte related to Pseudomonas mephitica and further investigated with a second highly similar protein from Burkholderia phytofirmans strain PsJN.Gene libraries of different endophytic bacteria isolated from potato were constructed in the pZero-2 vector by standard procedures (see supplemental material) and screened for HNL activity toward (R/S)-mandelonitrile using a colony-based colorimetric filter assay [67 mM (R/S)-mandelonitrile in 30 mM citratephosphate buffer (pH 3.5) at room temperature (RT)] (10). In the gene library of a strain related to Pseudomonas mephitica, HNL activity was detected. After subcloning and rescreening, the transformant exhibiting the strongest activity toward mandelonitrile was selected for further characterization. Sequencing revealed that the plasmid contained a 1,659-bp insert, which carried two open reading fram...