A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening.

Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.

Development of (R)-4-hydroxymandelonitrile synthesis in an aqueous-organic biphasic stirred tank batch reactor.

A relatively new hydroxynitrile lyase-catalyzed reaction was optimized to be suitable for rapid and efficient development of a full-scale production process. The conversion of 4-hydroxybenzaldehyde into (R)-4-hydroxymandelonitrile, catalyzed by Prunus amygdalus hydroxynitrile lyase, was carried out in a biphasic system of aqueous buffer (pH 5.5) and methyl tert-butyl ether and is described with a process model. The process model consists of a description of the reaction kinetics, mass transfer kinetics, and mass balances for both the aqueous and the organic phase. Values are determined for the equilibrium constant, the enzyme kinetic parameters, the lumped mass transfer coefficient for benzaldehyde, and the partition coefficients. By using estimated prices of enzyme and reactor use, the optimum aqueous phase volume fraction and required enzyme concentration were calculated at a temperature of 20 degrees C for a batch-operated stirred tank reactor. According to the process model it was possible to convert 90% of the 4-hydroxybenzaldehyde into (R)-4-hydroxymandelonitrile with 95% enantiomeric excess. The price optimum for this reaction was found at an aqueous phase volume of 17% of the total volume. The required enzyme concentration to meet the targets was 28.6 g/L aqueous phase. At the predicted optimum, the synthesis was performed experimentally and the results were in accordance with the simulation regarding the extent of conversion and the enantiomeric excess.

cis- and trans-1,2-diphenylaziridines: induction of xenobiotic-metabolizing enzymes in rat liver and mutagenicity in Salmonella typhimurium.

trans-Stilbene imine (trans-1,2-diphenylaziridine) is the nitrogen analog of trans-stilbene oxide, a potent inducer of several microsomal and cytosolic xenobiotic-metabolizing enzymes. Although the acute toxicity of cis- and trans-stilbene imines prevents their application at the usual dose for trans-stilbene oxide (400 mg/kg/day), it is apparent that the imines nevertheless potently induce several xenobiotic-metabolizing enzymes in rat liver. The IP administration of trans-stilbene imine resulted in statistically significant increases in the activities of aminopyrine N-demethylase, microsomal epoxide hydrolase, glutathione transferase (toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene and delta 5-androstene-3,17-dione) and UDP-glucuronosyltransferase (toward testosterone). cis-Stilbene imine was less potent in inducing these activities. Although trans-stilbene imine (total dose = 400 mg/kg) was more potent than trans-stilbene oxide (total dose = 1200 mg/kg) in inducing the activities of glutathione transferase (toward 1-chloro-2,4-dinitrobenzene) and UDP-glucuronosyltransferase (toward testosterone), both compounds belong to the class of substances which are more potent inducers of conjugating (phase II) enzymes. Because of their structural similarity with K-region arene imines which are potent mutagens, cis-stilbene imine and trans-stilbene imine were investigated for mutagenicity (reversion of his- strains of Salmonella typhimurium). cis-Stilbene imine and trans-stilbene imine were direct mutagens in the strain TA100. This result, and the finding that acenaphthene 1,2-imine efficiently reverts various strains of Salmonella typhimurium, demonstrates that not only K-region arene imines, but also other aziridines substituted at the two carbons with aromatic moieties, are mutagenic.