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Substrate Specificity of Mutants of the Hydroxynitrile Lyase from Manihot esculenta


  • Enzyme-Catalyzed Reactions, Part 47. Part 46: J. Roos, F. Effenberger, Tetrahedron: Asymmetry 2002, 13, 1855–1862.


Several tryptophan128-substituted mutants of the hydroxynitrile lyase from Manihot esculenta (MeHNL) are constructed and applied in the MeHNL-catalyzed addition of HCN to various aromatic and aliphatic aldehydes as well as to methyl and ethyl ketones to yield the corresponding cyanohydrins. The mutants (especially MeHNL-W128A) are in most cases superior to the wild-type (wt) enzyme when diisopropyl ether is used as the solvent. Substitution of tryptophan128 by an alanine residue enlarges the entrance channel to the active site of MeHNL and thus facilitates access of sterically demanding substrates to the active site, as clearly demonstrated for aromatic aldehydes, especially 3-phenoxybenzaldehyde. These experimental results are in accordance with the X-ray crystal structure of MeHNL-W128A. Aliphatic aldehydes, surprisingly, do not demonstrate this reactivity dependence of mutants on substrate bulkiness. Comparative reactions of 3-phenoxybenzaldehyde with wtMeHNL and MeHNL-W128A in both aqueous citrate buffer and a two-phase system of water/methyl tert-butyl ether again reveal the superiority of the mutant enzyme: 3-phenoxybenzaldehyde was converted quantitatively into a cyanohydrin nearly independently of the amount of enzyme present, with a space-time yield of 57 g L−1 h−1.