We established a simple HPLC method to determine the activity and stereochemistry of the chiral mandelonitrile synthesized from benzaldehyde and cyanide, and applied it to screen for hydroxynitrile lyase (HNL) activity of plant origin. A total of 163 species of plants among 74 families were examined for (R)-and (S)-HNL activities using the method. We discovered that homogenate of leaves of Baliospermum montanum shows (S)-HNL activity, while leaves and seeds from Passiflora edulis, and seeds from Eriobotrya japonica, Chaenomles sinensis, Sorbus aucuparia, Prunus mume, and Prunus persica show (R)-HNL activity. Partially purified (R)-HNLs from Passiflora edulis and Eriobotrya japonica acted not only on benzaldehyde but also on aliphatic ketone. The enantiomeric excess of (R)-methylpropylketone cyanohydrin synthesized from 2-pentanone using homogenate from leaves of Passiflora edulis was 87.0%, and that of (R)-mandelonitrile synthesized by homogenate from seeds of Eriobotrya japonica was 85.0%.
A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30-80% (NH(4))(2)SO(4) fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which suggested the presence of a carbohydrate side chain. The purified enzyme was a monomer with a molecular mass of 72 kDa as determined by gel filtration, and 62.3 kDa as determined by SDS-gel electrophoresis. The N-terminal sequence is reported. The enzyme was a flavoprotein containing FAD as a prosthetic group, and it exhibited a K(m) of 161 microM and a k(cat)/K(m) of 348 s(-1) mM(-1) for mandelonitrile. The optimum pH and temperature were pH 5.5 and 40 degrees C respectively. The enzyme showed excellent stability with regard to pH and temperature. Metal ions were not required for its activity, while activity was significantly inhibited by CuSO(4), HgCl(2), AgNO(3), FeCl(3), beta-mercaptoethanol, iodoacetic acid, phenylmethylsulfonylfluoride, and diethylpyrocarbonate. The specificity constant (k(cat)/K(m)) of the enzyme was investigated for the first time using various aldehydes as substrates. The enzyme was active toward aromatic and aliphatic aldehydes, and showed a preference for smaller substrates over bulky one.
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