S-selective hydroxynitrile lyase from a plant Baliospermum montanum: molecular characterization of recombinant enzyme.

A novel S-hydroxynitrile lyase (HNL) was purified from leaves of a plant, Baliospermum montanum, by ammonium sulfate fractionation and column chromatographies. Full-length cDNA and genomic DNA were cloned and sequenced. The latter contained two introns and one ORF encoding a 263-residue protein (subunit: 29.5 kDa). The hnl gene was expressed in Escherichia coli and the enzyme was characterized including detailed kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. The enzyme exhibited the highest specific activity (178 U/mg), k(cat) (98/s) and k(cat)/K(m) ratio for piperonal. k(cat)/K(m) ratio for aromatic aldehydes was much larger than those of aliphatic aldehydes and ketones. It was strongly inhibited by AgNO3, PMSF, phenol and methyl ethyl ketone, showed an optimum at pH 5, while having activity at range of 4-6.5. It exhibited stability at wide pH range 2.4-11, the highest activity at 20 °C, being active at 0-65 °C. The enzyme showed variations in residues involved in substrate pocket and substrate entrance channel compared to other S-selective HNLs, based on a model was built. C-terminal short truncations provided more enzyme production. Gel filtration revealed a 60-65 kDa molecular mass for this non-FAD enzyme and its C-terminally truncated forms using three buffer compositions, indicating dimeric structures.

Hydroxynitrile lyase from Passiflora edulis: Purification, characteristics and application in asymmetric synthesis of (R)-mandelonitrile.

A hydroxynitrile lyase from leaves of Passiflora edulis (PeHNL) was purified and characterized for the first time. The enzyme is a monomer of 15kDa and 18kDa by SDS-PAGE, and gel filtration, respectively. Asymmetric synthesis of (R)-mandelonitrile from benzaldehyde and acetone cyanohydrin in a biphasic system employing the PeHNL from rinds of P. edulis was carried out. Several parameters influenced the enantiomeric purity of the product and initial velocity of the reaction. Both pH and temperature were important parameters controlling the enantiomeric purity of the product. The optimum pH and temperature were pH 4 and 10°C, respectively. At the optimum pH and temperature, the spontaneous non-enzymatic reaction yielding the racemic mandelonitrile was almost completely suppressed. The PeHNL was stable (more than 80% residual activity after incubation for 12h) in the system of methyl-t-butyl ether (MTBE), dibutyl ether (DBE), hexane (HEX), and diisopropyl ether (DIPE) while diethyl ether (DEE) and ethyl acetate (EA) were not suitable solvents. The initial velocity was markedly affected by the type of organic solvent in the biphasic system, while high enantiomeric purity was obtained when organic solvents having logP lower than 3.5 were used. The highest initial velocity of reaction and enantiomeric purity of (R)-mandelonitrile were obtained in the biphasic system of DBE with the aqueous phase content of 30% (v/v). The optimum substrate concentrations were 250mM for benzaldehyde and 900mM for acetone cyanohydrin, and the optimum enzyme concentration was 26.7units/ml. The highest enantiomeric purity of (R)-mandelonitrile was successfully obtained with conversion and enantiomeric excess of 31.6% and 98.6%, respectively. The enzyme showed considerable reusability in batch reaction with high enantiomeric purity of product. Herein, we reported the characteristics of a unique (R)-PeHNL from leaves of P. edulis. The PeHNL from rinds had been isolated for the first time and the enzyme showed great ability in transcyanation of (R)-mandelonitrile with high e.e. in DBE as the co-organic solvent in a biphasic system.

Screening for new hydroxynitrilases from plants.

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%.