Isolation and characterization of multiple forms of mandelonitrile lyase from mature black cherry ( Prunus serotina Ehrh.) seeds

Abstract

Five multiple forms (forms 1–5) of mandelonitrile lyase (EC 4.1.2.10) which catalyze the decomposition of mandelonitrile to benzaldehyde and hydrogen cyanide have been extensively purified from seeds of black cherry ( Prunus serotina Ehrh.) by concanavalin A-Sepharose 4B chromatography and chromatofocusing. These forms are monomers which differ only slightly in molecular weight (57,000–59,000) and isoelectric point (4.58–4.63), but heterogeneity in their carbohydrate side-chains was suggested by concanavalin A-Sepharose 4B chromatography. The absorption spectra of the predominating forms 4 and 5 showed maxima of 278, 380, and 460 nm, indicative of flavoprotein character. Detailed comparative kinetic studies of forms 4 and 5 revealed few significant differences in behavior. Both proteins showed pH optima between 6.0 and 7.0, had identical K m values (0.17 m m) for ( R,S)-mandelonitrile, and retained similar activities upon storage at 4 and −20 °C. Neither form exhibited a metal ion requirement and both were affected similarly by metal salts, β-mercaptoethanol, and sulfhydryl reagents. Benzole acid, p-hydroxybenzyl alcohol, and benzyl alcohol inhibited both forms.