Catabolism of (/?)-Amygdalin and (/?)-Vicianin by Partially Purified ß-Glycosidases from Prunus serotina Ehrh. and Davallia trichomanoides Gary Kuroki. Pauline A. Lizotte, and Jonathan E. Poulton Department of Botany, University of Iowa, Iowa City, Iowa 52242 Z. Naturforsch. 39 c, 232-239 (1984); received October 3, 1983 Cyanogenic Disaccharides, /?-Glycosidases, Prunus serotina , Davallia trichomanoides , Amygdalin, Vicianin Mature black cherry (Prunus serotina Ehrh.) seeds accumulate high levels of the cyanogenic disaccharide (/?)-amygdalin. Extracts from these seeds contain two /?-glycosidases which have been identified and completely resolved by DEAE-cellulose ion-exchange chromatography. Amygdalin hydrolase hydrolyzed (ß)-amygdalin at an optimum pH of 5.5, releasing (ß)-pruna- sin and D-glucose. This enzyme showed highest activity towards (/?)-amygdalin and failed to hydrolyze (/?)-prunasin. linamarin, /?-gentiobiose and cellobiose. A distinct /?-glycosidase, prunasin hydrolase, displayed a pronounced preference for (/?)-prunasin, hydrolyzing this cyanogenic monosaccharide at an optimum pH of 6.5 to mandelonitrile and D-glucose. Prunasin hydrolase was inactive towards (^)-amygdalin, linamarin, and /?-gentiobiose. Both enzymes showed significant activity towards the artificial substrates /?-ONPGlu and /?-PNPGlu but did not hydrolyze x-PNPGlu. In view of the pronounced specificity of these enzymes towards endogenous cyanogens, it is concluded that upon disruption of black cherry seeds (/?)- amygdalin is catabolized to mandelonitrile in a stepwise manner (the sequential mechanism) by amygdalin hydrolase and prunasin hydrolase with (Ä)-prunasin serving as intermediate. Young fronds of Davallia trichomanoides are rich sources of (Ä)-vicianin (the /?-vicianoside of (/?)-mandelonitrile). A /?-glycosidase, vicianin hydrolase, has been partially purified from frond extracts by ion-exchange chromatography. At the optimum pH of 6.0, this enzyme showed highest hydrolytic activity with (fi)-vicianin, although both (/?)-amygdalin and (/?)-prunasin could be hydrolyzed at approximately 15% of the rate observed with (/?)-vicianin. It failed to hydrolyze /i-gentiobiose, cellobiose, linamarin and x-PNPGlu. Closer examination revealed that (/?)-vicianin and (/?)-amygdalin were hydrolyzed at the aglycone-disaccharide bond (the simultaneous mechanism) yielding mandelonitrile and the respective disaccharides vicianose and /?-gentiobiose. Introduction aglycone-disaccharide bond would release the 2 -hydroxynitrile and a disaccharide (the “simul­ The catabolism of cyanogenic glycosides is taneous” mechanism). Alternatively, the two sugar initiated by the cleavage of the carbohydrate moiety residues might be removed singly by stepwise by one or more /?-glycosidases [1], Although the hydrolysis with a cyanogenic monosaccharide acting catabolism of several cyanogenic monosaccharides as intermediate (the “sequential” mechanism). In is now well understood [ 2 ], the mode by which the latter case, the two hydrolytic steps might be cyanogenic disaccharides are hydrolyzed remains catalyzed by the same or by distinct /?-glycosidases. largely unclear. Theoretically, two distinct de- Previous reports in the literature [3-6] suggest that gradative pathways may exist for cyanogenic di­ both simultaneous and sequential pathways may saccharides, as shown in Fig. 1. Hydrolysis at the exist in Nature. In order to provide more informa­ tion about this subject, we have selected two cyanogenic plant tissues for closer examination, Abbreviations: PVP, polyvinylpolypyrrolidone; AH, amygdalin hydrolase; PH. prunasin hydrolase; VH, vicia­ namely. Primus serotina seeds and Davallia tricho­ nin hydrolase; /?-PNPGlu and /?-ONPGlu, para- and ortho- manoides fronds. These tissues accumulate high nitrophenyl-/?-D-glucopyranosides; /?-PNPXyl and concentrations of the cyanogenic disaccharides /?-ONPXyl, para- and o/7/;o-nitrophenyl-/?-D-xylopyrano- sides; /ÄPNPGal. p-nitrophenyl-/?-D-galactopyranoside; (/?)-amygdalin (the /?-gentiobioside of (/?)-mandelo- x-PNPGlu. /7-nitrophenyl-x-D-glucopyranoside. nitrile) and (/?)-vicianin (the /?-vicianoside of Reprint requests to Dr. J. E. Poulton. (/?)-mandelonitrile), respectively. Cyanogen-specific 0341-0382/84/0300-0232 S 01.30/0 //-glycosidases have been partially purified from G. Kuroki et al. • Hydrolysis of Cyanogenic Disaccharides 233 P sugar,-0-sugar2 R N=C—C—0—sugar.-O-sugar,------- !---- 2 . ------------- . NEEC-C-OH Hydroxynitrile ^ ^ + ^ ß-Glycosidase ^ Lyase (_j/ Cyanogenic Monosaccharide Fig. 1. The simultaneous (1) and sequential (II) mechanisms for catabolism of cyanogenic disaccharides. these plants and their behavior towards endogenous column (1.0x41 cm). The column was eluted with cyanogens and other glycosidic substrates in­ water-saturated «-butanol and fractions containing vestigated. This has allowed us to predict the vicianin were identified by the Feigl-Anger test [ 8 ], pathway by which the endogenous cyanogenic Vicianin was recrystallized from benzene:methanol disaccharides are catabolized upon tissue disrup­ ( 1 : 1, by vol.) and its purity was confirmed by thin tion. layer chromatography. Clorox was purchased from the Clorox Co., Oakland, Ca. Materials and Methods Plant materials Chemicals Mature black cherry (Prunus serotina Ehrh.) fruits were collected from Hickory Hill Park, Iowa City. Polyvinylpolypyrrolidone (PVP), /?-glucosidase The pits were removed and surface sterilized in 10% (type II, from almonds), simple sugars and chromo- Clorox, blotted dry and stored at 4°C until used. genic substrates were purchased from Sigma Davallia trichomanoides specimens were purchased Chemical Co., St. Louis, MO. Glycosidic substrates from Fountain Square Nurseries, Sacramento, Ca. were available from our laboratory collection. Silica gel K5 TLC plates (thickness, 250 p), 3MM chroma­ Purification and assay of amvgdalin hydrolase and tography paper and DEAE-cellulose were obtained prunasin hydrolase from Prunus serotina from Whatman Chemical Separation Ltd., Kent, U.K., Microcrystalline cellulose (Avicel) was pur­ Unless otherwise indicated, all stages were carried chased from Merck, Darmstadt, BRD. Vicianin was out at 4°C. Black cherry pits (approximately 35) obtained by grinding Vicia angustifolia seeds in were cut in half with a razor blade and the seeds liquid nitrogen with a mortar and pestle. The homogenized with 10 ml of buffer I, 0.5 g PVP, and powdered seeds were placed in boiling 70% 2.0 g glass beads in a mortar. The homogenate was methanol for 10 min and filtered through Whatman filtered through four layers of cheesecloth and grade 1 filter paper. The filtrate was extracted with centrifuged at 17600xg for 25 min. An aliquot an equal volume of petroleum ether. The methanolic (2.5 ml) of the supernatant liquid was chromatog­ fraction was evaporated to dryness, redissolved in raphed on a Sephadex G-25 column (1.5 x 8.3 cm) water, and applied to Whatman 3 MM chromatog­ which had been pre-equilibrated with buffer II. raphy paper. The paper was developed in «-buta­ Elution was carried out with this buffer. The eluate nol: acetic acid:H20 (4:1:5, by vol., upper phase). was applied to a DEAE-cellulose column (1.6x 10cm) After thoroughly drying the paper, the cyanogen which had been pre-equilibrated with buffer II. was located by the Feigl-Anger “sandwich” tech­ After washing the column excessively with buffer II, nique [7] employing the Davallia /?-glycosidase elution of bound proteins was accomplished with a preparation to release HCN. The glycoside was 0 -3 0 0 mM NaCl gradient in buffer II. Fractions eluted with methanol and applied to a cellulose containing amygdalin hydrolase (AH) and prunasin 234 G. Kuroki et al. ■ Hydrolysis of Cyanogenic Disaccharides hydrolase (PH) activity were pooled separately and strates. the enzyme (0.05 ml) was incubated with dialyzed against buffer II overnight. These enzyme 1 0 (imol chromogenic substrate (dissolved in 0 . 5 ml preparations were stored at 4 °C and used for assay H 20) and 0.2(imol sodium acetate-HCl buffer, of /?-glucosidase activity as described below. pH 6.0. in a total volume of 1.0 ml. After incubation The standard assay mixture for AH and PH at 30 °C for 1 h. the reaction was terminated by activity contained 3.75 (imol glycosidic substrate adding 2.0 ml of 0.2 M sodium borate-NaOH (dissolved in 0.15 ml H 20). 50 (imol citrate-phos- buffer, pH 9.8, and the developed colors were mea­ phate buffer (pH 5.5 for AH. and pH 6.5 for PH), sured at 400 nm. For other substrates, the standard 30 jal H 20, and 20 (il of diluted enzyme preparation assay mixture for //-glucosidase activity contained (diluted 1:7 with buffer II) in a total volume of 0.5 (.imol glycosidic substrate (dissolved in 20 |al 0.25 ml. After incubation at 30 °C for 5 min, the H 20), 10 (imol sodium acetate-HCl buffer, pH 6.0, reaction was terminated by transferring an aliquot and 20 (il of enzyme in a total volume of 50 jj.1. After (0.23 ml) to a test tube immersed in boiling water. incubation at 30 °C for 1 h. the reaction was After boiling the mixture for 30 s, it was placed on terminated by placing the reaction vial in boiling ice. Aliquots (0.2 ml) were removed and glucose water for 2 min and then on ice. After centrifuga­ production measured by the glucose oxidase tion, aliquots (40 (il) were removed and glucose procedure [9], The assay mixture for chromogenic production determined by the glucose oxidase substrates was essentially the same, but the reaction procedure [9]. was terminated