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Complete Article 528 Biochem. J. (1967) 103, 528 The Enzymic Hydrolysis of Amygdalin By D. R. HAISMAN AND D. J. KNIGHT The Fruit and Vegetable Preservation Research A88ociation, Chipping Campden, Glo8. (Received 27 July 1966) Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond f-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the ,B- glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components. Amygdalin, D(-)-mandelonitrile ,B-gentiobioside nitrile lyase was present in emulsin, and isolated and (Haworth & Wylam, 1923), is found in the tissues of characterized the enzyme. species of Prunu8, and is particularly abundant in Weidenhagen (1932) reviewed the enzymic the kemels. The kernels are also a rich source ofthe hydrolysis of amygdalin and, on the basis of his enzyme system, commonly known as emulsin, own kinetic measurements, concluded that the which attacks a wide variety of ,B-glycosidic bonds. hydrolysis was brought about by the action of only The enzymic hydrolysis of amygdalin was first one enzyme, ,-glucosidase, acting consecutively on observed by Wohler & Liebig (1837). Other early the 6-O-,B-D-glucopyranosyl-D-glucose bond and the studies with yeast extracts (Fischer, 1895) and aglucone-O-p-D-glucose bond. almond emulsin (Armstrong, Armstrong & Horton, Laterreviewers have castdoubt onWeidenhagen's 1908; Auld, 1908; Krieble, 1912) showed that the hypothesis of a single enzyme of low specificity able reaction took place in at least three steps: firstly the to hydrolyse a wide range of ,B-glycosidic bonds. In f-(1-6') bond of the gentiobiose portion of the view of the differences in specificity shown by amygdalin was split to yield D(-)-mandelonitrile ,B-glucosidase isolated from different sources (e.g. ,-glucoside, called prunasin, and glucose; next, almond and snail emulsin), Pigman (1944) suggested prunasin was hydrolysed to (+ )-mandelonitrile and that fl-glucosidase comprised a class of closely glucose; finally the (+ )-mandelonitrile was broken related enzymes all showing a specific ability to down to benzaldehyde and hydrocyanic acid. It hydrolyse ,B-glycosidic linkages. Veibel (1950) was thought that three different enzymes, amyg- examined the evidence on the specificities ofalmond dalin lyase, prunasin lyase and hydroxynitrile lyase emulsin and showed that the existence of a pure acted consecutively on the substrate molecule. homogeneous enzyme which would act on more than Armstrong, Armstrong & Horton (1912) found a one glycosidic type had not been proved. Jermyn high concentration of prunasin lyase in cherry- (1961), in a more general review, reached the same laurel leaves, which displayed only slight enzymic conclusion and, considering aglycone specificity, activity towards amygdalin. pointed out that the differences in the effect of The evidence for the existence of hydroxynitrile ortho-substitution in phenyl-p-glucosides on lyase stemmed mainly from observations on the enzymes from various sources precluded the influence of emulsin on the synthesis of (+)- existence of a single P-glucosidase of constant mandelonitrile from benzaldehyde and hydro- properties. cyanic acid (Feist, 1909; Krieble & Wieland, 1921; ,-Glucosidase activity in plant tissues is fre- Rosenthaler, 1922). Nordefeldt (1925) demon- quently assayed by measuring the rate ofhydrolysis strated that many of the earlier observations, made of a standard solution of salicin (Veibel, 1950; on unbuffered systems, were the result of changes in Baruah & Swain, 1957), and we have used the salicin pH when the emulsin was added to the reaction method for measuring the enzyme activity in the mixture. However, he also concluded that hydroxy- kernels of pluims. In such experimental work Vol. 103 ENZYMIC HYDROLYSIS OF AMYGDALIN 529 allowance must be made for the competitive effect obtained with the buffers and substrates used. Reducing of the natural substrate, amygdalin, which is the bromination time to 30sec. was found to minimize present in appreciable amounts. As the enzymic colour formation due to side reactions with the buffers, and hydrolysis of amygdalin is complex and appears to this time was adequate for the complete conversion of cyanide into cyanogen bromide. involve consecutive reactions, it was thought that Glucose could not be determined by the usual copper the inhibitory effect of amygdalin on the hydrolysis methods for reducing sugars because of interference from of salicin might be significant. To explore this the cyanide present, but an adaptation of the glucosazone possibility, the kinetics of the hydrolysis of amyg- method of Wahba, Hanna & El-Sadr (1956) was found to be dalin have been re-examined. The results agree with convenient and accurate. A portion of the reaction mixture the views of the earlier investigators, that different in 0-1 M-acetate buffer was pipetted into 3ml. of acetic acid, fi-glucosidases ofhigh specificity do exist. which stopped the enzyme reaction. The volume was made up to 8ml. with 0-1m-acetate buffer, 2ml. of 5% phenyl- hydrazine hydrochloride was added, and the solution was MATERIALS heated for lhr. in a boiling-water bath for full colour Enzyme. 3-Glucosidase preparation from almonds, development. The glucosazone showed maximum absorp- activity 1000 units/mg. (Baruah & Swain, 1957), was tion at 380m/u. Under the same conditions any benzalde- purchased from Koch-Light Laboratories Ltd. (Colnbrook, hyde present also formed a phenylhydrazone, absorption Bucks.). maximum 341m,. Extinctions were measured at both Stubstrate8. Mandelonitrile was obtained from B. Newton wavelengths against a reagent blank and calibration Maine Ltd. (North Walsham, Norfolk), estimated purity standards of glucose and benzaldehyde in 0-1M-acetate 89%. After distillation under reduced pressure, a colourless buffer were used in each set of determinations. The con- fraction, estimated purity 97%, was obtained. centrations ofglucose and benzaldehyde were calculated by (+ )-Salicin, amygdalin and gentiobiose were commercial the usual two-component analysis procedure (Knudson, samples. Meloche & Juday, 1940). Prunasin was prepared by the acid hydrolysis of In the degradation of amygdalin, benzaldehyde and amygdalin, by using the method of Caldwell & Courtauld hydrogen cyanide are produced in equimolar amounts, and (1907). It was recrystallized from ethyl acetate until found in our analyses the estimated benzaldehyde and cyanide pure by thin-layer chromatography. contents were in good agreement. To complete the analysis of the degradation products a method had to be devised for the estimation of mandelo- METHODS nitrile in the presence of hydrocyanic acid. Enzymic hydroly8e8. Hydrolyses were carried out in The formation ofcyanohydrins, as catalysed by enzymes, flasks in a shaking incubator bath maintained at 30°. All acids and other compounds, has been extensively studied, solutions were allowed to equilibrate in the bath before but there has been no methodical examination of their mixing. decomposition. As the formation of cyanohydrins is an Chromatography. Chromatographic separations were acid-catalysed reaction (Svirbely & Roth, 1953), the kinetics performed on thin layers of cellulose (Vomhof & Tucker, of the hydrolysis of mandelonitrile as a function of pH were 1965) or silica gel (Adachi, 1965), and the components were studied. It was found that the nitrile decomposes instan- identified either by treatment with silver nitrate and taneously at high pH values, so that it could be estimated in 0-5N-sodium hydroxide in ethanol, or by spraying with a solution by measuring the cyanide concentration before 0-5% thymol in 5% sulphuric acid in ethanol and heating and after alkaline hydrolysis. for 15min. at 1200. A stock solution of mandelonitrile was prepared by Electrophore8is. Electrophoretic separations were carried dissolving a catch weight ofabout 0-1g. in 250ml. ofdistilled out on cellulose acetate strips (Oxoid) with either acetate or water. Portions (2ml.) were immediately pipetted into barbitone-acetate buffers over the pH range 5-0-8-6. 50ml. of 0-2M-acetate or -citrate buffer or 80ml. of distilled Buffer concentrations between 0-025 and 0-1 M and potential water, and, after addition of hydrochloric acid or sodium gradients of about 20v/cm. were used. After drying, the hydroxide for the pH ranges 1-3 and 11-12, made up to a strips were stained by soaking overnight in 0-001% nigrosine final volume of 100ml. Blanks for each pH value were also in 2% acetic acid. prepared, and the pH value of each solution was checked Analy8e8. The expected products from the degradation of both before and after the experiment. amygdalin were prunasin, gentiobiose, glucose, mandelo- The samples were placed in an agitating water bath at nitrile, benzaldehyde andhydrogen cyanide, and thekinetics 300, and the time was recorded. Portions (5ml.) were of the degradation could best be followed if the concentra- withdrawn at intervals for estimation of their cyanide tions ofall
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