Prunasin hydrolases during fruit development in sweet and bitter almonds

Amygdalin is a cyanogenic diglucoside and constitutes the bitter component in bitter almond (Prunus dulcis). Amygdalin concentration increases in the course of fruit formation. The monoglucoside prunasin is the precursor of amygdalin. Prunasin may be degraded to hydrogen cyanide, glucose, and benzaldehyde by the action of the β-glucosidase prunasin hydrolase (PH) and mandelonitirile lyase or be glucosylated to form amygdalin. The tissue and cellular localization of PHs was determined during fruit development in two sweet and two bitter almond cultivars using a specific antibody toward PHs. Confocal studies on sections of tegument, nucellus, endosperm, and embryo showed that the localization of the PH proteins is dependent on the stage of fruit development, shifting between apoplast and symplast in opposite patterns in sweet and bitter cultivars. Two different PH genes, Ph691 and Ph692, have been identified in a sweet and a bitter almond cultivar. Both cDNAs are 86% identical on the nucleotide level, and their encoded proteins are 79% identical to each other. In addition, Ph691 and Ph692 display 92% and 86% nucleotide identity to Ph1 from black cherry (Prunus serotina). Both proteins were predicted to contain an amino-terminal signal peptide, with the size of 26 amino acid residues for PH691 and 22 residues for PH692. The PH activity and the localization of the respective proteins in vivo differ between cultivars. This implies that there might be different concentrations of prunasin available in the seed for amygdalin synthesis and that these differences may determine whether the mature almond develops into bitter or sweet.     

The enzymic hydrolysis of amygdalin

Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond beta-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 beta-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.     

Pattern of the Cyanide-Potential in Developing Fruits : Implications for Plants Accumulating Cyanogenic Monoglucosides (Phaseolus lunatus) or Cyanogenic Diglucosides in Their Seeds (Linum usitatissimum, Prunus amygdalus)

The absolute cyanide content of developing fruits was determined in Costa Rican wild lima beans (Phaseolus lunatus), oil flax (Linum usitatissimum), and bitter almonds (Prunus amygdalus). The cyanide potential (HCN-p) of the lima bean and the almond fruit began to increase shortly after anthesis and then stopped before fruit maturity. In contrast, the flax inflorescence had a higher HCN-p in absolute terms than the mature flax fruit. At all times of its development the bean fruit contained the monoglucosides linamarin and lotaustralin. The almond and the flax fruits contained, at anthesis, the monoglucosides prunasin, and linamarin and lotaustralin, respectively, while, at maturity, only the corresponding diglucosides amygdalin, and linustatin and neolinustatin, respectively, were present.     

Continuous spectrophotometric assays for beta-glucosidases acting on the plant glucosides L-picein and prunasin.

The neutral pH optimum beta-glucosidases of mammalian liver and almonds are each capable of hydrolyzing a number of plant glucosides, including L-picein (p-hydroxyacetophenone-beta-D-glucoside) and prunasin (D-mandelonitrile-beta-D-glucoside). Taking advantage of the marked differences in the spectra of the substrate/product pairs of L-picein/p-hydroxyacetophenone and prunasin/mandelonitrile, we have devised spectrophotometric assays that permit the continuous monitoring at pH 7.0 of p-hydroxyacetophenone (piceol) release from L-picein by guinea pig hepatic cytosolic beta-glucosidase and mandelonitrile from prunasin by almond beta-glucosidase. When L-picein hydrolysis was monitored at 320 nm and prunasin at 282 nm, the molar absorption coefficients determined for their products, namely piceol and mandelonitrile, were 3200 and 1360 M-1 cm-1, respectively. The kinetic parameter Km and Vmax values obtained using these spectrophotometric procedures for the guinea pig liver cytosolic beta-glucosidase acting on L-picein were 0.88 mM and 5.29 x 10(5) units/mg protein and for the almond beta-glucosidase acting on prunasin, Km 1.1 mM and Vmax 5.24 x 10(6) units/mg protein. These values agreed well with previously reported values obtained using less convenient, discontinuous assay procedures.     

Exolytic hydrolysis of toxic plant glucosides by guinea pig liver cytosolic beta-glucosidase.

We demonstrate that although the guinea pig liver cytosolic beta-glucosidase does not catalyze the hydrolysis of gentiobiose, it does hydrolyze, disaccharide-containing glycosides such as p-nitrophenyl-beta-D-gentiobioside (Glc beta 1----6Glc beta-pNP) and mandelonitrile-beta-D-gentiobioside (amygdalin). Furthermore, we establish that the enzyme attacks disaccharide glycosides exolytically; specifically, we document the exolytic deglucosylation of amygdalin and the generation of the intermediate monosaccharide glycoside mandelonitrile-beta-D-glucoside prior to the formation of the aglycone (mandelonitrile). We also show that the cytosolic beta-glucosidase catalyzes the hydrolysis of various phenolic (e.g. arbutin and salicin) and cyanogenic plant glucosides (e.g. prunasin). Using the everted gut-sack technique, we demonstrate that the plant glucosides, amygdalin, prunasin, and vicine, are transported across the small intestine of the guinea pig efficiently and without being hydrolyzed. Based on these data we speculate that the cytosolic beta-glucosidase may participate in biotransformation of toxic plant glucosides.     

Molecular markers for kernel bitterness in almond

Upon crushing, amygdalin present in bitter almonds is hydrolysed to benzaldehyde, which gives a bitter flavour, and to cyanide, which is toxic. Bitterness is attributable to the recessive allele of the Sweet kernel (Sk/sk) gene and is selected against in breeding programmes. Almond has a long intergeneration period due to its long juvenile phase, so breeders must wait 3 or 4 years to evaluate fruit traits in the field. For this reason, it is important to develop molecular markers to distinguish between sweet and bitter genotypes. The Sk gene is known to map to linkage group five (G5) of the almond genome, but its function is still undefined. Candidate genes involved in the amygdalin pathway have been mapped, but none of them were located to G5. We have saturated G5 with additional Simple Sequence Repeats (SSRs) using the progeny from the cross “R1000” × “Desmayo Largueta” and found six SSRs (UDA-045, EPDCU2584, CPDCT028, BPPCT037, PceGA025, and CPDCT016) closely linked to the Sk locus. The genotypes of four of these SSRs flanking the Sk locus, in a number of parents and a few seedlings of the CEBAS-CSIC almond breeding programme, allowed us to estimate the haplotypes of the parents, identifying the marker alleles adequate for an early and highly efficient selection against bitter genotypes. This analysis has established the usefulness of SSRs for screening populations of fruit trees such as almond by an easy, polymerase chain reaction-based method.     

Utilization of Amygdalin during Seedling Development of Prunus serotina

Cotyledons of mature black cherry (Prunus serotina Ehrh.) seeds contain the cyanogenic diglucoside (R)-amygdalin. The levels of amygdalin, its corresponding monoglucoside (R)-prunasin, and the enzymes that metabolize these cyanoglycosides were measured during the course of seedling development. During the first 3 weeks following imbibition, cotyledonary amygdalin levels declined by more than 80%, but free hydrogen cyanide was not released to the atmosphere. Concomitantly, prunasin, which was not present in mature, ungerminated seeds, accumulated in the seedling epicotyls, hypocotyls, and cotyledons to levels approaching 4 [mu]mol per seedling. Whether this prunasin resulted from amygdalin hydrolysis remains unclear, however, because these organs also possess UDPG:mandelonitrile glucosyltransferase, which catalyzes de novo prunasin biosynthesis. The reduction in amygdalin levels was paralleled by declines in the levels of amygdalin hydrolase (AH), prunasin hydrolase (PH), mandelonitrile lyase (MDL), and [beta]-cyanoalanine synthase. At all stages of seedling development, AH and PH were localized by immunocytochemistry within the vascular tissues. In contrast, MDL occurred mostly in the cotyledonary parenchyma cells but was also present in the vascular tissues. Soon after imbibition, AH, PH, and MDL were found within protein bodies but were later detected in vacuoles derived from these organelles.     

Development of the Potential for Cyanogenesis in Maturing Black Cherry (Prunus serotina Ehrh.) Fruits

Biochemical changes related to cyanogenesis (hydrogen cyanide production) were monitored during maturation of black cherry (Prunus serotina Ehrh.) fruits. At weekly intervals from flowering until maturity, fruits (or selected parts thereof) were analyzed for (a) fresh and dry weights, (b) prunasin and amygdalin levels, and (c) levels of the catabolic enzymes amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. During phase I (0-28 days after flowering [DAF]), immature fruits accumulated prunasin (mean: 3 micromoles/fruit) but were acyanogenic because they lacked the above enzymes. Concomitant with cotyledon development during mid-phase II, the seeds began accumulating both amygdalin (mean: 3 micromoles/seed) and the catabolic enzymes and were highly cyanogenic upon tissue disruption. Meanwhile, prunasin levels rapidly declined and were negligible by maturity. During phases II (29-65 DAF) and III (66-81 DAF), the pericarp also accumulated amygdalin, whereas its prunasin content declined toward maturity. Lacking the catabolic enzymes, the pericarp remained acyanogenic throughout all developmental stages.     

Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds

In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and d-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two β-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase.     

β-Glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular β-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 °C and 30 °C and slightly less at 35 °C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 °C) during an incubation period of 5 days. Supernatants of cultures grown at 25 °C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4–5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 °C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 °C and 30 °C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Received: 16 September 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997     

Possible involvement of polyphenols and polyamines in salt tolerance of almond rootstocks

Leaf physiological and biochemical adaptive strategies and more particularly the possible involvement of polyamines and polyphenols in salt stress tolerance were investigated. Three almond rootstocks (GN15, GF677 and bitter almond) were subjected to 0, 25, 50 and 75 mM NaCl for 30 days. The dry mass of leaves, stems and roots decreased with increasing salt concentration in the irrigation solution regardless of genotype. Photosynthetic assimilation rate decreased in the three almond rootstocks, but more so in GF677 and bitter almond. The accumulation of toxic ions was greater in the leaves than in the roots in all genotypes. GN15 accumulated less Na⁺ and Cl⁻ than GF677 and bitter almond. GF677 accumulated polyphenols, but had less anthocyanin and antioxidant activity in its leaves compared to bitter almond. It seems that GN15 was more able to tolerate the excess of toxic ions using anthocyanins which are abundant in its red leaves and free polyamines for a more efficient response to stress. However, most of the antioxidant activity was found in the leaves and was lower in the roots. Given that the upper part of the tree will be of a different cultivar after grafting, this advantage may not be relevant for the tree’s survival. GF677 showed a different antioxidant strategy; it maintained a stable carotenoids content and accumulated polyphenols in its leaves. The three rootstocks used different strategies to deal with the excess of salt in the growth medium.     

On the metabolism of amygdalin. 2. The distribution of beta-glucosidase activity and orally administered amygdalin in rats

The organs of 15-day-old rats had the highest capability to hydrolyze amygdalin and prunasin, and most of this activity is concentrated in the tissues of the small and large intestines. The activity decreased with age. In adult rats, the ability of the organs to hydrolyze prunasin is higher than that of amygdalin and is concentrated in the spleen, large intestine, and kidney (35.0, 15.0, and 8.9 micrograms prunasin hydrolyzed . h-1 . g tissue-1). Minced tissues of the liver, spleen, kidney, and stomach contain more hydrolytic capability than the homogenate of these organs, while the reverse is the case with the small and large intestines. When 30 mg amygdalin was orally administered to adult rats, its distribution after the 1st h was as follows: stomach (0.89 mg), small intestine (0.78 mg), spleen (0.36 mg), large intestine (0.30 mg), kidney (0.19 mg), liver (0.10 mg), and serum (5.6 micrograms/mL). At the end of the 2nd h, the highest amygdalin content was found in the large intestine (0.79 mg).     

Analysis of Bitter Almonds and Processed Products Based on HPLC-Fingerprints and Chemometry

In the processing field, there is a saying that “seed drugs be stir-fried”. Bitter almond (BA) is a kind of seed Chinese medicine. BA need be used after being fried. To distinguish raw bitter almonds (RBA) from processed products and prove the rationality of “seed drugs be stir-fried”, we analyzed the RBA and five processed products (scalded bitter almonds, fried bitter almonds, honey fried bitter almonds, bran fried bitter almonds, bitter almonds cream) using RP-HPLC fingerprints and chemometric methods. The similarity between RBA and processed products was 0.733∼0.995. Hierarchically clustered heatmap was used to evaluate the changes in components. Principal component analysis (PCA) was used for classification, and all samples are distinguished according to RBA and five processing methods. Six chemical markers were obtained by partial least squares discriminant analysis (PLS-DA). The content and degradation rate of amygdalin and β-glucosidase activity were determined. Compared with RBA, the content and degradation rate of amygdalin, and β-glucosidase activity were increased in bitter almonds cream. The content and degradation rate were decreased, and β-glucosidase was inactivated in other processed products. The above results showed that stir-frying had the best effect. The results showed that processing can ensure the stability of RBA quality, and the saying “seed drugs be stir-fried” is reasonable.     

Tissue and cellular localization of individual β‐glycosidases using a substrate‐specific sugar reducing assay

Traditional methods to localize β‐glycosidase activity in tissue sections have been based on incubation with the general substrate 6‐bromo‐2‐naphthyl‐β‐d ‐glucopyranoside. When hydrolysed in the presence of salt zinc compounds, 6‐bromo‐2‐naphthyl‐β‐d ‐glucopyranoside affords the formation of an insoluble coloured product. This technique does not distinguish between different β‐glycosidases present in the tissue. To be able to monitor the occurrence of individual β‐glycosidases in different tissues and cell types, we have developed a versatile histochemical method that can be used for localization of any β‐glycosidase that upon incubation with its specific substrate releases a reducing sugar. Experimentally, the method is based on hydrolysis of the specific substrate followed by oxidation of the sugar released by a tetrazolium salt (2,3,5‐triphenyltetrazolium chloride) that forms a red insoluble product when reduced. The applicability of the method was demonstrated by tissue and cellular localization of two β‐glucosidases, amygdalin hydrolase and prunasin hydrolase, in different tissues and cell types of almond. In those cases where the analysed tissue had a high content of reducing sugars, this resulted in strong staining of the background. This interfering staining of the background was avoided by prior incubation with sodium borohydride. The specificity of the devised method was demonstrated in a parallel localization study using a specific antibody towards prunasin hydrolase.     

Purification and Characterization of an Intracellular β-Glucosidase from the Methylotrophic Yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris beta-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate, gel filtration, and ion-exchange chromatography with Q-Sepharose and CM-Sepharose. The enzyme is a tetramer (275 kD) made up of four identical subunits (70 kD). The pH optimum is 7.3, and it is fairly stable in the pH range 5.5-9.5. The temperature optimum is 40 degrees C. The purified beta-glucosidase is effectively active on p-/o-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with Km values of 0.12, 0.22, and 0.096 mM and Vmax values of 10.0, 11.7, and 6.2 micromol/min per mg protein, respectively. It also exhibits different levels of activity against p-nitrophenyl-1-thio-beta-D-glucopyranoside, cellobiose, gentiobiose, amygdalin, prunasin, salicin, and linamarin. The enzyme is competitively inhibited by gluconolactone, p-/o-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), and glucose against p-NPG as substrate. o-NPF is the most effective inhibitor of the enzyme activity with Ki value of 0.41 mM. The enzyme is more tolerant to glucose inhibition with Ki value of 7.2 mM for p-NPG. Pichia pastoris has been employed as a host for the functional expression of heterologous beta-glucosidases and the risk of high background beta-glucosidase activity is discussed.     

A new HPLC-ELSD method to quantify indican in Polygonum tinctorium L. and to evaluate beta-glucosidase hydrolysis of indican for indigo production

A method to quantify the indigo precursor indican (indoxyl-beta-D-glucoside) in Polygonum tinctorium L. has been developed. Plant material was extracted in deionized water, and indican was identified and quantified using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Results confirmed that with this method it is possible to measure indican content in a short time, obtaining reliable and reproducible data. Using this method, leaf indican content was quantified every 15 days during the growing season (from May to October) in P. tinctorium crops grown in a field experiment in Central Italy. Results showed that indican increased along the growing season until flowering and was positively affected by photosynthetic active radiation (PAR). Indican is naturally hydrolyzed by native beta-glucosidase to indoxyl and glucose, the indoxyl yielding indigo. The activity of two enzymes, sweet almond beta-glucosidase and Novarom G preparation, were compared with P. tinctorium native beta-glucosidase to evaluate indigo production. Results showed that the ability to promote indigo formation increased as follows: almond beta-glucosidase 相似文献   

Purification and characterization of the NADH:ferredoxinBPH oxidoreductase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400

Mucor circinelloides LU M40 and Penicillium aurantiogriseum P 35 produce extracellular β-glycosidases that are active on the cyanogenic glycoside amygdalin. From the culture broths of M. circinelloides, only one β-glycosidase could be identified, while two different enzymes – both having amygdalase activity – were found in culture broths of P. aurantiogriseum. The study of the mechanism of hydrolysis of the β-bis-glycoside amygdalin with purified enzymes from the two organisms indicated a possible sequential (two-step) reaction. In all cases, the first step of hydrolysis from amygdalin to prunasin was very rapid, while the second step from prunasin to cyanohydrin was much slower. No cyanohydrin lyase activity was found in the culture broths of either fungus. Received: 16 May 1997 / Accepted: 11 September 1997     

Cyanogenesis in Cotoneaster-arten

The presence of the cyanogenic glycoside prunasin in leaves and fruits of Cotoneaster species was confirmed by GLC. In addition amygdalin was found in ripe fruits. The variation in prunasin and amygdalin was measured during development of the flowers and fruits of C. praecox and C. bullata. The importance of these findings for chemotaxonomy and physiology is discussed.     

Catalytic degradation of amygdalin by extracellular enzymes from Aspergillus niger

Amygdalin is a controversial anti-tumor natural product that has been used as an alternative cancer drug for many years. The anti-tumor mechanism and metabolism of amygdalin have been the focus of many studies. However, previous studies by our group demonstrated that amygdalin itself has no anti-tumor activity, but rather the active ingredients were determined to be amygdalin degradation products. To screen novel drugs with anti-tumor activity, the extracellular enzymes from Aspergillus niger were used to degrade amygdalin. Within 4 h of the catalytic reaction at 37°, amygdalin was rapidly degraded into four products. The products were then extracted and purified by column chromatography. By comparing the HPLC chromatograms, ¹H NMR, ¹³C NMR and MS data, the products were identified as mandelonitrile, prunasin, benzaldehyde and phenyl-(3,4,5-trihydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-acetonitrile (PTMT), a novel hydroxyl derivative of prunasin. Furthermore, pharmacology studies of these compounds demonstrated that 10 mg/kg of PTMT significantly suppressed the growth of S-18 tumor cells within 11 days in a concentration-dependent manner.     

A pseudo-β-glucosidase in Arabidopsis thaliana: Correction by site-directed mutagenesis, heterologous expression, purification, and characterization

Since At2g25630 is an intronless gene with a premature stop codon, its cDNA encoding the predicted mature beta-glucosidase isoenzyme was synthesized from the previously isolated Arabidopsis thaliana genomic DNA. The stop codon was converted to a sense codon by site-directed mutagenesis. The native and mutated cDNA sequences were separately cloned into the vector pPICZalphaB and expressed in Pichia pastoris. Only the cells transformed with mutated cDNA-vector construct produced the active protein. The mutated recombinant beta-glucosidase isoenzyme was chromatographically purified to apparent homogeneity. The molecular mass of the protein is estimated as ca. 60 kD by SDS-PAGE. The pH optimum of activity is 5.6, and it is fairly stable in the pH range of 5.0-8.5. The purified recombinant beta-glucosidase is effectively active on para-/ortho-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with K(m) values of 1.9, 2.1, 0.78 mM and k(cat) values of 114, 106, 327 nkat/mg, respectively. It also exhibits different levels of activity against para-/ortho-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), amygdalin, prunasin, cellobiose, gentiobiose, and salicin. The enzyme is competitively inhibited by gluconolactone and p-nitrophenyl-1-thio-beta-D-glucopyranoside with p-NPG, o-NPG, and 4-MUG as substrates. The enzyme is found to be very tolerant to glucose inhibition. The catalytic role of nucleophilic glutamic acid in the motif YITENG of beta-glucosidases and mutated recombinant enzyme is discussed.