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.     

Effects of the Gut microbiota on Amygdalin and its use as an anti-cancer therapy: Substantial review on the key components involved in altering dose efficacy and toxicity

Conventional and Alternative Medicine (CAM) is popularly used due to side-effects and failure of approved methods, for diseases like Epilepsy and Cancer. Amygdalin, a cyanogenic diglycoside is commonly administered for cancer with other CAM therapies like vitamins and seeds of fruits like apricots and bitter almonds, due to its ability to hydrolyse to hydrogen cyanide (HCN), benzaldehyde and glucose. Over the years, several cases of cyanide toxicity on ingestion have been documented. In-vitro and in-vivo studies using various doses and modes of administration, like IV administration studies that showed no HCN formation, point to the role played by the gut microbiota for the commonly seen poisoning on consumption. The anaerobic Bacteriodetes phylum found in the gut has a high β-glucosidase activity needed for amygdalin hydrolysis to HCN. However, there are certain conditions under which these HCN levels rise to cause toxicity. Case studies have shown toxicity on ingestion of variable doses of amygdalin and no HCN side-effects on consumption of high doses. This review shows how factors like probiotic and prebiotic consumption, other CAM therapies, obesity, diet, age and the like, that alter gut consortium, are responsible for the varying conditions under which toxicity occurs and can be further studied to set-up conditions for safe oral doses. It also indicates ways to delay or quickly treat cyanide toxicity due to oral administration and, reviews conflicts on amygdalin's anti-cancer abilities, dose levels, mode of administration and pharmacokinetics that have hindered its official acceptance at a therapeutic level.     

A β-glucosidase in feline kidney that hydrolyzes amygdalin (Laetrile)

A β-glucosidase has been demonstrated in cat, rat, and rabbit kidney tissue that catalyzes the hydrolytic cleavage of the terminal glucose residue of amygdalin (mandelonitrile-β-gentiobioside). The enzyme was partially purified from feline kidney and its properties were determined. Although the natural substrates for the enzyme are unknown at this time, this β-glucosidase requires an aryl or unsaturated alkyl aglycone moiety for catalytic function. The unusual species and tissue distribution of the enzyme have considerable implications for cancer chemotherapeutic trials with amygdalin (Laetrile).     

Immobilization of β-glucosidase from different sources on nylon tube

Cellulase from four different fungi and β-glucosidase from almonds were immobilized on the inner surface of nylon tubing. The highest values of β-glucosidase activity retention on the support were obtained when P. funiculosum and N. crassa were used as the enzyme source. A comparative study of the thermal stability referring to β-glucosidase activity was developed using free and immobilized enzymes. The most stable β-glucosidases (from P. funiculosum and A. niger) did not show an appreciable change in its thermal stability after immobilization. An important increase in thermal stability was observed when less stable β-glucosidases (from T. reesei, N. crassa and almonds) were immobilized.     

Bitterness in almonds

Bitterness in almond (Prunus dulcis) is determined by the content of the cyanogenic diglucoside amygdalin. The ability to synthesize and degrade prunasin and amygdalin in the almond kernel was studied throughout the growth season using four different genotypes for bitterness. Liquid chromatography-mass spectrometry analyses showed a specific developmentally dependent accumulation of prunasin in the tegument of the bitter genotype. The prunasin level decreased concomitant with the initiation of amygdalin accumulation in the cotyledons of the bitter genotype. By administration of radiolabeled phenylalanine, the tegument was identified as a specific site of synthesis of prunasin in all four genotypes. A major difference between sweet and bitter genotypes was observed upon staining of thin sections of teguments and cotyledons for beta-glucosidase activity using Fast Blue BB salt. In the sweet genotype, the inner epidermis in the tegument facing the nucellus was rich in cytoplasmic and vacuolar localized beta-glucosidase activity, whereas in the bitter cultivar, the beta-glucosidase activity in this cell layer was low. These combined data show that in the bitter genotype, prunasin synthesized in the tegument is transported into the cotyledon via the transfer cells and converted into amygdalin in the developing almond seed, whereas in the sweet genotype, amygdalin formation is prevented because the prunasin is degraded upon passage of the beta-glucosidase-rich cell layer in the inner epidermis of the tegument. The prunasin turnover may offer a buffer supply of ammonia, aspartic acid, and asparagine enabling the plants to balance the supply of nitrogen to the developing cotyledons.     

Functional cello-oligosaccharides production from the corncob residues of xylo-oligosaccharides manufacture

An integrated process has been developed, consisting of the “adsorption–separation” of cellulase enzymes to selectively remove β-glucosidase, and multi-stage enzymatic hydrolysis of corncob residues from xylo-oligosaccharides manufacture with the β-glucosidase deficient cellulase, aiming to obtain a high yield of cello-oligosaccharides production. After the “adsorption–separation” process, 79.50% of the endo-glucanase was retained in substrate, whereas 90.67% of β-glucosidase was removed with the separated liquid fraction, utilizing the different adsorbability of these enzymes to the substrate. A three-stage enzymatic hydrolysis of corncob residues with the β-glucosidase deficient cellulase was proposed in which the first, the second and the third stage were conducted for 6, 6 h and 12 h, respectively. Analysis indicated that the removal of hydrolysis products (glucose and cello-oligosaccharides) at each stage improved cello-oligosaccharides productivity and enzymatic hydrolysis yield. The cello-oligosaccharides yield and enzymatic hydrolysis yield in three-stage enzymatic hydrolysis were significantly improved to 51.78% and 75.56%, respectively, which were 36.00% and 25.10% higher than single-stage hydrolysis with original cellulase enzymes.     

Different exported proteins in E. coli show differences in the temporal mode of processing in vivo

A number of exported proteins in E. coli, both periplasmic proteins and proteins of the outer membrane, were examined to determine when removal of the “signal sequence” occurs in vivo. One protein was processed entirely cotranslationally (amp C β-lactamase) and one was processed entirely post-translationally (TEM β-lactamase). The others (maltose-binding protein, arabinose-binding protein, omp A protein, lam B protein and alkaline phosphatase) showed both modes of processing, although the amount of cotranslational processing varied considerably among the individual proteins of this class. When processing occurred cotranslationally, the proteolytic removal of the “signal” was a late event. For four of the proteins studied, processing was initiated only after the polypeptides had been elongated to approximately 80% of their full length.     

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.     

Influence of canopy fruit location on morphological,histochemical and biochemical changes in two oil olive cultivars

The influence of different irradiance conditions was evaluated under natural solar radiation by comparing well-exposed (in) and shaded fruit (out) in canopies of olive trees (Olea europaea L). Over a 2-year period, from 50 days after full bloom up to harvest time, “in” and “out” olive samples of two genotypes (“Frantoio Millennio” and “Coratina 5/19”) were periodically collected. Morphological, histochemical, and biochemical analysis were performed to study the changes on fruit morphometric traits, oil body accumulation, and β-glucosidase enzyme activity. Some parameters were modified by shading inside the canopy in which the proportion of incident photosynthetically active radiation intercepted by the crop was 47%. Shaded fruits developed at slow rate and were characterized by late darkgoing time, reduced size, with a tendency toward oblong shape. The rapid histochemical procedure proposed to estimate the oil body accumulation during fruit ripening showed that a reduced irradiance caused a decrease in oil body density. The canopy position influenced, in a different way, the β-glucosidase activity in relation to the fruit-ripening stage in both genotypes. These findings indicate that providing an adequate and uniform lighting of the olive canopy by careful choices of orchard management practices can be a key factor for several yield components.     

The mis-measure of manioc (Manihot esculenta,Euphorbiaceae)

The distinction between “bitter” and “sweet” (toxic and non-toxic) varieties of the cyanide-containing food crop manioc (Manihot esculenta, Euphorbiaceae) has a long tradition in the tropical forest areas of South and Central America where it was first cultivated. Yet this distinction has no taxonomic basis. The levels of cyanogenic glucosides found in manioc varieties not only vary widely, but do not correspond with any other known morphological or ecological feature. Nonetheless, these two “varieties” are commonly reported to have distinct geographical and cultural distributions and each is associated with a particular traditional food complex. This paper reviews the literature regarding the nature, distribution, and traditional uses of manioc varieties and concludes that (1) the geographical and cultural distribution of bitter and sweet varieties of manioc may not be as distinct as has been thought; (2) traditional categories of bitter and sweet manioc may stem more from culturally derived belief systems than from actual known levels of toxicity; and (3) the choice of complex and labor intensive processing methods usually associated with bitter manioc may not be required for detoxification but rather for the derived food products, particularly manioc flour.     

HPLC特征图谱结合含量测定的白芍及其炮制品的质量对比研究

建立白芍、炒白芍、酒白芍、硫熏白芍HPLC特征图谱,并结合多成分含量测定,为白芍、炒白芍、酒白芍和硫熏白芍的质量控制提供参考。采用Intersustain C₁₈(250 mm×4.6 mm,5μm)色谱柱,流动相为乙腈-0.1%醋酸水溶液,流速为每分钟1 mL,梯度洗脱,检测波长为230 nm,柱温为30℃,进样量为10μL。14批白芍、炒白芍、酒白芍和硫熏白芍的特征图谱,标定了6个共有峰,并均被指认,分别为没食子酸、儿茶素、芍药内酯苷、芍药苷、1,2,3,4,6-五没食子酰葡萄糖和苯甲酰芍药苷,而硫熏白芍标定7个共有峰,峰7为白芍硫熏后产生;且各色谱谱峰有较好的分离,但不同炮制品特征图谱存在一定差异;含量测定结果显示,白芍炒制、酒制及硫熏后,6种成分均有不同程度的变化;借助中药色谱指纹图谱相似度评价系统和SIMCA-P13.0软件对14批白芍、炒白芍、酒白芍和硫熏白芍进行相似度和正交偏最小二乘判别(OPLS-DA)分析,所建立的白芍和炮制品及硫熏品的质量评价方法稳定性、重复性好,可用于白芍、炒白芍、酒白芍和硫熏白芍的质量控制和评价。     

High level expression of extracellular secretion of a β-glucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris

A novel β-glucosidase gene (designated PtBglu3) from Paecilomyces thermophila was cloned and sequenced. PtBglu3 has an open reading frame of 2,557 bp, encoding 858 amino acids with a calculated molecular mass of 90.9 kDa. The amino acid sequence of the mature polypeptide shared the highest identity (70%) to a glycoside hydrolase (GH) family 3 characterized β-glucosidase from Penicillium purpurogenum. PtBglu3 without the signal peptides was cloned into pPIC9K vector and successfully expressed in Pichia pastoris as an active extracellular β-glucosidase (PtBglu3). High activity of 274.4 U/ml was obtained by high cell-density fermentation, which is by far the highest reported yield for β-glucosidase. The recombinant enzyme was purified to homogeneity with 3.3-fold purification and a recovery of 68.5%. The molecular mass of the enzyme was estimated to be 116 kDa by SDS-PAGE, and 198.2 kDa by gel filtration, indicating that it was a dimer. Optimal activity of the purified enzyme was observed at pH 6.0 and 65 °C, and it was stable up to 60 °C. The enzyme exhibited high specific activity toward pNP-β-D-glucopyranoside, cellooligosaccharides, gentiobiose, amygdalin and salicin, and relatively lower activity against lichenan and laminarin. The present results should contribute to improving industrial production of β-glucosidase.     

Acid β-Glucosidase: Enzymology and Molecular Biology of Gaucher Diseas

Abstract

Human lysosomal β-glucosidase (D-glucosyl-acylsphingo-sine glucohydrolase, EC 3.2.1.45) is a membrane-associated enzyme that cleaves the β-glucosidic linkage of glucosylcer-amide (glucocerebroside), its natural substrate, as well as synthetic β-glumsides. Experiments with cultured cells suggest that in vivo this glycoprotein requires interaction with negatively charged lipids and a small acidic protein, SAP-2, for optimal glucosylceramide hydrolytic rates. In vitro, detergents (Triton? X-100 or bile acids) or negatively charged gangliosides or phos-pholipids and one of several “activator proteins” increase hydrolytic rate of lipid and water-soluble substrates. Using such in vitro assay systems and active site-directed covalent inhibitors, kinetic and structural properties of the active site have been elucidated. The defective activity of this enzyme leads to the variants of Gaucher disease, the most prevalent lysosomal storage disease. The nonneuronopathic (type 1) and neuronopathic (types 2 and 3) variants of this inherited (autosomal recessive) disease but panethnic, but type 1 is most prevalent in the Ashkenazi Jewish population. Several missense mutations, identified in the structural gene for lysosomal β-glucosidase from Gaucher disease patients, are presumably casual to the specifically altered post-translational oligosaccharide processing or stability of the enzyme as well as the alterecA in vitro kinetic properties of the residual enzyme from patient tissues.     

Immobilization of β-glucosidase in fixed bed reactor and evaluation of the enzymatic activity

Adsorption of β-glucosidase from almonds, an enzyme with big molecular size (130?kDa, 6.7?nm molecular diameter), on mesoporous SBA-15 silica in fixed bed column was studied. Previously, zeta potential analysis confirmed that the electrostatic interactions between β-glucosidase and SBA-15 were the driving force of the immobilization process. The maximum difference in the zeta potential was 25?mV at pH 3.5. Adsorption isotherm was classified as an L3 (Langmuir type 3) curve according to the Giles classification and fitted to a double Langmuir equation. The adsorbed amount in a fixed bed column was around 3.5 times higher than the amount reached in the adsorption in batch. In addition, the β-glucosidase was strongly immobilized on SBA-15 with only 7?% of leaching in the washing step with buffer solution. Immobilized β-glucosidase was catalytically active in a continuous process, reaching 100?% substrate conversion and maintaining this activity level for more than 10?h without deactivation of the enzyme. Adsorption-desorption isotherms at 77?K before and after the adsorption were carried out, concluding that the adsorption of β-glucosidase was produced blocking the pore mouth, so that a part of the enzyme penetrates inside and another part stays outside the pore.     

Acrylamide in processed potato products: progress made and present status

Concerns related to higher levels of acrylamide in processed carbohydrate-rich foods, especially in fried potato products, are well known. This article provides updates on various aspects of acrylamide in processed potato products including mechanisms of acrylamide formation and health risks due to its intake. Levels of reducing sugars in potatoes are considered as a main factor contributing towards the formation of acrylamide in processed potato products. Useful approaches in lowering the levels of reducing sugars such as use of suitable varieties, storage methods, storage temperature and duration of storage are described and discussed. Importance and practical utility of various steps before and during the processing that can contribute in reducing the final concentration of acrylamide are highlighted. Progress made and present status of potato processing industry in India are part of this article. The article describes varietal improvement and spread of short-term and long-term storage technologies in India and their contribution towards round the year availability of processing-grade potatoes to the processing industries and how all this has helped in achieving reduced levels of acrylamide in chips and French fries. Outcome and implications of cold-induced sweetening tolerance in potatoes are presented along with other management practices and strategies that can lower the acrylamide levels in processed potato products. Future lines of work have been suggested to make the consumption of fried potato products safer.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.     

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.     

Indole alkaloid biosynthesis: Partial purification of “ajmalicine synthetase” from Catharanthus roseus

The “ajmalicine synthetase” system of $\text{Catharanthus roseus}$ has been partially purified from callus, seedlings and mature plants. On gel filtration of the cell-free extract, four β-D-glucosidase isozymes were observed in seedlings and plants. Only two were present in the callus. A protein peak at 55,000 daltons in all three materials was capable of synthesizing ajmalicine from tryptamine and secologanin in the presence of NADPH. This “ajmalicine synthetase” rapidly lost its ability to synthsize ajmalicine, but retained the β-glucosidase activity.     

β-Glucosidase activities and HCN liberation in unimbibed and imbibed seeds, and the induction of cocklebur seed germination by cyanogenic glycosides

In many seed species, the major source of HCN evolved during water imbibition is cyanogenic glycosides. The present investigation was performed to elucidate the role of endogenous cyanogenic glycosides in the control of seed germination and to examine the involvment of β-glucosidase in this process. All seed species used here contained some activities of β-glucosidase already in the dry state before imbibition. in the decreasing order of Malus pumila, Daucus carota, Hordeum vulgare, Chenopodium album and so on. β-Gluosidase activity in upper and lower seeds of cocklebur (Xanthium pennsylvanicum Wallr.) decreased with imbibition, and in lower seeds the activity disappeared when they germinated. On the contrary, in caryopses of rice (Oryza sativa L. cv. Sasanishiki) β-glucosidase increased during imbibition, and this increase continued even after germination. β-Glucosidase in cocklebur seeds was more active in the axial than in the cotyledonary tissue. Amygdalin, prunasin and linamarin could all serve as substrattes for the β-glucosidase(s) from both cocklebur and rice. Amygdalin, prunasin and linamarin as well as KCN, were effective in stimulating the germination of upper cocklebur seeds. The seeds evolved much more free HCN gas when they were exposed to the cyanogenic glycosides than when the glycosides were absent. Moreover, the application of the cyanogenic glycosides or of KCN caused accumulation of bound HCN in the seeds. Carbon monoxide, which stimulated cocklebur seed germination only slightly, did not cause accumulation of bound HCN. We suggest that a balance between the cytochrome and the alternative respiration pathways, which is adequate for germination (Esashi et al. 1987. Plant Cell Physiol. 28: 141–150), may be brought about by the action of endogenous HCN; a large portion of which is liberated from cyanogenic glycosides via the action of β-glucosidase. In addition to the partial suppression of the cytochrome path and unlike carbon monoxide, the HCN thus produced may act to supply cyanide group(s) to unknown compounds necessary for germination.     

Impact of processing conditions on the phytoprostanes profile of three types of nut kernels

The goal of this work was the identification and quantification of phytoprostanes (PhytoPs) in three types of nuts: “Walnut”, “Macadamia”, and “Pecan”. This study represents a first approach to the relationship between the quantitative and qualitative PhytoP profiles in the “Macadamia” and “Pecan” nuts subjected to fried salt or fried honey processing. The kernels were found to contain 9-F_1t-PhytoP, 9-epi-9-F_1t-PhytoP, 16-B₁-PhytoP, ent-16-B₁-PhytoP, 9-L₁-PhytoP, and ent-9-L₁-PhytoP. “Macadamia” fried salt nuts were the only ones that produced 9-epi-9-D₁-PhytoP and 9-D₁-PhytoP. The total PhytoP concentration in raw nuts was in the range of 5541–7830?ng kg^?1 fresh weight (FW); for most of the PhytoPs, the concentrations were lowest in raw walnuts, indicating that concentration of each PhytoP was influenced by the genotype. The frying process increased the total PhytoPs concentration to the range of 8903–33,727?ng kg^?1 FW. Therefore, this is the first work describing PhytoPs in nuts and reinforces the capacity of these compounds to act as biomarkers to monitor the processing treatments that influence the final quality of nuts.