Effects of a Lepidium sativum enzyme preparation on the degradation of glucosinolates

The effects of a crude enzyme extract prepared from Lepidium sativum seeds, on the degradation of three pure glucosinolates (allyl-, benzyl- and 2-phenethyl-) were investigated in the presence of the known enzyme co-factor, ascorbic acid. Isothiocyanates and nitriles were obtained but no thiocyanates. For maximum isothiocyanate formation there was an optimum concentration of ascorbic acid which varied directly with the concentration of substrate but was independent of the particular glucosinolate. Formation of isothiocyanate from any glucosinolate was linear with time but enzymic production of 2-phenethyl isothiocyanate was activated by ascorbic acid to a greater extent than for the other two glucosinolates studied. Isothiocyanate was still the major product even at low pH although the thioglucosidase was only weakly active. Nitrile formation was always erratic in the presence of ascorbic acid. In the absence of ascorbic acid thioglucosidase was still active although to a much lesser extent, but in these circumstances benzyl thiocyanate was an additional product. There is thus a thiocyanate-forming factor in the extract of L. sativum seeds which is inactivated in the presence of ascorbic acid. This factor did not cause the formation of thiocyanate from 2-phenethylglucosinolate.     

Studies on glucosinolate degradation in Lepidium sativum seed extracts

Analysis of Lepidium sativum seeds showed the presence of allyl, 2-phenethyl and benzyl glucosinolates, the first two being reported for the first time from this source. The effects of temperature, pH of the extraction medium and the length of time allowed for autolysis were assessed on the benzyl glucosinolate degradation products in seed extracts. In particulàr benzyl thiocyanate was not produced at higher temperatures but at ambient and lower temperatures it exceeded isothiocyanate. Nitrile was always the major product under the conditions studied, ever at pH levels as high as 7.4. Five new possible benzyl glucosinolate degradation products were detected and evidence is presented that benzaldehyde and benzyl alcohol could be secondary products formed thermally from isothocyanate and thiocyanate, respectively. Benzyl mercaptan and benzyl methyl sulphide also appear to be thermally produced.     

Benzylglucosinolate degradation in heat-treated Lepidium sativum seeds and detection of a thiocyanate-forming factor

Lepidium sativum seeds were dry heated at 125° for varying periods, and also for 30 min at various temperatures. Autolysates were then analysed for benzylglucosinolate degradation products. Whilst heating for 4 hr 20 min at 125° was sufficient to prevent formation of benzyl thiocyanate, just over 7.5 hr at 125° was required before benzyl isothiocyanate also ceased to be produced. This indicates the presence of a discrete, thiocyanate-forming factor in L. sativum seeds, separate from thioglucosidase. After 7.5 hr at 125°, benzyl cyanide continued to be formed, proving that it can be obtained (in relatively small amounts) directly from the glucosinolate even without the influence of any thioglucosidase. In general, isothiocyanate was the more favoured product of glucosinolate degradation following heat treatment of seeds, until the point of thioglucosidase inactivation was approached when nitrile formation took over. It is suggested that the thiocyanate-forming factor is an isomerase causing Z-E isomerization of the glucosinolate aglucone, but that only those glucosinolates capable of forming particularly stable cations are then able to undergo E-aglucone rearrangement to thiocyanate.     

Effects of metal ions on benzylglucosinolate degradation in Lepidium sativum seed autolysates

The effects of varying concentrations of Fe²⁺ (5 × 10^?5 ?5 × 10^?1 M) on benzylglucosinolate degradation in Lepidium sativum seed autolysates were investigated. Increased glucosinolate decomposition was observed over the whole range with a maximum effect at ca 6 × 10^?3 M Fe²⁺, at which point glucosinolate degradation was more than three times that obtained in the absence of added Fe²⁺ . Nitrile formation was especially enhanced in the presence of all concentrations of Fe²⁺ studied, and maximum amounts were obtained at ca 6 × 10^?3 M Fe²⁺ when a more than four-fold increase over quantities produced in the absence of Fe²⁺ was observed. Thiocyanate formation was also promoted with a maximum at ca 4 × 10^?3 M Fe²⁺, but isothiocyanate production was considerably reduced in allcases. It is suggested that Fe²⁺ inhibits isothiocyanate formation by interfering with the availability of ascorbic acid which is a proven co-factor for most thioglucosidase isoenzymes, but that an Fe²⁺-ascorbate complex might then be responsible for promoting enzymic production of nitrile. The effects of a limited range of concentrations of Fe³⁺ and Cu⁺ were also studied, and results related to those for Fe²⁺. The relevance of the findings to natural systems and to glucosinolate-containing foods is briefly discussed.     

Degradation of glucosinolates of Nasturtium officinale seeds

Nasturtium officinale contains four glucosinolates, the major representative being 2-phenethylglucosinolate. On autolysis of seeds or leaves, isothiocyanates were the main products of glucosinolate degradation but no thiocyanate was detected. The application of heat during extraction caused an increase in nitrile formation to dominance over isothiocyanates. A (benzyl) thiocyanate-forming extract of Lepidium sativum seeds did not provoke generation of any thiocyanate from glucosinolates of N. officinale (or Barbarea praecox), but it did impose accentuated nitrile-forming properties on the systems. The conclusion is reached that some glucosinolate-containing Cruciferae are predominantly nitrile-producing and some predominantly isothiocyanate-producing, all other factors being constant.     

Characterization of recombinant nitrile-specifier proteins (NSPs) of Arabidopsis thaliana: Dependency on Fe(II) ions and the effect of glucosinolate substrate and reaction conditions

Glucosinolates are plant secondary metabolites that are part of a plant defence system against pathogens and pests, the myrosinase-glucosinolate system, in which glucosinolates get activated by enzymic degradation through thioglucoside glucohydrolases called myrosinases. Epithiospecifier protein (ESP) and nitrile-specifier proteins (NSPs) divert myrosinase-catalyzed hydrolysis of a given glucosinolate from the formation of isothiocyanate to that of epithionitrile and/or nitrile. As the biological activity of glucosinolate hydrolysis products varies considerably, a detailed characterization of these specifier proteins is of utmost importance to understand their biological role. Therefore, the Arabidopsis thaliana AtNSP1, AtNSP2 and AtNSP5 and a supposed ancestor protein AtNSP-like1 were expressed in Escherichia coli and the activity of the purified recombinant proteins was tested in vitro on three highly different glucosinolates and compared to that of purified AtESP. As previously reported, only AtESP showed epithiospecifier activity on 2-propenylglucosinolate. We further confirmed that purified AtNSP1, AtNSP2 and AtNSP5, but not the ancestor AtNSP-like1 protein, show nitrile-specifier activity on 2-propenylglucosinolate and benzylglucosinolate. We now show for the first time that in vitro AtNSP1, AtNSP2 and AtNSP5 are able to generate nitrile from indol-3-ylmethylglucosinolate. We also tested the effect of different Fe(II) ion concentrations on the nitrile-specifier activity of purified AtNSP1, AtNSP2 and AtNSP5 on 2-propenylglucosinolate and benzylglucosinolate. AtNSP-related nitrile production was highly dependent on the presence of Fe(II) ions in the reaction assay. In the absence of added Fe(II) ions nitriles were only detected when benzylglucosinolate was incubated with AtNSP1. While AtNSP1 also exhibited overall higher nitrile-specifier activity than AtNSP2 and AtNSP5 at a given Fe(II) ion concentration, the pattern of nitrile formation in relation to Fe(II) ion concentrations depended on the AtNSP and the glucosinolate substrate. The pH of the solution also affected the reaction outcome, with a higher proportion of nitrile being produced at the higher pH for AtNSP2 and AtNSP5.     

Differing mechanisms of simple nitrile formation on glucosinolate degradation in Lepidium sativum and Nasturtium officinale seeds

Glucosinolates are sulphur-containing glycosides found in brassicaceous plants that can be hydrolysed enzymatically by plant myrosinase or non-enzymatically to form primarily isothiocyanates and/or simple nitriles. From a human health perspective, isothiocyanates are quite important because they are major inducers of carcinogen-detoxifying enzymes. Two of the most potent inducers are benzyl isothiocyanate (BITC) present in garden cress (Lepidium sativum), and phenylethyl isothiocyanate (PEITC) present in watercress (Nasturtium officinale). Previous studies on these salad crops have indicated that significant amounts of simple nitriles are produced at the expense of the isothiocyanates. These studies also suggested that nitrile formation may occur by different pathways: (1) under the control of specifier protein in garden cress and (2) by an unspecified, non-enzymatic path in watercress. In an effort to understand more about the mechanisms involved in simple nitrile formation in these species, we analysed their seeds for specifier protein and myrosinase activities, endogenous iron content and glucosinolate degradation products after addition of different iron species, specific chelators and various heat treatments. We confirmed that simple nitrile formation was predominantly under specifier protein control (thiocyanate-forming protein) in garden cress seeds. Limited thermal degradation of the major glucosinolate, glucotropaeolin (benzyl glucosinolate), occurred when seed material was heated to >120 °C. In the watercress seeds, however, we show for the first time that gluconasturtiin (phenylethyl glucosinolate) undergoes a non-enzymatic, iron-dependent degradation to a simple nitrile. On heating the seeds to 120 °C or greater, thermal degradation of this heat-labile glucosinolate increased simple nitrile levels many fold.     

Some glucosinolates of Farsetia aegyptia and Farsetia ramosissima

Air-dried leaves of Farsetia aegyptia and F. ramosissima have been analysed for their glucosinolates; the former was shown to contain at least six but chiefly allylglucosinolate, whilst the latter contains at least five but mainly but-3-enylglucosinolate with some 4-(methylthio)butylglucosinolate. Without the addition of extraneous thioglucosidase enzyme, both species gave predominantly nitrile degradation products of glucosinolates; but if extra enzyme were added, corresponding isothiocyanates became the major products instead. Varying the pH from the natural level for the plant also considerably affected the ratios of glucosinolate products.     

The effects of pH on glucosinolate degradation by a thioglucoside glucohydrolase preparation

An active thioglucoside glucohydrolase extract was prepared from commercial mustard powder and its effect on the degradation of two pure glucosinolates was investigated. During reaction in a distilled water medium the pH of the solution decreased markedly and the ratio of products (isothiocyanate and nitrile) varied considerably. After 20–30 min, when the pH had fallen to ca 5.6, isothiocyanate production ceased whilst nitrile continued to be produced and in amounts which increased linearly with time for at least 40 min. This behaviour can be correlated with the changing pH of the medium. In controlled pH experiments it was confirmed that nitrile formation is favoured at lower pH levels and that the ratio of nitrile to isothiocyanate is directly related to the hydrogen ion concentration of the medium. No reason could therefore be found for the observed formation of nitrile in some natural systems at pHs greater than 7.     

The ‘mustard oil bomb’: not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system

Glucosinolates are plant secondary metabolites that are hydrolysed by the action of myrosinases into various products (isothiocyanates, thiocyanates, epithionitriles, nitriles, oxazolidines). Massive hydrolysis of glucosinolates occurs only upon tissue damage but there is also evidence indicating metabolism of glucosinolates in intact plant tissues. It was originally believed that the glucosinolate–myrosinase system in intact plants was stable due to a spatial separation of the components. This has been coined as the ‘mustard oil bomb’ theory. Proteins that form complexes with myrosinases have been described: myrosinase-binding proteins (MBPs) and myrosinase-associated proteins (MyAPs/ESM). The roles of these proteins and their biological relevance are not yet completely known. Other proteins of the myrosinase enzyme system are the epithiospecifier protein (ESP) and the thiocyanate-forming protein (TFP) that divert the glucosinolate hydrolysis from isothiocyanate production to nitrile/epithionitrile or thiocyanate production. Some glucosinolate hydrolysis products act as plant defence compounds against insects and pathogens or have beneficial health effects on humans. In this review, we survey and critically assess the available information concerning the localization, both at the tissular/cellular and subcellular level, of the different components of the myrosinase enzyme system. Data from the model plant Arabidopsis thaliana is compared to that from other glucosinolate-producing Brassicaceae in order to show common as well as divergent features of the ‘mustard oil bomb’ among these species.     

Evidence implicating a novel thiol methyltransferase in the detoxification of glucosinolate hydrolysis products in Brassica oleracea L.

The physiological relevance of a novel thiol methyltransferase from cabbage, and its possible role in sulphur metabolism have been investigated. The enzyme was absent from the chloroplast, the site of sulphate reduction, and was localized in the cytosol. Potential substrates were initially screened on the basis of their ability to inhibit the methylation of iodide, a previously known substrate for the enzyme. Thiocyanate, 4,4 ′ ‐thiobisbenzenethiol, thiophenol, and thiosalicylic acid were identified as possible substrates. Methylation of these thiols by the purified enzyme using [Methyl‐³H]S‐adenosyl‐ L ‐methionine confirmed their nature as substrates. The purified enzyme strongly preferred thiocyanate as a methyl acceptor. The enzyme had K_m values of 11, 51, 250 and 746 mmol m ^{? 3} for thiocyanate, 4,4 ′ ‐thiobisbenzenethiol, thiophenol and thiosalicylic acid, respectively. The identity of methylthiocyanate as the product of thiocyanate methylation by the purified enzyme was confirmed by mass spectrometry. The enzyme was strictly associated with glucosinolate‐containing plants. Thiol substrates of the enzyme are known products of glucosinolate hydrolysis. Our observations indicate that this enzyme could be involved in the detoxification of reactive thiols produced upon glucosinolate degradation in these plants.     

The myrosinase-glucosinolate system, its organisation and biochemistry

The myrosinase-glucosinolate system is involved in a range of biological activities affecting herbivorous insects, plants and fungi. The system characteristic of the order Capparales includes sulphur-containing substrates, the degradative enzymes myrosinases, and cofactors. The enzyme-catalyzed hydrolysis of glucosinolates initially involves cleavage of the thioglucoside linkage, yielding D-glucose and an unstable thiohydroximate- O -sulphonate that spontaneously rearranges, resulting in the production of sulphate and one of a wide range of possible reaction products. The products are generally a thiocyanate, isothiocyanate or nitrile, depending on factors such as substrate, pH or availability of ferrous ions. Glucosinolates in crucifers exemplify components that are often present in food and feed plants and are a major problem in the utilization of products from the plants. Toxic degradation products restrict the use of cultivated plants, e.g. those belonging to the Brassicaceae. The myrosinase-glucosinolate system may, however, have several functions in the plant. The glucosinolate degradation products are involved in defence against insects and phytopathogens. and potentially in sulphur and nitrogen metabolism and growth regulation. The compartmentalization of the components of the myrosinase-glucosinolate system and the cell-specific expression of the myrosinase represents a unique plant defence system. In this review, we summarize earlier results and discuss the organisation and biochemistry of the myrosinase-glucosinolate system.     

Supercritical fluid chromatography as a method of analysis for the determination of 4-hydroxybenzylglucosinolate degradation products

In the present study analytical and preparative supercritical fluid chromatography (SFC) were used for investigation of myrosinase catalysed degradation of 4-hydroxybenzylglucosinolate (sinalbin). Sinalbin occurs as a major glucosinolate in seeds of Sinapis alba L., in various mustards and other food products. The degradation products were identified and quantified by analysis based on a developed SFC method using a bare silica column. Determinations comprised transformation products of sinalbin, produced both during degradation of isolated sinalbin, and during autolysis of meal from S. alba seeds. The conditions in the developed SFC method were used as basis for the preparative SFC procedure applied for isolation of the components prior to their identification by nuclear magnetic resonance (NMR) spectroscopy. Myrosinase catalysed sinalbin hydrolysis resulted in the reactive 4-hydroxybenzyl isothiocyanate as an initial product at pH values from 3.5 to 7.5 whereas 4-hydroxybenzyl cyanide was one of the major products at low pH values. 4-Hydroxybenzyl isothiocyanate was found to disappear from the aqueous reaction mixtures in a few hours, as it reacted easily with available nucleophilic reagents. 4-Hydroxybenzyl alcohol was found as the product from reaction with water, and with ascorbic acid, 4-hydroxybenzylascorbigen was produced.     

Toxicity of Glucosinolates and Their Enzymatic Decomposition Products to Caenorhabditis elegans

An aquatic 24-hour lethality test using Caenorhabditis elegans was used to assess toxicity of glucosinolates and their enzymatic breakdown products. In the absence of the enzyme thioglucosidase (myrosinase), allyl glucosinolate (sinigrin) was found to be nontoxic at all concentrations tested, while a freeze-dried, dialyzed water extract of Crambe abyssinica containing 26% 2-hydroxyl 3-butenyl glucosinolate (epi-progoitrin) had a 50% lethal concentration (LC₅₀) of 18.5 g/liter. Addition of the enzyme increased the toxicity (LC₅₀ value) of sinigrin to 0.5 g/liter, but the enzyme had no effect on the toxicity of the C. abyssinica extract. Allyl isothiocyanate and allyl cyanide, two possible breakdown products of sinigrin, had an LC₅₀ value of 0.04 g/liter and approximately 3 g/liter, respectively. Liquid chromatographic studies showed that a portion of the sinigrin decomposed into allyl isothiocyanate. The results indicated that allyl isothiocyanate is nearly three orders of magnitude more toxic to C. elegans than the corresponding glncosinolate, suggesting isothiocyanate formation would improve nematode control from application of glucosinolates.     

Light-dependent reduction of hydrogen peroxide by ruptured pea chloroplasts

Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H₂O₂-dependent oxidation of ascorbate and ascorbate-dependent reduction of H₂O₂. The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This indicates that the ascorbate peroxidase is a chloroplast enzyme. The pH optimum of ascorbate peroxidase activity was 8.2 and the K_m value for ascorbate was 0.6 millimolar. Pyrogallol, glutathione, and NAD(P)H did not substitute for ascorbate in the enzyme catalyzed reaction. The enzyme was inhibited by NaN₃, KCN, and 8-hydroxyquinoline but not ZnCl₂ or iodoacetate. The ascorbate peroxidase activity of sonicated chloroplasts was inhibited by light but not in the presence of substrate concentrations of ascorbate.     

Purification and characterization of pea cytosolic ascorbate peroxidase

The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves.     

Thermal degradation of glucosinolates

Three glucosinolates (allyl-, benzyl- and 2-phenethyl-) were shown to degrade thermally in a GC column to yield products identical with those obtained conventionally on enzymic decomposition, namely nitriles and isothiocyanates. Nitriles were formed more readily at 125° but the facility for isothiocyanate production varied slightly with the glucosinolate; 2-phenethylglucosinolate was the most labile of those studied yielding isothiocyanate at a column temperature of 150°. Temperature was confirmed as the cause of degradation by isolated heated-tube experiments. The results have significance both with regard to analytical methodology for glucosinolates and their products, and with regard to furthering understanding of the mechanisms of glucosinolate degradation.     

Volatile components of cocoa with particular reference to glucosinolate products

The aroma volatiles of raw, fermented and roasted cocoa beans were extracted and concentrated to valid essences using well-established techniques. Analysis by GC and GC/MS showed at least 84 components of which 13 were identified for the first time as cocoa volatiles. In total, ca 5,66 and 65 μg of aroma components were obtained per g of raw, fermented and roasted cocoa beans, respectively. The most abundant groups of volatiles from fermented beans were alcohols (ca40%w/w of the total volatiles) and esters (ca 32%), whilst those from roasted beans were pyrazines (ca 40%) and aldehydes (ca 23%). Trimethyl- and tetramethylpyrazine were also detected in fermented beans, and it is suggested that they contribute to the noticeable cocoa/chocolate aroma of fermented unroasted beans. Phenylacetonitrile, benzyl isothiocyanate and benzyl thiocyanate were all identified amongst cocoa volatiles, together showing the presence of precursor benzylglucosinolate in cocoa. Glucosinolate products were detected in roasted beans, and it seems likely that the enzyme thioglucoside glucohydrolase survived the conditions of roasting. Benzyl thiocyanate was detected only in raw beans, showing that the glucosinolate ‘thiocyanate–forming factor’ did not withstand conditions of fermentation