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.     

Glucosinolate profiling of Brassica rapa cultivars after infection by Leptosphaeria maculans and Fusarium oxysporum

The glucosinolate contents of two different cultivars of Brassica rapa (Herfstraap and Oleifera) infected with Leptosphaeria maculans and Fusarium oxysporum were determined. Infection triggered the accumulation of aliphatic glucosinolates (gluconapin, progoitrin, glucobrassicanapin and gluconapoleiferin) and indole glucosinolate (4-hydroxy-glucobrassicin) in Herfstraap and of two indole glucosinolates (glucobrassicin and 4-hydroxy-glucobrassicin) in Oleifera. While total and aliphatic glucosinolates decreased significantly in Oleifera, a large increase was observed in Herfstraap after fungal infection. The indole glucosinolate glucobrassicin accumulated in Oleifera at a higher rate than Herfstraap especially after infection with F. oxysporum. Apparently the interaction between fungus and B. rapa is cultivar and fungal species specific.     

Seasonal variation in the glucosinolate content of North American Brassica nigra and Dentaria species

Concentrations of glucosinolates in the leaves of the woodland cruciferous herbs Dentaria laciniata, D. diphylla and D. maxima declined during the 1974 growing season but generally equalled or exceeded the levels found in the foliage of Brassica nigra, a crucifer of neighbouring open habitats. The superior quality of Dentaria foliage for certain crucifer-feeding insects does not seem to result from an unusually low concentration of glucosinolates. The qualitative and quantitative compositions of glucosinolates in the leaves and rhizomes of D. maxima were found to be intermediate between the corresponding values for D. laciniata and D. diphylla. This finding is consistent with a hybrid origin for D. maxima.     

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.     

Isolation of glucosinolates and the identification of o-(α-l-rhamnopyranosyloxy)benzylglucosinolate from Reseda odorata

A method has been developed for the quantitative isolation of glucosinolates by ion-exchange chromatography and high voltage electrophoresis avoiding strongly alkaline and acidic conditions. The compounds were identified by ¹H and ¹³C NMR spectroscopy and through the products arising from enzymatic, acid and alkaline hydrolysis. The method is well suited for the isolation and identification of glucosinolates containing aglucone parts which produce non-volatile compounds on enzymatic hydrolysis. The method has been used in the isolation and identification of 2-hydroxy-2-methylpropylglucosinolate from Reseda alba, 2-hydroxy-2-phenylethylglucosinolate from R. luteola and a new glucosinolate, o-(α-l-rhamnopyranosyloxy)benzylglucosinolate, occurring in R. odorata. The glucosinolate content in different parts of this plant has been determined and the metabolism of glucosinolates is briefly discussed.     

Glucosinolates and amines in Reseda media

The content of glucosinolates and amines in green parts of Reseda media has been investigated. Benzylglucosinolate, 2-phenethylglucosinolate, and m-hydroxybenzylglucosinolate occur in appreciable amounts accompanied by minor amounts of other glucosinolates, benzylamine and m-hydroxybenzylamine. Isolation and identification of these compounds was made using ion-exchange chromatography, high voltage electrophoresis, GC, MS, and ¹³C-NMR spectroscopy. The glucosinolates were transformed into corresponding nitriles and isothiocyanates by thioglucoside glucohydrolase-catalysed hydrolysis and to the corresponding carboxylic acids by acid-catalysed hydrolysis. The content of glucosinolates and amines in leaves and inflorescences of R. media has been determined by UV-spectroscopy and GC.     

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.     

Glucosinolates of egyptian Capparis species

Capparis ovata var. palaestina Zoh., C. spinosa var. aegyptia Boiss. and C. spinosa var. deserti Zoh., were investigated for glucosinolates. Glucoiberin, glucocapparin, sinigrin, glucocleomin, glucocapangulin, glucobrassicin and neoglucobrassicin, in addition to two others, were isolated. Four of these viz. glucoiberin, sinigrin, glucobrassicin and neoglucobrassicin were detected for the first time in Capparis species. Comparative chromatographic analyses of the glucosinolates of the plants examined revealed qualitative differences.     

Seed glucosinolates of fourteen wild Brassica species

Seed glucosinolates have been determined for 14 wild Brassica species, by micro-scale GC analysis of silylated derivatives. Of these, 12 were investigated for the first time. The majority of taxa exhibited high alkenylglucosinolate levels, although prop-2-enylglucosinolate appears to be generally absent. Other known methionine-derived glucosinolates predominate in B. tournefortii, B. elongata and B. deflexa. Phenylalanine-derived 4-hydroxybenzylglucosinolate is characteristic of section Brassicaria plants and represents the first finding of this glucosinolate in authenticated Brassica material.     

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.     

Lunularic acid decarboxylase from the liverwort Conocephalum conicum

Some properties of a preparation of an enzyme, lunularic acid decarboxylase, from the liverwort Conocephalum conicum are described. The enzyme is normally bound and could be solubilized with Triton X-100; at least some of the bound decarboxylase activity appears to be associated with chloroplasts. For lunularic acid the enzyme has K_m 8.7 × 10^?5 M (pH 7.8 and 30°). Some substrate analogues have been tested but no other substrate was found. Pinosylvic acid is a competitive inhibitor for the enzyme, K_i 1.2 × 10^?4 M (pH 7.8 and 30°). No product inhibition was observed. Lunularic acid decarboxylase activity has also been observed with a cell-free system from Lunularia cruciata.     

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.     

High-performance liquid chromatography-based method to evaluate kinetics of glucosinolate hydrolysis by Sinapis alba myrosinase

Isothiocyanates (ITCs) are one of several hydrolysis products of glucosinolates, plant secondary metabolites that are substrates for the thioglucohydrolase myrosinase. Recent pursuits toward the development of synthetic non-natural ITCs have consequently led to an exploration of generating these compounds from non-natural glucosinolate precursors. Evaluation of the myrosinase-dependent conversion of select non-natural glucosinolates to non-natural ITCs cannot be accomplished using established ultraviolet–visible (UV–Vis) spectroscopic methods. To overcome this limitation, an alternative high-performance liquid chromatography (HPLC)-based analytical approach was developed where initial reaction velocities were generated from nonlinear reaction progress curves. Validation of this HPLC method was accomplished through parallel evaluation of three glucosinolates with UV–Vis methodology. The results of this study demonstrate that kinetic data are consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis–Menten and specific activity analyses. Consequently, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports.     

Distribution and chemotaxonomic significance of glucosinolates in certain Middle-Eastern cruciferae

Eighty-four collections of 51 species of Middle-eastern Cruciferae were analysed by paper and gas-liquid chromatography for seed glucosinolates. Thirty-two species were investigated for the first time. Thirty compounds were identified and three unknowns of limited occurrence were found, p-Hydroxybenzylglucosinolate was found in six species of various tribes. 4-Methylsulfinyl-3-butenyl, 3-methoxycarbonylpropyl, and p-rhamnopyranosyloxybenzyl glucosinolates, which are rare in the Cruciferae, were found in Sinapis aucheri two species of Erysimum and Thlaspi perfoliatum, respectively. The biosynthetic precursors of all but one of the 30 glucosinolates are protein amino acids and at least 20 glucosinolates are derived from methionine. The distribution of glucosinolates is very useful at the species and genus levels but may provide only minimal support to the tribal alliance of certain genera. It supports the removal of Sinapis aucheri from the genus, the recognition of Fibigia clypeata and F. macrocarpa as distinct species, and the alliance of Ochthodium with Euclidium and Arabis with Drabopsis.     

ω-Methylthioalkylglucosinolates and some oxidized congeners in seeds of Erysimum rhaeticum

The glucosinolates in seeds of Erysimum rhaeticum Schleich. ex DC. have been identified by structure analysis of their sulfur-containing enzymic hydrolysis products, comprising 5-methylthiopentyl and 6-methylthiohexyl isothiocyanate, the corresponding 3-hydroxylated isothiocyanates, and the sulfoxides and sulfones of the latter. The phytochemical results are evaluated in terms of their possible taxonomic significance.     

The Effects of Glucosinolates and Their Breakdown Products on Necrotrophic Fungi

Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.     

Pro-tRNA synthetase from Phaseolus aureus and Delonix regia

Pro-tRNA synthetase from Phaseolus aureus was photoinactivated in the presence of methylene blue or rose bengal. Pro and several imino acid analogues protected the enzyme against dye-mediated photoinactivation but ATP was ineffective. Together with kinetic data, this evidence suggested that a His-residue near the Pro-binding site was involved in the enzyme reaction. In the absence of methylene blue, Phaseolus enzyme was stable to light whilst that from Delonix was rapidly and reversibly photoinactivated. ATP as well as Pro, protected the Delonix enzyme against dye-independent photoinactivation. In the presence of methylene blue, the Delonix enzyme was more rapidly photoinactivated than in the absence of the dye. p-Chloromercuribenzoate (pCMB)-inhibited enzyme from both Phaseolus and Delonix was reactivated by sulphydryl reducing reagents. Reactivation of Delonix enzyme was markedly temperature-dependent whilst Phaseolus enzyme was reactivated equally efficiently at all temperatures tested. ATP, tRNA, Pro and several analogues of Pro, protected both the Phaseolus and Delonix enzymes against pCMB inhibition. The possible roles of the His-residue and SH group are discussed in relation to the known differences in substrate specificity between the Phaseolus and Delonix enzymes.     

The effect of enzyme addition on anaerobic digestion of JoseTall Wheat Grass

The effects of the addition of enzyme products containing cellulase, hemicellulase, and β-glucosidase to anaerobic digestion systems were studied using JoseTall Wheat Grass (wheat grass) as a model substrate. Anaerobic digestion tests were performed using batch reactors operated at 50 °C. The application of enzyme products in three digestion configurations were simulated and investigated: (1) enzyme addition to a single-stage digester, (2) pre-treatment of wheat grass with enzymes followed by a single-stage anaerobic digestion, and (3) enzyme addition to the first stage (hydrolysis and acidification) of a two-stage digestion system. The enzyme products showed positive effects on the solubilization of wheat grass when used alone to treat the wheat grass. However, no significant differences in biogas and methane yields, and volatile solids reduction resulted when the enzyme products were tested in the anaerobic digestion systems. This reveals that the microorganisms present in the inoculum were effective in carrying out the digestion of wheat grass. The types of microorganisms present in the inoculum were identified using 16S rRNA sequence analysis. A comparison of the sequences between the different inocula revealed that the prevalent operational taxonomic units were similar, but that the acidified inoculum contained a higher percentage of the species Thermotogae.     

Catalytic properties of the expressed acyclic carotenoid 2-ketolases from Rhodobacter capsulatus and Rubrivivax gelatinosus

Purple photosynthetic bacteria synthesize the acyclic carotenoids spheroidene and spirilloxanthin which are ketolated to spheroidenone and 2,2′-diketospirilloxanthin under aerobic growth. For the studies of the catalytic reaction of the ketolating enzyme, the crtA genes from Rubrivivax gelatinosus and Rhodobacter capsulatus encoding acyclic carotenoid 2-ketolases were expressed in Escherichia coli to functional enzymes. With the purified enzyme from the latter, the requirement of molecular oxygen and reduced ferredoxin for the catalytic activity was determined. Furthermore, the putative intermediate 2-HO-spheroidene was in vitro converted to the corresponding 2-keto product. Therefore, a monooxygenase mechanism involving two consecutive hydroxylation steps at C-2 were proposed for this enzyme. By functional pathway complementation studies in E. coli and enzyme kinetic studies, the product specificity of both enzymes were investigated. It appears that the ketolases could catalyze most intermediates and products of the spheroidene and spirilloxanthin pathway. This was also the case for the enzyme from Rba. capsulatus from which spirilloxanthin synthesis is absent. In general, the ketolase of Rvi. gelatinosus had a better specificity for spheroidene, HO-spheroidene and spirilloxanthin as substrates than the ketolase from Rba. capsulatus.     

Thermal stabilities of membrane-bound,solubilized, and artificially immobilized hydrogenase from Chromatium vinosum

Thermal inactivation (at 65 °C) of both membrane-bound and solubilized hydrogenase from Chromatium vinosum has been studied. Membrane binding greatly increases thermostability relative to the solubilized enzyme. Covalent attachment (but not simple adsorption) of the solubilized enzyme to ω-NH₂-alkyl-agaroses sharply increases thermal stability, which almost attains that of membrane-bound hydrogenase. It appears that there are at least two requirements for such stabilization—the enzyme should be in a hydrophobic medium and should be firmly bound to a hydrophobic support.