Myrosinase Activity Research Articles

Cloning of genes related to aliphatic glucosinolate metabolism and the mechanism of sulforaphane accumulation in broccoli sprouts under jasmonic acid treatment.

Cytochrome P450 79F1 (CYP79F1), cytochrome P450 83A1 (CYP83A1), UDP-glucosyltransferase 74B1 (UGT74B1), sulfotransferase 18 (ST5b) and flavin-containing monooxygenase GS-OX1 (FMOGS - OX1 ) are important enzymes in aliphatic glucosinolate biosynthesis. In this study, their full-length cDNA in broccoli was firstly cloned, then the mechanism of sulforaphane accumulation under jasmonic acid (JA) treatment was investigated. The full-length cDNA of CYP79F1, CYP83A1, UGT74B1, ST5b and FMOGS - OX1 comprised 1980, 1652, 1592, 1378 and 1623 bp respectively. The increase in aliphatic glucosinolate accumulation in broccoli sprouts treated with JA was associated with elevated expression of genes in the aliphatic glucosinolate biosynthetic pathway. Application of 100 µmol L(-1) JA increased myrosinase (MYR) activity but did not affect epithiospecifier protein (ESP) activity in broccoli sprouts, which was supported by the expression of MYR and ESP. Sulforaphane formation in 7-day-old sprouts treated with 100 µmol L(-1) JA was 3.36 and 1.30 times that in the control and 300 µmol L(-1) JA treatment respectively. JA enhanced the accumulation of aliphatic glucosinolates in broccoli sprouts via up-regulation of related gene expression. Broccoli sprouts treated with 100 µmol L(-1) JA showed higher sulforphane formation than those treated with 300 µmol L(-1) JA owing to the higher glucoraphanin content and myrosinase activity under 100 µmol L(-1) JA treatment. © 2016 Society of Chemical Industry.

Identification and Evolution of Functional Alleles of the Previously Described Pollen Specific Myrosinase Pseudogene AtTGG6 in Arabidopsis thaliana.

Myrosinases are β-thioglucoside glucohydrolases and serve as defense mechanisms against insect pests and pathogens by producing toxic compounds. AtTGG6 in Arabidopsis thaliana was previously reported to be a myrosinase pseudogene but specifically expressed in pollen. However, we found that AlTGG6, an ortholog to AtTGG6 in A. lyrata (an outcrossing relative of A. thaliana) was functional, suggesting that functional AtTGG6 alleles may still exist in A. thaliana. AtTGG6 alleles in 29 A. thaliana ecotypes were cloned and sequenced. Results indicate that ten alleles were functional and encoded Myr II type myrosinase of 512 amino acids, and myrosinase activity was confirmed by overexpressing AtTGG6 in Pichia pastoris. However, the 19 other ecotypes had disabled alleles with highly polymorphic frame-shift mutations and diversified sequences. Thirteen frame-shift mutation types were identified, which occurred independently many times in the evolutionary history within a few thousand years. The functional allele was expressed specifically in pollen similar to the disabled alleles but at a higher expression level, suggesting its role in defense of pollen against insect pests such as pollen beetles. However, the defense function may have become less critical after A. thaliana evolved to self-fertilization, and thus resulted in loss of function in most ecotypes.

Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3) β-Glucosidase with Myrosinase Activity from a Citrobacter Strain Isolated from Soil.

A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin as sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and gel filtration to give a pure protein of approximately 66 kDa. The N-terminal amino acid and internal peptide sequence of the purified protein were determined and used to identify the gene, which, based on InterPro sequence analysis, belongs to the family GH3, contains a signal peptide, and is a periplasmic protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out using protein extracts from cell-free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 mmol dm(-3) min(-1) mg(-1), respectively, with sinigrin as substrate. The optimum temperature and pH for enzyme activity were 25 °C and 6.0, respectively. The enzyme was marginally activated with ascorbate by a factor of 1.67.

Broccoli: An Insight into Formulation and Patentability Aspects

Broccoli is an edible plant which has many medicinal advantages. Chemical constituent of broccoli include sulforaphane, glucoiberin, glucoraphanin, carotenoids, myrosinase and vitamins, Sulforaphane being the most useful one. Sulforaphane is an isothiocyanate which is formed due to the activity of enzyme myrosinase on glucoraphanin. Broccoli can be used for treatment of cancer, skin disease, arthritis, diabetes, kidney disease, cholesterol, etc. This article highlights the global scenario of nutraceuticals and the present available formulations made from broccoli. This article also gives a brief knowledge about formulations of broccoli that have been researched. Research has been done but presently there are very less formulations that are available to the people. With the current growing populations and health trends, need for nutraceuticals like broccoli based products is increasing.

Editorial: Physiology and Cellular Mechanisms of Isothiocyanates and Other Glucosinolate Degradation Products in Plants.

Editorial: Physiology and Cellular Mechanisms of Isothiocyanates and Other Glucosinolate Degradation Products in Plants.

Enhancing growth, phytochemical constituents and aphid resistance capacity in cabbage with foliar application of eckol – a biologically active phenolic molecule from brown seaweed

Although foliar application of seaweed extracts on plant growth and development has and is extensively studied, reliable knowledge and understanding of the mode of action of particular compound(s) responsible for enhancing plant growth is lacking. A brown seaweed Ecklonia maxima is widely used commercially as a biostimulant to improve plant growth and crop protection. Eckol, a phenolic compound isolated from E. maxima has recently shown stimulatory effects in maize, indicating its potential use as a plant biostimulant. Cabbage is a widely cultivated vegetable crop throughout the world, which requires high input of fertilizers and is susceptible to several aphid borne diseases. This study was conducted to evaluate the effect of foliar application of eckol on the growth, phytochemical constituents and myrosinase activity (aphid resistance capacity) of commercially cultivated cabbage. Foliar application of eckol (10(-6) M) significantly enhanced shoot and root length, shoot and root fresh and dry weight, leaf area and leaf number. This treatment also showed a significant increase in photosynthetic pigments (chlorophyll 'a', chlorophyll 'b', total chlorophyll and carotenoid) compared to the untreated plants. The levels of protein, proline and iridoid glycosides were significantly higher in cabbage leaves with eckol treatment. All the control plants were severely infested with cabbage aphid (Brevicoryne brassicae) but no infestation was observed on the eckol-sprayed plants, which can be attributed to an increase in myrosinase activity. This study reveals dual effects (plant growth promoting and insect repelling) of eckol on cabbage plants that need further investigations both under field conditions and in other brassicaceous species.

Sulforaphane Bioavailability from Glucoraphanin-Rich Broccoli: Control by Active Endogenous Myrosinase.

Glucoraphanin from broccoli and its sprouts and seeds is a water soluble and relatively inert precursor of sulforaphane, the reactive isothiocyanate that potently inhibits neoplastic cellular processes and prevents a number of disease states. Sulforaphane is difficult to deliver in an enriched and stable form for purposes of direct human consumption. We have focused upon evaluating the bioavailability of sulforaphane, either by direct administration of glucoraphanin (a glucosinolate, or β-thioglucoside-N-hydroxysulfate), or by co-administering glucoraphanin and the enzyme myrosinase to catalyze its conversion to sulforaphane at economic, reproducible and sustainable yields. We show that following administration of glucoraphanin in a commercially prepared dietary supplement to a small number of human volunteers, the volunteers had equivalent output of sulforaphane metabolites in their urine to that which they produced when given an equimolar dose of glucoraphanin in a simple boiled and lyophilized extract of broccoli sprouts. Furthermore, when either broccoli sprouts or seeds are administered directly to subjects without prior extraction and consequent inactivation of endogenous myrosinase, regardless of the delivery matrix or dose, the sulforaphane in those preparations is 3- to 4-fold more bioavailable than sulforaphane from glucoraphanin delivered without active plant myrosinase. These data expand upon earlier reports of inter- and intra-individual variability, when glucoraphanin was delivered in either teas, juices, or gelatin capsules, and they confirm that a variety of delivery matrices may be equally suitable for glucoraphanin supplementation (e.g. fruit juices, water, or various types of capsules and tablets).

Abstract 913: Determination of individual isothiocyanates/indoles occurring as a result of glucosinolate degradation and their relation to biological potential of different Brassica plants

Abstract Glucosinolates (GLs), and particularly two classes of their degradation products - isothiocyanates (ITC) and indoles - released upon myrosinase catalyzed hydrolysis, belong to the most promising chemopreventive dietary components. Despite their wide use in research on chemopreventive mechanisms and industrial utilization in the production of e.g. dietary supplements, the methods of reliable characterization of myrosinase-GLs system have been underdeveloped. The biological potential ascribed to ITC and indoles, in the case of actual Brassica plant samples, is still assessed based on GL content, though it is well known that these two types of derivatives are not the only ones that are formed following myrosinase action. This means that the relationship between content and composition of bioactive Brassica ingredients and biological effect can not be properly assessed or predicted. The aim of our research was to optimize known or to develop novel methods that will ensure precise description of GLs-myrosinase system in any source, natural or processed, and to relate the rate of formation of ITC/indoles to biological effects observed. The methods optimized include: determinations of GLs by HPLC-ESI-MS [1], myrosinase activity by pH-static method [2], indoles by HPLC with fluorescent detection [3], as well as novel derivatization of ITC enabling their identification and quantitation by HPLC-DAD-MS. The developed methods were applied to characterize an array of Brassica samples and the results of chemical analyses were compared with determined cytotoxicity (MTT test), genotoxicity (comet assay) and capability of covalent DNA modification (restriction analysis [5]). GL conversion rate to ITC and indoles was found to differ significantly not only between Brassica species but also between individual plant parts and was independent of myrosinase activity. The yield of conversion of individual GLs to ITC was also not identical. The biological activity in tests used turned out to be mostly related to indoles, some of which were particularly toxic. The developed set of methods should ensure better and safer exploitation of chemopreventive potential of Brassica plants. 1. Kusznierewicz et al., J. Chromatography A, 1278: 108, 2013 2. Piekarska et al., Ind. Crops Prod., 50: 58, 2013 3. Pilipczuk et al., Food Anal. Meth., submitted 4. Pilipczuk et al., Quantitative and qualitative determination of isothiocyanates in plant extracts by HPLC after chemical derivatisation, manuscript in the final stage of preparation 5. Kolodziejski et al., Food Chem. Toxicol., 75: 118, 2015 Citation Format: Agnieszka Bartoszek, Barbara Kusznierewicz, Anna Piekarska, Dominik Kołodziejski, Tadeusz Pilipczuk, Izabela Koss-Mikołajczyk, Jacek Namieśnik. Determination of individual isothiocyanates/indoles occurring as a result of glucosinolate degradation and their relation to biological potential of different Brassica plants. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 913. doi:10.1158/1538-7445.AM2015-913

Effects of abscisic acid on glucosinolate content, isothiocyanate formation and myrosinase activity in cabbage sprouts

SummaryEffects of abscisic acid (ABA) on glucosinolate content, isothiocyanate formation and myrosinase activity in cabbage sprouts were investigated. Subsequently, their distributions in different organs were evaluated. ABA (50 μmol L−1) significantly increased glucosinolate content, isothiocyanate formation and myrosinase activity by 72.65%, 268.15% and 67.69%, respectively, in 5‐day‐old sprouts. Application of ABA alleviated the decrease rate of glucosinolate content and increased the myrosinase activity during germination. Besides, isothiocyanate formation rapidly increased after germinating for 3 days. The glucosinolate content and isothiocyanate formation reduced by 46.51% and 38.01%, respectively, in fluridon (Flu)‐treated sprouts. Cotyledon of ABA‐treated sprouts had the highest glucosinolate content and isothiocyanate formation, indicating that ABA was necessary to enhance the isothiocyanate formation in cabbage sprouts by increasing glucosinolate content and myrosinase activity.

Influence of the insecticides acetamiprid and carbofuran on arylamidase and myrosinase activities in the tropical black and red clay soils.

The objective of this study was to determine the effects of two insecticides, namely, acetamiprid and carbofuran on the enzymatic activities of arylamidase (as glucose formed from sinigrin) and myrosinase (as β-naphthylamine formed from L-leucine β-naphthylamide) in the black and red clay soils collected from a fallow groundnut (Arachis hypogaea L.) fields in the Anantapur District, Andhra Pradesh, India. The study was realized within the framework of the laboratory experiments in which the acetamiprid and carbofuran were applied to the soils at different doses (1.0, 2.5, 5.0, 7.5, 10.0 kg ha(-1)). Initially, the physicochechemical properties of the soil samples were analyzed. After 10 days of pesticide application, the soil samples were analyzed for the enzyme activities. Acetamiprid and carbofuran stimulated the arylamidase and myrosinase activities at lower concentrations after 10 days incubation. Striking stimulation in soil enzyme activities was noticed at 2.5 kg ha(-1), persists for 20 days in both the soils. Overall, higher concentrations (5.0-10.0 kg ha(-1)) of acetamiprid and carbofuran were toxic or innocuous to the arylamidase and myrosinase activities. Nevertheless, the outcomes of the present study clearly indicate that the use of these insecticides (at field application rates) in the groundnut fields (black and red clay soils) stimulated the enzyme (arylamidase and myrosinase) activities.

Effect of Se treatment on glucosinolate metabolism and health-promoting compounds in the broccoli sprouts of three cultivars

Broccoli sprouts are natural functional foods for cancer prevention because of their high glucosinolate (GSL) content and high selenium (Se) accumulation capacity. The regulation mechanism of Se on GSL metabolism in broccoli sprouts was explored. In particular, the effects of Se treatment (100μmol/L selenite and selenate) on the Se, sulfur (S), glucosinolate and sulforaphane contents; myrosinase activity and health-promoting compounds (ascorbic acid, anthocyanin, total phenolics and flavonoids) of three, 5day old, cultivars were investigated. The treatment did not influence the total GSL and ascorbic acid contents; significantly increased the myrosinase activity and sulforaphane, anthocyanin and flavonoids contents; and decreased the total phenolics content. The increase in sulforaphane during early growth can be primarily attributed to the increased myrosinase activity caused by Se treatment. Broccoli sprouts with suitable selenite and selenate concentrations, in the early growth days, could be desirable for improved human health.

Thermal and pressure stability of myrosinase enzymes from black mustard (Brassica nigra L. W.D.J. Koch. var. nigra), brown mustard (Brassica juncea L. Czern. var. juncea) and yellow mustard (Sinapsis alba L. subsp. maire) seeds

This study investigates the effects of temperature and pressure on inactivation of myrosinase extracted from black, brown and yellow mustard seeds. Brown mustard had higher myrosinase activity (2.75un/mL) than black (1.50un/mL) and yellow mustard (0.63un/mL). The extent of enzyme inactivation increased with pressure (600–800MPa) and temperature (30–70°C) for all the mustard seeds. However, at combinations of lower pressures (200–400MPa) and high temperatures (60–80°C), there was less inactivation. For example, application of 300MPa and 70°C for 10min retained 20%, 80% and 65% activity in yellow, black and brown mustard, respectively, whereas the corresponding activity retentions when applying only heat (70°C, 10min) were 0%, 59% and 35%. Thus, application of moderate pressures (200–400MPa) can potentially be used to retain myrosinase activity needed for subsequent glucosinolate hydrolysis.

Glucosinolates in collard greens grown under three soil management practices

Glucosinolates (GSLs, β-D-thioglucoside-N-hydroxysulfates) are polar compounds present in varying amounts in members of the Brassicaceae family. They suppress soil-borne pests due to the biofumigant properties of the highly toxic isothiocyanates present in Brassica vegetables. The objectives of this investigation were to: (1) assess variation in GSLs concentrations among collard plants grown under three soil management practices: sewage sludge (SS) mixed with native soil, chicken manure (CM) mixed with native soil, and no-mulch (NM) native soil, (2) quantify GSLs concentrations in collard roots, leaves, and stems at harvest for potential use of their crude extracts in plant protection, and (3) assess myrosinase activity in soil amended with CM and SS mixed with native soil. Separation of GSLs was accomplished by adsorption on a DEAE-Sephadex ion exchange resin using disposable pipette tips filled with DEAE, a weak base, with a net positive charge when ionized and exchange anions such as GSLs (hydrophilic plant secondary metabolites). Quantification of total GSLs was based on inactivation of collard endogenous myrosinase and liberation of the glucose moiety from the GSLs molecule by addition of standardized myrosinase and colorimetric determination of the liberated glucose moiety. Across all treatments, SS and CM increased soil organic matter content from 2.2% in native soil to 4.2 and 6.5%, respectively. GSLs concentrations were significantly greater in collard leaves (30.9 µmoles g−1 fresh weight) compared to roots and stems (7.8 and 1.2 µmoles g−1 fresh weight), respectively. Leaves of collard grown in soil amended with SS contained the greatest concentrations of GSLs compared to leaves of plants grown in CM and NM treatments. Accordingly, leaves of collard plants grown in soil amended with SS could play a significant role in sustainable agriculture as alternative tools for soil-borne disease management in conventional and organic agriculture.

Evaluating the impact of sprouting conditions on the glucosinolate content of Brassica oleracea sprouts

The glucosinolates content of brassica plants is a distinctive characteristic, representing a healthy advantage as many of these compounds are associated to antioxidant and anti-carcinogenic properties. Brassica sprouts are still an underutilized source of these bioactive compounds. In this work, four varieties of brassica sprouts (red cabbage, broccoli, Galega kale and Penca cabbage), including two local varieties from the North of Portugal, were grown to evaluate the glucosinolate profile and myrosinase activity during the sprouting. Also the influence of light/darkness exposure during sprouting on the glucosinolate content was assessed. Glucosinolate content and myrosinase activity of the sprouts was evaluated by HPLC methods. All sprouts revealed a higher content of aliphatic glucosinolates than of indole glucosinolates, contrary to the profile described for most of brassica mature plants. Galega kale sprouts had the highest glucosinolate content, mainly sinigrin and glucoiberin, which are recognized for their beneficial health effects. Penca cabbage sprouts were particularly richer in glucoraphanin, who was also one of the major compounds in broccoli sprouts. Red cabbage showed a higher content of progoitrin. Regarding myrosinase activity, Galega kale sprouts showed the highest values, revealing that the use of light/dark cycles and a sprouting phase of 7–9days could be beneficial to preserve the glucosinolate content of this variety.

Enhancement of glucosinolate and sulforaphane formation of broccoli sprouts by zinc sulphate via its stress effect

Broccoli sprouts have attracted much attention in recent years for their abundant phytochemicals such as glucosinolates and sulforaphane. In this study, zinc sulphate (ZnSO4) as a sulphur (S)-source was compared with potassium sulphate (K2SO4) and methionine (Met), significantly increased glucosinolates content and stimulated sulforaphane formation by enhancing myrosinase activity and gene expressions related to glucoraphanin biosynthesis and sulforaphane formation in broccoli sprouts. However, it resulted in a stress effect on sprout growth. Thus, ZnSO4 is a suitable S-source to improve sulforaphane formation in broccoli sprouts via its stress effect.

Plant defence responses in oilseed rape MINELESS plants after attack by the cabbage moth Mamestra brassicae.

The Brassicaceae family is characterized by a unique defence mechanism known as the 'glucosinolate-myrosinase' system. When insect herbivores attack plant tissues, glucosinolates are hydrolysed by the enzyme myrosinase (EC 3.2.1.147) into a variety of degradation products, which can deter further herbivory. This process has been described as 'the mustard oil bomb'. Additionally, insect damage induces the production of glucosinolates, myrosinase, and other defences. Brassica napus seeds have been genetically modified to remove myrosinase-containing myrosin cells. These plants are termed MINELESS because they lack myrosin cells, the so-called toxic mustard oil mines. Here, we examined the interaction between B. napus wild-type and MINELESS plants and the larvae of the cabbage moth Mamestra brassicae. No-choice feeding experiments showed that M. brassicae larvae gained less weight and showed stunted growth when feeding on MINELESS plants compared to feeding on wild-type plants. M. brassicae feeding didn't affect myrosinase activity in MINELESS plants, but did reduce it in wild-type seedlings. M. brassicae feeding increased the levels of indol-3-yl-methyl, 1-methoxy-indol-3-yl-methyl, and total glucosinolates in both wild-type and MINELESS seedlings. M. brassicae feeding affected the levels of glucosinolate hydrolysis products in both wild-type and MINELESS plants. Transcriptional analysis showed that 494 and 159 genes were differentially regulated after M. brassicae feeding on wild-type and MINELESS seedlings, respectively. Taken together, the outcomes are very interesting in terms of analysing the role of myrosin cells and the glucosinolate-myrosinase defence system in response to a generalist cabbage moth, suggesting that similar studies with other generalist or specialist insect herbivores, including above- and below-ground herbivores, would be useful.

Calcium mitigates the stress caused by ZnSO4 as a sulphur fertilizer and enhances the sulforaphane formation of broccoli sprouts

CaCl2 improved the growth and sulforaphane formation of broccoli sprouts under ZnSO4via enhancing key bioactive substances level, antioxidant capacity, myrosinase activity and related genes expression.

Effect of freezing methods on sulforaphane formation in broccoli sprouts

Freezing at −20 °C enhanced sulforaphane formation in broccoli sproutsviaincreasing myrosinase activity and decreasing sulforaphane nitrile formation.

The significance of glucosinolates for sulfur storage in Brassicaceae seedlings.

Brassica juncea seedlings contained a twofold higher glucosinolate content than B. rapa and these secondary sulfur compounds accounted for up to 30% of the organic sulfur fraction. The glucosinolate content was not affected by H2S and SO2 exposure, demonstrating that these sulfur compounds did not form a sink for excessive atmospheric supplied sulfur. Upon sulfate deprivation, the foliarly absorbed H2S and SO2 replaced sulfate as the sulfur source for growth of B. juncea and B. rapa seedlings. The glucosinolate content was decreased in sulfate-deprived plants, though its proportion of organic sulfur fraction was higher than that of sulfate-sufficient plants, both in absence and presence of H2S and SO2. The significance of myrosinase in the in situ turnover in these secondary sulfur compounds needs to be questioned, since there was no direct co-regulation between the content of glucosinolates and the transcript level and activity of myrosinase. Evidently, glucosinolates cannot be considered as sulfur storage compounds upon exposure to excessive atmospheric sulfur and are unlikely to be involved in the re-distribution of sulfur in B. juncea and B. rapa seedlings upon sulfate deprivation.

Influence of Temperature, Glucose, and Iron on Sinigrin Degradation by Salmonella and Listeria monocytogenes

Factors, including pH, temperature, glucose concentration, and iron compounds, affect the activity of plant myrosinase and, consequently, endogenous glucosinolate degradation. These factors also may affect glucosinolate degradation by bacterial myrosinase. Therefore, this study examined the effect of temperature (4 to 21°C), glucose (0.05 to 1.0%), and iron (10 mM ferrous or ferric) on sinigrin degradation by Salmonella or Listeria monocytogenes cocktails in Mueller-Hinton broth and the effect of sinigrin degradation on bacterial viability. The degradation of sinigrin by both pathogens increased with higher temperatures (21 > 10 > 4°C). Salmonella and L. monocytogenes cocktails hydrolyzed 59.1 and 53.2% of sinigrin, respectively, at 21°C up to 21 days. Both iron compounds significantly enhanced sinigrin degradation by the pathogens. On day 7, sinigrin was not detected when the Salmonella cocktail was cultured with ferrous iron or when the L. monocytogenes cocktail was cultured in Mueller-Hinton broth containing ferric iron. In contrast, ferric and ferrous iron inhibited the activity of 0.002 U/ml myrosinase from white mustard by 63 and 35%, respectively, on day 1. Salmonella and L. monocytogenes cocktails were able to degrade >80% of sinigrin at 0.05 and 0.1% glucose; however, 0.25 to 1.0% glucose significantly reduced sinigrin degradation. Although both pathogens significantly degraded sinigrin, the allyl isothiocyanate (AITC) recoverable was ≤6.2 ppm, which is not inhibitory to Salmonella or L. monocytogenes. It is probable that the gradual hydrolysis of sinigrin to form AITC either did not produce an inhibitory level of AITC or the AITC formed was unstable in the aqueous medium and rapidly decomposed to new compounds that were less bactericidal against the pathogens.