Myrosinase activity of cruciferous vegetables

Wilkinson, A. W.; Rhodes, M. J. C.; Fenwick, Roger

doi:10.1002/jsfa.2740350511

Cited by 61 publications

(34 citation statements)

References 23 publications

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“…The differences between mustard species in overall enzyme activity were not related to differences in protein content and hence, differences between species in specific activity prevailed. Variations in myrosinase enzyme activity within and between Brassica species have been reported previously and have been attributed to genetic and/or environmental factors (agronomic and climatic conditions) (Pocock et al, 1987;Wilkinson et al, 1984). In addition, Rask et al (2000) had also reported that the difference in thermal stability of myrosinase in Brassica plants was probably due to the existence of different isoforms of myrosinase, where some of them interact with myrosinase-binding proteins (a group of proteins found in Brassica plants) to form complexes that may improve stability.…”

Section: Resultsmentioning

confidence: 95%

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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

et al. 2015

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

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Section: Resultsmentioning

confidence: 95%

“…Myrosinase activity was measured according to the coupled enzymatic procedure with some modifications (Gatfield & Sand, 1983;Ghawi et al, 2012;Wilkinson, Rhodes, & Fenwick, 1984). A D-glucose determination kit was used (R-Biopharm Rhone, Heidelberg, Germany).…”

Section: Myrosinase Enzyme Activity Assaymentioning

confidence: 99%

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

et al. 2015

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“…The rarity of this type of inhibition has been ascribed to its potentially catastrophic consequences for a metabolic system [31] Most plant myrosinases are activated to some degree by ascorbate. Wilkinson and colleagues [33] reported a range of ascorbate concentrations of 0.7-5.0 mM for maximal activation of myrosinase in partially purified extracts of six crucifers. The enzyme from S. alba retains significant activity in the absence of ascorbate and is only activated by about 2-3-fold, whereas the enzymes from several other Brassica species show an 8-12-fold activation by ascorbate [23].…”

Section: Discussionmentioning

confidence: 99%

An unusual case of ‘uncompetitive activation’ by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings

Shikita

Fahey

Golden

et al. 1999

Biochemical Journal

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Myrosinase (thioglucoside glucohydrolase ; EC 3.2.3.1) is a plant enzyme that hydrolyses glucosinolates, principally to isothiocyanates. Myrosinase was purified to homogeneity in good yield from 8-day-old seedlings of Raphanus sati us (daikon) using a four-step procedure involving chromatographies on anion exchange, hydrophobic Phenyl-Sepharose, gel filtration and concanavalin A-Sepharose. In order to stabilize the enzyme and to avoid excessive peak broadening during chromatography, 30 % (v\v) glycerol was added to dialysis and chromatography buffers. The purified enzyme was eluted as a single peak from a gelfiltration sizing column with an apparent molecular mass of 120 kDa. The enzyme was resolved into two subunits with molecular masses of 61 and 62 kDa by SDS\PAGE. Ascorbic acid activated the purified enzyme more than 100-fold. The V max and K m values for the hydrolysis of allyl glucosinolate (sinigrin) were 2.06 µmol\min per mg of protein and 23 µM in the absence of ascorbate and 280 µmol\min per mg of protein and 250 µM in

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“…There is however, no GC, HPLC or HPCE method described up to date for the simultaneous determination of glucosinolates and their degradation products. Direct methods have been used both for determination of myrosinase activity and kinetic parameters, such as the determination of liberated acid, which is titrated with alkali using pH-stat apparatus (pHSA) [35], direct spectrophotometric assay (DSA) of glucosinolate degradation [12,36], the spectrophotometric coupled enzyme assay (SCEA) of liberated glucose [37,38] and the polarographic coupled assay (PCA) [39]. The pHSA, the DSA and the SCEA are used in routine analysis of myrosinase activity of Brassica extracts due to their being simple, fast and inexpensive.…”

Section: Introductionmentioning

confidence: 99%

Micellar electrokinetic capillary chromatography—Synchronous monitoring of substrate and products in the myrosinase catalysed hydrolysis of glucosinolates

Bellostas¹,

Sørensen²,

Sørensen³

2006

Journal of Chromatography A

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A micellar electrokinetic capillary chromatography (MECC) method has been developed for monitoring the myrosinase catalysed hydrolysis of 2-hydroxy substituted glucosinolates and the simultaneous formation of the corresponding degradation products (oxazolidine-2-thiones (OZTs) and nitriles). Glucosibarin ((2R)-2-hydroxy-2-phenylethylglucosinolate) was chosen as the model glucosinolate owing to the difficulties in determining hydrolysis rates of this type of substrates in traditional UV-assays. The method was afterwards validated with glucobarbarin ((2S)-2-hydroxy-2-phenylethylglucosinolate) and progoitrin ((2R)-2-hydroxybut-3-enylglucosinolate). Aromatic glucosinolates without a 2-hydroxy group in their side chains, such as glucotropaeolin (benzylglucosinolate) and gluconasturtiin (phenethylglucosinolate) were also tested. Formation of the glucosinolate hydrolysis products was monitored simultaneously at 206 nm and 230 nm. This allowed estimation of the extinction coefficient of the OZT derived from glucosibarin, which was found to be 18,000 M −1 cm −1 and 12,000 M −1 cm −1 at 206 nm and 230 nm, respectively. The developed method has limit of detection of 0.04 mM and 0.06 mM and limit of quantification of 0.2 mM and 0.3 mM for the glucosibarin derived OZT and nitrile, respectively. Linearity of the glucosinolate concentration was examined at six concentration levels from 2.5 mM to 100 mM and at 206 nm a straight line (R 2 = 0.9996) was obtained. The number of theoretical plates (N) at the optimal system conditions was 245,000 for the intact glucosibarin, 264,000 for the OZT and 252,000 for the nitrile.

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Myrosinase activity of cruciferous vegetables

Cited by 61 publications

References 23 publications

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

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

An unusual case of ‘uncompetitive activation’ by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings

Micellar electrokinetic capillary chromatography—Synchronous monitoring of substrate and products in the myrosinase catalysed hydrolysis of glucosinolates

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