Purification and Characterization of Broccoli (Brassica oleracea var

Nov 12, 2014 - Myrosinase from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A aff...
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Purification and Characterization of Broccoli (Brassica oleracea var. italica) Myrosinase (β-Thioglucosidase Glucohydrolase) Andrea Mahn,* Alejandro Angulo, and Fernanda Cabañas Department of Chemical Engineering, University of Santiago of Chile, Avenida Libertador Bernardo O’Higgins 3363, Estación Central, Santiago 9170019, Chile ABSTRACT: Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50−55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis−Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min−1, Km equal to 0.086 mM, and KI equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase. KEYWORDS: Brassica oleracea var. italica, myrosinase, purification process, kinetics

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INTRODUCTION Brassicaceae myrosinases (β-thioglucosidase glucohydrolase, EC 3.2.1.147) are glycoproteins that have multiple forms with different molecular masses, number of subunits (2−12, usually homodimer), subunit size (10−110 kDa),1,2 and carbohydrate content.3 These enzymes catalyze the hydrolysis of glucosinolates, a group of sulfur-containing secondary metabolites that are found in Brassiaceae plants. One of the hydrolysis products are isothiocyanates, also known as “mustard oils”, that are considered as powerful anticarcinogens.4 In broccoli, the most abundant glucosinolate is glucoraphanin, whose hydrolysis by myrosinase yields sulforaphane, a natural compound with demonstrated anticancer effects.5−7 In the plant, glucosinolates are physically segregated from myrosinase, and therefore, the basal content of sulforaphane in unprocessed broccoli is very low. Processing or chewing triggers the synthesis of sulforaphane, but because broccoli is mostly consumed cooked or boiled, myrosinase is usually inactive. As a consequence, there is a limited hydrolysis of glucosinolates and, accordingly, a low sulforaphane intake. On the other hand, myrosinase from gastrointestinal microflora also can convert glucosinolates into isothiocyanates; however, there is a marked variability in the conversion efficiency between individuals.8 Recently, Ghawi et al.9 suggested that the addition of exogenous myrosinase to processed broccoli would favor the hydrolysis of glucoraphanin and, therefore, the sulforaphane concentration. Broccoli myrosinase has been poorly characterized thus far, in terms of physicochemical properties and kinetic parameters. Some authors studied cabbage (Brassica oleracea var. Capitata) myrosinase and found a temperature stability range between 40 and 70 °C.10 It has to be noted that cabbage is very closely related to broccoli. Other authors studied the influence of intrinsic and extrinsic factors on the activity of myrosinase in fresh broccoli juice and reported an optimum pH range between 6.5 and 7.0 and an optimum temperature of 30 °C, © XXXX American Chemical Society

and they found that ascorbic acid and MgCl2 activate broccoli myrosinase.11 The denaturation kinetics of broccoli myrosinase in broccoli juice subjected to pressure/temperature treatments was also studied.12 They found that thermal inactivation occurred between 30 and 60 °C and pressure inactivation at 20 °C occurred between 200 and 450 MPa. Both denaturation kinetics were modeled by a consecutive step model. A firstorder inactivation kinetics was found when combined treatment was applied. Finally, a search in public protein databases yielded no information about the crystal structure of broccoli myrosinase and kinetic parameters, such as KM, turnover number, KI, etc. (UniProtKB, http://www.uniprot.org/; Brenda, www.brendaenzymes.org; and ENZYME, http://enzyme.expasy.org/). On the other hand, several authors have proposed diverse strategies for the purification of different Brassicaceae myrosinases. Myrosinase from 8-day-old daikon seedlings was purified by means of anion-exchange chromatography, followed by hydrophobic interaction chromatography, size-exclusion chromatography, and concanavalin A affinity chromatography.13 Myrosinase from crambe seeds has been isolated through a three-step process: concanavalin A affinity chromatography, cation-exchange chromatography, and sizeexclusion chromatography.3 Mustard seed myrosinase was partially purified and characterized in terms of temperature and pressure stability. 14 Purification was achieved by ammonium sulfate precipitation, followed by anion-exchange chromatography. Other authors isolated myrosinase from different sources: horseradish,15 Ethiopian mustard, and cauliflower.2 No reports about purification of broccoli myrosinase are currently available. Then, in this work, we Received: May 29, 2014 Revised: November 7, 2014 Accepted: November 12, 2014

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Protein Identification. Protein spots were excised directly from the SDS−PAGE gels and analyzed by matrix-assisted laser desorption ionization/time-of-flight (MALDI−TOF) at the Central Proteomics Facility of Sir William Dunn Pathology School at Oxford University. Samples were destained using a 25 mM ammonium bicarbonate in 50% acetonitrile/water (Mili-Q-grade) solution. Destained gel pieces were reduced with 10 mM dithiothreitol (DTT) and alkylated with 55 mM iodoacetamide. Reduced and alkylated gel pieces were washed 3 times with solution of 25 mM ammonium bicarbonate in 50% acetonitrile/water and dehydrated with 100% acetonitrile before adding 400 ng of Promega sequencing grade modified trypsin. Samples were left for 1 h at 4 °C to rehydrate. A 25 mM ammonium bicarbonate solution was added to cover the gel pieces, and the samples were incubated at 37 °C overnight. Digestion was stopped with 1 μL of acetic acid, and the supernatant was placed in new low binding microtubes. The gel pieces were covered with 50% acetonitrile/water with 2% formic acid solution and sonicated for 30 min to extract more peptides. The supernatant was added to new microtubes and dried down in a vacuum centrifuge. Samples were resuspended in 10 μL of 0.1% trifluoroacetic acid (TFA) and desalted using Millipore C18 ZipTips. Samples were eluted in 3 μL of 0.1% TFA in 50% acetonitrile/water solution, and 0.5 μL of the eluent was spotted onto the MALDI plate and left to air-dry. Dried samples were overlaid with 0.5 μL of MALDI matrix α-cyano-4-hydroxycinnamic acid and left to air-dry. Then, dried samples were analyzed on AB Sciex 4800 MALDI ToF−ToF. The instrument was calibrated using 4700 calibration mixture, and after that, samples were analyzed using mass spectrometry (MS) reflector positive (for MS spectra) and MS/MS 1 kV positive (for MS/MS spectra) methods. The data were searched by GPS explorer software, using Mascot search engine, National Center for Biotechnology Information (NCBI) non-redundant (nr) green plant database (NCBInr 20131208; Sept 30, 2014). Effect of pH and Temperature. The activity of myrosinase was determined at different pH (4.0, 5.0, 6.0, 7.0, and 8.0) and temperature (20, 40, 60, and 70 °C) conditions. We used 0.1 M acetate buffer to achieve pH 4.0 and 5.0 and 0.1 M phosphate buffer to achieve pH 6.0, 7.0, and 8.0. Activity assays were conducted using a thermostatic bath (Stuart, U.K.). All determinations were made in triplicate. Kinetic Characterization. The kinetic behavior of broccoli myrosinase was studied using sinigrin (Sigma-Aldrich, Schnelldorf, Germany) as the substrate at different initial concentrations (0.035, 0.050, 0.075, 0.100, 0.125, and 0.250 mM) at pH 7.0 and 40 °C. The experimental data were adjusted to Michaelis−Menten kinetic models, and the respective parameters were estimated. Analytical Determinations. Protein Concentration. The protein concentration was measured by the Bradford method using the Protein Quantification Kit-Rapid (Sigma-Aldrich, Schnelldorf, Germany). Absorbance at 595 nm was registered, and the protein concentration was estimated from a calibration curve made with bovine serum albumin (BSA) as the protein standard. Myrosinase Activity. Enzyme activity was assessed spectrophotometrically through the method described in the literature.18 A total of 1 mL of substrate buffer (33 mM sodium phosphate buffer at pH 7.4 containing 10 mM sinigrin, 3 mM MgCl, 0.55 mM ATP, 0.72 mM NADP, 3.5 units of hexokinase, and 1.75 units of glucose-6-phosphate dehydrogenase) was preincubated for a sufficient time (3−10 min) at 30 °C. Then, appropriate amounts of the enzyme were added. After mixing, the reaction was monitored at 346 nm. Myrosinase activity was calculated from the absorbance increasing rate, which was due to the formation of NADPH. A total of 1 unit of myrosinase activity was defined as the amount of enzyme that catalyzed the liberation of 1 μmol of glucose/min from sinigrin under the conditions described above. Results were expressed in units per milligram of protein. All chemicals were purchased from Sigma-Aldrich (Schnelldorf, Germany). All determinations were made in triplicate. SDS−PAGE. SDS−PAGE was performed in a mini-chamber (BioRad, Hercules, CA).17 Each gel (12% acrylamide) had 10 wells, and in each well, 20 μL of protein solution was loaded (14 μg of protein of crude extract and dialysis fraction and 7 μg of the affinity chromatography fraction that contains the pure enzyme). The gels

propose a process to purify broccoli myrosinase and also a partial characterization of the enzyme.

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MATERIALS AND METHODS

Plant Material. Broccoli (B. oleracea cv. Avenger) heads (3 days from harvesting) were purchased at the local market (Santiago, Chile) through a single supplier. The vegetables were immediately subjected to protein extraction. Protein Extract. Protein was extracted as previously described.16 Briefly, samples were pulverized with liquid nitrogen in a mortar until obtaining a homogeneous pulp. After that, 1 mL of buffer was added to 0.25 g of powder. The buffers were 0.1 M acetate at pH 5.0 and 0.1 M phosphate buffer at pH 6.0 and 7.0. The mixture was vortexed and then centrifuged at 15000g for 20 min at 4 °C. The supernatant was recovered for analysis. Purification. Purification Process Design. In a first stage, the crude broccoli extract was analyzed by sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) to visualize the complexity of the sample from which myrosinase was to be purified.17 The molecular mass of each band was estimated by comparison to fullrange rainbow molecular weight markers (12 000−225 000 Da) (GE Healthcare, Uppsala, Sweden). With this information, we performed a database search to characterize the main proteins that must be separated in the purification process. In parallel, the physicochemical properties of Brassicaceae myrosinases were obtained from databases and literature. The database searches were carried out in UniProtKB (http://www.uniprot.org). Finally, considering the characterization of the broccoli protein extract and also the information about purification processes reported for myrosinases from different sources, the purification process was designed. Ammonium Sulfate Precipitation. The protein extract was precipitated with ammonium sulfate at 55% saturation.17 This saturation percentage was chosen on the basis of literature.15 The mixture was agitated in a beaker for 10 min in an ice bath and then left at room temperature for 30 min. After that, the suspension was centrifuged at 7000g for 30 min, the precipitate was discarded because no activity was detected in it, and the supernatant was used in the subsequent purification steps. Dialysis. The supernatant from ammonium sulfate precipitation (5 mL) was subjected to dialysis against 500 mL of 20 mM Tris−HCl buffer at pH 8.0 plus 0.15 mM NaCl in 0.2 μm dialysis bags [Spectra/ Por molecular weight cut-off (MWCO) of 3500]. After 24 h of stirring at room temperature, the sample solution (10.5 mL) was recovered from the dialysis bag. Affinity Chromatography. Chromatographic runs were performed in a BioLogic LP system equipped with a fraction collector and the LP Data View software (Bio-Rad, Hercules, CA). A 1 mL column was packed with concanavalin A resin (GE Healthcare, Uppsala, Sweden). The column was washed and equilibrated (5 column volumes) with 20 mM Tris−HCl at pH 8.0 plus 0.15 mM NaCl (buffer A). The protein extract (5 mL) was loaded into the column and subsequently washed with buffer A (5 column volumes). Elution of myrosinase was obtained with an increasing gradient of methyl-α-D-mannopyranoside (SigmaAldrich, Schnelldorf, Germany) formed by mixing buffer A with buffer A added with 0.5 M methyl-α-D-mannopyranoside (buffer B). The elution gradient was 3.33% B/min. The flow rate was constant and equal to 0.5 mL/min. The experiments were conducted at room temperature. All buffers were filtered through 0.22 μm Millipore filters. Absorbance at 280 nm was registered. Myrosinase Characterization. Native Molecular Mass. The native molecular mass of broccoli myrosinase was determined by sizeexclusion chromatography. A 0.7 × 30 cm Econo-Column (Bio-Rad, Hercules, CA) was packed with 11.5 mL of Sephacryl 200-HR (SigmaAldrich, Schnelldorf, Germany). The calibration curve was built using the Gel Filtration Marker Kit for protein molecular weights of 29 000− 700 000 Da (Sigma-Aldrich, Schnelldorf, Germany). A total of 1 mL of the purified myrosinase fraction was loaded into the column and eluted with 50 mM Tris−HCl at pH 7.4 plus 0.15 M NaCl at 0.5 mL/ min. B

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were run at 120 V for 10 min and after that at 180 V until the advancing front reached the bottom of the gel. After that, the gels were soaked in a solution of 25% methanol and 7.5% acetic acid for 30 min, stained with Coomassie Brilliant Blue R-250 for 12 h (0.1% Coomassie Brilliant Blue R-250, 25% methanol, and 7.5% acetic acid), and destained in a solution of 25% methanol and 7.5% acetic acid. AccuRuler Prestained Protein Ladder (11 000−180 000 Da) (Lamda Biotech, Inc., Ballwin, MO) was used. The protein samples, except for the crude extract, were concentrated by acetone precipitation before SDS−PAGE.19 Image acquisition was performed with an ImageScanner II device (GE Healthcare, Uppsala, Sweden). All chemicals were analytical-grade and were purchased from Sigma-Aldrich (Schnelldorf, Germany).

protein extract is evident from Figure 1; besides, it is clear that the fraction composition changed after ammonium sulfate precipitation. In the chromatography fraction, three bands were detected, whose identity was elucidated through MS. Table 1 shows the results from the MS analysis performed on the three bands detected in SDS−PAGE. Bands 1 and 2 were identified as myrosinase from Brassica napus, with an acceptable statistical significance (scores higher than 120 and e-values lower than 10 × 10−5). These bands differ in their molecular size, in about 5 kDa. This difference can be attributed to different glycosylation states. Band 3 was identified as concanavalin A, which probably was released from the affinity resin. Figure 2 shows the concanavalin A affinity chromatogram. Two peaks eluted during the elution gradient, the smallest peak at 74% buffer B and the second peak at 95% buffer B, which agrees with the activity curve. The smallest peak showed no myrosinase activity and probably corresponds to one of the myrosinase bands detected in SDS−PAGE. Then, it is likely that this peak corresponds to an inactive isoform of myrosinase. The purification process proposed here was successful in purifying myrosinase from broccoli extract. Table 2 shows a summary of the purification process. The total myrosinase activity had only a small decrease (12.5%) with respect to the crude extract. Additionally, a high recovery was achieved, with a yield of 88%. Finally, purity increased 1328-fold. These values indicate that the process designed here is very efficient. This process is simpler than other processes proposed to purify myrosinases from other sources. For example, a four-step process was proposed to purify myrosinase from daikon seedlings, consisting of anion-exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, and a final concanavalin A affinity chromatography.13 The authors achieved a 49% recovery and a 230-fold purification. Another three-step process was also proposed, concanavalin A affinity chromatography, cation-exchange chromatography, and size-exclusion chromatography, for purifying crambe seed myrosinase.3 The authors reported a 4% yield and a purification factor of 40-fold. Another three-step process to purify myrosinase from horseradish consisted of ammonium sulfate precipitation, Q-sepharose, and concanavalin A affinity chromatography,15 resulting in a yield of 20% and a 34-fold purification. A two-step process was developed to separate myrosinase complexes from different Brassicaceae seeds, with a recovery between 18 and 48% and a maximum purification of 573-fold for Sinapis alba myrosinase.2 Then, the process proposed in the present work gave the highest recovery and the highest purification factor compared to the data available in the literature. Myrosinase Characterization. On the basis of the SDS− PAGE, myrosinase subunits have a molecular mass of 50−55 kDa. This value falls within the expected range of 10−110 kDa1 but differs slightly from the value of 62 kDa reported for myrosinases from Arabidopsis, Wasabia japonica, B. napus, and B. oleracea var. Capitata. The number of subunits in the native structure of broccoli myrosinase was determined by sizeexclusion chromatography (data not shown). The native molecular mass was equal to 157 kDa, and considering that each subunit weights 50−55 kDa, the native structure of broccoli myrosinase is composed of three subunits. It is very frequent that myrosinases form complexes with other proteins. These complexes exhibit a high molecular weight (200−600 kDa) and show myrosinase activity.2 In the

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RESULTS AND DISCUSSION Purification. The analysis of the proteins found in the crude broccoli extract yielded that there are approximately 21 main proteins, detected in the SDS−PAGE (shown in Figure 1), that

Figure 1. Image of SDS−PAGE that shows protein fractions along the purification process: lane 1, molecular weight ladder; lane 2, crude extract; lane 3, fraction after precipitation and dialysis; and lane 4, protein fraction after affinity chromatography.

must be eliminated in the purification process. The molecular weight of the monomers fluctuated between 93 and 19 kDa. According to the literature, among them, there are only two glycoproteins, β-galactosidase10 and myrosinase,2 which should be possible to separate through concanavalin A affinity chromatography. On the other hand, these two proteins differ considerably in their molecular weight. The molecular weight of most Brassicaceae myrosinases is about 62 kDa, while the molecular weight of β-galactosidase may vary between 5221 and 130 kDa,22 and it is usually 93 kDa in Brassicaceae.20 Besides, myrosinase usually forms homodimers, whereas β-galactosidase may form dimers or tetramers.23 As a consequence, a separation technique that exploits size differences would be adequate to purify broccoli myrosinase, such as ammonium sulfate precipitation. Hence, the purification process consisted of ammonium sulfate precipitation followed by concanavalin A affinity chromatography. An intermediate dialysis step was included to eliminate ammonium sulfate excess from the supernatant and also to perform a buffer change. Figure 1 shows SDS−PAGE that resolved the proteins present in the crude extract (second lane), the sample after ammonium sulfate precipitation and dialysis (third lane), and the active fraction obtained after concanavalin A affinity chromatography (fourth lane). The complexity of the crude C

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Table 1. Identification of Protein Bands by MS band

identity

score

e value

1

myrosinase (B. napus)

213

1.8 × 10−9

NCBInr access gi|127733

2

myrosinase (B. napus)

128

1.0 × 10−5

gi|127733

3

concanavalin A (Canavalia ensiformis)

1507

2.2 ×10−5

gi|167013346

case of broccoli, we can presume that such complexes were not formed. High-molecular-mass proteins or protein complexes are less soluble than small proteins; hence, if broccoli myrosinase forms complexes, they would have precipitated at 55% ammonium sulfate. However, the precipitate showed no myrosinase activity, but the supernatant did. Additionally, when forming complexes, myrosinase would still glycosylated, and therefore, the complexes should have been retained by the concanavalin A affinity column. However, this did not happen, because in SDS−PAGE, only myrosinase and concanavalin A (released from the affinity column) were detected after affinity chromatography. If there were complexes with other proteins, we should have detected different bands corresponding to the proteins bound to myrosinase. Figure 3 shows myrosinase activity at different pH (Figure 3a) and temperature (Figure 3b) conditions. Myrosinase remained active at acidic pH and showed a decrease in its activity as pH increased, showing a significant loss of activity at pH above 5.0. Myrosinase was active in the temperature range from 20 to 70 °C, agreeing with the literature.24,25 The optimum temperature at pH 6.0 was around 40 °C. The optimum pH would be below 4.0, differing from the optimum pH reported for other myrosinases. Crambe seed myrosinase has an optimum pH equal to 7.5,3 and the optimum pH of horseradish myrosinase is 5.7,15 both using sinigrin as the substrate. Figure 4 shows myrosinase kinetics on sinigrin. Myrosinase exhibits substrate inhibition, because there is a maximum initial activity at 0.25 mM sinigrin, and at higher sinigrin concentrations, the initial reaction rate decreased drastically. This result agrees with another study25 about the effect of

Figure 2. Chromatogram of the concanavalin A affinity step. The protein elution curve (flat line) is overlapped with the myrosinase activity curve (circles).

Table 2. Purification of Myrosinase from Broccoli Extract

stage crude extract ammonium sulfate precipitation dialysis concanavalin A affinity chromatography

total protein content (mg)

total activity (units)

specific activity (unit mg−1)

yield (%)

608.8 302.2

2.65 2.65

0.0044 0.0088

100

2.0

56.5 0.4

1.16 2.32

0.0205 5.8000

44 88

4.6 1318.2

matched peptide FSFAWSR GVNQGGLDYYHK DYADLCFK IGPVMITRWFLPFDESDPASIEAAER LTYDNSRGEFLGPLFVEDK GIYYVMDYFK YGDPLIYVTENGFSTPSSENREQAIADYKR EKGVNVR FGLSYVNW EDLDDR FSFAWSR GVNQGGLDYYHKLIDALLEK IGPVMITRWFLPFDESDPASIEAAER GRYPDIMR LTYDNSRGEFLGPLFVEDK GIYYVMDYFK--YGDPLIYVTENGFSTPSSENR EKGVNVR XDTIVAVELD TYPNTDIGDP SYPHIGIDIK NMQNGKVGT HIIYNSVDKR VGLSASTGLY KETNTILSWS HETNALHFMF QFSKDQKDL ILQGDATTGTDGNLELTRVSSNGSPQGSSVGR SPDSHPADGIAFFISNIDSSIPSGSTGRLLGLFPDAX

purification factor (fold)

D

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has a Km value of 2.06 μmol min−1 mg−1 and a Vmax equal to 23 μM,13 whereas the kinetic parameters reported for horseradish myrosinase were 0.128 mM and 0.624 μmol min −1 , respectively.15 Despite these authors not considering substrate inhibition, our values are of the same order of magnitude. 0.246[S]

V=

0.086 + [S] +

[S]2 0.368

(1)

In summary, broccoli myrosinase was successfully purified by means of a rationally designed process that consists of ammonium sulfate precipitation, followed by concanavalin A affinity chromatography. The process resulted in an 88% recovery and a 1318-fold purification, the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular weight of 50−55 kDa, and the native structure is composed of three subunits. The maximum myrosinase activity was achieved at pH 4.0 and 40 °C. Broccoli myrosinase is subjected to substrate inhibition, with kinetic parameters that coincide in order of magnitude with those reported for other myrosinases.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 56-2-27181833. Fax: 56-2-27181800. E-mail: [email protected]. Funding

This work was funded by Fondecyt Grant 1130384. Notes

Figure 3. Myrosinase stability at different pH and temperature: (a) myrosinase stability between pH 4.0 and 8.0 at 40 °C and (b) myrosinase stability between 20 and 70 °C at pH 6.0.

The authors declare no competing financial interest.

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REFERENCES

(1) James, D. C.; Rossiter, J. T. Development and characteristics of myrosinase in Brassica napus during early seedling growth. Physiol. Plant. 1992, 82, 163−170. (2) Bellostas, N.; Petersen, I. L.; Sørensen, J. C.; Sørensen, H. A fast and gentle method for the isolation of myrosinase complexes from brassicaceous seeds. J. Biochem. Biophys. Methods 2008, 70, 918−925. (3) Bernardi, R.; Finiguerra, M. G.; Rossi, A. A.; Palmieri, S. Isolation and biochemical characterization of a basic myrosinase from ripe Crambe abyssinica seeds, highly specific for epi-progoitrin. J. Agric. Food Chem. 2003, 51, 2737−2744. (4) Sivakumar, G.; Aliboni, A.; Bacchetta, L. HPLC screening of anticancer sulforaphane from important European Brassica species. Food Chem. 2007, 104, 1761−1764. (5) Quazi, A.; Pal, J.; Maitah, M.; Fulciniti, M.; Pelluru, D.; Nanjappa, P.; Lee, S.; Batchu, R.; Prasad, M.; Bryant, C.; Rajput, S.; Gryaznov, S.; Beer, D.; Weaver, D.; Munshi, N.; Goyal, R.; Shammas, M. Anticancer activity of a broccoli derivative, sulforaphane, in Barrett adenocarcinoma: Potential use in chemoprevention and as adjuvant in chemotherapy. Transl. Oncol. 2010, 3, 389−399. (6) Devi, J. R.; Thangam, E. B. Mechanisms of anticancer activity of sulforaphane from Brassica oleracea in HEp-2 human epithelial carcinoma cell line. Asian Pac. J. Cancer Prev. 2012, 13, 2095−2100. (7) Vyas, A. R.; Singh, S. V. Functional relevance of D,L-sulforaphanemediated induction of vimentin and plasminogen activator inhibitor-1 in human prostate cancer cells. Eur. J. Nutr. 2014, 53, 843−852. (8) Fahey, J. W.; Wehage, S. L.; Holtzclaw, W. D.; Kensler, T. W.; Egner, P. A.; Shapiro, T. A.; Talalay, P. Protection of humans by plant glucosinolates: Efficiency of conversion of glucosinolates to isothiocyanates by the gastrointestinal microflora. Cancer Prev. Res. 2012, 5, 603−611. (9) Ghawi, S. K.; Methven, L.; Niranjan, K. The potential to intensify sulforaphane formation in cooked broccoli (Brassica oleracea var.

Figure 4. Initial reaction rate of myrosinase at different initial sinigrin concentrations.

broccoli blanching on sulforaphane synthesis. The authors suggest that a decrease in myrosinase activity at temperatures above 57 °C may be explained by substrate inhibition, owing to the higher release of glucosinolates from the vegetal matrix. The behavior of broccoli myrosinase differs from that exhibited by horseradish and Raphanus sativus myrosinases, which showed no substrate inhibition.13,15 The kinetic parameters Vmas, Km, and KI were estimated from the Michaelis−Menten model with substrate inhibition (eq 1). Here, V is the initial reaction rate, and [S] is the substrate concentration. The maximum activity predicted by the model occurred at 0.18 mM sinigrin [Smax]. Myrosinase from R. sativus E

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Journal of Agricultural and Food Chemistry

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dx.doi.org/10.1021/jf504957c | J. Agric. Food Chem. XXXX, XXX, XXX−XXX