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Studies regarding the myrosinase enzymatic activity from black mustard seeds
(Brassica nigra)

Article in Journal of Food Agriculture and Environment · January 2009

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Meri-Rastilantie 3 B, FI-00980 Journal of Food, Agriculture & Environment Vol.7 (1) : 44-47. 2009 www.world-food.net
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e-mail: [email protected]

Studies regarding the myrosinase enzymatic activity from black mustard

(Brassica nigra) seeds
Daniela Stoin *, Paul Pirsan, Florina Radu, Mariana-Atena Poiana, Ersilia Alexa and Diana Dogaru
Banat’s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului 119, 300645 - Timisoara, Romania.
*e-mail: [email protected], [email protected], [email protected]

Received 12 October 2008, accepted 5December 2008.

Abstract
The aim of this study consists in the presentation of a simple and fast method for myrosinase activity quantification towards sinigrin from the
aqueous extracts obtained from black mustard (Brassica nigra) seeds as raw material (SM) or crushed down (CM). The myrosinase activity was
determined under different pH (5-8.5), temperature (T = 25-85°C) and reaction time (τ = 30-390 minutes) conditions, according to the
spectrophotometric method and was expressed depending on the concentration of the glucose resulted from enzymatic reaction. The optimum
parameters corresponding to the maximum myrosinase activity in the aqueous extracts of black mustard were the following: pH 7, T 55°C and τ 210
minutes for the crushed down mustard samples and τ 240 minutes for the mustard seeds samples. The myrosinase activity is significantly influenced
by the physical state of the vegetable material (raw or crushed down), this values being of 116.3510 µg glucose/g.min for CM in comparison to the
85.0660 µg glucose/g.min for SM. The enzymatic hydrolysis reaction of the sinigrin takes place with heat releasing.

Key words: Brassica nigra, glucosionolates (GLS), sinigrin (SIN), myrosinase (MYR), enzymatic activity.

Introduction
Glucosinolates (GLS) are natural compounds belonging to As a result of their high variety and bioactivity, GLS are suited
S-glycosides class, being sulphated esters of the S-(β-D- to be used in the pharmaceutical industry, but they also have a
glucopyranosile)-metanethyohydroxaminic, C-substituted. GLS series of applications in the food industry. The interest in GLS has
are formed in plants from amino acid precursors upon some started from several observations: these compounds are involved
biochemical reactions. In vegetable products, these compounds in the plants defensive mechanism, have a protective role against
are found as glycosylated form and passed/transformed into active carcinogen chemical agents and they interfere with the sulphur
pharmaceutical-dynamic form as a result of the enzymatic metabolism and regulation of plant development. Also, the
degradations 2, 8, 16, 19. Until now, GLS have been identified only in researches performed reflect the chemopreventive effect of GLS
dicotyledonous plants from the Brassicaceae, Capperaceae, on different human cancer types 12, 18. The GLS known as sinigrin
Resedaceae, Moringoceae and Tovaraceae families, rarely (SIN) (thio-β-glucopyranosyl-1-N-sulphate-2-propenylimidate) is
appearing in some species of the Limantroceae, Caricaceae, in significant content in mustard (Armoracia rusticana) seeds 3, 4.
Gyrostemonaceae, Salvodoraceae and Euphorbiaceae families 5, 6, MYR enzymes (thioglucoside-GLS) are a group of isoenzymes
8
. GLS are intravacuolarly located in the cells, and upon grinding, that catalyses the GLS hydrolysis (naturally occurring
breaking or cutting tissues they get in touch with the myrosinase thioglucosides). Usually MYR enzyme physically occurs from GLS,
(MYR), enzyme that determines the division of the glucose (GLU) but upon wounding of plant (e.g. during cooking, mastication or
rest. A variety of volatile compounds (isothiocyanates, damage of the insects) it catalyses their hydrolysis to obtain mainly
thiocyanates, nitriles, hydroxinitriles, epithyonitriles) with isothiocyanates, GLU and bisulphate. The reaction involves an
antimicrobial and phytotoxic properties are released upon initial hydrolysis of the breakdown products: β-D-glucose and
enzymatic hydrolysis along with GLU 3, 4, 13, 14. thiohydroximate-O-sulphonate. MYR enzyme catalyzed hydrolysis
Among GLS degradation products, the isothiocyanates are of GLS initially involves the cleavage of the thioglucoside linkage
responsible for the “spicy” and hot taste. These compounds are yielding D-glucose and thiohydroximate-O-sulphonate. This
found in mustard, horseradish, cabbage and broccoli. Unlike their intermediation rapidly rearranges resulting in the production of
precursors, characterized by a neutral or weak activator effect sulphate and thiocyanate, isothiocyanate or nitrite depending on
towards microorganisms development, the hydrolysis products different factors such as substrate, pH, temperature and
have proved to be powerful inhibitors of the microbial flora 15. The availability of ferrous ions 9, 17. The enzymatic hydrolysis of the
GLS content is higher in black mustard seeds and horseradish SYN is influenced by a series of factors like reaction medium pH,
roots (over 10% by dry weight) than in the other constituent parts reaction temperature, the necessary speed for reaching the
of the Brassicaceae 11, 14, 15.

44 Journal of Food, Agriculture & Environment, Vol.7 (1), January 2009

equilibrium and the presence of some inhibitor/promoter temperature. Every 30 minutes, a sample was taken from each lot.
substances 1, 9, 10, 17. The enzymatic hydrolysis reaction was stopped by adding 2 ml
Starting from these observations, our study aims to describe a 20% TCA solution each. The samples were passed through PTFE
simple and fast method for the MYR quantification on SIN present 0.45 µm filtrating membranes, stored in test tubes with stoppers
in black mustard (Brassica nigra) seeds as raw material (SM) or and maintained in refrigerating conditions until analysis. The
crushed down (CM). The study was performed under different experiments were performed three times each.
pH, temperature and reaction time conditions. The MYR enzymatic
activity was expressed depending on the concentration of GLU Spectrophotometric determination of the MYR activity: The
released upon enzymatic reaction. The o-toluidine MYR (EC 3.2.3.1) enzymatic activity was determined by the
spectrophotometrical method was used for determination of method of Al-Turky and Dick 1. According to this method, “one
GLU 1, 10. unit of enzymatic activity represents the enzyme amount that
hydrolyses the sinigrin, releasing 1µg glucose/g.min under the
Materials and Methods presented analysis conditions”. MYR activity determination is
Samples: The black mustard, Brassica nigra (L.), was harvested based on the reaction between GLU and o-toluidine that forms a
from Didactic Experiment Station of Banat’s University of blue-greenish complex that exhibits maximum absorbance at λ =
Agricultural Sciences and Veterinary Medicine, Timisoara. The 630 nm. In order to prepare the standard curve A=f(conc GLU)
seeds were harvested and conditioned (dust and metal impurities from the GLU stock solution (5 mg/ml), standard solutions with
removal, liquid nitrogen freezing and storage at -66°C until the concentrations in the range of 0.1-1.5 mg/ml were prepared.
analysis). From these solutions, 100 µl were taken and passed in graduated
test tubes of 10 ml. One ml of 3% TCA and 5 ml of o-toluidine
Determination of the pH influence on the MYR activity: The pH reagent solution were added. After shaking, the samples were put
influence on enzymatic reaction was established by suspending into a thermostatic water bath at boiling point and maintained for
the vegetable material (1 g from CM and SM) in 10 ml solution of 13 minutes until a blue-greenish coloration appeared. The samples
phosphate buffer for pH 5-7.5 and borate buffer for pH 8 and 8.5, were cooled to the room temperature and the absorbance was
respectively. The samples were put into shaker at 35°C and read against the blank sample containing o-toluidine and 3% TCA
continuously shaken for 30 minutes. Afterwards, in order to stop solution prepared under the same conditions. For each standard
the MYR activity, 2 ml of 20% trichloracetic acid solution (TCA) solution three determinations were made. The standard curve
was added in each sample. The resulted reaction mixture was equation is Y = 0.6368 + 0.0129X and the regression coefficient
centrifuged at 6000 rot/min for 10 minutes. From each supernatant, R2 = 0.9983.
5 ml sample was taken and passed through filtrating membranes,
and the clear samples were stored in test tubes with stoppers and Results and Discussion
stored in refrigeration conditions (4-6°C) until analysis. For each The influence of pH on MYR activity from Brassica nigra: The
pH value, the experiments were performed three times each. pH effect on MYR activity is shown in Fig. 1 and the results are
according to the literature 1, 9, 10, 17 . The MYR activity is affected
Determination of the reaction temperature effect on the MYR by the reaction medium pH especially due to the enzyme protein
activity: The temperature effect on MYR activity was studied nature. The corresponding optimum pH to MYR activity in white
through this parameter variation in the range of 25-85°C, the mustard is in the range of 4.5-4.9 17. The study of the pH influence
reaction time being constantly maintained at 30 minutes. Vegetable on enzyme activity was done in the range of 5-8.5 because at
material (1 g from CM and SM) was suspended in 10 ml phosphate higher pH there is interference of desulphatase enzyme that
buffer pH 7. The samples were put into a shaker at the generally exhibits optimum activity at an alkaline pH, the action
corresponding temperatures and continuously stirred for 30 substrate being common 7.
minutes. Afterwards, the samples were submitted to the same From the experimental data shown in Fig. 1, it can be seen that
operations described above. For each reaction temperature, the MYR is active in a wide pH range, the enzymatic activity
experiments were performed three times each. continuously increasing from pH 5 (SM 38.90%; CM 46.56%) to
pH 7 (SM 73.11%; CM 100%), and after that it’s activity starts
The kinetics study of the SIN enzymatic hydrolyze reaction: The decreasing, remaining practically constant at pH above 8 (SM
enzymatic hydrolysis process was studied in vitro using as a 36.81%; CM 46.84%). The MYR activity is also significantly
substrate the SIN from the vegetable material taken into study,
CM - crushed down mustard
and not the SIN purified 1. The SIN enzymatic hydrolysis reaction SM - mustard seeds
kinetics was performed under the following experimental 130
116.35
conditions: pH 7, reaction time τ 30-390 minutes and reaction 120
(µ g glucose/g.min)
Enzyme activity

110
temperature T 25-75°C. The concentration of glucose resulted 100 97.54 98.7
from the reaction was taken into sight parameter. The reaction 90 85.29 85.07
order and the speed constant were experimentally determined from 80
70 65.43 76.2 77.74 66.64
the graphical representations ln(conc GLU) = f(time). The activation 60
54.18 63.92
55.5 54.51
energy was determined according to Arrhenius equation 19. One g 50 49.17
vegetable material (SM and CM) was weighed and 10 ml 40 45.25 42.84
5 5.5 6 6.5 7 7.5 8 8.5
phosphate buffer pH 7 was added. The samples were hermetically
pH
closed and put into a shaker at the corresponding reaction Figure 1. Phosphate buffer pH effect on MYR activity.

Journal of Food, Agriculture & Environment,Vol.7 (1), January 2009 45

influenced by the physical state of the vegetable material (raw or CM crushed down mustard, T 45ºC
CM crushed down mustard, T 55ºC
crushed down), this values being of 116.3510 µg glucose/g.min SM mustard seeds, T 45ºC
for CM, in comparison to 85.0660 µg glucose/g.min for SM. SM mustard seeds, T 55ºC
350

(µ g glucose/g.min)
Enzyme activity
325
The influence of temperature on MYR activity from Brassica 300
nigra: The temperature effect on MYR activity is shown in Fig.2. 275
The temperature directly influences the speed of the hydrolysis 250
enzymatic reaction, that is, along with the increasing of the 225
200
temperature, the MYR activity increases also, but this increase is
175
limited by the thermal stability of the molecule of protein nature of
30 60 90 120 150 180 210 240 270 300 330 360 390
that enzyme 20. From the experimental data, it can be appreciated Time (min)
that MYR exhibits maximum activity at temperatures in the range Figure 3. Incubation time influence on MYR activity.
of 45-55°C. Also, it can be seen that at high temperatures 75-85°C
too, MYR still exhibits activity even if very weak (for SM 27.21%),
results that are contrary to the data obtained by Ludikhuyze and reaches the equilibrium, fact proved by almost constantly
van Doom 9, 17, who mentioned that MYR is inhibited at maintaining of the MYR activity value.
temperatures higher than 75°C. A possible explanation for the It can be noticed that in the case of the mustard, the linear
obtained results could be that in this case the experiments took variation of the enzyme activity with the reaction time is
place by using as a substrate the SIN from the aqueous extracts significantly influenced by the preparing step of vegetable material
and not the SIN isolated and purified, thus being possible that but also by the reaction temperature, that is, the increasing of the
activity is a result of the desulphatase enzyme, thermostable to quantity of the released GLU in the reaction. A possible explanation
temperatures of 90-95°C, enzyme that uses SIN as a substrate, for this behavior can be given if we take into account Bones 2
too. The data presented in Fig. 2 have led to obtaining some researches regarding the localization of the substrate and enzyme
isotherm curves resembling those obtained by Sharma and Garg13, in the subcellular compartments of the same vegetable cell, but
who used purified MYR from Brassica juncea. It can be also of the morphological forms of the enzyme 9. Thus, if the
appreciated that the enzymatic hydrolysis reaction of the SIN assumption is that the MYR is generally located in the cell wall,
takes place with heat releasing, in all situations the reaction the mechanical disruption of the cell membrane will determine the
enthalpy exhibiting negative values (at 55°C SM 1,214,771 J/ shortening of the time for reaction initiation that will determine
mol.K; CM 1,333,768 J/mol.K). Also, the variation in inverse the starting of the hydrolysis almost in a snapshot and thus, the
proportion of the reaction enthalpy with the reaction temperature quantity of GLU produced is almost double (124.43 g glucose/
confirm the above mentioned assumptions that MYR exhibits g.min for CM in comparison with 76.2 g/g.min in the case of SM).
optimum activity at moderate temperature. In exchange, the MYR activity initially increases no matter the
reaction temperature value, and once the value of 45°C is exceeded,
CM crushed down mustard the MYR activity is very slow (124.43 g glucose/g.min at 25°C and
SM mustard seeds 228.50 g glucose/g.min at 45°C, respectively, only 278.10 g glucose/
325
285.82 g.min at 55°C) at the same reaction time. The function linear form
(µ g glucose/g.min)

275 252.26 of enzymatic activity/reaction time in the range of 180 minutes

Enzyme activity

254.14 239.64
225 indicates that the process of GLU formation is a first order reaction,
175 189.75 at least for this time range.
171.53
110.39 130.92
Incubation time higher than 210 minutes caused a deviation of
125 92.1
109.37 80.69 the reaction speed from the linearity, suggesting that the
75 86.98 hydrolysis reaction of the SIN became limited, the reaction products
61.2 66.71
25 (GLU, allyl-isothiocyanate and possibly thiocyanate and nitriles)
25 35 45 55 65 75 85 becoming feeding source for the possible microorganisms present
T (ºC)
in the aqueous extracts or accumulating. From the allure of the
Figure 2. Temperature effect on MYR activity. presented curves in Fig. 3, it may be concluded that a reaction
time of maximum 180-200 minutes is enough for testing the sinigrin-
myrosinase system potential. From the mathematical expression
The influence of reaction time on MYR activity from Brassica of the enzymatic hydrolysis reaction speed, it was ascertained
nigra: The reaction time effect on MYR activity is shown in Fig. 3. that except for the values obtained at 25°C, R2<0.95 for SM, the
The study of the reaction time influence on MYR activity was step determined by the speed represents a first order reaction.
performed in temperature range of 25-75°C, but significant results
were obtained especially in 45-55°C. From Fig. 3 it can be seen Conclusions
that the curves form remains the same for the entire studied time The study of the myrosinase activity from Brassica nigra has led
range. For example, the MYR activity in the case of the mustard to the conclusion that in general, the sinigrin-myrosinase system
not submitted to grinding operations, increases almost linearly is influenced by a variety of medium factors, among the most
with the incubation time until 128-180 minutes, reaches a maximum important being the pH, temperature and reaction time. Upon
in the range of 210-240 minutes and after that slowly decreases to enzymatic hydrolysis of the sinigrin at optimum parameters (pH 7;
270 minutes, and after this time the enzymatic hydrolysis reaction reaction temperature T 55°C; incubation time τ 210-240 minutes)

46 Journal of Food, Agriculture & Environment, Vol.7 (1), January 2009

 19
results a significant quantity of D-glucose that may be Yen, G. and Wei, Q. 1993. Myrosinase activity and total glucosinolate
spectrophotometrically determined and used as a measure for the content of cruciferous vegetables, and some properties of cabbage
enzymatic activity of the myrosinase from Brassica nigra. The myrosinase in Taiwan. J. Sci. Food. Agric. 61:471–475.
20
presented method is fast, accurate and can be used in almost all Jiang, Z.-T., Li, R. and Yu, J.C. 2006. Pungent components from
thioglucosides in Armoracia rusticana grown in China, obtained by
laboratories.
enzymatic hydrolysis. Food Technol. Biotechnol. 44(1):41–45.

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Journal of Food, Agriculture & Environment,Vol.7 (1), January 2009 47

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