Copyright © 2012 by Modern Scientific Press Company, Florida, USA
International Journal of Modern Biochemistry, 2012, 1(1): 1-17
International Journal of Modern Biochemistry
Journal homepage: www.ModernScientificPress.com/Journals/IJBioChem.aspx
ISSN: 2169-0928
Florida, USA
Article
Purification and Characterization of a Cysteine Protease from
the Bulb of Common Onion Allium cepa L. (cv. Red Creole)
Uche Samuel Ndidi *, Humphrey Chukwuemeka Nzelibe
Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.:
+2348088698667.
Article history: Received 18 April 2012, Received in revised form 11 May 2012, Accepted 14 May
2012, Published 16 May 2012.
Abstract: Cysteine proteases (E.C. 3.4.22) are used extensively in the food industry.
However, it would be of benefit to find additional sources of cysteine proteases with
potentially better useful properties. Therefore, the present work was carried out to purify
and characterize the enzyme from one of the Nigerian common onion cultivars (Red
Creole). The enzyme had overall purification fold and purification yield values of 15.94
and 9.25%, respectively. The enzyme had a single band of 22 kDa by SDS-PAGE, which
suggests relative purity. Characteristically, the optimum pH and temperature are 4 and 45
oC, respectively. It was stable at pH range 4-6 and temperature range 20 - 50
oC. Eighty-
nine percent of the enzyme activity remained after 1 h time-dependent inactivation at 45
oC. The Michaelis-Menten constant (KM) and the maximum velocity (Vmax) using casein
as substrate were found to be 6.74 mg/mL and 0.86 U/mL, respectively. The enzyme also
showed more affinity towards casein (KM = 6.74 mg/mL) than other examined substrates.
Some of the examined metal cations, which include Li+, Mg
2+ and Mn
2+ activated the
enzyme, while Ca2+
, Cu2+
, Hg2+
and Zn2+
inhibited the enzyme activity. Mercuric ion
(Hg2+
), known to be an inhibitor of cysteine proteases exhibited the highest inhibition
(34.79%). The proteolytic activity of the enzyme was inhibited by thiol-specific inhibitor,
ethyl iodoacetate (5 mM), in a mixed inhibition pattern. The characteristics of this enzyme
provide basis for its use in various food industries.
Keywords: Allium cepa; cysteine protease; chromatography; assay; enzyme; inhibition.
Int. J. Modern Biochem. 2012, 1(1): 1-17
Copyright © 2012 by Modern Scientific Press Company, Florida, USA
2
1. Introduction
Allium cepa Linne (onion) belongs to the family of Liliaceae (lily) and is known to have
originated from the former Mesopotamia and Iraq. It is grown mostly in the Northern part of Nigeria.
The popularity of Allium plants as spices as well as the reputation of onion as a medicinal plant
stimulated a lot of scientific investigations. With the advent of modern spectroscopic and
chromatographic techniques, the molecular basis for the odor, taste and biological activity of the onion
bulb has been investigated by the phytochemists (Block et al., 1993). The numerous works on onion
notwithstanding, little or nothing is known about the enzymes in onion (Lin and Yao, 1995a).
Proteases, of all the enzymes, remain the dominant enzyme type because of their extensive application
in the detergent, food and dairy industries (Kirk et al., 2002).
Several researches have been conducted on proteases, which include but not limited to
structural characterization, elucidation of mechanism of action, kinetics, sequencing and cloning of
genes (Fahmy et al., 2004). The activities of proteases have been reported in several plant materials.
Some reports are on legumes (Fischer et al., 2000; Yu and Greenwood, 1994), cereals (Wang et al.,
2003; Waters and Dalling, 1983), vegetables (Lin and Chan, 1990) and apricot and grapes (Ninomiya
et al., 1981). There are also reports on bulbs (Lin and Yao, 1995a & b). Aspartic proteinase and some
aminopeptidase activities are present in ungerminated seeds and some of these enzymes have been
purified and cloned (Beers et al., 2004; Sarkkinen et al., 1992; Weideranders, 2003). Cysteine
proteinases (Shutov and Vaintraub, 1987) and carboxypeptidase (Dunaevsky and Belozersky, 1989)
are expressed in germinating and post-germinating seeds.
The applications of plant proteases are too numerous to mention. For instance, proteases are
used in the alcohol, flour, milling and baking industries (Haarasilta and Pullinen, 1992). Papain, a plant
cysteine protease, is used in a process developed for solubilising fish and fish offal for animal feed
(Shahidi and Kamil, 2001), and traditionally used in chillproofing of beer (Moll, 1987). Papain,
bromelain and ficin are used in the tenderization of meat on commercial scale (Schwimmer, 1981).
However, the market for the latter two plant proteases, quantitatively, seems to be much smaller than
the market for papain (Adler-Nissen, 1994).
Cysteine proteases as reviewed above are used extensively in the food industry, and it would be
of interest to find additional sources of cysteine proteases with potentially useful properties or modes
of action. Therefore, in the present work we described the purification and characterization of a
cysteine protease from one of the Nigerian common onion cultivars (Red Creole).
2. Materials and Methods
2.1. Chemicals
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All chemicals were of analytical grade purchased from British Drug House (BDH Chemicals
Ltd, Poole, England), Hopkins and Williams (Essex, England) or Sigma Chemicals Co. St. Louis,
England.
2.2. Plant Material and Instruments
The onion bulbs used in this study were purchased by July 2010 from the Institute for
Agriculture Research (IAR), Zaria, Kaduna state, Nigeria. They were identified and validated at the
herbarium unit of the Department of Biological Sciences, Ahmadu Bello University, Zaria, Nigeria.
The voucher number as deposited at the herbarium unit is 900157. The instruments used for this study
were obtained in the Department of Biochemistry, Ahmadu Bello University, Zaria. The research was
carried out in the same department in 2010 and it took about six months to purify and characterize the
enzyme.
2.3. Preparation of Crude Extract
The crude protease extraction was carried out according to Lin and Yao (1995a) with a slight
modification. Ten grams of fresh onion was ground in a mortar in the presence of the extraction buffer
(30 mL) at a low temperature (4°C). It was sieved into a flat-bottomed flask using a muslin cloth and
then centrifuged at 10,000 g for 20 min. The supernatant liquid was collected as the crude extract and
immediately subjected to an assay of cysteine protease activity. The extraction buffer contains sodium
acetate buffer (100 mM, pH 5.0); sodium azide (0.2 g) was added as a preservative; cysteine (30 mM)
as reducing agent to activate the cysteine protease (Rao et al., 1998); EDTA (30 mM) was added as an
inhibitor of other metalloenzymes in the onion and polyvinylpyrolidine (1%) for the removal of
polyphenols.
2.4. Cysteine Protease Assay
Protease activity was determined according to Fahmy et al. (2004), who applied the method of
Dominguez and Cejudo (1996).
2.5. Protein Determination and Buffers
Protein concentration was quantified by the Biuret method (Layne, 1957). The buffers were
prepared according to Gomori (1955) and the final pH was confirmed with a pH meter.
2.6. Purification of Cysteine Protease
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Fifty millimolar of sodium acetate buffer, pH 6.0 containing 30 mM dithiothreitol and 30 mM
EDTA was used for the various steps in the purification.
Step 1: Partial Purification with Ammonium Sulphate
The partial fractionation of proteins was carried out according to the method of Ram et al.
(1986).
Step 2: Chromatography on DEAE-Cellulose
The dialysed sample (10 mL) was applied to a column of DEAE-cellulose (60 x 1.6 cm i.d.)
pre-equilibrated with 50 mM sodium phosphate buffer, pH 6.0 containing 2 mM cysteine and 1 mM
EDTA. The adsorbed material was eluted with stepwise gradient concentrations of sodium chloride
ranging from 0.0 M to 0.4 M prepared in the buffer earlier mentioned. The flow rate was 25 mL/h and
5-mL fractions were collected. The fractions containing the enzyme activity were pooled.
Step 3: Chromatography on Sephadex G-100
The pooled active fractions from DEAE-cellulose column chromatography were applied to a
Sephadex G-100 column (60 x 1.2 cm i.d.) previously equilibrated with 50 mM sodium phosphate
buffer, pH 6.0, containing 2 mM cysteine and 1 mM EDTA. The column was developed at a flow rate
of 12 mL/h and 3-mL fractions were collected.
2.7. Polyacrylamide Gel Electrophoresis
Electrophoresis under denaturing conditions was performed in 12.5% (w/v) acrylamide disc gel
according to the method of Laemmli (1970) using a Tris-glycine buffer, pH 8.3. Protein bands were
located by staining with Coomassie Brilliant Blue R-250.
2.8. Molecular Weight Determination
The molecular weight was estimated by SDS-polyacrylamide gel electrophoresis. Molecular
weight markers for SDS-PAGE were obtained from Sigma Chemical Co. (St Louis, MO). SDS
markers: BSA (molecular weight, 66 kDa), ovalbumin (molecular weight, 45 kDa), glyceraldehyde-3-
phosphate dehydrogenase (molecular weight, 36 kDa), carbonic anhydrase (molecular weight, 29
kDa), trypsinogen (molecular weight, 24 kDa), soybean trypsin inhibitor (molecular weight, 20 kDa)
and α–lactalbumin (molecular weight, 14 kDa).
2.9. Statistical Analysis
The analysis was carried out in triplicates for all determinations except where otherwise stated
and the results of the triplicate were expressed as mean ± standard error of mean (SEM). The SPSS
17.0 for windows Computer Software Package was used for the student t-test to compare the means of
Int. J. Modern Biochem. 2012, 1(1): 1-17
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5
various cations and inhibitors against the control. The level of significant difference was determined at
P < 0.05.
3. Results
3.1. Purification of the Cysteine Protease from Allium cepa L
The results of the purification of Allium cepa L. cysteine protease are summarized in Table 1.
The crude extract contained approximately 3.5301 enzyme units with a specific activity of 1.2959
unit/mg protein. Purification of the crude extract with ammonium sulphate, followed by DEAE-
cellulose chromatography and Sephadex G-100 chromatography resulted in a 15.94-fold purification
of cysteine protease with a 9.25% recovery. From the elution profile of the chromatography on DEAE-
cellulose column (Fig. 1), it can be visualized that three active peaks were eluted according to their
elution order. The final purified preparation was obtained by gel filtration on a Sephadex G-100
column (Fig. 2) and only one active peak emerged (20.6582 unit/mg protein).
3.2. Homogeneity and Molecular Weight
The electrophoretic behavior, under denaturing conditions of samples from gel filtration
purification step using Sephadex G-100 chromatography and DEAE-cellulose chromatography were
shown in Plate I. One band each was detected on the gels from both Sephadex G-100 (Lane B) and
DEAE-cellulose chromatography (Lane C) which indicated the homogeneity of the final preparation.
The molecular weight of the cysteine protease was estimated to be 22 kDa.
Table 1. Purification scheme for Allium cepa L. cysteine protease
Purification step Total
protein (mg)
Total
activity a
Specific activity
(mg-1
protein)
Purification
fold
Yield
(%)
Crude extract 2.7240 3.5301 1.2959 1.0000 100.0000
Ammonium sulphate
fractionation
1.3470 2.4845 1.8445 1.4200 70.3800
DEAE-cellulose 0.0360 0.4967 13.7972 10.6400 14.0700
Sephadex G-100 0.0158 0.3264 20.6582 15.9400 9.2500 Note:
a One unit of protease activity was defined as the amount of enzyme that hydrolyses 1 mg casein per hour under
standard assay conditions.
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Figure 1. A typical elution profile for the chromatography of Allium cepa L. (cv. Red creole) cysteine
protease on DEAE-cellulose column (60 x 1.6 cm i.d.) previously equilibrated with 50 mM sodium
phosphate buffer, pH 6 containing 30 mM cysteine and 30 mM EDTA at a flow rate of 25 mL/h and 5
mL fractions were collected.
3.3. Characterization of Cysteine Protease
The cysteine protease had an optimum pH 4.0 in sodium acetate buffer (Fig. 3). The effect of
pH on the stability of the protease was also carried out as shown in Fig. 4. The enzyme was
preincubated at various pH values for 30 min prior to substrate addition. The enzyme was stable in the
pH range 4 to 6. The enzyme exhibited a temperature optimum at 45 °C (Fig. 5). The effect of
temperature on the stability of protease is shown in Fig. 6. The enzyme was stable up to 50 °C and
afterwards drastically lost 54 and 80% of its activity at 60 and 92 °C, respectively. The time course for
the loss of the protease activity was maintained at 45 °C as shown in Fig. 7. The protease activity was
lost relatively slowly, retaining 89% activity after incubation for 1 h. Although, the difference in loss
of activity was rather high (18%) between 90 min and 100 min incubation, the enzyme lost
approximately 67% of its activity after 2 h. The Michaelis-Menten constant was estimated to be 6.74
mg casein/mL and the maximum velocity was estimated to be 0.86 U/mL (Fig. 8). A study of substrate
specificity for cysteine protease was made using 3 mg/mL of different substrates (Fig. 9). The affinity
Int. J. Modern Biochem. 2012, 1(1): 1-17
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7
of the substrate for the enzyme was decreased in the order of casein (KM 6.74 mg casein/mL), gelatin
(KM 13.767 mg gelatin/mL) and haemoglobin (KM 18.767 mg haemoglobin/mL). The effect of metal
cations on protease is shown in Fig. 10. All the examined cations whose bars are significantly lower
than the control had inhibitory effects on the protease and vice versa, and the different superscripts
depict significant difference. The effect of different reagent inhibitors on the Allium cepa cysteine
protease was examined (Fig. 11). It was found out that the enzyme was inhibited by thiol-blocking
agent, ethyl iodoacetate (48.72%) whose bar is significantly lower than the control. A study of
inhibition kinetics shows that ethyl iodoacetate caused a mixed inhibition (Fig. 12).
Figure 2. Gel filtration of Allium cepa L. (cv. Red creole) cysteine protease DEAE-cellulose fraction
on Sephadex G-100 (60 x 1.2 cm i.d.). The column was equilibrated with 50 mM sodium phosphate
buffer, pH 6.0 containing 30 mM cysteine and 30 mM EDTA at a flow rate of 12 mL/h and 3 mL
fractions were collected.
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Plate I. Electrophoretic patterns for Allium cepa L. cysteine protease, 10 μg protein of the purified
enzyme was loaded on the gel. SDS-PAGE for molecular weight determination: Lane A: Marker
proteins; Lane B: Band from Sephadex G-100 purification step; Lane C: Band from DEAE-cellulose
purification step
Figure 3. The pH optimum for Allium cepa cysteine protease. Each point represents the average of
three experiments ± SE.
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9
Figure 4. Effect of pH on Allium cepa cysteine protease stability. The enzyme was preincubated at
different pH values for 30 min prior to substrate addition, adjusted to and maintained at pH 5. The
cysteine protease assay was carried out as given in the text. Each point represents the average of three
experiments ± SE.
Figure 5. Temperature optimum for Allium cepa cysteine protease. Each point represents the average
of three experiments ± SE.
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Figure 6. Effect of temperature on Allium cepa cysteine protease stability. The enzyme was
preincubated at different temperatures for 30 min prior to substrate addition, adjusted to and
maintained at 30 oC. The cysteine protease assay was carried out as given in the text. Each point
represents the average of three experiments ± SE.
Figure 7. Time course of inactivation of cysteine protease activity of Allium cepa incubated at 45 oC.
The1.0 mL aliquots of the purified sample of Allium cepa were withdrawn at different time intervals,
cooled to and maintained at 30 oC, and the cysteine protease assay was carried out as given in the text.
Int. J. Modern Biochem. 2012, 1(1): 1-17
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Figure 8. Lineweaver-Burk plot relating Allium cepa cysteine protease reaction velocities to casein
concentration. KM was calculated as mg casein/mL.
Figure 9. Double reciprocal plots showing the effect of casein, gelatin and haemoglobin
concentrations on the activity of cysteine protease.
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Figure 10. Effect of metal cations on Allium cepa cysteine protease. Enzymes were preincubated for
15 min at 37 oC with 2 mM of listed cations prior to substrate addition. Activity without added metal
cations was taken as 100%. Cations with different superscripts from the control differ significantly
from it. Each bar represents the average of three experiments ± standard error (SE). Error bars
represents SEs.
Figure 11. Effect of different compounds on Allium cepa cysteine protease. Enzymes were
preincubated for 15 min at 37 oC with 5 mM of listed compounds prior to substrate addition. Activity
without added compounds was taken as 100%. Inhibitors with different superscripts from the control
differ significantly from it. Each bar represents the average of three experiments ± standard error (SE).
Error bars represents SEs. DTT = Dithiothreitol, STI = Soybean Trypsin Inhibitor, 2-ME = 2-
Mercaptoethanol, EDTA = Ethylenediaminetetraacetic acid, PMSF = Phenylmethylsulphonylfluoride,
IA = ethyl Iodoacetate.
0
20
40
60
80
100
120
140
160
Control PMSF Cysteine 2-ME EDTA DTT IA STI
Inhibitor (5 mM each)
% R
ela
tiv
e a
cti
vit
y
a a b b a b b a
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Figure 12. Kinetics of inhibition for Allium cepa cysteine protease by IA. Plot of reciprocal
concentration of casein (mg).
4. Discussion
The purification procedure of Allium cepa L. cysteine protease yields an essentially
homogenous preparation with an overall recovery of 9.25%. Similar results have been reported in
some cases (Fahmy et al., 2004; Usha and Singh, 1996). The estimated molecular mass of Allium cepa
L. cysteine protease (22 kDa) was similar with respect to some other molecular masses from plants (12
kDa-36 kDa) (De Barros and Larkins, 1990; Jinka et al., 2009). However, it is smaller than the
molecular masses of cysteine proteases from barley (29 kDa-37 kDa) (Koehler and Ho, 1990; Zhang
and Jones, 1996), resting wheat grains (40 kDa-50 kDa) (Dominguez and Cejudo, 1995), and
germinating wheat grains (~60 kDa) (Fahmy et al., 2004). The molecular weight of an enzyme seems
to be related to origin or function of the enzyme and the action of a high molecular weight enzyme
generally occurs by complicated convertible processes (Akuzawa and Okitani, 1995).
The pH optimum of Allium cepa L. cysteine protease was 4, suggesting that it acts in an acidic
cellular compartment such as the vacuole as reported by Sutoh et al. (1999). This value agrees with the
pH optima of other plant cysteine proteases (pH 3.8-4.6) (Asano et al., 1999; Jinka et al., 2009; Muntz
and Shutov, 2002; Zhang and Jones, 1996). Allium cepa L. cysteine protease had a temperature
optimum at 45 °C and was stable up to 50 °C. A temperature-activity profile for barley aleurain
(cysteine protease) showed optimal activity in the range of 25-35 °C (Fahmy et al., 2004).
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The only potent inhibitor for Allium cepa L. protease was ethyl iodoacetate, where 5 mM of
ethyl iodoacetate inhibited 48.72% proteolytic activity and this would be considered specific for
cysteine proteases. According to Sutoh et al. (1999), inhibitors such as para-hydroxymercuribenzoate,
iodoacetate, E-64, para-chloromercuribenzoate and iodoacetamide inhibit cereal cysteine proteases,
varyingly. The inhibitors specific for serine-(PMSF) and metallo-(EDTA) proteases had no inhibitory
effect on the enzyme. Sulfhydryl reagents, 2-mercaptoethanol, cysteine and dithothreitol stimulated the
activity of the enzyme. Soybean cysteine proteases were exhibited in the presence of a sulfhydryl
reagent, such as 2-mercaptoethanol (Asano et al., 1999). Allium cepa L. cysteine protease resists
inhibition by proteinaceous inhibitor, soybean trypsin inhibitor, which is present in protein-rich foods
such as soybeans.
Some of the examined metal cations, which include Hg2+
, Ca2+
, Cu2+
and Zn2+
inhibited the
enzyme with 65.21, 13.64, 48.55 and 22.61% inhibition, respectively. This is similar to the metal ions
inhibition in T. aestivum cysteine protease (Fahmy et al., 2004). However, barley cysteine protease
EP1 was inhibited completely by Cu2+
, Zn2+
, or Hg2+
while EP2 was not inhibited by Cu2+
or Zn2+
and
was stimulated 20% by Ca2+
(Miller and Huffaker, 1981). The winged-bean acidic protease has also
been reported to be strongly inhibited by Hg2+
(Usha and Singh, 1996).
Casein was discovered to have the highest affinity for the enzyme followed by gelatin and
hemoglobin. This differs from that obtained by Fahmy et al. (2004), as gelatin was shown to be a
better substrate than both casein and hemoglobin; and haemoglobin was a better substrate when
compared with casein. Zhang and Jones (1996) also reported different affinities for different protein
substrates. Allium cepa L. cysteine protease has KM value of 6.743 mg casein/mL, which is not
compared easily with KM values from other sources since most of the other sources used different
substrates. However, it can be said to have lower affinity towards casein compared to the affinity of
cysteine protease from T. aestivum towards azocasein (KM 2.8 mg azocasein/mL) (Fahmy et al., 2004)
and much lower than that from barley proteases (0.21-0.47 mg azocasein/mL) (Miller and Huffaker,
1981).
The properties of this enzyme are similar to those of cysteine proteases used in food industries.
Papain, bromelain and ficin have broader pH-activity profile ranging from 5 to 9 and stable to heat but
are known to be inactivated by oxidizing agents and by exposure to air (Fahmy et al., 2004). However,
the latter two are slightly less thermostable (Adler-Nissen, 1994). Digestive enzymes such as cysteine
proteases which work optimally at pH 4.0 and temperature at 25 oC are used in fish industry (Gildberg
and Raas, 1979; Stenfansson, 1988).
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15
5. Conclusions
In conclusion, most of the prerequisites for industrial utilization of enzymes are emphasized for
the obtained Allium cepa L. cysteine protease in the present study. It has good storage stability as it has
its highest stability at 50 °C and a broad acidic pH (pH 3-5). Therefore, Allium cepa L. cysteine
protease have properties, which offer potential for food industries.
Potential Conflicts of Interest
The authors declare no conflict of interest.
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Author’s Contributions
Ndidi U. S. carried out the purification, SDS gels, enzyme assays and characterization and
drafted the manuscript. Nzelibe H. C. participated in project conception, design, coordination and
supervision and helped to draft the manuscript. All authors read and approved the final manuscript.