purification and characterization of a cysteine protease from

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.

http://www.modernscientificpress.com/Journals/IJBioChem.aspx

Int. J. Modern Biochem. 2012, 1(1): 1-17


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



3

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



4

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



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.



6

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



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.



8

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.



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.



10

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.



11

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.



12

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



13

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).



14

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).



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.

purification and characterization of a cysteine protease from

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