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Agricultural Science Research Journals Vol. 2(8), pp. 434-440, August 2012 Available online at http://www.resjournals.com/ARJ ISSN-L:2026-6073© 2012 International Research Journals

Full Length Research Paper

Isolation and characterization protease enzyme from leguminous seeds

M. Akhtaruzzaman 1 , *N.H.M. Rubel Mozumder 2 , Ripa Jamal 3 , Atikur Rahman 4 and Tanjina Rahman 3

1 Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh 2 Department of Food Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur- 5200, Bangladesh

3 Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh 4 Department of Food Processing and Preservation, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200, Bangladesh.

*Corresponding Author’s Email: rubel.infs@gmail.com

ABSTRACT

The present study was conducted to isolate and characterize the proteases from seven leguminous seeds: soybean, lentil, black gram, green gram, bengal gram, groundnut and pea bean. We elaborated the easy procedure for isolation of protease from leguminous seeds by using (NH 4 ) 2 SO 4 precipitation. The effect of pH and temperature of protease activity were determined. This study revealed that the protein concentration of crude extract ranged between 2.215 to 2.40 mg/ml in which groundnut was highest protein concentration (2.40 mg/ml) and lentil accounted was lowest concentration (2.21 mg/ml). The pH profile of proteases showed maximum specific activity at pH 7.5 to 9.0 with one main peak at pH 9.0. Among the seeds, bengal gram showed highest specific activity (0.007659 U/mg of protein) at pH 7.5 but lowest activity was observed in pea bean (0.001681 U/mg of protein) at pH 9.0. The temperature vs. specific and catalytic activity of all proteases relationship demonstrated a symmetrical distribution with one main peak and optimum at 37 o C except black gram which showed two main peaks at 37 o C and 70 o C respectively. The paper concludes that leguminous seeds can be source of proteases for industrial purposes.

Key Words. Protease activity, Characterization, Industrial purposes, leguminous seeds.

INTRODUCTION

Proteases are one of the largest groups of industrial enzymes that catalyze the hydrolytic reactions by cleaving peptide bonds in protein. Proteases may be classified as two major groups; exopeptidase and endopeptidase based on their ability to degrade N- or C- terminal peptide bond. Endopeptidases, which have more potent industrial applications than exo-peptidases, can be divided into four types (aspartic, cysteine, metallo and serine protease) on the basis of their active site and sensitivity to various inhibitors (Al- sherhi and Mostafa, 2004; Sandhya et al., 2004). Proteases have been used in processing of various foods such as calf rennet or chymosin in cheese making in which chymosin hydrolyze

the specific peptide bond (the Phe-15-Met 106 bond) to generate park-k-Casein and macro peptides (Smit et al., 2005). They are also used in preparation of soy sauce and other hydrolysates (Wang & Wang, 2004) and used of papain for meat tenderization (Soper, 1998). The bitterness of protein hydrolysate in soybean is a major barrier of uses as a food and health care product. The intensity of bitterness is increased due to the number of hydrophobic bonds present in the protein. The proteases, which can cleave hydrophobic bond into amino acids, are valuable in debittering of protein hydrolysates in soy- based products (Rao et al., 1998). In leather processing steps such as soaking, dehairing and baiting, the use of

proteases as alternatives to chemical detergent powder has proved successful modification to remove protein and bloodstain from the skin and improving environmental pollution (Nadafi and Deobagkar, 2005). In pharmaceutical industry, they offer a gentle and selective debridement, supporting the natural healing process in the successful local management of skin ulcerations by the efficient removal of necrotic material (Sjodahl et al.,

2002).

Proteases are mainly obtained from microbial sources for industrial purposes Some examples of microbial proteases include: microbial rennin like enzyme from Mucor miechi, pepsin like acid protease from Aspergillus spp. and Rhizopus spp., acid protease from thermophilic Penicillium sp. and a neutral metalloprotease, Pseu-216A from Aspergillus oryzae (Kumar et al., 2005; Tremacoldi et al., 2004). Though microbial protease are the predominant source of industrial enzyme due to their broad biochemical diversity, rapid growth of microorganisms and limited space required for cell cultivation, it involves advanced technology in the field of biotechnology and microbiology ( Rao et al., 1998). In our country, there are few opportunities to do a research on microbial enzyme for industrial purposes. In view of unrestricted availability, the plant sources would be a possible alternative of microbial and animal proteases. Beside this, to study the mechanism of protein mobilization process and in-vitro degradation of protein, several studies have been conducted on purification and characterization of proteases and peptidases, some of which occur only transiently in germinating leguminous seeds (Ashton, 1976; Davy et al., 1998; Shutov and Vaintraub, 1987). The understanding of such facts influenced us to conduct this study. In the present study, we tried to establish an easy procedure for the isolation and characterization of protease enzyme obtained from leguminous seeds.

METHODS AND MATERIALS

Materials

The present study was conducted in the laboratory of the Institute of Nutrition and Food Science, University of Dhaka (DU) on seven kinds of leguminous seeds collected from six market places of Dhaka city. These were soybean (Glycine max), lentil (Lens esculenta), black gram (Vigna mungo), green gram (Vigna radiate), bengal gram (Cicer arietinum,) groundnut (Arachis hypogaea) and pea bean (Phaseolus vulgaris). Bovine serum albumin (BSA), casein, hemoglobin and Folin ciocalteau reagent (FCR) were collected from E. Merk (Germany) and Tricholoacetic acid (TCA) from BDH (England). The other chemicals and reagents used in this experiment were analytical grade and obtained from Institute of Nutrition and Food Science (INFS), University of Dhaka without further purification.

Methods

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Isolation and preparation of protease enzyme (crude extract)

The seeds (soybean, lentil, black gram, green gram, bengal gram, groundnut and pea bean), approximately

fifty grams (50g) in amounts, were washed separately and then soaked in distilled water at room temperature

for overnight germination. After that, all the seeds except

soybean and groundnut were ground by electric

homogenizer without using acetone, because of their low

fat content. Soybean and groundnut were homogenized

with cold acetone to remove the fat. Then the homogenates were finely powdered in a pre-chilled mortar and mixed with chilled 10mMTris-HCl buffer at pH 8.0 containing 2M NaCl for 3 hours. The extracted mixtures were filtered through gauge and filtrates were centrifuged at 10000 rpm for 10 minutes below 4 o C. The

collected supernatant was used for the estimation of extracellular concentration of protein and further purifications.

(NH 4 ) 2 SO 4 precipitation: The collected supernatants were saturated with 50% solid (NH 4 ) 2 SO 4 for overnight precipitation. After precipitation, they were centrifuged at 10000 rpm for 30 min below 4 o C. The collected precipitated were dissolved in 10 nm Tris-HCl buffer (pH 8) and dialyzed against the same buffer and finally centrifuged at 5000 rpm for 10 min. The supernatant was used as crude enzyme for the assay of specific activity of enzyme and characterization.

Protein measurement

Protein concentration was determined by the method of Lowry et al., 1951 using bovine serum albumin (BSA) as

standard protein. The amount of the soluble protein was calculated from the standard curve as mg of protein per

ml of test samples.

Assay of protease enzyme: Determination of catalytic activity of protease

Assay of protease was determined by the method of Anson (1938). The reaction mixture consisted of 250 µl of crude enzyme solution (supernatant) obtained by centrifugation and 1 ml of substrate (2% hemoglobin for acidic pH and 2% casein for alkaline pH in various buffers) was incubated at 37 o C for 30 minutes. The reaction was stopped by the addition of 500 µl of 10% trichloroacetic acid (TCA). For the control, the substrate was precipitated with 500 µl 10% TCA before adding the enzyme solution and then treated as described above. The resulting precipitate was removed by centrifugation and collected 300 µl supernatant was added to the 2.5 ml

436 Agric. Sci. Res. J.

Table 1. Concentration of protease (crude extract) from leguminous seeds

Common Name

Botanical Name

volume of (NH 4 ) 2 SO 4 precipitation (ml)

Protein Concentration (mg/ml)

Soybean

Glycine max

97.0

2.35

Lentil

Lens esculenta

98.0

2.21

Black gram

Vigna mungo

97.0

2.30

Green gram

Vigna radiata

97.0

2.33

Bengal gram

Cicer arietinum

97.0

2.39

Groundnut

Arachis hypogaea

97.5

2.40

Pea bean

Phaseolus vulgaris

97.0

2.38

cu-alkaline solution using vortex and allowed to stand for 15 minutes. The mixture was then similarly mixed well with 250 µl of double times diluted folin ciocalteau reagent (FCR). The absorbance was measured at 660 nm by spectrophotometer. The activity of proteases is designated as endopeptidases activity since casein (Kunitz, 1946) or haemoglobin (Bergmeyer, 1984) used as a substrate. The protease activity was expressed as the difference of absorbance at 660 nm between the control sample and the test sample. A unit of protease activity is defined as the amount of enzyme required to release TCA- soluble fragment giving a blue colour equivalent to one microgram of product under the same condition of assay.

Calculation of specific activity

Specific activity of the protease was calculated by the activity of protease per milligram of protein per minute specifically dividing the determined protease activity values on the protein content results (Equation 1) Specific activity= Absorbance at 660 nm X (Protein

concentration)

-1

X (min) -1 ……… (1)

Effect of pH and temperature specific activity of proteases: Partial characterization

Optimum temperature and pH of protease activity were determined using various temperatures (10 o C, 20 o C, 37 o C, 50 o C and 70 o C) and pH (2.0, 4.0, 7.5, 9.0 and

11.0).

RESULTS AND DISCUSSIONS

The present study investigated the protease activity obtained from seven leguminous seeds by establishing an easy assay system using two substrates: casein for alkaline protease and haemoglobin for acidic protease. In this study, the most widely used ammonium sulfate fractionation was carried out directly from the crude extract of germinating seeds and maximum amount of

protease enzyme was recovered in the precipitation obtained by fractionating with 50% (NH 4 ) 2 SO 4 saturation. The protein concentrations of extracellular proteases isolated from seven leguminous seeds were shown in Table 1. The concentration of extracellular soluble protein ranged between 2.215-2.40 mg/ml in which groundnut showed highest protein concentration (2.40 mg/ml) and lentil accounted lowest concentration (2.21 mg/ml). Dahot M U. (1992) investigated on proteases present in some plant seeds and found the ranged of protein concentration between 1.10-2.76 mg/ml with highest value in soybean seed (Glycine max) (2.76 mg/ml) which is in a good agreement with our value reported for soybean seeds (2.35 mg/ml). Ericson and Chrispeels (1973) observed that the storage proteins of leguminous seeds have recently been shown to be glycoproteins containing both mannose and glucosamine. The effect of different pH (2.0, 4.0, 5.0, 7.5, 9.0 and 11.0) on the specific proteases activity (Units/mg) of germinated leguminous seeds using casein (alkaline protease) and haemoglobin (acidic protease) as a substrate was depicted in Table 2. All the enzymes showed maximum specific activity at pH 7.5 and 9.0 when they were treated by 2% casein. From the table, Bengal gram showed highest specific activity (0.007659 U/mg of protein) at pH 7.5 but lowest activity was observed in pea bean (0.001681 U/mg of protein) at pH 9.0. The second highest specific activity was observed in green gram, (0.005722 U/ mg protein) that was lower than the previous studies (Dahot MU, 1992; Lin and Yao, 1996; Rahman et al., 2007; Vidyavati et al. 1983). With this assay system, the pH vs. protease (catalytic) activity relationship was depicted by line graph in Figure 1. The pH profiles showed one main peak at 9.0. A very similar situation (pH maxima at 8.4 and 9.2) was also reported by Evans et al., 2011 for the extract of palm weevil (Rhynchophorous palmarum).The results from effect of pH indicate that the alkaline proteases involved in all seeds were more potent than the acidic proteases. This alkaline protease may be playing an important role in industrial food applications such as production of soy sauce, digestion of soy bean protein and in leather and detergent industries (Kamini et al., 2004; Nadafi and

Table 2. Effect of pH on specific activity (U/mg) of proteases at 37 0 C

Akhtaruzzaman et al

437

Sample

Specific protease activity (U/mg) at different pH

 
 

2.0

4.0

7.5

9.0

11.0

Soybean

0.001986

0.002553

0.002723

0.005106

0.002156

Lentil

0.001204

0.002287

0.003612

0.003070

0.001505

Black gram

0.001101

0.003362

0.003536

0.005217

0.001159

Green gram

0.000572

0.004063

0.004578

0.005722

0.001030

Bengal gram

0.001116

0.007476

0.007659

0.005523

0.001283

Ground nut

0.00027

0.002222

0.002666

0.002167

0.000722

Pea bean

0.000448

0.001232

0.001569

0.001681

0.001457

Table 3. Effect of temperatures on specific activity (U/mg) of proteases at pH 4.0 and pH 9.0 isolated from leguminous seeds

Sample

Temperature ( 0 C)

pH 4.0

pH 9.0

 

Specific activity

Specific activity

(U/mg)

(U/mg)

Soybean

10

0.001191

0.000511

20

0.002156

0.000965

37

0.002723

0.005106

50

0.003574

0.001702

70

0.001021

0.001135

Lentil

10

0.000361

0.000783

20

0.000722

0.001204

37

0.002287

0.003070

50

0.001746

0.001023

70

0.000903

0.000542

Black gram

10

0.000638

0.001043

20

0.001739

0.001449

37

0.003536

0.005217

50

0.002203

0.001739

70

0.000522

0.001217

Green gram

10

0.001946

0.001602

20

0.003891

0.002518

37

0.004578

0.005722

50

0.005036

0.004464

70

0.002289

0.002232

Bengal gram

10

0.000614

0.000725

20

0.001116

0.004351

37

0.007755

0.005523

50

0.003347

0.002957

70

0.000725

0.000391

Ground nut

10

0.000500

0.001000

20

0.001000

0.000889

37

0.002222

0.002167

50

0.001722

0.001111

70

0.000556

0.001056

Pea bean

10

0.000168

0.000560

20

0.001008

0.000672

37

0.001232

0.001681

50

0.000728

0.001176

70

0.00448

0.000392

438 Agric. Sci. Res. J.

0.16 Soybean 0.14 Lentil 0.12 0.1 Black gram 0.08 0.06 Green gram 0.04 Bengal gram
0.16
Soybean
0.14
Lentil
0.12
0.1
Black gram
0.08
0.06
Green gram
0.04
Bengal gram
0.02
0
Ground nut
2
4
7.5
9
11
PH
Catalytic (protease) activity (Units/ml)

Figure 1. Effect of pH on catalytic activity of protease at 37 0 C.

0.16 0.14 0.12 Soy bean 0.1 Lentil 0.08 Black gram Green gram 0.06 Bengal gram
0.16
0.14
0.12
Soy bean
0.1
Lentil
0.08
Black gram
Green gram
0.06
Bengal gram
0.04
Ground nut
Pea bean
0.02
0
10
20
37
50
70
Temperature 0 (oC)
Temperature (
C)
Catalytic activity (Units/ml)

Figure 2a. Effect of temperature on catalytic activity of protease at pH 4.0

Deobagkar, 2005). The effect of temperature on specific protease activity at pH 4.0 and 9.0 from leguminous seeds was studied and presented in Table 3. At pH 4.0, all the enzymes in the present study demonstrated maximum specific

activity at 37 o C except soybean and green gram; both showed maximum activity at 50 o C. But the specific activity of protease from all leguminous seeds expressed their maximum activity at 37 o C and pH 9.0. Dahot MU, 1992 investigated on plant protease and found that the

Akhtaruzzaman et al

439

0.12 0.1 0.08 0.06 0.04 0.02 0 10 20 37 50 70 Catalytic activity (Units/ml)
0.12
0.1
0.08
0.06
0.04
0.02
0
10
20
37
50
70
Catalytic activity (Units/ml)

Temperature (oC)

Soy bean37 50 70 Catalytic activity (Units/ml) Temperature (oC) Lentil Black gram Green gram Bengal gram Ground

Lentil37 50 70 Catalytic activity (Units/ml) Temperature (oC) Soy bean Black gram Green gram Bengal gram

Black gram20 37 50 70 Catalytic activity (Units/ml) Temperature (oC) Soy bean Lentil Green gram Bengal gram

Green gram20 37 50 70 Catalytic activity (Units/ml) Temperature (oC) Soy bean Lentil Black gram Bengal gram

Bengal gram20 37 50 70 Catalytic activity (Units/ml) Temperature (oC) Soy bean Lentil Black gram Green gram

Ground nut20 37 50 70 Catalytic activity (Units/ml) Temperature (oC) Soy bean Lentil Black gram Green gram

Pea bean37 50 70 Catalytic activity (Units/ml) Temperature (oC) Soy bean Lentil Black gram Green gram Bengal

Figure 2b. Effect of temperature on catalytic activity of protease at pH 9.0

optimum temperature for enzyme reaction was 35 o C, which was a good consistent with our results but Evans et al., 2011 reported optimum temperature for the palm weevil was 23 o C on 0.03% casein. Kamini et al., 2004 and Aoki et al., 2004 have shown that the protease activity with optimum temperature less than 20 o C is considered as a cold protease. So, the proteases from present study can be excellent source for industrial purposes where requires low or mild (optimum) temperature as a vital important factors in food processing steps. The effect of temperature on catalytic activity of protease (temperature vs. catalytic activity relationship) at pH 4.0 and 9.0 was mapped out at Figure 2a and Figure 2b. From Figure 2a, the protease enzyme from soybean seed showed a symmetrical distribution with one main peak at 37 o C but enzyme from black gram showed two main peaks at 37 o C and 70 o C. This characteristic indicates that enzymes are ampholytes (having carboxyl and amino group) and undergo change in respect to solubility, osmotic pressure, viscosity etc. The change in enzymatic activity with varying pH levels is due to changes in ionization of the enzyme, the substrate or the enzyme substrate complex (Gerald Reed, 1975). All the enzymes showed their symmetrical distribution curve at 37 o C under pH 9.0 (Figure 2b) whereas catalytic activity from pea bean was less significant. 0

CONCLUSION

In present study, we extracted the proteases from overnight imbibed leguminous seeds and tried to make

an easy assay of protease activity procedure for the leguminous seeds using hemoglobin and casein as substrate. Using this easy procedure during the germination, it seems to be important to purify and characterize these acidic and alkaline proteases from the leguminous seeds. The study clearly expressed that all the protease enzymes isolated from seeds were edible and showed higher activities in both acidic and alkaline conditions. They have the potentiality in our food industries.

ACKNOWLEDGEMENTS

This work was supported through a grant provided by University Grant Commission of Bangladesh (UGC).

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