Akhtaruzzaman Et Al

8/19/2019 Akhtaruzzaman Et Al

http://slidepdf.com/reader/full/akhtaruzzaman-et-al 1/7

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

Full Length Research Paper

Isolation and characterization protease enzyme fromleguminous seeds

M. Akhtaruzzaman1, *N.H.M. Rubel Mozumder 2, Ripa Jamal3, Atikur Rahman4 and Tanjina Rahman3

1Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh

2Department of Food Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur-

5200, Bangladesh3Institute 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: [email protected]

ABSTRACT

The present study was conducted to isolate and characterize the proteases from seven leguminousseeds: soybean, lentil, black gram, green gram, bengal gram, groundnut and pea bean. We elaboratedthe easy procedure for isolation of protease from leguminous seeds by using (NH4)2SO4 precipitation.The effect of pH and temperature of protease activity were determined. This study revealed that theprotein concentration of crude extract ranged between 2.215 to 2.40 mg/ml in which groundnut washighest 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 7.5but 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 withone main peak and optimum at 37

oC except black gram which showed two main peaks at 37

oC and 70

oC

respectively. The paper concludes that leguminous seeds can be source of proteases for industrialpurposes.

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 bycleaving peptide bonds in protein. Proteases may beclassified as two major groups; exopeptidase andendopeptidase based on their ability to degrade N- or C-terminal peptide bond. Endopeptidases, which have morepotent industrial applications than exo-peptidases, can bedivided into four types (aspartic, cysteine, metallo andserine protease) on the basis of their active site andsensitivity to various inhibitors (Al- sherhi and Mostafa,2004; Sandhya et al., 2004). Proteases have been usedin processing of various foods such as calf rennet orchymosin 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 sauceand other hydrolysates (Wang & Wang, 2004) and usedof papain for meat tenderization (Soper, 1998). Thebitterness of protein hydrolysate in soybean is a majobarrier of uses as a food and health care product. Theintensity of bitterness is increased due to the number ofhydrophobic bonds present in the protein. The proteaseswhich can cleave hydrophobic bond into amino acids, arevaluable in debittering of protein hydrolysates in soy-based products (Rao et al., 1998). In leather processingsteps such as soaking, dehairing and baiting, the use of



proteases as alternatives to chemical detergent powderhas proved successful modification to remove protein andbloodstain from the skin and improving environmentalpollution (Nadafi and Deobagkar, 2005). Inpharmaceutical industry, they offer a gentle and selectivedebridement, supporting the natural healing process in

the successful local management of skin ulcerations bythe efficient removal of necrotic material (Sjodahl et al., 2002).Proteases are mainly obtained from microbial sources forindustrial purposes Some examples of microbialproteases include: microbial rennin like enzyme fromMucor miechi , pepsin like acid protease from Aspergillusspp. and Rhizopus spp., acid protease from thermophilicPenicillium sp. and a neutral metalloprotease, Pseu-216Afrom Aspergillus oryzae (Kumar et al., 2005; Tremacoldiet al., 2004). Though microbial protease are thepredominant source of industrial enzyme due to theirbroad biochemical diversity, rapid growth ofmicroorganisms and limited space required for cellcultivation, it involves advanced technology in the field ofbiotechnology and microbiology ( Rao et al., 1998). In ourcountry, there are few opportunities to do a research onmicrobial enzyme for industrial purposes.

In view of unrestricted availability, the plant sourceswould be a possible alternative of microbial and animalproteases. Beside this, to study the mechanism of proteinmobilization process and in-vitro degradation of protein,several studies have been conducted on purification andcharacterization of proteases and peptidases, some ofwhich occur only transiently in germinating leguminousseeds (Ashton, 1976; Davy et al., 1998; Shutov andVaintraub, 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 isolationand characterization of protease enzyme obtained fromleguminous seeds.

METHODS AND MATERIALS

Materials

The present study was conducted in the laboratory of theInstitute of Nutrition and Food Science, University ofDhaka (DU) on seven kinds of leguminous seeds

collected from six market places of Dhaka city. Thesewere soybean (Glycine max ), lentil (Lens esculenta),black gram (Vigna mungo), green gram (Vigna radiate),bengal gram (Cicer arietinum,) groundnut ( Arachishypogaea) and pea bean (Phaseolus vulgaris). Bovineserum albumin (BSA), casein, hemoglobin and Folinciocalteau reagent (FCR) were collected from E. Merk(Germany) and Tricholoacetic acid (TCA) from BDH(England). The other chemicals and reagents used in thisexperiment were analytical grade and obtained fromInstitute of Nutrition and Food Science (INFS), Universityof Dhaka without further purification.

Akhtaruzzaman et al 435

Methods

Isolation and preparation of protease enzyme (crudeextract)

The seeds (soybean, lentil, black gram, green gram

bengal gram, groundnut and pea bean), approximatelyfifty grams (50g) in amounts, were washed separatelyand then soaked in distilled water at room temperaturefor overnight germination. After that, all the seeds excepsoybean and groundnut were ground by electrichomogenizer without using acetone, because of their lowfat content. Soybean and groundnut were homogenizedwith cold acetone to remove the fat. Then thehomogenates were finely powdered in a pre-chilledmortar and mixed with chilled 10mMTris-HCl buffer at pH8.0 containing 2M NaCl for 3 hours. The extractedmixtures were filtered through gauge and filtrates werecentrifuged at 10000 rpm for 10 minutes below 4

oC. The

collected supernatant was used for the estimation oextracellular concentration of protein and furthepurifications.

(NH4)2SO4 precipitation: The collected supernatantswere saturated with 50% solid (NH4)2SO4 for overnighprecipitation. After precipitation, they were centrifuged a10000 rpm for 30 min below 4

oC. The collected

precipitated were dissolved in 10 nm Tris-HCl buffer (pH8) and dialyzed against the same buffer and finallycentrifuged at 5000 rpm for 10 min. The supernatant wasused as crude enzyme for the assay of specific activity ofenzyme and characterization.

Protein measurement

Protein concentration was determined by the method ofLowry et al., 1951 using bovine serum albumin (BSA) asstandard protein. The amount of the soluble protein wascalculated from the standard curve as mg of protein perml of test samples.

Assay of protease enzyme: Determination of catalyticactivity of protease

Assay of protease was determined by the method o Anson (1938). The reaction mixture consisted of 250 µl ocrude enzyme solution (supernatant) obtained bycentrifugation and 1 ml of substrate (2% hemoglobin foacidic pH and 2% casein for alkaline pH in variousbuffers) was incubated at 37

oC for 30 minutes. The

reaction was stopped by the addition of 500 µl of 10%trichloroacetic acid (TCA). For the control, the substratewas precipitated with 500 µl 10% TCA before adding theenzyme solution and then treated as described aboveThe resulting precipitate was removed by centrifugationand 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 (NH4)2SO4 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 2.30Green 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 for15 minutes. The mixture was then similarly mixed wellwith 250 µl of double times diluted folin ciocalteaureagent (FCR). The absorbance was measured at 660nm by spectrophotometer. The activity of proteases isdesignated as endopeptidases activity since casein(Kunitz, 1946) or haemoglobin (Bergmeyer, 1984) usedas a substrate. The protease activity was expressed asthe difference of absorbance at 660 nm between thecontrol sample and the test sample. A unit of proteaseactivity is defined as the amount of enzyme required torelease TCA- soluble fragment giving a blue colourequivalent to one microgram of product under the samecondition of assay.

Calculation of specific activity

Specific activity of the protease was calculated by theactivity of protease per milligram of protein per minute

specifically dividing the determined protease activityvalues on the protein content results (Equation 1)Specific activity= Absorbance at 660 nm X (Proteinconcentration)

-1 X (min)

-1……… (1)

Effect of pH and temperature specific activity ofproteases: Partial characterization

Optimum temperature and pH of protease activity weredetermined using various temperatures (10

oC, 20

oC,

37oC, 50

oC and 70

oC) and pH (2.0, 4.0, 7.5, 9.0 and

11.0).

RESULTS AND DISCUSSIONS

The present study investigated the protease activityobtained from seven leguminous seeds by establishingan easy assay system using two substrates: casein foralkaline protease and haemoglobin for acidic protease. Inthis study, the most widely used ammonium sulfatefractionation was carried out directly from the crudeextract of germinating seeds and maximum amount of

protease enzyme was recovered in the precipitationobtained by fractionating with 50% (NH4)2SO4 saturation.

The protein concentrations of extracellular proteasesisolated from seven leguminous seeds were shown inTable 1. The concentration of extracellular soluble proteinranged between 2.215-2.40 mg/ml in which groundnushowed highest protein concentration (2.40 mg/ml) andlentil accounted lowest concentration (2.21 mg/ml). DahoM U. (1992) investigated on proteases present in someplant seeds and found the ranged of proteinconcentration between 1.10-2.76 mg/ml with highesvalue in soybean seed (Glycine max ) (2.76 mg/ml) whichis in a good agreement with our value reported forsoybean seeds (2.35 mg/ml). Ericson and Chrispeels(1973) observed that the storage proteins of leguminousseeds have recently been shown to be glycoproteinscontaining both mannose and glucosamine.

The effect of different pH (2.0, 4.0, 5.0, 7.5, 9.0 and11.0) on the specific proteases activity (Units/mg) o

germinated leguminous seeds using casein (alkalineprotease) and haemoglobin (acidic protease) as asubstrate was depicted in Table 2. All the enzymesshowed maximum specific activity at pH 7.5 and 9.0when they were treated by 2% casein. From the tableBengal gram showed highest specific activity (0.007659U/mg of protein) at pH 7.5 but lowest activity wasobserved in pea bean (0.001681 U/mg of protein) at pH9.0. The second highest specific activity was observed ingreen gram, (0.005722 U/ mg protein) that was lowethan the previous studies (Dahot MU, 1992; Lin and Yao1996; Rahman et al., 2007; Vidyavati et al. 1983). Withthis assay system, the pH vs. protease (catalytic) activity

relationship was depicted by line graph in Figure 1. ThepH profiles showed one main peak at 9.0. A very similarsituation (pH maxima at 8.4 and 9.2) was also reportedby Evans et al., 2011 for the extract of palm weevi(Rhynchophorous palmarum).The results from effect opH indicate that the alkaline proteases involved in alseeds were more potent than the acidic proteases. Thisalkaline protease may be playing an important role inindustrial food applications such as production of soysauce, digestion of soy bean protein and in leather anddetergent industries (Kamini et al., 2004; Nadafi and




Table 2. Effect of pH on specific activity (U/mg) of proteases at 370C

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 ( C) pH 4.0 pH 9.0

Specific activity(U/mg)

Specific activity(U/mg)

Soybean 1020375070

0.0011910.0021560.0027230.0035740.001021

0.0005110.0009650.0051060.0017020.001135

Lentil 1020375070

0.0003610.0007220.0022870.0017460.000903

0.0007830.0012040.0030700.0010230.000542

Black gram 10

20375070

0.000638

0.0017390.0035360.0022030.000522

0.001043

0.0014490.0052170.0017390.001217

Green gram 1020375070

0.0019460.0038910.0045780.0050360.002289

0.0016020.0025180.0057220.0044640.002232

Bengal gram 1020375070

0.0006140.0011160.0077550.0033470.000725

0.0007250.0043510.0055230.0029570.000391

Ground nut 1020375070

0.0005000.0010000.0022220.0017220.000556

0.0010000.0008890.0021670.0011110.001056

Pea bean 1020375070

0.0001680.0010080.0012320.0007280.00448

0.0005600.0006720.0016810.0011760.000392




0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

2 4 7.5 9 11

C a t a l y t i c ( p r o t e a s e ) a c t i v i t y ( U n i t s / m l )

PH

Soybean

Lentil

Black gram

Green gram

Bengal gram

Ground nut

Figure 1. Effect of pH on catalytic activity of protease at 370C.

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

10 20 37 50 70

C a t a l y t i c a c t i v i t y ( U n i t s / m l )

Temperature (oC)

Soy bean

Lentil

Black gram

Green gram

Bengal gram

Ground nut

Pea bean

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 studiedand presented in Table 3. At pH 4.0, all the enzymes inthe present study demonstrated maximum specific

activity at 37oC except soybean and green gram; both

showed maximum activity at 50oC. But the specific

activity of protease from all leguminous seeds expressedtheir maximum activity at 37

oC and pH 9.0. Dahot MU

1992 investigated on plant protease and found that the

Temperature (0C)




0

0.02

0.04

0.06

0.08

0.1

0.12

10 20 37 50 70

C a t a l y t i c a c t i v i t y ( U n i t s / m l )

Temperature (oC)

Soy bean

Lentil

Black gram

Green gram

Bengal gram

Ground nut

Pea bean

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

optimum temperature for enzyme reaction was 35oC,

which was a good consistent with our results but Evanset al., 2011 reported optimum temperature for the palmweevil was 23

oC on 0.03% casein. Kamini et al., 2004

and Aoki et al., 2004 have shown that the proteaseactivity with optimum temperature less than 20

oC is

considered as a cold protease. So, the proteases frompresent study can be excellent source for industrialpurposes where requires low or mild (optimum)temperature as a vital important factors in foodprocessing steps.

The effect of temperature on catalytic activity ofprotease (temperature vs. catalytic activity relationship) atpH 4.0 and 9.0 was mapped out at Figure 2a and Figure2b. From Figure 2a, the protease enzyme from soybeanseed showed a symmetrical distribution with one mainpeak at 37

oC but enzyme from black gram showed two

main peaks at 37oC and 70

oC. This characteristic

indicates that enzymes are ampholytes (having carboxyland amino group) and undergo change in respect tosolubility, osmotic pressure, viscosity etc. The change in

enzymatic activity with varying pH levels is due tochanges in ionization of the enzyme, the substrate or theenzyme –substrate complex (Gerald Reed, 1975). All theenzymes showed their symmetrical distribution curve at37

oC under pH 9.0 (Figure 2b) whereas catalytic activity

from pea bean was less significant. 0

CONCLUSION

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

an easy assay of protease activity procedure for theleguminous seeds using hemoglobin and casein assubstrate. Using this easy procedure during thegermination, it seems to be important to purify andcharacterize these acidic and alkaline proteases from theleguminous seeds. The study clearly expressed that althe protease enzymes isolated from seeds were edibleand showed higher activities in both acidic and alkalineconditions. They have the potentiality in our foodindustries.

ACKNOWLEDGEMENTS

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

REFERENCES

Al-sherhri MA, Mostafa SY (2004). Products form and

properties of protease produced by Bacillus licheniformisBiological science 7(9):1631-1635.

Anson ML (1938). The Estimation of pepsin, Tripsin, Papainand cathepsin with Hemoglobin, Journal of GeneraPhysiology, 22, 79-89.

Aoki H, Nazmul HMd, Matsno K, Hagiwara T, Watabe S (2004)Partial Purification of Protease that are Generated byProcessing of the Northern Shrimp (Pandalus. borealis) andwhich can Tenderize Beef. International Journal of FoodScience and Technology 39 (5): 471- 480.

Ashton F (1976). Plant Endopeptidases, In Plant ProteolyticEnzymes, Vol- 1, Michael. Tremacoldi C.R, Watanabe N.Kand Carmona E.C. 2004. Production of extracellular acid




proteases by Aspergillus clavatus, World J. Microbiol.Biotechnol. 20: 639 –642.

Bergmeyer HU (edi) (1985). Method of Enzymatic Analysis, 3rd

edition, , Verlag Chemie, Vol.2 (1985): pp. 208-216,227-230.

Dahot MU (1992). Investigation of protease in plant seeds.Journal of Islamic Academy of Sciences 5:4, 241-244.

Davy A, Svendsen I, Sorensen SO, Sorensen MB, Rouster J,

Meldal M, Simpson DJ, Cameron-Mills V (1998). Substratespecificity of barley cysteine endoproteases EP-A and EP-B.Plant Physiol. 117: 255- 261.

Egwim Evans C (2011). Partial Characterization of Proteaseactivity from Rhynchophorus palmarum (Palm Weevil).

African Journal of Food Science and Technology, Vol. 2(6)pp. 140-145.

Ericson MC, Chrispeels MJ (1973). Isolation andcharacterization of glucosamine-containing storageglycoproteins from the cotyledons of Phaseolusaureus. PlantPhysiol. 52: 98-104.

Gerald Reed (1975). Enzymes in Food Processing” 2nd

Edition, Academic Press, London

Gupta R, Beg QK and Lorenz P (2002). Bacterial AlkalineProtease: Molecular Approaches and Industrial Applications,

Appl. Microbiol. and Biotechnology. 59(1): 15-32.Kamini NR, Hemachander C, Mala JGS, Puvanakrishnan R

(2004). Microbial Enzyme Technology as an Alternative toConventional Chemicals in Leather Industry. Financial TimeInformation Ltd Asia Intelligence Wire. Feb.

Kumar S, Sharma NS, Saharan MR, Singh R (2005).Extracellular acid protease from Rhizopus oryzae: Purificationand characterization, Process Biochem. 40:1701 –1705.

Kunitz, M (1946). Crystalline soybean trypsin inhibitors 11.General properties. Journal of General Physiology 30:291-310.

Lowry OH, Rosebrough NJ, Farr AF, Randall, RJ (1951).Protein measurement with the Folin Phenol Reagent, Journalof Biological chemistry. 193, 265-275.

Nadafi MF, Deobagkar D (2005). Potential Application ofProtease Isolated from Pseudomonas aeruginosa PD100.Electronic journal of biotechnology 8(2).

Rahman M, Mahbubar L, Arjumand BM, Rahman MM,Shahjadee UF (2007). Changes of the Enzymes ActivityDuring Germination of Different Green gram Varieties. Bangladesh J. Sci. Ind. Res. 42(2): 213-216.

Rao MB, AM Tanksale, MS Ghatge, Deshpande VV (1998).Molecular and biotechnological aspects of microbialproteases. Microbiol. Mol. Biol. Rev. 62:597-635.

Sandhya C, Sumantha A, Pandey A (2004). Proteases InEnzyme Technology, A. Pandey, C. Webb, C.R. Soccol, CLarroche (Eds.), Asiatech Publishers Inc., New Delhi, Indiapp. 312 –325.) .

Scalet M, Alpi A, Picciarelli P (1984). Proteolytic activities in Alfalfa (Medicago sativa L.) leaves. J Plant Physiol, 116:133145.

Shutov AD, Vaintraub IA (1987). Degradation of storageproteins in germinating seeds. Phytochemistry, 26: 15571566.

Smit G, Smit BA, Engels WJM (2005). Flavor formation by lacticacid bacteria and biochemical flavor profiling of cheeseproducts. FEMS Microbiol Rev., 29: 591-610.

Sjodahl J, Emmer A, Vincent I, Roeraade J (2002)Characterization of Proteinase from Antartic Krill (Euphausiasuperba). Protein Expression and Purification 26(1): 1.

Soper R (1998). Biological Science (low price ed) CambridgeUK. Pp 507-510.

Vidyavathi U, Shivaraj B, Pattabiraman TN (1883). Proteases ingerminating finger millet (Eleusine coracana) seeds . Biosci.Vol. 5, Number 3, , pp. 219 –224.

Wang L, Wang YJ (2004). Rice starch isolation by neutra

protease and high-intensity ultrasound, J. Cereal Sci. 39291 –296.

Yaw- Huei Lin, Wen_Hsiang Yao (1996). Green gram (Vignaradiate L. Wilczek) contains some high proteolytic activitiesalready before germination. Bot Bull. Acad. Sin(37):1-7.

Documents

Akhtaruzzaman Et Al