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Isolation and characterization of trypsin inhibitors (Kunitz Soybean Trypsin Inhibitor, Bowman-Birk Inhibitor) in Soybean.

Introduction

Soybean is one of the important food resources that can combat diseases ascribed to mal- and under nutrition in developing countries[8]. On the other hand, raw soybean meals in the diet have been reported to cause growth retardation, impaired nutrient utilization, increased pancreatic secretion and pancreas enlargement in some monogastric animals and humans[3]. Serine proteinase inhibitors are considered to account for a significant part of these effects of raw soybeans[7]. These proteinase inhibitors are widespread in the plant kingdom, their physiological roles including the regulation of endogenous proteinases during seed dormancy, the reserve protein mobilization, and the protection against the proteolytic enzymes of parasites and insects[12]. Moreover, they may also act as storage or reserve proteins. Plant serine proteinase inhibitors are grouped into Soybean (Kunitz), Bowman-Birk, potato I and II, and squash families. Several other inhibitor families, such as barley, ragi 1 and 2, and thaumatin, were also suggested[9,1].

Trypsin is one of the three principal digestive proteinases in the digestive fract of animals and humans[3]. In the digestive process, trypsin acts with the other proteinases to break down dietary protein molecules to their component peptides and amino acids. Kunitz Soybean Trypsin Inhibitor (KSTI) and Bowman -Birk Inhibitor (BBI) are the two major trypsin inhibitors in soybeans. These inhibitors are large, tightly folded proteins that are not completely deactivated during ordinary cooking[5]. Soybean processors have worked hard to get antinutrients (e.g. trypsin inhibitors) out of the finished product, particularly soybean protein isolate (SPI) which is the key ingredient in most soybean foods that imitate meat and dairy products, including baby formulas and some brands of soybean milk. Much of the trypsin inhibitor content can be removed through high-temperature processing, but not all[5]. But high-temperature processing performed to inhibit or denaturate the inhibitor activity, and on the other hand, has unfortunate side-effect of denaturing the other proteins in soybean that they are rendered largely ineffective[15].

The main part of the present study was devoted to isolation, purification and characterization of trypsin inhibitors in soybean. The results of this study can be used for improving of soybean products quality by help to remove most of the trypsin inhibitors from the soybean products in food industry.

Material and methods

Lipid Extraction

30 gr soybean seeds were grounded by use of a food processor grinder. Therefore super critical fluid extraction (SFE) was used for defating of soybean meal.

Crude Extraction

Ten g defatted meal from soybean was homogenized in 30 mL deionized water by Ultra-Turrax homogenisation for 2 6 min with 0.5 min intervals, resulting in an effective time of extraction of 2'6 min. The homogenized meal slurry was centrifuged for 20 min (3000g). Supernatant was kept and sediment was reextracted again by using 20 mL deionized water and was centrifuged for 20 min (3000g). The two supernatants were combined and pH was adjusted to 3 by HCl and was left at 4[degrees]C. After 48 hour pH of the combined supernatants was readjusted to 3 and was centrifuged again for 20 min (13000 g). Centrifuged supernatant was used for determination of protein content, enzyme inhibitor activity and purification of enzyme inhibitors.

Gel Filtration

Column (100'2.5 cm; Sephadex G-75, swollen 12-15) was used for gel filtration. For preparing the column the Sephadex G-75 powder, deionized water and sodium azid (for microb elimination) were mixed and were left it 24 h in room temperature. Swelled gel was degassed and was added to the column which then was allowed to settle. The column was equilibrated with deionized water at a flow rate of 40 mL h-1. 30 mL crude extract was added to the column and was eluted with deionized water (flow rate: 40 mL h-1, fraction size: 10 mL, detection: 280 nm).

Obtained fractions were tested for enzyme inhibitor activity and relevant fractions were combined in 4 groups.

Flash Chromatography

The crude extract was filtered prior to application to the SP-sepharose cation exchange column used for this part of experiments. With this column it was possible to apply a large sample volume in a short time and thereby group-separated on the basis of differences in charges. After washing the column with ethanol and then water followed by buffer exchange with buffer A (0.01 M CH3CO2Na, pH 3.0), the strongly acidic cation-exchanger (ZetaPrep SP, KE) was equilibrated (flow rate 4 mL min-1) with 100 mL buffer A. Buffer exchanged sample was pumped through the column at a flow rate of 4 mL min-1. The column was eluted with buffer A (flow rate: 4mL min-1, fraction size: 10mL, detection: 280 nm) until the UV-signal was decreased significantly. Gradient elution was started with 250 mL of each of buffers A and B (0.01 M Na2HPO4, pH 8.3) in a gradient mixer.

Finally obtained fractions were tested for enzyme activity and relevant fractions were combined. Protein content and total enzyme activity was determined.

Enzyme Activity

For determine of trypsin inhibitor activity [DELTA]A / min was measured by the spectrophotometer at time drive and detection of 410 nm. Afterwards inhibitor activity was calculated by using the following formulas:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

Polymer Liquid Chromatography

Superdex 75 (3000-7000) column (Pharmacia) was used in this part of experiment. 200 mL sample was added and was eluted with 0.02 M NaH2PO4 + 0.05 M NaCl, pH 6.9 (flow rate: 1 mL min-1, fraction size: 1 mL, detection: 214 nm).

Protein Assay

Absorbance value/mL at 280 nm was used to determine protein content that was calculated by using the following equilibrium:

Pr otein content (mg [l.sup.-1]) =

(Absorbance value (280nm)/ ml) x Dilution factor

Isoelectric Focusing

IEF (polyacrylamide gels, pH 3.5-9.5) on the Pharmacia Phast Gel Electrophoretic SystemTM was performed as described in Serensen et al[14].

The samples were solubilized in deionized water and prefocusing and focusing conditions were according to table 1.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

SDS-PAGE (Polyacrylamide gels) on the Pharmacia Phast Gel Electrophoretic System was performed to investigate the result of inhibitor separations. The samples were denatured with SDS. 10-50 mL sample was mixed with 200 mL denaturation buffer (0.2 g dithiothreitol, 2.0 mL SDS (10%), 1.0 mL glycerol (87%), 0.2 mL bromphenol blue (0.1%), 2.0 mL TRIS (0.02 M, pH 6.8) + deionized water to a total volium at 10 mL). This mixture was boiled for 5 min and was let to cool.

To preparation of buffer strips, 100 mL of the following solution: 0.2 M tricine, 0.2 M TRIS, 0.55% SDS and 2.0% agarose (pH 8.1), was heated under magnetic stirring, and boiled for 5-6 min and was let to cool by placing the solution in a 56[degrees]C water bath. After the solution had reached 56[degrees]C, it was transferred to the buffer strips template that had placed on a heated, horizontal surface.

Staining and destaining procedure was based on the pharmacia Phast Gel Developing Device.

High Performance Capillary Electrophoresis

Trypsin Inhibitor Assay

Trypsin inhibitor assay (Detection 410 nm, Tris-HCl as buffer) in both ELIZA test and cuvet was done with using Na-benzoyl-L-arginine-4-nitroanilide (L-BAPA) as a substrate. Reaction of substrate with trypsin is as follow:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

Results

Lipid Extraction

8.15 g soybean powder free of lipid was obtained from 10 g grounded soybean after lipid extraction by Supercritical fluid technique.

Crude Extraction

After crude extraction processes 46 mL protein solution in water without carbohydrates was gathered from the supernatant part of centrifuge tubes. Crude extract contained 833.06 mg protein (Table 2). Thus, water soluble proteins contain 8.33% of soybean dry matter.

Gel Filtration

In figure 1 the result of gel filtration according to molecular weight is showed. ELIZA test of fractions (Table 3) showed that there were four peaks

(Peak1 (f.24-31), Peak2 (f.32-38), Peak3 (f.39-41) and Peak4 (f.42- 45), respectively) related to trypsin inhibitor in the middle part of curve.

Flash Chromatography

By using this method it was found four peaks with trypsin inhibitor activity according to Ion exchange separation on the basis of differences in charges. Trypsin inhibition was observed in the A.) fraction 43-45, B.) fraction 29-35, C.) fraction 36-49 and D.) fraction 31-35 of FC (fig. 2).

Enzyme Activity

Inhibitor activity and total activity were measured for all fractions and results are showed in table 4. The highest amount of activity (U/mL) were observed in peak 3 F.C, peak2 F.C, peak 2 G-75 and peak 3 G-75 ,respectively.

Fast Polymer Liquid Chromatography

By using FPLC also it were found four peaks with trypsin inhibitor activity according to molecular weight. The curves are showed in figure 3. Here peak 4 has three parts (a, b & b[cents]). ELIZA test was performed for all resulting fractions (data not shown). Trypsin inhibition was observed in the A.) fraction14 -15, B.) fraction14 -16, C.) fraction15 -17 and D.) fraction [a (17-19), b (14-15) & bc (16-17) of FPLC (fig. 3).

Protein Content

Protein content for all fractions after each purification step was measured and results are showed in table 2. The highest amount of protein (mg/ml) was observed in crude extract and peak 4bc FPLC had minimum amount of protein. During purification processes maximum amount of protein was measured in peak 4 G-75 and peak 3 G-75, respectively.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Isoelectric Focusing

Separation was done by use of pH gradient 3.5 -9.3 according to isoelectric focusing point and results were showed in figure 4 Gels A -D. As it is clear in gel pictures soybean trypsin inhibitor was separated at pi -Value = 4.55 in different peaks. There was no trypsin inhibitor in F.C peak 4a, FPLC peak 4a and FPLC peak 4b[cents] (Gel D). FPLC accomplished inhibitor separation better than F.C and F.C did this job better than gel filtration technique.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Separation was done according to subunit size (negative charges and molecular weight) and denaturation gradient was 8-25 (i.e. 6-150 Kilo Dalton). In figure 5 gels A -C show the results. By considering standard lines at left and right borders of gel it is possible to recognize which bands are related to KSTI (MW=22 KD) and which one are related to BBI (MW=7.8 KD).

High Performance Capillary Electrophoresis

Separation was done and all electropherograms for different peaks according to immigration time and pick area under curve are presented in figure 6. The location of KSTI and BBI in each electropherogram could be estimated by a simple comparison between standard KSTI as a reference electropherogram with inferred results from different peaks. Immigration time for BBI and KSTI were about 14.567 and 15.032 minutes after beginning of experiment.

The highest amount of KSTI was observed at peak 2 G-75 (0.8 mg/ml), peak 3 G-75 (0.47 mg/ml) and peak 2 FC (0.44 mg/ml) respectively. BBI was separated at peak 3 G-75 just before KSTI.

Discussion

Lipids consist of 19.6% of soybean dry matter[14]. In this study 8.15 g soybean powders obtained after lipid extraction of 10 g grounded-soybean meal by supercritical fluid technique. Thus18.5% (1.85 g) lipid was removed from sample and SFE was efficient method for lipid extraction. There is 35-45% protein in soybean, which 10% of them are albumins and 90% are globulins. Most of the soybean protein fraction contains soluble proteins and enzymes. During crude extraction steps soluble proteins and related enzymes were separated after starch (carbohydrate) precipitation. Protein assay showed that water soluble proteins contain 8.33% of soybean dry matter. By using gel filtration technique it is possible to separate protein fractions according to molecular weight. Results of spot test for gel filtration fractions in ELISA plate showed that there are some inhibition regions in the middle part of curve (Figure 1). It means soybean trypsin inhibitors should be among those related fractions. Those tubes were selected and the relevant fractions combined and finally classified in four groups (peak1, peak2, peak3 and peak4). These fractions all together contained 157.44 mg protein (Table 2). Thus, about 81% (675.62 mg) of proteins of crude extract removed by gel filtration and led to more purification of trypsin inhibitor. For more inhibitor purification flash chromatography technique was used. By using this method separation has been done base on the release of the exchangeable ions by removal or change of their net charge or removal of the charges on an acidic cation-exchanger. Thus, a partial purification of soybean trypsin inhibitor was made and the relevant fractions mixed together followed by spot test again and used in FPLC for more purification processes. Protein content of resulted fractions of FC was 139.8 mg (Table 2). FC method removed 11.2% (17.64 mg) of proteins that was present in GF fractions. Inhibitor activity test showed that peak 3 F.C, peak2 F.C, peak 2 G-75 and peak 3 G-75 had the highest amount of activity (U/ml), respectively. Further purification became feasible by FPLC technique according to molecular weight and different peaks were resulted with inhibitor activity. Here also results were confirmed by spot test. Protein assay results showed that all peaks in different purification systems contain protein with the exception of peak 4b[cents] FPLC but peak 4 G-75 and peak 3 G-75, had maximum amount of protein among all fractions. These fractions contained 2.889 mg protein (Table 2). Thus, 86.96% (136.911 mg) of proteins of FC fractions removed by FPLC and led to extremely pure trypsin inhibitor. With comparing the percent of removed protein in each step it is concluded that efficiency of purification for different methods were as follow: FPLC> GF> FC. All above mentioned purification techniques have been used by many workers on different organisms in different studies in order to separation and purification of trypsin inhibitor because of good accuracy and reasonable yield[2]. For getting an idea about separation accuracy during different purification processes all fractions were tested with inhibitor activity by IEF technique which could be use also as a detection method according to isoelectric focusing point. Figure 4 (Parts A-D) shows the results of IEF for all peaks during purification processes. As it is clear gel filtration has made a rough purification and several compact bands appeared from inhibitor fractions. Then F.C. has created a partial purification and there are a few bands in the gel. Finally after FPLC a sharp separation resulted. Otherwise FPLC can separate two kind of trypsin inhibitor from each other (KSTI & BBI) according to molecular weight. Another possibility is SDS-PAGE technique which could be use as an identification method according to subunit size. In this work similar results (Figure 5) were also observed which means rough separation was made by gel filtration and more resolution was made by F.C and FPLC, respectively. Generally, it should be recommended that combination of these techniques could be an optimum approach to purify trypsin inhibitor from different sources.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

There are many articles about identification and characterization of trypsin inhibitor in different organisms and most of them have utilized from IEF or SDS-PAGE as analytical methods[6,5,11,10,4,13,16]. HPCE represents a modern analytical technique derived principally from traditional electrophoresis, but with references to chromatography as well[14]. This technique was adapted for determination and characterization of KSTI and BBI from soybean. In this study by considering standard KSTI as a reference electropherogram and comparison of inferred results from different peaks with standard it is easy to recognize which peaks are related to KSTI and BBI according to immigration time and pick area under curve (figures 6). So by using this technique different peaks were analyzed. It is concluded that peak 2 G 75 (0.8 mg/ml), peak 3 G-75 (0.47 mg/ml) and peak 2 FC (0.44 mg/ml) had the highest amount of KSTI, respectively, whereas BBI was seen at peak 3 G-75 just before KSTI.

[FIGURE 6 OMITTED]

References

[1.] Ascenzi, P., M. Ruoppolo, A. Amoresano, P. Pucci, R. Consonni, L. Zetta, S. Pascarella, F. Bortolotti and E. Menegatti, 2000. Characterisation of low molecular mass trypsin isoinhibitors from oil-rape (Brassica napus var. oleifera) seed, Eur. J. Biochem., 267: 6486-6492.

[2.] Beirao, L.H., M.I. Mckintoch, T. Evanilda and D. Cesar, 2001. Purification and characterization of trypsin-like enzyme from the pyloric caeca of cod (Gadus morhua) II. Brazilian Arch. Biol. Techn., 44(1): 33-40.

[3.] Birk, Y., 1996. BBI-The trypsin and chymotrypsin inhibitor from soybeans: Friend or Foe? Second international symposium on the role of soy in preventing and treating chronic disease Brussells, Belgium, September 15-18.

[4.] De Meester, P., P. Brick, L.F. Lloyd, D.M. Blow and S. Onesti, 1998. Structure of the Kunitz-type soybean trypsin inhibitor (STI): implication for the interactions between members of the STI family and tissue-plasminogen activator. Acta Crystallogr D Biol Crystallogr, 54: 589-597.

[5.] Duranti, M., A. Barbiroli, A. Scarafoni, G. Tedeschi and P. Morazzoni, 2003. One-step purification of Kunitz soybean trypsin inhibitor. Protein Expr. Purif., 30: 167-170.

[6.] Franco, O.L., S.C. Dias, C.P. Magalhaes, A.C. Monteiro, C. Bloch, F.R. Melo, O.B. OliveiraNeto, R.G. Monnerat and M.F. Grossi-de-Sa, 2004. Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis). Phytochemistry, 65: 81-89.

[7.] Olli, J.J., K. Hjelmeland and A. Krogdahl, 1994. Soybean trypsin inhibitors in diets for Atlanticsalmon (SaZmo salar, L): effects on nutrient digestibilities and trypsin in pyloric caecahomogenate and intestinal content. Comp. Biochem. Physiol., 109A(4): 923-928.

[8.] Kumar, V., A. Rani, C. Tindwani and M. Jain, 2003. Lipoxygenase isozymes and trypsin inhibitor activities in soybean as influenced by growing location, Food Chemistry, 83: 79-83.

[9.] Laskowski, M.J. and I. Kato, 1980. Protein inhibitors of proteinases, Annu. Rev. Biochem., 49: 593-626.

[10.] Quirce, S., M. Fernandez-Nieto, F. Polo and J. Sastre, 2002. Soybean trypsin inhibitor is an occupational inhalant allergen. J Allergy Clin. Immunol., 109: 178.

[11.] Roychaudhuri, R., G. Sarath, M. Zeece and J. Markwell, 2003. Reversible denaturation of the soybean Kunitz trypsin inhibitor. Arch. Biochem Biophys, 412: 20-26.

[12.] Ruoppolo, M., A. Amoresano, P. Pucci, S. Pascarella, F. Polticelli, M. Trovato, E. Menegatti and P. Ascenzi, 2000. Characterization of five new low-molecular-mass trypsin inhibitors from white mustard (Sinapis alba L.) seed. Eur. J. Biochem., 267: 6486-6492.

[13.] Song, H.K. and S.W. Suh, 1998. Kunitz-type soybean trypsin inhibitor revisited: refined structure of its complex with porcine trypsin reveals an insight into the interaction between a homologous inhibitor from Erythrina caffra and tissue-type plasminogen activator. J Mol Biol., 275: 347-363.

[14.] Serensen, H., S. Serensen, C. Bjergegaard and S. Michaelsen, 1999. Chromatography and capillary electrophoresis in food analysis, Pubblyshed by The Royal Society of Chemistry.

[15.] Wallace, G.M., 1971. Studies on the processing and properties of soymilk, Journal of Science and Food Agriculture, 22: 526-535.

[16.] Zhao, Y., M.A. Botella, L. Subramanian, X.M. Niu, S.S. Nielsen, R.A. Bressan and P.M. Hasegawa, 1996. Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive s proteinase inhibitory activities than a constitutive homolog. Plant Physiol., 111: 12991306.

Corresponding Author

Farzad Nazari, Department of Horticultural Science, College of Agriculture, University of Kurdistan, Sanandaj, Iran TeleFax: +98 87166220553; E-mail: fnazari56@yahoo.com

(1) Hamid Reza Roosta, (2) Taimoor Javadi and (2) Farzad Nazari

(1) Department of Horticultural Science, College of Agriculture, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran

(2) Department of Horticultural Science, College of Agriculture, University of Kurdistan, Sanandaj, Iran

Hamid Reza Roosta, Taimoor Javadi and Farzad Nazari; Isolation and characterization of trypsin inhibitors (Kunitz soybean trypsin inhibitor, Bowman-birk inhibitor) in soybean
Table 1: Prefocusing and focusing conditions

Prefocusing          2000 V   2.5 mA   1.8 W   15[degrees]C   75 Vh
Sample application   200 V    2.5 mA   1.8 W   15[degrees]C   75 Vh
Focusing             2000 V   2.5 mA   1.8 W   15[degrees]C   210 Vh

Table 2: Protein content and total protein for soybean extracted
fractions during different purification processes

                                                               Total
                    Volume   Dilution   Absorbance   Protein   Protein
Sample              (ml)     factor     / ml         (mg/ml)   (mg)

Crude extract       46       10         1.811        18.11     833.06
Peak 1 G-75         60       1          0.635        0.635     38.1
Peak 2 G-75         55       1          0.756        0.756     41.58
Peak 3 G-75         21       1          1.180        1.180     24.78
Peak 4 G-75         30       1          1.766        1.766     52.98
Peak 1 F.C.         20       1          0.426        0.426     8.52
Peak 2 F.C.         67       1          0.561        0.561     37.587
Peak 3 F.C          125      1          0.509        0.509     63.625
Peak 4 F.C a        10       1          0.707        0.707     7.07
Peak 4 F.C b        40       1          0.575        0.575     23
Peak 1 FPLC         2        1          0.417        0.417     0.834
Peak 2 FPLC         3        1          0.406        0.406     1.218
Peak 3 FPLC         2        1          0.403        0.403     0.806
Peak 4a FPLC        3        1          0.001        0.001     0.003
Peak 4b FPLC        2        1          0.014        0.014     0.028
Peak 4bcents FPLC   2        1          -0.011       0         0

Table 3: ELISA test for gel filtration of crude extract-fraction
results

     1        2        3        4        5        6

A    0.896    0.691    0.676    0.662    0.716    0.844
B    0.844    0.698    0.697    0.580    0.466    0.408
C    0.174    0.153    0.157    0.149    0.144    0.140
D    0.153    0.149    0.163    0.152    0.149    0.154
E    0.843    0.146    0.215    0.169    0.335    0.685
F    0.849    0.665    0.697    0.592    0.714    0.824

     7        8        9        10       11       12

A    0.913    1.00     0.853    0.142    0.914    0.903
B    0.282    0.190    0.229    0.234    0.201    0.171 *
C    0.131    0.133    0.139    0.134    0.145    0.152
D    0.160    0.163    0.185    0.809    0.917    0.908
E    0.836    0.915    0.919    0.975    0.937    0.987
F    0.875    0.868    0.857    0.880    0.923    0.886

* Bold digits show low levels of absorbance suppose to contain
trypsin inhibitor.

Table 4: Enzyme activity (U/mL) and total activity (U) for soybean
trypsin inhibitor during different purification processes

                  Dilution
Sample            factor         Volume (ml)    DA / min

Trypsin (Ref.)    1              0.05           0.1006
Crude Extract     100            46             0.357
Peak 1 G-75       1              60             0.0926
Peak 2 G-75       10             55             0.951
Peak 3 G-75       10             21             0.603
Peak 4 G-75       1              30             0.0744
Peak 1 F.C.       1              20             0.0767
Peak 2 F.C.       10             67             0.940
Peak 3 F.C        10             125            0.985
Peak 4 F.C a      1              10             0.0927
Peak 4 F.C b      1              40             0.0772

                  Activity       Total
Sample            (U/ml)         activity(U)

Trypsin (Ref.)    0              0
Crude Extract     67.4           3100,04
Peak 1 G-75       0              0
Peak 2 G-75       22,66          1246,3
Peak 3 G-75       13,28          257,88
Peak 4 G-75       0              0
Peak 1 F.C.       0              0
Peak 2 F.C.       22,28          1492,76
Peak 3 F.C        23,47          2933,75
Peak 4 F.C a      0              0
Peak 4 F.C b      0              0
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Title Annotation:Original Article
Author:Roosta, Hamid Reza; Javadi, Taimoor; Nazari, Farzad
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:Jan 1, 2011
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