INVESTIGATION OF ISOLATION CONDITIONS AND ION-EXCHANGE PURIFICATION OF PROTEIN COAGULATION COMPONENTS FROM COMMON BEAN SEED

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1 APTEFF, 38, 1-19 (27) UDC: : :66.97: DOI:1.2298/APT7383A BIBLID: (27) 38, 3-1 Original scientific paper INVESTIGATION OF ISOLATION CONDITIONS AND ION-EXCHANGE PURIFICATION OF PROTEIN COAGULATION COMPONENTS FROM COMMON BEAN SEED Mirjana G. Antov, Marina B. Š iban, Slavica R. Adamovi and Mile T. Klašnja Investigation of an extraction procedure of protein coagulants from common bean seed regarding concentration of NaCl and ph was performed. High values of protein concentration and coagulation activity in crude extract (9.19 g/l and 23.9%, respectively) were obtained when the extraction was performed using.5 mol/l NaCl and water as solvent, which represents an advantage for economic and environmental reasons. Crude extract of common bean seed was purified by precipitation at two different percentages of (NH 4 ) 2 SO 4 saturation, followed by batch ion-exchange chromatography. The highest obtained coagulation activity, 45%, was determined in fraction that was elu ted at 1.75 mol/l NaCl from resin loaded with proteins precipitated upon 8-1% (NH 4 ) 2 SO 4 saturation. High values of coagulation activity showed by some eluates suggest their application as natural coagulant for water purification. KEYWORDS: Natural coagulant, common bean seed, extraction and purification, proteins, coagulation activity INTRODUCTION In treatment of turbid water, aluminium sulphate and other synthetic chemicals are commonly used. However, their application may have considerable disadvantages, the most important being possible negative effect on consumers' health. Namely, several studies reported that some chemical coagulants (e.g. aluminium salts) remain in tap water after coagulation and may induce Alzheimer s disease and similar health problems (1, 2). Moreover, for monomers of some synthetic organic polymers (e.g. acryl amide) it has been shown to possess neurotoxic and carcinogenic properties (3). On the other hand, sludge disposal to the environment are of concern. That is why considerable attention is focused on the development of new coagulants, preferably from natural and renewable sources, which have to be safe for human health as well as biodegradable. Dr. Mirjana G. Antov, Assoc. Prof., Dr. Marina B. Š iban, Assist. Prof., Slavica Adamovi, B.Sc., Dr. Mile T. Klašnja, Prof., University of Novi Sad, Faculty of Technology, 21 Novi Sad, Bulevar Cara Lazara 1, Serbia, mantov@uns.ns.ac.yu 3

2 In some rural tropical areas, natural coagulants of plant and mineral origin were traditionally used for domestic purposes in water treatment even before the advent of chemical coagulants. Nowadays, most of the reports describe natural coagulants from the seed of Moringa oleifera (4-7), Nirmali seed (Strychnos potatorium), maize (Zee mays) (8), mesquite bean (Prosopis juliflora) and Cactus latifaria (9). These natural coagulants can be used alone or as a substitution for chemical coagulants and flocculants (1), and are applied for reducing turbidity and microorganisms in water (11), for water softening (6) and for dewatering of sludges (12). Our previous studies indicated the ability of extract from common bean (Phaseolus vulgaris) seed to act as a natural coagulant (13). The choice of material was made on the basis of protein content in seed (2-25%), considering several reports' about the protein nature of coagulant agents from plant extracts (7, 11-14). Several active coagulation fractions were partially purified from crude extract of common bean seed and corresponding optimal coagulation doses were determined (15). The objective of this study, as the next step in our investigations, was to establish the most appropriate conditions for seed extraction considering concentration of NaCl and ph, then the level of salt saturation during the natural coagulant precipitation, and finally elution conditions in batch ion-exchange procedure of separation and purification. 4 EXPERIMENTAL Optimization of extraction parameters The Phaseolus vulgaris seed was ground to a fine powder by using a laboratory mill and sieved through.4 mm sieve. The fraction with particle size less than.4 mm was used in experiments. Ten grams of seed powder was suspended in 1 l of water,.5 mol/l NaCl or 1mol/l NaCl. For optimization of ph in extraction procedure, seed powder was suspended in.5 mol/l NaCl in.1 mol/l phosphate buffer at ph values 6, 7 and 8, respectively. The suspensions were stirred using a magnetic stirrer for 1 min to extract the coagulation active compounds and then filtered through a rugged filter paper. The obtained filtrates were referred to as crude extracts. Protein precipitation from crude extracts Crude extracts were saturated to -8% or 8-1% by adding (NH 4 ) 2 SO 4 and centrifuged at 35 rpm for 1 min. Precipitates were resuspended in.1 mol/l phosphate buffer ph 7.5 and dialysed overnight against deionised water at 4 o C. Elution conditions in batch ion-exchange procedure A further purification step was ion-exchange chromatography in batch mode of operation: dialysed samples were loaded on an anion-exchange resin Amberlite IRA 9Cl, the resin was rinsed with phosphate buffer, and active components were eluted by stepwise increasing NaCl concentrations:.25,.5,.75, 1., 1.25, 1.5, 1.75 and 2 mol/l NaCl. Total protein in collected fractions was monitored by measuring the absorbance at 28 nm. For each fraction coagulation activity was determined.

3 Preparation of turbid water Turbid water for coagulation tests was prepared by adding 1 g of kaolin to 1 l of tap water. The suspension was stirred for 1 h to achieve uniform dispersion of kaolin particles, and then it was allowed to remain for 24 h for completing hydration of the particles. This suspension was used as the stock solution. Turbid water with 5 mg/l kaolin (about 35 nephelometric turbidity units - NTU) was prepared by diluting 5 ml of stock suspension to 1 ml using tap water just before the coagulation test. The initial ph of the synthetic water was adjusted to 9. with 1mol/l NaOH solution, in accordance with previous investigations (13, 14). Coagulation test Jar test was carried out to evaluate coagulation activity of the eluates with coagulation active components. A volume of 3 ml of turbid water was filled in four 5 ml bakers. Each coagulant was added to the beaker at a same dose (.1 ml) and the content was stirred at 2 rpm for 2 minutes. The mixing speed was then reduced to 8 rpm and was kept for 3 minutes. Then, the suspensions were left to allow sedimentation. After 1 hour of sedimentation, an aliquot of 15 ml of clarified sample was collected from the middepth of the beaker and residual turbidity was measured. The residual turbidity of sample was RT S. The same coagulation test was performed with no coagulant as the blank. The residual turbidity in the blank was RT B. Coagulation activity was calculated as follows: Coagulation activity (%) = 1 (RT B RT S )/RT B [1] Analytical methods Turbidity was measured using a nephelometer (VOS 4) and it was expressed in NTU (16). Protein concentration in crude extracts was determined by Kjeldahl method. Total protein in collected fractions was monitored by measuring the absorbance at 28 nm (JENWAY 645 UV/Vis). RESULTS AND DISCUSSION For the optimization of an extraction procedure, coagulation active components from common bean seed were extracted using water or two solutions having different concentration of NaCl in water. Protein concentration, as well as coagulation activity differed between obtained extracts (Fig. 1). Presence of NaCl in the extraction solution yielded higher concentrations of crude protein in extracts in comparison to water extract. The highest protein concentration, determined by Kjeldahl method, was found in crude extract of common bean seed obtained by.5 mol/l NaCl extraction. In the same extract, the highest coagulation activity was measured 23.9%. Since the amount of protein and corresponding coagulation activity were found to be highest in crude extract obtained using.5 mol/l NaCl, this salt concentration was used in further experiments of extraction optimization. 5

4 Coagulation activity (%) Protein (g/l).5 1. Extraction solutions (mol/l NaCl in water) Coagulation activity (%) Protein (g/l) Fig. 1. Coagulation activity and protein concentration of crude extracts of common bean seed proteins extracted with water and different concentrations of NaCl In the next experiments, extractions of common bean seed were performed using.5 mol/l NaCl in phosphate buffer at different ph values and obtained extracts were evaluated by their protein concentrations and coagulation activities (Fig. 2). Coagulation activity (%) Protein (g/l) water ph 6 ph 7 ph 8 Extraction solutions (.5 mol/l NaCl in water or.1 mol/l phosphate buffer) Coagulation activity (%) Protein (g/l) Fig. 2. Coagulation activity and protein concentration of crude extracts of common bean seed proteins extracted by.5 mol/l NaCl in water or.1 mol/l phosphate buffer at different ph values Concentration of crude protein in extracts decreased slightly with the increase in ph of extraction solution. The same trend was observed considering coagulation activities of crude extracts. Protein concentrations in extracts obtained using.5 mol/l NaCl in water or in phosphate buffer, ph 6, were estimated to be 9.19 and 9.84 g/l, respectively. The corresponding coagulation activities of these two extracts were found to be 23.9 and 23.%, respectively. Considering the fact that the amount of extracted crude proteins and their coagulation activity varied a little in two extraction protocols, further experiments were carried out using NaCl solution with water as a solvent. 6

5 Crude extract in.5 mol/l water solution of NaCl was saturated to -8% in the first step, and then to 1% by adding (NH 4 ) 2 SO 4. Two samples of precipitated proteins were dialysed and loaded to previously activated ion-exchange resin. Elution of common bean seed coagulation proteins was carried out with gradually increasing concentration of NaCl. It is well known from the theory of ion-exchange chromatography that different proteins can be eluted from resin by varying the ionic strength. Concentrations of NaCl, used for resin elution, were varied in the range of mol/l and the obtained fractions were collected for further analyses. Protein contents, expressed as absorbance at 28 nm, in eluted fractions are shown at Fig. 3. Results revealed that content of protein, that were precipitated after 8% (NH 4 ) 2 SO 4 saturation, varied in the fractions eluted from the resin. According to the measured absorbance, protein content was considerably higher when.25 and.5 mol/l NaCl was applied for elution in comparison to the fraction obtained by elution at higher salt concentrations. On the contrary, protein content in the fraction from resin loaded with protein precipitate obtained after 8-1% saturation, was similar, yet expectedly lower than in the fraction of proteins from the previously examined sample. 2 1,5 A28 1,5,5 1 1,5 2 2,5 Protein fractions eluted at different c(nacl) (mol/l) ammonium sulphate saturation -8% ammonium sulphate saturation 8-1% Fig. 3. Protein content in eluted fractions of common bean seed proteins precipitated from crude extract by saturation in (NH 4 ) 2 SO 4 up to 8% or 8-1% In the next experiments coagulation activities of protein fractions obtained by eluation with increasing concentrations of NaCl were measured (Fig. 4). Collected protein fractions eluted at gradually increased ionic strength showed different coagulation efficiencies when applied at the same dose. All fractions of proteins that precipitated above 8% (NH 4 ) 2 SO 4 showed higher coagulation activities compared with those corresponding to proteins precipitated up to 8%. Maximal obtained coagulation activities were eluated at 1 and 2 mol/l NaCl for protein sample saturated to 8% and amounted to 29.5 and 3.5%, respectively. The highest obtained coagulation activity, 45%, was determined in the fraction that was eluted at 1.75 mol/l NaCl from the resin loaded with proteins precipitated after 8-1% (NH 4 ) 2 SO 4 saturation. It is worth to note that all fractions expressing high coagulation activity had a relatively low protein content, proving the purification of proteins that have characteristics of natural coagulants. 7

6 6 Coagulation activity (%) 4 2,5 1 1,5 2 2,5 Protein fractions eluted at different c(nacl) (mol/l) ammonium sulphate saturation -8% ammonium sulphate saturation 8-1% Fig. 4. Coagulation activity in eluted fractions of common bean seed proteins precipitated from crude extract by saturation in (NH 4 ) 2 SO 4 to 8% or 8-1% 8 CONCLUSION Optimization of an extraction procedure of protein coagulants from common bean seed regarding concentration of NaCl and ph was performed. High values of protein concentration and coagulation activity in crude extract were obtained when extraction was performed using.5 mol/l NaCl and water as solvent, which represents an advantage for economic and environmental reasons. Crude extract of common bean seed was purified by precipitation at two different percentages of (NH 4 ) 2 SO 4 saturation and batch ion-exchange chromatography. The highest obtained coagulation activity in fractions corresponded to low protein content, proving the purification of proteins that have characteristics of natural coagulants, extracted from common bean seed. High values of coagulation activity showed by some eluates suggest their application as natural coagulant for water purification. REFERENCES 1. Martyn, C. N., D. J. P. Barker, C. Osmond, E. C. Harris, J. A. Edwardson and R. F. Lacey: Geographical relation between Alzheimer's disease and aluminium in drinking water. The Lancet, 1 (1989) Letterman, R.D. and R.W. Pero: Contaminants in polyelectrolytes used in water treatment. J. Am. Wat. Wks. Assoc. 82 (199) Mallevialle, J., A. Brichet and F. Fiessinger: How safe are organic polymers in water treatment. J. Am. Wat. Wks. Assoc. 76 (1984) Gassenschmidt, U., D. K. Jany, B. Tauscher and H. Niebergall: Isolation and characterization of a flocculating protein from Moringa oleifera Lam. Biochem. Biophys. Acta 1243 (1995) Okuda, T., A. U. Baes, W. Nishijima and M. Okada: Improvement of extraction method of coagulation active components from Moringa oleifera seed. Wat. Res. 33, 15 (1999)

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8 ,, -.. Received 3 May 27 Accepted 19 July 27 1