Moringa

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Water Research 39 (2005) 2338–2344 www.elsevier.com/locate/watres

A simple purification and activity assay of the coagulant protein from Moringa oleifera seed Kebreab A. Ghebremichaela, K.R. Gunaratnab, Hongbin Henrikssonc, Harry Brumerc, Gunnel Dalhammarb, a

Department of Land and Water Resources Engineering, Brinellva¨gen 32, Royal Institute of Technology (KTH), 100 44 Stockholm, Sweden b Division of Applied Environmental Microbiology, Department of Biotechnology, Royal Institute of Technology (KTH), Albanova University Centre, 106 91 Stockholm, Sweden c Mass Spectrometry Laboratory, Department of Biotechnology, Royal Institute of Technology (KTH), Albanova University Centre, 106 91 Stockholm, Sweden Received 24 February 2004; received in revised form 15 February 2005; accepted 22 March 2005 Available online 25 May 2005

Abstract Use of extracts from Moringa oleifera (MO) is of great interest for low-cost water treatment. This paper discusses water and salt extraction of a coagulant protein from the seed, purification using ion exchange, its chemical characteristics, coagulation and antimicrobial properties. The coagulant from both extracts is a cationic protein with pI greater than 9.6 and molecular mass less than 6.5 kDa. Mass spectrometric analysis of the purified water extract indicated that it contained at least four homologous proteins, based on MS/MS peptide sequence data. The protein is thermoresistant and remained active after 5 h heat treatment at 95 1C. The coagulant protein showed both flocculating and antibacterial effects of 1.1–4 log reduction. With samples of high turbidity, the MO extract showed similar coagulation activity as alum. Cecropin A and MO extract were found to have similar flocculation effects for clay and microorganisms. Simple methods for both the purification and assay of MO coagulating proteins are presented, which are necessary for large-scale water treatment applications. r 2005 Elsevier Ltd. All rights reserved. Keywords: Moringa oleifera; Salt/water extraction; Antimicrobial; Coagulation activity assay; Purification; Characterization

1. Introduction Aluminium and iron salts are the most commonly used coagulants in water treatment. The cost and environmental side effects of these compounds has increased interest in the use of organic coagulants derived from plant material, such as Moringa oleifera Corresponding author. Tel.: +46 0 8 5537 8300; fax: +46 0 8 5537 8468. E-mail address: [email protected] (G. Dalhammar).

(MO) seed (Jahn, 1986, 1988; Olsen, 1987; Sutherland et al., 1994; Muyibi and Evison, 1995a, b). MO extracts have been shown to have large effects on turbidity removal (92–99% reduction) (Jahn, 1986; Muyibi and Evison, 1995a). Water treated with MO seed extract produces less sludge volume compared to alum (Ndabigengesere and Narasiah, 1998). An additional benefit of using coagulants derived from MO is that a number of useful products may be extracted from the seed. In particular, edible and other useful oils may be extracted before the coagulant is fractionated. Residual solids may

0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2005.04.012

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be used as animal feed and fertilizer, while the shell of the seed may be activated and used as an adsorbent. The coagulant is thus obtained at extremely low or zero net cost. The main concern in using MO for water treatment is the significant increase in organic load (Ndabigengesere and Narasiah, 1998; Okuda et al., 2001b). Jahn (1988) reported that water treated with crude MO extract should not be stored for more than 24 h. The crude extract is therefore not generally suitable for large water supply systems where the hydraulic residence time is very high. Two approaches may allow the use of MO seed in such systems. Adsorption can be used to remove the organic load from the crude seed extract. Alternatively, the active coagulating component may be extracted from the seed and used in pure or semi-pure form, thus reducing the total amount of organic material added to the treatment process. The extraction of coagulants from MO has been described previously, although it is somewhat unclear what the exact nature of the compounds are. Several reports have described the main water-extractable component as proteinaceous. It was described as a water-soluble protein with a net positive charge (Nkhata, 2001), as dimeric cationic proteins with molecular mass of 12–14 kDa and isoelectric point (pI) between 10 and 11 (Ndabigengesere et al., 1995). Others reported a molecular mass of 6.5 kDa and a pI greater than 10 (Gassenschmidt et al., 1995). On the other hand, Okuda et al. (2001a) reported that the active component from an aqueous salt extraction was not a protein, polysaccharide or lipid, but an organic polyelectrolyte with molecular weight of about 3.0 kDa. This suggests that the water and salt extract may be of different nature. The varying reports on the nature and properties of the coagulant protein from MO thus necessitate further study. Additionally, currently used purification methods involve multiple steps, which complicate the use of MO seed extracts in large-scale treatment applications. In the present study, a simple, scalable purification method and a convenient coagulation activity assay have been developed which allow the straightforward comparison of the characteristics and coagulation properties of water and salt MO extracts relative to alum. In addition, the antimicrobial properties of MO extract against a few gram-positive and gram-negative bacteria are discussed.

2. Materials and methods 2.1. Water sample Kaolin clay (10 g) was added to 1 L tap water, stirred for 30 min and allowed to settle for 24 h to allow complete hydration. Desired turbidity was obtained by dilution.

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2.2. Extraction Dried MO seeds were obtained from Senegal and stored at room temperature. The seeds were shelled just before the extraction and the kernel was ground using a kitchen blender. Oil was extracted with 95% ethanol and the solids were dried at room temperature. Samples (5%, w/v) were prepared from the dried solids using distilled water or 0.5 M NaCl solution, stirred for 30 min and filtered through Whatman paper No. 3 and 0.45 mm fiberglass. The filtrate is termed the crude extract. Aqueous aluminium sulphate (alum) was prepared as a 5% (w/v) solution.

2.3. Purification MO coagulant protein (MOCP) was purified using a High-trap CM FF 1 mL cation exchanger column on an A¨kta explorer (Pharmacia Biotech). A flow rate of 2 mL min 1 was applied and 1 mL samples were collected with an automatic fraction collector. The column was equilibrated with 50 mM ammonium acetate solution, pH 7 (solution A). Step elution using 1 M ammonium acetate, pH 7 (solution B) was performed as follows. Crude water extract (CWE): 0–35% B over 1 min, hold for 10 min, then 35–100% B over 15 min. Crude salt extract (CSE): 0–35% B over 1 min, hold for 10 min, then 35–65% B over 5 min, hold for 10 min, 65–85% B over 10 min, hold for 10 min, then to 100% B over 5 min. Coagulation activity was measured for each fraction and the active fractions were collected for further analysis.

2.4. Characterization Native PAGE was carried out according to Hultmark et al. (1983) using a Mini-PROTEAN 2 apparatus (BioRad). After electrophoresis, the 0.5 cm gel pieces were cut horizontally and the protein was eluted into either milli-Q water or 50 mM phosphate buffer. The coagulation activity of each fraction was measured. Protein content was estimated by Bradford method (Bradford, 1976) with bovine serum albumin as a standard. Molecular mass was determined by SDS-PAGE on a 10% mini gel and tris-tricine SDS-PAGE. The pI was determined from isoelectric focusing (IEF) (Model 111 mini IEF cell, BioRad) run with ampholytes in the pH range 8–10. Markers with a pI range from 4.45 to 9.6 were used. Thermal resistance of the coagulant protein was studied by incubating crude extracts at temperatures ranging from 60 to 100 1C for 0.5 to 5 h. Samples were filtered through 0.45 mm fiber glass and tested for coagulation activity.

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2.5. MS analysis Mass spectrometric analysis was carried out on a QTOFTM II mass spectrometer fitted with a nano Z spray source (Waters Corporation, Micromass MS Technologies, Manchester, UK). The instrument was operated at a resolution of 410,000 full width at half maximum (FWHM). Mass calibration was obtained over the m/z range 50–2000 using a solution of NaI (1.5 mg/mL) in 1:1 2-propanol/water. Peptides from in-gel trypsin digestions of SDS-PAGE samples were desalted and transferred to 1:1 acetonitrile/ water containing 0.1% formic acid using C-18 ZipTipTM units (Millipore) according to the manufacture’s instructions. Samples were introduced into the mass spectrometer from borosilicate nanospray needles (source voltage 1000 V). The quadrupole mass filter of the QTOFTM II was operated in a wide band pass (RF only) mode when collecting time of flight MS data. For collision-induced dissociation MS/MS analysis, a quadrupole resolution of ca. 2 m/z was used when selecting precursor ions. Maximum EntropyTM 3 (MaxEnt3) was used to simplify the raw combined MS/MS data by deconvolution of isotopic and charge state information to produce centroid spectra containing only monoisotopic, single-charged peaks. De novo sequencing was performed using PepSeqTM.

path, HELLMA) for absorbance measurements. The reduction in absorbance relative to the control defines coagulation activity. 2.7. Antimicrobial tests An antimicrobial study was performed using Escherichia coli D31 (obtained from H. G. Boman, Institute of Microbiology, University of Stockholm, Sweden) and K12, Pseudomonas aeruginosa (obtained from SMI Karolinska Institute, Stockholm Sweden) and Bacillus thuringiensis Bt7 and Bt75 (Siden et al., 1979). Cultures were grown in 0.2 M phosphate Buffered Lauryl Broth (LBB) medium at 37 1C overnight with continuous shaking. Cultures were centrifuged and the pellet was resuspended in 50 mM phosphate buffer or a 1:10 dilution of LBB to OD620 of 0.2. MOCP and cecropin A were added to the suspension culture, mixed well and incubated at 37 1C for 2 h. Cell aggregations were analysed under the microscope (Olympus BX51 with AnalySIS) and images were captured using a Sony PC 120 camera. For growth inhibition tests, samples from the incubated culture were diluted 10- to 106-fold. Aliquots were uniformly spread on nutrient agar plates and incubated at 37 1C overnight, followed by colony counting.

3. Results and discussion 2.6. Coagulation activity assay 3.1. Purification Optimum coagulant dosage is commonly estimated from jar test analysis using 1 or 2 L volume beakers equipped with mechanical stirrers. Since jar test analysis requires large volumes of sample and coagulant dosage, it may not be convenient for studying and comparing large number of samples. In order to facilitate the biochemical studies, it was necessary to develop a small volume coagulation assay. This was performed by measuring optical density (OD) of clay suspension at 500 nm (OD500) in a 1 mL semi-micro plastic cuvette (10  4  45 mm, Sarsted Aktiengesellschaft & Co, Germany). This method not only reduces the volume of clay suspension sample and coagulant dosage requirements, but also makes simultaneous analysis of large number of samples possible. It is suitable to easily screen out active and non-active coagulants and to observe settling characteristics of the flocs by continuously recording OD500. Coagulant solutions (10 mL) were added to high turbidity 1 mL clay suspension (250–300 NTU) in the 1 mL cuvette and homogenized instantly. This was allowed to settle for 1 h and absorbance was measured at 500 nm using a UV–Visible spectrophotometer (Cary 50 Bio). In order to reduce the background effect, a sample volume of 200 mL from the top was transferred to a quartz glass cuvette (type 105.200-QS, 10 mm light

The high pI value of MOCP has been advantageously used for simple purification using a cation exchange resin. The absorbance spectrum at 280 nm of the bound protein and corresponding coagulation activity are shown in Fig. 1. Salt and water extract samples showed three protein peaks (a, b and c) where peaks b and c showed good coagulation activity. The presence of two active peaks may arise from the heterogeneity of one or more active proteins in the extract. The two fractions had similar molecular mass distributions by SDS-PAGE (Fig. 2a), but showed differences in pI (result not shown), as expected from their differential elution from the cation exchange resin. It is interesting to note that the salt extract contains a larger proportion of peak c, which implies that this fraction is more tightly bound to the matrix, perhaps due to stronger ionic interactions, which can only be disrupted under conditions of high ionic strength. These results indicate that a single fractionation step is sufficient to remove the majority of proteins from crude MO extracts to produce a sample enriched in coagulating proteins. The use of standard cation exchange chromatography, which is well established in batch and continuous flow applications, will allow the facile scale-up of the method to meet the needs of large volume applications.

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Similar coagulation activity was observed in samples purified by native PAGE. In native PAGE, coagulation activity was found in two gel-extracted fractions close to the end of the gel phase. Fractions from native PAGE

0.8

(a)

c

Salt extract

121

0.7 0.6

101

0.5

81

0.4 61

a

0.3

b

41

Activity

Absorbance at 280nm (mAbs)

141

21

UV-280

0.2

Activity

0.1

1 0.0 144 154 165 176 186 197 208 218 229 240 250 Elution volume (mL) 180

0.8

a

160

Water extract

0.7

b

140

0.6

120

0.5

100

0.4

80 0.3

60

UV-280

c 40

Activity

0.1

20

and IEX had similar molecular weight (Fig. 2b) and other properties. 3.2. Characterization of MOCP Bradford method and absorbance at 280 nm showed that the coagulants from both water and salt extracts were proteins with similar characteristics. Others reported that the coagulant from water extract is a cationic protein (Nkhata, 2001; Ndabigengesere et al., 1995; Gassenschmidt et al., 1995). Our finding is at variance with results of Okuda et al. (2001a), who stated the active component from salt extract as an organic compound, which was neither protein, polysaccharide nor lipid. The molecular mass of purified MOCP was less than 6.5 kDa, as determined by SDS-PAGE with Coomassie staining (Fig. 2a) and tris-tricine SDS-PAGE (data not shown). Earlier studies have reported molecular weights of 6.5 and 12–14 kDa (Ndabigengesere et al., 1995; Gassenschmidt et al., 1995). It was also observed that the two active peaks from the cation exchange column had different pI values, both of which were above 9.6. The coagulant protein is thermoresistant and remained active after 5 h heat treatment at 95 1C. Coagulation efficiencies of the heat-treated samples were slightly higher than the raw samples. For boiling times of 30 min to 5 h reduction in absorbance ranged from 79–87% whereas that of the raw sample was 79%. Such a high thermal stability renders it easy to process and handle and heat treatment could be used to remove oil before the purification process. One of the options for reducing organic load in water treatment from the use of MO is carbon adsorption. In such cases heat treatment could be used to break down large molecules for better adsorption.

0.0

0

(b)

0.2

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62

67

73

78

84

89

95

100

Fig. 1. High-Trap CM FF IEX chromatography and coagulation activity for salt extract (a) and water extract (b). Ammonium acetate buffer (50 mM) (pH 7) was used for equilibration and it was eluted with step gradient up to 1 M concentration of the same buffer.

3.3. MS sequence analysis From the SDS-PAGE and IEF analysis, it is clear that each protein fraction obtained after cation exchange chromatography does not consist of a single, homogeneous protein, but is a mixture of proteins with similar

Fig. 2. SDS-PAGE images of the various fractions from IEX and native PAGE. (a) molecular weight determination using BioRad protein marker and cecropin A. The two active protein peaks (peaks b and c) show the same size (b) Compares sizes of protein samples from IEX (peak c) and native PAGE (lower fraction).

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3.4. Coagulation study 3.4.1. Small volume assay In this study we developed a simple and quick method to test coagulation activity with very small sample volumes (1 mL) and coagulant dosage. This method can be generally applied for easy screening of active coagulants from large number of samples. A coagulation kinetics study could also be easily performed to monitor settling rate behaviour of different coagulants. Such data can be used to distinguish active and nonactive coagulants in a short time period. A significant difference in attenuance (OD500) was observed between the coagulants and the control already in 10–20 min (Fig. 3a). Attenuance measurements were made either directly on the 1 mL cuvette or by taking a 200 mL sample to a quartz glass cuvette. For comparison and screening purposes the direct measurement could be used with satisfactory results, such as for identifying the active coagulant fractions in a large number of samples. When

Table 1 Electrospray ionization—MS/MS determination of the sequences of peptides from the trypsin digested protein Observed peptide mass

Observed charge state

Peptide sequence

1268.62 2087.09 2100.18 2122.12 2130.28

+2 +3 +2, +3 +2, +3 +2, +3

ANPPVQPDFQR QAVQLTHQQQGQAGPLQVR QAVQLAHQQQGQVGPQQVR QAVQLETQQQGQVGPQQVR QAVQLTHQQQGQVGPQQVRa

a

This is identical to the sequence published by Gassenschmidt et al. (1995).

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

High turbidity

Control Alum CWE Cecropin A 0

5

(a)

10 20 25 35 45 60 100 135 145 Time, min

0.7 Absorbance at 500 nm, Abs

physical characteristics. To further analyse the composition of these mixtures, MS/MS experiments were performed to obtain peptide sequence information from the samples after SDS-PAGE. As is shown in Table 1, a number of peptide sequences were obtained which are similar or identical to the known sequence of an MOCP (Gassenschmidt et al., 1995). It has been reported that more than one protein family with flocculation activity is present in MO seed (Gassenschmidt et al., 1995). Indeed, ongoing genomics projects around the world continue to demonstrate that plants express many closely related proteins during different developmental stages (Dong et al., 2004) so it is not unexpected that MO produces a range of sequence variants of seed storage proteins with coagulant activity. Further work on the genome and proteome of MO is clearly necessary to identify the exact nature.

Absorbance at 500 nm, Abs

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Low turbidity

0.6 0.5 0.4 0.3 0.2

Control Alum CWE

0.1 0.0 0

(b)

5

10 20 25 35 45 60 100 135 145 Time, min

Fig. 3. Coagulation kinetics of MO, cecropin A and alum for high- and low-initial turbidity waters.

the clear solution from the top is transferred to a quartz glass cuvette, the background effect is reduced thereby a higher reduction in attenuance could be observed. 3.4.2. Comparative studies A jar test study of the CSE and the CWE revealed that coagulation activity is higher in the CSE, which has been reported previously (Ghebremichael and Hultman, 2004; Okuda et al., 1999). Possible explanations for this observation are: (a) when the CSE was mixed with water or low molar buffer solution, precipitates were observed. The precipitates could act as nuclei for floc formation. (b) The amount of protein was higher (two-fold) in the CSE than in the CWE. Although the protein concentration in CSE was higher than in CWE, the amount of protein bound to the ion-exchange matrix during purification was similar in both extracts. In the purified form, both extraction methods showed similar coagulation activity and physico-chemical properties. This was also observed in samples from batch cation exchange purification experiments, where the eluted proteins from the water and salt extracts had similar protein content and coagulation activity. Further characterization and comparative tests were then carried out using only samples from the CWE.

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We compared flocculation caused by the CWE and alum both by the standard jar analysis and the small volume method developed. At high initial turbidity (250–300 NTU), both coagulants showed similar activity whereas at low initial turbidity (76–110 NTU), alum showed a higher settling rate and lower final turbidity than the CWE (Fig. 3b). Result of kinetic experiments using CWE (15 mg/L), alum (15 mg/L) and cecropin A (5 mg/L) showed similar coagulation activity for high initial turbidity (Fig. 3a). We observed that there was no difference in coagulation activity before and after oil extraction. Thus, it is possible to obtain the coagulant protein from oil processing residue. As cecropin A is a small antimicrobial peptide (4 kDa) with high pI, it is of interest to compare it with MOCP. Cecropin A showed similar coagulation activity to both MOCP and alum. This is the first such report to our knowledge of this effect, which suggests that a number of other small, basic peptides from plants and animals could be examined for such purposes. There are several areas where this result could be applied, the most appealing of which is to develop efficient coagulation in water and wastewater treatment systems using locally available materials.

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negative and gram-positive bacteria strains were selected. The effect of MOCP and cecropin A on cell aggregation are shown in Fig. 4. MOCP was able to aggregate both E. coli (D31) and B. thuringiensis (Bt7 and Bt75). On the other hand cecropin A was able to aggregate E. coli (D31) but not B. thuringiensis (Bt7) (Fig. 4). From viable counts using MOCP, we observed 1.1 log reduction in E. coli (D31) and 4 log reduction in E. coli (K12), B. thuringiensis (Bt7, Bt75) and P. aeruginosa. In the case of E. coli (K12) and P. aeruginosa reduction in viable counts was observed without noticeable cell aggregation. Cecropin A did not show aggregation or reduction in viable count for B. thuringiensis (Bt7). Studies have reported that B. thuringienesis inhibits the activity of cecropin A by a protease that degrades the peptide (Dalhammar, 1987). We could find similarities between MOCP and cecropin A although there was no resemblance in the sequence data. Further studies are required to establish the true nature of MOCP and the mechanism of antimicrobial action.

4. Conclusion 3.5. Antimicrobial effect A preliminary study of the antimicrobial effects of MOCP compared to cecropin-A was conducted in terms of cell aggregation and growth inhibition. A few gram-

MOCP has been purified from the native source using a simple and straightforward technique, which can be readily applied for water treatment in areas where MO is available. A key advantage of the purification is that it reduces the organic load in treatment systems without

Fig. 4. Flocculation effect of MOCP and cecropin A for D31 and Bt7. Images a, b and c represent control, MOCP treated and cecropin A treated samples of D31, respectively. The corresponding images for Bt7 are shown in d, e and f, respectively. Note that cecropin A did not aggregate Bt7 (f).

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requiring more complex protein production methods, such as recombinant (heterologous) expression. Moreover, the high thermostability of MOCP and its ability to reduce microbial populations contributes to its attractiveness as a flocculant. Similar results using cecropin A indicate that plants and animals are fruitful sources of proteins and peptides for water and wastewater treatment applications.

Acknowledgments The authors are grateful to Per Dalhammar and Jaen Ping for their assistance in the laboratory work and useful discussions. We would also like to extend our gratitude to Kaj Kauko for microscopic analysis. Funding for the purchase of mass spectrometry equipment was from the Wallenberg Consortium North.

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