Isolation of napin from Brassica nigra seeds and coagulation activity to turbid pond water Open Access

Poor drinking water is one of the major challenges for the growing world population. In the developing countries of the world, the drinking water source might be contaminated as a result of waterborne diseases. There are two steps protocols for drinking water purification ‘i.e., clarification and disinfection’. Mostly, aluminum sulfate (alum) is used as a flocculating agent and is being used for clarification of drinking water in developed countries; but the use of alum for clarification of drinking is more efficient and relatively cost effective (Teh & Wu 2014; Rocha et al. 2019). The disinfection of drinking water is attained by chlorine-based compounds. However, continued usage of chemicals produces Alzheimer's disease related to the autonomous and central nervous system (Crisponi et al. 2011), reproductive impairments, and different types of cancer by releasing physiologically incompatible reactive species or by-products into water (Mariotto et al. 2011; Sharavanan et al. 2020). Moreover, treatment of highly turbid water requires some proteolytic additives along with alum, making it an expensive process. Therefore, use of safe, traditional or conventional water treatment agents from natural sources becomes necessary. The use of natural coagulants in clarification of turbid water dates back several millennia (Muthusaravanan et al. 2018).

Plant coagulants possess charged particles suggesting a coagulation mechanism of adsorption or neutralization of charges (Arunkumar et al. 2019). Water-soluble proteins have a high efficacy towards wastewater treatment and turbidity removal (Okuda et al. 1999; Sengupta et al. 2012). These proteins act as a natural cationic polyelectrolyte for water treatment. The mechanism of coagulation with proteins involves charge neutralization and adsorption of the colloidal particles (Bhuptawat et al. 2007). These coagulant proteins bind to mainly negatively charged particles like bacteria, silt, clay, etc., suspended in colloidal form in turbid water (Kwaambwa & Maikokera 2007).

Moringa oleifera is used as a natural coagulant for water treatment. Conversely for Moringa oleifera plant, the seeds of other plant Brassica genus species have been traditionally used to remove the turbidity of pond drinking water (Bodlund et al. 2013, 2014). Brassica nigra is used for various aspects of food, medicine, vegetable and industrial products throughout the world. The Brassica nigra plant is enriched with many pharmacologically active phytochemicals, such as flavonoids, alkaloids terpenoids and tannins (Shafaghat 2010; Tomar & Shrivastava 2014). The use of natural plant coagulants does not show the noxious effects after treatment as for alum, where reports link it to Alzheimer's disease (Yin 2010). The aqueous extract from plant coagulants is known to have charged particles, indicating a coagulation mechanism of adsorption or neutralization of charges. However, the use of crude extracts often results in some disadvantages like incorporation of unwanted organic matter, which serves as a nutrient source for microorganisms and adds the need for another purification technique.

The primary objective and novelty of this study is to isolate the natural coagulant napin, a seed storage protein from Brassica nigra seeds that showed coagulation activity against a synthetic kaolin solution and turbid pond water. Napin protein was isolated and purified through size-exclusion chromatography. The effect of various parameters coagulant doses and initial turbidity on the coagulation process (turbidity removal) was investigated.

Napin protein from seed extract of Brassica nigra was purified and identified through gel electrophoresis, N-terminal amino acid sequencing, fast protein liquid chromatography (FPLC), dialysis and spectroscopic techniques.

Seeds (10 g) of Brassica nigra were ground with a mortar and pestle to a powder form. The fine seed powder was homogenized in 100 ml of 25 mM phosphate buffer (pH 7.0). The mixture was stirred continuously for 3 hours at 25 °C. After stirring, the sample was centrifuged (Ogawa 6470) at 10,000 rpm for 30 minutes and the pellet was discarded. The supernatant was filtered through a Whatman filter paper (pore size 8 μm; EW-06648-46) to remove any particulate matter. The clear seed extract (70 ml) was subjected to 30, 50 and 70% ammonium sulfate precipitation by stepwise slow addition of salt with constant stirring using a magnetic stirrer at 4 °C. The precipitated proteins were separated at low centrifugation speed of 3,000 rpm for 4 min. The supernatant was removed and the resulting pellet was re-dissolved in 10 ml of the same phosphate buffer and dialyzed thoroughly in 25 mM phosphate buffer (pH 7.0) at 4 °C with gentle stirring. The desalted napin was further purified by injecting the solution onto a pre-equilibrated HiLoad 16/60 Superdex 200 column (GE Healthcare, ÄKTA prime plus). The protein was eluted with the same phosphate buffer at a flow rate of 1.0 ml/min. Absorbance of the eluents was recorded at a wavelength of 280 nm. The concentration of purified protein was quantified using NanoDrop 2000c system (Thermo Scientific, peqLab, Germany).

The fractions with maximum protein content were analyzed and combined after running 12% SDS-PAGE (E-VS10-SYS, omniPAGE mini-System, Germany) (Laemmli 1970). The common procedure was acquired for the preparation of the 12% gel to visualize the protein samples. Non-reduced (without β-mercaptoethanol) and reduced samples (β-mercaptoethanol) were heated at 95 °C for 5 minutes in the heating block to denature the samples. The gel was stained with Coomassie Brilliant Blue R-250 (CBB R-250) dye to visualize the protein bands.

For coagulation activity, a turbid water sample was prepared as a 1% kaolin solution using tap water. The solution was stirred for 90 min and settled for 24 hours to allow complete hydration of the particle. The upper turbid layer of the solution was separated and used for coagulation activity test.

The coagulation activity test was carried out with kaolin synthetic clay solution as described by the Ghebremichael method (Ghebremichael et al. 2005). The Brassica nigra (Bn) seed extract (10 μl) was added to the clay solution to make a final volume of 1 ml with clay solution, mixed instantly and the initial turbidity at 500 nm (A500) was measured using a spectrophotometer. The solution was allowed to settle for 60 minutes and thereafter at A500 was measured again. The percentage of coagulation activity was calculated using the following formula: [(t0 − t)/t0)*100], where t0 is the A500 measured instantly after the sample has been homogenized and t is the A500 measured after 60 or 90 min. Different concentrations of napin protein were tested for coagulation activity. Kaolin clay solution was used as a negative control and Moringa oleifera (Mo) seed extract was used as a positive control in the whole experiment, because it contains natural organic polymers. Coagulant water-soluble proteins and polysaccharides are present in the Mo seed extract (Chandrashekar et al. 2020).

Polyvinylidene difluoride (PVDF) membrane (Standard Millipore Immobilon 0.45 micrometer membrane) blot was prepared according to the standard protocol (Towbin et al. 1979). The transfer of protein bands was performed at 200 mA for 4 h. An N-terminal amino acid sequence was obtained applying the peptide and protein sequencing system (Applied Biosystems Edman sequencer 476, Germany) using Edman degradation methodology (Hewick et al. 1981). A search was performed by applying Protein BLAST in the UniProtKB protein database (http://www.uniprot.org/blast/) for related alignment sequences and homologous sequences.

In order to compare the coagulation activity of napin and Moringa oleifera (Mo) seed extract against turbid pond water, it was added to the water. Turbid water was collected from a pond near Bhobtian Chock, Lahore, Pakistan to measure the coagulation activity. Mo seed extract and napin protein 55 μg were added separately in 1 ml turbid pond water. Initial absorbance was measured at 500 nm (A500) using a UV-visible spectrophotometer, t = 0 and at every 60 min time interval up to 3 h. Turbid pond water alone served as the control and had an initial absorbance of around 1.5 NTU (nephelometric turbidity units).

The aqueous extract was used as coagulant material. The aqueous extract contained macromolecules such as lipids, proteins and carbohydrates. The water-soluble coagulant proteins were isolated and characterized from an aqueous crude extract of plant materials for the purpose of purifying drinking water (Pritchard et al. 2009). The object of this experiment is to isolate the protein from the seed of Brassica nigra and to test its coagulation potential. The lipid content of the seeds was defatted by using petroleum ether and the defatted seeds were used for a further isolation process (Subramaniam et al. 2008).

The total water-soluble protein content of the defatted Brassica nigra seed was determined using the NanoDrop 2000c system and it was found to be around 40 mg/g (Table 1). Crouch & Sussex (1981) reported that water-soluble proteins in the B. napus seeds ranged from 55 to 80 mg/g. The protein content of the seed extract varies with plant species, protein detection method and the experimental conditions carried out. Napin was precipitated by ammonium sulfate from the crude extract of Brassica nigra seeds. Napin was present in the supernatant after 50% (w/v) (NH₄)₂SO₄ saturation constant with impure form. The supernatant of 50% saturation constant was used for further purification of napin. Napin was precipitated at 70% ammonium sulfate saturation constant and pellet was dissolved in the extraction buffer (25 mM phosphate buffer; pH 7.0). The dissolved pellet was extensively dialyzed to remove any further salt traces and subjected to size-exclusion chromatography to obtain the highly purified protein fractions. Ultimately, an optimized combination of ammonium sulfate precipitation along with chromatographic steps provided a > 95% pure napin solution from seeds of Brassica nigra, as judged by SDS-PAGE analysis. The gel filtration chromatogram showed two absorbance peaks and the corresponding SDS-PAGE showed that peak 1 (Figure 1(b), lane L3) contained high-molecular-weight proteins, while napin was found in peak 2 (napin) (Figure 1(a) and 1(b)). Napin fractions with maximum purity were stored at 4 °C. Further SDS-PAGE analysis showed the splitting of napin (16 kDa) into two daughter fragments of 11 and 5 kDa upon addition of β-mercaptoethanol. The 5 kDa fragment was not visible by 12% SDS-PAGE. This result showed that napin has inter-chain disulfide bonds (Figure 1(b), L1 and L2). Similarly, the napins from Brassica juncea have molecular weights ranging from 12 to 13 kDa and are composed of two polypeptides of 9 and 4 kDa (Venkatesh & Rao 1988). Sinapis alba napin possesses a molecular weight of 14.6 kDa and is composed of two polypeptide chains of 9.5 and 5 kDa (Menéndez-Arias et al. 1987). The purification steps resulted in purification by 20-fold with a 5% yield (Table 1) from 1 g of Brassica nigra seed powder.

Brassica nigra seed extract, peak 2 fractions (napin) showed the coagulation activity that changed the turbid water into a solid or semi-solid form. Brassica nigra seed extract and napin revealed approximately 80 and 85% coagulation activity, respectively, after 120 min and was compared to 87% of M. oleifera (Mo) seed extract; whereas clay solution alone showed 28% activity. The time kinetics of the coagulation activity of Brassica nigra seed extract and napin protein in comparison with Mo and kaolin (clay) are shown in Figure 2. The peak I fraction exhibited 18% coagulation activity. Moreover, for different concentrations from 10–300 μg/ml of Brassica nigra seed extract, the peak 2 fraction (napin) was tested for coagulation activity. The highest coagulation activity was observed between 60 and 70 μg/ml protein concentration (Figure 3). It was observed that coagulation activity decreased after 70 μg/ml protein concentration because of the failure of flocculation formation. This occurs because the polymers of the natural coagulant will cover the whole surface of the colloidal particles. Therefore, there is no place for the end chain to stick to and the flocculation process does not take palace. Therefore colloidal particles become stable and are unable to bind further with the natural coagulant (Srawaili 2008; Udayakumar et al. 2021).

There are other species of Brassicaceae such as mustard varieties and other plants like Jatropha curcas and Phaseolus vulgaris showed coagulation activity and are considered to be potential water treatment agents (Antov et al. 2010; Abidin et al. 2013). However, the coagulation activity depends on the seed nature: turbidity of water, extraction procedure and ion present in the water sample (Ghebremichael et al. 2005). This study showed that napin was identified as a coagulant agent and compared with synthetic turbid kaolin clay solutions prepared with tap water.

Napin was isolated from crude extracts of Brassica nigra seeds in 100 ml of 25 mM phosphate buffer; pH 7.0. The 15 amino acid peptide (RIPKCRKEFQQAQHL) was obtained from the N-terminus of the 16 kDa protein band through Edman degradation (Niall 1973). Analysis of this sequence via PROTEIN BLAST, National Center for Biotechnology Information (NCBI) showed maximum homology with other known napin proteins of the Brassicaceae family (Table 2). The sequence showed 100% amino acid identity with that of Napin3 from Brassica napus and Allergen Sin a 1 from Sinapis alba. It also showed 86% sequence alignment results between the identified peptide and Napin3 (P80208, 2SS3_BRANA) from Brassica napus which showed the 15 residue matches, indicating its highly homology (Figure 4(a)). Further, sequence alignment between the two seed storage proteins of Moringa oleifera (P24303, MO2X_MOROL) and Napin3 (P80208, 2SS3_BRANA) of Brassica napus showed a match of 23 residues (Figure 4(b)), indicating its plausible association to possess synergetic coagulation activity in Brassica nigra. Suarez et al. (2005) reported a hypothetical model of MO2.1 coagulant protein using Napin3 as a homology (Suarez et al. 2005). These results showed that seed storage protein has the coagulation potential and these proteins are commonly present in the species of Brassicaceae.

The efficiency of napin was tested against turbid pond water and compared the with coagulation activity of Mo aqueous extract. Turbid pond water alone was used as a control. Napin and Mo showed coagulation activity against turbid water collected from a pond. Interestingly, napin showed good coagulation activity of approximately 85% compared with Mo that showed 80% against pond water (Figure 5). While the higher coagulation activity of Mo of 87% showed against synthetic kaolin clay was compared to napin coagulation activity, as shown in Figure 1. It was indicated that there was possible variability in efficiencies of plant coagulant proteins according to the initial turbidity, nature of clay and other suspended particles.

However, it could be speculated that napin from Brassica nigra is a 16 kDa protein, which has high sequence similarity with Napin3 from Brassica napus, as shown in Table 1. Okuda et al. (2001) reported that the coagulation activity of natural coagulant agent depends on the types of seeds and extraction methods. The reduction in the absorbance may be due to the coagulant activity between the charged particle and the suspended charge particles (Ndabigengesere et al. 1995). The 12 and 6.5 kDa proteins from Moringa oleifera possessed coagulation activity in turbid water (Gassenschmidt et al. 1995). Napin3 is homologous with MO2.1 protein, thereby it might be that napin protein has a synergistic effect of coagulation activity.

This study result showed that napin protein is responsible for coagulation activity against turbid pond water.

A natural coagulant napin protein was isolated, purified and identified from seeds of Brassica nigra. It was identified as the active natural coagulant protein with a molecular weight of 16 kDa. It was used to treat a synthetic clay solution and turbid pond water to reduce the turbidity. Maximum removal of turbidity was obtained with pure napin protein, which was isolated by size-exclusion chromatography. Coagulation assay results showed that napin removed 87 and 85% turbidity from synthetic clay solution and turbid pond water, respectively. These results are promising in respect to the isolation and purification of napin protein and which has high efficacy in turbidity removal in turbid pond water. This simple and greener alternative for water treatment is viable and sustainable in the present era of climate change and unchecked environmental conditions.

All relevant data are included in the paper or its Supplementary Information.