Chemical and Enzymatic Protein Cross-Linking To Improve Flocculant

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Chemical and Enzymatic Protein Crosslinking to Improve Flocculant Properties Matthew Essandoh, Rafael A. Andres Garcia, and Christine Marin Nieman ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02395 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on September 2, 2018

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ACS Sustainable Chemistry & Engineering

Chemical and Enzymatic Protein Crosslinking to Improve Flocculant Properties Matthew Essandoh*; Rafael A. Garcia, Christine M. Nieman

United States Department of Agriculture Agricultural Research Service Eastern Regional Research Center Biobased and Other Animal Coproducts Research Unit 600 East Mermaid Lane, Wyndmoor, PA 19038, USA

Editor: Please include this statement “Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer”

Corresponding Author *Tel: (215) 836-6909; fax: (215) 233-6795; email: [email protected]

Full postal address Eastern Regional Research Center Biobased and Other Animal Coproducts Research Unit 600 East Mermaid Lane Wyndmoor, PA 19038 USA

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ABSTRACT Some proteins promote the flocculation of aqueous suspensions, but proteins are much smaller than modern polymeric flocculants, and they are often not as potent. In this study, two globular proteins, hemoglobin (Hb) and bovine serum albumin (BSA), were crosslinked through either chemical or enzymatic means. The degree of crosslinking was approximately 95%. Compared to the native proteins, the crosslinked proteins had a 20-30 fold increase in molecular weight and a reduced isoelectric point. Crosslinked proteins appear to have lost the secondary structures present in their native counterparts, and do not go through organized structural transitions upon heating. The crosslinked Hb sample showed a higher peak clarification efficiency (KCE = 2.96) compared to its BSA counterpart, indicating its potential to be used as a flocculant for water clarification. Interestingly, native BSA has no flocculant activity (KCE = 0, 0% clarification) but enzymatically crosslinked BSA has substantial activity (peak KCE = 1.85, or ~98% clarification).

KEYWORDS Hemoglobin, bovine serum albumin, protein crosslinking, biodegradable flocculant, SDS-PAGE, size exclusion chromatography, bridging mechanism


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INTRODUCTION Flocculants are substances that promote the aggregation and removal of colloidal particles. Many widely used flocculants are synthetic polymers with very high molecular weights, often in the range of 1-60 million Daltons. The molecular weights of such flocculants are known to be important to their function.1 These flocculants can have high efficiency, but they are nonbiodegradable and are of environmental and health concern.2 Bio-based flocculants have attracted a lot of attention in coagulation/flocculation processes. They have the advantages of being produced from renewable resources, low toxicity, and good biodegradability. Chitosan, a polysaccharide derived from crustacean shell waste, has been one of the most widely adopted bio-based flocculants,3 although it has limitations. Failure of chitosan flocculants in an erosion control study4 was partially attributed to relatively low MW of chitosan compared to synthetic polymeric flocculants. Proteins are being increasingly recognized for their potential as bio-based flocculants.5-8 Proteins derived from agricultural by-products can be inexpensive, and some such as hemoglobin from slaughterhouse blood, display good flocculant properties. Slaughterhouse blood is an under-utilized by-product of meat production. Hemoglobin (Hb) is the most abundant blood protein, and it is normally found only in the cytoplasm of red blood cells. Hb has been shown by the authors’ research group to be a good bio-based alternative to synthetic polymer flocculants.9 It is a tetrameric protein consisting of two alpha (α) and two beta (β) subunits, with a total of 574 amino acids and a total molecular weight of ~65 kDa. Serum albumins are the most abundant proteins in blood plasma, and the second most abundant proteins in blood overall.10 In contrast to Hb, bovine serum albumin (BSA) does not have good flocculant properties.11 It consists of a single peptide chain, and an overall molecular weight (~66 kDa) similar to Hb. 3 ACS Paragon Plus Environment


Intermolecular crosslinking joins proteins into higher molecular weight complexes. Crosslinking between proteins can be incited by chemical crosslinker, heat, alkaline conditions, enzyme, or photo-oxidative treatment.12 Glutaraldehyde (1,5-pentanedial) is a homobifunctional reagent with the formula CH2(CH2CHO)2. Glutaraldehyde (GTA) has the capability of forming both inter- and intra- molecular protein crosslinks. Different side chain functional groups of proteins including amine, phenol, thiol and imidazole are all capable of reacting with glutaraldehyde.13 The chemical may react with proteins in different ways including aldol condensation or Michael-type addition.14 GTA can link the protein amino groups although the actual mechanism by which GTA crosslinks proteins is still not known.14 This chemical has been used by various authors to crosslink soy protein,15 cocoa protein,16 and castor bean protein,17 among others. Crosslinking with GTA is common because of its high reactivity and low-cost. Transglutaminases (TGase) are a family of enzymes that have been found in microorganisms, mammals and plants.18 TGase has the ability to form both intra- and intermolecular protein crosslinks. This enzyme initiates acyl transfer reaction between the γcarboxamide groups of protein-bound glutamine residues acting as acyl donor and primary amines (including the amino group of lysine) as acyl acceptors. Nucleophilic attack by a lysyl εamino group at the carbonyl moiety of the thioacyl-moiety intermediate lead to the formation of isopeptide-crosslinked proteins.18 When the acyl acceptor is the ε-amino group of peptide-bound lysine, the resulting product is an ε-(γ-glutamyl) lysine crosslinked product. Recently, fermented tiger nut milk crosslinked with mTGase has been studied for its ability to improve the physicochemical and microbiological properties of the crosslinked product.19 The crosslinked product showed higher molecular weight compared to the native protein. Other authors20 have shown that crosslinking collagen with proteins (casein, keratin and soy protein isolate) using mTGase can


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enhance its mechanical properties and thermal stability. The crosslinked complexes were found to have higher molecular weight than the original protein. The present study examines the hypothesis that proteins crosslinked to form high molecular weight complexes will better mimic high molecular weight synthetic flocculants, and have improved flocculant performance, relative to native protein. The study utilizes one protein with good flocculant properties (Hb) and one with poor flocculant properties (BSA), and attempts to crosslink each through two different methods (using GTA and TGase). The crosslinked products are characterized and tested for their ability to flocculate a model suspension.

EXPERIMENTAL SECTION Materials. Chemicals used for the study include bovine hemoglobin (lyophilized powder), bovine serum albumin, glutaraldehyde (25% wt. in water), dithiothreitol (DTT), and protein standard mixture which were obtained from MilliporeSigma (St. Louis, MO). Microbial transglutaminase was obtained from Ajinomoto USA, Inc. (Teaneck, NJ). Water was purified to a resistance of 18 megohm-cm using a Barnstead E-pure system. Protein crosslinking by GTA. In general, the substrate protein was dissolved at 1% (m/v) in 100 mM sodium phosphate buffer, pH 7.2. GTA was then added to a final concentration of 0.00625% to 0.4%. The reaction was then mixed briefly and left to incubate at room temperature for 2 h. Protein crosslinking by TGase. A stock solution of 5000 mg/mL of TGase solution was prepared. The substrate protein was dissolved at 0.5% (m/v) in 100 mM sodium phosphate buffer, pH 7.2, with TGase concentration of 0 to 20 mg/mL. Dithiothreitol (DTT) was added to 5 ACS Paragon Plus Environment


the protein solution at a final concentration of 12.5 mM DTT for the cleavage of disulfide bonds. The DTT solution was prepared fresh and used immediately. The reaction was mixed gently and incubated at 40 oC for 3 h. Degree of crosslinking. The degree of crosslinking was determined by following the work of Djoullah et al.21 with some slight modification. In summary, crosslinked protein samples were dissolved at 1% in a solution of 2% SDS in 0.1 M phosphate buffer, pH 8. The samples were then centrifuged for 10 min at 4000 × g. The preparation of o-phthaldialdehyde (OPA) reagent was carried out according to a published protocol and used immediately.22 Two mL of OPA reagent was added to 50 µL of the supernatant taken after centrifugation. The samples were then vortexed to ensure adequate mixing before allowing them to sit at room temperature undisturbed for 5 min. The degree of crosslinking was calculated by comparing the UV absorbance at 340 nm to that of a blank solution that was prepared in the same manner without the addition of the sample. Size Exclusion Chromatography (SEC). Samples for SEC analyses were first filtered using nylon 66 membrane (pore size = 0.45 µm) before being transferred into 300 µL polypropylene (12 x 32 mm) snap neck vials. The HPLC instrument (Waters 2695, Waters Corporation, Milford, MA) was programmed to inject 15 µL of the samples using 0.05% sodium azide and 0.1 M sodium sulfate in 0.1 M phosphate buffer, pH 7.2, as the mobile phase. Samples were run for 20 min at a flow rate of 0.75 mL/min and the eluted compounds were detected at 280 nm. Eluted SEC peaks were compared to a standard calibration graph for the estimation of the molecular weight. The column used was the TSKgel UltraSW Aggregate, 3 µm, 7.8 mm ID x 30 cm (Millipore Sigma, Saint Louis, MO, USA). All data generated were analyzed with Empower software (Waters Corporation). 6 ACS Paragon Plus Environment

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Electrophoresis. Molecular weight determination via sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) was done by mixing the crosslinked samples with sample buffer containing 10 mM Tris-HCl, 1 mM EDTA (pH 8.0), 2.5% (w/v) sodium dodecyl sulfate, 713 mM 2-mercaptoethanol and 0.1% (w/v) bromophenol blue. This mixture was heated for 5 min at 80 oC. Samples were loaded onto a PhastGel gradient 4-15 gel. After the run, the gel was fixed with a 1:1 mixture of glutaraldehyde and water for 30 min, and stained with Coomassie blue for 30 min before destaining for 15 min with a 3:1:6 mixture of methanol, acetic acid and water. Circular dichroism. Secondary structure content changes occurring after protein crosslinking were studied with a circular dichroism (CD) spectrometer (Model 420, Biomedical Inc., Lakewood, NJ). The samples were first filtered with nylon 66 membrane (pore size = 0.45 µm) and run on the spectrometer at 25 oC, bandwidth 1 nm, path length 1 mm, and averaging time 5 s. A blank was also prepared for baseline correction. Samples were then run in the far UV range (190 to 250 nm). Differential Scanning Calorimetry (DSC). Lyophilized samples were dissolved in a degassed aqueous solution at concentration of 50-90 mg/mL. Then, 750 µL was measured into the ampoules for DSC analysis using a multi-cell differential scanning calorimeter (TA Instruments, Newcastle, DE). Samples were run at 1oC/min from 20 to 120 oC. Zeta Potential Measurement. Sample buffer was prepared using nanopure water from pH 2 to 9 containing 0.2 M KCl. The pH was adjusted using 0.1 N HCl or 0.1 N NaOH. About 5 mg of the sample was dissolved in 5 mL of the buffer and vortexed until well mixed. It was then incubated at room temperature for about 30 min. The supernatant obtained was transferred into a sample cell and the zeta potential analyzed using Zetasizer Nano Z (Malvern Instrument Inc., 7 ACS Paragon Plus Environment


Westborough, MA). Samples were run using an equilibration time of 2 min and a temperature of 25 oC. Flocculant testing. Native and crosslinked samples were all tested as flocculants. Kaolin suspension was prepared by dispersing 3 g (or 1 g in the case of BSA) of kaolin in 1L of MalicMES-Tris buffer (25 mM, pH 5.5). Twenty-four mL of kaolin suspension was dispensed into a glass vial and its initial turbidity measured using a turbidimeter (2100AN IS, Hach, Loveland, CO). The glass vials containing the suspension were inserted into the turbidimeter and particles present in the vials causes the incident light beam to scatter light. The scattered light is then quantified relative to a traceable standard. The amount of sample doses ranged from 0 to 40 mg protein/g kaolin. The amount of BSA and its crosslinked products in each dose was directly based on the dry weight of the lyophilized sample. In the case of native Hb and its crosslinked product, the concentration of hemoglobin needed was determined by the alkaline heamatin D575 method.23,24 The flocculants were then added to the suspension before shaking for 1 min at a speed of 400 rpm using an orbital shaker. This was then followed with slower shaking at 200 rpm for 15 min. The samples were then left undisturbed in a temperature control incubator at 20 ± 1 °C. Final turbidity of the samples was measured at 1, 3 and 5 h of incubation time. A control was also prepared without the addition of the flocculant and all treatments were carried out in triplicates. The ability of native and crosslinked samples to flocculate suspensions of clay was estimated using Kaolin Clarification Effectiveness (KCE):25

(..) =


(1)

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Standard deviation of KCE results was calculated using the equation 2 below: =

. √

+

(2)

where the standard deviations of the initial and final turbidity measurements are represented as "#

and "$ , respectively and n is the number of replicates. Unless otherwise stated, flocculation

studies and other analyses involve the utilization of 0.05% GTA Hb, 0.2% GTA BSA and 10 mg/mL TGase BSA

RESULTS AND DISCUSSION Synthesis of crosslinked proteins using GTA. There is currently no consensus on the actual mechanism by which this reaction occurs. Understanding of the reaction is complicated by the several monomeric, dimeric and polymeric forms of GTA that can exist in solution at different temperature, pH and concentration.14 In the case of Hb, lower concentrations of GTA (0.00625% to 0.025%) did not show any crosslinking as measured by the SEC while gel formation was observed at higher concentrations (>0.1% GTA). The concentration of GTA was therefore kept at 0.05% to achieve Hb polymerization without gel formation. Enzymatic crosslinking of protein using TGase. A simplified scheme for the reaction of TGase with proteins is shown in Scheme 1. The major step for this reaction is the interaction of the active site of TGase with γ-carboxamide group of glutamine residue of the protein to yield thioacyl-moiety.18 The thioacyl-moiety intermediate that is formed then reacts with an amine group to form an isopeptide amide bond. Crosslinking of BSA was achieved as measured by SEC. However, no crosslinking was observed in the case of Hb. Other authors have found that 9 ACS Paragon Plus Environment


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crosslinking Hb using TGase was not possible either in the presence or absence of DTT.26 It is possible that most of the available sites are buried and not exposed to react with the TGase enzyme. Px-(CH2)2-CO-NH2 + H2N-(CH2)4-Py TGase Px-(CH2)2-CO-NH-(CH2)4-Py Scheme 1: Reaction of protein with TGase to yield ɛ-(ɣ-glutamyl) lysine crosslink, isopeptide amide bond. Degree of crosslinking. The formula below was used to calculate the degree of crosslinking: )

%(&) = 1 − * , 100 ) +

(3)

where %(&) refers to the degree of crosslinking, and ./ and .& refer to the absorbance of the

control and the crosslinked sample at time 0, respectively, at a wavelength of 340 nm. Both GTA

Hb and TGase BSA produced a %(&) of ~95%, meaning that the majority of the reaction had gone

almost to completion. However, GTA BSA sample was sparingly soluble during the degree of crosslinking experiment and therefore its %(&) could not be determined. Even the use of sodium dodecyl sulfate and β-mercaptoethanol to increase solubility was not successful. Size-exclusion chromatography. Figure 1a displays the chromatogram obtained after crosslinking the BSA with different concentrations of GTA from 0% to 0.4%. Increasing the concentration of GTA increases the molecular weight of the polymerized sample. The increase in molecular weight of the polymerized protein compared to the BSA was about 20- to 30-fold.


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(b)

(a)

0.14

0.18

Native Hb

Native BSA

0.10

0.025% GTA 0.05% GTA 0.075% GTA

0.06

0.1% GTA 0.2% GTA 0.4% GTA

0.02

0.00625% GTA

0.14

Absorbance (AU)

Absorbance (AU)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.0125% GTA 0.025% GTA

0.10

0.05% GTA

0.06 0.02 -0.02

-0.02 5

10 15 Retention time (min)

5

20


20

Figure 1. Chromatogram for size-exclusion chromatography of native and polymerized protein samples with different concentrations of GTA. Experimental conditions used for the crosslinking include a protein concentration of 10 mg/mL, a reaction time of 2 h, and 0.1 M sodium phosphate buffer (pH 7.2) as the reaction solvent at ambient temperature.

In the case of Hb, increasing the concentration of GTA (> 0.05%) led to the formation of polymerized molecules (Figure 1b). However, concentrations that are > 0.1% GTA gelatinized the samples and they could not be filtered. The polymerized protein formed has a high molecular mass, with most of the crosslinked samples above the exclusion limit of the column. As explained previously, this reaction is complex with GTA, possibly, utilizing many different available reaction sites on Hb. It has been reported that the reaction of Hb with GTA can give a molecular mass greater than 1000 kDa.27 Figure 2 shows the chromatogram obtained when different concentrations of TGase (0-20 mg/mL) were reacted with BSA in the presence of DTT for 3 h. The samples studied show the



presence of the BSA (~13.57 min), TGase (~14.88 min) and DTT (~17.55 min). The use of 0.5 mg/mL TGase did not produce any crosslinking as the chromatogram obtained was similar to the control (0 mg/mL). The first appearance of polymerized BSA was observed at 5 mg/mL TGase concentration. Increasing the concentration of the TGase from 5 to 20 mg/mL does not alter the pattern of the chromatogram obtained, except that the intensity of the polymerized sample (> 2000 kDa) formed in the reaction mixture increased.

0.09

0.07 Absorbance (AU)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.05

0.03

0.01

-0.01 5


0 mg/mL TGase 5 mg/mL 20 mg/mL

20

0.5 mg/mL 10 mg/mL

Figure 2. Chromatogram for size-exclusion chromatography of BSA and polymerized BSA samples with different concentrations of TGase. Experimental conditions used for the crosslinking include a protein concentration of 5 mg/mL, DTT concentration of 12.5 mM, a reaction time of 3 h, and 0.1 M sodium phosphate buffer (pH 7.2) as the reaction solvent at ambient temperature.


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Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). SDSPAGE has been used widely for the separation of protein mixtures. The proteins were prepared in a denaturing and reducing environment using sodium dodecyl sulfate, heat, and βmercaptoethanol. The SDS-bound proteins, which had acquired a negative charge, were separated mainly on their size. In Figure 3a, polymerized BSA samples larger than 300 kDa can be seen (lanes 3 and 4), as shown by the red arrows on the right of the gel. In Figure 3b, the addition of 5, 10 and 20 mg/mL TGase concentration did show crosslinking of the BSA protein in lanes 4, 5, and 6, respectively. Polymerized proteins were seen with higher molecular weight compounds (>300 kDa) that were too large to enter the gel as shown by the red arrow on the right of the gels. The SDS-PAGE results show that TGase crosslinking of BSA protein was successful and corroborates the size-exclusion chromatography results.

Figure 3. SDS-PAGE analysis of BSA crosslinked protein using different (a) GTA and (b) TGase concentrations. Bands from top to bottom indicate lower molecular weight proteins to higher molecular weight proteins. Red arrow on the right indicates protein too large to enter gel. The bands in Figure 3a are lane 1 (protein molecular weight marker), lane 2 (BSA) and lanes 3-4 13 ACS Paragon Plus Environment


refer to the addition of 0.1% and 0.2% GTA, respectively. In Figure 3b the bands refer to protein molecular weight marker (lane 1), BSA (lane 2) while lanes 3-6 refer to the addition of 0, 5, 10 and 20 mg/mL TGase, respectively

SDS-PAGE analysis of native and polymerized Hb samples are shown in Figure 4. Lanes 1 to 6 represent protein molecular weight marker, native Hb and addition of 0.00625%, 0.0125%, 0.025% and 0.05% GTA, respectively. All samples show the presence of ~16 kDa protein with the exception of the 0.05% GTA sample. The ~16 kDa band is due to the presence of monomeric Hb obtained during subunit dissociation of the tetrameric Hb in the denaturing environment. The crosslinked proteins (red arrow on the right) in lane 6 indicate Hb can be successfully polymerized using GTA.

Figure 4. SDS-PAGE analysis of Hb crosslinked protein using different GTA concentration. Bands from top to bottom indicates lower molecular weight proteins to higher molecular weight proteins. Red arrow on the right of the gel indicates polymerized protein that was too large to enter gel. 14 ACS Paragon Plus Environment

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Circular Dichroism (CD) Spectroscopy. The conformational changes on the secondary structure of native and crosslinked samples have been studied by using circular dichroism spectropolarimeter. Circular dichroism graphs obtained for the native and crosslinked samples are displayed in Figure 5. The CD analysis was carried out in a region that is very sensitive to the secondary structure changes of protein (190 to 250 nm). Two negative bands at 222 nm and 208 nm were seen, typical of protein rich in alpha helix for both BSA and Hb samples. However, upon crosslinking, the secondary structure of these native proteins were destroyed, leading to more denatured or disordered protein. Similar CD graphs were also reported for both native and modified proteins (BSA and Hb) in past studies.28, 24 It is possible that the disordering of the protein folding exposes functional groups that would otherwise be buried, and allows them to participate in the binding of suspended particles.

150 GTA BSA BSA TGase BSA

100 Ellipticity (mdeg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GTA Hb Hb

50 0 -50 -100 190

210

230

250

Wavelength (nm)

Figure 5. CD spectra of native (Hb and BSA) and crosslinked proteins (GTA Hb, GTA BSA, TGase BSA). Traces for the all crosslinked proteins are superimposed about 0 mdeg. Samples (0.2 mg/mL) prepared in 10 mM phosphate buffer, pH 7.2, were run at 25 oC in a 1 mm cuvette from 190 to 250 nm. 15 ACS Paragon Plus Environment


Differential Scanning Calorimetry (DSC). Differential scanning calorimetry was carried out to study the influence of the crosslinking on the thermal stability of the proteins. Endotherm peaks are seen for both Hb and BSA (Figure S1). The high denaturing temperature observed compared to what is normally seen in the literature might be due to the higher concentrations (90 mg/mL) of the samples used for the analysis. It has been found that the denaturing temperature of BSA increases with increasing concentration of sample used.29 Of particular importance is the broader nature of the BSA peak that starts to denature around 65 oC. None of the crosslinked samples showed any sign of denaturing under the range of temperature studied. These observations, as well as the CD results, are consistent with the crosslinked proteins being in a disordered state. Denaturation may affect flocculant performance by changing the number of charged groups exposed to the solvent, or by increasing the “surface area” of the protein. Zeta Potential Measurement. At the isoelectric point, the total number of positive charges from protein amino groups and negative charges from the carboxylic groups are equivalent. At low pH (less than the isoelectric point of the protein) the protein is positively charged while at high pH (greater than the isoelectric point of the protein) the protein is negatively charged. Isoelectric points and zeta potential measurements obtained for the native and crosslinked proteins are displayed in Table 1 and Figure 6, respectively. After crosslinking, the isoelectric point of both proteins decreased. This is as a result of the reduction or utilization of the free amino groups during crosslinking. For example, positively charged amino groups are converted to isopeptide amide bonds during crosslinking.


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Table 1. Isoelectric point (pI) of kaolin, native and polymerized proteins Material

pI

Hb BSA

7.3 3.6

GTA Hb

3.0

TGase BSA

2.7

GTA BSA

2.5

Kaolin

2.1

30 20 Zeta potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10 0 -10 -20 -30 -40 2

4

Hb GTA Hb

6 pH BSA GTA BSA

8

10

TGase BSA kaolin

Figure 6. Zeta potential as a function of pH for kaolin, native and crosslinked proteins.

Application of crosslinked protein as flocculant. Results from the flocculation studies are presented in terms of Kaolin Clarification Efficiency (KCE), a logarithmic scale which is convenient for comparing the completeness of water clarification; removal of 99.0% of the original turbidity results in a KCE of 2, while removal of 99.9% of the original turbidity results



in a KCE of 3. The ability of the crosslinked samples to be used as flocculants were tested. The results of the flocculation studies are presented in Figure 7. Both the BSA and crosslinked GTA BSA samples show little to no flocculation activity (Figure 7a). It is possible that BSA is not an active flocculant primarily because of its low isoelectric point (pI = 3.6), and even after crosslinking (GTA BSA), there is no enhancement in its flocculation activity. Inability to GTA BSA to promote flocculation is surprising and the reason is currently unknown and will be investigated in detail later. However, lower solubility of the GTA BSA sample was observed in the kaolin suspension.

2

(b)

3

(a) 1.5

2.5

1 BSA GTA BSA TGase BSA

0.5

Mean KCE

Mean KCE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

2 1.5

Hb GTA Hb

1 0.5

-0.5

0 0

10

20

30

40

0

Flocculant dose (mg/g kaolin)

10 20 30 40 Flocculant dose (mg/g kaolin)

Figure 7. Kaolin clarification efficiency (KCE) of native and crosslinked proteins. Experimental conditions: settling time (5 h), flocculant dose (0 to 40 mg/g kaolin), temperature (20 oC), and pH (5.5). Error bars are ±1 standard deviation, from triplicate measurements.


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In the case of native Hb (peak KCE = 2.24), clarification of kaolin suspension was observed (Figure 7b). At pH 5.5, kaolin is negatively charged. At this pH native Hb (pI = 7.3) carries an overall positive charge, but has local regions of both positive and negative charges. There is, therefore, an electrostatic attraction between positively charged region of the Hb molecules and the negatively charged kaolin particles. Flocculation results from the neutralization of kaolin’s surface charges, resulting in a lessening of the electrostatic repulsion between kaolin particles. Recently, charge neutralization was also realized to be the main flocculation mechanism employed by native and methylated Hb.24 TGase BSA (peak KCE = 1.85, see Figure 7a) and GTA Hb (peak KCE = 2.96, see Figure 7b) showed substantial increase in their clarification efficiencies despite their low isoelectric points. Although both carry an overall negative charge at pH 5.5, these are high molecular weight polymerized proteins and therefore a portion of the protein can adsorb to two or more particles simultaneously, physically “bridging” the particles together and promoting flocculation. Here, the polymer will generally adopt an extended configuration with loops and tails that go beyond the electrical double layer, leading to ‘linking’ of the polymers with the particles.30 This is known in the literature as a bridging mechanism. Our results indicate the potential of using polymerized proteins as flocculants for the clarification of colloidal suspensions during the water treatment process. Chemical oxygen demand (COD) was used as measure of the pollution load after treating the kaolin suspension with the flocculant. To estimate how much COD is being added by the flocculant, native and crosslinked proteins were tested together with the control. The COD values of the supernatant after the 5 h flocculation experiment were measured using the Hach Ultra High Range COD vials and DR 3900 spectroscopy (Hach, Loveland, CO) by following the 19 ACS Paragon Plus Environment


manufacturer’s protocol. The COD values after the addition of the flocculants were found to be between 4713 and 4947 mg/L compared to the control (4903 mg/L), see Table 2. The COD change were between 0.55% and 3.88% indicating the presence of the flocculant slightly changes the COD content of the suspension. The results obtained in the study would be very valuable to help minimize the impact the residual native and crosslinked flocculants could have on wastewater treatment process.

Table 2: Characteristics of the supernatant after flocculation with native and crosslinked proteins. Flocculation was done using a settling time of 5 h, flocculant dose of 20 mg/g kaolin and a temperature of 20 oC. A control (using only kaolin suspension) was also done in parallel

pH COD, mg/L COD change (%)

Control 5.71 4903

TGase BSA 5.93 4930 0.55

Hb

GTA Hb

5.69 4947 0.90

5.68 4713 3.88

CONCLUSIONS Hb and BSA proteins have been successfully polymerized using both enzymatic and chemical means. Electrophoresis, size exclusion chromatography, zeta potential measurement, circular dichroism and differential scanning calorimetry studies confirmed that these polymerized samples exhibit high molecular weight (~2000 kDa), lower isoelectric points and disordered structures. This study shows that polymerized proteins can be used as effective flocculants, considering the fact that commercial polymeric flocculants are of environmental concern. The findings are an advance towards the substitution of synthetic polymer flocculants with biopolymer-based alternatives. 20 ACS Paragon Plus Environment

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ASSOCIATED CONTENT Supporting Information Additional data (DSC thermogram) AUTHOR INFORMATION Corresponding Author Email: [email protected] ORCID Matthew Essandoh: 0000-0002-3117-9141 Notes The authors declare no competing financial interest

REFERENCES 1. Li, H.; O'Shea, J.-P.; Franks, G. V. Effect of molecular weight of poly(N-isopropyl acrylamide) temperature-sensitive flocculants on dewatering. AlChE J. 2009, 55 (8), 2070-2080. 2. Lee, C. S.; Robinson, J.; Chong, M. F. A review on application of flocculants in wastewater treatment. Process Saf. Environ. Prot. 2014, 92 (6), 489-508. 3. Renault, F.; Sancey, B.; Badot, P. M.; Crini, G. Chitosan for coagulation/flocculation processes – An eco-friendly approach. Eur. Polym. J. 2009, 45 (5), 1337-1348. 4. Orts, W. J.; Sojka, R. E.; Glenn, G. M. Biopolymer additives to reduce erosion-induced soil losses during irrigation. Ind. Crop. Prod. 2000, 11 (1), 19-29. 5. Piazza, G.; Garcia, R. Proteins and peptides as renewable flocculants. Bioresour. Technol. 2010, 101 (15), 5759-5766. 6. Essandoh, M.; Garcia, R. A.; Nieman, C. M.; Bumanlag, L. P.; Piazza, G. J.; Zhang, C. Practical limitations of the dilute acid hydrolysis method for solubilizing meat and bone meal protein. ACS Sustain. Chem. Eng. 2017, 5 (12), 11652-11659. 7. Seki, H., Suzuki, A. Flocculation of diatomite by methylated egg albumin. J. Colloid Interface Sci. 2003, 263, 42-46. 8. Bonilla, S.; Allen, D. G. Flocculation with lysozyme: A non-enzymatic application. Can. J. Chem. Eng. 2015, 94, 231-237. 9. Piazza, G. J.; Lora, J. H.; Garcia, R. A. Flocculation of kaolin and lignin by bovine blood and hemoglobin. J. Chem. Technol. Biotechnol. 2015, 90, 1419-1425. 10. Carter, D. C.; Ho, J. X. Structure of serum albumin. Adv. Protein Chem. 1994, 45, 153-203. 11. Piazza, G. J.; Nunez, A.; Garcia, R. A. Identification of highly active flocculant proteins in bovine blood. Appl. Biochem. Biotechnol. 2012, 166, 1203-1214. 12. Gerrard, J. A. Protein-protein crosslinking in food: methods, consequences, applications. Trends Food Sci. Technol. 2002, 13 (12), 391-399. 13. Habeeb, A. F. S. A.; Hiramoto, R. Reaction of proteins with glutaraldehyde. Arch. Biochem. Biophys. 1968, 126 (1), 16-26. 21 ACS Paragon Plus Environment


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For Table of Contents Use Only

SEC of polymerized protein

Application

Performance Mean KCE

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.5 Protein 0.5

Polymerized protein

-0.5 5

10

15

20

Ret. time (min)

0 15 30 45 Flocculant dose (mg/g)

Synopsis Polymerized proteins generated through enzymatic and chemical crosslinking were effective as flocculants.


Chemical and Enzymatic Protein Cross-Linking To Improve Flocculant

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