Separation and Purification Technology 138 (2014) 28–33
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Recovery of Bacillus cereus cyclodextrin glycosyltransferase using ionic liquid-based aqueous two-phase system Hui Suan Ng a, Chien Wei Ooi b, Pau Loke Show c,i, Chin Ping Tan d, Arbakariya Ariff e, Mohd Noriznan Moktar f, Eng-Poh Ng g, Tau Chuan Ling h,⇑ a
Department of Food Science and Nutrition, Faculty of Applied Sciences, UCSI University, UCSI Heights, 56000 Cheras, Kuala Lumpur, Malaysia Discipline of Chemical Engineering, School of Engineering, Monash University Malaysia, 47500 Bandar Sunway, Selangor, Malaysia Department of Chemical and Environmental Engineering, Faculty of Engineering, University of Nottingham Malaysia Campus, Jalan Broga, Semenyih 43500, Selangor Darul Ehsan, Malaysia d Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia e Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia f Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia g School of Chemical Sciences, Universiti Sains Malaysia, Minden 11800, Malaysia h Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia i Manufacturing and Industrial Processes Division, Faculty of Engineering, Centre for Food and Bioproduct Processing, University of Nottingham Malaysia Campus, Jalan Broga, Semenyih 43500, Selangor Darul Ehsan, Malaysia b c
a r t i c l e
i n f o
Article history: Received 15 May 2014 Received in revised form 21 August 2014 Accepted 27 September 2014 Available online 18 October 2014 Keywords: Imidazolium Aqueous two-phase system Cyclodextrin glycosyltransferase Bacillus cereus Purification
a b s t r a c t Ionic liquids-based aqueous two-phase system (ILATPS) offers immediate phase separation and thus reduces the overall processing time, is significantly advantageous as compared to conventional ATPS such as polymer/polymer ATPS and polymer/salt ATPS. In this study, ILATPSs composed of imidazolium-based ionic liquid (IL) and salt were experimentally evaluated for their efficiencies in recovering Bacillus cereus cyclodextrin glycosyltransferase (CGTase) from fermentation broth. The phase-forming behavior of 1ethyl-3-methylimidazolium tetrafluroborate, (Emim)BF4/sodium citrate ILATPS and (Emim)BF4/sodium carbonate ILATPS were first studied by constructing the binodal curves. Effects of the ILs concentration, pH value, feedstock loading, and addition of sodium chloride (NaCl) on the recovery of CGTase in ILATPS were investigated. The optimum conditions for the recovery of CGTase were obtained in an ILATPS consisting of 35% (w/w) (Emim)BF4, 18% (w/w) sodium carbonate and 3% (w/w) NaCl. Experimental results showed that 78% of CGTase could be recovered in the IL-rich phase in single-step purification with a purification fold (PF) of 15.4. The high PF indicates that this ILATPS is feasible to be applied in the recovery and separation of CGTase from the fermentation broth. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Ionic liquids (ILs) are potential green extraction solvents for various biomaterials due to their unique features such as high chemical and physical stability, negligible vapor pressure, and also non-flammability. The relatively lower viscosity of ILs as compared to the polymer-based phase-forming reagents (e.g. PEGs, dextran, etc.) offers rapid phase separation in ATPS construction [1–4]. Besides, ILs is suggested to be an attractive ATPS-forming component because of the ability to ensure a clean manufacturing process due to the negligible vapor pressure [5]. ILATPSs have been used in the extraction of small organic molecules, proteins and other bio⇑ Corresponding author. Tel.: +60 3 79674104; fax: +60 3 79674178. E-mail address:
[email protected] (T.C. Ling). http://dx.doi.org/10.1016/j.seppur.2014.09.038 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.
materials [3,5–7]. ILATPSs are capable of overcoming the limitations of conventional ATPSs by adopting a more environmentally benign ATPS. Other advantages of these ILATPSs include enzyme stabilization, high selectivity of product or substrate in ATPS, and negligible emulsion formation [5]. However, ILs is rather expensive as compared to other ATPS phase-forming components, thus the recycling of ILs is significantly important for the sustainable downstream processing of the biomaterials [8]. The extremely low vapor pressure featured by ILs enables them to be easily recycled with least energy consumption, and thus minimizes the cost of the ILATPSs [9]. ILATPSs have become more attractive when several strategies for recycling of ILs have been established (e.g. ultrafiltration [10], dialysis, pervaporation [11] and supercritical CO2), where the potency of recycling of ionic liquids have been affirmed. Recent studies also showed that ILs can be recycled through electrodialysis (membrane
H.S. Ng et al. / Separation and Purification Technology 138 (2014) 28–33
separation) and different types of distillations such as vacuum evaporation, column distillation and molecular distillation [12–14]. ILATPSs are usually formed by using hydrophilic ILs instead of hydrophobic ILs because hydrophobic ILs are relatively more expensive and environmentally disadvantageous as compared to hydrophilic ILs. Hydrophilic ILs are unable to be directly applied in the ATPS formation due to the complete mutual solubility. They often required the presence of an aqueous solution of water-structuring salts (i.e. promoters for the water structure) or known as kosmotropes in order to be salted out as a separated aqueous phase in ATPS [15,16]. Citrate salt was suggested as a promising candidate for promoting for the phase separation of ILATPS owing to its biodegradability and non-toxicity. It can be readily disposed into biological wastewater treatment plants, results in least environmental impact factor [17]. Cyclodextrin glycosyltransferase, CGTase (E.C. 2.4.1.19) is a hydrolytic enzyme used in the starch hydrolysis for the synthesis of cyclodextrins (CDs) [18]. Bacterial CGTase often involved in the production of three major types of CDs, namely a-CD, b-CD and c-CD at different ratio. Besides, large-ring CDs consisted of nine and more glucose units can also be produced by CGTase [19]. CDs are cyclic oligosaccharides contain an apolar cavity that is capable to structure inclusion complexes with various guest molecules and changed their physiochemical properties [20]. CDs are widely utilized in various industries owing to the unique structure of CDs and their derivatives [20–22]. As a consequence of the increasing demands of CDs in various industries, it is vital to enhance the recovery and purification strategies of CGTase. In previous studies, PEG/citrate ATPS and ethylene oxide-propylene oxide (EOPO)/phosphate ATPS were developed for the recovery of Bacillus cereus CGTase from fermentation broth with PF of 16.3 and 13.1 respectively [23,24]. Though PEG/citrate ATPS showed high PF of CGTase, it required longer settling time and centrifugation process to attain the phase separation which is disadvantageous for large-scale production of CGTase. Besides, recycling of PEG often coupled with complicated procedures that increase the overall operation costs. Thus, application of ILATPS in the recovery of B. cereus CGTase was deployed in this study with an aim to improve the existing polymer/salt ATPS in the context of cost-efficiency. Moreover, it was suggested that the ILATPS is able to improve the purification efficiency of CGTase through enhancement of CGTase selectivity and preservation of the biological activity in ILATPS [5]. At present, there are very limited studies reporting on the use of ILATPS for the purification of protein. To date, reports on the purification of fermented B. cereus CGTase using the ILATPS are still unavailable. In this study, 1-ethyl-3-methylimidazolium tetrafluroborate, (Emim)BF4 has been selected as the phase-forming component in ILATPS for the recovery of CGTase. The effects of different types of salts on the phase separation and partition coefficient of CGTase have been investigated. The feasibility and purification efficiency of the ILATPSs on the recovery of B. cereus CGTase were studied. The working parameters of ATPS such as concentrations of phase components, feedstock loading, addition of neutral salt, NaCl have been evaluated for the optimum recovery of CGTase.
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were of analytical grades except for (Emim)BF4 which was of chemical grade. 2.2. B. cereus cultivation and CGTase production B. cereus was cultivated in a 500-mL shake flask containing 100 mL of the medium as described in a previous publication [23]. A 10% inoculated culture medium was grown under aerobic condition at 37 °C for 30 h with continuous agitation (250 rpm). The crude feedstock containing CGTase was prepared by subjecting the fermentation broth to centrifugation at 4000 rpm for 15 min. The supernatant was stored at 4 °C. 2.3. CGTase activity assay CGTase activity was evaluated spectrophotometrically (550 nm) using phenolphthalein method [25,26] with some modifications. 25 lL of enzyme sample (either the crude enzyme or the enzyme samples withdrawn from ILATPS) was added into 750 lL substrate solution [1% (w/v) starch in 0.05 M Tris–HCl buffer pH 8.0] and incubated at 55 °C for 15 min. 375 lL of 0.15 M NaOH was then added into the reaction mixture for enzyme deactivation. Finally, 100 lL of 0.02% (w/v) phenolphthalein reagent was added. The amount of the b-CD produced was determined by using a standard curve of b-CD. One unit of CGTase activity was defined as the amount of CGTase producing 1 lmol of b-CD per min under the assay conditions. 2.4. Bicinchoninic acid assay (BCA assay) Total protein concentration was measured using BCA assay [27] and calculated using a bovine serum albumin (BSA) standard curve. In a microtiter plate, 200 lL of BCA working reagent was added into the well containing 50 lL samples. The microtiter plate was then incubated at 37 °C for 30 min followed by the measurement of absorbance at the wavelength of 562 nm. To avoid the interference of IL, a blank sample (i.e. an ILATPS containing same phase composition without the feedstock) was prepared. 2.5. Ilatps 2.5.1. Phase diagram Binodal curves, denoting the two-phase formation as described by Albertsson [1] were constructed based on turbidometric titration method [28]. Known concentrations of IL and salts were mixed to form several ILATPSs in each tube with different total composition. The resulting mixture was turbid initially due to the immiscibility of the two phase-forming components. Distilled water was added drop-wise into the mixture and mixed well until the turbidity was clear. Disappearance of the turbidity indicated the attainment of critical point at which the resulted mixture was homogeneous. The binodal node was determined by measuring the weight of distilled water added for the turbidity to disappear. The final concentrations of IL and salts were calculated and the binodal curves of ILATPSs were plotted at varying concentrations of ILs and salts.
2. Materials and method 2.1. Materials (Emim)BF4 (CAS: 143314-16-3) was obtained from Fluka Co. (USA). Bicinchoninic acid solution (BCA), a-, b-, and c-CDs were purchased from Sigma Chemical Co. (MO, USA). Phenolphthalein, sodium chloride, sodium citrate and sodium carbonate were purchased from Merck (Darmstadt, Germany). All of these chemicals
2.5.2. Partitioning of the enzyme CGTase A 10-g of ILATPS was prepared by transferring appropriate amounts of IL, salts, 20% (w/w) crude CGTase and distilled water into a 15-mL centrifuge tube. The mixture was then gently stirred and followed by a centrifugation at 2000 rpm for 1 min for complete phase separation. VR of each ILATPS was measured. Samples from top and bottom phases were analyzed using CGTase activity assay and BCA assay.
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2.5.3. Determination of partition coefficient, selectivity, purification fold and yield Distribution of CGTase and total protein in both phases was denoted by the partition coefficient of CGTase (Ke) (1) and partition coefficient of protein (Kp) (2), respectively:
AT AB PT Kp ¼ PB
Ke ¼
ð1Þ ð2Þ
where AT and AB represent the CGTase activity (U/mL) obtained in top and bottom phases of the ILATPS, respectively. PT and PB represent the total protein concentrations (mg/mL) in top phase and bottom phase of ILATPS, respectively. Selectivity (S) of the ILATPS was defined as the ratio of Ke to Kp (3):
S¼
Ke Kp
CGTase activity Total protein concentration
ð4Þ
Purification fold (PF) of the CGTase in the IL-rich phase was measured as the ratio of the SA in the IL-rich phase (SAI) to the SA in the original feedstock (SAc) (5):
PF ¼
SAI SAC
ð5Þ
Yield (YT) of CGTase recovered in IL-rich phase was evaluated by using Eq. (6):
Y T ð%Þ ¼
Phase components
Concentration, %(w/w)
Relative activity
Ionic liquid, (Emim)BF4
30 50 80
0.97 0.78 0.32
Sodium citrate
20 40 60
0.99 0.85 0.57
Sodium carbonate
20 40 60
0.98 0.92 0.68
ð3Þ
Specific activity of CGTase, SA (U/mg) was calculated as the ratio of CGTase activity to the total protein concentration (4):
SA ¼
Table 1 Effect of phase components on the enzyme activities of B. cereus CGTase. Effects of (Emin)BF4, sodium citrate and sodium carbonate on the B. cereus CGTase enzyme activities were investigated at varying concentrations. The crude CGTase was incubated in each phase components at room temperature for 1h prior to the CGTase enzyme assay. A control with incubation of CGTase in the 0.05 M Tris-HCl buffer (pH 8.0) was prepared for the evaluation of the CGTase relative activity in each sample.
100 1 þ V1R K e
ð6Þ
where VR is the ratio of the volume of top phase to the volume of the bottom phase in ILATPS. 2.5.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) analysis SDS–PAGE analysis was carried out according to the procedures as described previously with some modification [29,30]. Page Blue™ Protein Staining Solution was used and the staining process was performed under gentle agitation for 1 h. Protein bands were visualized using a gel documentation system (Alphaimager, Alpha Innotec GmbH, Germany) after de-stained with distilled water for 5 min.
gested that IL is able to improve the enzyme stability though high concentrations of ILs will decrease the enzyme activity [4,5,32,33]. CGTase activity was not considerably affected by the presence of sodium citrate and sodium carbonate, except at high concentration of salts (Table 1). However, protein precipitations were observed in high concentration of salts [e.g. 60% (w/w)], result in the loss of CGTase activities. The occurrence of protein precipitation was mainly resulted from the salting-out effect. Salting-out effect was observed when CGTase has reached its maximum solubility in highly concentrated salt solutions. High salt concentration corresponds to lesser water molecules to interact with CGTase and eventually leads to precipitation of CGTase [4]. As a result, ILATPS is developed at lower concentrations of (Emim)BF4 and salts for the recovery of CGTase. 3.2. Phase diagram of (Emim)BF4/salts ILATPSs Phase diagrams define the biphasic compositions which are important for the partitioning and purification processes of enzymes. Phase diagrams of (Emim)BF4/citrate ILATPS and (Emim)BF4/carbonate ILATPS that differ in the types of anions present were constructed (Fig. 1). Kosmotropic ions such as citrate and carbonate anions promote the biphasic system formation as they possess high water affinity that is able to attract water molecules towards them by forming strong intermolecular interaction. It was observed that the binodal curve of (Emim)BF4/citrate is closer to the origin, indicating stronger phase-separation ability as compared to that of (Emim)BF4/carbonate (Fig. 1). The immiscibility
3. Results and discussion 3.1. Effects of phase components on CGTase enzyme activity Concentrations of IL and salts used in the ATPS formation are the factors affecting the stability of CGTase throughout the enzyme recovery process [31]. To evaluate the effect of each phase component on the B. cereus CGTase stability, crude CGTase was mixed with IL or salts at varying concentrations. The results are shown in Table 1. CGTase activity was stable at lower concentrations [30% (w/w) and 50% (w/w)] of IL but it was suppressed by high concentration of IL, (Emim)BF4 [80% (w/w)] with a relative activity of CGTase of 0.32 (Table 1). This indicates that the lower concentrations of (Emim)BF4 were preferred for the CGTase recovery. Loss of biological activity of CGTase and protein precipitation could be minimized at lower concentrations of (Emim)BF4. Several literatures on the applications of ILATPS on the protein purification sug-
Fig. 1. Phase diagrams of ILATPSs. Phase diagrams of two ILATPSs were constructed at 298 K. The binodal curves of (Emim)BF4/citrate ILATPS ( ) and (Emim)BF4/ carbonate ILATPS ( ) were plotted.
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of (Emim)BF4 and salts (i.e. citrate and carbonate) was due to the salting-out effect that could be explained by Gibbs energies of hydration (DGhyd) [4]. Citrate ion which has more negative DGhyd value (DGhyd = 2315 kJ/mol) than carbonate ion (DGhyd = 1300 kJ/mol) shows ability to attract more water molecules towards them [4,34]. Thus, the concentrations of salts and IL required for the formation of (Emim)BF4/citrate ILATPS were lower. Formation of ion-hydration complexes was found when water molecules were excluded from the IL-rich phase, thereby promoting the ILATPS formation [4].
Table 3 Partitioning of CGTase in different phase composition of (Emim)BF4/carbonate ILATPS. Partitioning of CGTase in (Emim)BF4/carbonate ILATPS was investigated. The concentrations of (Emim)BF4 were varied from 30% (w/w) to 38% (w/w) while the concentrations of sodium carbonate were varied from 15% (w/w) to 27% (w/w). Partitioning behavior of CGTase in (Emim)BF4/carbonate ILATPS were summarized in terms of selectivity and PF. Phase composition
3.4. Partitioning of CGTase in ILATPS The Ke and PF of CGTase in ILATPS were determined at different concentrations of IL and carbonate salts as depicted in Table 3. The Table 2 Partitioning of CGTase in ILATPSs. The phase composition for both (Emim)BF4/citrate and (Emim)BF4/carbonate were fixed at 20% (w/w)/25% (w/w). The partitioning behavior of CGTase was evaluated in each ILATPS. The relative activity of CGTase in each phase component solution was determined as the ratio of CGTase activity in the phase component to the crude enzyme activity. Salts
Concentration of (Emim)BF4/salt, % (w/w)
pH
Selectivity
PF
Sodium citrate Sodium carbonate
20/25 20/25
9.12 9.38
2.46 4.63
1.78 4.32
PF
Sodium carbonate, % (w/w)
30
15 18 21 24 27
1.01 7.56 5.05 1.14 0.55
3.12 6.79 4.43 0.99 0.34
35
15 18 21 24 27
6.12 9.66 5.73 2.59 2.37
9.82 11.75 8.81 5.46 2.82
38
15 18 21 24 27
0.93 6.38 5.31 5.04 0.03
0.43 1.67 4.83 3.97 0.03
3.3. Selection of types of salts for ILATPS The Ke and PF of CGTase in (Emim)BF4/citrate ILATPS and (Emim)BF4/carbonate ILATPS were determined. The concentrations of IL and salts for both the ILATPSs were fixed at 25% (w/w) and 20% (w/w), respectively. The pHs for both ATPSs were 9.12 and 9.38, respectively (Table 2). Table 2 shows the Ke, selectivity and PF of CGTase in both types of ILATPSs. Based on the results obtained, CGTase showed significantly higher selectivity (4.63) and PF (4.32) in (Emim)BF4/carbonate ILATPS. In addition, protein precipitation at the interphase was observed in (Emim)BF4/citrate ILATPS. It was postulated that the low selectivity (2.46) and PF (1.78) of CGTase obtained from (Emim)BF4/citrate ILATPS could be attributed to the loss of protein due to the precipitation. The pH of an ATPS is often the main factor governing the partitioning of a target protein in ATPS. The changes in pH in ILATPS might affect the electrostatic interaction between the phase components and the exposed groups of target protein. Moreover, the net charge of protein that driven the partition of target protein is greatly affected by the pH of the ILATPS. The protein would be more negatively charged as the pH of the ILATPS increased [35]. In the developed ILATPSs with pH of 9.12 and 9.38, respectively (Table 2), CGTase (isoelectric point, pI = 6.9) [36] was negatively charged in both the ILATPSs, resulting in a good partitioning of CGTase towards the IL-rich phase as indicated in Table 2. The negatively charged characteristics exhibited by CGTase at these pHs caused a strong repulsion force exerted by the salt-rich phase, thereby facilitated the partitioning of CGTase into the IL-rich phase [37]. IL-rich phase is relatively neutral as compared to the salt-rich phase because the (Emim)BF4 possess a pH of 7. The partition of CGTase to the IL-rich phase was enhanced at these alkaline pHs when negatively charged CGTase interacted with the cation (i.e. (Emim)+) in IL-rich phase, driving more of the CGTase to the IL-rich phase. ILATPSs formed at extreme pHs (pH > 10) were not suitable for the partitioning of proteins because they may denature the protein or lead to the unfolding of the target protein [37]. In view of all these, a better partition efficiency of CGTase was observed in (Emim)BF4/carbonate ILATPS and thus (Emim)BF4/carbonate ILATPS was selected for the recovery of CGTase.
Selectivity
(Emim)BF4, % (w/w)
optimum partitioning of CGTase was observed at phase composition comprised of 18% (w/w) carbonate salt and 35% (w/w) IL. Highest selectivity (9.66) and PF (11.75) were exhibited in this optimum ILATPS (Table 3). Overall results indicated that the partition efficiency of CGTase in (Emim)BF4/carbonate ILATPS is higher in lower concentrations of carbonate and IL (Table 3). An increase in concentration of IL corresponded to an increase in the relative hydrophobicity [38]. The increase in hydrophobic attraction in ILrich phase for CGTase enhanced the partition of CGTase towards the IL-rich phase. Same phenomenon was observed in increased salt concentration, where the partition efficiency of CGTase increases with increasing salt concentration. The salting-out strength of a salt was often associated with the concentration of salt in the ILATPS. A high concentration of carbonate salt resulted in higher salting-out effect of CGTase, which repelled the CGTase towards the IL-rich phase, thereby enhanced the partition efficiency of CGTase in ILATPS. However, precipitated protein was observed at interphase at high concentration of IL (i.e. 38% (w/ w)) and high concentration of carbonate salts (i.e. 24% (w/w) and 27% (w/w)). The protein precipitation would result in denaturing and unfolding of CGTase and eventually led to loss of CGTase activity [27].
3.5. Effects of feedstock load on partitioning of CGTase in (Emim)BF4/ carbonate ILATPS To achieve the maximum yield of CGTase from the ILATPS recovery, the effect of amount of feedstock load on the partition efficiency of CGTase was evaluated in ILATPSs with varying feedstock load [i.e. 10–25% (w/w)]. As shown in Fig. 2, the maximum recovery of CGTase in ILATPS was achieved with 15% (w/w) feedstock load with that exhibited the highest YT (90.12%) and PF (11.08). Higher concentration of feedstock load often accompanied with high percentage of impurities. The presence of the impurities might alter the phase compositions and the electrostatic potential of ILATPS where they reacted with the phase components through electrostatic interactions, thereby reduced the partition affinity of CGTase towards the IL-rich phase. As a result, the partition efficiency of CGTase in ILATPS has been reduced. In addition, higher feedstock load would result in loss of CGTase at interphase precipitation due to the excluded-volume effect [21]. All these explained
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1
2
kDa
3
4
100 70 55
CGTase
40 35 25 15
10 Fig. 2. Effect of crude load on partitioning of CGTase in (Emim)BF4/carbonate ILATPS. Effect of crude load on the partitioning of CGTase in (Emim)BF4/carbonate ILATPS was investigated. (Emim)BF4/carbonate ILATPS composed of 35% (w/w) (Emim)BF4 and 18% (w/w) sodium carbonate was used in this experiment. PF ( ) and YT ( ) were determined according to Eqs. (4) and (5) respectively.
the reduction of CGTase recovery in higher concentration of feedstock loaded into the ILATPS. 3.6. Addition of NaCl on partitioning of CGTase in (Emim)BF4/ carbonate ILATPS As mentioned earlier, the main driving forces for the partitioning of CGTase in ILATPS were attributed with the electrostatic and hydrophobic interaction between the CGTase and phase components. In view of these, the partitioning of CGTase was believed to be improved by the increase in the electrostatic potential of ILATPS. Addition of neutral salts (e.g. NaCl) into the ILATPS permitted the increase of electrostatic potential difference as described in other literature [39]. Fig. 3 shows the partition efficiency of CGTase in different ILATPS varied with concentration of additional salt, NaCl. The optimum condition of CGTase partitioning is attained with additional 3% (w/w) NaCl into the developed ILATPS (Fig. 3). Improved YT (96.23%) and PF (13.86) were observed in this optimum ILATPS.
Fig. 4. SDS–PAGE analysis on the recovery of CGTase. The purity of partitioned CGTase was assessed by a 12% SDS–PAGE analysis. Sample of recovered CGTase was obtained in the IL-rich phase in the (Emim)BF4/carbonate ILATPS composed of 35% (w/w) (Emim)BF4, 18% (w/w) sodium carbonate, 3% (w/w) NaCl and 15% (w/w) of crude load. Molecular mass of standard protein marker ranged from 7 to 175 kDa. SDS–PAGE – Lanes 1 and 3: protein molecular markers; lane 2: crude feedstock; lane 4: IL-rich phase.
Presence of NaCl resulted in larger electrostatic potential difference in ILATPS, thereby promoting the migration of CGTase towards the IL-rich phase. Addition of salt was suggested to affect the water structuring salt (i.e. carbonate), leading to alteration of phase composition of ILATPS, where it affected the partitioning of CGTase [1,40]. Besides, a phase inversion was observed in the ILATPS added with NaCl due to the modification of phase density.
3.7. CGTase recovery from ILATPS (SDS–PAGE analysis) The maximum recovery of CGTase was achieved in ILATPS comprised of 35% (w/w) IL, 18% (w/w) carbonate salt, 15% (w/w) loaded feedstock with addition of 3% (w/w) of NaCl. Partitioned enzyme present in IL-rich phase could be recovered by dialysis or distillation. The purity of CGTase was evaluated by SDS–PAGE and the profile was depicted in Fig. 4. Characteristic multiple bands were observed in crude feedstock (lane 2), indicating the presence of unwanted proteins in the crude feedstock. In general, B. cereus CGTase has a molecular mass in the range of 33–103 kDa [36]. Sample from ILrich phase (lane 4) contained a significant single band, which was corresponded to a molecular mass of 55 kDa, suggesting the high purification efficiency of ILATPS on CGTase. No loss of enzyme activity was observed along the purification process of CGTase.
4. Conclusion
Fig. 3. Effect of NaCl on partitioning of CGTase in (Emim)BF4/carbonate ILATPS. Effect of NaCl addition on the partitioning of CGTase in (Emim)BF4/carbonate ILATPS was investigated. (Emim)BF4/carbonate ILATPS composed of 35% (w/w) (Emim)BF4 and 18% (w/w) sodium carbonate with 15% (w/w) crude load was used in this experiment. PF ( ) and YT ( ) were determined according to Eq. (4) and (5) respectively.
(Emim)BF4 was shown to be successfully applied in IL-salt based ATPS formation. Optimum recovery of B. cereus CGTase from fermentation broth was achieved in (Emim)BF4/carbonate ILATPS at 35% (w/ w) of (Emim)BF4 and 18% (w/w) of sodium carbonate with the addition of 3% (w/w) of NaCl and 15% (w/w) of crude loading. The results signify that ILATPS is an effective approach for the CGTase purification and recovery in a single operation with a relatively high PF (13.86) and YT (96.23%). ILATPS possessing the advantages of environmental benign, ease of recycling and possibility to increase the enzyme stability are proved to be a promising strategy for the recovery of CGTase. Thus, the application of ILATPS in large-scale industrial CGTase downstream processes is highly recommended.
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Acknowledgements This work was funded by Ministry of Higher Education (MOHE), Malaysia through the Fundamental Research Grant Scheme (FP0052013B and FRGS/1/2013/SG05/UNIM/02/1) and Ministry of Science, Technology and Innovation (Malaysia, MOSTI-02-02-12-SF0256).
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