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Partitioning of β-galactosidase in aqueous two-phase systems containing polyethyleneglycol and phosphate salts
Fluid Phase Equilibria 385 (2015) 147–152
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Fluid Phase Equilibria j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / fl u i d
Partitioning of b-galactosidase in aqueous two-phase systems containing polyethyleneglycol and phosphate salts Gholam Khayati a, *, Masumeh Anvari b , Nushin Shahidi a a b
Department of Chemical Engineering, Faculty of Engineering, University of Guilan, Rasht, P.O. Box 41625-3756, Iran Department of Microbiology, Islamic Azad University, Rasht Branch, P.O. Box 41335-3516, Rasht, Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 July 2014 Received in revised form 31 October 2014 Accepted 4 November 2014 Available online 6 November 2014
Purification of a desired protein from fermentation broth containing a wide variety of biomolecules is one of the major challenges in the biotechnology industry. One possible strategy for this challenge, an aqueous two-phase extraction (APTE) can be used as the primary downstream processing for partial purification of industrial enzymes. In the present study, the feasibility of utilizing ATPE for the partitioning behavior of b-galactosidase (from Aspergilus niger) was investigated. The results showed that the PEG2000/(NH4)2HPO4 system was suitable for the extraction of b-galactosidase from cell free fermentation broth of A. niger PTCC 1050. In the following, multilevel factorial design was used to evaluate the effects of three independent variables (PEG to salt weight ratio, fermentation broth loaded mass and system pH) on b-galactosidase extraction. The results indicated that PEG to salt weight ratio and fermentation broth loaded mass was the major contributory factors for b-galactosidase partitioning. Among the different conditions studied, a phase system having a composition of 15.24% (w/w) PEG2000 and 17.14% (w/w) (NH4)2HPO4 at pH 7 with loaded fermentation broth of 5% (w/w) gave the best overall results in term of purification degree. ã 2014 Elsevier B.V. All rights reserved.
Keywords:
b-galactosidase Aqueous two-phase system Poly ethylene glycol Multilevel factorial design
1. Introduction
b-galactosidase is one of the principal enzymes that has important applications [1]. This enzyme is mainly used to hydrolyze lactose into glucose and galactose. Problems of lactose are due to three main areas: health, food technology and environment. Concerning the health issue, lactose becomes a problem when there is insufficient intestinal b-galactosidase enzyme and lactose is passed into the blood, finally appearing in the urine and in the large intestine resulting in several disorders [2]. Lactose intolerant people are restricted to the lactose amount they can ingest daily. Because of these problems, the reduction of lactose in milk and dairy products is of prime importance and the enzyme b-galactosidase is commercially used for this purpose [3,4]. From the food technology point of view, the high lactose content in non-fermented milk products such as ice-c ream and condensed milk, can lead to excessive lactose crystallization resulting in products with a mealy, sandy or gritty texture. Again, the use of b-galactosidase enzyme prior to the condensing operation can reduce the lactose content to a point where lactose is no longer a problem [2]. Moreover, glucose and galactose expand
* Corresponding author. Tel.: +98 9111329514. E-mail address: [email protected] (G. Khayati). 0378-3812/$ – see front matter ã 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fluid.2014.11.003
the use of lactose in food because of a remarkable increase in sweetness and solubility [5]. New applications of b-galactosidase enzyme such as the recovery of biologically active oligosaccharides from milk have also been described in literature [6]. One of the major challenges in the biotechnology industry is the purification of a desired protein from a fermentation broth containing a wide variety of biomolecules [7]. Partitioning using aqueous two-phase systems (ATPS) has proved to be a valuable tool and mild technique for separating and purifying mixtures of biomolecules. These systems can form a gentle environment for enzymes and other biologically active proteins, therefore, the extraction in the systems has the advantages of high capacity, high activity yields and easy to scale up. These systems have many applications for protein purification in biochemistry and biotechnology [8,9]. The partitioning of biomolecules in ATPS is influenced by pH, temperature, surface properties, size and concentrations of the biomolecules and the types of employed polymers and salts [10]. In the present study, at first, the phase-forming salt was selected, and then molecular weight of PEG was varied. After the selection of phase-forming salt and molecular weight of PEG, the influence of process parameters such as PEG to salt weight ratio, loaded fermentation broth and pH of the system on b-galactosidase partitioning behavior was studied by multilevel factorial design and, the results were presented as well as discussed in the following sections.
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2. Materials and methods 2.1. Chemicals All chemicals used were of analytical grade. Aspergillus niger PTCC5010 was obtained from the Iranian Research Organization for Science and Technology (IROST). 2.2. Fermentation conditions The pre-culture medium was nutrient broth containing (per liter): 2.0 g yeast extract; 5.0 g peptone; 5.0 g NaCl and 1.0 g beef extract sterilized at 121 C for 15 min. Submerged fermentation was carried out in 250 ml shake-flasks with 50 ml medium containing the following constituents (g/L): glucose 5.0; lactose 40.0; peptone 0.25; yeast extract 0.5; (NH4)2SO4 3.0; KH2PO4 1.0 and K2HPO4 3.0. Sugars were sterilized separately. After 72 h of incubation, cells were harvested by centrifugation at 7000 rpm for 20 min. The filtrate was used as crude enzyme.
Fig. 1. Effect of phase composition in PEG4000/salt ATPS on the purification factor of b-galactosidase in top phase and selectivity. 1: 7.27%PEG4000 + 21.81% (NH 4)2HPO4; 2: 14.54%PEG4000 + 16.36% (NH 4)2HPO4; 3: 21.81%PEG4000 + 10.91 (NH 4)2HPO4; 4: 29.09%PEG4000 + 5.45% (NH 4)2HPO4; 6: 7.27%PEG4000 + 21.81% K2HPO4; 7: 14.54%PEG4000 + 16.36% K2HPO4; 8: 21.81%PEG4000 + 10.91 K2HPO4; 9: 29.09%PEG4000 + 5.45% K2HPO4.
C p;t C p;b
2.3. Preparation of aqueous two-phase systems
Kp ¼
All aqueous two-phase systems were done in 15 ml graduated centrifuge tubes at equal volume of salt and PEG solution in different conditions. To study the effects of PEG molecular weight and types of salt on the partitioning of b-galactosidase from cell free fermentation broth using ATPS, different salts including diammonium hydrogen phosphate and di-potassiom hydrogen phosphate were mixed with different PEG2000, 4000 and 8000 in aqueous system. The glass tube was shaken vigorously by turning upside-down followed by centrifuged for 10 min at 7000 rpm to assist phase separation. After phase separation and visual estimation of the top and bottom phase, the volumes of the phases were used to estimate the volume ratio. The samples of the top and bottom phases were carefully withdrawn by using a plastic syringe with short and long needle, respectively. Aliquots from each phase were analyzed to determine the enzyme activity and protein concentration. All experiments were run in duplicate at room temperature (24 1 C).
where Cp,t and Cp,b are concentrations of protein in top and bottom phase, respectively. The partition coefficient for b-galactosidase activities (Ke) in the aqueous two-phase systems were defined as:
2.4. Analytical methods
b-galactosidase assay was done spectrophotometrically. The enzyme activity was essentially determined according to the method described by Nagy et al. [11]. Protein concentration was measured by the method of Bradford [12] using bovine serum albumin as a standard. Results were evaluated by a few parameters including: protein partition coefficient in the aqueous two-phase systems was defined as [8]:
Ke ¼
(1)
At Ab
(2)
where At and Ab are enzyme activities in top and bottom phase, respectively. The partition coefficient (Ke) is used to quantify the degree of separation reached in an extraction process. Yield (%) of b-galactosidase in top phase was also calculated based on the partition coefficient to study the efficiency of the system by using the following equation. Yð%Þ ¼
100 ðV R K e Þ1 þ 1
(3)
The (VR) was defined as the volume ratio of the top phase to the bottom phase (Vt/Vb). Selectivity (S) was calculated as the ratio of the b-galactosidase enzyme partition coefficient (Ke) to the protein partition coefficient Kp. S¼
Ke At C p;b ¼ K p Ab C p;t
(4)
Table 1 Effect of phase composition in PEG4000/salt ATPS on partitioning of b-galactosidase at 24 C. Phase composition (%, w/w)
Ke
Kp
Y (%)
VR
7.27%PEG4000 + 21.81% (NH 4)2HPO4 14.54%PEG4000 + 16.36%(NH 4)2HPO4 21.81%PEG4000 + 10.91(NH 4)2HPO4 29.09%PEG4000 + 5.45%(NH 4)2HPO4 7.27%PEG4000 + 21.81% K2HPO4 14.54%PEG4000 + 16.36%K2HPO4 21.81%PEG4000 + 10.91 K2HPO4 29.09%PEG4000 + 5.45%K2HPO4
6.10 3.25 2.67 2.75 4.48 2.29 1.86 1.48
0.23 0.08 0.12 0.11 4.12 0.84 0.46 0.77
58.46 64.07 80.85 91.68 48.91 60.07 75.61 90.86
0.23 0.54 1.57 4.00 0.21 0.65 1.66 6.69
Fig. 2. Effect of PEG molecular weight in PEG/(NH4)2HPO4 ATPS on the yield (Y%) and partition coefficients of b-galactosidase (Ke).
G. Khayati et al. / Fluid Phase Equilibria 385 (2015) 147–152
The purification factor in the top phase (PFtop) was defined as PFtop ¼
SAt SAi
(5)
where SAt and SAi are specific activities in top and crude enzyme, respectively. Also, specific activity represents the ratio of the enzyme activity to the protein concentration in a sample. SA ¼
A Cp
(6)
where A and Cp are the enzyme activity and the total protein concentration in each phase, respectively. 3. Results and discussion Theoretical predictions of biomolecule extraction using ATPS could be difficult. This was because of the additives and/or impurities’ type and concentration in aqueous solution directly affected both system features and solute properties [9]. For the efficacy of aqueous two-phase extraction (ATPE), suitable phase system selection is very important. In ATPSs, the most significant and difficult step is to choose the appropriate type and composition of the system in terms of achieving the efficient extraction of the biomolecule [13]. It is well known that as a result of several factors such as type of phase forming salt, PEG molecular weight, concentration of salt and polymer, tie line length, volume ratio and ionic composition of the phases, partition and distribution behaviors of compounds in ATPSs are considered as very complex phenomena and therefore, inferring a general rule is not possible [14,15]. Choosing the phase forming components types for the distribution of the biomolecule is the second most important step following the selection of the phase system type as polymer– salt [16]. In this study, polymer–salt systems were selected because of some advantages they had such as lower cost and gentler environment besides lower viscosity in comparison to polymer– polymer systems. 3.1. Selection of phase forming salt The choice of phase-forming salt has a direct effect on separation and extraction of a given biomolecule in ATPE due to its significant influence on system environment [17]. Also, the selection of salt employed in the extraction system is one of the key points of this technique [18]. To ensure the efficiency of the extraction, the partitioning of b-galactosidase from A. niger was carried out by employing various phase forming salts, namely diammonium hydrogen phosphate and di-potassium hydrogen
Fig. 3. Effect of PEG molecular weight in PEG/(NH4)2HPO4 ATPS on the selectivity and specific b-galactosidase activity in top phase.
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phosphate with PEG4000 at different concentrations in order to arrive at the most suitable aqueous two-phase system. The PEG/ phosphate system is able to extract macromolecules from fermentation broth [19]. The results were summarized in Table 1. The distribution of the protein and b-galactosidase in ATPS was reported by Kp and Ke, respectively. In our results, the Kp in K2HPO4 system was much larger than that in (NH4)2HPO4 system, which was due to the greater salting-out power of K+ ions than of NH4+ ions [20]. Theoretically, high Kp value along with high Ke indicates that most of proteins as well as target one were more partitions to the top phase, while high Ke with low Kp implies that especially the target molecule (enzyme) was more partitioned into the top phase [21]. As shown in Table 1, all of the Ke in the systems are larger than 1, which indicates that b-galactosidase enzyme in the crude extract is preferably distributed on the top phase. The low Kp values indicate that the contaminant proteins and other undesirable components could shift to the bottom phase. In general, partitioning of the total proteins and enzyme to the bottom and
Table 2 Multilevel factorial experimental design and responses. Run
Factor levels
Response (PFtop)
WPEG/Wsalt
Loaded mass (%w/w)
System pH
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
0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 2 2 2 2 2 2 2 2 2 2 2 2 5.33 5.33 5.33 5.33 5.33 5.33 5.33 5.33 5.33 5.33 5.33 5.33
5 5 5 10 10 10 15 15 15 20 20 20 5 5 5 10 10 10 15 15 15 20 20 20 5 5 5 10 10 10 15 15 15 20 20 20 5 5 5 10 10 10 15 15 15 20 20 20
7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9
22.85 7.34 26.84 21.44 8.37 17.51 21.79 10.58 37.02 25.76 15.26 16.56 38.15 26.74 36.75 24.98 7.67 28.62 14.41 8.58 21.87 14.92 10.11 19.96 19.48 24.93 12.42 28.28 22.04 8.06 14.86 12.07 5.44 15.64 13.21 4.45 16.73 26.28 5.96 12.25 11.33 4.56 10.23 15.01 4.69 8.99 10.12 4.02
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top phases is dependent on their isoelectric points [19]. It is certain that one of the most important requirements of ATPS for an effective purification is that the contaminant proteins should not be extracted in the same phase with the target enzyme. Therefore along with a high Ke value, a low Kp value is desirable as well. Table 1 also shows that the yield of b-galactosidase in the top phase, with both salts were decreased when increase of salt concentration from 5.45 to 21.81 (%w/w). It can be found that increase in salt quantity caused the salting-out effect [22]. Enhancement of salt quantity provided the higher proportion of salt-rich bottom phase leading to practically reduced of VR. Johansson [23] reported that the partitioning of a protein is influenced by the presence of salts and this effect is enhanced with increasing of the protein net charge. It is found that di-ammonium hydrogen phosphate salt has shown higher results in terms of selectivity and purification factor in top phase in compared to that of di-potassium hydrogen
phosphate (Fig. 1). The results showed that the partitioning of b-galactosidase were strongly dependent on the type of salts. This was mainly due to the favorable biocompatible phase environment by the ammonium salt and hence, this system was selected for further studies. 3.2. Selection of polymer molecular weight It has been demonstrated that the molecular weight of PEG affects its distribution in the two phases and polymer–protein interactions [8]. Therefore, in order to select a suitable PEG molecular weight for the extraction of b-galactosidase from A. niger, ATPE was performed with different molecular weights of PEG (MW 2000, 4000 and 8000) at ambient temperature. The enzyme partition coefficients, yield of b-galactosidase in the top phase, selectivity and specific b-galactosidase activity in the top phase are shown as the functions of PEG molecular weight in Figs. 2 and 3. The results showed that the partitioning of b-galactosidase in PEG/ (NH4)2HPO4 system was strongly dependent on the molecular weight of the PEG. The yield (Y%) and partition coefficients of b-galactosidase (Ke) were found to decrease with the increase of PEG molecular weight (Fig. 2). About 16.5% decrease in extraction yield and 51.4% decrease in partition coefficient of b-galactosidase was observed. At the same time, it can be seen that volume ratio decreased with an increase in molecular weight of PEG (not shown). In other words, the volume of the bottom phase increased with an increase in PEG molecular weight, which promotes selective partitioning of the enzyme to the bottom phase which is its preferential phase. Also the selectivity plot shows that the values of S decreased sharply with the increase of PEG molecular weight from 2000 to 8000. While SAtop of b-galactosidase was found to decrease gradually with the increase of PEG molecular weight (Fig. 3). This means that the b-galactosidase increasingly partitions to the bottom phase as the PEG molecular weight increases. Similar trends were found by the other researchers [24–26] and could be attributed to two different effects. The most obvious was the increase of the top phase hydrophobicity. In fact as PEG chain length increases (with increasing molecular weight) there will be less hydroxyl groups for the same concentration of the polymer and so the hydrophobicity of the polymer-rich phase (top) increases [27]. On the other hand, the increase of the chain length will also cause the reduction of the free volume [28,29], meaning less space available for the b-galactosidase. The increase of PEG molecular weight had facilitated the partitioning of b-galactosidase to the bottom phase. It can be concluded from the Fig. 3 that maximum selectivity of b-galactosidase (S = 50.05) was observed when the smallest PEG had been used. We suggest that the lower PEG molecular weight is more favorable for b-galactosidase partitioning as the best system was obtained at PEG2000/(NH4)2HPO4. After selecting the type of salt and molecular weight of PEG, a multilevel factorial design of experiments was chosen for thorough investigation of the effects Table 3 Analysis of variance of the regression parameters for the multilevel factorial design. Source value
Fig. 4. Main effects of the variable for the purification factor of b-galactosidase in top phase based on the multilevel factorial design results.
WPEG/ Wsalt Loaded mass (%w/w) System pH WPEG/Wsalt loaded mass WPEG/Wsalt pH Loaded mass pH Error
D. F.
Sum of square Mean square
Fvalue
p-value
3 3 2 9
846.09 649.95 207.14 763.41
282.03 216.65 103.57 84.82
15.84 12.17 5.82 4.76
0.000 0.000 0.011 0.002
6 6 18
1379.95 127.58 320.52
229.99 21.26 17.81
12.92 1.19
0.000 0.353
G. Khayati et al. / Fluid Phase Equilibria 385 (2015) 147–152
Fig. 5. The relationship between the calculated purification factor of b-galactosidase in top phase and experimental data.
of the different factors and their interdependence for extraction of b-galactosidase from cell free fermentation broth of A. niger. 3.3. Effects of independent variables on the b-galactosidase partitioning Multilevel factorial design was used to illustrate the effect of PEG2000 to (NH4)2HPO4 weight ratio, loaded fermentation broth and system pH on the extraction of b-galactosidase from the fermentation supernatant of A. niger. The physical values are shown in Table 2. On the basis of this design, 48 experiments were carried out and the dependent parameter (the purification factor in top phase) was determined for each experiment (Table 2). The main effects of each parameter are presented in Fig. 4, which serve as a measure to indicate the contribution individual variable on the b-galactosidase partitioning. This was estimated based on the averages of measurements made at the level of each factor. The effects of PEG2000 to (NH4)2HPO4 weight ratio on partitioning of b-galactosidase were shown in Fig. 4A. The results of Fig. 4A show that weight ratio of PEG to salt had significant influence on the purification factor of b-galactosidase in top phase.
151
Purification factor in top phase was found to increase initially until weight ratio of PEG2000 to salt = 0.89% (w/w). At this point, the purification factor reached its highest values of 21.89. With further increase in PEG2000 to salt weight ratio, a gradually decrease was observed in the PFtop, since the enzyme activity in the top phase decreased with the increase of polymer concentration. For example, increasing the PEG to salt weight ratio from 0.89 to 5.33% (w/w) resulted in decrease in PFtop of b-galactosidase from 21.89 to 10.85 in ATPS. This decrease in the b-galactosidase PFtop is due to the influence of volume exclusion [30], which increases with an increase in polymer concentration. Also, based on the partitioning theory [31], the increase of PEG to salt weight ratio will result in the increase of viscosity and the interfacial tension between the two phases of the ATPS, hence the resistance increase for transferring of b-galactosidase molecules into the top PEG phase. However, the volume of top phase will increase (VR value increased) with an increase of PEG to salt weight ratio, so the activity recovery will change consequently. The effect of the loaded crude broth on the b-galactosidase partitioning in the PEG2000/(NH4)2HPO4 ATPS is shown in Fig. 4B. The amount of added crude broth to the ATPS is important because the loaded feedstock can change the partitioning behavior of the target protein and the phase VR [18]. Based on the results, loaded crude broth of 5% (w/w) had the maximum capacity on the 10 g of ATPS. The PFtop of b-galactosidase for the 5% (w/w) crude load ATPS was 22.87. In addition, the increase of the loaded crude broth reduced the ATPE performance and the value of PFtop to 13.25. Such behavior can be explained by the increasing accumulation of precipitate at the interface, showing the loss of b-galactosidase with other contaminants in the purification. To study the influence of pH on b-galactosidase partitioning for the selected phase system [PEG2000/(NH4) 2HPO4], experiments were performed in the pH range 7.0–9.0 and the results are presented in Fig. 4C. It is well known that biomolecules partition in ATPSs is obviously influenced by pH of the system and generally system pH affects the partitioning behavior of protein by changing the charge of the target protein. Also, it affects the ratio of the charged species present in the extract by altering the surface characteristics of the so-called species or the ion composition [32].
Fig. 6. Normal probability plot of residual values for the purification factor of b-galactosidase in top phase.
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The highest PFtop (19.42) was obtained in the system pH of 7.0 (Fig. 4C). This could be due to the net charge of the enzyme surface compared to their isoelectric points. b-galactosidase from Aspergillus spp. are acidic enzymes, with isoelectric points of 4.0–5.0. As the pH of the system increases above the isoelectric point, the b-galactosidase surface charge becomes negative. As a general rule, negatively charged proteins prefer the top phase in PEG–salt systems, while positively charged ones normally partition in the bottom phase due to electrostatic attraction as a result of charge distribution [21]. As a result, b-galactosidase was concentrated in the top PEG-rich phase. Subsequently, the PFtop values decreased as pH is increased from 7.0 to 8.0. This is probably due to inappropriate state of the charge in enzyme active site at the alkaline condition. Chaiwut et al. [33] have also observed similar effects for protease partitioning in PEG4000/sulphate salt ATPS. They reported that at alkaline (9.0) condition the protease recovery were clearly decreased in comparison with the neutral pH (7.0). At pH of 8–9, a negligible change of enzyme partitioning (PFtop) was observed (i.e., 14.35 and 16.54, respectively).
that b-galactosidase tended to partitioning in the upper phase in all the evaluated aqueous two-phase systems. It could be found that the b-galactosidase partitioning was strongly dependent on the type and concentration of salt. Also, the results showed that the lower PEG molecular weight is favorable for the extraction process. In summary, aqueous two-phase extraction is a promising technique for the separation of b-galactosidase from cell free fermentation broth with the PEG2000/(NH4)2HPO4 system. In this study, an ATPS comprising 15.24% (w/w) of PEG2000 and 17.14% (w/w) of (NH4)2HPO4 at pH 7 with fermentation broth loaded mass of 5% (w/w) was suggested as optimum condition with PFtop of 38.17. References [1] [2] [3] [4] [5]
3.4. Analysis of the experimental design
[6]
The purification factor of b-galactosidase in top phase was found to range from 4.02 to 38.17 in response to the variation in the experimental conditions (Table 2). The results from the analysis of the experimental design are shown in Table 3. The analysis of data was generated using Minitab statistical software version 15. The degree of significance of each factor is represented in Table 3 by its p-value; when a factor has a p-value of less than 0.05 it influences the process in a significant way for a confidence level of 0.95. The obtained results show that all of factors were significant (pvalue < 0.05). The results indicated that PEG2000 to (NH4)2HPO4 weight ratio and crude broth loaded mass were the major contributing factors to b-galactosidase partitioning. The quality of the experimental design was evaluated on the basis of determination coefficient (R2). Determination Coefficient (R2) is defined as the ratio of the explained variation to the total variation and used to measure the degree of fitness. In this case, the R2 value was 0.925. Joglekar and May [34] suggested that R2 should be at least 0.80 for a good fitness of the model. This implies that 92.5% of the variations for the b-galactosidase partitioning are explained by the independent variables and only 7.5% of the total variations in the response are not explained by the model. The high value of R2 (0.925) demonstrated a high degree of agreement between the experimental observations and predicted values (Fig. 5) [35]. The residues were also examined for normal distribution. Fig. 6 shows the normal probability plot of residual values. It could be seen that the experimental points were reasonably aligned, thus suggesting normal distribution.
[7] [8] [9]
4. Conclusions The aqueous two-phase systems (ATPS) can be utilized for the extraction of b-galactosidase from cell free fermentation broth of A. niger PTCC 1050 since the obtained results in this study showed
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Fluid Phase Equilibria j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / fl u i d
Partitioning of b-galactosidase in aqueous two-phase systems containing polyethyleneglycol and phosphate salts Gholam Khayati a, *, Masumeh Anvari b , Nushin Shahidi a a b
Department of Chemical Engineering, Faculty of Engineering, University of Guilan, Rasht, P.O. Box 41625-3756, Iran Department of Microbiology, Islamic Azad University, Rasht Branch, P.O. Box 41335-3516, Rasht, Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 July 2014 Received in revised form 31 October 2014 Accepted 4 November 2014 Available online 6 November 2014
Purification of a desired protein from fermentation broth containing a wide variety of biomolecules is one of the major challenges in the biotechnology industry. One possible strategy for this challenge, an aqueous two-phase extraction (APTE) can be used as the primary downstream processing for partial purification of industrial enzymes. In the present study, the feasibility of utilizing ATPE for the partitioning behavior of b-galactosidase (from Aspergilus niger) was investigated. The results showed that the PEG2000/(NH4)2HPO4 system was suitable for the extraction of b-galactosidase from cell free fermentation broth of A. niger PTCC 1050. In the following, multilevel factorial design was used to evaluate the effects of three independent variables (PEG to salt weight ratio, fermentation broth loaded mass and system pH) on b-galactosidase extraction. The results indicated that PEG to salt weight ratio and fermentation broth loaded mass was the major contributory factors for b-galactosidase partitioning. Among the different conditions studied, a phase system having a composition of 15.24% (w/w) PEG2000 and 17.14% (w/w) (NH4)2HPO4 at pH 7 with loaded fermentation broth of 5% (w/w) gave the best overall results in term of purification degree. ã 2014 Elsevier B.V. All rights reserved.
Keywords:
b-galactosidase Aqueous two-phase system Poly ethylene glycol Multilevel factorial design
1. Introduction
b-galactosidase is one of the principal enzymes that has important applications [1]. This enzyme is mainly used to hydrolyze lactose into glucose and galactose. Problems of lactose are due to three main areas: health, food technology and environment. Concerning the health issue, lactose becomes a problem when there is insufficient intestinal b-galactosidase enzyme and lactose is passed into the blood, finally appearing in the urine and in the large intestine resulting in several disorders [2]. Lactose intolerant people are restricted to the lactose amount they can ingest daily. Because of these problems, the reduction of lactose in milk and dairy products is of prime importance and the enzyme b-galactosidase is commercially used for this purpose [3,4]. From the food technology point of view, the high lactose content in non-fermented milk products such as ice-c ream and condensed milk, can lead to excessive lactose crystallization resulting in products with a mealy, sandy or gritty texture. Again, the use of b-galactosidase enzyme prior to the condensing operation can reduce the lactose content to a point where lactose is no longer a problem [2]. Moreover, glucose and galactose expand
* Corresponding author. Tel.: +98 9111329514. E-mail address: [email protected] (G. Khayati). 0378-3812/$ – see front matter ã 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fluid.2014.11.003
the use of lactose in food because of a remarkable increase in sweetness and solubility [5]. New applications of b-galactosidase enzyme such as the recovery of biologically active oligosaccharides from milk have also been described in literature [6]. One of the major challenges in the biotechnology industry is the purification of a desired protein from a fermentation broth containing a wide variety of biomolecules [7]. Partitioning using aqueous two-phase systems (ATPS) has proved to be a valuable tool and mild technique for separating and purifying mixtures of biomolecules. These systems can form a gentle environment for enzymes and other biologically active proteins, therefore, the extraction in the systems has the advantages of high capacity, high activity yields and easy to scale up. These systems have many applications for protein purification in biochemistry and biotechnology [8,9]. The partitioning of biomolecules in ATPS is influenced by pH, temperature, surface properties, size and concentrations of the biomolecules and the types of employed polymers and salts [10]. In the present study, at first, the phase-forming salt was selected, and then molecular weight of PEG was varied. After the selection of phase-forming salt and molecular weight of PEG, the influence of process parameters such as PEG to salt weight ratio, loaded fermentation broth and pH of the system on b-galactosidase partitioning behavior was studied by multilevel factorial design and, the results were presented as well as discussed in the following sections.
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2. Materials and methods 2.1. Chemicals All chemicals used were of analytical grade. Aspergillus niger PTCC5010 was obtained from the Iranian Research Organization for Science and Technology (IROST). 2.2. Fermentation conditions The pre-culture medium was nutrient broth containing (per liter): 2.0 g yeast extract; 5.0 g peptone; 5.0 g NaCl and 1.0 g beef extract sterilized at 121 C for 15 min. Submerged fermentation was carried out in 250 ml shake-flasks with 50 ml medium containing the following constituents (g/L): glucose 5.0; lactose 40.0; peptone 0.25; yeast extract 0.5; (NH4)2SO4 3.0; KH2PO4 1.0 and K2HPO4 3.0. Sugars were sterilized separately. After 72 h of incubation, cells were harvested by centrifugation at 7000 rpm for 20 min. The filtrate was used as crude enzyme.
Fig. 1. Effect of phase composition in PEG4000/salt ATPS on the purification factor of b-galactosidase in top phase and selectivity. 1: 7.27%PEG4000 + 21.81% (NH 4)2HPO4; 2: 14.54%PEG4000 + 16.36% (NH 4)2HPO4; 3: 21.81%PEG4000 + 10.91 (NH 4)2HPO4; 4: 29.09%PEG4000 + 5.45% (NH 4)2HPO4; 6: 7.27%PEG4000 + 21.81% K2HPO4; 7: 14.54%PEG4000 + 16.36% K2HPO4; 8: 21.81%PEG4000 + 10.91 K2HPO4; 9: 29.09%PEG4000 + 5.45% K2HPO4.
C p;t C p;b
2.3. Preparation of aqueous two-phase systems
Kp ¼
All aqueous two-phase systems were done in 15 ml graduated centrifuge tubes at equal volume of salt and PEG solution in different conditions. To study the effects of PEG molecular weight and types of salt on the partitioning of b-galactosidase from cell free fermentation broth using ATPS, different salts including diammonium hydrogen phosphate and di-potassiom hydrogen phosphate were mixed with different PEG2000, 4000 and 8000 in aqueous system. The glass tube was shaken vigorously by turning upside-down followed by centrifuged for 10 min at 7000 rpm to assist phase separation. After phase separation and visual estimation of the top and bottom phase, the volumes of the phases were used to estimate the volume ratio. The samples of the top and bottom phases were carefully withdrawn by using a plastic syringe with short and long needle, respectively. Aliquots from each phase were analyzed to determine the enzyme activity and protein concentration. All experiments were run in duplicate at room temperature (24 1 C).
where Cp,t and Cp,b are concentrations of protein in top and bottom phase, respectively. The partition coefficient for b-galactosidase activities (Ke) in the aqueous two-phase systems were defined as:
2.4. Analytical methods
b-galactosidase assay was done spectrophotometrically. The enzyme activity was essentially determined according to the method described by Nagy et al. [11]. Protein concentration was measured by the method of Bradford [12] using bovine serum albumin as a standard. Results were evaluated by a few parameters including: protein partition coefficient in the aqueous two-phase systems was defined as [8]:
Ke ¼
(1)
At Ab
(2)
where At and Ab are enzyme activities in top and bottom phase, respectively. The partition coefficient (Ke) is used to quantify the degree of separation reached in an extraction process. Yield (%) of b-galactosidase in top phase was also calculated based on the partition coefficient to study the efficiency of the system by using the following equation. Yð%Þ ¼
100 ðV R K e Þ1 þ 1
(3)
The (VR) was defined as the volume ratio of the top phase to the bottom phase (Vt/Vb). Selectivity (S) was calculated as the ratio of the b-galactosidase enzyme partition coefficient (Ke) to the protein partition coefficient Kp. S¼
Ke At C p;b ¼ K p Ab C p;t
(4)
Table 1 Effect of phase composition in PEG4000/salt ATPS on partitioning of b-galactosidase at 24 C. Phase composition (%, w/w)
Ke
Kp
Y (%)
VR
7.27%PEG4000 + 21.81% (NH 4)2HPO4 14.54%PEG4000 + 16.36%(NH 4)2HPO4 21.81%PEG4000 + 10.91(NH 4)2HPO4 29.09%PEG4000 + 5.45%(NH 4)2HPO4 7.27%PEG4000 + 21.81% K2HPO4 14.54%PEG4000 + 16.36%K2HPO4 21.81%PEG4000 + 10.91 K2HPO4 29.09%PEG4000 + 5.45%K2HPO4
6.10 3.25 2.67 2.75 4.48 2.29 1.86 1.48
0.23 0.08 0.12 0.11 4.12 0.84 0.46 0.77
58.46 64.07 80.85 91.68 48.91 60.07 75.61 90.86
0.23 0.54 1.57 4.00 0.21 0.65 1.66 6.69
Fig. 2. Effect of PEG molecular weight in PEG/(NH4)2HPO4 ATPS on the yield (Y%) and partition coefficients of b-galactosidase (Ke).
G. Khayati et al. / Fluid Phase Equilibria 385 (2015) 147–152
The purification factor in the top phase (PFtop) was defined as PFtop ¼
SAt SAi
(5)
where SAt and SAi are specific activities in top and crude enzyme, respectively. Also, specific activity represents the ratio of the enzyme activity to the protein concentration in a sample. SA ¼
A Cp
(6)
where A and Cp are the enzyme activity and the total protein concentration in each phase, respectively. 3. Results and discussion Theoretical predictions of biomolecule extraction using ATPS could be difficult. This was because of the additives and/or impurities’ type and concentration in aqueous solution directly affected both system features and solute properties [9]. For the efficacy of aqueous two-phase extraction (ATPE), suitable phase system selection is very important. In ATPSs, the most significant and difficult step is to choose the appropriate type and composition of the system in terms of achieving the efficient extraction of the biomolecule [13]. It is well known that as a result of several factors such as type of phase forming salt, PEG molecular weight, concentration of salt and polymer, tie line length, volume ratio and ionic composition of the phases, partition and distribution behaviors of compounds in ATPSs are considered as very complex phenomena and therefore, inferring a general rule is not possible [14,15]. Choosing the phase forming components types for the distribution of the biomolecule is the second most important step following the selection of the phase system type as polymer– salt [16]. In this study, polymer–salt systems were selected because of some advantages they had such as lower cost and gentler environment besides lower viscosity in comparison to polymer– polymer systems. 3.1. Selection of phase forming salt The choice of phase-forming salt has a direct effect on separation and extraction of a given biomolecule in ATPE due to its significant influence on system environment [17]. Also, the selection of salt employed in the extraction system is one of the key points of this technique [18]. To ensure the efficiency of the extraction, the partitioning of b-galactosidase from A. niger was carried out by employing various phase forming salts, namely diammonium hydrogen phosphate and di-potassium hydrogen
Fig. 3. Effect of PEG molecular weight in PEG/(NH4)2HPO4 ATPS on the selectivity and specific b-galactosidase activity in top phase.
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phosphate with PEG4000 at different concentrations in order to arrive at the most suitable aqueous two-phase system. The PEG/ phosphate system is able to extract macromolecules from fermentation broth [19]. The results were summarized in Table 1. The distribution of the protein and b-galactosidase in ATPS was reported by Kp and Ke, respectively. In our results, the Kp in K2HPO4 system was much larger than that in (NH4)2HPO4 system, which was due to the greater salting-out power of K+ ions than of NH4+ ions [20]. Theoretically, high Kp value along with high Ke indicates that most of proteins as well as target one were more partitions to the top phase, while high Ke with low Kp implies that especially the target molecule (enzyme) was more partitioned into the top phase [21]. As shown in Table 1, all of the Ke in the systems are larger than 1, which indicates that b-galactosidase enzyme in the crude extract is preferably distributed on the top phase. The low Kp values indicate that the contaminant proteins and other undesirable components could shift to the bottom phase. In general, partitioning of the total proteins and enzyme to the bottom and
Table 2 Multilevel factorial experimental design and responses. Run
Factor levels
Response (PFtop)
WPEG/Wsalt
Loaded mass (%w/w)
System pH
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
0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 2 2 2 2 2 2 2 2 2 2 2 2 5.33 5.33 5.33 5.33 5.33 5.33 5.33 5.33 5.33 5.33 5.33 5.33
5 5 5 10 10 10 15 15 15 20 20 20 5 5 5 10 10 10 15 15 15 20 20 20 5 5 5 10 10 10 15 15 15 20 20 20 5 5 5 10 10 10 15 15 15 20 20 20
7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9
22.85 7.34 26.84 21.44 8.37 17.51 21.79 10.58 37.02 25.76 15.26 16.56 38.15 26.74 36.75 24.98 7.67 28.62 14.41 8.58 21.87 14.92 10.11 19.96 19.48 24.93 12.42 28.28 22.04 8.06 14.86 12.07 5.44 15.64 13.21 4.45 16.73 26.28 5.96 12.25 11.33 4.56 10.23 15.01 4.69 8.99 10.12 4.02
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top phases is dependent on their isoelectric points [19]. It is certain that one of the most important requirements of ATPS for an effective purification is that the contaminant proteins should not be extracted in the same phase with the target enzyme. Therefore along with a high Ke value, a low Kp value is desirable as well. Table 1 also shows that the yield of b-galactosidase in the top phase, with both salts were decreased when increase of salt concentration from 5.45 to 21.81 (%w/w). It can be found that increase in salt quantity caused the salting-out effect [22]. Enhancement of salt quantity provided the higher proportion of salt-rich bottom phase leading to practically reduced of VR. Johansson [23] reported that the partitioning of a protein is influenced by the presence of salts and this effect is enhanced with increasing of the protein net charge. It is found that di-ammonium hydrogen phosphate salt has shown higher results in terms of selectivity and purification factor in top phase in compared to that of di-potassium hydrogen
phosphate (Fig. 1). The results showed that the partitioning of b-galactosidase were strongly dependent on the type of salts. This was mainly due to the favorable biocompatible phase environment by the ammonium salt and hence, this system was selected for further studies. 3.2. Selection of polymer molecular weight It has been demonstrated that the molecular weight of PEG affects its distribution in the two phases and polymer–protein interactions [8]. Therefore, in order to select a suitable PEG molecular weight for the extraction of b-galactosidase from A. niger, ATPE was performed with different molecular weights of PEG (MW 2000, 4000 and 8000) at ambient temperature. The enzyme partition coefficients, yield of b-galactosidase in the top phase, selectivity and specific b-galactosidase activity in the top phase are shown as the functions of PEG molecular weight in Figs. 2 and 3. The results showed that the partitioning of b-galactosidase in PEG/ (NH4)2HPO4 system was strongly dependent on the molecular weight of the PEG. The yield (Y%) and partition coefficients of b-galactosidase (Ke) were found to decrease with the increase of PEG molecular weight (Fig. 2). About 16.5% decrease in extraction yield and 51.4% decrease in partition coefficient of b-galactosidase was observed. At the same time, it can be seen that volume ratio decreased with an increase in molecular weight of PEG (not shown). In other words, the volume of the bottom phase increased with an increase in PEG molecular weight, which promotes selective partitioning of the enzyme to the bottom phase which is its preferential phase. Also the selectivity plot shows that the values of S decreased sharply with the increase of PEG molecular weight from 2000 to 8000. While SAtop of b-galactosidase was found to decrease gradually with the increase of PEG molecular weight (Fig. 3). This means that the b-galactosidase increasingly partitions to the bottom phase as the PEG molecular weight increases. Similar trends were found by the other researchers [24–26] and could be attributed to two different effects. The most obvious was the increase of the top phase hydrophobicity. In fact as PEG chain length increases (with increasing molecular weight) there will be less hydroxyl groups for the same concentration of the polymer and so the hydrophobicity of the polymer-rich phase (top) increases [27]. On the other hand, the increase of the chain length will also cause the reduction of the free volume [28,29], meaning less space available for the b-galactosidase. The increase of PEG molecular weight had facilitated the partitioning of b-galactosidase to the bottom phase. It can be concluded from the Fig. 3 that maximum selectivity of b-galactosidase (S = 50.05) was observed when the smallest PEG had been used. We suggest that the lower PEG molecular weight is more favorable for b-galactosidase partitioning as the best system was obtained at PEG2000/(NH4)2HPO4. After selecting the type of salt and molecular weight of PEG, a multilevel factorial design of experiments was chosen for thorough investigation of the effects Table 3 Analysis of variance of the regression parameters for the multilevel factorial design. Source value
Fig. 4. Main effects of the variable for the purification factor of b-galactosidase in top phase based on the multilevel factorial design results.
WPEG/ Wsalt Loaded mass (%w/w) System pH WPEG/Wsalt loaded mass WPEG/Wsalt pH Loaded mass pH Error
D. F.
Sum of square Mean square
Fvalue
p-value
3 3 2 9
846.09 649.95 207.14 763.41
282.03 216.65 103.57 84.82
15.84 12.17 5.82 4.76
0.000 0.000 0.011 0.002
6 6 18
1379.95 127.58 320.52
229.99 21.26 17.81
12.92 1.19
0.000 0.353
G. Khayati et al. / Fluid Phase Equilibria 385 (2015) 147–152
Fig. 5. The relationship between the calculated purification factor of b-galactosidase in top phase and experimental data.
of the different factors and their interdependence for extraction of b-galactosidase from cell free fermentation broth of A. niger. 3.3. Effects of independent variables on the b-galactosidase partitioning Multilevel factorial design was used to illustrate the effect of PEG2000 to (NH4)2HPO4 weight ratio, loaded fermentation broth and system pH on the extraction of b-galactosidase from the fermentation supernatant of A. niger. The physical values are shown in Table 2. On the basis of this design, 48 experiments were carried out and the dependent parameter (the purification factor in top phase) was determined for each experiment (Table 2). The main effects of each parameter are presented in Fig. 4, which serve as a measure to indicate the contribution individual variable on the b-galactosidase partitioning. This was estimated based on the averages of measurements made at the level of each factor. The effects of PEG2000 to (NH4)2HPO4 weight ratio on partitioning of b-galactosidase were shown in Fig. 4A. The results of Fig. 4A show that weight ratio of PEG to salt had significant influence on the purification factor of b-galactosidase in top phase.
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Purification factor in top phase was found to increase initially until weight ratio of PEG2000 to salt = 0.89% (w/w). At this point, the purification factor reached its highest values of 21.89. With further increase in PEG2000 to salt weight ratio, a gradually decrease was observed in the PFtop, since the enzyme activity in the top phase decreased with the increase of polymer concentration. For example, increasing the PEG to salt weight ratio from 0.89 to 5.33% (w/w) resulted in decrease in PFtop of b-galactosidase from 21.89 to 10.85 in ATPS. This decrease in the b-galactosidase PFtop is due to the influence of volume exclusion [30], which increases with an increase in polymer concentration. Also, based on the partitioning theory [31], the increase of PEG to salt weight ratio will result in the increase of viscosity and the interfacial tension between the two phases of the ATPS, hence the resistance increase for transferring of b-galactosidase molecules into the top PEG phase. However, the volume of top phase will increase (VR value increased) with an increase of PEG to salt weight ratio, so the activity recovery will change consequently. The effect of the loaded crude broth on the b-galactosidase partitioning in the PEG2000/(NH4)2HPO4 ATPS is shown in Fig. 4B. The amount of added crude broth to the ATPS is important because the loaded feedstock can change the partitioning behavior of the target protein and the phase VR [18]. Based on the results, loaded crude broth of 5% (w/w) had the maximum capacity on the 10 g of ATPS. The PFtop of b-galactosidase for the 5% (w/w) crude load ATPS was 22.87. In addition, the increase of the loaded crude broth reduced the ATPE performance and the value of PFtop to 13.25. Such behavior can be explained by the increasing accumulation of precipitate at the interface, showing the loss of b-galactosidase with other contaminants in the purification. To study the influence of pH on b-galactosidase partitioning for the selected phase system [PEG2000/(NH4) 2HPO4], experiments were performed in the pH range 7.0–9.0 and the results are presented in Fig. 4C. It is well known that biomolecules partition in ATPSs is obviously influenced by pH of the system and generally system pH affects the partitioning behavior of protein by changing the charge of the target protein. Also, it affects the ratio of the charged species present in the extract by altering the surface characteristics of the so-called species or the ion composition [32].
Fig. 6. Normal probability plot of residual values for the purification factor of b-galactosidase in top phase.
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The highest PFtop (19.42) was obtained in the system pH of 7.0 (Fig. 4C). This could be due to the net charge of the enzyme surface compared to their isoelectric points. b-galactosidase from Aspergillus spp. are acidic enzymes, with isoelectric points of 4.0–5.0. As the pH of the system increases above the isoelectric point, the b-galactosidase surface charge becomes negative. As a general rule, negatively charged proteins prefer the top phase in PEG–salt systems, while positively charged ones normally partition in the bottom phase due to electrostatic attraction as a result of charge distribution [21]. As a result, b-galactosidase was concentrated in the top PEG-rich phase. Subsequently, the PFtop values decreased as pH is increased from 7.0 to 8.0. This is probably due to inappropriate state of the charge in enzyme active site at the alkaline condition. Chaiwut et al. [33] have also observed similar effects for protease partitioning in PEG4000/sulphate salt ATPS. They reported that at alkaline (9.0) condition the protease recovery were clearly decreased in comparison with the neutral pH (7.0). At pH of 8–9, a negligible change of enzyme partitioning (PFtop) was observed (i.e., 14.35 and 16.54, respectively).
that b-galactosidase tended to partitioning in the upper phase in all the evaluated aqueous two-phase systems. It could be found that the b-galactosidase partitioning was strongly dependent on the type and concentration of salt. Also, the results showed that the lower PEG molecular weight is favorable for the extraction process. In summary, aqueous two-phase extraction is a promising technique for the separation of b-galactosidase from cell free fermentation broth with the PEG2000/(NH4)2HPO4 system. In this study, an ATPS comprising 15.24% (w/w) of PEG2000 and 17.14% (w/w) of (NH4)2HPO4 at pH 7 with fermentation broth loaded mass of 5% (w/w) was suggested as optimum condition with PFtop of 38.17. References [1] [2] [3] [4] [5]
3.4. Analysis of the experimental design
[6]
The purification factor of b-galactosidase in top phase was found to range from 4.02 to 38.17 in response to the variation in the experimental conditions (Table 2). The results from the analysis of the experimental design are shown in Table 3. The analysis of data was generated using Minitab statistical software version 15. The degree of significance of each factor is represented in Table 3 by its p-value; when a factor has a p-value of less than 0.05 it influences the process in a significant way for a confidence level of 0.95. The obtained results show that all of factors were significant (pvalue < 0.05). The results indicated that PEG2000 to (NH4)2HPO4 weight ratio and crude broth loaded mass were the major contributing factors to b-galactosidase partitioning. The quality of the experimental design was evaluated on the basis of determination coefficient (R2). Determination Coefficient (R2) is defined as the ratio of the explained variation to the total variation and used to measure the degree of fitness. In this case, the R2 value was 0.925. Joglekar and May [34] suggested that R2 should be at least 0.80 for a good fitness of the model. This implies that 92.5% of the variations for the b-galactosidase partitioning are explained by the independent variables and only 7.5% of the total variations in the response are not explained by the model. The high value of R2 (0.925) demonstrated a high degree of agreement between the experimental observations and predicted values (Fig. 5) [35]. The residues were also examined for normal distribution. Fig. 6 shows the normal probability plot of residual values. It could be seen that the experimental points were reasonably aligned, thus suggesting normal distribution.
[7] [8] [9]
4. Conclusions The aqueous two-phase systems (ATPS) can be utilized for the extraction of b-galactosidase from cell free fermentation broth of A. niger PTCC 1050 since the obtained results in this study showed
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