Dependence of chymosin and pepsin partition coefficient with phase volume and polymer pausidispersity in polyethyleneglycol–phosphate aqueous two-phase system

Colloids and Surfaces B: Biointerfaces 51 (2006) 80–85

Dependence of chymosin and pepsin partition coefficient with phase volume and polymer pausidispersity in polyethyleneglycol–phosphate aqueous two-phase system Dar´ıo Spelzini, Guillemo Pic´o, Beatriz Farruggia ∗ Faculty of Biochemical and Pharmaceutical Sciences, CONICET, FonCyT and CIUNR, National University of Rosario, Suipacha 570 (S2002RLK), Rosario, Argentina Received 21 June 2005; received in revised form 28 February 2006; accepted 5 March 2006 Available online 25 April 2006

Abstract The influence of the phase volume ratio and polymer pausidispersity on chymosin and pepsin partition in polyethylenglycol–phosphate aqueous two-phase systems was studied. Both proteins showed a high affinity for the polyethylenglycol rich phase with a partition coefficient from 20 to 100 for chymosin and from 20 to 180 for pepsin, when the polyethyleneglycol molecular mass in the system varied between 1450 and 8000. The partition coefficient of chymosin was not affected by the volume phase ratio, while the pepsin coefficient showed a significant decrease in its partition coefficient with the increase in the top/bottom phase volume ratio. © 2006 Published by Elsevier B.V. Keywords: Partition; Pepsin; Chymosin; Aqueous two-phase system

1. Introduction Pepsin is an acidic protease widely used in food and pharmaceutic industry; it has a single polypeptidic enzymatic chain of 324 amino acid residues. Its molecular weight is 35,000. It is the principal proteolytic enzyme of vertebrate gastric juice and the optimum activity pH and isoelectric point is 1.0 [1]. Chymosin is a neonatal gastric aspartic protease of commercial importance in the cheese industry. It is more stable at pH values between 5.3 and 6.3; at pH 3–4, the enzyme loses its activity due to autodegradation, while at alkaline pH above 9.8, this loss is due to an irreversible conformation change. Chymosin has a single polypeptide enzymatic chain of 323 amino acid residues with a low content of basic residues and rich in dicarboxylic acid residues. It has a molecular weight of 35.0 kDa [2]. Chymosin has the enzymatic capacity to clot milk.

Abbreviations: CHY, chymosin; Mw, average molecular weight; PEP, pepsin; PEG1450, PEG3350, PEG6000 and PEG8000, polyethylenglycol of average molecular mass 1450, 3350, 6000 and 8000, respectively ∗ Corresponding author. Tel.: +54 4314314643; fax: +54 4314804598. E-mail address: [email protected] (B. Farruggia). 0927-7765/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.colsurfb.2006.03.023

The downstream processing of enzymes and macromolecules generally requires purification techniques that preserve their biological activity. The conventional procedures include salt precipitation, chromatography, dialysis and filtration; therefore, these methods result in a loss of biological activity with a concomitant poor yield of the final product. It has been demonstrated that aqueous two-phase systems (ATPS) are a good method to be applied as a first purification step, since such systems allow removal contaminant. They are also simple and inexpensive methods [3]. ATPS are formed when two flexible chain polymers or one polymer and a salt are mixed; macromolecules are partitioned between the two phases and a partition coefficient (K) can be defined as: K=

[P]T [P]B

(1)

where [P]T and [P]B are the macromolecule concentration in the top and bottom phases, respectively. The partition coefficient of a protein depends on different factors such as the molecular mass of the polymer and of the macromolecule, pH, presence of salts and ions in the medium, macromolecular hydrophobicity, etc. All these factors are intrinsically dependent on the macromolecule and ATPS. Therefore,

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the knowledge of better conditions for the macromolecule recovery from a liquid–liquid extraction process depends on a previous study of the system variables to make the extraction optimal. The efficiency of a liquid–liquid extraction method to purify a target protein can be evaluated through the recovery of the target protein which is tabulated as the percent yield (y%) and it can be calculated from the Eq. (2): ytop (%) =

100 1 + (1/RK)

(2)

where ytop (%) is the macromolecule recovery in the top phase and R = VT /VB , VT and VB being the top and the bottom phase volumes, respectively. The above equation predicts that the top/bottom volume ratio play an important role in the recovery capacity determination of ATPS for a target macromolecule in a liquid–liquid extraction method in one phase. The partition mass balance allows us to demonstrate that the R-value directly determines the percent recovery of the target protein in one phase. Therefore, it is possible to increase the recovery capacity in a liquid–liquid extraction process by using an adequate top/bottom volume ratio. In the literature, there are many reports that refer to the effect of the different experimental conditions about the protein partition coefficient with the aim of achieving that the target protein is present in one of the phases. These studies have been performed with a phase volume ratio close to the unity. Another important variable in the medium is the molecular mass of the polymer, however the available commercial PEGs are narrow disperse, therefore, only a molecular mass of discrete value can be assayed. Previous reports [4] have demonstrated that if the PEG molecular mass is varied in a regular way, the protein partition coefficient variation can be modulated. The goal of this work was to make a previous determination of the best experimental conditions on the target protein recovery by manipulating of the volume top–bottom ratio and the polymer molecular mass dispersivity. In order to do this, we studied the partition features of two acid proteases: pepsin and chymosin, both present in the stomach mucose we calculated the recovery in one phase in order to compare the validity of the model to apply it in the near future. This method could be used to isolate these proteins from a complex natural product. 2. Materials and methods 2.1. Chemicals Polyethyleneglycols of the following average molecular mass: 1450 (PEG1450), 3350 (PEG3350), and 8000 (PEG8000) and pepsin (PEP) were purchased from Sigma Chemical Co. and used without further purification. PEG of molecular mass 6000 (PEG6000) was purchased from Merck, and used without further purification. Chymosin (CHY) was gently donated by Chr. Hansen (Quilmes, Argentina) produced by fermentacion of Aspergillus niger. It was previously dialysed against buffer 50 mM sodium phosphate pH 7, and concentrated by ultradialysis. Its purification was determined by PAGE electrophoresis, the content of pure enzyme proved to be greater than 96%.

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2.2. Preparation of the aqueous two-phase systems Two-phase systems were prepared from stock solutions as 40% (w/w) PEG polymers, 25% (w/w) potassium phosphate, pH 7.0, were mixed in order to obtain the total system compositions of binodial diagrams previously reported [5]. Systems of 40 g were prepared, stirred for equilibration for 2 h and the phases were then allowed to settle overnight at 8 ◦ C. After phase separation, 2 ml of each phase was mixed to reconstitute the two-phase systems in which the protein partition was assayed. In order to speed up phase separation, low-speed centrifugation was used after a gentle mixing of the system components. 2.3. Determination of the protein partition coefficient (K) Increasing aliquots (2–15 ␮l) of concentrated protein solution were added to the performed two-phase systems containing 2 ml of each equilibrated phase, the total volume change of each phase being negligible. After mixing by inversion for 30 min and leaving it to settle for at least 2 h, the system was centrifuged at low speed for the two-phase separation. Samples were withdrawn from the separated phases, and after dilution, the protein content in each phase was determined by measuring the protein native fluorescence emission at 340 nm, while the excitation was made at 295 nm. Equally diluted samples from identical phase systems without protein were used as blanks, which had been prepared at the same time. In the protein concentration range assayed, a plot of [P]top versus [P]bottom showed linear behaviour, K-value being its slope. All the determinations were carried out at 8 ◦ C. 2.4. Preparation of ATPS with different PEG phase pausidispersity The effect of polymer pausidispersity on protein partitioning behaviour was evaluated using different groups of two-phase systems. Each group was prepared with two PEGs of different molecular weight and potassium phosphate. Different mixtures of PEG compositions were obtained by increasing the amount of one of the PEGs and parallely decreasing the amount of the other polymer; the total mass of PEG and salt remaining constant. wPEG(A) and wPEG(B) stand for the feed compositions in weight fraction of each PEG in the mixture: mPEG(A) wPEG(A) = mPEG(A) + mPEG(B) (3) mPEG(B) wPEG(B) = mPEG(A) + mPEG(B) where mPEG is the mass of each PEG A or B of different molecular mass. Therefore, the weight–average molecular weight Mw can be calculated according to: Mw = MPEG(A) wPEG(A) + MPEG(B) wPEG(B) Mw = (MPEG(A) − MPEG(B) )wPEG(A) + MPEG(B)

(4)

where MPEG(A) and MPEG(B) are the average molecular weights of narrow standard polyethyleneglycol fractions, thus considered monodisperse systems. It should be noted that the feeds

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Table 1 PEGs compositions and Mw for the ATPS obtained mixing PEGs of two different molecular masses PEG Mw system

1450 (% (w/w))

8000 (% (w/w))

1 2 3 4 5 6 7 8 9 10 11

15.62 14.06 12.50 10.93 9.37 7.81 6.25 4.69 3.12 1.59 0

0 1.56 3.12 4.69 6.25 7.81 9.37 10.93 12.50 14.06 15.62

PEG total concentration: 15.62% (w/w), phosphate total concentration: 12.80%.

of all the assayed systems have the same total PEG concentration but a different average molecular weight. Table 1 shows the range used for of PEGs compositions and the corresponding Mw. 2.5. Experimental designs and statistical analysis The influence of the variables molar mass of PEG and top–bottom volume ratio (R) on the protein recovery (y%) in the top phase was evaluated according to a 22 factorial design with 4 repetitions at the central point as reported previously [14]. The “Mathematica” (version 5.0) and Sigma Plot (v. 8) software’s were used for regression and graphical analyses of the data obtained. 3. Results and discussion 3.1. Effect of PEG molecular mass on the protein partition CHY and PEP were partitioned at 8 ◦ C in different systems containing PEG–phosphate with increasing molecular mass of PEG as shown Fig. 1. In order to compare the effect of the different molecular masses of the polymer, the PEG and phosphate concentrations were chosen at the same distance from the critical point of the binodial diagrams [5]. Both proteins showed an anomalous higher partition coefficient compared with other proteins of similar molecular mass and isoelectric point. The observed partition coefficient values for both protein were between 20 and 180. Other proteins of similar molecular mass such as ovoalbumin (Mw 44,000) has a lower partition coefficient, including the small lisozyme (Mw 12,000), whose partition cefficient in PEG–phosphate systems has a value between 5 and 10 [6]. The above finding suggests that both proteins have high hydrophobic surface character which induces a significant protein–polymer interaction. Another finding is the dependence of the partition coefficient with the PEG molecular mass as is shown in Fig. 1: the partitioning of CHY was slightly decreased for PEG of molecular masses between 3350 and 8000, while an increase in K value was observed at PEG3350 and PEG6000 for PEP, as well as a decreaseat PEG8000, which suggests greater interaction of PEG with the PEP rather than the CHY. The PEG

Fig. 1. Partition coefficient dependence with the PEG molecular mass for CHY (䊉) and PEP (); temperature 8 ◦ C, pH 7.0. Composition of system (concentration % (w/w)) was ATPS PEG1450-Pi (potassium phosphate: 15.17, PEG1450: 18.64, water: 66.19); ATPS PEG3350-Pi (potassium phosphate: 12.30, PEG3350: 13.70, water: 74.00); ATPS PEG6000-Pi (potassium phosphate: 12.98, PEG6000: 14.94, water: 72.08); ATPS PEG8000-Pi (potassium phosphate: 11.70, PEG8000: 15.50, water: 72.80).

observed effect on a protein can be due to a fine balance between two opposing factors [7]: (1) The PEG exclusion effect on the protein, which acts only by a change in the free volume available in the rich PEG phase, is a consequence on the protein transfer to the other phase when the PEG molecular mass increases [7]. The excluded volume theory shows that the PEG concentration or its greater molecular mass induces a diminution of the protein solubility in the phase where the protein is situated, thus favouring the PEG transfer to the salt rich PEG. (2) The PEG–protein binding is produced through the hydrophobic area of the protein exposed to the solvent. This last effect will depend on the chemical structure of the protein. Proteins, with great hydrophobic surface area exposed to solvent have the possibility of interacting with PEG. Arakawa and Timasheff [8] examined the interaction between PEG and beta lactoglobulin as a function of PEG molecular mass; the preferential exclusion increases with an increase in the PEG size. This result argues in favour of the steric exclusion as the factor to determine the interaction of PEG with proteins. The opposed PEG effect on PEP can be better understood in terms of the PEG solution behavior. It has been demonstrated that PEG, which is a flexible molecule, can adquire compact structure stabilized by intramolecular hydrophobic bonds. The PEG compact structure has a lower interaction with the solvent than the fully extended ones; this allows the PEG molecule to interact with the protein domain. 3.2. Effect of top/bottom phase volume ratio on the CHY and PEP partition Some partition studies have been done with a phase volume ratio close to one, optimization of purification could be achieved by manipulating this volume ratio trying to find the best experimental conditions where the protein recovery is maximal.

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equation that drives the protein partition [3]. However, this equation does not predict how the phase volume influences on the partition coefficient, according to Eq. (2), the phase volume influences on the protein recovery in one of the phases. However, a reason for this may be due to the fact that the variation of the phase volume induces a change in the protein activity coefficient due to a change on the polymer–protein interaction, possibly this fact is due to the variation of the excluded volume that has been suggests previously [11,12], and therefore this results in a partition coefficient modification. We have analysed two variables: K and R, and their influence on the recovery of the protein in the top phase, where the protein has preference. Applying Eq. (2), the percentage recovered (y%) as a function of the R was plotted as shown in Fig. 3A and B. The recovery of PEP in the top phase was not influenced by the R and no significant difference was observed between the different PEG molecular masses. The recovery of CHY in the top phase was better in the PEG3350 system. For R equal to unity, an important recovery, between 95 and 99% for all the PEGs was observed to the exception of PEG1450 and PEG8000, where a decreasing of recovery was observed for PEP. 3.3. Effect of the polymer molecular mass pausidispersivity on the partition Fig. 2. Effect of the top/bottom ratio on the partition coefficient of CHY (A) and PEP (B). Temperature 8 ◦ C, pH 7.0.

According to Eq. (2), the protein recovery in the top phase depends on the partition coeficient and the volume ratio. Fig. 2A and B show the effect of the variation of top/bottom ration on the CHY and PEP partition coefficient. The change in the phase volume ratio within the range from 0.1 to 1.0 influenced on the partition coefficient. The volume of the bottom phase was taken as a constant value of 2 ml, while the top phase volume was varied since the protein has affinity for this phase. Thus, this allows us to analyse how the volume of the PEG rich phase influences on the target protein recovery. Fig. 2A shows that the CHY partition coefficent was slightly affected by the volume ratio variation in the case of PEG1450 and PEG3350, while no significant difference was observed for the other PEGs. PEP partition coefficient was significantly affected by the increase in the R-value, which induced an important decrease in the partition. This effect was higher in the case of PEG1450 and PEG3350. Marcos et al. [9] studied the effect of phase volume ratio on the extraction of penicilin acylase; the partition coefficient of the enzyme decreased with the increase in the volume ratio. These results agree with those obtained by Huddleston et al. [10] for the extraction of pure bovine albumin in a PEG–phosphate system. They also reported that the protein partition coefficient remains constant for ATPS where the differences between the two-phase concentrations are lower. The partition coefficient depends on the experimental variables such as pH, salt concentration, molecular mass of the polymer. These variable effects have been described by the state

In previous works [13,15], we have isolated alpha-1-antitrypsin from human serum plasma and separated it from albumin – the most abundant plasma protein (albumin/antitrypsin

Fig. 3. Dependence of the protein recovery for CHY (A) and PEP (B) with the top/bottom ratio. y% values has been calculated from the results of Fig. 2A and B applying Eq. (2).

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concentration ratio 20:1) – by using partitioning in aqueous two-phase systems of polyethyleneglycol–phosphate. In a system of polyethyleneglycol 600–phosphate both proteins have a tendency to be partitioned to the polymer rich phase, while in a system formed by polyethyleneglycol 1000–phosphate, the protein target tends to go to the phosphate rich phase. However, a selective separation between the partitioning coefficient values of both proteins was observed for a system formed by phosphate and a mixture of PEG600 and PEG1000. We examined the effect of the polyethyleneglycol molecular mass polydispersivity variation on the partitioning coefficient of CHY and PEP to apply this variable to improve the ATPS selectivity. Fig. 4A and B show the influence of the relative composition of PEGs mixture of molecular mass between 1450 and 8000 on the partition coefficient of CHY and PEP. A decrease of K can be seen when the PEG molecular mass increases for the proteins in agreement with the predicted general effect of the polymer molecular mass on the protein partitioning. These curves allows us to have a better idea about the molecular mass effect on a protein partition in order to determine the best polymer molecular mass value for a protein recovery in a liquid–liquid extraction method. It could be used to isolate and purify a protein from a complex mixture. Mayerhoff et al. [14], studied the molecular mass effect of the xylose reductase for liquid–liquid extraction. They found that the best experimental

Table 2 R

PEG Mw

y (%)

R

PEG Mw

y (%)

Quimosin 0.24 0.4 0.6 0.8 1.0

1450 1450 1450 1450 1450

91.2 95.0 96.4 98.5 97.4

0.24 0.4 0.6 0.8 1.0

3350 3350 3350 3350 3350

95.0 96.6 98.3 98.9 99.1

0.24 0.4 0.7 1.0

6000 6000 6000 6000

87.8 95.2 96.7 97.2

0.24 0.4 0.6 0.8 1.0

8000 8000 8000 8000 8000

87.3 89.2 93.1 94.6 96.6

Pepsin 0.2 0.4 0.6 0.8 1.0

1450 1450 1450 1450 1450

97.1 98.0 97.6 93.7 84.7

0.2 0.4 0.6 0.8 1.0

3350 3350 3350 3350 3350

94.7 97.9 97.1 97.3 97.8

0.2 0.4 0.6 0.8 1.0

6000 6000 6000 6000 6000

92.8 95.5 93.5 96.7 97.9

0.2 0.4 0.6 0.8 1.0

8000 8000 8000 8000 8000

92.4 97.1 96.0 91.4 93.4

recovery was for the 744 and 937 PEG molecular mass. It is known that those PEG molecular masses are not commercialy availables. However, by mixing two PEGs, it is possible to get a polymer phase that acts as if the medium had this molecular mass value. 3.4. The influence of R and Mw on the protein recovery Pic´o et al. [15] demostrated the vality to used PEG mixtures of different molecular mass to influent the partition coefficent of a protein and allows a better protein separation. Table 2 shows the experimental results for the assays of recovery of the protein in the top phase. A regression analysis was carried out to fit mathematical model to the experimental data to determine an optimal region for the responses studied. This analysis relates the percentage of recovery y(%) with the polymer molecular mass pausidispersivity (Mw) and the top/bottom phase volume ratio. The mathematical model that best fitted the experimental data can described by the following second grade equation: y(%) = h + aMw + bR + cMw2 + dR2

(5)

Fig. 5A and B shows the reponse surface for y(%) obtained by the mathematical model assayed. The analysis of variance shows that for the y(%) responses, the model adjusted was adeTable 3 R and Mw values for CHY and PEP

Fig. 4. Effect of the pausidispersivity on the CHY (A) and PEP (B) partition coefficient. Temperature 8 ◦ C, pH 7.0. Composition of systems view Table 1.

Protein

Mw

R

y (%)

CHY PEP

3496 3906

0.85 0.45

99.5 97.5

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the PEGs used to make the mixtures can be considered to be monodisperse according to the manufacturer information. By mixing PEGs of two different molecular mass, it is possible to obtain a pausidisperse mixture which behaves as a PEG of molecular mass 3496 or 3906, the PEG Mw values neccessary to obtain a greater efficiency in the protein recovery for CHY and PEP, respectively. Experiments are being carried out in progress in our laboratory to apply methods to obtain a concentrated of both protein from the natural product where they are present. Acknowledgements This work was supported by a grant from FoNCyT No. 06-12476/02, CONICET. We thank Mar´ıa Robson, Susana Spirandelli and Marcela Culasso for the language correction of the manuscript and Chr. Hansen Argentina because this company gently donated their CHY-MAX product for this work. References

Fig. 5. Response surface plot for the optimization of the CHY (A) and PEP (B) recovery in the top phase.

quate with high value of r2 and significant values of P (>99%) confidence level. Table 3 shows the values of R and Mw which predicts a better protein recovery. PEG obtained from industrial synthesis, has a molecular mass which remains narrowly disperse. Thus, it can be assumed that

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