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NaCit aqueous two-phase systems: Structural and functional approach
Protein Expression and Purification 129 (2017) 25e30
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Partitioning of xylanase from Thermomyces lanuginosus in PEG/NaCit aqueous two-phase systems: Structural and functional approach Dana B. Loureiro, Mauricio Braia, Diana Romanini, Gisela Tubio* n, Instituto de Procesos Biotecnolo gicos y Químicos (IPROBYQ) CONICET, Facultad de Ciencias Laboratorio de Fisicoquímica Aplicada a la Bioseparacio Bioquímicas y Farmac euticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 August 2016 Received in revised form 25 August 2016 Accepted 9 September 2016 Available online 10 September 2016
The structure and catalytic activity of xylanase from Thermomyces lanuginosus were studied in different media (containing polyethylene glycol -PEG- or salt) at different temperatures. The aim was to study how the native structure of the enzyme is affected to understand the partitioning behavior of xylanase in PEG/ sodium citrate (PEG/NaCit) aqueous two-phase systems. The presence of PEGs of different molar masses slightly altered the native structure of xylanase, although its catalytic activity was not affected. All the polymers assayed protect the native structure (and catalytic activity) of xylanase against temperature, except for PEG1000. Surface hydrophobicity experiments showed that xylanase favorable interacts with PEGs. Partitioning experiments confirmed this result and demonstrated that PEG1000/NaCit is the best system to partition xylanase from Thermomyces lanuginosus, since the Kp was 17.7 ± 0.3. © 2016 Elsevier Inc. All rights reserved.
Keywords: Thermomyces lanuginosus Xylanase Characterization Aqueous two-phase systems
1. Introduction Xylanase (EC 3.2.1.8), also known as endo-1,4-b-D-xylanase, is an enzyme that catalyzes the hydrolysis of the glycosidic linkage (b1,4) of xylan, the second most abundant natural polysaccharide leading to the formation of xylose, xylobiose and others sugars [1]. This enzyme has many biotechnological applications in different industries: bioconversion of lignocellulose in biofuels, replacing chlorine-based chemicals in the bleaching of pulp and clarification of juice among others [2e4]. Therefore, it is necessary to produced large quantities of xylanase using a fast, easy to scale up, and a low cost process. Usually, the purification of an industrial enzyme is the most important step since it represents at least 60% of the total cost of the process. Obviously, it highly depends on the purification level that needs to be achieved. Aqueous two-phase systems (ATPSs) have been used for the purification of many industrial enzymes [5e8]. These systems are formed by two immiscible phases, one composed of a polymer and the other composed of a polymer or salt [9] and the protein will distribute between both phases according to system and partitioned-biomolecule properties. In preliminary works, polyethylene glycol/sodium citrate (PEG/NaCit) ATPSs were found to be
* Corresponding author. E-mail address: [email protected] (G. Tubio). http://dx.doi.org/10.1016/j.pep.2016.09.003 1046-5928/© 2016 Elsevier Inc. All rights reserved.
more suitable for isolating xylanase from Aspergillus niger and bovine pancreatic trypsin than conventional PEG/phosphate systems due to several advantages such as the biodegradability and non-toxicity of the citrate anion [10,11]. ATPSs are mainly used as a first extractive step since they present many advantages: low cost of reagents and equipment, ease to scale up, high yield and velocity, capability of being reused in a cyclic process and capability of concentration [12,13]. On the other hand, it is very important to study the catalytic activity and the native structure of the enzyme since the interaction with the polymer or the salt may induce conformational changes that lead to the loss of activity or denature the protein. By studying PEG and salt-protein interaction through some physico-chemical techniques like fluorescence and circular dichroism can be explained the effect of cosolutes on xylanase partitioning behavior via its influence on the water structure. Fluorescence emission spectra of a protein are sensitive to changes in its environment thus; the fluorescence signal can be used as an optical probe to analyze the effect of cosolutes on the tertiary structure of the protein [14]. Also, fluorescence probes such as 1(anilino)-naphthalene-8-sulfonate (ANS) can be used to quantify protein hydrophobicity, to monitor conformational changes in biological macromolecules and to study protein binding sites. In this particular case, it can be very useful to predict how the xylanase will distribute in the ATPS [15]. On the other hand, CD spectra of proteins are particularly sensitive to changes in the secondary
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structure [16]. The aim of this work is to study the effect of PEGs and NaCit on the native structure of xylanase from Thermomyces lanuginosus and its partitioning in PEG/NaCit two-phase systems.
triplicate. The data were collected and corrected using the software provided by the instrument manufacturer. The CD data were plotted as mean residue ellipticity ([q]MRE, expressed as deg. cm2 dmol1) vs wavelength. The [q]MRE was calculated by Eq. (2) [16]:
2. Materials and methods
½qMRE ¼
2.1. Chemicals Xylanase from Thermomyces lanuginosus (Xyl) was purchased from Sigma Aldrich. A solution of the enzyme was prepared in 50 mM citrate buffer pH 5.3 and centrifuged. The supernatant was used as a source of xylanase. Sodium citrate (NaCit) was supplied by Cicarelli. Solution of the salt was prepared at a concentration of 25% (w/w) and pH 5.20. Beechwood xylan, xylose, polyethylene glycols of average molar masses (Da): 1000, 2000, 4600 and 8000 (PEG1000, PEG2000, PEG4600, PEG8000), and 1-anilinonaphthalene-8-sulfonate (ANS) were purchased from Sigma Aldrich and used without further purification. 2.2. Methodologies 2.2.1. Determination of Xyl activity Xyl activity was determined by the 3,5-dinitrosalicilic acid (DNS) method [17] measuring the amount of reducing sugars liberated when beechwood xylan (1% w/w) and Xyl were mixed in 50 mM sodium citrate buffer pH 5.3, according to Bailey et al. [18]. Solutions of substrate alone and enzyme alone were used as controls. Each sample was incubated at 50 C for 10 min. Then 1 mL of DNS reagent was added to each tube, and the samples were boiled for 10 min. The absorbance was measured at 560 nm. Xyl activity was expressed as U.mL1 (mmol xylose. mL1. min1) and calculated by Eq. (1):
U m$moles$xylose Fd ¼ mL 10 min Vx
(1)
where Fd is the dilution factor, Vx is the volume of the aliquot and 10 min is the incubation time. All the experiments were performed in triplicate. 2.2.2. Fluorescence spectra of Xyl in the presence of PEGs and NaCit at different temperatures Fluorescence emission spectra of Xyl were recorded after incubating the enzyme for 10 min at five different temperatures: 20, 40, 50, 60 and 70 C, in the absence and presence of PEGs and NaCit at pH 5.20. PEGs of different molar masses were used: 1000, 2000, 4600 and 8000. The presence of both PEGs and NaCit was assayed at two different final concentrations: 5% (w/w) and 10% (w/w). The spectra were performed using an Aminco Bowman Series 2 spectrofluorometer using a thermostated quartz cuvette of 1 cm pathlength, by exciting the protein (0.23 mM) at 280 nm. The data were recorded between 290 and 430 nm. The bandwith was 4 nm. The scanning rate was 3 nm. seg1 and the data was recorded every 0.1 nm with a slit of 0.1 nm. All the experiments were performed in triplicate and corrected using a blank without enzyme. 2.2.3. Circular dichroism spectra of Xyl in the presence of PEGs Circular dichroism spectra (CD) of Xyl at a final concentration of 0.23 mM were performed in a Jasco J-810 spectropolarimeter using a thermostated cuvette with a 1 mm path length. The scan rate was 50 nm min1 and the bandwidth was 1 nm. Repetitive scanning of eight cycles was used. All the measurements were performed in
MRW qobs 10 d c
(2)
where qobs is the observed ellipticity (expressed in degrees), d is the path length (cm) and c is the protein concentration (g.mL1). The Mean Residue Weight (MRW) for the peptide bond was calculated as MRW ¼ M/(N-1), where M is the molar mass of the polypeptide chain (in Da) and N the number of amino acids in the chain. The number of peptide bonds is N-1. All CD spectra were corrected using a blank without enzyme. 2.2.4. Measurements of the surface hydrophobicity of Xyl (S0) The surface hydrophobicity of Xyl was determined using the optical method reported by Haskard [19] in which ANS is used as the fluorescence probe. A 120 mM ANS solution, prepared in 50 mM phosphate buffer pH 6.00, was titrated with a 9 mM Xyl solution. The initial volume was 2500 mL. The concentration of protein varied from 0 to 0.34 mM. The excitation and emission wavelengths were 382 and 466 nm respectively with a bandwidth of 4 nm. The fluorescence intensity of ANS was measured after each addition of the protein solution and plotted against the concentration of Xyl. Finally, the data set was fitted to a linear regression model and the S0 of Xyl was determined from the initial slope of the straight line. All the experiments were performed at 25, 50 and 65 C and in the absence and presence of PEGs and NaCit. The temperature was controlled by using a HAAKE DC3 bath and measured with a thermocouple immersed inside the cuvette. The temperature accuracy was 0.01 C. 2.2.5. Preparation of the aqueous two-phase system To prepare the ATPSs, stock solutions of the phase components e PEG of different molar masses 40% (w/w) and NaCit 20% (w/w) of a given pH - were mixed according to the binodal diagram previously obtained in our laboratory [10,20]. The suitable pH of the salt solution was adjusted by the addition of sodium hydroxide. The ATPSs were mixed with a rotary mixer at 20 rpm for 30 min and then, left for 24 h at 25 C to allow phase separation. Finally, 1 mL of each phase was mixed to reconstitute several two-phase systems in which the protein partition was assayed. For a given PEG molar mass the compositions corresponding to four different tie lines were assayed. They were numbered (from 1 to 4) according to their increasing tie line length (TLL) which was calculated by applying the Eq. (3):
TLL ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ½DPEG2 þ ½DNaCit2
(3)
where: [DPEG] and [DNaCit] are the differences between the concentration of PEG and NaCit in the top and bottom phases expressed in % w/w. The total system compositions and the TLL selected for this work are shown in Table 2. 2.2.6. Determination of the partition coefficient (Kp) Partitioning behavior of Xyl was analyzed by dissolving a given amount of protein (10.4 mM total system concentration) in the twophase systems containing 1 mL of each equilibrated phase. Small aliquots of the protein stock solution were added to the systems in order to make the change of the total volume of each phase negligible. After mixing by inversion for 1 min and leaving it
D.B. Loureiro et al. / Protein Expression and Purification 129 (2017) 25e30
27
Table 1 Effect of temperature on xylanase fluorescence spectra in 50 mM potassium phosphate buffer pH 6.00 and 10% (w/w) of PEG1000, PEG8000 and NaCit. T ( C)
Buffer alone
PEG molar mass 1000
20 40 50 60 70
Salt 8000
NaCit
lmax
If
lmax
If
lmax
If
lmax
If
344.2 344.2 344.2 344.2 342.9
6.318 5.882 5.643 5.414 4.428
342.9 342.9 342.9 342.9 341.6
5.873 5.681 5.638 5.177 4.353
342.9 342.9 342.9 342.9 342.9
5.658 5.623 5.727 5.612 4.872
344.2 344.2 344.2 344.2 342.9
5.155 4.954 4.828 4.697 4.206
If: maximum fluorescence intensity observed at lmax. The protein concentration was 0.23 mM in all cases.
Table 2 Composition of aqueous two-phase systems formed by PEGs of different molar masses and sodium citrate pH 5.20. Temperature 25 C. TL
1 2 3 4
Total system composition (% w/w) PEG1000
NaCit
PEG2000
NaCit
PEG4600
NaCit
PEG8000
NaCit
17.29 18.00 18.43 19.00
13.32 14.00 14.50 14.90
17.29 18.00 18.43 19.00
13.32 14.00 14.50 14.90
10.41 10.90 11.73 11.83
10.25 10.51 10.97 11.03
9.63 11.69 14.32 15.15
9.50 9.52 9.22 9.36
to settle for at least 120 min, the system was centrifuged at 2000 rpm for 5 min for the two-phase separation. Samples were withdrawn from the separated phases and after the appropriate dilution (with the equilibrated phase free from protein), protein activity in each phase was determined. The partition coefficient was calculated by Eq. (4):
Kp ¼
½Xyl activityT F ½Xyl activityB
(4)
where [Xyl activity]T and [Xyl activity]B are the enzyme activities of the partitioned protein in the PEG-rich and salt-rich phases, respectively. The effect of the phase composition on the enzyme activity was considered. A correction factor (F) was calculated as the ratio between the activities of reference solutions (of known enzyme concentration) in each phase. Temperature was maintained constant at 25 C and controlled within ±0.1 C.
Fig. 1. Fluorescence spectra of xylanase from Thermomyces lanuginosus in 50 mM potassium phosphate buffer pH 6.00 and presence of 5% (w/w) salt and PEGs: NaCit; PEG1000; PEG2000; PEG4600 and PEG8000. Temperature 20 C.
3.2. Circular dichroism spectra of Xyl Figs. 2 and 3 show the effect of the presence of PEGs at 20 C and the effect of temperature on the CD spectra of Xyl, respectively. According to previous reports, the CD spectrum of xylanase shows that the enzyme has predominantly b-sheet structure [21]. It presents a positive band at 197e201 nm and a negative band at 215e225 nm with its characteristic features. A weakening of the band at 220 nm can be seen, although the protein never lost its secondary structure completely. PEG1000 produced the largest decrease in the intensity of the band at 220 nm, while an increase in the temperature gradually modified the spectra of the protein. On the other hand, as can be seen in Fig. 4, PEG1000 protects the native structure of Xyl against temperature between 20 and 50 C, since no changes of the band at 220 nm are observed. At 70 C the protein lost all the secondary structure indicating that PEG1000 and high temperature cooperatively affect the native structure of Xyl. More important, PEG2000, 4600 and 8000 protect the secondary structure of Xyl even at 60 C and 70 C (data not shown). 3.3. Catalytic activity of xylanase Fig. 5 shows the effect of the temperature on the Xyl catalytic activity in the absence and presence of PEGs. In the presence of
3. Results and discussion 3.1. Effect of the presence of PEGs and NaCit on the fluorescence spectra of Xyl Fig. 1 shows no significant shifts of the emission peak position (344 nm) in the different media, suggesting that the environment of the tryptophan residues of the protein did not suffer conformational changes. This indicates that no modification in the tertiary structure of the protein is induced by the presence of PEG or NaCit. Table 1 shows the effect of temperature on the position and the intensity of the fluorescence emission peaks in the absence and presence of PEG1000 and PEG8000 and NaCit. It can be seen that the presence of any of the cosolutes did not produce a significant change in the emission peak position. Besides, Table 1 shows that the maximum fluorescence intensity disminishes when temperature increases, suggesting that this loss of energy could be due to vibrational processes favored by the increase in temperature. The same effect was observed for PEG2000 and 4600 (data not shown).
Fig. 2. Circular dichroism spectra of xylanase from Thermomyces lanuginosus (0.23 mM) in 50 mM potassium phosphate buffer pH 6.00 and presence of 5% (w/w) PEGs: PEG1000; PEG2000; PEG4600 and PEG8000. Temperature 20 C.
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D.B. Loureiro et al. / Protein Expression and Purification 129 (2017) 25e30
presence of PEG8000, Xyl activity remained constant between 20 and 60 C; losing only 20% of the initial activity at 70 C. These results agree with those obtained by circular dichroism and indicate that the interaction between Xyl and PEGs of high molar masses did not affect the enzymatic activity and protects the native structure of the protein against thermal denaturation. 3.4. Surface hydrophobicity of Xyl (S0)
Fig. 3. Circular dichroism spectra of xylanase from Thermomyces lanuginosus (0.23 mM) at different temperatures: 20 C; 40 C; 50 C; 60 C and 70 C in 50 mM in potassium phosphate buffer pH 6.00.
Fig. 6 shows the change of the surface hydrophobicity of Xyl at 25, 50 and 65 C and in the presence of different cosolutes. It can be seen that in the presence of NaCit, S0 was higher than in buffer alone. This effect is due to the high hydration of the salt ions, which helps to unmask hydrophobic domains on the surface of the protein. These domains interact with ANS, enhancing the fluorescence. In the presence of PEGs, S0 decreased its value (except for PEG1000 at 65 C), compared to the enzyme in buffer alone. When Xyl interacts with the polymer, hydrophobic clusters on the surface of the protein became less exposed, thus preventing ANS from interacting with them. This causes a decrease of the ANS fluorescence and hence, S0 value. These results indicate that Xyl favorable interacts with PEG1000 at 25 C and PEG2000, 4600 and 8000 at all temperatures assayed. In the case of PEG1000 at 65 C, it can be seen that S0 is higher than in buffer alone (and higher than the other PEGs). It is important to remember that circular dichroism spectra showed that between 60 C and 70 C, the enzyme started to lose its native structure. Therefore, an increase of S0 indicates that buried hydrophobic clusters are getting exposed and interact with ANS. These results are also consistent with the measures of catalytic activity. 3.5. Determination of the partition coefficient (Kp)
Fig. 4. Circular dichroism spectra of xylanase from Thermomyces lanuginosus (0.23 mM) in presence of 5% (w/w) PEG1000 at different temperatures: 20 C; 40 C; 50 C; 60 C and 70 C.
Fig. 7 shows the influence of PEG molar mass on Xyl partition coefficients in PEG/NaCit systems at different TLL (25 C). It can be seen that Kp was always higher than one, indicating that the enzyme preferentially partitions into the PEG-rich phase. This is due to favorable interactions between Xyl and PEG. These results agree with the surface hydrophobicity experiments. Also, the
Fig. 5. Xylanase activity (U/mL) at different temperatures, in 50 mM sodium citrate buffer pH 5.3 and presence of PEGs with different molar masses: PEG1000; PEG2000; PEG4600 and PEG8000.
PEG1000, it can be seen that the activity at 60 C diminished about five fold with respect to the initial activity at 20 C and at 70 C the catalytic activity is completely lost. On the other hand, in the
Fig. 6. Surface hydrophobicity of xylanase from Thermomyces lanuginosus in 50 mM potassium phosphate buffer pH 6.00 and presence of 5% (w/w) NaCit and PEGs of different molar masses at 25, 50 and 65 C.
D.B. Loureiro et al. / Protein Expression and Purification 129 (2017) 25e30
29
that xylanase increases its hydrophobicity in the presence of salts due to exposure of inner hydrophobic clusters. Also, it can be seen that xylanase favorable interacts with PEG2000, 4600 and 8000 at any temperature. These results validate the use of PEG/salt aqueous two-phase systems to study the partition of xylanase from T. lanuginosus. The enzyme preferentially partition into the PEG-rich phase and showed the highest values of Kp in the systems formed by PEG1000 (Kp 17.7 ± 0.3). Acknowledgments
Fig. 7. Partition of xylanase from Thermomyces lanuginosus in PEG/NaCit pH 5.20 twophase system. Temperature 25 C.
partitioning behavior of the enzyme did not significantly changed as the tie line length increased. In general, the highest values of Kp were obtained in the ATPSs formed by PEG1000 and 2000. This behavior was more pronounced in PEG1000/NaCit than in PEG2000/NaCit systems, since the Kp was 17.7 ± 0.3. This is due to the excluded volume effect: an increase of the PEG molar mass induces a decrease in the protein solubility in the phase where the polymer is situated. According to these results, the excluded volume effect also drives the Xyl partition between the two phases. In the systems with PEG4600 and 8000 the partition coefficients decreased, which is consistent with the increase of the PEG excluded volume in such a way that an increase in molar mass induced the transfer of the protein to the opposite phase [22,23]. 4. Conclusion The structure and catalytic activity of xylanase was studied in different media and conditions. This is very important for validating the use of aqueous two-phase extraction as an initial step in a purification protocol. Partitioning of proteins in aqueous twophase systems highly depends on experimental conditions, chemical nature of polymers and/or salts and physical-chemical properties of the protein. Experiments are not only focused on improving the recovery and purity of the enzyme but also on preserving its native structure and catalytic activity. In this work, the structure and catalytic activity of xylanase in the presence of NaCit and PEGs was studied in order to establish the basis for the use of PEG/NaCit aqueous two-phase systems. Xylanase was able to retain its secondary structure and more than 70% of its catalytic activity at 60 C and 50% at 70 C. The interaction of xylanase with PEG2000, 4600 and 8000 protects the enzyme from thermal denaturation: xylanase retained all its catalytic activity at 60 C. At 70 C, PEG4600 showed the best results, yielding 84% of the initial catalytic activity. On the other hand, the interaction with PEG1000 completely destabilized the native structure of the enzyme at 70 C; hence the catalytic activity was lost. All these results are consistent with the circular dichroism experiments. The most important evidence is the loss of secondary structure when the enzyme interacted with PEG1000, showing the characteristic signal of random coil structure at 70 C. Fluorescence experiments did not show changes in the environment of the tryptophan residues of the protein. This indicates that the interaction of xylanase with the PEGs did not affect its tertiary structure. Surface hydrophobicity experiments showed
This work was supported by a Grant from Agencia Nacional de n Científica y Tecnolo gica (PICT2013-1730), CONICET Promocio (PIP551/2012) and SeCyT-UNR BIO338. We would like to thank the staff from the English Department (Facultad de Ciencias Biouticas, UNR) for the language correction of the químicas y Farmace manuscript. References [1] T. Collins, C. Gerday, G. Feller, Xylanases, xylanase families and extremophilic xylanases, FEMS Microbiol. Rev. 29 (2005) 3e23. [2] M.L.T.M. Polizeli, A.C.S. Rizzatti, R. Monti, H.F. Terenzi, J.A. Jorge, D.S. Amorim, Xylanases from fungi: properties and industrial applications, Appl. Microbiol. Biotechnol. 67 (2005) 577e591. [3] J. Robert, B.A.L. Whitehurst, in: B.A.L.a.A.J. Taylor (Ed.), Enzymes in Food Technology, CRC Press LLC, 2002. [4] A. Wolfgang, Industrial enzymes, in: A. Wolfgang (Ed.), Enzymes in Industry. Production and Applications, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim: Leiden, 2007. [5] J.G.L.F. Alves, L.D.A. Chumpitaz, L.H.M. da Silva, T.T. Franco, A.J.A. Meirelles, Partitioning of whey proteins, bovine serum albumin and porcine insulin in aqueous two-phase systems, J. Chromatogr. B Biomed. Sci. Appl. 743 (2000) 235e239. [6] S. Yang, Z. Huang, Z. Jiang, L. Li, Partition and purification of a thermostable xylanase produced by Paecilomyces thermophila in solid-state fermentation using aqueous two-phase systems, Process Biochem. 43 (2008) 56e61. [7] Z. Gu, C.E. Glatz, Aqueous two-phase extraction for protein recovery from corn extracts, J. Chromatogr. B 845 (2007) 38e50. [8] E. Stratikos, P.G.W. Gettins, Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 Å and full insertion of the reactive center loop into b-sheet A, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 4808e4813. [9] B.Y. Zaslavsky, in: Marcel Dekker, Inc (Ed.), Aqueous Two-phase Partitioning, Physical Chemistry and Bioanalytical Applications, 1995. New York. [10] D.B. Loureiro, D. Romanini, G. Tubio, Structural and functional analysis of Aspergillus niger xylanase to be employed in polyethylene glycol/salt aqueous two-phase extraction, Biocatal. Agric. Biotechnol. 5 (2016) 204e210. rez, D.B. Loureiro, B.B. Nerli, G. Tubio, Optimization of pancreatic trypsin [11] R.L. Pe extraction in PEG/citrate aqueous two-phase systems, Protein Expr. Purif. 106 (2015) 66e71. [12] A. Glyk, T Fau Scheper, S. Beutel, PEG-salt aqueous two-phase systems: an attractive and versatile liquid-liquid extraction technology for the downstream processing of proteins and enzymes, Appl. Microbiol. Biotechnol. (2015) (1432e0614 (Electronic)). [13] R. Hatti-Kaul, Aqueous two-phase systems - a general overview, Mol. Biotechnol. 19 (2001) 269e277. [14] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, vol. 1983, Plenum Press, 1983, p. 233. Spring Str. New York, NY 10013. [15] J. Lamba, P.S. Hasija, V. Aggarwal, T.K. Chaudhuri, Monitoring protein folding and unfolding pathways through surface hydrophobicity changes using fluorescence and circular dichroism spectroscopy, Biochem. Mosc. 74 (2009) 393e398. [16] S.M. Kelly, T.J. Jess, N.C. Price, How to study proteins by circular dichroism, Biochim. Biophys. Acta (BBA) - Proteins Proteom. 1751 (2005) 119e139. [17] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. 31 (1959) 426e428. [18] M.J. Bailey, P. Biely, K. Poutanen, Interlaboratory testing of methods for assay of xylanase activity, J. Biotechnol. 23 (1992) 257e270. [19] C.A. Haskard, E.C.Y. Li-Chan, Hydrophobicity of bovine serum albumin and ovalbumin determined using uncharged (PRODAN) and anionic (ANS-) fluorescent probes, J. Agric. Food Chem. 46 (1998) 2671e2677. , B. Nerli, Liquid-liquid equilibria of aqueous two[20] G. Tubio, L. Pellegrini, G. Pico phase systems containing poly(ethylene glycols) of different molecular weight and sodium citrate, J. Chem. Eng. Data 51 (2006) 209e212. [21] K. Gruber, G. Klintschar, M. Hayn, A. Schlacher, W. Steiner, C. Kratky, Thermophilic xylanase from Thermomyces lanuginosus: high-resolution x-ray
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structure and modeling studies, Biochemistry 37 (1998) 13475e13485. [22] S. Saravanan, J.R. Rao, B.U. Nair, T. Ramasami, Aqueous two-phase poly(ethylene glycol)epoly(acrylic acid) system for protein partitioning: influence of molecular weight, pH and temperature, Process Biochem. 43 (2008) 905e911.
[23] G.G. George, D.P.S. Oliveira, I.C. Roberto, M. Vitolo, A. Pessoa Jr., Partition behavior and partial purification of hexokinase in aqueous two-phase polyethylene glycol/citrate systems, in: J.W. Lee, Brian H. Davison, Mark Finkelstein, James D. McMillan (Eds.), Biotechnology for Fuels and Chemicals, Humana Press, New York, 2003, pp. 787e797.
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Partitioning of xylanase from Thermomyces lanuginosus in PEG/NaCit aqueous two-phase systems: Structural and functional approach Dana B. Loureiro, Mauricio Braia, Diana Romanini, Gisela Tubio* n, Instituto de Procesos Biotecnolo gicos y Químicos (IPROBYQ) CONICET, Facultad de Ciencias Laboratorio de Fisicoquímica Aplicada a la Bioseparacio Bioquímicas y Farmac euticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 August 2016 Received in revised form 25 August 2016 Accepted 9 September 2016 Available online 10 September 2016
The structure and catalytic activity of xylanase from Thermomyces lanuginosus were studied in different media (containing polyethylene glycol -PEG- or salt) at different temperatures. The aim was to study how the native structure of the enzyme is affected to understand the partitioning behavior of xylanase in PEG/ sodium citrate (PEG/NaCit) aqueous two-phase systems. The presence of PEGs of different molar masses slightly altered the native structure of xylanase, although its catalytic activity was not affected. All the polymers assayed protect the native structure (and catalytic activity) of xylanase against temperature, except for PEG1000. Surface hydrophobicity experiments showed that xylanase favorable interacts with PEGs. Partitioning experiments confirmed this result and demonstrated that PEG1000/NaCit is the best system to partition xylanase from Thermomyces lanuginosus, since the Kp was 17.7 ± 0.3. © 2016 Elsevier Inc. All rights reserved.
Keywords: Thermomyces lanuginosus Xylanase Characterization Aqueous two-phase systems
1. Introduction Xylanase (EC 3.2.1.8), also known as endo-1,4-b-D-xylanase, is an enzyme that catalyzes the hydrolysis of the glycosidic linkage (b1,4) of xylan, the second most abundant natural polysaccharide leading to the formation of xylose, xylobiose and others sugars [1]. This enzyme has many biotechnological applications in different industries: bioconversion of lignocellulose in biofuels, replacing chlorine-based chemicals in the bleaching of pulp and clarification of juice among others [2e4]. Therefore, it is necessary to produced large quantities of xylanase using a fast, easy to scale up, and a low cost process. Usually, the purification of an industrial enzyme is the most important step since it represents at least 60% of the total cost of the process. Obviously, it highly depends on the purification level that needs to be achieved. Aqueous two-phase systems (ATPSs) have been used for the purification of many industrial enzymes [5e8]. These systems are formed by two immiscible phases, one composed of a polymer and the other composed of a polymer or salt [9] and the protein will distribute between both phases according to system and partitioned-biomolecule properties. In preliminary works, polyethylene glycol/sodium citrate (PEG/NaCit) ATPSs were found to be
* Corresponding author. E-mail address: [email protected] (G. Tubio). http://dx.doi.org/10.1016/j.pep.2016.09.003 1046-5928/© 2016 Elsevier Inc. All rights reserved.
more suitable for isolating xylanase from Aspergillus niger and bovine pancreatic trypsin than conventional PEG/phosphate systems due to several advantages such as the biodegradability and non-toxicity of the citrate anion [10,11]. ATPSs are mainly used as a first extractive step since they present many advantages: low cost of reagents and equipment, ease to scale up, high yield and velocity, capability of being reused in a cyclic process and capability of concentration [12,13]. On the other hand, it is very important to study the catalytic activity and the native structure of the enzyme since the interaction with the polymer or the salt may induce conformational changes that lead to the loss of activity or denature the protein. By studying PEG and salt-protein interaction through some physico-chemical techniques like fluorescence and circular dichroism can be explained the effect of cosolutes on xylanase partitioning behavior via its influence on the water structure. Fluorescence emission spectra of a protein are sensitive to changes in its environment thus; the fluorescence signal can be used as an optical probe to analyze the effect of cosolutes on the tertiary structure of the protein [14]. Also, fluorescence probes such as 1(anilino)-naphthalene-8-sulfonate (ANS) can be used to quantify protein hydrophobicity, to monitor conformational changes in biological macromolecules and to study protein binding sites. In this particular case, it can be very useful to predict how the xylanase will distribute in the ATPS [15]. On the other hand, CD spectra of proteins are particularly sensitive to changes in the secondary
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structure [16]. The aim of this work is to study the effect of PEGs and NaCit on the native structure of xylanase from Thermomyces lanuginosus and its partitioning in PEG/NaCit two-phase systems.
triplicate. The data were collected and corrected using the software provided by the instrument manufacturer. The CD data were plotted as mean residue ellipticity ([q]MRE, expressed as deg. cm2 dmol1) vs wavelength. The [q]MRE was calculated by Eq. (2) [16]:
2. Materials and methods
½qMRE ¼
2.1. Chemicals Xylanase from Thermomyces lanuginosus (Xyl) was purchased from Sigma Aldrich. A solution of the enzyme was prepared in 50 mM citrate buffer pH 5.3 and centrifuged. The supernatant was used as a source of xylanase. Sodium citrate (NaCit) was supplied by Cicarelli. Solution of the salt was prepared at a concentration of 25% (w/w) and pH 5.20. Beechwood xylan, xylose, polyethylene glycols of average molar masses (Da): 1000, 2000, 4600 and 8000 (PEG1000, PEG2000, PEG4600, PEG8000), and 1-anilinonaphthalene-8-sulfonate (ANS) were purchased from Sigma Aldrich and used without further purification. 2.2. Methodologies 2.2.1. Determination of Xyl activity Xyl activity was determined by the 3,5-dinitrosalicilic acid (DNS) method [17] measuring the amount of reducing sugars liberated when beechwood xylan (1% w/w) and Xyl were mixed in 50 mM sodium citrate buffer pH 5.3, according to Bailey et al. [18]. Solutions of substrate alone and enzyme alone were used as controls. Each sample was incubated at 50 C for 10 min. Then 1 mL of DNS reagent was added to each tube, and the samples were boiled for 10 min. The absorbance was measured at 560 nm. Xyl activity was expressed as U.mL1 (mmol xylose. mL1. min1) and calculated by Eq. (1):
U m$moles$xylose Fd ¼ mL 10 min Vx
(1)
where Fd is the dilution factor, Vx is the volume of the aliquot and 10 min is the incubation time. All the experiments were performed in triplicate. 2.2.2. Fluorescence spectra of Xyl in the presence of PEGs and NaCit at different temperatures Fluorescence emission spectra of Xyl were recorded after incubating the enzyme for 10 min at five different temperatures: 20, 40, 50, 60 and 70 C, in the absence and presence of PEGs and NaCit at pH 5.20. PEGs of different molar masses were used: 1000, 2000, 4600 and 8000. The presence of both PEGs and NaCit was assayed at two different final concentrations: 5% (w/w) and 10% (w/w). The spectra were performed using an Aminco Bowman Series 2 spectrofluorometer using a thermostated quartz cuvette of 1 cm pathlength, by exciting the protein (0.23 mM) at 280 nm. The data were recorded between 290 and 430 nm. The bandwith was 4 nm. The scanning rate was 3 nm. seg1 and the data was recorded every 0.1 nm with a slit of 0.1 nm. All the experiments were performed in triplicate and corrected using a blank without enzyme. 2.2.3. Circular dichroism spectra of Xyl in the presence of PEGs Circular dichroism spectra (CD) of Xyl at a final concentration of 0.23 mM were performed in a Jasco J-810 spectropolarimeter using a thermostated cuvette with a 1 mm path length. The scan rate was 50 nm min1 and the bandwidth was 1 nm. Repetitive scanning of eight cycles was used. All the measurements were performed in
MRW qobs 10 d c
(2)
where qobs is the observed ellipticity (expressed in degrees), d is the path length (cm) and c is the protein concentration (g.mL1). The Mean Residue Weight (MRW) for the peptide bond was calculated as MRW ¼ M/(N-1), where M is the molar mass of the polypeptide chain (in Da) and N the number of amino acids in the chain. The number of peptide bonds is N-1. All CD spectra were corrected using a blank without enzyme. 2.2.4. Measurements of the surface hydrophobicity of Xyl (S0) The surface hydrophobicity of Xyl was determined using the optical method reported by Haskard [19] in which ANS is used as the fluorescence probe. A 120 mM ANS solution, prepared in 50 mM phosphate buffer pH 6.00, was titrated with a 9 mM Xyl solution. The initial volume was 2500 mL. The concentration of protein varied from 0 to 0.34 mM. The excitation and emission wavelengths were 382 and 466 nm respectively with a bandwidth of 4 nm. The fluorescence intensity of ANS was measured after each addition of the protein solution and plotted against the concentration of Xyl. Finally, the data set was fitted to a linear regression model and the S0 of Xyl was determined from the initial slope of the straight line. All the experiments were performed at 25, 50 and 65 C and in the absence and presence of PEGs and NaCit. The temperature was controlled by using a HAAKE DC3 bath and measured with a thermocouple immersed inside the cuvette. The temperature accuracy was 0.01 C. 2.2.5. Preparation of the aqueous two-phase system To prepare the ATPSs, stock solutions of the phase components e PEG of different molar masses 40% (w/w) and NaCit 20% (w/w) of a given pH - were mixed according to the binodal diagram previously obtained in our laboratory [10,20]. The suitable pH of the salt solution was adjusted by the addition of sodium hydroxide. The ATPSs were mixed with a rotary mixer at 20 rpm for 30 min and then, left for 24 h at 25 C to allow phase separation. Finally, 1 mL of each phase was mixed to reconstitute several two-phase systems in which the protein partition was assayed. For a given PEG molar mass the compositions corresponding to four different tie lines were assayed. They were numbered (from 1 to 4) according to their increasing tie line length (TLL) which was calculated by applying the Eq. (3):
TLL ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ½DPEG2 þ ½DNaCit2
(3)
where: [DPEG] and [DNaCit] are the differences between the concentration of PEG and NaCit in the top and bottom phases expressed in % w/w. The total system compositions and the TLL selected for this work are shown in Table 2. 2.2.6. Determination of the partition coefficient (Kp) Partitioning behavior of Xyl was analyzed by dissolving a given amount of protein (10.4 mM total system concentration) in the twophase systems containing 1 mL of each equilibrated phase. Small aliquots of the protein stock solution were added to the systems in order to make the change of the total volume of each phase negligible. After mixing by inversion for 1 min and leaving it
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27
Table 1 Effect of temperature on xylanase fluorescence spectra in 50 mM potassium phosphate buffer pH 6.00 and 10% (w/w) of PEG1000, PEG8000 and NaCit. T ( C)
Buffer alone
PEG molar mass 1000
20 40 50 60 70
Salt 8000
NaCit
lmax
If
lmax
If
lmax
If
lmax
If
344.2 344.2 344.2 344.2 342.9
6.318 5.882 5.643 5.414 4.428
342.9 342.9 342.9 342.9 341.6
5.873 5.681 5.638 5.177 4.353
342.9 342.9 342.9 342.9 342.9
5.658 5.623 5.727 5.612 4.872
344.2 344.2 344.2 344.2 342.9
5.155 4.954 4.828 4.697 4.206
If: maximum fluorescence intensity observed at lmax. The protein concentration was 0.23 mM in all cases.
Table 2 Composition of aqueous two-phase systems formed by PEGs of different molar masses and sodium citrate pH 5.20. Temperature 25 C. TL
1 2 3 4
Total system composition (% w/w) PEG1000
NaCit
PEG2000
NaCit
PEG4600
NaCit
PEG8000
NaCit
17.29 18.00 18.43 19.00
13.32 14.00 14.50 14.90
17.29 18.00 18.43 19.00
13.32 14.00 14.50 14.90
10.41 10.90 11.73 11.83
10.25 10.51 10.97 11.03
9.63 11.69 14.32 15.15
9.50 9.52 9.22 9.36
to settle for at least 120 min, the system was centrifuged at 2000 rpm for 5 min for the two-phase separation. Samples were withdrawn from the separated phases and after the appropriate dilution (with the equilibrated phase free from protein), protein activity in each phase was determined. The partition coefficient was calculated by Eq. (4):
Kp ¼
½Xyl activityT F ½Xyl activityB
(4)
where [Xyl activity]T and [Xyl activity]B are the enzyme activities of the partitioned protein in the PEG-rich and salt-rich phases, respectively. The effect of the phase composition on the enzyme activity was considered. A correction factor (F) was calculated as the ratio between the activities of reference solutions (of known enzyme concentration) in each phase. Temperature was maintained constant at 25 C and controlled within ±0.1 C.
Fig. 1. Fluorescence spectra of xylanase from Thermomyces lanuginosus in 50 mM potassium phosphate buffer pH 6.00 and presence of 5% (w/w) salt and PEGs: NaCit; PEG1000; PEG2000; PEG4600 and PEG8000. Temperature 20 C.
3.2. Circular dichroism spectra of Xyl Figs. 2 and 3 show the effect of the presence of PEGs at 20 C and the effect of temperature on the CD spectra of Xyl, respectively. According to previous reports, the CD spectrum of xylanase shows that the enzyme has predominantly b-sheet structure [21]. It presents a positive band at 197e201 nm and a negative band at 215e225 nm with its characteristic features. A weakening of the band at 220 nm can be seen, although the protein never lost its secondary structure completely. PEG1000 produced the largest decrease in the intensity of the band at 220 nm, while an increase in the temperature gradually modified the spectra of the protein. On the other hand, as can be seen in Fig. 4, PEG1000 protects the native structure of Xyl against temperature between 20 and 50 C, since no changes of the band at 220 nm are observed. At 70 C the protein lost all the secondary structure indicating that PEG1000 and high temperature cooperatively affect the native structure of Xyl. More important, PEG2000, 4600 and 8000 protect the secondary structure of Xyl even at 60 C and 70 C (data not shown). 3.3. Catalytic activity of xylanase Fig. 5 shows the effect of the temperature on the Xyl catalytic activity in the absence and presence of PEGs. In the presence of
3. Results and discussion 3.1. Effect of the presence of PEGs and NaCit on the fluorescence spectra of Xyl Fig. 1 shows no significant shifts of the emission peak position (344 nm) in the different media, suggesting that the environment of the tryptophan residues of the protein did not suffer conformational changes. This indicates that no modification in the tertiary structure of the protein is induced by the presence of PEG or NaCit. Table 1 shows the effect of temperature on the position and the intensity of the fluorescence emission peaks in the absence and presence of PEG1000 and PEG8000 and NaCit. It can be seen that the presence of any of the cosolutes did not produce a significant change in the emission peak position. Besides, Table 1 shows that the maximum fluorescence intensity disminishes when temperature increases, suggesting that this loss of energy could be due to vibrational processes favored by the increase in temperature. The same effect was observed for PEG2000 and 4600 (data not shown).
Fig. 2. Circular dichroism spectra of xylanase from Thermomyces lanuginosus (0.23 mM) in 50 mM potassium phosphate buffer pH 6.00 and presence of 5% (w/w) PEGs: PEG1000; PEG2000; PEG4600 and PEG8000. Temperature 20 C.
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presence of PEG8000, Xyl activity remained constant between 20 and 60 C; losing only 20% of the initial activity at 70 C. These results agree with those obtained by circular dichroism and indicate that the interaction between Xyl and PEGs of high molar masses did not affect the enzymatic activity and protects the native structure of the protein against thermal denaturation. 3.4. Surface hydrophobicity of Xyl (S0)
Fig. 3. Circular dichroism spectra of xylanase from Thermomyces lanuginosus (0.23 mM) at different temperatures: 20 C; 40 C; 50 C; 60 C and 70 C in 50 mM in potassium phosphate buffer pH 6.00.
Fig. 6 shows the change of the surface hydrophobicity of Xyl at 25, 50 and 65 C and in the presence of different cosolutes. It can be seen that in the presence of NaCit, S0 was higher than in buffer alone. This effect is due to the high hydration of the salt ions, which helps to unmask hydrophobic domains on the surface of the protein. These domains interact with ANS, enhancing the fluorescence. In the presence of PEGs, S0 decreased its value (except for PEG1000 at 65 C), compared to the enzyme in buffer alone. When Xyl interacts with the polymer, hydrophobic clusters on the surface of the protein became less exposed, thus preventing ANS from interacting with them. This causes a decrease of the ANS fluorescence and hence, S0 value. These results indicate that Xyl favorable interacts with PEG1000 at 25 C and PEG2000, 4600 and 8000 at all temperatures assayed. In the case of PEG1000 at 65 C, it can be seen that S0 is higher than in buffer alone (and higher than the other PEGs). It is important to remember that circular dichroism spectra showed that between 60 C and 70 C, the enzyme started to lose its native structure. Therefore, an increase of S0 indicates that buried hydrophobic clusters are getting exposed and interact with ANS. These results are also consistent with the measures of catalytic activity. 3.5. Determination of the partition coefficient (Kp)
Fig. 4. Circular dichroism spectra of xylanase from Thermomyces lanuginosus (0.23 mM) in presence of 5% (w/w) PEG1000 at different temperatures: 20 C; 40 C; 50 C; 60 C and 70 C.
Fig. 7 shows the influence of PEG molar mass on Xyl partition coefficients in PEG/NaCit systems at different TLL (25 C). It can be seen that Kp was always higher than one, indicating that the enzyme preferentially partitions into the PEG-rich phase. This is due to favorable interactions between Xyl and PEG. These results agree with the surface hydrophobicity experiments. Also, the
Fig. 5. Xylanase activity (U/mL) at different temperatures, in 50 mM sodium citrate buffer pH 5.3 and presence of PEGs with different molar masses: PEG1000; PEG2000; PEG4600 and PEG8000.
PEG1000, it can be seen that the activity at 60 C diminished about five fold with respect to the initial activity at 20 C and at 70 C the catalytic activity is completely lost. On the other hand, in the
Fig. 6. Surface hydrophobicity of xylanase from Thermomyces lanuginosus in 50 mM potassium phosphate buffer pH 6.00 and presence of 5% (w/w) NaCit and PEGs of different molar masses at 25, 50 and 65 C.
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29
that xylanase increases its hydrophobicity in the presence of salts due to exposure of inner hydrophobic clusters. Also, it can be seen that xylanase favorable interacts with PEG2000, 4600 and 8000 at any temperature. These results validate the use of PEG/salt aqueous two-phase systems to study the partition of xylanase from T. lanuginosus. The enzyme preferentially partition into the PEG-rich phase and showed the highest values of Kp in the systems formed by PEG1000 (Kp 17.7 ± 0.3). Acknowledgments
Fig. 7. Partition of xylanase from Thermomyces lanuginosus in PEG/NaCit pH 5.20 twophase system. Temperature 25 C.
partitioning behavior of the enzyme did not significantly changed as the tie line length increased. In general, the highest values of Kp were obtained in the ATPSs formed by PEG1000 and 2000. This behavior was more pronounced in PEG1000/NaCit than in PEG2000/NaCit systems, since the Kp was 17.7 ± 0.3. This is due to the excluded volume effect: an increase of the PEG molar mass induces a decrease in the protein solubility in the phase where the polymer is situated. According to these results, the excluded volume effect also drives the Xyl partition between the two phases. In the systems with PEG4600 and 8000 the partition coefficients decreased, which is consistent with the increase of the PEG excluded volume in such a way that an increase in molar mass induced the transfer of the protein to the opposite phase [22,23]. 4. Conclusion The structure and catalytic activity of xylanase was studied in different media and conditions. This is very important for validating the use of aqueous two-phase extraction as an initial step in a purification protocol. Partitioning of proteins in aqueous twophase systems highly depends on experimental conditions, chemical nature of polymers and/or salts and physical-chemical properties of the protein. Experiments are not only focused on improving the recovery and purity of the enzyme but also on preserving its native structure and catalytic activity. In this work, the structure and catalytic activity of xylanase in the presence of NaCit and PEGs was studied in order to establish the basis for the use of PEG/NaCit aqueous two-phase systems. Xylanase was able to retain its secondary structure and more than 70% of its catalytic activity at 60 C and 50% at 70 C. The interaction of xylanase with PEG2000, 4600 and 8000 protects the enzyme from thermal denaturation: xylanase retained all its catalytic activity at 60 C. At 70 C, PEG4600 showed the best results, yielding 84% of the initial catalytic activity. On the other hand, the interaction with PEG1000 completely destabilized the native structure of the enzyme at 70 C; hence the catalytic activity was lost. All these results are consistent with the circular dichroism experiments. The most important evidence is the loss of secondary structure when the enzyme interacted with PEG1000, showing the characteristic signal of random coil structure at 70 C. Fluorescence experiments did not show changes in the environment of the tryptophan residues of the protein. This indicates that the interaction of xylanase with the PEGs did not affect its tertiary structure. Surface hydrophobicity experiments showed
This work was supported by a Grant from Agencia Nacional de n Científica y Tecnolo gica (PICT2013-1730), CONICET Promocio (PIP551/2012) and SeCyT-UNR BIO338. We would like to thank the staff from the English Department (Facultad de Ciencias Biouticas, UNR) for the language correction of the químicas y Farmace manuscript. References [1] T. Collins, C. Gerday, G. Feller, Xylanases, xylanase families and extremophilic xylanases, FEMS Microbiol. Rev. 29 (2005) 3e23. [2] M.L.T.M. Polizeli, A.C.S. Rizzatti, R. Monti, H.F. Terenzi, J.A. Jorge, D.S. Amorim, Xylanases from fungi: properties and industrial applications, Appl. Microbiol. Biotechnol. 67 (2005) 577e591. [3] J. Robert, B.A.L. Whitehurst, in: B.A.L.a.A.J. Taylor (Ed.), Enzymes in Food Technology, CRC Press LLC, 2002. [4] A. Wolfgang, Industrial enzymes, in: A. Wolfgang (Ed.), Enzymes in Industry. Production and Applications, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim: Leiden, 2007. [5] J.G.L.F. Alves, L.D.A. Chumpitaz, L.H.M. da Silva, T.T. Franco, A.J.A. Meirelles, Partitioning of whey proteins, bovine serum albumin and porcine insulin in aqueous two-phase systems, J. Chromatogr. B Biomed. Sci. Appl. 743 (2000) 235e239. [6] S. Yang, Z. Huang, Z. Jiang, L. Li, Partition and purification of a thermostable xylanase produced by Paecilomyces thermophila in solid-state fermentation using aqueous two-phase systems, Process Biochem. 43 (2008) 56e61. [7] Z. Gu, C.E. Glatz, Aqueous two-phase extraction for protein recovery from corn extracts, J. Chromatogr. B 845 (2007) 38e50. [8] E. Stratikos, P.G.W. Gettins, Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 Å and full insertion of the reactive center loop into b-sheet A, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 4808e4813. [9] B.Y. Zaslavsky, in: Marcel Dekker, Inc (Ed.), Aqueous Two-phase Partitioning, Physical Chemistry and Bioanalytical Applications, 1995. New York. [10] D.B. Loureiro, D. Romanini, G. Tubio, Structural and functional analysis of Aspergillus niger xylanase to be employed in polyethylene glycol/salt aqueous two-phase extraction, Biocatal. Agric. Biotechnol. 5 (2016) 204e210. rez, D.B. Loureiro, B.B. Nerli, G. Tubio, Optimization of pancreatic trypsin [11] R.L. Pe extraction in PEG/citrate aqueous two-phase systems, Protein Expr. Purif. 106 (2015) 66e71. [12] A. Glyk, T Fau Scheper, S. Beutel, PEG-salt aqueous two-phase systems: an attractive and versatile liquid-liquid extraction technology for the downstream processing of proteins and enzymes, Appl. Microbiol. Biotechnol. (2015) (1432e0614 (Electronic)). [13] R. Hatti-Kaul, Aqueous two-phase systems - a general overview, Mol. Biotechnol. 19 (2001) 269e277. [14] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, vol. 1983, Plenum Press, 1983, p. 233. Spring Str. New York, NY 10013. [15] J. Lamba, P.S. Hasija, V. Aggarwal, T.K. Chaudhuri, Monitoring protein folding and unfolding pathways through surface hydrophobicity changes using fluorescence and circular dichroism spectroscopy, Biochem. Mosc. 74 (2009) 393e398. [16] S.M. Kelly, T.J. Jess, N.C. Price, How to study proteins by circular dichroism, Biochim. Biophys. Acta (BBA) - Proteins Proteom. 1751 (2005) 119e139. [17] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. 31 (1959) 426e428. [18] M.J. Bailey, P. Biely, K. Poutanen, Interlaboratory testing of methods for assay of xylanase activity, J. Biotechnol. 23 (1992) 257e270. [19] C.A. Haskard, E.C.Y. Li-Chan, Hydrophobicity of bovine serum albumin and ovalbumin determined using uncharged (PRODAN) and anionic (ANS-) fluorescent probes, J. Agric. Food Chem. 46 (1998) 2671e2677. , B. Nerli, Liquid-liquid equilibria of aqueous two[20] G. Tubio, L. Pellegrini, G. Pico phase systems containing poly(ethylene glycols) of different molecular weight and sodium citrate, J. Chem. Eng. Data 51 (2006) 209e212. [21] K. Gruber, G. Klintschar, M. Hayn, A. Schlacher, W. Steiner, C. Kratky, Thermophilic xylanase from Thermomyces lanuginosus: high-resolution x-ray
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