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Partitioning of Geotrichum candidum Lipase from fermentative crude extract by aqueous two-phase system of polyethylene glycol and sodium citrate
Accepted Manuscript Partitioning of Geotrichum candidum Lipase from fermentative crude extract by Aqueous two-phase system of polyethylene glycol and sodium citrate Cristina Junqueira Mazzeu, Elisa Zaparoli Ramos, Marcello Henrique da Silva Cavalcanti, Daniela Battaglia Hirata, Luciano Sindra Virtuoso PII: DOI: Reference:
S1383-5866(15)30244-6 http://dx.doi.org/10.1016/j.seppur.2015.09.069 SEPPUR 12597
To appear in:
Separation and Purification Technology
Received Date: Accepted Date:
8 September 2015 25 September 2015
Please cite this article as: C.J. Mazzeu, E.Z. Ramos, M.H.d. Cavalcanti, D.B. Hirata, L.S. Virtuoso, Partitioning of Geotrichum candidum Lipase from fermentative crude extract by Aqueous two-phase system of polyethylene glycol and sodium citrate, Separation and Purification Technology (2015), doi: http://dx.doi.org/10.1016/j.seppur. 2015.09.069
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Partitioning of Geotrichum candidum Lipase from fermentative crude extract by Aqueous two-phase system of polyethylene glycol and sodium citrate
Cristina Junqueira Mazzeu, Elisa Zaparoli Ramos, Marcello Henrique da Silva Cavalcanti, Daniela Battaglia Hirata, Luciano Sindra Virtuoso*
Colloid Chemistry Group, Chemistry Institute, Universidade Federal de Alfenas (UNIFAL-MG), Rua Gabriel Monteiro da Silva, 700, Zip Code, 37130-000, Alfenas-MG, Brazil
*To whom correspondence should be addressed. Tel: +55-35-3299-1260. Fax: +55-35 3299 1384. E−mail: [email protected]
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Abstract The partitioning behaviour of the crude extracellular lipase of Geotrichum candidum (GCL) were studied in aqueous two-phase systems (ATPS) formed by mixtures of poly(ethylene oxide) (PEO) (1500 or 4000 or 6000 g mol-1) or triblock copolymer [(EO)11(PO)16(EO)11] (L35) (1900 g mol-1), where EO and PO are ethylene oxide and propylene oxide, respectively, + sodium citrate + water. The partition ratio for the proteins present in the crude broth (which is predominantly lipase) in the different ATPS were investigated as a function of the pH, PEO molar mass, phases hydrophobicity, tie-line length and temperature. The proteins were preferentially partitioned into the salt-rich phase in all of the systems studied with partition ratios ranging between 0.025 and 0.7. The thermodynamic parameters of transfers of the proteins between phases were obtained and shows that the transfer of proteins to the bottom phase is entropic driven, however negative values obtained for the enthalpy of the transfer of proteins the bottom to the top phase (∆ = -12.12 to -7.10 kJ mol-1 in the temperature range of 5.0 to 45.0 oC, respectively) constitutes evidence of the strong interactions between the proteins presents in the crude broth and the PEO chains in the top phase of the ATPS. The ATPS formed for a mixture of PEO 1500 g mol-1 + sodium citrate + water at pH 7.0 was optimised using the responsesurface methodology. The optimized conditions for the partition of proteins of crude broth were: 19.0 % w/w + 12.0 % w/w and 17.6 % w/w + 14.0 % w/w of PEO 1500 g mol-1 and sodium citrate, respectively at pH 7.0. In these conditions were obtained the highest enzymatic concentrations in the bottom phase, with Kp < 0.15.
Keywords: Geotrichum candidum lipase; aqueous two-phase systems; partition ratio; thermodynamic parameters.
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1. Introduction
Aqueous two-phase systems (ATPS) can be formed by a great variety of combinations of chemical substances. However, the combinations of polyethylene glycol (PEO)/salt (e.g., citrate, phosphate and sulphate) and PEO/dextran are the most commonly utilised systems for liquidliquid extraction [1-3]. These systems have several advantages, such as being highly selective, nontoxic and easily scalable and having a low interfacial tension and reagents that can be recycled. Additionally, they are able to maintain the biological activity of molecules, e.g., proteins and enzymes [4,5]. The distribution of the biomolecules between the two phases are influenced by a variety of factors, such as the concentration of the phase-forming polymer and salt, the hydrophobicity, the type and molar mass of the polymer, the salt nature, the pH and the temperature of the system [6,7]. The use of PEO/salt systems for extraction and purification of lipases is well documented in the literature [2-8]. Lipases are enzymes (triacylglycerol ester hydrolases, EC 3.1.1.3) that catalyse the hydrolysis of oils and fats to free fatty acids, acylglycerols and glycerol. In organic media, they are able to catalyse esterification and transesterification reactions [9,10]. These enzymes can be applied in several industrial systems, such as flavours and fragrances, emulsifiers, cosmetics, agrochemicals and detergent, as well as to the synthesis of biopolymers and biodiesel and the production of pharmaceuticals [10-12]. Geotrichum candidum lipase has been recognised to have a specificity for unsaturated fatty acids with a double bond(s) at the cis-9 position, such as oleic and linoleic acids [13,14]. For this reason, this lipase is being investigated for industrial applications (e.g., the production of specialty chemicals from fats and oils) and the preparation of enantiomerically pure compounds [15].
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While there previously published works about using ATPS in the study of extraction/purification processes of lipases is important to note that these lipases are different in size, shape, and other characteristics and are obtained by fermentation processes involving other microorganisms [16-18]. In this sense, so far, there has been no report in the literature on the use of ATPS for the purification of the extract fermentation of extracellular lipase of Geotrichum candidum. For this reason, the objective of this work was to study and predict the lipase partition by PEO / citrate system.
2. Materials and methods
2.1 Materials The reagents used in the production of the lipase phase were: Olive oil (0.4% acidity) from Carbonell (Córdoba, Spain) was purchased at local market. Refined cottonseed oil was purchased from Campestre Indústria e Comércio de Óleos Vegetais (São Bernardo do Campo, SP, Brazil). Gum Arabic and anhydrous ethanol (purity > 99.5% m/m) were purchased from Synth® (São Paulo, Brazil). All other chemical reagents and solvents were supplied by Synth® and Vetec Química Ltd. (São Paulo, SP, Brazil).The reagents used in the purification step were poly (ethylene glycol) with average molar mass of 1500, 4000 and 6000 g mol-1 (Aldrich, USA); the triblock copolymer L35, (EO)11(PO)16(EO)11 (Aldrich, USA) with an average molar mass 1900 g mol-1; sodium citrate (Synth, Brazil); Coomassie Brilliant Blue G250 (Sinopharm Chemical Reagent, China); and phosphoric acid (Vetec, Brazil). Water purified by a Milli-Q II (Millipore, USA) was used to prepare all of the aqueous solutions.
2.2 Microorganisms
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Geotrichum candidum strain NRRL Y-552 was kindly acquired from Fundação André Tosello (Campinas, SP, Brazil). The stock cultures were maintained at 4.0 °C in a medium of 2% w/v malt extract, 0.5% w/v peptone and 1.5% w/v agar (MEA agar plates).
2.3 Inoculum The G. candidum strains were initially grown on Sabourand dextrose agar (SDA) plates and incubated at 30.0 °C for 48 h. A colony of 5 mm in diameter was transferred into a Erlenmeyer flask (1000 mL) containing 100 mL of a medium consisting of peptone (2 % w/v), yeast extract (0.1 % w/v), olive oil (1 % w/v), NaNO3 (0.05 % w/v) and MgSO4 (0.05 % w/v) at pH 7.0. It was incubated at 30.0 °C in a rotary shaker (250 rpm) (Solab, SL 221, Brazil) for 24 h.
2.4 Lipase production Lipase production was performed by incubating 10% v/v inoculum in 90 mL of culture medium containing 3% w/v peptone, 0.05% w/v yeast extract, 0.05% w/v NaNO3, 1.5% w/v cottonseed oil and 0.05% w/v MgSO4. The system was kept under agitation at 28.0 °C in a rotary shaker (250 rpm) (Solab, SL 221, Brazil) pH 7.0 for 72 h. The experiments were conducted twice, and samples were collected at intervals of 12h for analysis of the hydrolytic activity of the lipase, cell concentration and pH.
2.5 Polyacrylamide gel electrophoresis SDS-PAGE was performed according to Laemmli [19], using 5% and 12% polyacrylamide for the stacking and resolving gels, respectively. Broad range molar mass standards (Protein Ladder 10-220 kDa from Life Technologies®) were used for the mass determinations. The gels were stained with Coomassie Brilliant Blue R-250.
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2.6 Enzyme assay
The hydrolytic activity of the free enzyme was determined by the hydrolysis of olive oil, as modified by the Soares et al. methodology [20]. First, 5 mL of an emulsion (5% gum arabic and 5% olive oil) in 100 mM phosphate buffer pH 7.0 was placed into a 125 mL conical flask. Next, 1 mL of the fermented broth containing the enzyme was added to this mixture, and it was incubated for 10 min at 37.0 °C. The reaction was stopped by the addition of 5 mL of acetone/ethanol 1:1 (v/v), and the liberated fatty acids were titrated with a 0.02 M NaOH solution to the transition point (indicated by a slight pink tinge), using phenolphthalein as the indicator. One unit of lipase activity was defined as the amount of enzyme that liberates 1 µmol of fatty acid per minute under the conditions described.
2.7 Partitioning experiments
The ATPS formed by PEO (1500 or 4000 or 6000 g mol-1) + sodium citrate + water or L35 (1900 g mol-1) + sodium citrate + water were prepared in five different compositions in accordance with the phase diagrams described in the literature [21-23]. After mixing the stock solutions, the systems were vigorously agitated and then left to rest for 24 h in a Vortex (Phoenix, AP 56, Brazil) at a controlled temperature of 25.0 °C in a thermostatic bath (SOLABBR, with an uncertainty of 0.1 °C). Aliquots of the top and bottom phase were collected with the aid of syringes and a total of 3.0 g of each phase was mixed with 100 µL of fermented broth into three tubes. These tubes were agitated for approximately 2 min, centrifuged (Centribio, Brazil) for 15 min and then transferred to the thermostatic bath at 25.0 °C (or another controlled temperature) and incubated for approximately 24 h to obtain thermodynamic equilibrium. Each resulting phase was carefully collected with a syringe, leaving a layer of 5 mm
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thickness above and below the interface. To study the effect of pH on the proteins partition, acidic or basic systems were obtained using the inventory of polymer and salt solutions at pH values adjusted by the addition of small aliquots of concentrated NaOH or HCl solution, respectively. The studies of the partition of biomolecules were performed in triplicate, and the quantification of the biomolecules in both phases was performed by measuring the absorbance at 595 nm according to the method of Bradford with a UV-VIS spectrometer (Shimadzu UV-2401 PC, USA). Then, the partition ratio (Kp) in the top phase, divided by the corresponding value in the bottom phase, is described by Eq. (1): ୮
ୟ ౩ౢ౫౪
ሾୗ୭୪୳୲୭ሿ
ୟా ౩ౢ౫౪
ሾୗ୭୪୳୲୭ሿా
.
(1)
where and ሾܗܜܝܔܗ܁ሿ ܂are, respectively, the activity and the concentration of the analytic in the different phases (T= top or B=bottom).The protein partition ratio (Kp) was determined, and the relative standard deviation was, in general, less than 8%.
2.8 Thermodynamic functions The partition ratios were determined at five different temperatures, and the dependency of lnKp on 1/T was approximated by a polynomial expression, as represented by Eq. 2 [23],
.
(2)
Then, by applying the nonlinear Van’t Hoff equation (Eq. 3), the standard molar enthalpy of was calculated: transfer of proteins between phases ∆
∆ 2 .
(3)
The standard molar Gibbs free energy of transfer ∆ and the standard molar entropy of transfer ∆ associated with the proteins partition of the crude broth were
determined according to Eqs. (4) and (5): RTlnK
∆
(4)
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∆
∆ ∆ೝ ೝ
.
(5)
In Eqs. (2), (3), (4) and (5), Kp is the protein partition ratio, T is the absolute temperature, R is the ideal gas constant and a, b, c, d, … are the parameters of the adjustment from Eq. (3).
2.9 Factorial design for the partitioning process A specific ATPS system was optimised using the response-surface methodology. An 11 full factorial design with three replicates at the points centred was employed to evaluate the components utilised in the aqueous two-phase systems. Star points were added to the experimental design when necessary to compose second-order models. Eleven experiments were performed in a random order. The levels of each independent variable were selected based on the importance of the experiments. The parameters and their levels were the PEO concentration (17.6-27.4 % w/w) and the citrate sodium concentration (11.2-16.8 % w/w). The protein partition ratio was taken as the response of the design experiment. The results from the experimental design were analysed using the software Statistica version 5.0 (StatSoft Inc., USA).
3. Results and Discussion
3.1 Lipase production The highest enzymatic activity was observed at 60 h (22.4 U/mL) in this case, the concentration of protein (0.124 g/mL) was relatively low, and the pH was maintained at 5.5 during the 72 h of fermentation. The fermented broth was filtered, and the microorganisms were harvested. The supernatants were used as the enzyme extract because Geotrichum candidum produces extracellular lipase.
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The SDS–PAGE analysis is shown in Figure 1. The lipase of Geotrichum candidum (GCL) was confirmed for only a single band homogeneous (lane 2). In general, the molar mass of microbial lipases are variable, ranging from 12 to 76 kDa [25]. The fermented broth contains multiple impurities present in the culture medium but revealed the presence just of a single band of protein. Jacobsen et al. [26] proved that G. candidum produced a very few proteins besides the lipases when were used a synthetic medium for fermentation. Charton & Macrae [27] reported the same fact for produced lipases from G. candidum CMICC 335426 using a synthetic medium. For this reason it is possible to affirm that the protein showed in electrophoresis was the produced lipase, justifying in present work, the studies of the partition of biomolecules based in the quantification of the proteins. Insert Figure 1. 3.2 Effect of the molar mass Insert Figure 2. To study the effect of the PEO molar mass on the partitioning of proteins of the crude broth experiments were performed in various ATPS conditions by varying the molar mass of PEO (1500 or 4000 or 6000 g mol-1) and sodium citrate in five different tie lines. After mixing the components at a specific global composition, the systems spontaneously separated into two isotropic transparent phases, with the top and bottom phase being enriched in macromolecules and salt, respectively. Figure 2 shows that the partitioning of proteins in the PEO/citrate system was substantially dependent on the molar mass of the PEO. The Kp increase from 0.15 to 0.30 with the the increase of PEO molar mass. In similar studies of the porcine pancreatic lipase (PPL) partition in ATPS formed by PEO/potassium phosphate at various pH values, Bassani et al. [28] observed that the enzyme was preferentially partitioned into the polymer-rich phase in systems with a PEO molar mass of 4000-8000, whereas for PEO with 10000 molar mass, it
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was concentrated in the phosphate-rich phase. The increase in lnK with the tie-line length (TLL) was related to a polymer–protein interaction due to the hydrophobic character of the PPL. In the present study, with the decrease of the molar mass of the polymer, there was a reduction in the lipase partition ratio, which indicates an increase in the enzyme concentration in the bottom phase of ATPS used in this study. This effect observed was contrary to the exclusionary effect observed in lipases partition produced by other microorganisms. The TLL is commonly used as a variable that determines the processes of solute partition. It is an thermodynamic parameter that expresses the difference between the intensive thermodynamic properties of the two phases of the ATPS. The difference between the intensive thermodynamic properties is enhanced with the increase in the value of TLL, that is calculated according to Eq. (6): !! "# # # # ,
(6)
where $ polymer concentration in the top phase, $ polymer concentration in the bottom phase, $ salt concentration in the top phase and $ salt concentration in the bottom phase in % w/w. Generally, the uneven solute distribution will increase with increasing TLL values. In these studies of the molar mass effect, the proteins partition ratios were determined for five different TLLs for all of the ATPSs. Values of partition ratios significantly lower than 1 of proteins, for example 0.2, in the studied systems indicate the high potential of two-phase
extraction with ATPS in the purification of Geotrichum candidum Lipase (GCL).
3.3 Hydrophobic effect Insert Figure 3.
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Figure 3 shows the Kp of proteins of the crude broth versus TLL in systems formed by PEO 1500 g mol-1 + sodium citrate + water and L35 1900 g mol-1 + sodium citrate + water. As seen, the partition ratio decreases when the hydrophobicity of the top phase increases. L35 is a difunctional triblock copolymer surfactant terminating in primary hydroxyl groups that forms thermodynamically stable micelles with increasing copolymer concentration and/or solution temperature. The difference in the hydrophobicity between the copolymerrich phase and the PEO-rich phase in these ATPSs is due to the existence of propylene oxide segments in the L35 macromolecules, which is composed of 50% by weight of a poly(propylene glycol) (PPG) block and the remaining 50% consisting of two PEO blocks. The PPG segments exhibit weak molecular interactions with water molecules [29]. Studies performed by Alexandridis and Alan Hatton [30] showed that the micellisation process of PEO-PPO-PEO copolymers in water is endothermic and driven by a decrease in the polarity of the ethylene oxide (EO) and propylene oxide (PO) segments as the temperature increases and by the entropy gain in water when monomers aggregate to form micelles. The micelles have hydrodynamic radii of approximately 10 nm and aggregation numbers on the order of 50. The aggregation number is thought to be independent of the copolymer concentration and to increase with temperature. The hydrophobic nature of the PO group induces aggregation of the macromolecules into nanostructures with cores consisting of only the PPG block and a corona region consisting of water and PEO blocks. The decrease in the values of proteins partition ratios in the systems formed by the L35 copolymer indicates that this enzyme has lower tendency to solubilise in the hydrophobic core of the micelles, showing a volume-exclusion effect slightly more pronounced than that observed for systems with PEO 1500, especially with increasing TLL.
3.4 pH effect
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To study the influence of pH on proteins of the crude broth partition experiments were performed in various ATPS with PEO of different molar mass (1500, 4000 or 6000 g mol-1), sodium citrate and water, experiments were performed in the pH range of 3.0 to 9.5, and the results are presented in Figure 4. The results show that the partition ratio does not show a strong dependence on the PEO molar mass at various pH values, except at pH 4 for the ATPS formed by PEO 4000 and 1500 and at pH = 6.5 for the ATPS formed by PEO 6000. In the system with PEO 4000, a large increase in the value of Kp was observed at approximately 0.2 to 0.6. At this pH value, the Geotrichum candidum Lipase (GCL) is near its isoelectric point (PIGCL = 4.3) and therefore is slightly positively charged. This result was due to a marked increase in the concentration of proteins in the system interface; that is, there was not migration of the proteins to the upper layer. Analyses performed on the interface of the systems studied at various pH values showed that the relative proportion of the enzyme in the interface region at pH 4.0 was approximately 15%, whereas it was below 2% at the other points. For systems consisting of PEO 1500 and 6000, the values of Kp changed from 0.1 to 0.2 at pH values of 4 and 6.5, respectively. Insert Figure 4.
3.5 Effect of temperature on the partition Insert Figure 5. The effect of temperature on proteins of the crude broth partition has been studied in systems formed by PEO 4.000/sodium citrate in the range of 5.0 to 45.0 °C. The partition ratio decreases with increasing system temperature and increases with increasing TLL, as shown in Fig. 5a. It has been reported that as the temperature increases, the PEO structure becomes more extended and, as a result, its preferential interaction with the protein decreases by decreasing the Kp [31]. With increasing temperature, the effect of prolonging the PEO backbone thereby 12
increasing the volume-exclusion effect, leading to a decrease in Kp with increasing temperature, and lower Kp values were observed at 45.0 °C. In Fig. 5b, a nonlinear relationship between ∆ and TLL is observed. Thermodynamically, the ∆ energy could be split into two contributions, the enthalpic ∆ and entropic ∆ contributions. The analysis of the
proteins of the crude broth partitioning at various temperatures also makes it possible to indirectly determine the heat when the enzyme is partitioned between the two phases. To investigate the contribution of the enthalpic molecular interaction to the proteins transference, we calculated the ∆ ! %" using the nonlinear form of the classic Van’t Hoff approximation, Eqs. (2) and (3) truncated in the third term. Additionally, the Gibbs energy (∆ ! &" and the entropic change ∆ ! '" were calculated by applying Eqs. (4) and (5). To this proposal, the values of proteins partition ratios in the five different temperatures, with TLL of approximately 49.69 ± 1.45% were used for calculated the ∆ ! %" . The values of the nonlinear fit of the Van't Hoff equation are shown in Table 1. The values of ∆ ! %" and of ∆ ! '" obtained in this study are consistent with those found by Bassani et al [28]. The thermodynamic parameters of transfers of the proteins between phases shows that the transfer of proteins to the bottom phase is entropic driven, however negative values obtained for the enthalpy of the transfer of proteins the bottom to the top phase (∆ = -12.12 to -7.10 kJ mol-1 in the range of 5.0 to 45.0 oC, respectively) constitutes evidence of the strong interactions between the proteins presents in the crude broth and the PEO chains in the top phase of the ATPS. Insert Table 1. The natural logarithms of the observed values for Kp fit a polynomial quadratic plot vs. 1/T leading to the thermodynamics parameters of transfer listed in Table 1. As is evident in this table, the partitioning coefficient decreases as the temperature increases, indicating that the proteins transfer from the citrate-rich phase to the macromolecule-rich phase is an exothermic
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process. These data provide evidence of strong interactions between the proteins and the PEO in the top phase of the ATPS used in the studies on partition. However, the process of transferring the proteins for the salt-rich phase predominates, which constitutes evidence that the entropic term is the driving force for the process of the proteins of the crude broth partition.
3.6 Optimisation and validation of the partition ratio of the protein The most efficient ATPS system for proteins of the crude broth partition was the PEO 1500 g mol-1 + sodium citrate + water at pH 7.0. This biphasic system was chosen for optimisation of the process of proteins partition. The results shown in Table 1 indicated that the partition ratio of proteins varied from 0.15 (assays 1 and 5) to 0.24 (centre points). A good correspondence between the experimental and predicted values was observed, meaning that the experimental model obtained from the factorial design adequately explains most of the variations in the response [32]. Table 2 shows the regression coefficient for the model that predicts the partition ratio of proteins as a function of the PEO and sodium citrate concentrations. According to this table, all of the terms were statistically significant at the 95% confidence level. Analysis of variance (ANOVA) was used to evaluate the adequacy of the fitted model (Table 3). Based on ANOVA, a second-order model (Eq. 7) describing the partition ratio of proteins was established. The pure error, calculated from the central points, was very low, indicating good reproducibility of the experimental data. Based on the F-test, the model is predictive because the calculated F-test value (76.32) was higher than the listed F value (5.05). Additionally, the coefficient of determination (R2 = 98.7%) was very good. Therefore, the coded model expressed by Eq. 7 was used to obtain the response surfaces and the contour plots (Fig. 7). Insert Table 2 and 3. Insert Table 4.
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Kp = 0.233 + 0.008 x1 - 0.032 x12 + 0.006 x2 – 0.028 x22 – 0.016 x1·x2 (7) The proteins of the crude broth preferentially partitioned to the bottom phase, evidenced by the fact that the Kp values were found to be Kp < 1. By studying the variations in the concentrations of PEO and salt used in the system, it was possible to determine the optimal concentration range of the components of the systems to achieve lower Kp values or a higher concentration of lipase in the bottom phase. For the validation of the proposed model, the conditions of assay 1 were reproduced, which had obtained a lower partition ratio of proteins (19 % w/w of PEO 1500 g mol-1 and 12 % w/w of sodium citrate). The predicted value 0.1377 was obtained from equation 7, and the experimental value was 0.1489 ± 0.07. Therefore, the factorial design and response-surface methodology were appropriate to describe the partitioning of lipase in the aqueous two-phase system studied.
4. Conclusions The lipase of Geotrichum candidum is a hydrophilic enzyme with a molar mass of 60 kDa and
an isoelectric pH of 4.3 that is of interest in biotechnological processes for the production of biodiesel. In this work, the GCL partition ratio was investigated as a function of the system pH, the polymer molar mass, the hydrophobicity, the tie-line length and the temperature, with the goal of improving the selectivity of the ATPS and determining the optimal conditions for separation. The results showed a higher influence of the PEO molar mass, TLL and temperature of the system on the partition ratio of proteins of the crude broth. The data obtained provide evidence of strong interactions between the proteins and the PEO in the top phase of the ATPS. However, the low partition ratio found in the ATPS formed for PEO of various molar masses suggests that the proteins transfer process in this systems is entropically driven.
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Acknowledgements The authors wish to acknowledge financial support from the National Counsel of Technological and Scientific Development (CNPq) and the Foundation for Research Support of the Minas Gerais State (FAPEMIG).
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7. J. M. Jahim, F. A. A. Mutalib, N. Anuar, M. Markom, Optimisation of lipase extraction from industrial preparation using aqueous two phase PEO6000/phosphate system. Asian J. Exp. Sci, vol. 22, N 1 (2008) 33-42. 8. Y. Zhou, C. Hu, N. Wang, W. Zhang, X. Yu, Purification of porcine pancreatic lipase by aqueous two-phase systems of polyethylene glycol and potassium phosphate. Journal of Chromatography B, vol. 926 (2013) 77– 82. 9. A. A. Mendes, H. F. Castro, R. L. C. Giordano, Enzymatic synthesis of biodiesel from vegetable oils using microbial lipases immobilized on resin affinity by multipoint covalent attachment. Journal of Molecular Catalysis B: Enzymatic, vol. 68 (2011) 109-115. 10. F. Hasan, A. Ali Shah, A. Hameed, Methods for detection and characterization of lipases: A comprehensive review. Enzyme and Microbial Technology, vol. 39 (2006) 235-251. 11. K. C. Santos, D. M. J. Cassimiro, M. H. M. Avelar, D. B. Hirata, H. F. Castro, FernándezLafuente, R.; Mendes, A. A. Characterization of the catalytic properties of lipases from plant seeds for the production of concentrated fatty acids from different vegetable oils Industrial Crops and Products, vol. 49 (2013) 462– 470. 12. R. Gupta, N. Gupta, P. Rathi, Bacterial lipases: an overview of production, purification and biochemical properties Appl Microbiol Biotechnol, vol. 64 (2004) 763–781. 13. Y. Shimada, A. Sugihara, T. Nagao, Y. Tominaga, Induction of Geotrichum candidum Lipase by Long-Chain Fatty Acids. Journal of fermentation and bioengineering, vol. 74 N. 2 (1992) 77-80. 14. M. W. Baillargeon, R. G. Bistline Jr, P. E. Sonnet, Evaluation of strains of Geotrichum candidum for lipase production and fatty acid specificity. Appl Microbiol Biotechnol, vol. 30 (1989) 92-96.
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15. M. Zarevucka, Z. Kejik, D. Samana, Z. Wimmera, K. Demnerov, Enantioselective properties of induced lipases from Geotrichum. Enzyme and Microbial Technology, vol. 37 (2005) 481–486. 16. Anvari, Masumeh. Extraction of lipase from Rhizopus microsporus fermentation culture by aqueous two-phase partitioning. Biotechnology & Biotechnological Equipment ahead-ofprint (2015): 1-9. 17. Aradhana, Dayal, et al. Optimization of Rhizopus niveus Lipase Partitioning by an Aqueous Biphasic System. Chemical Engineering & Technology, vol. 37.7 (2014): 1191-1197. 18. Snellman, Erick A., Elise R. Sullivan, and Rita R. Colwell. Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG‐1. European Journal of Biochemistry vol. 269.23 (2002): 5771-5779. 19. U. K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, vol. 277 (1970) 680-685. 20. C. M. F. Soares, H. F. De Castro, F. F. Moraes, G. M. Zanin, Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica. Appl. Biochem. Biotechnol., vol. 77 (1999) 745. 21. R. M. Oliveira, J. S. D. R. Coimbra, L. A. Minim, L. H. M. da Silva, M. P. Ferreira Fontes, Liquid–liquid equilibria of biphasic systems composed of sodium citrate + polyethylene (glycol) 1500 or 4000 at different temperatures. J Chem. Eng. Data, vol. 53 (2008) 895-899. 22. J. P. Martins, A. B. Mageste, M. D. C. Hespanhol da Silva, L. H. M. da Silva, P. D. R. Patrício, J.S.D.R. Coimbra, L. A. Minim, Liquid-liquid equilibria of an aqueous two-phase system formed by a triblock copolymer and sodium salts at different temperatures. J ChemEng Data, vol. 54 (2009) 2891-2894. 23. C. P. Carvalho, J. S. R. Coimbra, I. A. F. Costa, L. A. Minim, M. C. Maffia, L. H. M. Silva, Influence of the temperature and type of salt on the phase equilibrium of PEO 1500+
18
potassium phosphate and PEO 1500+ sodium citrate aqueous two-phase systems. Quim.Nova, vol. 31 (2008) 209-213. 24. R. I. Boysen Y. Wang H. H. Keah M. T. W. Hearn, Observations on the origin of the nonlinear van’t Hoff behaviour of polypeptides in hydrophobic environments. Biophys Chem., vol. 77(2-3) (1999) 79–97. 25. M. Holmquist, Insights into the molecular basis for fatty acyl specificities of lipases from Geotrichum candidum and Candida rugosa. Chemistry and physics of lipids, vol. 93 (1998) 57-65. 26. T. Jacobsen, J. Olsen, K. Allermann, O. M. Poulsen, J. Hau, Production, partial purification, and immunochemical characterization of multiple forms of lipase from Geotrichum candidum. Enzyme Microb Technol 1989; 11: 90-95. 27. E. Charton, A.R. Macrae, Substrate specificities of lipases A and B from Geotrichum candidum CMICC 335426. Biochimica et Biophysica Acta 1992: V.1123, 59-64. 28. G. Bassani, B. Farruggia, B. Nerli, D. Romanini, G. Picó, Porcine pancreatic lipase partition in potassium phosphate–polyethylene glycol aqueous two-phase systems. Journal of Chromatography B, 859(2) (2007) 222-228. 29. M. Svensson, K. Berggren, A. Veide, F. Tjerneld, Aqueous two-phase systems containing self-associating
block
copolymers:
Partitioning
of
hydrophilic
and
hydrophobic
biomolecules. Journal of Chromatography A, vol. 839 (1999) 71. 30. P. Alexandridis, A. Hatton, T. Poly (ethylene oxide)-poly(propylene oxide )-poly (ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling. Colloids and Surfaces A: Physicochemical and Engineering Aspects, vol. 96 (1995) I 46.
19
31. H. D. Nucci, B. Nerli, G. Pico, Comparison between the thermodynamic features of a 1antitrypsin and human albumin partitioning in aqueous two-phase systems of polyethyleneglycol–dextran. Biophy Chem; vol. 89 (2001) 219–29. 32. Y. Q. Ling, H. L. Nie, S. N. Su, Branford-White, C., & Zhu, L. M. Optimization of affinity partitioning conditions of papain in aqueous two-phase system using response surface methodology. Separation and Purification Technology, 73(3) (2010) 343-348.
20
Table 1 – Average thermodynamic parameters of the protein transfer obtained by nonlinear
Van’t Hoff equation, 2.66747 3250.96515 668879.359 , in ATPS formed by PEO 4000 + sodium citrate + water at five different temperatures with TLL fixed in approximately 49.69±1.45%. TLL
50.26 49.68 50.93 48.77 48.82 * Equations
Temperature (K) 278.15 283.15 298.15 308.15 318.15
Kp 0.44589 0.31472 0.20364 0.14460 0.11377
(2-5) were used to obtain these results.
ΔtrGo (kJ mol-1) 1.87 2.72 3.94 4.95 5.75
ΔtrHo (kJ mol-1) -12.13 -11.42 -9.44 -8.23 -7.10
ΔtrSo (J mol-1) -50.31 -49.94 -44.91 -42.80 -40.38
Table 2 – Matrix of factorial design used to investigate the influence of the independent variables on the partition ratio.
Coded (actual) variables Assay 1 2 3 4 5 6 7 8 9 10 11
Partition ratio (Kp)
PEO (% m/m)
Salt (% m/m)
Experimental
Predicted
-1 (19) +1 (26) -1 (19) +1 (26) -1,41 (17.6) 1,41 (27.4) 0 (22.5) 0 (22.5) 0 (22.5) 0 (22.5) 0 (22.5)
-1 (12) -1 (12) +1 (16) +1 (16) 0 (14) 0 (14) -1,41 (11.2) +1,41(16.8) 0 (14) 0 (14) 0 (14)
0.15 ± 0.05 0.19 ±0.04 0.19 ±0.04 0.17 ± 0.03 0.15 ±0.03 0.18 ±0.04 0.17 ±0.04 0.18 ±0.04 0.24 ±0.02 0.23 ±0.03 0.24 ±0.02
0.14 0.20 0.19 0.17 0.16 0.18 0.17 0.19 0.23 0.23 0.23
21
Table 3 – Estimated coefficients, standard error, t-values and p-values for the partition ratio obtained in factorial design. Coefficients of regression
Standard errors
t-values
p-values
Media
0.233
0.0029
80.66
0.000000
x1
0.008
0.0018
4.47
0.006584
-0.032
0.0021
-14.96
0.000024
0.006
0.0018
3.13
0.025896
-0.028
0.0021
-13.30
0.000043
-0.016
0.0025
-6.306
0.001476
x1
2
x2 x2
2
x1.x2
Table 4 – Analysis of variance (ANOVA) for the model that represents the partition ratio obtained Source
Sum of squares
Degree of freedom
Mean squares
F-value
p-value
Regression
0.009541
5
0.001908
76.32
0.000102
Residues
0.000125
5
0.000025
Lack of fit
0.000085
Pure Error
0.000040
Total R2 = 98.70%; F5;5;0.05= 5.05
0.009666
10
22
Figures
Figure 1.SDS–PAGE analysis of lipase from Geotrichumcandidum NRRL Y-552. The molar mass of the standard protein marker ranged between 30 and 66 kDa. SDS–PAGE – lane MM: protein molecular markers; and fermented crude broth.
1
Figures
Figure 2.Effect of the PEOmolar mass on the partition ratio of protein (Kp) as a function of the tie-line length (TLL) values in PEO/sodium citrate at 25.0 °C and pH = 7 (○), PEO 1500 (▲), PEO 4000 and (■) PEO 6000.
0,40
0,35
0,30
Kp
0,25
0,20
0,15
0,10
0,05 25
30
35
40
45
50
55
60
65
TLL
1
Figures
Figure 3. Effect on the hydrophobic lipase partition ratio as a function of the tie line length (TLL) at 25.0 °C and pH 7.0: (□) PEO 1500 + sodium citrate + water (▲) and L35 1900 + sodium citrate + water. 0,30
0,25
Kp
0,20
0,15
0,10
0,05
0,00 25
30
35
40
45
50
55
60
65
TLL
1
Figures
Figure 4. pH effect on the lipase partition ratio in the systems PEO (Δ) 6000, (■) 4000 or (●) 1500 + sodium citrate + water at 25.0 °C.
0,7
0,6
0,5
Kp
0,4
0,3
0,2
0,1
0,0 2
3
4
5
6
7
8
9
10
pH
1
Figures
Figure 5.(a)Protein partition ration versus TLL and (b) transfer free energy ∆𝑡𝑟 𝐺𝑚𝑜 of protein versus TLL in ATPS formed by a mixture of PEO 4000 + sodium citrate + water at temperatures of ( ■ ) 5.0 °C, ( ○ ) 10.0°C, ( ▲ ) 25.0 °C, ( □ ) 35.0 °C and ( ● ) 45.0 °C and pH 7.0.
0,70 0,65 0,60 0,55 0,50 0,45
0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 32
34
36
38
40
42
44
46
48
50
52
54
56
58
60
CLA
(a) 9 8 7
trGm
6
o
Kp
0,40
5 4 3 2 1 35
40
45
50
TLL
(b)
55
60
62
Figures
Figure 6. Response surface (a) and contour curve (b) for Kp as a function of the percentage of PEO and the percentage of salt.
(a)
(b)
Highlights •
Partition of the lipase of the Geotrichum candidum has been studied in ATPS.
•
The lipase partition was investigated as a function of five variables.
•
The enzyme was preferentially partitioned into the salt rich phase in all systems.
•
The protein transfer process is entropic driven.
•
Was performed optimization of the ATPS using response surface methodology.
23
S1383-5866(15)30244-6 http://dx.doi.org/10.1016/j.seppur.2015.09.069 SEPPUR 12597
To appear in:
Separation and Purification Technology
Received Date: Accepted Date:
8 September 2015 25 September 2015
Please cite this article as: C.J. Mazzeu, E.Z. Ramos, M.H.d. Cavalcanti, D.B. Hirata, L.S. Virtuoso, Partitioning of Geotrichum candidum Lipase from fermentative crude extract by Aqueous two-phase system of polyethylene glycol and sodium citrate, Separation and Purification Technology (2015), doi: http://dx.doi.org/10.1016/j.seppur. 2015.09.069
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Partitioning of Geotrichum candidum Lipase from fermentative crude extract by Aqueous two-phase system of polyethylene glycol and sodium citrate
Cristina Junqueira Mazzeu, Elisa Zaparoli Ramos, Marcello Henrique da Silva Cavalcanti, Daniela Battaglia Hirata, Luciano Sindra Virtuoso*
Colloid Chemistry Group, Chemistry Institute, Universidade Federal de Alfenas (UNIFAL-MG), Rua Gabriel Monteiro da Silva, 700, Zip Code, 37130-000, Alfenas-MG, Brazil
*To whom correspondence should be addressed. Tel: +55-35-3299-1260. Fax: +55-35 3299 1384. E−mail: [email protected]
1
Abstract The partitioning behaviour of the crude extracellular lipase of Geotrichum candidum (GCL) were studied in aqueous two-phase systems (ATPS) formed by mixtures of poly(ethylene oxide) (PEO) (1500 or 4000 or 6000 g mol-1) or triblock copolymer [(EO)11(PO)16(EO)11] (L35) (1900 g mol-1), where EO and PO are ethylene oxide and propylene oxide, respectively, + sodium citrate + water. The partition ratio for the proteins present in the crude broth (which is predominantly lipase) in the different ATPS were investigated as a function of the pH, PEO molar mass, phases hydrophobicity, tie-line length and temperature. The proteins were preferentially partitioned into the salt-rich phase in all of the systems studied with partition ratios ranging between 0.025 and 0.7. The thermodynamic parameters of transfers of the proteins between phases were obtained and shows that the transfer of proteins to the bottom phase is entropic driven, however negative values obtained for the enthalpy of the transfer of proteins the bottom to the top phase (∆ = -12.12 to -7.10 kJ mol-1 in the temperature range of 5.0 to 45.0 oC, respectively) constitutes evidence of the strong interactions between the proteins presents in the crude broth and the PEO chains in the top phase of the ATPS. The ATPS formed for a mixture of PEO 1500 g mol-1 + sodium citrate + water at pH 7.0 was optimised using the responsesurface methodology. The optimized conditions for the partition of proteins of crude broth were: 19.0 % w/w + 12.0 % w/w and 17.6 % w/w + 14.0 % w/w of PEO 1500 g mol-1 and sodium citrate, respectively at pH 7.0. In these conditions were obtained the highest enzymatic concentrations in the bottom phase, with Kp < 0.15.
Keywords: Geotrichum candidum lipase; aqueous two-phase systems; partition ratio; thermodynamic parameters.
2
1. Introduction
Aqueous two-phase systems (ATPS) can be formed by a great variety of combinations of chemical substances. However, the combinations of polyethylene glycol (PEO)/salt (e.g., citrate, phosphate and sulphate) and PEO/dextran are the most commonly utilised systems for liquidliquid extraction [1-3]. These systems have several advantages, such as being highly selective, nontoxic and easily scalable and having a low interfacial tension and reagents that can be recycled. Additionally, they are able to maintain the biological activity of molecules, e.g., proteins and enzymes [4,5]. The distribution of the biomolecules between the two phases are influenced by a variety of factors, such as the concentration of the phase-forming polymer and salt, the hydrophobicity, the type and molar mass of the polymer, the salt nature, the pH and the temperature of the system [6,7]. The use of PEO/salt systems for extraction and purification of lipases is well documented in the literature [2-8]. Lipases are enzymes (triacylglycerol ester hydrolases, EC 3.1.1.3) that catalyse the hydrolysis of oils and fats to free fatty acids, acylglycerols and glycerol. In organic media, they are able to catalyse esterification and transesterification reactions [9,10]. These enzymes can be applied in several industrial systems, such as flavours and fragrances, emulsifiers, cosmetics, agrochemicals and detergent, as well as to the synthesis of biopolymers and biodiesel and the production of pharmaceuticals [10-12]. Geotrichum candidum lipase has been recognised to have a specificity for unsaturated fatty acids with a double bond(s) at the cis-9 position, such as oleic and linoleic acids [13,14]. For this reason, this lipase is being investigated for industrial applications (e.g., the production of specialty chemicals from fats and oils) and the preparation of enantiomerically pure compounds [15].
3
While there previously published works about using ATPS in the study of extraction/purification processes of lipases is important to note that these lipases are different in size, shape, and other characteristics and are obtained by fermentation processes involving other microorganisms [16-18]. In this sense, so far, there has been no report in the literature on the use of ATPS for the purification of the extract fermentation of extracellular lipase of Geotrichum candidum. For this reason, the objective of this work was to study and predict the lipase partition by PEO / citrate system.
2. Materials and methods
2.1 Materials The reagents used in the production of the lipase phase were: Olive oil (0.4% acidity) from Carbonell (Córdoba, Spain) was purchased at local market. Refined cottonseed oil was purchased from Campestre Indústria e Comércio de Óleos Vegetais (São Bernardo do Campo, SP, Brazil). Gum Arabic and anhydrous ethanol (purity > 99.5% m/m) were purchased from Synth® (São Paulo, Brazil). All other chemical reagents and solvents were supplied by Synth® and Vetec Química Ltd. (São Paulo, SP, Brazil).The reagents used in the purification step were poly (ethylene glycol) with average molar mass of 1500, 4000 and 6000 g mol-1 (Aldrich, USA); the triblock copolymer L35, (EO)11(PO)16(EO)11 (Aldrich, USA) with an average molar mass 1900 g mol-1; sodium citrate (Synth, Brazil); Coomassie Brilliant Blue G250 (Sinopharm Chemical Reagent, China); and phosphoric acid (Vetec, Brazil). Water purified by a Milli-Q II (Millipore, USA) was used to prepare all of the aqueous solutions.
2.2 Microorganisms
4
Geotrichum candidum strain NRRL Y-552 was kindly acquired from Fundação André Tosello (Campinas, SP, Brazil). The stock cultures were maintained at 4.0 °C in a medium of 2% w/v malt extract, 0.5% w/v peptone and 1.5% w/v agar (MEA agar plates).
2.3 Inoculum The G. candidum strains were initially grown on Sabourand dextrose agar (SDA) plates and incubated at 30.0 °C for 48 h. A colony of 5 mm in diameter was transferred into a Erlenmeyer flask (1000 mL) containing 100 mL of a medium consisting of peptone (2 % w/v), yeast extract (0.1 % w/v), olive oil (1 % w/v), NaNO3 (0.05 % w/v) and MgSO4 (0.05 % w/v) at pH 7.0. It was incubated at 30.0 °C in a rotary shaker (250 rpm) (Solab, SL 221, Brazil) for 24 h.
2.4 Lipase production Lipase production was performed by incubating 10% v/v inoculum in 90 mL of culture medium containing 3% w/v peptone, 0.05% w/v yeast extract, 0.05% w/v NaNO3, 1.5% w/v cottonseed oil and 0.05% w/v MgSO4. The system was kept under agitation at 28.0 °C in a rotary shaker (250 rpm) (Solab, SL 221, Brazil) pH 7.0 for 72 h. The experiments were conducted twice, and samples were collected at intervals of 12h for analysis of the hydrolytic activity of the lipase, cell concentration and pH.
2.5 Polyacrylamide gel electrophoresis SDS-PAGE was performed according to Laemmli [19], using 5% and 12% polyacrylamide for the stacking and resolving gels, respectively. Broad range molar mass standards (Protein Ladder 10-220 kDa from Life Technologies®) were used for the mass determinations. The gels were stained with Coomassie Brilliant Blue R-250.
5
2.6 Enzyme assay
The hydrolytic activity of the free enzyme was determined by the hydrolysis of olive oil, as modified by the Soares et al. methodology [20]. First, 5 mL of an emulsion (5% gum arabic and 5% olive oil) in 100 mM phosphate buffer pH 7.0 was placed into a 125 mL conical flask. Next, 1 mL of the fermented broth containing the enzyme was added to this mixture, and it was incubated for 10 min at 37.0 °C. The reaction was stopped by the addition of 5 mL of acetone/ethanol 1:1 (v/v), and the liberated fatty acids were titrated with a 0.02 M NaOH solution to the transition point (indicated by a slight pink tinge), using phenolphthalein as the indicator. One unit of lipase activity was defined as the amount of enzyme that liberates 1 µmol of fatty acid per minute under the conditions described.
2.7 Partitioning experiments
The ATPS formed by PEO (1500 or 4000 or 6000 g mol-1) + sodium citrate + water or L35 (1900 g mol-1) + sodium citrate + water were prepared in five different compositions in accordance with the phase diagrams described in the literature [21-23]. After mixing the stock solutions, the systems were vigorously agitated and then left to rest for 24 h in a Vortex (Phoenix, AP 56, Brazil) at a controlled temperature of 25.0 °C in a thermostatic bath (SOLABBR, with an uncertainty of 0.1 °C). Aliquots of the top and bottom phase were collected with the aid of syringes and a total of 3.0 g of each phase was mixed with 100 µL of fermented broth into three tubes. These tubes were agitated for approximately 2 min, centrifuged (Centribio, Brazil) for 15 min and then transferred to the thermostatic bath at 25.0 °C (or another controlled temperature) and incubated for approximately 24 h to obtain thermodynamic equilibrium. Each resulting phase was carefully collected with a syringe, leaving a layer of 5 mm
6
thickness above and below the interface. To study the effect of pH on the proteins partition, acidic or basic systems were obtained using the inventory of polymer and salt solutions at pH values adjusted by the addition of small aliquots of concentrated NaOH or HCl solution, respectively. The studies of the partition of biomolecules were performed in triplicate, and the quantification of the biomolecules in both phases was performed by measuring the absorbance at 595 nm according to the method of Bradford with a UV-VIS spectrometer (Shimadzu UV-2401 PC, USA). Then, the partition ratio (Kp) in the top phase, divided by the corresponding value in the bottom phase, is described by Eq. (1): ୮
ୟ ౩ౢ౫౪
ሾୗ୭୪୳୲୭ሿ
ୟా ౩ౢ౫౪
ሾୗ୭୪୳୲୭ሿా
.
(1)
where and ሾܗܜܝܔܗ܁ሿ ܂are, respectively, the activity and the concentration of the analytic in the different phases (T= top or B=bottom).The protein partition ratio (Kp) was determined, and the relative standard deviation was, in general, less than 8%.
2.8 Thermodynamic functions The partition ratios were determined at five different temperatures, and the dependency of lnKp on 1/T was approximated by a polynomial expression, as represented by Eq. 2 [23],
.
(2)
Then, by applying the nonlinear Van’t Hoff equation (Eq. 3), the standard molar enthalpy of was calculated: transfer of proteins between phases ∆
∆ 2 .
(3)
The standard molar Gibbs free energy of transfer ∆ and the standard molar entropy of transfer ∆ associated with the proteins partition of the crude broth were
determined according to Eqs. (4) and (5): RTlnK
∆
(4)
7
∆
∆ ∆ೝ ೝ
.
(5)
In Eqs. (2), (3), (4) and (5), Kp is the protein partition ratio, T is the absolute temperature, R is the ideal gas constant and a, b, c, d, … are the parameters of the adjustment from Eq. (3).
2.9 Factorial design for the partitioning process A specific ATPS system was optimised using the response-surface methodology. An 11 full factorial design with three replicates at the points centred was employed to evaluate the components utilised in the aqueous two-phase systems. Star points were added to the experimental design when necessary to compose second-order models. Eleven experiments were performed in a random order. The levels of each independent variable were selected based on the importance of the experiments. The parameters and their levels were the PEO concentration (17.6-27.4 % w/w) and the citrate sodium concentration (11.2-16.8 % w/w). The protein partition ratio was taken as the response of the design experiment. The results from the experimental design were analysed using the software Statistica version 5.0 (StatSoft Inc., USA).
3. Results and Discussion
3.1 Lipase production The highest enzymatic activity was observed at 60 h (22.4 U/mL) in this case, the concentration of protein (0.124 g/mL) was relatively low, and the pH was maintained at 5.5 during the 72 h of fermentation. The fermented broth was filtered, and the microorganisms were harvested. The supernatants were used as the enzyme extract because Geotrichum candidum produces extracellular lipase.
8
The SDS–PAGE analysis is shown in Figure 1. The lipase of Geotrichum candidum (GCL) was confirmed for only a single band homogeneous (lane 2). In general, the molar mass of microbial lipases are variable, ranging from 12 to 76 kDa [25]. The fermented broth contains multiple impurities present in the culture medium but revealed the presence just of a single band of protein. Jacobsen et al. [26] proved that G. candidum produced a very few proteins besides the lipases when were used a synthetic medium for fermentation. Charton & Macrae [27] reported the same fact for produced lipases from G. candidum CMICC 335426 using a synthetic medium. For this reason it is possible to affirm that the protein showed in electrophoresis was the produced lipase, justifying in present work, the studies of the partition of biomolecules based in the quantification of the proteins. Insert Figure 1. 3.2 Effect of the molar mass Insert Figure 2. To study the effect of the PEO molar mass on the partitioning of proteins of the crude broth experiments were performed in various ATPS conditions by varying the molar mass of PEO (1500 or 4000 or 6000 g mol-1) and sodium citrate in five different tie lines. After mixing the components at a specific global composition, the systems spontaneously separated into two isotropic transparent phases, with the top and bottom phase being enriched in macromolecules and salt, respectively. Figure 2 shows that the partitioning of proteins in the PEO/citrate system was substantially dependent on the molar mass of the PEO. The Kp increase from 0.15 to 0.30 with the the increase of PEO molar mass. In similar studies of the porcine pancreatic lipase (PPL) partition in ATPS formed by PEO/potassium phosphate at various pH values, Bassani et al. [28] observed that the enzyme was preferentially partitioned into the polymer-rich phase in systems with a PEO molar mass of 4000-8000, whereas for PEO with 10000 molar mass, it
9
was concentrated in the phosphate-rich phase. The increase in lnK with the tie-line length (TLL) was related to a polymer–protein interaction due to the hydrophobic character of the PPL. In the present study, with the decrease of the molar mass of the polymer, there was a reduction in the lipase partition ratio, which indicates an increase in the enzyme concentration in the bottom phase of ATPS used in this study. This effect observed was contrary to the exclusionary effect observed in lipases partition produced by other microorganisms. The TLL is commonly used as a variable that determines the processes of solute partition. It is an thermodynamic parameter that expresses the difference between the intensive thermodynamic properties of the two phases of the ATPS. The difference between the intensive thermodynamic properties is enhanced with the increase in the value of TLL, that is calculated according to Eq. (6): !! "# # # # ,
(6)
where $ polymer concentration in the top phase, $ polymer concentration in the bottom phase, $ salt concentration in the top phase and $ salt concentration in the bottom phase in % w/w. Generally, the uneven solute distribution will increase with increasing TLL values. In these studies of the molar mass effect, the proteins partition ratios were determined for five different TLLs for all of the ATPSs. Values of partition ratios significantly lower than 1 of proteins, for example 0.2, in the studied systems indicate the high potential of two-phase
extraction with ATPS in the purification of Geotrichum candidum Lipase (GCL).
3.3 Hydrophobic effect Insert Figure 3.
10
Figure 3 shows the Kp of proteins of the crude broth versus TLL in systems formed by PEO 1500 g mol-1 + sodium citrate + water and L35 1900 g mol-1 + sodium citrate + water. As seen, the partition ratio decreases when the hydrophobicity of the top phase increases. L35 is a difunctional triblock copolymer surfactant terminating in primary hydroxyl groups that forms thermodynamically stable micelles with increasing copolymer concentration and/or solution temperature. The difference in the hydrophobicity between the copolymerrich phase and the PEO-rich phase in these ATPSs is due to the existence of propylene oxide segments in the L35 macromolecules, which is composed of 50% by weight of a poly(propylene glycol) (PPG) block and the remaining 50% consisting of two PEO blocks. The PPG segments exhibit weak molecular interactions with water molecules [29]. Studies performed by Alexandridis and Alan Hatton [30] showed that the micellisation process of PEO-PPO-PEO copolymers in water is endothermic and driven by a decrease in the polarity of the ethylene oxide (EO) and propylene oxide (PO) segments as the temperature increases and by the entropy gain in water when monomers aggregate to form micelles. The micelles have hydrodynamic radii of approximately 10 nm and aggregation numbers on the order of 50. The aggregation number is thought to be independent of the copolymer concentration and to increase with temperature. The hydrophobic nature of the PO group induces aggregation of the macromolecules into nanostructures with cores consisting of only the PPG block and a corona region consisting of water and PEO blocks. The decrease in the values of proteins partition ratios in the systems formed by the L35 copolymer indicates that this enzyme has lower tendency to solubilise in the hydrophobic core of the micelles, showing a volume-exclusion effect slightly more pronounced than that observed for systems with PEO 1500, especially with increasing TLL.
3.4 pH effect
11
To study the influence of pH on proteins of the crude broth partition experiments were performed in various ATPS with PEO of different molar mass (1500, 4000 or 6000 g mol-1), sodium citrate and water, experiments were performed in the pH range of 3.0 to 9.5, and the results are presented in Figure 4. The results show that the partition ratio does not show a strong dependence on the PEO molar mass at various pH values, except at pH 4 for the ATPS formed by PEO 4000 and 1500 and at pH = 6.5 for the ATPS formed by PEO 6000. In the system with PEO 4000, a large increase in the value of Kp was observed at approximately 0.2 to 0.6. At this pH value, the Geotrichum candidum Lipase (GCL) is near its isoelectric point (PIGCL = 4.3) and therefore is slightly positively charged. This result was due to a marked increase in the concentration of proteins in the system interface; that is, there was not migration of the proteins to the upper layer. Analyses performed on the interface of the systems studied at various pH values showed that the relative proportion of the enzyme in the interface region at pH 4.0 was approximately 15%, whereas it was below 2% at the other points. For systems consisting of PEO 1500 and 6000, the values of Kp changed from 0.1 to 0.2 at pH values of 4 and 6.5, respectively. Insert Figure 4.
3.5 Effect of temperature on the partition Insert Figure 5. The effect of temperature on proteins of the crude broth partition has been studied in systems formed by PEO 4.000/sodium citrate in the range of 5.0 to 45.0 °C. The partition ratio decreases with increasing system temperature and increases with increasing TLL, as shown in Fig. 5a. It has been reported that as the temperature increases, the PEO structure becomes more extended and, as a result, its preferential interaction with the protein decreases by decreasing the Kp [31]. With increasing temperature, the effect of prolonging the PEO backbone thereby 12
increasing the volume-exclusion effect, leading to a decrease in Kp with increasing temperature, and lower Kp values were observed at 45.0 °C. In Fig. 5b, a nonlinear relationship between ∆ and TLL is observed. Thermodynamically, the ∆ energy could be split into two contributions, the enthalpic ∆ and entropic ∆ contributions. The analysis of the
proteins of the crude broth partitioning at various temperatures also makes it possible to indirectly determine the heat when the enzyme is partitioned between the two phases. To investigate the contribution of the enthalpic molecular interaction to the proteins transference, we calculated the ∆ ! %" using the nonlinear form of the classic Van’t Hoff approximation, Eqs. (2) and (3) truncated in the third term. Additionally, the Gibbs energy (∆ ! &" and the entropic change ∆ ! '" were calculated by applying Eqs. (4) and (5). To this proposal, the values of proteins partition ratios in the five different temperatures, with TLL of approximately 49.69 ± 1.45% were used for calculated the ∆ ! %" . The values of the nonlinear fit of the Van't Hoff equation are shown in Table 1. The values of ∆ ! %" and of ∆ ! '" obtained in this study are consistent with those found by Bassani et al [28]. The thermodynamic parameters of transfers of the proteins between phases shows that the transfer of proteins to the bottom phase is entropic driven, however negative values obtained for the enthalpy of the transfer of proteins the bottom to the top phase (∆ = -12.12 to -7.10 kJ mol-1 in the range of 5.0 to 45.0 oC, respectively) constitutes evidence of the strong interactions between the proteins presents in the crude broth and the PEO chains in the top phase of the ATPS. Insert Table 1. The natural logarithms of the observed values for Kp fit a polynomial quadratic plot vs. 1/T leading to the thermodynamics parameters of transfer listed in Table 1. As is evident in this table, the partitioning coefficient decreases as the temperature increases, indicating that the proteins transfer from the citrate-rich phase to the macromolecule-rich phase is an exothermic
13
process. These data provide evidence of strong interactions between the proteins and the PEO in the top phase of the ATPS used in the studies on partition. However, the process of transferring the proteins for the salt-rich phase predominates, which constitutes evidence that the entropic term is the driving force for the process of the proteins of the crude broth partition.
3.6 Optimisation and validation of the partition ratio of the protein The most efficient ATPS system for proteins of the crude broth partition was the PEO 1500 g mol-1 + sodium citrate + water at pH 7.0. This biphasic system was chosen for optimisation of the process of proteins partition. The results shown in Table 1 indicated that the partition ratio of proteins varied from 0.15 (assays 1 and 5) to 0.24 (centre points). A good correspondence between the experimental and predicted values was observed, meaning that the experimental model obtained from the factorial design adequately explains most of the variations in the response [32]. Table 2 shows the regression coefficient for the model that predicts the partition ratio of proteins as a function of the PEO and sodium citrate concentrations. According to this table, all of the terms were statistically significant at the 95% confidence level. Analysis of variance (ANOVA) was used to evaluate the adequacy of the fitted model (Table 3). Based on ANOVA, a second-order model (Eq. 7) describing the partition ratio of proteins was established. The pure error, calculated from the central points, was very low, indicating good reproducibility of the experimental data. Based on the F-test, the model is predictive because the calculated F-test value (76.32) was higher than the listed F value (5.05). Additionally, the coefficient of determination (R2 = 98.7%) was very good. Therefore, the coded model expressed by Eq. 7 was used to obtain the response surfaces and the contour plots (Fig. 7). Insert Table 2 and 3. Insert Table 4.
14
Kp = 0.233 + 0.008 x1 - 0.032 x12 + 0.006 x2 – 0.028 x22 – 0.016 x1·x2 (7) The proteins of the crude broth preferentially partitioned to the bottom phase, evidenced by the fact that the Kp values were found to be Kp < 1. By studying the variations in the concentrations of PEO and salt used in the system, it was possible to determine the optimal concentration range of the components of the systems to achieve lower Kp values or a higher concentration of lipase in the bottom phase. For the validation of the proposed model, the conditions of assay 1 were reproduced, which had obtained a lower partition ratio of proteins (19 % w/w of PEO 1500 g mol-1 and 12 % w/w of sodium citrate). The predicted value 0.1377 was obtained from equation 7, and the experimental value was 0.1489 ± 0.07. Therefore, the factorial design and response-surface methodology were appropriate to describe the partitioning of lipase in the aqueous two-phase system studied.
4. Conclusions The lipase of Geotrichum candidum is a hydrophilic enzyme with a molar mass of 60 kDa and
an isoelectric pH of 4.3 that is of interest in biotechnological processes for the production of biodiesel. In this work, the GCL partition ratio was investigated as a function of the system pH, the polymer molar mass, the hydrophobicity, the tie-line length and the temperature, with the goal of improving the selectivity of the ATPS and determining the optimal conditions for separation. The results showed a higher influence of the PEO molar mass, TLL and temperature of the system on the partition ratio of proteins of the crude broth. The data obtained provide evidence of strong interactions between the proteins and the PEO in the top phase of the ATPS. However, the low partition ratio found in the ATPS formed for PEO of various molar masses suggests that the proteins transfer process in this systems is entropically driven.
15
Acknowledgements The authors wish to acknowledge financial support from the National Counsel of Technological and Scientific Development (CNPq) and the Foundation for Research Support of the Minas Gerais State (FAPEMIG).
5. References
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7. J. M. Jahim, F. A. A. Mutalib, N. Anuar, M. Markom, Optimisation of lipase extraction from industrial preparation using aqueous two phase PEO6000/phosphate system. Asian J. Exp. Sci, vol. 22, N 1 (2008) 33-42. 8. Y. Zhou, C. Hu, N. Wang, W. Zhang, X. Yu, Purification of porcine pancreatic lipase by aqueous two-phase systems of polyethylene glycol and potassium phosphate. Journal of Chromatography B, vol. 926 (2013) 77– 82. 9. A. A. Mendes, H. F. Castro, R. L. C. Giordano, Enzymatic synthesis of biodiesel from vegetable oils using microbial lipases immobilized on resin affinity by multipoint covalent attachment. Journal of Molecular Catalysis B: Enzymatic, vol. 68 (2011) 109-115. 10. F. Hasan, A. Ali Shah, A. Hameed, Methods for detection and characterization of lipases: A comprehensive review. Enzyme and Microbial Technology, vol. 39 (2006) 235-251. 11. K. C. Santos, D. M. J. Cassimiro, M. H. M. Avelar, D. B. Hirata, H. F. Castro, FernándezLafuente, R.; Mendes, A. A. Characterization of the catalytic properties of lipases from plant seeds for the production of concentrated fatty acids from different vegetable oils Industrial Crops and Products, vol. 49 (2013) 462– 470. 12. R. Gupta, N. Gupta, P. Rathi, Bacterial lipases: an overview of production, purification and biochemical properties Appl Microbiol Biotechnol, vol. 64 (2004) 763–781. 13. Y. Shimada, A. Sugihara, T. Nagao, Y. Tominaga, Induction of Geotrichum candidum Lipase by Long-Chain Fatty Acids. Journal of fermentation and bioengineering, vol. 74 N. 2 (1992) 77-80. 14. M. W. Baillargeon, R. G. Bistline Jr, P. E. Sonnet, Evaluation of strains of Geotrichum candidum for lipase production and fatty acid specificity. Appl Microbiol Biotechnol, vol. 30 (1989) 92-96.
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15. M. Zarevucka, Z. Kejik, D. Samana, Z. Wimmera, K. Demnerov, Enantioselective properties of induced lipases from Geotrichum. Enzyme and Microbial Technology, vol. 37 (2005) 481–486. 16. Anvari, Masumeh. Extraction of lipase from Rhizopus microsporus fermentation culture by aqueous two-phase partitioning. Biotechnology & Biotechnological Equipment ahead-ofprint (2015): 1-9. 17. Aradhana, Dayal, et al. Optimization of Rhizopus niveus Lipase Partitioning by an Aqueous Biphasic System. Chemical Engineering & Technology, vol. 37.7 (2014): 1191-1197. 18. Snellman, Erick A., Elise R. Sullivan, and Rita R. Colwell. Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG‐1. European Journal of Biochemistry vol. 269.23 (2002): 5771-5779. 19. U. K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, vol. 277 (1970) 680-685. 20. C. M. F. Soares, H. F. De Castro, F. F. Moraes, G. M. Zanin, Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica. Appl. Biochem. Biotechnol., vol. 77 (1999) 745. 21. R. M. Oliveira, J. S. D. R. Coimbra, L. A. Minim, L. H. M. da Silva, M. P. Ferreira Fontes, Liquid–liquid equilibria of biphasic systems composed of sodium citrate + polyethylene (glycol) 1500 or 4000 at different temperatures. J Chem. Eng. Data, vol. 53 (2008) 895-899. 22. J. P. Martins, A. B. Mageste, M. D. C. Hespanhol da Silva, L. H. M. da Silva, P. D. R. Patrício, J.S.D.R. Coimbra, L. A. Minim, Liquid-liquid equilibria of an aqueous two-phase system formed by a triblock copolymer and sodium salts at different temperatures. J ChemEng Data, vol. 54 (2009) 2891-2894. 23. C. P. Carvalho, J. S. R. Coimbra, I. A. F. Costa, L. A. Minim, M. C. Maffia, L. H. M. Silva, Influence of the temperature and type of salt on the phase equilibrium of PEO 1500+
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potassium phosphate and PEO 1500+ sodium citrate aqueous two-phase systems. Quim.Nova, vol. 31 (2008) 209-213. 24. R. I. Boysen Y. Wang H. H. Keah M. T. W. Hearn, Observations on the origin of the nonlinear van’t Hoff behaviour of polypeptides in hydrophobic environments. Biophys Chem., vol. 77(2-3) (1999) 79–97. 25. M. Holmquist, Insights into the molecular basis for fatty acyl specificities of lipases from Geotrichum candidum and Candida rugosa. Chemistry and physics of lipids, vol. 93 (1998) 57-65. 26. T. Jacobsen, J. Olsen, K. Allermann, O. M. Poulsen, J. Hau, Production, partial purification, and immunochemical characterization of multiple forms of lipase from Geotrichum candidum. Enzyme Microb Technol 1989; 11: 90-95. 27. E. Charton, A.R. Macrae, Substrate specificities of lipases A and B from Geotrichum candidum CMICC 335426. Biochimica et Biophysica Acta 1992: V.1123, 59-64. 28. G. Bassani, B. Farruggia, B. Nerli, D. Romanini, G. Picó, Porcine pancreatic lipase partition in potassium phosphate–polyethylene glycol aqueous two-phase systems. Journal of Chromatography B, 859(2) (2007) 222-228. 29. M. Svensson, K. Berggren, A. Veide, F. Tjerneld, Aqueous two-phase systems containing self-associating
block
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biomolecules. Journal of Chromatography A, vol. 839 (1999) 71. 30. P. Alexandridis, A. Hatton, T. Poly (ethylene oxide)-poly(propylene oxide )-poly (ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling. Colloids and Surfaces A: Physicochemical and Engineering Aspects, vol. 96 (1995) I 46.
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31. H. D. Nucci, B. Nerli, G. Pico, Comparison between the thermodynamic features of a 1antitrypsin and human albumin partitioning in aqueous two-phase systems of polyethyleneglycol–dextran. Biophy Chem; vol. 89 (2001) 219–29. 32. Y. Q. Ling, H. L. Nie, S. N. Su, Branford-White, C., & Zhu, L. M. Optimization of affinity partitioning conditions of papain in aqueous two-phase system using response surface methodology. Separation and Purification Technology, 73(3) (2010) 343-348.
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Table 1 – Average thermodynamic parameters of the protein transfer obtained by nonlinear
Van’t Hoff equation, 2.66747 3250.96515 668879.359 , in ATPS formed by PEO 4000 + sodium citrate + water at five different temperatures with TLL fixed in approximately 49.69±1.45%. TLL
50.26 49.68 50.93 48.77 48.82 * Equations
Temperature (K) 278.15 283.15 298.15 308.15 318.15
Kp 0.44589 0.31472 0.20364 0.14460 0.11377
(2-5) were used to obtain these results.
ΔtrGo (kJ mol-1) 1.87 2.72 3.94 4.95 5.75
ΔtrHo (kJ mol-1) -12.13 -11.42 -9.44 -8.23 -7.10
ΔtrSo (J mol-1) -50.31 -49.94 -44.91 -42.80 -40.38
Table 2 – Matrix of factorial design used to investigate the influence of the independent variables on the partition ratio.
Coded (actual) variables Assay 1 2 3 4 5 6 7 8 9 10 11
Partition ratio (Kp)
PEO (% m/m)
Salt (% m/m)
Experimental
Predicted
-1 (19) +1 (26) -1 (19) +1 (26) -1,41 (17.6) 1,41 (27.4) 0 (22.5) 0 (22.5) 0 (22.5) 0 (22.5) 0 (22.5)
-1 (12) -1 (12) +1 (16) +1 (16) 0 (14) 0 (14) -1,41 (11.2) +1,41(16.8) 0 (14) 0 (14) 0 (14)
0.15 ± 0.05 0.19 ±0.04 0.19 ±0.04 0.17 ± 0.03 0.15 ±0.03 0.18 ±0.04 0.17 ±0.04 0.18 ±0.04 0.24 ±0.02 0.23 ±0.03 0.24 ±0.02
0.14 0.20 0.19 0.17 0.16 0.18 0.17 0.19 0.23 0.23 0.23
21
Table 3 – Estimated coefficients, standard error, t-values and p-values for the partition ratio obtained in factorial design. Coefficients of regression
Standard errors
t-values
p-values
Media
0.233
0.0029
80.66
0.000000
x1
0.008
0.0018
4.47
0.006584
-0.032
0.0021
-14.96
0.000024
0.006
0.0018
3.13
0.025896
-0.028
0.0021
-13.30
0.000043
-0.016
0.0025
-6.306
0.001476
x1
2
x2 x2
2
x1.x2
Table 4 – Analysis of variance (ANOVA) for the model that represents the partition ratio obtained Source
Sum of squares
Degree of freedom
Mean squares
F-value
p-value
Regression
0.009541
5
0.001908
76.32
0.000102
Residues
0.000125
5
0.000025
Lack of fit
0.000085
Pure Error
0.000040
Total R2 = 98.70%; F5;5;0.05= 5.05
0.009666
10
22
Figures
Figure 1.SDS–PAGE analysis of lipase from Geotrichumcandidum NRRL Y-552. The molar mass of the standard protein marker ranged between 30 and 66 kDa. SDS–PAGE – lane MM: protein molecular markers; and fermented crude broth.
1
Figures
Figure 2.Effect of the PEOmolar mass on the partition ratio of protein (Kp) as a function of the tie-line length (TLL) values in PEO/sodium citrate at 25.0 °C and pH = 7 (○), PEO 1500 (▲), PEO 4000 and (■) PEO 6000.
0,40
0,35
0,30
Kp
0,25
0,20
0,15
0,10
0,05 25
30
35
40
45
50
55
60
65
TLL
1
Figures
Figure 3. Effect on the hydrophobic lipase partition ratio as a function of the tie line length (TLL) at 25.0 °C and pH 7.0: (□) PEO 1500 + sodium citrate + water (▲) and L35 1900 + sodium citrate + water. 0,30
0,25
Kp
0,20
0,15
0,10
0,05
0,00 25
30
35
40
45
50
55
60
65
TLL
1
Figures
Figure 4. pH effect on the lipase partition ratio in the systems PEO (Δ) 6000, (■) 4000 or (●) 1500 + sodium citrate + water at 25.0 °C.
0,7
0,6
0,5
Kp
0,4
0,3
0,2
0,1
0,0 2
3
4
5
6
7
8
9
10
pH
1
Figures
Figure 5.(a)Protein partition ration versus TLL and (b) transfer free energy ∆𝑡𝑟 𝐺𝑚𝑜 of protein versus TLL in ATPS formed by a mixture of PEO 4000 + sodium citrate + water at temperatures of ( ■ ) 5.0 °C, ( ○ ) 10.0°C, ( ▲ ) 25.0 °C, ( □ ) 35.0 °C and ( ● ) 45.0 °C and pH 7.0.
0,70 0,65 0,60 0,55 0,50 0,45
0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 32
34
36
38
40
42
44
46
48
50
52
54
56
58
60
CLA
(a) 9 8 7
trGm
6
o
Kp
0,40
5 4 3 2 1 35
40
45
50
TLL
(b)
55
60
62
Figures
Figure 6. Response surface (a) and contour curve (b) for Kp as a function of the percentage of PEO and the percentage of salt.
(a)
(b)
Highlights •
Partition of the lipase of the Geotrichum candidum has been studied in ATPS.
•
The lipase partition was investigated as a function of five variables.
•
The enzyme was preferentially partitioned into the salt rich phase in all systems.
•
The protein transfer process is entropic driven.
•
Was performed optimization of the ATPS using response surface methodology.
23