Mixed Reverse Micelles Facilitated Downstream Processing of Lipase Involving Water-Oil-Water Liquid Emulsion Membrane Saibal Bhowal Dept. of Food Engineering, CSIR-Central Food Technological Research Inst., Mysore, Karnataka 570 020 India
B. S. Priyanka and Navin K. Rastogi Dept. of Food Engineering, CSIR-Central Food Technological Research Inst., Mysore, Karnataka 570 020 India Academy of Scientific and Innovative Research, CSIR-Central Food Technological Research Inst., Mysore, Karnataka 570 020 India DOI 10.1002/btpr.1941 Published online July 3, 2014 in Wiley Online Library (wileyonlinelibrary.com)
Our earlier work for the first time demonstrated that liquid emulsion membrane (LEM) containing reverse micelles could be successfully used for the downstream processing of lipase from Aspergillus niger. In the present work, we have attempted to increase the extraction and purification fold of lipase by using mixed reverse micelles (MRM) consisting of cationic and nonionic surfactants in LEM. It was basically prepared by addition of the internal aqueous phase solution to the organic phase followed by the redispersion of the emulsion in the feed phase containing enzyme, which resulted in globules of water-oil-water (WOW) emulsion for the extraction of lipase. The optimum conditions for maximum lipase recovery (100%) and purification fold (17.0-fold) were CTAB concentration 0.075 M, Tween 80 concentration 0.012 M, at stirring speed of 500 rpm, contact time 15 min, internal aqueous phase pH 7, feed pH 9, KCl concentration 1 M, NaCl concentration 0.1 M, and ratio of membrane emulsion to feed volume 1:1. Incorporation of the nonionic surfactant (e.g., Tween 80) resulted in remarkable improvement in the purification fold (3.1–17.0) of the lipase. LEM containing a mixture of nonionic and cationic surfactants can be successfully used for the enhancement in the activity recovery and purification fold during downstream C 2014 American Institute of Chemical Engineers Biotechprocessing of enzymes/proteins. V nol. Prog., 30:1084–1092, 2014 Keywords: liquid emulsion membrane, reverse micelles, lipase, downstream processing, cationic surfactant, nonionic surfactant
Introduction Lipases are triacylglycerol acylhydrolases (E.C. 3.1.1.3), which are ubiquitous enzymes of considerable physiological significance and industrial potential. These are the supplest biocatalysts, which bring about a wide range of bioconversion reactions. In eukaryotes, lipases are involved in various stages of lipid metabolism including fat digestion, absorption, reconstitution, and lipoprotein metabolism. In plants, lipases are found in energy reserve tissues. The prospect of lipase utilization encompasses many areas such as the oleochemical, detergent, leather, cosmetics, and perfume as well as organic industries including environmental management, biomedical, and biosensor applications.1 Lipase catalyzed transesterification, hydrolysis, and esterification are the important class of reactions for food technology applications in fats and oil, dairy, pharmaceuticals, and bakery industry.2,3 Most of the enzymes for commercial applications require a certain degree of purity depending upon the final application in industries. The purification strategies employed, Correspondence concerning this article should be addressed to N. K. Rastogi at [email protected] or [email protected]. 1084
should be rapid, inexpensive, high-yielding, and pliable to large-scale operations. They should have the potential for continuous product recovery, with a relatively high capacity and selectivity for the desired product. Various purification strategies such as membrane separation processes, aqueous two-phase systems, reverse micellar extraction, hydrophobic interaction chromatography, immuno-purification, etc. have been used for the separation of lipase.4,5 Liquid emulsion membrane (LEM) technology has been extensively examined for potential application in diverse field such as the removal of contaminants from waste water, recovery of heavy metal ions,6 applications in foodstuffs, cosmetics, pharmaceuticals, lubricants, laundry, and cleaning,7 and extraction of products of fermentation.8 The process of LEM may be considered to be an alternative separation technique and its application for the recovery of the fermented products has grown rapidly.9 It offers several advantages such as ease of operation, less energy requirement, large interfacial area, low cost factors, and a single stage operation combining extraction and stripping.10 Generally, the proteins are denatured on exposure to organic liquids, and hence the usual LEMs were not found to be appropriate for the extraction of protein/enzyme. C 2014 American Institute of Chemical Engineers V
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However, there are various reports for the extraction of protein using reverse micelles as carrier in bulk liquid membrane or LEM, respectively.11 The reverse micelles mediated transport in liquid membrane is an important research field for the future for the downstream processing of biomolecules.12 Bhavya et al.13 demonstrated for the first time that the LEM containing reverse micelles can be successfully used for the downstream processing of lipase. In this work, cationic surfactant reverse micelles were evenly dispersed in organic membrane phase. These micelles act as a carrier for the transport of lipase. The optimum activity recovery and purification fold were found to be 78.6% and 3.1-fold, respectively. Encouraged with the earlier work and with a view to enhance the extent of recovery and purification of lipase, the MRM consisting of cationic and nonionic surfactants were used to improve the recovery of lipase in LEM. The presence of reverse micelles in organic solvent provides a biocompatible environment for biomolecules.14 Reverse micellar extraction consisting of MRM was shown to be more efficient in case of reverse micellar extraction of b-glucosidase,15 lipase,16,17 as well as for other model system.18,19 The main objective of this work is to increase the extent of extraction of lipase by incorporating MRMs into LEM to facilitate the migration of lipase through the organic liquid membrane for the downstream processing of lipase. The effect of various operating parameters on the extraction process has also been discussed.
Theoretical Background The reverse micelles assisted transport of lipase through liquid membrane consisted of the following steps. At first lipase is solubilized into Cetyltrimethyl ammonium bromide (CTAB) reverse micelles and then these reverse micelles enriched with lipase are transported through the liquid membrane. Finally, lipase is released into internal aqueous phase. Electrostatic and hydrophobic interactions between proteins and micelles are mainly responsible for the extraction of lipase into reverse micelles. The surfactant head groups for cationic surfactant (CTAB) are positively charged, which induce attractive force on the negatively charged proteins. The negative charges on protein are achieved by adjusting pH values higher than the isoelectric point (pI) of the enzyme13 (Figure 1a). The extent of the extraction using reverse micelles assisted transport is limited due to the size of the micelles formed in the liquid membrane. The inclusion of nonionic surfactant along with cationic surfactant results in the increase in the size of the micelles by the incorporation of nonionic surfactant molecules between the cationic surfactant (Figure 1b). These MRM result in decrease in the surface charge density, which in turn lead to the increase in the diameter of the micelles (Figure 1c,d). The increase in diameter of the MRM can be inferred from the water content of reverse micelles.20 Hemavathi et al.15 and Moilanen et al.20 have demonstrated that MRM consisting of both ionic and nonionic surfactant resulted in an increased size of the reverse micelles as compared to the micelles containing only ionic surfactant.
Materials and Methods Materials CTAB was procured from Himedia Laboratories Pvt., Mumbai; isooctane from Sisco Research laboratories,
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Mumbai; Triton X-100, gum arabic, and acetonitrile were procured from Ranbaxy Chemicals, Mumbai. Sorbitonmonooleate (Span 80) from Loba Chemie Pvt., Mumbai; Liquid paraffin light, pNp (para-nitrophenol) and pNpA (para-nitrophenol acetate) from SD Fine Chemicals, New Delhi; isopropanol and Tween 80 (polyoxyethylene sorbitan monooleate), potassium dihydrogenphosphate, tris-buffer, sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium chloride, sodium chloride, and HCl from Merck Ltd. Mumbai. All the chemicals used were of AR grade. Preparation of crude enzyme extract Fermented rice bran containing lipase was obtained from M/s. Zeus Biotech Laboratory, Mysore after cultivation of Aspergillus niger by solid-state method. The enzyme was extracted from rice bran (5 g) using 50 ml of 0.2 M TrisHCl (pH 9). The mixture was stirred using magnetic stirrer at 200 rpm for 2 h at 25 6 2 C.21 The suspension was filtered through a muslin cloth and centrifuged at 5000 rpm for 15 min at 4 C (REMI Compufuge). The clear supernatant was used as a crude extract (feed). The isoelectric point of lipase was reported to be 4.2.13,22 Enzyme extraction using liquid emulsion membrane The extraction of lipase from aqueous solution (feed) using LEM involves four steps namely formation of LEM, extraction of lipase, separation of emulsion, and deemulsification. The formation of LEM (first step) involved suspension of surfactants [CTAB, 0.05–0.1 M; Tween 80, 0–0.025 M; and Span 80, 0.18 M] in a mixture of organic phase consisting of isooctane and paraffin oil (1:1 ratio) using a high speed homogenizer (7000 rpm, 15 min). To this mixture, internal aqueous phase (0.05–0.15 M KCl prepared using 0.2 M sodium phosphate buffer, pH 6–8) was added dropwise and mixed using homogenizer (12,000 rpm, 30 min). It resulted in the formation of stable WOW emulsion. The volume ratio of organic membrane phase to internal aqueous phase was maintained at 1.125. In the second step, the emulsion thus obtained was mixed with the enzyme extract (feed) containing NaCl (0.025–0.15 M in 0.2 M Tris-HCl, pH 9.0) for the extraction of lipase and stirred using a magnetic stirrer (350–550 rpm, 5– 25 min). The volume ratio of emulsion membrane phase to aqueous feed phase (enzyme extract) was varied from 0.6 to 1.4. In the third step, the mixture was allowed to settle in a separation funnel resulting in the separation of two phases. The upper phase consisted of the emulsion with the lipase entrapped in the reverse micelles within the emulsion while the crude extract being denser, is settled in the lower phase. In the fourth step, the emulsion enriched with lipase was destabilized by the addition of isopropanol (0.1 ml/ml of emulsion) and centrifugation (5000 rpm for 5 min) to recover lipase. The activity of lipase and protein content was estimated in lower aqueous phase. These steps for the extraction of lipase have been schematically presented in Figure 2. Enzyme assay The activity of the enzyme extracted from the feed solution was determined by spectrophotometric method using para-nitrophenyl acetate (pNPA) as a substrate. Liberation of p-nitro-phenol (pNP) was measured at 410 nm using UV–
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Figure 1. (a–d) Schematic representation of the mechanism of lipase transport in the mixed reverses micelles in liquid emulsion membrane, (e–g) chemical structure of surfactants used in present work.
visible spectrophotometer (Shimadzu, Model UV-200S, Japan). One unit of lipase activity was defined as the amount of enzyme required to liberate 1.0 mmol of pNP.22,23 Lipase activity ðl=ml=min Þ 5
Micromol of pNP released ðVtÞ 3100 Vc3t3Ve
(1)
where Vt, Vc, and Ve are the total volume, the volume used in colorimetric determination and the volume of
enzyme extract, respectively; t is the incubation time in minutes. Estimation of protein content Protein content was estimated by measuring absorbance at 595 nm using spectrophotometer and was expressed in mg/ml according to Bradford.24
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Figure 2. Schematic representation of the steps in the mechanism of lipase transport.
Estimation of activity recovery and purification fold Activity recovery and purification fold of lipase were calculated as per the following equations. Lextract 3100 (2) Lcrude Specific activity after extraction PLextract extract Purification fold 5 (3) Specificactivity of crude PLcrude crude Lipase activity recovery ð%Þ5
where Lcrude and Lextract are lipase activities in crude and after extraction, respectively. Pcrude and Pextract are the protein concentrations in the crude and after extraction, respectively. Estimation of water content in reverse micelles Water content (Wo) in reverse micelles in organic phase is defined as the molar ratio of solubilized water to surfactants. The water content in the organic phase was measured by Karl–Fischer coulometer (Metler Toledo DL32, Germany) after mixing emulsion with the crude extract. For MRM system of CTAB and nonionic surfactant water content is given by the following equation: ðWaterÞ Water content ðWo Þ 5 ðCTABÞ1ðnon2ionicÞ
Gel electrophoresis To confirm the purity of lipase extracted by LEM, aliquot of the extracted solution was subjected to SDS–PAGE with
12% gel. It was performed using electrophoresis unit (Genei, Bangalore) as per the procedure described by Deuscher.25 Samples were diluted in a sample buffer and heated at 100 C for 5 min. Electrophoresis was run at 50 V, 12.5 mA, for about 3–4 h. The gels were stained using a silver stain.26
Results and Discussion The transport of the target biomolecule across the liquid membrane is dependent on the ability of the desired species to partition into the membrane phase and its rate of diffusion through the membrane. The extraction of the target biomolecule (lipase) from the external aqueous phase to the internal aqueous phase in the emulsion globule depends on intrinsic (concentration and type of the surfactant, aqueous phase pH, and ionic strength) and extrinsic (treatment ratio, contact time, and stirring speed) parameters related with the aqueous and organic phases as well as the biomolecule itself. The extraction of the target biomolecule can be optimized by suitably varying these parameters. The effect of these parameters is discussed in the following sections. Effect of nonionic surfactant concentration The effect of the concentration of nonionic surfactant for fixed concentration of ionic surfactant (CTAB, 0.1, 0.075, and 0.05 M) on the activity recovery and purification fold of lipase is presented in Figure 3a,b. The increase in concentration of nonionic surfactant for the all the concentrations of ionic surfactant resulted in increase in activity recovery and purification fold up to a certain extent and beyond which
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attributed to the accumulation of excess surfactant molecules at the membrane surface leading to swelling of membrane thereby increasing the transport resistance. Effect of feed phase pH
Figure 3.
Effect of nonionic-surfactant concentration for certain concentrations of CTAB (䊏 0.01 M, w 0.075 M, and 䊏 0.050 M) on (a) activity recovery, and (b) purification fold; and (c) water content (Wo) during extraction of lipase. Span 80 concentrations was maintained at 0.18 M. pH of aqueous feed phase and internal aqueous phase was 9.0 and 7.0, respectively. NaCl and KCl concentration was maintained at 0.1 and 1 M, respectively. The volume ratio of emulsion phase to aqueous feed phase (enzyme extract) was 1.0. Contact time and speed was 15 min and 450 rpm, respectively.
both these parameters were decreased. For 0.1, 0.075, and 0.05 M concentrations of CTAB, the maximum activity recovery and purification fold were found when the concentration of nonionic surfactant were 0.005, 0.012, and 0.025 M, respectively. Among these values the highest activity recovery (100%) and purification fold (17.0) were found when the concentrations of CTAB and nonionic surfactant were 0.075 and 0.012 M, respectively (Figure 3a,b). Addition of nonionic surfactant increases the diameter of the reverse micelles for the efficient extraction of the biomolecule. The increase in the diameter of the reverse micelles after incorporation of nonionic surfactant was evidenced by the increase in water content (Wo) of the micelles (Figure 3c). Furthermore, MRM by combining CTAB (0.075 M) and tween 80 (0.020 M) resulted in highest Wo values (Figure 3c). The increase in the concentration of nonionic surfactant beyond certain extent resulted in decrease in the activity recovery and purification fold of lipase, which may be
The variations of activity recovery and purification fold with feed phase pH (8.0–10.0) have been presented in Figure 4a. The activity recovery and the purification fold were found to increase from 63.3% to 100% and 4.3–17.0, respectively with an increase in feed phase pH from 8.0 to 9.0 (Figure 4a) due to increase in negative charge on the target molecule in second stage of titration curve in which removal of proton takes place from the 1NH3-group of amino acids present in the enzyme (lipase). Further increase in pH resulted in decrease in activity recovery and purification fold. The net charge on the enzyme/protein molecule to be extracted plays an important role in its extraction. The main driving force behind lipase extraction is the strong electrostatic interactions between the biomolecule and the surfactant head group.14 The increase in feed phase pH above the isoelectric point of lipase (4.2), the charge on the biomolecule becomes negative. Marini et al.27 and Feng et al.28 have also indicated negative charges on lipase above its isoelectric point. The increase in pH 8.0– 9.0 may result in increase in the extent of negative charge on the target biomolecule, which led to stronger electrostatic interaction, thereby increase in extraction of target biomolecule.29,30 Further increase in feed phase pH from 9.0 should have resulted in higher activity recovery and purification fold, but the enzyme stability studies indicating that lipase is most stable in the pH range from 7.0 to 9.0.22 Electrostatic interactions are widely believed to be the primary forces controlling the pH-dependent phenomena. One or more of the catalytic residues are titratable residues (e.g., Lys, Arg, His, Glu, Asp, Tyr, Cys, C-terminal, and N-terminal) that carry charge above or below a certain pH value. The charge carried by these residues is dictated by several factors such as dielectric constant and ionic strength of the surrounding solvent and the proximity of other charged residues. The direct result of a pH change is a modification in the equilibrium concentrations of the protonated and deprotonated forms of the titratable residues and the most pronounced consequence of this modification is a corresponding change in the average charge of the titratable residues. Thus, pH is of key importance for enzyme activity.31 This increase in enzyme activity recovery may also be due to structural rearrangements of the selective biomolecular interaction of hydrophobic patches present in lipase.32,33 This phenomenon could be analogs to interfacial activation.34 Effect of internal aqueous phase pH The variations of activity recovery and purification fold with an increase in pH of internal aqueous phase from 6.0 to 8.0 have been presented in Figure 4b. The increase in pH from 6.0 to 7.0 resulted in an increase in the activity recovery and purification fold from 63 to 100% and from 4.8 to 17.0, respectively. Further increase in pH up to 8.0 resulted in decrease in the activity recovery and purification fold to 54.5% and 3.3, respectively. The activity recovery of lipase (100%) and purification fold (17) were found to be highest when the internal aqueous phase pH was 7.0 (Figure 4b). Unlike feed phase pH, the internal aqueous phase pH should be below the isoelectric point of the target biomolecule (lipase, isoelectric point 4.2) so that the internal aqueous
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Figure 4. (a) Effect of feed phase pH, (b) internal aqueous phase pH, (c) NaCl concentration, and (d) KCl concentration on activity recovery (•) and purification fold (䉫) during extraction of lipase. Span 80, CTAB and tween 80 concentration was maintained at 0.18, 0.075, and 0.012 M, respectively. The volume ratio of emulsion phase to aqueous feed phase (enzyme extract) was 1.0. Contact time and speed was 15 min and 450 rpm, respectively. pH of internal aqueous phase was 7.0 for (a, c, d), pH of aqueous feed phase was 9.0 for (b, c, d). NaCl and KCl concentration was maintained at 0.1 and 1 M for (a, b, d) and for (a, b, c), respectively.
phase pH should result in minimum electrostatic interaction between the target biomolecule (lipase) and surfactant head groups leading to easy delivery of target biomolecule to the internal aqueous phase. However, the enzyme stability study indicated that the enzyme is stable above pH 7.0. Hence, the activity recovery and purification fold was found to be maximum when the pH of internal aqueous phase was 7.0.13,22 Effect of NaCl concentration The variations of activity recovery and purification fold with feed phase ionic strength (NaCl concentration 0.025– 0.20) have been presented in Figure 4c. The activity recovery and the purification fold were found to increase from 65.6 to 100% and 2.1–17.0, respectively with an increase in NaCl concentration in feed phase from 0.025 to 0.10 M (Figure 4c). Further increase in NaCl concentration resulted in decrease in both the parameters. The ionic strength of feed phase is mainly responsible for the interaction between biomolecule and surfactant head as well as between the two surfactant heads. At optimum ionic strength (0.10 M NaCl concenration), the interaction between the biomolecule and surfactant head enhances and the repulsive force between the two surfactant heads reduces. The balance of these forces results in the formation of the stable reverse micelles. The higher ionic strength (more than 0.10 M NaCl concentration) results in reduction of electrostatic repulsion between charged head groups of the surfactants thereby decreasing the size of reverse micelles (squeezing out effect), consequently the enzymes/proteins molecules of larger size are excluded (size exclusion effect). High amount of salt also promotes the interaction of salt ion with the surfactant, which in turn reduces the interaction between surfactant head and biomolecules leading to reduced debye length.22
This phenomenon results in the reduction of interaction between biomolecule and the surfactant head group and is termed as electrostatic screening effect.29,35 Effect of KCl concentration Potassium salt (larger K1 ions) was preferentially used for internal aqueous phase during the extraction of lipase because it can cause more solubilization of enzymes/proteins as compared to sodium ions (smaller Na1 ions). Moreover, K1 is water structure breaking salt (chaotropes) where as Na1 is a water structure making salt (kosmotropes).30 The studies on the effect of KCl concentration present in internal aqueous phase on the activity recovery and purification factor of lipase indicated that increase in KCl concentration from 0.5 to 1.0 M resulted in increase in the activity recovery of lipase from 48.9 to 100%, purification fold from 2.1 to 17.0, respectively (Figure 4d). The increase in KCl concentration beyond 1.0 M resulted in decreased in both the parameters. At optimum KCl concentration (1.0 M), the activity recovery (100%) of lipase and purification fold (17.0) was highest. These values were minimum when the KCl concentration was lowest (0.05 M) or highest (1.50 M). At lower concentration (0.05 M), the concentration was not enough to influence the interaction between the charged enzyme/protein and surfactant head as well as between the two surfactant heads. Whereas at higher concentration (1.50 M KCl), the activity recovery of lipase and purification fold was lower because of the dominance of hydrophobic interactions, aggregation, and precipitation of protein.5,22 Effect of treatment ratio The volume ratio of the emulsion to the feed phase (Vemulis defined as the treatment ratio. The effect of
sion/Vfeed)
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purification fold was found to reduce because of higher proportion of the more viscous emulsion phase leading to improper mixing of phases.13 Generally, increase in the treatment ratio should increase the percentage extraction of lipase. But, higher stirring speed is required for higher treatment ratio due to the increase in viscosity of the mixture. If the speed is constant then it will reduce the extraction due to improper mixing of phases. The treatment ratio 1:1 resulted in maximum extraction of imidazole from dilute aqueous solutions,36 alcohol from aqueous solution10 and lipase from crude extract.13 Effect of stirring speed The stirring speed was found to have a profound effect in the rate of mass transfer of biomolecule through LEM. The increase in stirring from 350 to 450 rpm resulted in the increase in activity recovery and purification fold from 78 to 100% and 2.0–17.0, respectively (Figure 5b). Further increase in stirring speed resulted in decrease in activity recovery and purification fold. It may explained on the fact that the increase in the stirring speed up to a certain extent (450 rpm) results in the formation of smaller sized emulsion droplets leading to enhanced surface area for mass transfer.10 Further increase in stirring speed adversely affects stability of emulsion globules resulting in breakage, which might have resulted in the decrease in activity recovery and purification fold. Effect of contact time
Figure 5.
(a) Effect of treatment ratio, (b) stirring speed, and (c) stirring time on activity recovery (•) and purification fold (䉫) during extraction of lipase. Span 80, CTAB and tween 80 concentration was maintained at 0.18, 0.075, and 0.012 M, respectively. pH of aqueous feed phase and internal aqueous phase was 9.0 and 7.0, respectively. NaCl and KCl concentration was maintained at 0.1 M and 1 M, respectively. Contact time and speed were 15 min and 450 rpm, respectively for (a), treatment ratio and contact time were maintained at 1 and 15 min, respectively for (b). Treatment ratio and stirring speed were maintained at 1 and 450 rpm, respectively for (c).
treatment ratios from 0.6 to 1.4 on the activity recovery and purification fold of lipase has been presented in Figure 5a. The increase in ratio from 0.6 to 1.0 was found to increase the activity recovery and purification fold of lipase from 63.4% to 100% and 1.9–17.0, respectively. Further increase in treatment ratio resulted in decrease in the activity recovery and purification fold to 69.4% and 2.8, respectively. The increase in the treatment ratio up to an optimum values (1.0) results in increase in the number of available droplets of emulsion phase in aqueous feed phase leading to increase in the interfacial surface area per unit volume of the feed solution, which leads to higher rate of extraction. Whereas, at higher treatment ratio the activity recovery and
The variations of activity recovery and purification fold over a range of contact time from 5 to 25 min have been presented in Figure 5c. The activity recovery and the purification fold were found to increase from 83.3% to 100% and 2.0–17.0, respectively with an increase in stirring time from 5 to 15 min (Figure 5c). Beyond 15 min, the activity recovery and purification fold were found to decrease to 71.7% and 2.7, respectively. The increase in activity recovery and purification fold up to a certain contact time (15 min in the present case) may be attributed to the effective and efficient contact of feed phase containing lipase with LEM leading to higher mass transfer. The prolonged contact time might have resulted in membrane rupture or diffusion of extracted lipase released back into the feed phase.11 SDS–PAGE analysis The purity of lipase obtained by LEM was examined with SDS–PAGE analysis with 12% gel. The SDS–PAGE profile is depicted in Figure 6, which indicated molecular weight markers (Lane 1), crude lipase extract (Lane 2), and lipase extracted using LEM (Lane 3). The crude lipase indicated large number of overlapping bands. Lipase after LEM extraction indicated a prominent dark band with the molecular mass of 43 kDa (Lane 3). A closer look at the Figure 6, Lane 2 and 3 indicates that a large number of contaminating proteins were not extracted into LEMs from the feed. However, some other proteins close to the molecular weight of lipase were still present. Because of this reason, the purification fold was only 17 though the activity recovery was 100%. The micellar assisted purification of lipase using LEM may be regarded as primary purification technique and depending upon the requirement purification fold may be
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Figure 6. SDS–PAGE analysis of reverse micellar extracted lipase from Aspergillus niger. Lane 1: molecular marker; Lane 2: crude lipase and Lane 3: lipase after liquid emulsion membrane extraction.
increased by integrating this process with conventional purification techniques.
Conclusion LEM containing MRM (cationic surfactant, CTAB, and nonionic surfactant, tween 80) was shown for the first time to enhance the activity recovery (from 60 to 100%) and purification factor (from 3.1 to 17.0) for lipase. The optimized conditions were established for the extraction of lipase, which was found to vary depending upon the surfactants concentration (0.075 M CTAB and 0.012 M Tween 80), feed phase (pH 9) and NaCl concentration (0.1 M), internal aqueous phase pH (7.0), and KCl concentration (1.0 M), volume ratio of membrane emulsion to feed volume (1.0), stirring speed (500 rpm), and stirring time (15 min). At these optimum conditions the enzyme activity recovery and purification fold were found to be 100% and 17.0, respectively. LEM containing MRM can be successfully used for the downstream processing of lipase, if the challenges such as use of alcohol for the destabilization of emulsion and stability of the liquid membranes are suitably addressed.
Acknowledgments The authors thank Director, CSIR-Central Food Technological Research Institute (CFTRI), Mysore for encouragement. Thanks are also due to Dr. Mukesh Kapoor, Department of Protein Chemistry and Technology, CFTRI for his useful advice.
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activity and stability of lipase immobilized onto woolen cloth. Colloids Surf B: Biointerfaces. 2013;102:526–533. Tonova K, Lazarova Z. Reversed micelle solvents as tools of enzyme purification and enzyme-catalyzed conversion. Biotechnol Adv. 2008;26:516–532. Nandini KE, Rastogi NK. Single step purification of lactoperoxidase from whey involving reverse micelles-assisted extraction and its comparison with reverse micellar extraction. Biotechnol Prog. 2010;26:763–771. Petersen MTN, Fojan P, Petersen SB. How do lipases and esterases work: the electrostatic contribution. J Biotechnol. 2011;85: 115–147. Brzozowski AM, Derewenda U, Derewenda ZS, Dodson GG, Lawson DM, Turkenburg JP, Bjorkling F, Huge-Jensen B, Patkar SA, Thim L. A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature 1991;351:491–494.
Biotechnol. Prog., 2014, Vol. 30, No. 5 33. Derewenda U, Brzozowski AM, Lawson DM, Derewenda ZS. Catalysis at the interface: the anatomy of a conformational change in a triglyceride lipase. Biochemistry 1992;31:1532– 1541. 34. Jaeger KE, Dijkstra BW, Reetz MT. Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol. 1999;53: 315–351. 35. Deak NA, Murphy PA, Johnson LA. Effects of NaCl concentration on salting-in and dilution during salting-out on soy protein fractionation. J Food Sci. 2006;71:C247–C54. 36. Venkatesan S, Begum KM. Emulsion liquid membrane pertraction of imidazole from dilute aqueous solutions by Aliquat-336 mobile carrier. Desalination 2009;236:65–77. Manuscript received Jun. 5, 2013, and revision received May 10, 2014.
B. S. Priyanka and Navin K. Rastogi Dept. of Food Engineering, CSIR-Central Food Technological Research Inst., Mysore, Karnataka 570 020 India Academy of Scientific and Innovative Research, CSIR-Central Food Technological Research Inst., Mysore, Karnataka 570 020 India DOI 10.1002/btpr.1941 Published online July 3, 2014 in Wiley Online Library (wileyonlinelibrary.com)
Our earlier work for the first time demonstrated that liquid emulsion membrane (LEM) containing reverse micelles could be successfully used for the downstream processing of lipase from Aspergillus niger. In the present work, we have attempted to increase the extraction and purification fold of lipase by using mixed reverse micelles (MRM) consisting of cationic and nonionic surfactants in LEM. It was basically prepared by addition of the internal aqueous phase solution to the organic phase followed by the redispersion of the emulsion in the feed phase containing enzyme, which resulted in globules of water-oil-water (WOW) emulsion for the extraction of lipase. The optimum conditions for maximum lipase recovery (100%) and purification fold (17.0-fold) were CTAB concentration 0.075 M, Tween 80 concentration 0.012 M, at stirring speed of 500 rpm, contact time 15 min, internal aqueous phase pH 7, feed pH 9, KCl concentration 1 M, NaCl concentration 0.1 M, and ratio of membrane emulsion to feed volume 1:1. Incorporation of the nonionic surfactant (e.g., Tween 80) resulted in remarkable improvement in the purification fold (3.1–17.0) of the lipase. LEM containing a mixture of nonionic and cationic surfactants can be successfully used for the enhancement in the activity recovery and purification fold during downstream C 2014 American Institute of Chemical Engineers Biotechprocessing of enzymes/proteins. V nol. Prog., 30:1084–1092, 2014 Keywords: liquid emulsion membrane, reverse micelles, lipase, downstream processing, cationic surfactant, nonionic surfactant
Introduction Lipases are triacylglycerol acylhydrolases (E.C. 3.1.1.3), which are ubiquitous enzymes of considerable physiological significance and industrial potential. These are the supplest biocatalysts, which bring about a wide range of bioconversion reactions. In eukaryotes, lipases are involved in various stages of lipid metabolism including fat digestion, absorption, reconstitution, and lipoprotein metabolism. In plants, lipases are found in energy reserve tissues. The prospect of lipase utilization encompasses many areas such as the oleochemical, detergent, leather, cosmetics, and perfume as well as organic industries including environmental management, biomedical, and biosensor applications.1 Lipase catalyzed transesterification, hydrolysis, and esterification are the important class of reactions for food technology applications in fats and oil, dairy, pharmaceuticals, and bakery industry.2,3 Most of the enzymes for commercial applications require a certain degree of purity depending upon the final application in industries. The purification strategies employed, Correspondence concerning this article should be addressed to N. K. Rastogi at [email protected] or [email protected]. 1084
should be rapid, inexpensive, high-yielding, and pliable to large-scale operations. They should have the potential for continuous product recovery, with a relatively high capacity and selectivity for the desired product. Various purification strategies such as membrane separation processes, aqueous two-phase systems, reverse micellar extraction, hydrophobic interaction chromatography, immuno-purification, etc. have been used for the separation of lipase.4,5 Liquid emulsion membrane (LEM) technology has been extensively examined for potential application in diverse field such as the removal of contaminants from waste water, recovery of heavy metal ions,6 applications in foodstuffs, cosmetics, pharmaceuticals, lubricants, laundry, and cleaning,7 and extraction of products of fermentation.8 The process of LEM may be considered to be an alternative separation technique and its application for the recovery of the fermented products has grown rapidly.9 It offers several advantages such as ease of operation, less energy requirement, large interfacial area, low cost factors, and a single stage operation combining extraction and stripping.10 Generally, the proteins are denatured on exposure to organic liquids, and hence the usual LEMs were not found to be appropriate for the extraction of protein/enzyme. C 2014 American Institute of Chemical Engineers V
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However, there are various reports for the extraction of protein using reverse micelles as carrier in bulk liquid membrane or LEM, respectively.11 The reverse micelles mediated transport in liquid membrane is an important research field for the future for the downstream processing of biomolecules.12 Bhavya et al.13 demonstrated for the first time that the LEM containing reverse micelles can be successfully used for the downstream processing of lipase. In this work, cationic surfactant reverse micelles were evenly dispersed in organic membrane phase. These micelles act as a carrier for the transport of lipase. The optimum activity recovery and purification fold were found to be 78.6% and 3.1-fold, respectively. Encouraged with the earlier work and with a view to enhance the extent of recovery and purification of lipase, the MRM consisting of cationic and nonionic surfactants were used to improve the recovery of lipase in LEM. The presence of reverse micelles in organic solvent provides a biocompatible environment for biomolecules.14 Reverse micellar extraction consisting of MRM was shown to be more efficient in case of reverse micellar extraction of b-glucosidase,15 lipase,16,17 as well as for other model system.18,19 The main objective of this work is to increase the extent of extraction of lipase by incorporating MRMs into LEM to facilitate the migration of lipase through the organic liquid membrane for the downstream processing of lipase. The effect of various operating parameters on the extraction process has also been discussed.
Theoretical Background The reverse micelles assisted transport of lipase through liquid membrane consisted of the following steps. At first lipase is solubilized into Cetyltrimethyl ammonium bromide (CTAB) reverse micelles and then these reverse micelles enriched with lipase are transported through the liquid membrane. Finally, lipase is released into internal aqueous phase. Electrostatic and hydrophobic interactions between proteins and micelles are mainly responsible for the extraction of lipase into reverse micelles. The surfactant head groups for cationic surfactant (CTAB) are positively charged, which induce attractive force on the negatively charged proteins. The negative charges on protein are achieved by adjusting pH values higher than the isoelectric point (pI) of the enzyme13 (Figure 1a). The extent of the extraction using reverse micelles assisted transport is limited due to the size of the micelles formed in the liquid membrane. The inclusion of nonionic surfactant along with cationic surfactant results in the increase in the size of the micelles by the incorporation of nonionic surfactant molecules between the cationic surfactant (Figure 1b). These MRM result in decrease in the surface charge density, which in turn lead to the increase in the diameter of the micelles (Figure 1c,d). The increase in diameter of the MRM can be inferred from the water content of reverse micelles.20 Hemavathi et al.15 and Moilanen et al.20 have demonstrated that MRM consisting of both ionic and nonionic surfactant resulted in an increased size of the reverse micelles as compared to the micelles containing only ionic surfactant.
Materials and Methods Materials CTAB was procured from Himedia Laboratories Pvt., Mumbai; isooctane from Sisco Research laboratories,
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Mumbai; Triton X-100, gum arabic, and acetonitrile were procured from Ranbaxy Chemicals, Mumbai. Sorbitonmonooleate (Span 80) from Loba Chemie Pvt., Mumbai; Liquid paraffin light, pNp (para-nitrophenol) and pNpA (para-nitrophenol acetate) from SD Fine Chemicals, New Delhi; isopropanol and Tween 80 (polyoxyethylene sorbitan monooleate), potassium dihydrogenphosphate, tris-buffer, sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium chloride, sodium chloride, and HCl from Merck Ltd. Mumbai. All the chemicals used were of AR grade. Preparation of crude enzyme extract Fermented rice bran containing lipase was obtained from M/s. Zeus Biotech Laboratory, Mysore after cultivation of Aspergillus niger by solid-state method. The enzyme was extracted from rice bran (5 g) using 50 ml of 0.2 M TrisHCl (pH 9). The mixture was stirred using magnetic stirrer at 200 rpm for 2 h at 25 6 2 C.21 The suspension was filtered through a muslin cloth and centrifuged at 5000 rpm for 15 min at 4 C (REMI Compufuge). The clear supernatant was used as a crude extract (feed). The isoelectric point of lipase was reported to be 4.2.13,22 Enzyme extraction using liquid emulsion membrane The extraction of lipase from aqueous solution (feed) using LEM involves four steps namely formation of LEM, extraction of lipase, separation of emulsion, and deemulsification. The formation of LEM (first step) involved suspension of surfactants [CTAB, 0.05–0.1 M; Tween 80, 0–0.025 M; and Span 80, 0.18 M] in a mixture of organic phase consisting of isooctane and paraffin oil (1:1 ratio) using a high speed homogenizer (7000 rpm, 15 min). To this mixture, internal aqueous phase (0.05–0.15 M KCl prepared using 0.2 M sodium phosphate buffer, pH 6–8) was added dropwise and mixed using homogenizer (12,000 rpm, 30 min). It resulted in the formation of stable WOW emulsion. The volume ratio of organic membrane phase to internal aqueous phase was maintained at 1.125. In the second step, the emulsion thus obtained was mixed with the enzyme extract (feed) containing NaCl (0.025–0.15 M in 0.2 M Tris-HCl, pH 9.0) for the extraction of lipase and stirred using a magnetic stirrer (350–550 rpm, 5– 25 min). The volume ratio of emulsion membrane phase to aqueous feed phase (enzyme extract) was varied from 0.6 to 1.4. In the third step, the mixture was allowed to settle in a separation funnel resulting in the separation of two phases. The upper phase consisted of the emulsion with the lipase entrapped in the reverse micelles within the emulsion while the crude extract being denser, is settled in the lower phase. In the fourth step, the emulsion enriched with lipase was destabilized by the addition of isopropanol (0.1 ml/ml of emulsion) and centrifugation (5000 rpm for 5 min) to recover lipase. The activity of lipase and protein content was estimated in lower aqueous phase. These steps for the extraction of lipase have been schematically presented in Figure 2. Enzyme assay The activity of the enzyme extracted from the feed solution was determined by spectrophotometric method using para-nitrophenyl acetate (pNPA) as a substrate. Liberation of p-nitro-phenol (pNP) was measured at 410 nm using UV–
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Figure 1. (a–d) Schematic representation of the mechanism of lipase transport in the mixed reverses micelles in liquid emulsion membrane, (e–g) chemical structure of surfactants used in present work.
visible spectrophotometer (Shimadzu, Model UV-200S, Japan). One unit of lipase activity was defined as the amount of enzyme required to liberate 1.0 mmol of pNP.22,23 Lipase activity ðl=ml=min Þ 5
Micromol of pNP released ðVtÞ 3100 Vc3t3Ve
(1)
where Vt, Vc, and Ve are the total volume, the volume used in colorimetric determination and the volume of
enzyme extract, respectively; t is the incubation time in minutes. Estimation of protein content Protein content was estimated by measuring absorbance at 595 nm using spectrophotometer and was expressed in mg/ml according to Bradford.24
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Figure 2. Schematic representation of the steps in the mechanism of lipase transport.
Estimation of activity recovery and purification fold Activity recovery and purification fold of lipase were calculated as per the following equations. Lextract 3100 (2) Lcrude Specific activity after extraction PLextract extract Purification fold 5 (3) Specificactivity of crude PLcrude crude Lipase activity recovery ð%Þ5
where Lcrude and Lextract are lipase activities in crude and after extraction, respectively. Pcrude and Pextract are the protein concentrations in the crude and after extraction, respectively. Estimation of water content in reverse micelles Water content (Wo) in reverse micelles in organic phase is defined as the molar ratio of solubilized water to surfactants. The water content in the organic phase was measured by Karl–Fischer coulometer (Metler Toledo DL32, Germany) after mixing emulsion with the crude extract. For MRM system of CTAB and nonionic surfactant water content is given by the following equation: ðWaterÞ Water content ðWo Þ 5 ðCTABÞ1ðnon2ionicÞ
Gel electrophoresis To confirm the purity of lipase extracted by LEM, aliquot of the extracted solution was subjected to SDS–PAGE with
12% gel. It was performed using electrophoresis unit (Genei, Bangalore) as per the procedure described by Deuscher.25 Samples were diluted in a sample buffer and heated at 100 C for 5 min. Electrophoresis was run at 50 V, 12.5 mA, for about 3–4 h. The gels were stained using a silver stain.26
Results and Discussion The transport of the target biomolecule across the liquid membrane is dependent on the ability of the desired species to partition into the membrane phase and its rate of diffusion through the membrane. The extraction of the target biomolecule (lipase) from the external aqueous phase to the internal aqueous phase in the emulsion globule depends on intrinsic (concentration and type of the surfactant, aqueous phase pH, and ionic strength) and extrinsic (treatment ratio, contact time, and stirring speed) parameters related with the aqueous and organic phases as well as the biomolecule itself. The extraction of the target biomolecule can be optimized by suitably varying these parameters. The effect of these parameters is discussed in the following sections. Effect of nonionic surfactant concentration The effect of the concentration of nonionic surfactant for fixed concentration of ionic surfactant (CTAB, 0.1, 0.075, and 0.05 M) on the activity recovery and purification fold of lipase is presented in Figure 3a,b. The increase in concentration of nonionic surfactant for the all the concentrations of ionic surfactant resulted in increase in activity recovery and purification fold up to a certain extent and beyond which
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attributed to the accumulation of excess surfactant molecules at the membrane surface leading to swelling of membrane thereby increasing the transport resistance. Effect of feed phase pH
Figure 3.
Effect of nonionic-surfactant concentration for certain concentrations of CTAB (䊏 0.01 M, w 0.075 M, and 䊏 0.050 M) on (a) activity recovery, and (b) purification fold; and (c) water content (Wo) during extraction of lipase. Span 80 concentrations was maintained at 0.18 M. pH of aqueous feed phase and internal aqueous phase was 9.0 and 7.0, respectively. NaCl and KCl concentration was maintained at 0.1 and 1 M, respectively. The volume ratio of emulsion phase to aqueous feed phase (enzyme extract) was 1.0. Contact time and speed was 15 min and 450 rpm, respectively.
both these parameters were decreased. For 0.1, 0.075, and 0.05 M concentrations of CTAB, the maximum activity recovery and purification fold were found when the concentration of nonionic surfactant were 0.005, 0.012, and 0.025 M, respectively. Among these values the highest activity recovery (100%) and purification fold (17.0) were found when the concentrations of CTAB and nonionic surfactant were 0.075 and 0.012 M, respectively (Figure 3a,b). Addition of nonionic surfactant increases the diameter of the reverse micelles for the efficient extraction of the biomolecule. The increase in the diameter of the reverse micelles after incorporation of nonionic surfactant was evidenced by the increase in water content (Wo) of the micelles (Figure 3c). Furthermore, MRM by combining CTAB (0.075 M) and tween 80 (0.020 M) resulted in highest Wo values (Figure 3c). The increase in the concentration of nonionic surfactant beyond certain extent resulted in decrease in the activity recovery and purification fold of lipase, which may be
The variations of activity recovery and purification fold with feed phase pH (8.0–10.0) have been presented in Figure 4a. The activity recovery and the purification fold were found to increase from 63.3% to 100% and 4.3–17.0, respectively with an increase in feed phase pH from 8.0 to 9.0 (Figure 4a) due to increase in negative charge on the target molecule in second stage of titration curve in which removal of proton takes place from the 1NH3-group of amino acids present in the enzyme (lipase). Further increase in pH resulted in decrease in activity recovery and purification fold. The net charge on the enzyme/protein molecule to be extracted plays an important role in its extraction. The main driving force behind lipase extraction is the strong electrostatic interactions between the biomolecule and the surfactant head group.14 The increase in feed phase pH above the isoelectric point of lipase (4.2), the charge on the biomolecule becomes negative. Marini et al.27 and Feng et al.28 have also indicated negative charges on lipase above its isoelectric point. The increase in pH 8.0– 9.0 may result in increase in the extent of negative charge on the target biomolecule, which led to stronger electrostatic interaction, thereby increase in extraction of target biomolecule.29,30 Further increase in feed phase pH from 9.0 should have resulted in higher activity recovery and purification fold, but the enzyme stability studies indicating that lipase is most stable in the pH range from 7.0 to 9.0.22 Electrostatic interactions are widely believed to be the primary forces controlling the pH-dependent phenomena. One or more of the catalytic residues are titratable residues (e.g., Lys, Arg, His, Glu, Asp, Tyr, Cys, C-terminal, and N-terminal) that carry charge above or below a certain pH value. The charge carried by these residues is dictated by several factors such as dielectric constant and ionic strength of the surrounding solvent and the proximity of other charged residues. The direct result of a pH change is a modification in the equilibrium concentrations of the protonated and deprotonated forms of the titratable residues and the most pronounced consequence of this modification is a corresponding change in the average charge of the titratable residues. Thus, pH is of key importance for enzyme activity.31 This increase in enzyme activity recovery may also be due to structural rearrangements of the selective biomolecular interaction of hydrophobic patches present in lipase.32,33 This phenomenon could be analogs to interfacial activation.34 Effect of internal aqueous phase pH The variations of activity recovery and purification fold with an increase in pH of internal aqueous phase from 6.0 to 8.0 have been presented in Figure 4b. The increase in pH from 6.0 to 7.0 resulted in an increase in the activity recovery and purification fold from 63 to 100% and from 4.8 to 17.0, respectively. Further increase in pH up to 8.0 resulted in decrease in the activity recovery and purification fold to 54.5% and 3.3, respectively. The activity recovery of lipase (100%) and purification fold (17) were found to be highest when the internal aqueous phase pH was 7.0 (Figure 4b). Unlike feed phase pH, the internal aqueous phase pH should be below the isoelectric point of the target biomolecule (lipase, isoelectric point 4.2) so that the internal aqueous
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Figure 4. (a) Effect of feed phase pH, (b) internal aqueous phase pH, (c) NaCl concentration, and (d) KCl concentration on activity recovery (•) and purification fold (䉫) during extraction of lipase. Span 80, CTAB and tween 80 concentration was maintained at 0.18, 0.075, and 0.012 M, respectively. The volume ratio of emulsion phase to aqueous feed phase (enzyme extract) was 1.0. Contact time and speed was 15 min and 450 rpm, respectively. pH of internal aqueous phase was 7.0 for (a, c, d), pH of aqueous feed phase was 9.0 for (b, c, d). NaCl and KCl concentration was maintained at 0.1 and 1 M for (a, b, d) and for (a, b, c), respectively.
phase pH should result in minimum electrostatic interaction between the target biomolecule (lipase) and surfactant head groups leading to easy delivery of target biomolecule to the internal aqueous phase. However, the enzyme stability study indicated that the enzyme is stable above pH 7.0. Hence, the activity recovery and purification fold was found to be maximum when the pH of internal aqueous phase was 7.0.13,22 Effect of NaCl concentration The variations of activity recovery and purification fold with feed phase ionic strength (NaCl concentration 0.025– 0.20) have been presented in Figure 4c. The activity recovery and the purification fold were found to increase from 65.6 to 100% and 2.1–17.0, respectively with an increase in NaCl concentration in feed phase from 0.025 to 0.10 M (Figure 4c). Further increase in NaCl concentration resulted in decrease in both the parameters. The ionic strength of feed phase is mainly responsible for the interaction between biomolecule and surfactant head as well as between the two surfactant heads. At optimum ionic strength (0.10 M NaCl concenration), the interaction between the biomolecule and surfactant head enhances and the repulsive force between the two surfactant heads reduces. The balance of these forces results in the formation of the stable reverse micelles. The higher ionic strength (more than 0.10 M NaCl concentration) results in reduction of electrostatic repulsion between charged head groups of the surfactants thereby decreasing the size of reverse micelles (squeezing out effect), consequently the enzymes/proteins molecules of larger size are excluded (size exclusion effect). High amount of salt also promotes the interaction of salt ion with the surfactant, which in turn reduces the interaction between surfactant head and biomolecules leading to reduced debye length.22
This phenomenon results in the reduction of interaction between biomolecule and the surfactant head group and is termed as electrostatic screening effect.29,35 Effect of KCl concentration Potassium salt (larger K1 ions) was preferentially used for internal aqueous phase during the extraction of lipase because it can cause more solubilization of enzymes/proteins as compared to sodium ions (smaller Na1 ions). Moreover, K1 is water structure breaking salt (chaotropes) where as Na1 is a water structure making salt (kosmotropes).30 The studies on the effect of KCl concentration present in internal aqueous phase on the activity recovery and purification factor of lipase indicated that increase in KCl concentration from 0.5 to 1.0 M resulted in increase in the activity recovery of lipase from 48.9 to 100%, purification fold from 2.1 to 17.0, respectively (Figure 4d). The increase in KCl concentration beyond 1.0 M resulted in decreased in both the parameters. At optimum KCl concentration (1.0 M), the activity recovery (100%) of lipase and purification fold (17.0) was highest. These values were minimum when the KCl concentration was lowest (0.05 M) or highest (1.50 M). At lower concentration (0.05 M), the concentration was not enough to influence the interaction between the charged enzyme/protein and surfactant head as well as between the two surfactant heads. Whereas at higher concentration (1.50 M KCl), the activity recovery of lipase and purification fold was lower because of the dominance of hydrophobic interactions, aggregation, and precipitation of protein.5,22 Effect of treatment ratio The volume ratio of the emulsion to the feed phase (Vemulis defined as the treatment ratio. The effect of
sion/Vfeed)
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purification fold was found to reduce because of higher proportion of the more viscous emulsion phase leading to improper mixing of phases.13 Generally, increase in the treatment ratio should increase the percentage extraction of lipase. But, higher stirring speed is required for higher treatment ratio due to the increase in viscosity of the mixture. If the speed is constant then it will reduce the extraction due to improper mixing of phases. The treatment ratio 1:1 resulted in maximum extraction of imidazole from dilute aqueous solutions,36 alcohol from aqueous solution10 and lipase from crude extract.13 Effect of stirring speed The stirring speed was found to have a profound effect in the rate of mass transfer of biomolecule through LEM. The increase in stirring from 350 to 450 rpm resulted in the increase in activity recovery and purification fold from 78 to 100% and 2.0–17.0, respectively (Figure 5b). Further increase in stirring speed resulted in decrease in activity recovery and purification fold. It may explained on the fact that the increase in the stirring speed up to a certain extent (450 rpm) results in the formation of smaller sized emulsion droplets leading to enhanced surface area for mass transfer.10 Further increase in stirring speed adversely affects stability of emulsion globules resulting in breakage, which might have resulted in the decrease in activity recovery and purification fold. Effect of contact time
Figure 5.
(a) Effect of treatment ratio, (b) stirring speed, and (c) stirring time on activity recovery (•) and purification fold (䉫) during extraction of lipase. Span 80, CTAB and tween 80 concentration was maintained at 0.18, 0.075, and 0.012 M, respectively. pH of aqueous feed phase and internal aqueous phase was 9.0 and 7.0, respectively. NaCl and KCl concentration was maintained at 0.1 M and 1 M, respectively. Contact time and speed were 15 min and 450 rpm, respectively for (a), treatment ratio and contact time were maintained at 1 and 15 min, respectively for (b). Treatment ratio and stirring speed were maintained at 1 and 450 rpm, respectively for (c).
treatment ratios from 0.6 to 1.4 on the activity recovery and purification fold of lipase has been presented in Figure 5a. The increase in ratio from 0.6 to 1.0 was found to increase the activity recovery and purification fold of lipase from 63.4% to 100% and 1.9–17.0, respectively. Further increase in treatment ratio resulted in decrease in the activity recovery and purification fold to 69.4% and 2.8, respectively. The increase in the treatment ratio up to an optimum values (1.0) results in increase in the number of available droplets of emulsion phase in aqueous feed phase leading to increase in the interfacial surface area per unit volume of the feed solution, which leads to higher rate of extraction. Whereas, at higher treatment ratio the activity recovery and
The variations of activity recovery and purification fold over a range of contact time from 5 to 25 min have been presented in Figure 5c. The activity recovery and the purification fold were found to increase from 83.3% to 100% and 2.0–17.0, respectively with an increase in stirring time from 5 to 15 min (Figure 5c). Beyond 15 min, the activity recovery and purification fold were found to decrease to 71.7% and 2.7, respectively. The increase in activity recovery and purification fold up to a certain contact time (15 min in the present case) may be attributed to the effective and efficient contact of feed phase containing lipase with LEM leading to higher mass transfer. The prolonged contact time might have resulted in membrane rupture or diffusion of extracted lipase released back into the feed phase.11 SDS–PAGE analysis The purity of lipase obtained by LEM was examined with SDS–PAGE analysis with 12% gel. The SDS–PAGE profile is depicted in Figure 6, which indicated molecular weight markers (Lane 1), crude lipase extract (Lane 2), and lipase extracted using LEM (Lane 3). The crude lipase indicated large number of overlapping bands. Lipase after LEM extraction indicated a prominent dark band with the molecular mass of 43 kDa (Lane 3). A closer look at the Figure 6, Lane 2 and 3 indicates that a large number of contaminating proteins were not extracted into LEMs from the feed. However, some other proteins close to the molecular weight of lipase were still present. Because of this reason, the purification fold was only 17 though the activity recovery was 100%. The micellar assisted purification of lipase using LEM may be regarded as primary purification technique and depending upon the requirement purification fold may be
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Figure 6. SDS–PAGE analysis of reverse micellar extracted lipase from Aspergillus niger. Lane 1: molecular marker; Lane 2: crude lipase and Lane 3: lipase after liquid emulsion membrane extraction.
increased by integrating this process with conventional purification techniques.
Conclusion LEM containing MRM (cationic surfactant, CTAB, and nonionic surfactant, tween 80) was shown for the first time to enhance the activity recovery (from 60 to 100%) and purification factor (from 3.1 to 17.0) for lipase. The optimized conditions were established for the extraction of lipase, which was found to vary depending upon the surfactants concentration (0.075 M CTAB and 0.012 M Tween 80), feed phase (pH 9) and NaCl concentration (0.1 M), internal aqueous phase pH (7.0), and KCl concentration (1.0 M), volume ratio of membrane emulsion to feed volume (1.0), stirring speed (500 rpm), and stirring time (15 min). At these optimum conditions the enzyme activity recovery and purification fold were found to be 100% and 17.0, respectively. LEM containing MRM can be successfully used for the downstream processing of lipase, if the challenges such as use of alcohol for the destabilization of emulsion and stability of the liquid membranes are suitably addressed.
Acknowledgments The authors thank Director, CSIR-Central Food Technological Research Institute (CFTRI), Mysore for encouragement. Thanks are also due to Dr. Mukesh Kapoor, Department of Protein Chemistry and Technology, CFTRI for his useful advice.
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