Ind. Eng. Chem. Res. 2005, 44, 7469-7473
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Lipoxygenase-1 Mass-Transfer Coefficient in Aqueous Two-Phase System Using Spray Extraction Column Vahid Arsalani,† Khosrow Rostami,*,‡ and Azadeh Kheirolomoom§ Azad University, Department of Science and Research, Ashrafee Esfahani Express Way, Hesarak, Post Office Box, 14515-775, Tehran, Iran, Biotechnology Center, Iranian Research Organization for Science and Technology, 71 Forsat Street, Tehran-15819, Iran, and Sharif University of Technology, Department of Petroleum and Chemical Engineering, Post Office Box 11365-8639, Tehran, Iran
Extraction of lipoxygenase-1 was investigated by using the aqueous two-phase system formed by sodium sulfate-poly(ethylene glycol)-buffer. The extraction was performed in a 47-mm inner diameter spray column, operating in a semibatch manner. The effects of dispersed-phase flow rate and phase compositions on fractional dispersed-phase hold up (D) and volumetric masstransfer coefficient (KDa) were respectively studied. It was found that the hold-up and volumetric mass-transfer coefficient increased with an increase in dispersed-phase velocity and decreased with increasing phase composition. Correlations have been developed by nonlinear regression for estimation of fractional dispersed-phase hold up and volumetric mass-transfer coefficients. Introduction Lipoxygenases (EC 1.13.11.12) are enzymes which catalyze the specific addition of molecular oxygen to polyunsaturated fatty acids (containing the cis,cis-1,4pentadiene moiety) yielding the 1,3-cis,trans-diene-5hydroperoxides. Among the fatty acids with this functionality, linoleic and linolenic acids are the best known substrates for the lipoxygenase-1 (LOX-1) isoenzyme. The hydroperoxide products have been recognized as versatile reaction intermediates in the production of different fine chemicals Gardner.1 Further, lipoxygenases are considered to have significant physiological functions and are becoming increasingly interesting in studies of asthma and related allergies by Israedl,2 Wenzell,3 and Kane.4 Aqueous two-phase systems (ATPSs) are composed of two water-soluble polymers, such as poly(ethylene glycol) (PEG) and dextran (DX), or a polymer and a salt, usually PEG and phosphate or sulfate in water. ATPS have a number of advantages over commonly used extractive techniques. Some of these advantages are summarized as follows: High water content of both the phases (85-95%, w/w), which provides high compatibility and indicates minor degradation of biomolecules. Low interfacial tension results in high mass transfer and ease of scale up. Furthermore, relative high capacity and yield, and the possibility of polymer recycling, are the very few advantages of ATPS to be used as extractive phases and fermentation medium, respectively. However, Albertson5 and Hustedt et al.6 have used ATPS on the large scale for purification of biomacromolecules. A wide variety of biological systems, such as proteins, nucleic * To whom correspondence should be addressed. Tel/Fax: +98-21-8838350. E- mail:
[email protected]. † Azad University, Department of Science and Research, Ashrafee Esfahani Express Way, Hesarak, Post Office Box, 14515-775, Tehran, Iran. ‡ Biotechnology Center, Iranian Research Organization for Science and Technology, 71 Forsat Street, Tehran-15819, Iran. § Sharif University of Technology, Department of Petroleum and Chemical Engineering, Post Office Box 11365-8639, Tehran, Iran.
acids, microorganisms, and animal and plant cells, have been successfully separated using ATPS.7 To achieve a desirable extraction, the product should be preferentially partitioned in favor of top and the interfering substance into the lower phase. The value of the partition coefficient relies on the physicochemical properties of the species and the other interacting molecules with the system. The observed partition coefficient is a result of van der Waals, hydrogen bond, hydrophobic effect, and ionic interactions between the species. Therefore, the partition coefficient is influenced by many factors, such as concentration and molecular mass of PEG, type and concentration of salts forming ATPS, temperature, and pH. The range of partition coefficients in ATPS for proteins is from 0.01 to 100. However the mechanism governing the partition (m) of biological materials is still not very clear.8 ATP extraction is usually carried out at room temperature. The variation of distribution coefficient with 1 or 2 °C of changing the temperature normally does not affect the yield and the purity of the product. However, the distribution coefficient is higher at lower temperatures. Further, enough attention should be paid during test performance that viscosity should not lead to complication. Spray columns are widely used in liquid-liquid extraction provided not more than about four theoretical stages required. Spray columns are easy to operate, clean, and construct. Rostami et al.9 have addressed a review of spray column. Materials and Methods Materials. Linoleic acid and Tween 20 were analytical grade and purchased from Sigma & Merck companies. PEG (number average molecular mass 4000) and sodium sulfate were also purchased from Merck Co. All other reagents were analytical grade. Equipment. A spray column of internal diameter of 47 mm was used to investigate the effects of superficial dispersed-phase velocity and phase composition on D and KDa. The distributor consisted of 18 holes of 1 mm in diameter arranged on a squared pitch and chamber length of 100 mm. The schematic diagram of the spray column has been presented in Figure 1.
10.1021/ie058001t CCC: $30.25 © 2005 American Chemical Society Published on Web 08/10/2005
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Ind. Eng. Chem. Res., Vol. 44, No. 19, 2005 Table 1. Phase Composition of System Used: Sodium Sulfate-PEG(4000)-Buffer composition
Figure 1. Schematic diagram of experimental setup.
Substrate Preparation. The substrate stock solution (10 mM solution of linoleic acid and Tween 20 in deionized water) was prepared according to the procedure described by Axelrod10 and used throughout the present study. Preparation of Crude Enzyme. A 20-kg bag of soybeans was purchased from the local market, stored at a cool and dry place, and the required quantity was used from time to time. Major care was taken to keep the seed-crushing temperature under 40 ( 2 °C. Initially industrial n-hexane was used to defat the flour; as a result LOX-1 with extremely poor activity was obtained. Therefore, analytical grade of n-hexane was used to defat the flour. To defat the soybean flour, the procedure of Axelrod10 was followed. However, it was found out that double centrifugation at 10 000 g was more effective than using centrifugation followed by cheesecloth separation to recover the enzyme. For partial purification of LOX-1, Agnes’s11 procedure with certain modification was used. Soybean meal extract was heated under optimum conditions in a water bath of 70 °C for 5 min at pH 5.2 with an ionic strength of 0.05 M and then cooled to room temperature using an ice bath and centrifuged at 10 000 g for 20 min at room temperature, and then the clarified extract was taken to 23% saturation with ethanol. The pellet obtained by centrifugation (10 000 g, 20 min) was suspended in minimal amount of borate buffer (pH 9, 0.2 M). The resulting enzyme solution was stored frozen in -20 °C without adding any stabilizer. The loss of activity for a period of three months was about 3%. The initial reading for each set of experiment was observed and used. Enzyme Assay. The method of Axelrod,10 which has been modified by Sigma Company, was used. The LOX-1 activity was measured spectrophotometrically. The reaction medium consisted of 0.1 mL of sodium linoleate substrate, 0.1 mL of enzyme, and 2.8 mL of borate buffer (pH 9).The enzyme reaction was initiated by the addition of enzyme solution. A control solution, containing all the components except the enzyme, was run in tandem with these trials. The LOX-1 activity was expressed as the increase in absorbance at 234 nm on a Shimadzu 2101 continuous spectrophotometer. Triplicate observation was performed and the average result has been reported. Preparation of Phase Composition. The ATPS was prepared according to the procedure given by
system no.
% PEG
% sodium sulfate
% buffer
1 2 3 4
13.6 15.0 17.83 14.4
6.5 8.0 7.83 10.87
79.9 77.0 74.34 74.33
Rostami.12 The predetermined weighed quantities of PEG and sodium sulfate were added to a weighed quantity of buffer. The entire mixture was stirred for about 4 h to equilibrate. The phases were allowed to settle overnight, and then each phase was carefully removed and filtered to get two clean phases, salt-rich phase (SRP) and PEG-rich phase (PRP). In this case the pH of the phases was adjusted to 5.7 using sodium acetate. The equilibrium diagram for this system has been reported by Pathak.13 The system compositions used in the present work have been given in Table 1. Experimental Procedure. In all the experiments, the solute, LOX-1, was dissolved in the PRP and transferred to the SRP. LOX-1 was dissolved in the PRP at a predetermined activity level, and the stock solution was stored in a glass overhead tank. The extent of deactivation of enzyme was found to be negligible during the time of experiment (less than 3 h). The flow of PRP was monitored using a precalibrated rotameter. When the level of PRP was above the nozzles of sieve plate distributor, the SRP was gently added along the wall of the column and the addition was completed in less than 20 s. The volume of SRP was predetermined so that the desired height of SRP in the column, 400 mm, was achieved. The flow of PRP continued for a period that was always 10 times greater than the residence time of the PRP. This duration was provided for ensuring a steady state with respect to the dispersion characteristics. The PRP droplets coalesced at the top of the SRP, and the coalesced layer was removed continuously through the overflow outlets. All contacting experiments were carried out at 24 ( 2 °C. A schematic diagram of the experimental setup has been presented in Figure 1. Measurement of Hold Up and Calculation of Mass-Transfer Coefficient. The PRP hold up was measured due to height expansion (∆H) with respect to each superficial dispersed-phase velocity. The clear liquid height (HC) and the height of dispersion (HD) were measured, and then D has been obtained by the following eq
D ) (HD - HC)/HD
(1)
The height of the SRP was 400 mm in all experiments. The partition coefficient, m, was calculated as
m ) CT/CB
(2)
where CT and CB are the activities of LOX-1 in PRP and SRP at equilibrium, respectively. On the basis of the measured inlet and outlet activities of LOX-1 in the PRP, the average activity of LOX-1 in the SRP was estimated from material balance. The mass balance for LOX-1 over a differential height, dH, of the column gives
LdCp ) KDa(Cp - mCs)S dH
(3)
Ind. Eng. Chem. Res., Vol. 44, No. 19, 2005 7471
Figure 2. Effect of superficial dispersed-phase velocity on dispersed-phase hold up using various phase compositions.
The values of partition coefficient, m, of LOX-1 were low, and hence the value of mCs has been found to be always less than 2% of the minimum value of Cp in the column at any time of the experiment. Therefore, the value of mCs, which can be assumed to be constant with respect to height and the extent of mixing in the continuous phase, is, thus, unimportant as far as the estimation of mass transfer coefficient is concerned. The average C value was estimated on the basis of material balance (total enzyme extracted divided by the volume of SRP). Further, it has been assumed that the dispersed phase moves in the plug flow manner. The dispersed-phase Peclet number has been found to be always greater than 20 using the procedures of Laddha,14 Wijffels and Rietema,15 and Joshi.16 In addition, the extent of LOX-1 extraction from the PRP was less than 25%. Therefore, the extent of axial mixing is also unimportant and substitution of boundary conditions (H ) 0, Cp ) Ci, H ) HD, Cp ) C0) gives
KDa ) (L/vD) ln[(Ci - mCs)/(C0 - mCs)]
(4)
Reproducibility of D and KDa measurements at high dispersed velocity was within 10%. Results and Discussion Effect of Superficial Dispersed Phase Velocity. The effect of superficial dispersed-phase velocity, vD, in the range of 0.16-1.12 mm s-1 and phase composition presented in Table 1 were studied. The values of D and KDa were found to increase with an increase in vD as has been depicted in Figures 2 and 3, respectively. Kumar and Hartland17 have shown that the drop size decreases with an increase in orifice velocity. Therefore, an increase in vD increased the number of drops and resulted in an increase in the values of D and KDa. Similar effects are observed in other multiphase reactors such as bubble columns, slurry reactors, air-lift, loop reactors, and fluidized beds reported by Treybal.18 For spherical drops, effective liquid-liquid interfacial area is given by
a ) 6D/dP
(5)
where D was measured by eq 1. The above equation
Figure 3. Effect of superficial dispersed-phase velocity on dispersed-side mass-transfer coefficient using various phase composition.
indicates that a varies linearly with D. As a result, KDa also increases with an increase in D. The pressure drop (∆P) per unit length increases with a decrease in D and an increase in the energy dissipation rate, which produce drops of smaller size. The smaller drops have lower slip velocity, which will result in the higher values of D. Zuber and Findly19 proposed eq 6 to understand this phenomena
vD/D ) Co1vD + Ci1
(6)
We know that Co1 shows the radial hold-up profile and that Ci1 indicates the rise velocity of drops. Knowledge of terminal rise velocity of drops, pressure drop, Co1, and Ci1 is necessary to predicate hold up. Since the values of Co1 and Ci1 are independent of column height and diameter, they can be used for estimation of hold up and pressure drop.20 Further, at all superficial dispersedphase velocities, a small region of packed drops (flooding region) was observed at the top of the column. The height of this region increased dramatically above a vD value of 1 mm s-1. Because of this problem, the maximum values of vD were always around 1 mm s-1. Effect of Phase Composition. In the present work, the PEG-sodium sulfate-buffer (pH 5.7) system was used. The system compositions and their physical properties are given in Tables 1 and 2,21 respectively. It can be seen from Table 2 that, with an increase in the system composition, the interfacial tension, density difference, and the phase viscosities increase. The combined effect of these properties is that the average drop size and the drop velocity increase with an increase in the system composition. Therefore, D and KDa decrease with an increase in the system composition. The experimental results have been shown in Figures 2 and 3, respectively. Controlling Resistance to Mass Transfer. In the sodium sulfate-PEG-LOX-1 system, the PRP viscosity was at least 9 times higher than that of SRP. Greankoplis22 has proposed the following equation for determination of enzyme and protein, diffusion coefficient as the value of diffusivity depends on viscosity
DD ) 9.4 × 10-15θ/µD(MA)1/3
(7)
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Ind. Eng. Chem. Res., Vol. 44, No. 19, 2005
Table 2. Physical Properties of the Phase System Sodium Sulfate-PEG(4000)-Buffer, Temperature ) 24 ( 2 °C viscosity, mPa s
density, kg/m3
system no.
PRP (µD)
SRP (µc)
PRP (FD)
SRP (FC)
interfacial tension, mN/m (σ)
pH
percentage tie line (% t)
partition coefficient (m)
1 2 3 4
11.60 22.80 28.90 35.50
1.25 1.35 1.36 1.44
1071 1072 1073 1082
1115 1138 1150 1162
0.06 0.15 0.24 0.55
5.7 5.7 5.7 5.7
18.0 25.6 34.0 38.4
0.048 0.035 0.0305 0.0305
Equation 7 presents that the diffusivity varies inversely with viscosity. In addition, an increase in the viscosity enhances the film thickness or the contact time of two phases. The combined effect of these factors result in lower values of true mass transfer coefficient kD on the dispersed side (PRP side) as compared with that on the salt phase side. Again, the true overall mass transfer coefficient KD is given by
Correlations. The fractional dispersed-phase hold up D and dispersed-side mass-transfer coefficient KDa were correlated by nonlinear least-squares regression analysis as functions of the liquid-phase physical properties group obtaining the following equations
1/KD ) m/kC + 1/kD
Moreover, the interfacial tension, the density difference, and the phase viscosities increase also with an increase in the phase composition. Therefore, D and KDa data can be also correlated with length of the tie line as the parameter. However the hold-up values and overall mass transfer coefficients are correlated with the physical properties group Np
(8)
The values of distribution coefficient m were in the range 0.0305-0.048 as can be seen from Table 2 for the sodium sulfate-PEG- LOX-1 systems. Thus the continuous phase resistance to mass transfer will be at least 80 times lower than that for the dispersed phase. The combined effect of these factors results in lower values of true mass transfer coefficient kD for the PEGrich dispersed-phase side compared with those for the SRP side. All the above factor is that the controlling resistance to mass transfer is offered by the PEG-rich dispersed phase.
Np ) (σ9Fc2/g µc2∆F)
D ) 0.07vD0.39(Np)-1.02
(9)
(10)
with r2 ) 0.99, standard error of estimate ) 0.0169
KDa ) 2.96 × 10-4vD1.52(Np)-0.06
(11)
with r2 ) 0.96, standard error of estimate ) 0.0001. The parity plots for eqs 10 and 11 are presented in Figures 4 and 5. Conclusion A PEG-salt ATPS consisting of PEG (number average molecular mass 4000) and sodium sulfate was successfully employed for extraction of the LOX-1 enzyme. The values of D and KDa were found to increase with an increase in superficial dispersed-phase velocity. The values of D and KDa were found to decrease with an increase in the tie-line length. The dispersed PRP offers resistance to mass transfer. The correlations developed for D and KDa are good estimates for the scale up of such equipments. Figure 4. Parity plot of dispersed-phase hold up.
Acknowledgment This work was partially supported by Biotechnology Center of Iranian Research Organization for Science and Technology (IROST). Thanks to Dr. F. Chamani, Agnes SZ Nemeth of Hungary, Zamanizadeh, M. A. Doust and Mr. Safafar of Oil Seeds Research and Development Company. Nomenclature
Figure 5. Parity plot of dispersed-side mass-transfer coefficient.
a ) effective interfacial area, m2 m-3 CB ) concentration of enzyme in bottom phase, lipoxyganase mL-1 Ci ) inlet concentration of enzyme in PRP, lipoxyganase mL-1 Co ) outlet concentration of enzyme in PRP, lipoxyganase mL-1 CP ) concentration of enzyme in PRP, lipoxyganase mL-1 Cs ) concentration of enzyme in SRP, lipoxyganase mL-1
Ind. Eng. Chem. Res., Vol. 44, No. 19, 2005 7473 CT ) concentration of enzyme in top phase, lipoxyganase mL-1 D ) column inner diameter, m DD ) diffusion coefficient of enzyme, m2 s-1 HC ) clear liquid height, m HD ) height of dispersion at steady state, m kC ) true heavy SRP mass-transfer coefficients, m s-1 kD ) true light-phase mass-transfer coefficient, m s-1 kD a ) dispersed-side mass-transfer coefficient, s-1 KD ) overall true mass-transfer coefficient, m s-1 L ) volumetric flow rate of PRP, m3 s-1 m ) partition coefficient of enzyme, dimensionless MA ) molar mass of enzyme, kg kmol-1 NP ) physical property group, dimensionless S ) cross sectional area of the column, m2 T ) operating temperature, °C V ) volume of dispersion and continuous phase, mL vD ) superficial dispersed-phase velocity, mm s-1 Subscripts B ) bottom phase C ) continuous phase D ) dispersed phase P ) physical s ) SRP T ) top phase Greek Symbols D ) fractional dispersed-phase hold-up, dimensionless µ ) viscosity, Pas F ) density, kg m-3 ∆H ) HD - HC height difference before and after dispersion, mm ∆F ) density difference between the phases, kg m-3 σ ) interfacial tension, m N m-1 θ ) temperature, K
Literature Cited (1) Gardner, H. W. Lipoxygenase as a Versatile Biocatalyst. J. Am. Oil Chem. Soc. 1996, 73, 1347. (2) Israedl, E.; Cohn, J.; Dube, L.; Drazen, J. M. Effect of Treatment With Zileuton, A. 5-Lipoxygenase Inhibitor in Patients with Asthma. A Randomized Controlled Trial. J. Am. Med. Asoc. 1996, 275, 931. (3) Wenzell, S. E.; Kamada, A. K. Zileuton: the First 5-Lipoxygenase Inhibitor for the Treatment of Asthma. Ann. Pharmacother. 1996, 30, 858. (4) Kane, G. C.; Police, M.; Kim C. J.; Cohn, J.; Dwworski, R. T.; Murray, J. J.; Sheller, J. R.; Fish, J. E.; Peters, S. P. A Controlled Trial of the Effect of the 5-Lipoxygenase Inhibitor, Zileuton on Lung Inflammation Produced by Segmental Antigen Callenge in Human Beings. J. Alleerg. Clin. Immunol. 1996, 97, 646.
(5) Albertson, P. A. Partition of Cell Particles and Macromolecules; Wiley: New York, 1971. (6) Hustedt, H.; Kroner, K. H.; Kula, M. R. Partitioning in Aqueous Two Phase Systems; Walter, H., Brooks, E. D., Fisher. D., Eds.; Academic Press: New York, 1985. (7) Albertson, P. A. Partition of Cell Particles and Macromolecules, 3rd ed.; Wiley: New York, 1985. (8) Ufuk, G. Partitioning of Bovine Serum Albumin in an Aqueous Two-Phase System: Optimization of Partition Coefficient. J. Chromatogr., B 2000, 743, 259. (9) Rostami Jafarabad, Kh.; Kale, D. D.; Joshi, J. B. Effect of Viscosity and Drag Reducing Agent on Mass Transfer Coefficient in Liquid-Liquid Spray Column. Solvent Extr. Ion Exch. 1990, 8, 669. (10) Axelrod, B.; Cheesbrough, T. M.; Iaakso, S. Lipoxygenase from Soybean. Methods Enzymol. 1981, 71, 441. (11) Anges, N. Sz.; Szajani, B. Sz; Marczy, J. Sz. A Simple and Rapid Method Enhancing of Lipoxygenase-1 to Lipoxygenase2+Lipoxygenase-3 Isoenzyme Activity Ratio in Soybean Meal Extracts. Biotechnol. Tech. 1998, 12, 389. (12) Rostami Jafarabad, Kh.; Sawant, S. B.; Joshi, J. B. Enzyme and Protein Mass Transfer Coefficient in Aqueous Two-Phase Systems-1. Spray Extraction Columns. Chem. Eng. Sci. 1992, 47, 1, 57. (13) Pathak, S. P.; Sudha, S.; Sawant, S. B.; Joshi, J. B. New Salt-PEG Systems for Two Phase Aqueous Extraction. Biochem. Eng. J. 1991, 46, B31-B34. (14) Laddha, G. S.; Krishman, T. R.; Vishwanathan, S.; Vedaiyan, S.; Degaleesan, T. E.; Hoelscher, H. E. A. I. Some Performance Characteristics of Liquid-Phase Spray Column. Chem. Eng. J. 1976, 22, 456. (15) Wijffels, J. B.; Rietema, K. Flow Patterns in Axial Mixing in Liquid-Liquid Spray Columns. Trans. Inst. Chem. Eng. 1972, 50, 224. (16) Joshi, J. B. Gas Dispersion in Bubble Columns. Chem. Eng. J. 1982, 24, 213. (17) Kumar, A.; Hartland, S. Prediction of Drop Size Produced by a Multiorifice Distributor. Trans. Inst. Chem. Eng. 1982, 60, 35. (18) Treybal, R. E Mass Transfer Operations; McGraw-Hill: New York, 1980. (19) Zuber, N.; Findlay, J. A. Average Volumetric Concentration in Two Phase Flow System. ASME J. Heat Transfer 1965, 87, 453. (20) Joshi, J. B.; Ranade, V. V.; Gharat, S. D.; Lele, S. S. Sparged Loop Reactors. Can. J. Chem. Eng. 1990, 68, 705. (21) Pawar, P. A.; Rostami, Kh.; Sawant, S. B.; Joshi, J. B. Enzyme Mass Transfer Coefficient in Aqueous Two Phase Systems: Spray Extraction Columns. Chem. Eng. Commun. 1993, 122, 151. (22) Geankoplis, C. J. Transport Processes: Momentum, Heat and Mass; Allyn and Bacon: London, 1983.
Received for review January 3, 2005 Revised manuscript received May 23, 2005 Accepted June 8, 2005 IE058001T
