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Ind. Eng. Chem. Res. 2009, 48, 1574–1578
Some Studies of Soybean Lipoxygenase-II Leaching Employing a Stirred Tank Reactor Khosrow Rostami,*,† Hamid Bekhteyari,‡ Armita Farahmand,§ Sohyla Azarian,† and Nishi Kazuhiko| Biotechnology Center, Iranian Research Organization for Science and Technology, P.O. Box 15815-3538, Tehran 15814, Iran, Department of Science and Research, DiVision of Agricultural Sciences, Azad UniVersity, Ashrafee Esfahani Express Way, Hesarak, P.O. Box 14515-775, Tehran, Iran, International Center for Science & High Technology & EnVironmental Sciences, Kerman, P.O. Box 76315-117, Iran, and Faculty of Engineering, DiVision of Materials Science and Chemical Engineering, Yokohama National UniVersity, 79-5, Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan
In the present work, a procedure for leaching of lipoxygenase-II from soybean flour has been developed. The effect of operational parameters such as temperature, mixing speed, pH, sodium salts, leaching period, and reactor scale on enzyme extraction was studied. It has been found that analytical grade n-hexane is more advantageous for defatting of soybean flour than is industrial grade. Further, lipoxygenase-II was leached from defatted soybean flour by using sodium phosphate buffer of 0.05 M strength of pH 6.5. It has also been observed that by increasing the medium temperature from 4 to about 25 ( 1 °C, higher enzyme could be leached. The effect of a wide range of agitator speeds on the enzyme leached was investigated. It has been found that at 5 rps, a considerable amount of lipoxygenase-II could be leached. A vector diagram of the agitated tank and scale-up at 5 and 1.67 rps using CFD has been presented, respectively. Lsqnonlin Matlab version 6.5 was used to analyze the data statistically. A correlation has been developed. 1. Introduction Lipoxygenases (EC 1.13.11.12) are produced from animals, microorganisms, and plants. Lipoxygenase has several applications in a number of industrial processes, such as catalyzing the peroxidation of fatty acids that contain cis,cis-1,4-pentadiene moieties.1-3 Lipoxygenases are multifunctional enzymes, which catalyze at least three different types of reactions: (I) dioxygenation of lipid substrates (dioxygenase reaction), (II) secondary conversion of hydroperoxy lipids (hydroperoxidase reaction), and (III) formation of epoxy leukotrienes (leukotriene synthesis reaction).1 Lipoxygenases govern a significant key role in various development stages including seeding, germinating, vegetative growth, signaling in wounding, and induction of cell death.4,5 Moreover, lipoxygenase can be produced from plant, animal, and microbial sources. Production of lipoxygenase from microbial sources at present is not very popular.6 However, literature does not address a detailed procedure for leaching of lipoxygenase from plant sources; therefore, it was desirable to attempt a systematic investigation of it.7,8 Soybean meal remaining after oil leaching contains a number of important components such as proteins, vitamins, phospholipids, and an important potential enzyme such as lipoxygenase.9-11 Also, hydroperoxide lyase, hydroperoxide peroxygenase, and hydroperoxide isomerase exist in soybean meal.3,12 Lipoxygenase appears to be regiostereoselective1 and has a preference to oxygenate certain polyunsaturated fatty acids, producing conjugated unsaturated fatty acid hydroperoxides.2 These and other biochemical and enzymatic properties would enable the employment of the lipoxygenase for the manufacture of specific * To whom correspondence should be addressed. Tel./fax: +98-2188838350. E-mail:
[email protected]. † Iranian Research Organization for Science and Technology. ‡ Azad University. § International Center for Science & High Technology & Environmental Sciences. | Yokohama National University.
products.13 Most of the work published on lipoxygenase deals with its deactivation to produce seeds having lower off-flavor.2 Furthermore, CFD code (R-Flow Co. Ltd. of Japan was used for calculation) of 5 rps stirring speed has been presented for both scales. 2. Materials and Methods 2.1. Soybean Seed, Chemicals, and Reagents. Soybean seed of William’s variety was purchased from a local market in a bag of 12 kg, which was stored at the appropriate place, and the required quantity for grinding and oil leaching was used. Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, disodium tetrabourate, boric acid, Tween 20, sodium acetate, acetic acid, orthophosphoric acid, bovine serum albumin (Fraction V), and n-hexane were purchased from Merck. Coomassie Brilliant Blue G250 procured from Fluka of Switzerland and corn meal agar (BBL) were used. Ethanol and N2 were bought from a local market. Further, archidonic acid and linoleic acid were purchased from Sigma Co. 2.2. Preparation of Soybean Flour. 2.2.1. Milling and Defatting of Soybean Seed. The soybean seed was washed, dried, and sieved to obtain seeds of similar size. The soybean seed was milled at such condition that temperature of milling did not rise above 40 °C to avoid enzyme deactivation. The temperature of milling was always in the range of 40 ( 1 °C by adding n-hexane into the chamber of the milling machine. After the flour was cooled to room temperature, solvent was extracted to obtain defatted flour. For defatting soybean flour, 200 g of soybean flour was introduced into a reflux unit containing at least 2000 mL of n-hexane, using the immersion type of solvent extraction.7 At the end of each batch, after decanting, fresh n-hexane was added to the partially treated milled flour. This procedure was followed in defatting soybean flour unless n-hexane color was unchanged or mentioned otherwise. The defatted flour was allowed to air-dry overnight,
10.1021/ie801154z CCC: $40.75 2009 American Chemical Society Published on Web 12/16/2008
Ind. Eng. Chem. Res., Vol. 48, No. 3, 2009 1575 Table 1. Effect of Different Sodium Salts of 0.05 M on Enzyme Leaching no. 1 2
Figure 1. Schematic diagram of the experimental setup.
so that the leftover n-hexane evaporated, was packed airtight and stored at the appropriate condition, and used for enzyme leaching within a month. 2.3. Experimental Details. 2.3.1. Experimental Apparatus. The schematic diagram of the experimental setup of the stirred tank reactor has been presented in Figure 1. A double bladed propeller impeller used as mixer was made of glass. The impeller was mounted on a shaft, which was driven by Stuart Scientific SS3 A.C motor. The 4 baffled stirred tank reactor was jacketed to control its temperature during operation. Water of specified temperature was circulated in the jacket of the reactor by a pump to maintain the reactor temperature at a predetermined point. The water bath was used to maintain the reactor temperature as desired. The total volume of the doublejacketed glass-made stirred reactor was 150 mL. The reactor inside diameter and height were 50 and 80 mm, respectively. In addition, a double-jacketed glass-made vessel of 625 mL was used to investigate the effect of scale. The vessel was stirred with a doubled bladed propeller of standard dimension (DT/DI: 3:1) located at 1/3DT of the tank’s bottom. pH measurement was carried out using Horiba (F-12), made in Japan. A Shimadzu UV-2101 PC spectrophotometer (continuous) was used to analyze the enzyme activity. 2.3.2. Experimental Procedure. 2.3.2.1. Leaching of Lipoxygenase-II. Seven grams of soybean flour was introduced into a buffered doubled distilled water of pH 6.5, maintained at 25 ( 1 °C, and at appropriate intervals leaching was terminated and the stirred tank content was poured into centrifuge tubes, balanced, and centrifuged at 16 300g for 10 min and maintained at 4 °C. The supernatant was used for lipoxygenase-Π activity measurements, and the precipitate was discarded. The excess supernatant was divided into a number of 3 mL vials and frozen at -20 °C without addition of any stabilizer to be available for further studies. The procedure presented by Nemeth was used to prepare sodium archidonic.8 The effect of temperature, buffer, agitator speed, operational duration, and geometrical scale-up on activity of leached enzyme was studied. Effect of Temperature. The effect of temperature on leaching of lipoxygenase-II was conducted at 4 and 10 °C, and thereafter at 20, 30, and 40 °C successively. In 70 mL of distilled water buffered with 0.05 M sodium phosphate buffer of pH 6.5 was added 7 g of defatted flour. The leaching was carried out in stirred tank reactor at a speed of 5 rps for 10 min. The content of the stirred tank reactor after termination was centrifuged at 16 300g for 10 min at 4 °C, and the supernatant was used for enzyme activity determination. To study the effect of each temperature point on the enzyme leaching, a fresh experiment at a specified temperature was run. Effect of Buffer. The effect of different sodium phosphate buffer on the activity of leached enzyme was studied. The aforementioned amount of soybean defatted flour was introduced into 70 mL of 0.05 M strength of different sodium buffer such
3
buffer type sodium sodium sodium sodium sodium sodium sodium sodium sodium
acetate acetate phosphate phosphate phosphate phosphate phosphate borate borate
pH
enzyme activity (U mL-1)
5.5 6.0 6.5 6.8 7.0 7.5 8 8.5 9.0
3230 3530 6100 5270 4460 3875 2360 1730 1230
as sodium acetate, sodium phosphate, and sodium borate, where the medium pH and activity are presented in Table 1. Leaching of the enzyme in the respective experiment was carried out under specified conditions as mentioned earlier for 10 min. Effect of Agitation. The aforementioned system and stirred tank reactor were used to examine the effect of a wide range of impeller speed from 1.66 to 8.33 rps, respectively, on the enzyme leached. The stirred tank reactor was operated for 10 min, and the medium temperature was maintained at 25 °C; a fresh medium was used for each set of experiment performed. Effect of Operational Period. The effect of a wide range of operational period from 5 to 40 min on the enzyme leaching was studied employing the aforesaid medium and stirred tank reactor agitated at 5 rps, and the medium temperature was maintained at about 25 °C. For each set of experiments, fresh defatted flour under specified conditions was used. Effect of Geometrical Scale-Up. The effect of agitator speed was determined using 70 g of defatted flour added to 500 mL of 0.05 M of sodium phosphate buffered medium of pH 6.5, which was maintained at about 25 °C. 2.4. Analytical Analysis. 2.4.1. Lipoxygenase-II Activity Measurement and Calculation. Lipoxygenase-Π activity was measured using the Axelrod method.7 The activity measurement was followed using a blank solution of 2.9 and 0.1 mL of buffer and substrate at 238 nm for 5 min using a UV-2101 PC Shimadzu spectrophotometer; the maximum linear rate for both the test and the blank was obtained. The enzyme medium temperature in a quartz cuvette for assay was about 25 °C and had pH 6.1. The enzyme activity has been expressed in terms of 0.001 changes in A238 per min at pH 6.1 having 0.2 M strength and 25 °C, where 0.1 mL of supernatant was added in a 3.0 mL reaction volume. Therefore, one unit of enzyme is defined as the amount of enzyme that catalyzes the formation of 1 µmol of hydroperoxides of archidonic acid per min at 25 °C and pH 6.1. The Bradford method for protein estimation was used.14 3. Results and Discussion 3.1. Effect of Different Sodium Buffer. It has been depicted in Table 1 that slightly acidic or nearly neutral buffers were more suitable for extraction of lipoxygenase-II than were acidic and or alkaline buffers. Phosphate buffer of pH 6.5 resulted in the highest enzyme extraction as compared to the other environment. 3.2. Effect of Temperature. The result has been presented in Figure 2. It can be seen as the temperature was increased from 10 to 25 °C that the activity of leached enzyme has enhanced. As the temperature was increased from 25 to 28 °C, it had no remarkable effect on the enzyme extracted. However, further increasing the temperature led to an adverse effect on the activity of enzyme leached. 3.3. Effect of Agitator Speed. It has been illustrated in Figure 3a that as the stirrer speed was increased from 1.66 to 5
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Figure 2. Effect of temperature on leaching of lipoxygenase-II. Figure 4. Effect of operational period on leaching of lipoxygenase-II.
calculation for obtaining the vector diagram, the equations of continuity and motion and the k-ε model as the turbulent model were used. Equation of continuity: ∂F ) + (V · ∇)F + F ∇ · V ) 0 ∂t
(2)
Equation of motion:
( ∂V∂t + (V · ∇)V) ) - ∇ p + ∇ ((µ + µ ) ∇ V) + F(g)
F
Figure 3. (a) Effect of stirrer speed on leaching of lipoxygenase-II. (b) Vector diagram of the vertical plane in agitated tank at a stirrer speed of 5.00 rps.
rps, the activity of the leached enzyme also increased. The mass transfer in the form of enzyme activity from the soybean flour (solid) to the buffer (liquid) has been increased as a result of stirrers’ higher speed.15,16 The solid-liquid mass transfer coefficient in turbulent flow stirred tank has been represented by the following equation.17-19 Sh ) 2 + A(ε1 ⁄ 3dp3 ⁄ 4/ν)BSc1 ⁄ 3
(1)
where A and B are constants, Sh is the Sherwood number, and Sc is the Schmidt number. ε is usually proportional to the third power of the stirrer speed in the turbulent flow stirred tank; the mass transfer coefficient kL increases by the Bth power of the agitation speed. The vector diagram of the vertical plane in agitated tank at 5 rps has been presented in Figure 3b. In
e
(3)
where V is the vector of velocity, t is time, F is density, p is pressure, g is gravity, and µ and µe are the viscosity and the Eddy viscosity, respectively. The values of the water density and viscosity at 25 °C were used. It can be observed that a strong axial flow has been developed in the stirred tank reactor, which corresponds to sound dispersion in the system. Therefore, the increase in the mass transfer coefficient may be the cause of the increase in the enzyme activity of the enzyme. Furthermore, the physicochemical property of defatted soybean flour may also contribute to the increase in the enzyme activity.20 Also, increasing the agitation speed did not have any significant effect on the enzyme leaching, which may be due to the high mass transfer rate at 5 rps, and the enzyme concentration might have reached equilibrium concentration within 10 min of operation. The slight decrease of activity at large agitation speed may be due to the deactivation of enzyme at the gas-liquid interface.21 3.4. Effect of Operational Period. The result has been depicted in Figure 4. It can be seen that as the leaching period was increased from 5 to 10 min of operation, consequently higher leaching of enzyme has been achieved. However, during 5 min of initial operation, the amount of enzyme leached was low and inconsistent, and it appears the mixture of defatted flour and buffer might have not been well equilibrated. Further, increasing the extraction period more than 10 min had no remarkable effect on leaching of enzyme. 3.5. Correlation Developed. Hence, it was desirable to develop a generalized correlation with the above parameters to correlate the observed data. The correlation of the proposed correlation equals 0.98 and is as follows: U ) 0.0001t0.3T 0.17r0.91
(4)
where r is agitator speed (rps), t is operational period (s), T is the operating temperature (K), and U is the enzyme activity (U/mL). The correlation (4) agreement is effective (SD 6.95), and the correlation captures the effect of parameters. It can be concluded that the agitation speed has a governing effect on
Ind. Eng. Chem. Res., Vol. 48, No. 3, 2009 1577
Figure 6. Impellers tip velocity relation and lipoxygenase-II activity.
presented in Figure 5b, which was developed using CFD at 1.67 rps. It can be observed that the axial flow through the whole tank was generated even at a low stirrer speed. This result is due to the geometrical similarity of both tanks. Although the liquid volume increased about 10 times, the area of gas-liquid interface increased only 2.5 times. It is thought the relative decrease in the interfacial area to the volume prevented the enzyme deactivation at the gas-liquid interface.21 It is usually useful for the industries to determine the scale-up factor to characterize the operational conditions. When the scale-up factor is the same, an identical performance is achieved. The mass transfer of solid-liquid is influenced by several factors22 such as flow pattern,23 shear of impeller,24 and complex behavior of dissipation energy.25-27 Figure 6 shows the relation between tip speed of impeller and the enzyme activity. It can be observed that as the tip speed was increased to 0.15 m/s, the enzyme activity increased. 4. Conclusions
Figure 5. (a) Effect of stirrer speed on leaching of lipoxygenase-II in a scaled-up stirred tank reactor. (b) Vector diagram of the vertical plane in a scaled-up tank at a stirrer speed of 1.67 rps.
leaching of lipoxygenase-2. The present phenomena may be due to the solid-liquid mass transfer rate. 3.6. Effect of Geometrical Scale-Up. 3.6.1. Effect of Agitation Speed. 70 g of defatted flour was added to 500 mL of 0.05 M of sodium phosphate buffered medium of pH 6.5, which was maintained at about 25 °C. The effect of agitation on lipoxygenase-II leaching in the range of 1.66-8.33 rps was investigated. At appropriate intervals, the experiment was terminated to centrifuge the reactor content, and in a step ahead the supernatant was analyzed for lipoxygenase-II. The result has been illustrated in Figure 5a. It can be seen as the agitator speed was increased to about 3 rps the amount of lipoxygenaseII leached increased. Further, increasing agitation speed higher than about 3 rps had no significant effect on the enzyme extracted. This value is about 5 rps in the case of smaller tank. The vector diagram of the scaled-up stirred tank has been
In the present work, an effective and simple procedure for extraction of lipoxygenase-II from soybean has been presented. During the experimental operation, it was realized that analytical grade n-hexane is less harmful to the enzyme than is the industrial grade, which was used for the defatting process. It has been observed that sodium phosphate buffer of 0.05 M of pH 6.5 has an appreciable effect on enzyme leaching. Buffers of acidic or alkaline nature have no considerable effect on enzyme leaching. It has been found that by increasing the medium temperature from 10 to 25 °C, the amount of enzyme leached has increased. Increasing the impeller speed from 1.66 to 5 rps, the amount of enzyme leached has increased, indicating the rate of mass transfer is increased. Further, it has been found that within 10 min of operation, a maximum amount of lipoxygenase II could be leached. Also, the effect of geometrical scale-up such as mixer speed on lipoxygenase-II leaching shows that due to the well-developed velocity profile in the stirred tank, Figure 5b at lower speed, say 3 rps, the same amount of enzyme has been leached. It is realized by setting up the agitation speed on the basis of the tip speed equal to 0.15 m/s that the same amount of enzyme has been leached in the scaled-up stirred tank. Acknowledgment We thank the International Center for Science & High Technology & Environmental Sciences, Kerman-Iran, and IROST for providing financial support for this work. Thanks are due to Dr. Agnes Nemeth, SZ, for her guidance in the analysis of lipoxygenase-II.
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Nomenclature A ) a constant B ) a constant dp ) particle diameter, m kL ) liquid side mass transfer coefficient, m/s g ) acceleration due to gravity, m s-2 r ) agitator speed, rps t ) operational period, s T ) operating temperature, K U ) enzyme activity, U/mL V ) velocity, m/s Sc ) the Schmidt number, dimensionless Sh ) the Sherwood number, dimensionless
Greek Symbols ε ) the dissipation energy, w µ ) viscosity, Pa · s µe ) Eddy viscosity, Pa · s ν ) kinematics viscosity, m2/s F ) density, kg/m3
Literature Cited (1) Bornscheuer, U. T. Enzymes In Lipid Modification; Wiley-VCH Publication: New York, 2000; Vol. 309. (2) Lius, K. Soybeans: Chemistry, Technology and Utilization; Kluwer Academic Publisher: Norwell, MA, 1997. (3) Doehlert, D. C.; Wicklow, D. T.; Gardner, H. W. Evidence implicating the lipoxygenase pathway in providing resistance to soybeans against Aspergillus FlaVus. Phytopathology 1993, 83, 1473. (4) Porta, H.; Roach-Sosa, M. Plant lipoxygenases physiological and molecular features. Plant Physiol. 2002, 15. (5) Siedow, J. N. Plant lipoxygenase: structure and function. Annu ReV. Plant Phys. 1991, 42, 145. (6) Bisakawski, B.; Kermasha, S.; Klopfenstein, M. L. Partially purified lipoxygenase from Fusarium Oxysproum: Characterization and kinetic studies. Process Biochem. 1995, 30, 261. (7) Axelrod, B.; Cheesbrough, T. M.; Laakso, S. Methods in Enzymology; Academic Press: New York, 1981; Vol. 71, p 441. (8) Nemeth, A. S. Z.; Szajani, B. J.; Marczy, S. Z.; Simon, M. L. A simple and rapid method for enhancing of lipoxygenase-1 to lipoxygenase-2 and lipoxygenase-3 isoenzyme activity ratio in soybean meal extracts. Biotechnol. Tech. 1998, 12, 389. (9) Patterson, H. B. W. Handling and Storage of Oil Seeds, Oils, Fats and Meal; Elsevier Applied Science: New York, 1989. (10) Semon, M.; Patterson, M.; Wyborney, P. Soybean Oil; www.wsu. edu.
(11) Institute of Food Research, Soya; www.ifr.bbsrc.uk. (12) Gardner, H. W.; Weisleder, D.; Plattner, R. D. Hydroperoxide lyase and other hydroperoxide metabolizing activity in tissues of soybean, Glycine Max. Plant Physiol. 1991, 97, 1059. (13) Folic, V. A.; Nivar-Aristy, R. A.; Kragewski, M.; Prinyawiwatkul, W.; Marshall, W. E. Lipoxygenase contributes to the oxidatiox of lipids in human atherosclerotic plaques. J. Chin. InVest. 1995, 96, 504. (14) Bradford, M. M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248. (15) Nagata, S. Mixing, Principles and Application; Wiley: Kodansha, Tokyo, 1975. (16) Nienow, A. W. Dissolution mass transfer in a turbine agitated baffled vessel. Can. J. Chem. Eng. 1969, 47, 248. (17) Miller, D. N. Scale-Up of agitated vessels. Mass transfer from suspended solute particles. Ind. Eng. Chem. Process Des. DeV. 1971, 10, 365. (18) Levins, D. M.; Glsdtonbury, J. R. Particle-liquid hydrodynamics and mass transfer in a stirred vessel. Trans. Inst. Chem. Eng. 1972, 50, 132. (19) Kikuchi, K.; Tadakuma, Y.; Sugawara, T.; Ohashi, H. Effect of inert particle concentration on mass transfer between particles and liquid in solid-liquid two-phase up-flow through vertical tubes and in stirred tanks. J. Chem. Eng. Jpn. 1987, 20, 134. (20) Riaz, M. N. Soy Applications in Food; CRC/Taylor and Francis: Boca Raton, FL, 2006; p 19. (21) Patill, N. S.; Ghadge, R. S.; Sawant, S. B.; Joshi, J. B. Lipase deactivation at gas- liquid interface and its subsequent reactivation. AIChE J. 2000, 46, 1280. (22) Hixson, A. W.; Baum, S. J. Agitation mass transfer coefficients in liquid-solid agitation systems. Ind. Eng. Chem. 1941, 33, 478. (23) Kaminoyama, M.; Saito, F.; Kamiwano, M. Flow analogy of pseudoplastic liquid in geometrically similar stirred vessels based on numerical analysis. J. Chem. Eng. Jpn. 1990, 23, 214. (24) Niesmak, G. Feststoffverteilung und Leistungsbedarf geru¨hrter Suspensionen. Chem.-Ing.-Tech. 1983, 55, 318. (25) Yoo, H. S.; Hill, D. F.; Balchandar, S.; Adrian, R. J.; Ha, M. Y. Reynolds number scaling of flow in a Rushtan turbine stirred tank. Part 1Mean flow, circular jet and tip vortex scaling. Chem. Eng. Sci. 2005, 60, 3169. (26) Derksen, J. J.; Doelmann, M. S.; Van Den Akker, H. E. A. Three dimensional LDA measurements in the impeller region of a turbulent stirred tanks. Exp. Fluids 1999, 27, 522. (27) Lamberto, D. J.; Alvarez, M. M.; Muzzio, F. J. Experimental and computational investigation of laminar flow structure in a stirred tank. Chem. Eng. Sci. 1999, 54, 919.
ReceiVed for reView July 27, 2008 ReVised manuscript receiVed November 10, 2008 Accepted November 12, 2008 IE801154Z
