at SciVerse ScienceDirect LWT - Food Science and Technology 50 (2013) 519e525 Contents lists available LWT - Food Science and Technology journal homepage: www.elsevier .com/locate/ lwt Characterization of immobilized phospholipase A1 on magnetic nanoparticles for oil degumming application Dianyu Yu a,b, Ying Ma a,*, Sophia J. Xue c, Lianzhou Jiang b, John Shi c,** a School of Food Science and Engineering, Harbin Institute of Technology, Harbin 150090, China b School of Food Science and Technology, Northeast Agricultural University, Harbin 150090, China cGuelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, Ontario N1G 5C9, Canada a r t i c l e i n f o Article history: Received 23 April 2012 Received in revised form 17 August 2012 Accepted 20 August 2012 Keywords: Phospholipase A1 Immobilization Magnetic Fe3O4/SiOx-g-P(GMA) nanoparticles Oil degumming Soybean oil * Corresponding author. Tel.: þ86 451 86282903. ** Corresponding author. Tel.: þ1 519 780 8035. E-mail addresses: maying@hit.edu.cn (Y. Ma), john 0023-6438/$ e see front matter Crown Copyright � http://dx.doi.org/10.1016/j.lwt.2012.08.014 a b s t r a c t Phospholipase A1 (PLA1) was immobilized onto magnetic Fe3O4/SiOx-g-P(GMA) nanoparticles. The properties of immobilized PLA1 were compared to free PLA1 in order to assess the feasibility of utilizing the immobilized enzyme for degumming of soybean oil. The immobilized PLA1 process retained 2066.67 m/g activity with 64.7% immobilization efficiency. Compared to free PLA1 process, the immo- bilized PLA1 had a broader pH-activity profile of pH 4.5e6.5 and was remarkably stable at 45 �Ce55 �C for 7 h. The optimum temperature for the immobilized and free PLA1 was 60 �C and 50 �C, respectively. After 10 cycles through the soybean oil degumming process at 55 �C, the immobilized PLA1 still possessed more than 80% of its initial activity. The water degumming process was carried out at 55 �C and pH 6.0. The residual phosphorus content was 9.6 mg/kg, which is to meet oil safety standard and suitable for the physical refining of soybean oil. Crown Copyright � 2012 Published by Elsevier Ltd. All rights reserved. 1. Introduction Degumming process is the first stage in the crude vegetable oil refining process and serves to remove phospholipids and muci- laginous gums from the crude oil. The phospholipids in the crude oil can cause oil discoloration and off-flavors. Therefore, the removal of the phospholipids is required when producing high- quality refined oil. The traditional degumming processes used to remove phospholipids are water degrumming (Jahani, Alizadch, Pirozifard, & Qudsevali, 2008), acid treatment (Dijkstra, 2010), and ultrafiltration (Singh, Rezac, & Pfromm, 2009). These tradi- tional methods do not remove all phospholipids and as a result they do not achieve the low phosphorus levels (<10 mg/kg) required for industrial applications. Although the traditional water degumming process is effective for hydratable phosphatides (those phospho- lipids that absorb water, swell and become insoluble in the oil phase and are removed), a significant amount of nonhydratable phosphatides (NHP) will remain in the oil. Normally, about 90% of the phosphatides in soybean oil are hydratable phosphatides and the residual NHP remaining after .shi@agr.gc.ca (J. Shi). 2012 Published by Elsevier Ltd. All water degumming process can be easily removed during chemical refining process. However, when the soybean seeds are seriously damaged, the hydratable phosphatide levels could be reduced by 50% (Rossell & Pritchard, 1991). Although the NHP can be removed simultaneously with the free fatty acids (FFA) by chemical refining process, a greater amount of acid must be used, which results in high cost for waste water treatment (O’Brien, Farr, & Wan, 2000, p. 155). Other technical and environmental demerits of chemical refining process include low yields, nutrient loss, high energy consumption, large amount of water consumption, large disposal of highly polluted waste, etc. Some physical refining processes have gained popularity as more environmentally friendly treatments with lower operating costs. Nonetheless a small amount of NHP will remain in the degummed soybean oil even after physical refining process (O’Brien et al., 2000, p. 155). It is therefore important that a new approach be developed for the complete removal of phosphatides from vegetable oils. Phospholipase A1 (PLA1) has been successfully reduce phos- phorus levels to less than 10 mg/kg on the degumming process of vegetable oils (Manjula, Jose, Divakar, & Subramanian, 2011; Sheelu, Kavitha, & Fadnavis, 2008; Yang, Wang, Yang, Mainda, & Guo, 2006; Yang, Zhou, Yang, Wang, & Wang, 2008). Phospholi- pase A1 (PLA1) hydrolyzes the acyl group of phospholipids at the sn- 1 position, liberating the fatty acid, and producing 2-acyl-1- lysophospholipid (Richmond & Smith, 2011). Most studies on the rights reserved. Delta:1_given name Delta:1_surname Delta:1_given name Delta:1_surname Delta:1_given name mailto:maying@hit.edu.cn mailto:john.shi@agr.gc.ca www.sciencedirect.com/science/journal/00236438 http://www.elsevier.com/locate/lwt http://dx.doi.org/10.1016/j.lwt.2012.08.014 http://dx.doi.org/10.1016/j.lwt.2012.08.014 http://dx.doi.org/10.1016/j.lwt.2012.08.014 D. Yu et al. / LWT - Food Science and Technology 50 (2013) 519e525520 degumming of crude vegetable oils are based on the free PLA1 (Manjula et al., 2011; Yang et al., 2006, 2008). However, the free PLA1 is very sensitive to pH and temperature, and cannot be easily reused. Therefore, the immobilization of PLA1 is a promising tech- nology and has a large impact on the industrial scale degumming operations. Recently the immobilization technology for enzymes has expanded into nanomaterials such as nanopolymers (Arica, Öktem, Öktem, & Tuncel, 1999; Cabrera-Padilla et al., 2012) and nano- particles (Jiang et al., 2009; Khoshnevisan et al., 2011; Lee et al., 2009; Lei et al., 2009; Lei et al., 2011; Liu et al., 2011). The nano- particle can provide a larger surface area for the attachment of enzymes, leading to higher enzyme loading per unit mass of particles (Khoshnevisan et al., 2011). However, for industrial applications, the immobilized enzymes are very difficult to sepa- rate from the reaction medium via centrifugation or filtration. Thus, the magnetic nanoparticles have been used as alternative immo- bilized enzymes because they can be easily and rapidly separated from the reaction medium when placed in a magnetic field. The magnetic nanoparticles are also increased loading and improved stability of the immobilized biomolecules (Bahar & Celebi, 2000; Khoshnevisan et al., 2011; Lei et al., 2009; Lei et al., 2011; Liu et al., 2011; Zeng, Luo, & Gong, 2006). Sheelu et al. (2008) used the immobilized Lecitase (phospholipase A1 on gelatin crosslinked with glutaraldehyde) to degum rice bran oil without loss of enzyme activity even after six recycles. In our previous work, the Lecitase� Ultra immobilized on calcium alginate- chitosan with a high fixa- tion level but the immobilized enzyme could only be used for four cycles (Yu et al., 2012). The aim of this work was to immobilize PLA1 onto magnetic Fe3O4/SiOx-g-P (GMA) nanoparticles and achieve complete enzyme reusability through the unique separating capability of the magnetic Fe3O4/SiOx-g-P (GMA) nanoparticles when placed in a magnetic field. The conditions for immobilizing PLA1 onto the magnetic nanoparticles were determined, and the thermal stability, the effects of pH and the reusability of immobilized PLA1 were investigated when used to degum soybean oil. 2. Materials and methods 2.1. Materials The phospholipase A1 (Lecitase� Ultra) was obtained from Novozymes A/S (Bagsvaerd, Denmark). The water-degummed soybean oil with a phosphorus content of 138 mg/kg was supplied by the Fukang Oil and Fat Co. (Harbin, China). Glycidyl methacrylate (GMA) were purchased from Sigma Aldrich Chemie GmbH (Japan); g-aminopropyltriethoxysilane (APTES, 98 g/100 g), copper (I) chloride, copper (II) chloride, tetraethoxysilane (TEOS, 98 g/100 g) and dimethylformamide (DMF) were purchased from Sinopharm Chemical Reagent Co. Ltd.(China); 2,2-bipyridine (Bpy) and ammonium hydroxide (25 g/100 g wet-base) were obtained from Tianjing Chemical Reagent Company (China); triethylamine (TEA), ferric chloride hexahydrate (FeCl3$6H2O), ferrous chloride tetrahydrate (FeCl2$4H2O), and other chemicals and solvents were analytical grade, and obtained from Sinopharm Chemical Reagent Co. Ltd.(China). All other chemicals were analytical grade. 2.2. Preparation of Fe3O4/SiOx-g-P(GMA) nanoparticles 2.2.1. Preparation of Fe3O4 nanoparticles The magnetic Fe3O4 nanoparticles were prepared by using the conventional co-precipitation method (Lei et al., 2011). FeCl3$6H2O and FeCl2$4H2O were dissolved in deionized water under nitrogen, and the molar ratio of Fe3þ/Fe2þ in the solution was maintained at 1.5:1. The solution was transferred into a three-necked flask equipped with a mechanical stirrer, and placed into a 60 �C water bath. A solution of 25 g/100 g ammonium hydroxide was added slowly to the solution with constant stirring at 40 rpm until the mixture reached pH 10. The temperature was increased to 80 �C and the reaction continued for an additional 1 h. After 1 h the precipitate was extracted with a magnet, rinsed with double distilled water until pH 7.0 and then dried under vacuum for 24 h at room temperature. 2.2.2. Preparation of Fe3O4/SiOx nanoparticles Coating magnetic nanoparticles with silica (Fe3O4/SiOx) was based on the procedure of Deng, Wang, Hu, Yang, and Fu (2005) with some modifications. Two grams of Fe3O4 nanoparticle was added to a solution containing 160 mL alcohol, 40 mL water and 5 mL ammonium hydroxide (25 g/100 g). The dispersion was homogenized by ultrasonic vibration for 1 h, and then 12 mL tet- raethyl orthosilicate was slowly added with constant stirring (40 rpm) for 12 h to allow the silica to form on the surface of the magnetic nanoparticles through hydrolysis and condensation of tetraethyl orthosilicate. The precipitate was extracted with a magnet and washed with double distilled water until pH 7.0. The washed precipitate was soaked in 1 mol/L HCl for 12 h, separated with a magnet, washed with double distilled water until pH 7.0 and then dried under vacuum for 24 h at room temperature. The formed particles are the magnetic Fe3O4/SiOx nanoparticles. 2.2.3. Preparation of Fe3O4/SiOx-g-APTES nanoparticles The preparation procedures for the APTES-modified Fe3O4/SiOx nanoparticles was based on the method of Lei et al. (2011). One gram of Fe3O4/SiOx nanoparticles was dispersed into 60 mL of ethanol under ultrasonic vibration for 1 h, then 6.0 mL of ammo- nium hydroxide (25 g/100 g) was added, and sonicated for an additional 10 min to homogenize the mixture. Under continuous mechanical stirring (20 rpm), 4.0 mL of APTES was slowly added and then allowed to react for 8 h at 50 �Cwith constant stirring. The resultant product was separated with a magnet, thoroughly washed with ethanol and deionized water until pH 7.0, and then were dried under vacuum for 24 h at room temperature. 2.2.4. Preparation of magnetic Fe3O4/SiOx-g-P(GMA) nanoparticles Following the method of Lei et al. (2011), 2 g of Fe3O4/SiOx-g- APTES were dispersed into 30mL of toluene containing 4mL of TEA under electromagnetic stirring in an ice bath. After cooling the mixture to 0 �C, a solution of chloroacetyl chloride (4 mL) and toluene (8 mL) was added slowly into the dispersoid. The mixture was stirred under an electromagnetic stirrer for 10 h at room temperature. The nanoparticles were separated with a magnet, washed with toluene and ethanol thoroughly, and then were dried under vacuum for the subsequent polymerization step. For the preparation of the GMA polymer on the Fe3O4/SiOx surface, 0.5 g of the prepared nanoparticles as described above was dispersed into 20 mL of DMF/water (1:1, mL:mL). Nitrogen was bubbled into themixture for 30min in order to remove oxygen, and then [GMA]/[CuCl]/[CuCl2]/[Bpy] (100:1:0.2:2) was added. The mixture was incubated for 12 h at room temperature under continuous mechanical stirring (20 rpm) to form the poly (GMA)- grafted Fe3O4/SiOx surface. After the reaction, the poly (GMA)- grafted Fe3O4/SiOx nanoparticles was extracted thoroughly with acetone for 48 h in order to ensure the complete removal of the adhered and physically adsorbed polymer, and then, the sample was dried under vacuum for 24 h at room temperature. The final product is the magnetic Fe3O4/SiOx-g-P (GMA) nanoparticles. The scanning electron microscope (SEM) image indicated that the morphology of the particles is approximately spherical with D. Yu et al. / LWT - Food Science and Technology 50 (2013) 519e525 521 a diameter of about 250 nm. Based on X-ray Diffractometer anal- ysis, the pure magnetic nanoparticles has a typical Fe3O4 crystal phase. It also exhibited saturation magnetization of 9.92 Am2/kg and the FT-IR spectra confirmed the formation of the magnetic Fe3O4/SiOx-g-P (GMA) nanoparticles. 2.3. Immobilization of phospholipase A1 on magnetic nanoparticle The coupling reaction used to immobilize PLA1 onto the magnetic nanoparticles was carried out under different conditions to determine the optimum conditions for immobilization. The preliminary trials investigated changes the pH of the reaction mixture for pH 4.5e7.5 with 0.5 intervals, and changes to the immobilization time for 3e10 h, and changes to the amount of PLA1 for 250e750 U/mL. The magnetic nanoparticles were soaked in sodium and potassium phosphate buffers (0.1 mol/L, pH 7.0) for 24 h, and then separated from the buffer with a magnet. One gram of the activated magnetic nanoparticles was added to PLA1 solution and the mixture was shaken for 5 h at 45 �C. The magnetic nano- particles with immobilized PLA1were subsequently separatedwith a magnet. The supernatant was decanted, and the particles were washed three more times with phosphate buffer to remove the unbound PLA1. The immobilized PLA1 was collected and dried under vacuum for 24 h at room temperature. The activity of the dried immobilized enzyme was measured with the standard assay procedure (AOCS, 1997). 2.4. Enzyme activity assay The activity of PLA1 was determined using the method described by Sheelu et al. (2008) with some modifications. A soybean lecithin emulsion was prepared by dispersing 4 g of soybean lecithin in 100 mL of disodium hydrogen phosphateecitric acid buffer (0.01 mol/L, pH 5.0), and shaking for 5 min in a 50 �C reciprocating water bath (180 rpm). The enzyme solution was prepared by diluting 1 mL of commercial PLA1 in 10 mL of phosphate-citric buffer. Four milliliters of the diluted enzyme solutionwas added to the warmed lecithin emulsion and incubated for 15 min. The hydrolysis reactionwas stopped by adding 60 mL of alcohol. The long chain fatty acids released by PLA1 were neutral- ized with addition of 0.05mol/L NaOH tomaintain a fixed pH of 5.0. The enzyme activity was expressed as micromoles of NaOH consumed per min which is equivalent to 1 mmol/L of fatty acid released per min under the assay conditions. 2.5. Effect of pH and temperature on the activities of free and immobilized enzymes An emulsion consisting of 4 g/100 g soybean lecithin in 0.1mol/L phosphate buffer with pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 were used to determine the optimum pH of the free and immobilized PLA1 enzymes, respectively. The optimum tempera- tures of the free PLA1 at pH 5.0 and immobilized PLA1 at pH 6.0 was determined in a 4 g/100 g lecithin emulsion (0.1 mol/L acetate buffer) under 8 different temperatures (40, 45, 50, 55, 60, 65, 70, and 75 �C). The highest activity measured under the corresponding pH or temperature was designated as 100%, and the activities at all the remaining pH and temperatures were values proportional to that highest activity. 2.6. Thermal stabilities of free and immobilized enzymes The thermal stabilities of the free and immobilized enzymes were determined according to our pervious study (Yu et al., 2012). The thermal treatments were conducted by incubating the enzymes in 0.1 mol/L phosphate buffer (pH 5.5) at different temperatures of 40e60 �C at 5 �C intervals for 6 h. At 1 h intervals, an aliquot of the incubationmixturewas removed and immediately cooled to ambient temperature in a container of ice water. The residual enzyme activities were then measured. The activity was defined as the value proportional to the initial activity (100%). 2.7. Reusability of immobilized enzymes The determination of reusability of immobilized enzyme was used from pervious study (Yu et al., 2012). The immobilized enzyme was placed into soybean oil at pH 6.0 at 55 �C and 60 �C conditions, respectively. The hydrolysis of the phospholipids for 7 h was monitored over 10 cycles. After each cycle, the immobilized enzyme was washed with 0.1 mol/L phosphate buffer at pH 6.0 and then reused. The residual enzyme activity after each cycle was defined as the value proportional to the original activity (100%). 2.8. Batch degumming process of soybean oil Batch degumming of soybean oil was performed with free and immobilized enzymes according the method from our pervious study (Yu et al., 2012). Three hundred grams of water-degummed soybean oil and 0.36 mL citric acid (45 g/100 g) were added to a 500 mL fiber reinforced plastics container, heated to 80 �C for 20min under stirring at 500 rpm (HYJ30mixer, HuilongMixers Co., Ltd. China). The oil was cooled to 60 �C under running cold water, and then adjusted to pH 5.5 and 6.0 with 1 mol/L NaOH. Distilled water of 6 mL was added to the oil and stirred at 30 rpm for 20 min. The free or the immobilized enzyme solutionwas added to the oil at a dose rate of 250 U/kg (oil mass). The mixture was incubated with continuous stirring at 60 rpm for 7 h at 55 �C and 60 �C, respec- tively. The samples were taken at 1 h intervals for phosphorus analysis. The residual phosphorus content in the oil phase was assayed by the AOCS method of 12e55 (AOCS, 1997). 2.9. Statistical analysis All experiments were performed in triplicate. The data are re- ported as means standard deviations. One-way ANOVA was per- formed using SPSS 14 Statistical software (SPSS Inc., Chicago, IL.). Differences were considered to be significant at p � 0.05, according to Duncan’s Multiple Range Test. 3. Results and discussion 3.1. Immobilization of PLA1 During the immobilization procedure, the effects of time and pH on the activity of the immobilized PLA1 were determined and the results shown in Fig. 1A and B. Data shows that the process at pH of 6.0 and for reaction time of 5 h can obtain 65% and 67% immobi- lization efficiencies. When the pH value was higher or lower than 6.0, the immobi- lization efficiency was lower. The activity of the free enzyme, the effectiveness of enzyme utilization, and the operational stability of the immobilized enzyme might be influenced by immobilization conditions. The change in pH value can influence the electrical charges on the surface of the magnetic nanoparticle and limit the interactions between epoxy group on the surface of magnetic particle and amino groups of PLA1 (Lee et al., 2009; Zeng et al., 2006). Protein denaturation can occur under conditions of excess acid or alkali (Liu et al., 2011). The immobilization efficiency significantly increased with treatment time until it reached a maximum at 5 h, then steadily declined over the next 3 h. The 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 20 30 40 50 60 70 80 90 100 R el at iv e Ac tiv ity (% ) pH Fig. 2. pHeactivity profiles of free and immobilized PLA1 (- free PLA1, C immobilized PLA1). 3 4 5 6 7 8 30 35 40 45 50 55 60 65 70 Im m ob iliz at io n Ef fic ie nc y (% ) Time (h) A 4.5 5.0 5.5 6.0 6.5 7.0 7.5 25 30 35 40 45 50 55 60 65 70 Im m ob iliz at io n Ef fic ie nc y (% ) pH B 300 400 500 600 700 800 30 40 50 60 70 80 90 100 Concentration of Initial PLA1 (U/mL) Im m ob iliz at io n Ef fic ie nc y (% ) Activity of Im m obilized PLA 1 (U/g)1400 1500 1600 1700 1800 1900 2000 C Fig. 1. Effect of immobilization time (A) and pH (B) on the immobilization efficiency of immobilized PLA1; Effect of initial concentration of PLA1 on the immobilization effi- ciency and activity of immobilized PLA1 (C) (: immunization efficiency, - activity of immobilized PLA1). D. Yu et al. / LWT - Food Science and Technology 50 (2013) 519e525522 longer immobilization times could result in a greater load of PLA1 molecules on the surface of the support and limit substrate diffu- sion due to steric inhibition (Lei et al., 2011; Liu et al., 2011). The initial concentration of enzyme can also greatly affect the activity of the immobilized enzyme. In order to find the optimal concentration of PLA1, the immobilization of various concentration range of PLA1 from 250 to 750 U/mL on 1.0 g magnetic nanoparticle was investigated and the results are shown in Fig. 1C. The highest immobilization efficiency is 98.32% at the lowest concentration of PLA1 of 250 U/mL. Increasing the concentration of PLA1 resulted in a significant decrease in immobilization efficiency. However, the activity of the immobilized PLA1 increases with increasing PLA1 concentration. The highest activity was observed with concentra- tion of 550 U/mL. Further increases in PLA1 concentration caused a decrease in PLA1 activity that followed a corresponding decrease in immobilization. Moreover, the saturation and multilayering of the enzyme on the surface could cause intermolecular steric hindrance to substrate diffusion at the higher ratio of enzyme/support (Lei et al., 2011; Liu et al., 2011). When the ratio of enzyme/support is low, the enzyme is easily fixed on the surface of the support due to less steric hindrance and more active sites (Wang et al., 2011). Although, the immobilization efficiency was higher at low ratio of enzyme/ support, the small amount of enzyme loaded on the support contributed to the lower activity of the immobilized PLA1. The enzymatic aggregation on the surface of magnetic nanoparticles could block the active sites of the enzyme. Based on the activity of immobilized PLA1 and immobilization efficiency, as show in Fig. 1C, the optimal conditions of immobilization of PLA1 are immobiliza- tion time of 5 h, pH of 6.0, and concentration of 550 U/mL. Under such conditions, the activity of immobilized PLA1 was 2066.67 U/g support (dried base), and the immobilization efficiency was 64.7%. 3.2. Effect of pH on the activities of free and immobilized enzymes Fig. 2 shows the pH e activity profiles of the free and the immobilized enzymes from pH 4.0 to 7.5 at 50 �C. The maximum activity for free PLA1 occurred at pH 5.0. This indicates that the free enzyme hydrolyzes phospholipids under acid conditions. The optimum pH of immobilized enzyme was pH of 6.0. The immobi- lized enzymemaintained more than 90% activities at pH of 4.5e6.5. At pH of 4.5 and 5.5, the activities of free enzyme are 80.6% and 85.2%, respectively. The activity of the free PLA1 dramatically decreased at pH values above or below pH 5.0. These results suggest that the free PLA1 was more sensitive to environmental pH compared to the immobilized enzyme. The results also show that the optimum pH of immobilized PLA1 shifted toward the neutral region, although the optimum pH value of free PLA1 is in basic condition. The phenomenon of shifting pH region might be due to the immobilized enzymemolecules preserved from conformational alterations induced by pH changes (Madoery, Gattone, & Fidelio, 1995). The covalent immobilization of PLA1 by reacting epoxy groups on the surface of magnetic particle, with the amino groups of PLA1., resulted in an abundance of amino groups on the carriers. After the proton transfers to the amino acid and residues at the 0h 1h 2h 3h 4h 5h 6h 7h 0 10 20 30 40 50 60 70 80 90 100 110 R el at iv e Ac tiv ity (% ) Time A D. Yu et al. / LWT - Food Science and Technology 50 (2013) 519e525 523 active site, it becomes less hindered (Lee et al., 2009; Zeng et al., 2006). The amino groups on the surface can prevent the uniform distribution of hydrogen ions between the surface and the bulk solution, and can produce a microenvironment around the enzy- me(Bayramoglu, Tunali, & Arica, 2007). The immobilization of enzyme on a cationic carrier usually causes the optimum pH to shift to the acidic range whereas the immobilization on an anionic carrier causes a shift to the basic range (Kharrat, Ali, Marzouk, Garouri, & Karra-Chaabouni, 2011). The immobilization of PLA1 had high stability over a broader pH range. These results are in agreement with other reports that showed a shift in optimum pH value and an increase in activity when the enzyme was immobi- lized (Arica et al., 1999; Kharrat et al., 2011; Khoshnevisan et al., 2011; Lee et al., 2009; Madoery et al., 1995; Wang et al., 2011; Zeng et al., 2006). 3.3. Effect of temperature on the activities of free and immobilized enzymes The results of the effect of temperature for free and immobilized PLA1 are shown in Fig. 3. The temperature optima for the free PLA1 and the immobilized PLA1 were 50 and 60 �C, respectively. For the free PLA1, an increase in temperature above its optimum point resulted in lower activities due to thermal denaturation. The immobilized PlA1 retained more than 90% of its initial activity over a range of temperatures from 50 �C to 65 �C, and 80% of its initial activity at 75 �C. After the increase of the temperature from 60 �C to 65 �C, the immobilized enzyme can still retain high activity because immobilization process can increase stability and form the enzymeesubstrate complex which can hinder the access of substrates to the active site (Bornscheuer, 2000). The immobiliza- tion of PLA1 on magnetic particle can protect the active site of PLA1, and also inhibit unfolding of the enzyme at high temperatures, compared to the free enzyme (Jiang et al., 2009). Our results sug- gested that the immobilization of PLA1 onto magnetic support increases its thermostability and will extend its technological applications. There are many advantages to run bioprocesses at elevated temperatures such as higher diffusion rates, lower substrate viscosities, increased reactant solubility, and reduced risk of microbial contamination. These results confirm that enzymes can be made to withstand harsh processing condition by using different immobilization techniques (Jiang et al., 2009; Kharrat et al., 2011; Khoshnevisan et al., 2011; Lee et al., 2009; Wang et al., 2011; Zhang, Gao, & Gao, 2010) 40 45 50 55 60 65 70 75 80 30 40 50 60 70 80 90 100 R el at iv e Ac tiv ity (% ) Temperature (oC) Fig. 3. Temperature e activity profiles of free and immobilized PLA1 (- free PLA1, C immobilized PLA1). 3.4. Thermal stability of free enzyme and immobilized enzymes The thermal stability of the free and the immobilized enzymes is shown in Fig. 4A and B. The free enzymes loss their activity at 40 �C and only 57.3% activity were remained after 7 h. However, only 3.1% activity remained after 7 hwhen tested at its optimum temperature (50 �C). Soybean oil degumming process with free PLA at 50 �C requires about 5 h to reach phosphorus residual levels less than 10 mg/kg, and only retained 26.8% of the free enzyme. The immo- bilized enzyme process could retain 89.6% and 78.5% of its initial activity after 7 h treatment at 50 �C and 55 �C, respectively, but only 39.6% activity remained at its optimum temperature (60 �C) after 6 h (Fig. 4B). These results suggest that the immobilization signif- icantly improves the stability of enzyme against heat denaturation. Increase of thermal stability has been reported for a number of other immobilized enzymes (Arica et al., 1999; Kharrat et al., 2011; Sheelu et al., 2008;Wang et al., 2011; Zeng et al., 2006). Because the PLA1 was fixed on the surface of the magnetic particle through reaction with the epoxy group, the magnetic polymer network could inhibit conformational changes in the enzyme molecule, which will decrease denaturation and lead to improved thermo- stability (Arica et al., 1999; Kharrat et al., 2011; Lee et al., 2009). So the increased thermostability of the immobilized enzyme would extend its life span and broaden its potential applications. 0h 1h 2h 3h 4h 5h 6h 7h 30 40 50 60 70 80 90 100 110 R el at iv e Ac tiv ity (% ) Time B Fig. 4. Thermal stability of free PLA1(A) and immobilized PLA1 (B) (- at 45 �C, C at 50 �C, :at 55 �C, ;at 60 �C). Table 1 The residual phosphorus contents (mg/kg) after degumming by free and immobi- lized PLA1 in batch process. Time (h) Residual phosphorus contents (mg/kg) Free PLA1 (50 �C) Immobilized PLA1 (55 �C) Immobilized PLA1 (60 �C) 1 68.2 � 2.2c 125.5 � 2.6a 118.7 � 2.8b 2 36.2 � 2.4c 104.2 � 2.7a 88.6 � 3.0b 3 12.4 � 0.5c 53.2 � 2.2a 49.5 � 1.9b 4 10.1 � 0.3c 26.8 � 0.5a 21.8 � 0.6b 5 8.7 � 0.4c 16.3 � 0.7a 13.1 � 0.3b 6 7.9 � 0.3c 11.0 � 0.5a 10.5 � 0.4a 7 7.0 � 0.3c 9.6 � 0.2a 9.0 � 0.1b Values (mean � SD, n ¼ 3) in the same raw followed by different letters are significantly different at p < 0.05. D. Yu et al. / LWT - Food Science and Technology 50 (2013) 519e525524 3.5. Reusability of immobilized enzymes The operational stability of the immobilized PLA1 was tested by recycling in water degumming oil. The activity of the first cycle was defined as 100%. The activity of the immobilized enzyme versus the number of cycles is shown in Fig. 5. The activity of the immobilized PLA1 decreased continuously with increasing number of cycles. When the reaction temperature was 55 �C, the immobilized PLA1 retained 90.3% and 80.3% of their initial activity after 7 and 10 recycles, respectively. The significant decrease in activity could be due to agglomeration and loss of Fe3O4/SiOx-g-P(GMA) nano- particles during repeated separation and redispersion (Wang et al., 2011). At 60 �C, the activities of the immobilized PLA1 decreased to 73% and 61.2% after 7 and 8 recycles respectively. The lower activ- ities at 60 �C are most likely the results of increased heat dena- turation of the immobilized enzyme at the higher temperature. Therefore, the use of high processing temperatures will adversely impact the reusability of the immobilized PLA1. Similar recycle studies using immobilized PLA1 in a rice bran oil medium showed no loss in enzyme activity (�5%) after 10 recycles, compared to an aqueous buffer medium where less than 20% of its original activity remained after 10 recycles. This slow loss of enzyme activity in the aqueous system was attributed to leaching (Sheelu et al., 2008). In our previous study, the PLA1 was immobilized on calcium alginate- chitosan, and that immobilized PLA1 retained 80% of its initial activity after 4 recycles (Yu et al., 2012). The current results indicate that immobilization of PLA1 on magnetic Fe3O4/SiOx-g-P (GMA) nanoparticles significantly improves its operational stability. 3.6. Batch soybean oil degumming process by free and immobilized PLA1 The free and immobilized PLA1 were used to reduce the phos- pholipid content in the soybean oil in the lab batch scale. The initial phosphorus content in water-degummed soybean oil was 138 mg/ kg. For the free PLA1 process, the optimum degumming conditions were at pH 5.0, and 50 �C. For the immobilized PLA1 process, the degumming conditions were pH 6.0 with incubation temperatures of 55 �C and 60 �C. The hydrolysis time and residual phosphorus levels are showed in Table 1. The free PLA1 process rapidly reduced the phosphorus content to 10 mg/kg after 4e5 h. The phosphorus content after 7 h was 7.0 mg/kg. However the change in phosphorus content after 5e7 h treatment was not significant due to the drop in free PLA1 activity 0 1 2 3 4 5 6 7 8 9 10 11 55 60 65 70 75 80 85 90 95 100 105 110 R el at iv e A ct iv ity (% ) Fig. 5. Reusability of immobilized PLA1 (- at 55 �C, C at 60 �C). (Fig. 4A). When the phospholipids were hydrolyzed by the immo- bilized PLA1 process at 55 �C and 60 �C for 7 h, the residual phos- phorus contents were 9.6 mg/kg and 9.0 mg/kg, respectively. Soybean oil with this phosphorus level is suitable for physical refining. Although the immobilized PLA1 process exhibited similar degumming profiles at 55 �C and 60 �C, the immobilized PLA1 process at 50 �C has the advantages of better operational stability (Fig. 5). A number of other studies have used free phospholipase A1 to reduce the phospholipid content of vegetable oil to a final phosphorus level less than 10 mg/kg (Clausen, 2001; Yang et al., 2006, 2008). Sheelu et al. (2008) used immobilized PLA1 (immo- bilized Lecitase in gelatin hydrogel) process to degum rice bran oil, and reduced the phosphorus content from 400 mg/kg to 50e 70 mg/kg in one cycle. After charcoal treatment and de-waxing, a second enzymatic treatment, brought the phosphorus content to less than 5 mg/kg (Sheelu et al., 2008). 4. Conclusions PLA1 was successfully immobilized in magnetic Fe3O4/SiOx-g- P(GMA) nanoparticles. The immobilized PLA1 showed better stability over a broad range of temperature and pH, compared to the free PLA1. In a batch oil degumming process, the final residual phosphorus content was reduced to less than 10 mg/kg after 7 h with immobilized PLA1. The immobilized PLA1 can be reused 7 times without a significant loss in the enzyme activity. 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(2008). Insight into the enzymatic degumming process of soybean oil. Journal of the American Oil Chemists’ Society, 85, 421e425. Yu, D. Y., Jiang, L. Z., Li, Z. L., Shi, J., Xue, J., & Yukio, K. (2012). Immobilization of phospholipase A1 and its application in soybean oil degumming. Journal of the American Oil Chemist’s Society, 89, 649e656. Zeng, L., Luo, K., & Gong, Y. (2006). Preparation and characterization of dendritic composite magnetic particles as a novel enzyme immobilization carrier. Journal of Molecular Catalysis B: Enzymatic, 38, 24e30. Zhang, S., Gao, S., & Gao, G. (2010). Immobilization of b-galactosidase onto magnetic beads. Applied Biochemistry and Biotechnology, 160, 1386e1393. Characterization of immobilized phospholipase A1 on magnetic nanoparticles for oil degumming application 1. Introduction 2. Materials and methods 2.1. Materials 2.2. Preparation of Fe3O4/SiOx-g-P(GMA) nanoparticles 2.2.1. Preparation of Fe3O4 nanoparticles 2.2.2. Preparation of Fe3O4/SiOx nanoparticles 2.2.3. Preparation of Fe3O4/SiOx-g-APTES nanoparticles 2.2.4. Preparation of magnetic Fe3O4/SiOx-g-P(GMA) nanoparticles 2.3. Immobilization of phospholipase A1 on magnetic nanoparticle 2.4. Enzyme activity assay 2.5. Effect of pH and temperature on the activities of free and immobilized enzymes 2.6. Thermal stabilities of free and immobilized enzymes 2.7. Reusability of immobilized enzymes 2.8. Batch degumming process of soybean oil 2.9. Statistical analysis 3. Results and discussion 3.1. Immobilization of PLA1 3.2. Effect of pH on the activities of free and immobilized enzymes 3.3. Effect of temperature on the activities of free and immobilized enzymes 3.4. Thermal stability of free enzyme and immobilized enzymes 3.5. Reusability of immobilized enzymes 3.6. Batch soybean oil degumming process by free and immobilized PLA1 4. Conclusions Acknowledgments References