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Preliminary application of light-pH sensitive recycling aqueous two-phase systems to purification of lipase
Process Biochemistry 45 (2010) 598–601
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Preliminary application of light-pH sensitive recycling aqueous two-phase systems to purification of lipase Xian Li, Junfen Wan, Xuejun Cao * State Key Laboratory of Bioreactor Engineering, Department of Biochemical Engineering, East China University of Science & Technology, 130 Meilong Road, Shanghai 200237, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 May 2009 Received in revised form 10 November 2009 Accepted 12 November 2009
One key problem of aqueous two-phase systems (ATPS) is that phase-forming polymers could not be recycled efficiently. This results in high cost and environmental pollution. In this study, we introduced novel aqueous two-phase systems which are composed by pH-sensitive polymer PADB and light-sensitive polymer PNNC. PNNC is enriched in the top phase while PADB is found in the bottom phase. And recoveries of two-phase-forming polymers can both reach over 96%. This aqueous two-phase system was used for purification of lipase from its crude material. The influences of various process parameters such as concentration of the phase-forming polymer, system pH, different types and concentrations of neutral salts on partitioning of lipase are evaluated. It has been found that partition coefficient of pure lipase could reach 0.061 under optimized conditions. Lipase from crude material was purified with 83.7% recovery and a purification factor of approximately 18 folds. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: pH-sensitive polymer Light-sensitive polymer Aqueous two-phase systems Purification Recovery Lipase
1. Introduction Aqueous two-phase systems (ATPS) were developed by Albertsson in 1950s [1,2]. Then the application study has been extended into areas of bio-separation and bio-catalysis. However, one key problem of ATPS is that most phase-forming copolymers could not be recycled effectively at low cost. Traditional ATPS are based on polyethylene glycol (PEG)/dextran, or PEG/potassium phosphate. However, high cost and environmental pollution of these systems limits their application in biotechnology industry. So few reports on the application of this technique are available. In recent years, many scientists have made their great efforts on exploring recyclable copolymers. It was reported that ethylene oxide–propylene oxide copolymers (EOPO) could be recycled by temperature [3]. Then random copolymer of EO and PO modified with a hydrophobical aliphatic C14H29-group (HM-EOPO) was synthesized by Johansson et al. [4]. Hasan et al. [5] synthesized an anion polymer with N,N-diallyl-N-carboethoxymethylammonium chloride as a monomer to form aqueous two-phase systems, which was a pH-sensitive polymer and would precipitate in 0.1 M HCl. Kumar et al. [6] synthesized a temperature-sensitive polymer, poly-N-isopropyl-acrylamide [poly(NIPAM)] to form ATPS. In our laboratory, Wei et al. [7] synthesized a novel pH-sensitive polymer (PABC) copolymerized by 2-(dimethylamino) ethyl methacrylate (DMAEMA), tert-butyl methacrylate (tBMA) and methyl methac-
* Corresponding author. Fax: +86 21 64252695. E-mail address: [email protected] (X. Cao). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.11.008
rylate (MMA) as monomers. It can form ATPS with polyethylene glycol of molecular mass 20,000 (PEG20000). PABC could be recovered by adjusting isoelectric point (pI) to 8.4, with recovery of 95%. Meanwhile, Kong et al. [8] synthesized the visible lightsensitive polymer PNBC which was copolymerized by using Nisopropylacrylamide (NIPA), n-butyl acrylate, and chlorophyllin sodium copper salt (CHL) as monomers. The ATPS were formed by the PNBC and dextran with molecular mass 20,000 (DEX20000). Over 95% of the PNBC could be recycled. Recently, Wang et al. [9] synthesized two novel polymers called PADB and PNNC which are pH-sensitive and light-sensitive, respectively. They could form novel ATPS. PADB could be recovered by adjusting pH to its isoelectric point (pI = 4.1) and PNNC by laser at 488 nm. Their recoveries can reach more than 97%. High recovery means that they could be re-utilized for several tens of times. Lipase is an important enzyme due to its wide applications in fats and oleo chemical industry, production of biodegradable polymers, textile industry, detergent industry, diagnostic tool and food industry, etc. [10]. Especially, the lipase will play a key role in bio-diesel production. Conventionally, lipase is purified by various steps such as ammonium sulphate fractionation and chromatography, etc. However, these methods could not be used in large volume and dilute crude material solution. So it is necessary to develop a purification process in large scale in industry. In our laboratory, a pH-sensitive PADB polymer and a lightsensitive PNNC polymer were prepared by co-workers, and they could form ATPS each other. In this study, the novel PADB–PNNC ATPS were attempted to purify lipase from its crude material. And recycle times of the novel PADB–PNNC ATPS were investigated after
X. Li et al. / Process Biochemistry 45 (2010) 598–601
extraction. Concentration of the phase-forming polymer, system pH, different types and concentrations of neutral salts were also investigated in the process of extraction. Some interesting results have been obtained.
599
Lipase recovery in the bottom phase y (%) was also calculated according to the given equation
yð%Þbottom ¼
100 1 þ RK
(3)
where R = VT/VB, VB and VT are the bottom and top phase volume, and K is the lipase partition coefficient.
2. Materials and methods 2.1. Materials
The purification factor (PF) was calculated as
N-(2-(dimethylamino) ethyl)-methacrylamide was purchased from Wanduofu Chemical Co. (Zibo, Shandong Province, China). Acrylic Acid (AA), n-butyl methacrylate (BMA) and 2-20 -azoisobutyronitrile (AIBN) were obtained from Ling Feng Chemical Co. (Shanghai, China). N-vinyl-2-pyrrolidone (NVP) and chlorophyllin sodium copper salt (CHL) were from Sigma. Benzene and n-hexane were from Fei Da Co. (Shanghai China). N-isopropylacrylamide was synthesized by reacting acryloyl chloride with isopropyl amine and crystallized from hexane solution [11]. Commercial porcine pancreatic lipase (lipase) was purchased from Shanghai KaiYang Biotechnology Co. Ltd. Crude enzyme material was from Suzhou Dongwu Biochemical Products Company (Jiangsu province, China). All chemicals were of analytical grade and were used without further purification.
PF ¼
½Actbottom ½Actoriginal
(4)
[Act]bottom and [Act]top are the lipase specific activity in the bottom phase and in original mixture, respectively. 2.2.3.4. Polyacrylamide gel electrophoresis. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the following method [12]. 30% polyacrylamide slab gel was prepared and electrophoresis was run at 120 V, 12.5 mA, for 2–3 h. The gel was stained with the solution containing Coomassie Brilliant Blue R250 of 0.05% (w/v), 50% (v/v) methanol and 12% (v/v) acetic acid. The gel was distained using the same buffer without Coomassie Brilliant Blue.
2.2. Methods 2.2.1. Synthesis of P (AA-co-DMAEMA-co-BMA) (PADB) and P (NIPA-co-NVP-co-CHL) (PNNC) PADB and PNNC were synthesized according to the methods described by Wang et al. [9]. 2.2.2. Recycling of copolymers 2.2.2.1. Recovery of PADB copolymer. The dissolved copolymer solution was adjusted pH to pI (4.1) to produce a precipitate and then the precipitate was separated from solution by centrifugation, and dried in the vacuum drier. 2.2.2.2. Light recovery of PNNC copolymer. The dissolved copolymer solution was recovered by irradiation with a 488 nm laser (model 5500A ion laser equipment from Ion laser Technology). And during light irradiation, the temperature of PNNC solution is controlled in 258C with water bath, and kept below the LCST (31.8 8C) of PNNC. The precipitate of PNNC was shrinked into a clot and can be taken out from the solution with a tweezers, and then dried in the vacuum drier. 2.2.3. Determination of lipase 2.2.3.1. Assay of lipase activity. The activity of lipase was determined by olive oil hydrolysis. A 100 ml olive oil emulsion was prepared by mixing 25 ml olive oil and 75 ml polyvinyl alcohol (PVA) solution. The assay mixture consisted of 4 ml emulsion, 5 ml phosphate buffer (pH 7.5) and 1 ml free enzyme (1.0 mg/ml). Oil hydrolysis was carried out at 40 8C for 15 min in a water bath. The reaction was stopped by the addition of 15 ml of ethanol solution (95%). The released fatty acid in the reaction was determined by titration with 50 mM NaOH solution. One lipase unit was expressed as release of 1 mmol fatty acid per minute in the assay conditions, and the specific activity was expressed as lipase units per mg protein. Activity was calculated by the following formula:
X¼
ðB AÞ c 50 n 0:05t
3. Results and discussion 3.1. Effect of PNNC concentration in top phase on the partitioning of lipase Concentration of PADB is fixed at 5% (w/w), and PNNC concentration range investigated was 7–10% (w/w). Fig. 1 shows the effect of PNNC concentration on lipase partition coefficient. From Fig. 1, it could be seen that partitioning of pure lipase and crude lipase can be altered by change PNNC concentration. Partition coefficient of pure lipase decreases sharply with increase of PNNC concentration from 7% (w/w) to 10%. This indicates that lipase bias to bottom with increase of PNNC concentration. The result can be explained by Donnan effect. PNNC is a polymer with negative charges in top phase and it can repulse lipase with negative charges in top phase to bottom phase. Compare with pure lipase, crude material contains many proteins. Some proteins have also negative charge as lipase. Therefore, lipase and the contaminants have same charge property. As a result, partition coefficient of lipase decreases slowly with the increase of PNNC concentration due to competition of contaminant proteins. As we can see from Fig. 1, with the increase of PNNC concentration, the partition coefficients of crude enzymes decrease slowly from 0.98 to 0.90.
(1)
where X is the enzyme activity, B the NaOH volume consumed by sample, A the NaOH volume consumed by blank sample, c the NaOH concentration, t the reaction time and n the dilution factor. 2.2.3.2. Determination of protein concentration. When proteins partition reached equilibrium in ATPS, bottom phase was taken out by a syringe. After PADB in it was recovered by adjusting pH to 4.1, protein concentration in the supernatant was determined by the absorbance at 280 nm. 2.2.3.3. Determination of partition coefficient (K). Lipase was dissolved in ATPS with 1 mg/ml total systems. Samples were taken out from two phases and Lipase activity in each phase was determined by aforementioned method. The partition coefficient was defined as
K¼
½Acttop ½Actbottom
(2)
where [Act]top and [Act]bottom are activity of partitioned lipase in the top phase and bottom phases, respectively.
Fig. 1. Partition coefficient of lipase affected by PNNC concentration.
X. Li et al. / Process Biochemistry 45 (2010) 598–601
600
3.2. Effect of pH on the partitioning of lipase Considering PADB solubility and lipase stability, pH range investigated was 7.0–8.0. pH was controlled by using phosphate buffer system. The volume change of top and bottom phases was negligible within the pH range. Fig. 2 shows the effect of pH on partition coefficient. For pure enzyme, K decreases sharply from 3.5 to 0.142 in the range of 7.0–7.5. The minimum K = 0.142 appears at pH 7.5. And then it increased slowly from 7.5 to 8.0. Contrarily, change of crude enzyme partition coefficient is not so much. Most of points distribute around 1.0. The minimum K is 0.61 at pH 7.4. This could also be attributed to charge effect (Donnan effect). Lipase was negatively charged protein (pI = 5.0) at pH 7.0–8.0. PNNC has negative charge above pH 7.0. With pH increasing, both PNNC and PADB have more and more negative charges. But this effect is more significant for PNNC. The more charges PNNC carries, the stronger repulsive force between the polymer and protein are. So much pure Lipase transfers into bottom phase which leads to the decrease of partition coefficient. At pH 7.5, apparently lipase shows lowest partition coefficient. If pH continually increases, partition coefficient slight increases. The result may be a complex balance between charges on polymers and charges on lipase. Crude material contains many impurities with unknown pI. Some are above 7.0 and some are below 7.0. At pH 7.0 partition coefficient is around 1.0. With pH increasing, contaminants, lipase, PNNC and PADB have competition balance among them. Finally, we can see the curve shown in Fig. 2.
ammonium salts. At the pH values, lipase has a negative net charge. Most of partitioning coefficient values is more than 1.0. For different positive ions, the partition coefficients decreased according to the following order: NH4+ < Na+ < K+. And these nine curves show this trend. According to Fig. 3, we choose four salts with better partitioning effect for lipase. They are listed as followed: NaBr, K2SO4, KCl and KBr. we find that all partition coefficients are less than 1.0. We chose four conditions with 50 mM NaBr, 40 mM K2SO4, 40 mM KCl and 20 mM KBr, which had brilliant effect on protein partitioning, to investigate partition of lipase due to better partition coefficients of pure lipase in these four systems. The results are listed in Table 1.
3.3. Effect of neutral salts and their concentrations on partition of lipase The addition of neutral salts to ATPS can manipulate partition of proteins. Changes of the salt type and concentration can produce an electrical potential difference between the two phases due to uneven partition of ions. The effect of nine neutral salts (KCl, KBr, KI, K2SO4, NaCl, Na2SO4, NH4Cl, (NH4)2SO4) on partitioning for pure lipase is investigated from 10 to 80 mM concentrations. The results are shown in Fig. 3a–c. Salts could lead to electric potential difference between two phases, which influences on partition of charged biological macromolecules. For pure enzyme, the effects of potassium salts on partition of lipase are much better than that of sodium salts and
Fig. 2. Partition coefficient of lipase affected by pH.
Fig. 3. Partition coefficient of lipase affected by different salts. (a) Partition coefficient of lipase affected by sodium salts; (b) partition coefficient of lipase affected by potassium salts; and (c) partition coefficient of lipase affected by ammonium salts.
X. Li et al. / Process Biochemistry 45 (2010) 598–601 Table 1 Partition coefficient and specific activity of crude enzyme in phase systems composed of four different salt (sodium bromide, potassium sulphate, potassium chloride, potassium bromide) solutions. Each experiment was performed in duplicate. The results are expressed as the mean of triplicate readings, which have an estimated error of 10%. NaBr (50 mM)
K2SO4
KCl (40 mM)
KBr (20 mM)
0.265 0.431 135
0.061 0.584 270
(40 mM) Pure enzyme Crude enzyme Specific activity (U/mg)
0.38 0.112 135
0.192 0.418 180
601
trend, lipase has the largest possibility partitioning to the bottom phase. That is why it has the best partition result in ATPS with 20 mM KBr. 3.5. Recycling of copolymers After extraction of ATPS, two copolymers PNNC and PADB were recovered by laser at 488 nm for PNNC and pH at 4.1 for PADB. 97.8% and 96.7% recovery related to initial amount of the two polymers at fifth cycles.
p.s. The original lipase specific activity is 15 U/mg.
4. Conclusions
According to Table 1, the optimal condition for partitioning lipase was observed in 20 mM potassium bromide system, which had a partition coefficient of 0.061 for pure lipase and the highest lipase specific activity of 270 U/mg for crude lipase. The results indicate that lipase has more satisfactory partition behavior at lower concentrations of potassium salt. Generally speaking, different types of salts have different ions partition in the two phases. This results in an electrostatic potential difference over the interface [13].
A novel ATPS composed by 5% (w/w) PADB pH-sensitive polymer and 10% (w/w) PNNC light-sensitive polymers were firstly applied for purification of lipase. The partition coefficient of pure lipase could reach 0.061 at the concentration of 5% (w/w) PADB, 10% (w/w) PNNC and 20 mM KBr at pH 7.5. Under the same condition, lipase was purified from crude lipase material with recovery of 83.7% and purification factor of 18. Partition coefficient of lipase could be effectively controlled by using types and concentration of some special inorganic salts. The system is composed by light-sensitive PNNC and pH-sensitive PADB. PADB could be recycled by adjusting the pH to its isoelectric point (pI = 4.1). Light-sensitive PNNC can be recycled by laser irradiation at 488 nm. This novel ATPS show potential application in biotechnology industry due to low cost and no any environmental problem.
3.4. SDS-PAGE After partition in ATPS reached equilibrium, bottom phase was taken out and PADB was recovered by adjusting pH to 4.1. The supernatant was used for SDS-PAGE analysis. The result is shown in Fig. 4. Fig. 4 showed that some contaminants are removed by ATPS partition. Especially, KBr shows obvious effect. In Fig. 4, lanes 1–5 show the partition effect after added KBr in the system. From it, we can see that the fifth lane has the best partition effect among these five lanes because of fewer impurities in electrophoresis bands. In the crude material, some proteins with pI above 7.0 such as trypsin and other proteins are influenced by the charged polymer. They transfer to the top phase and could be separated from crude material. For the other impurities such as chymotrypsin and pancreatic kininogenase, their molecular weights are lower than lipase. So lipase has larger space volume. This leads to less binding force with PNNC. To the negatively charged protein, its partition coefficient decreases according to the following order: Na+ < K+ and Cl < Br. So summarizing this
Fig. 4. SDS-PAGE analysis of purified lipase. Samples from bottom phase were procured from ATPS with 5% (w/w) PADB and 10% (w/w) PNNC. Each sample contains different salts. Lane 1: ATPS with 50 mM NaBr; lane 2: ATPS with 40 mM K2SO4; lane 3: ATPS with 40 mM KCl; lane 4: sample of crude enzyme dissolved in Na2HPO4–NaH2PO4 buffer; lane 5: ATPS with 20 mM KBr; and lane 6: Marker.
Acknowledgements This project was supported by Natural Scientific Foundation of China (20474016), and the National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204). Authors very appreciate for above National funds. References [1] Albertsson PA. Partition of cell particles and macromolecules, 3rd ed., New York: Wiley; 1986. [2] Albertsson PA. Partition of cell particles and macromolecules, 4th ed., New York: Wiley; 1986. [3] Berggren K, Johansson HO, Tjerneld F. Effects of salts and the surface hydrophobicity of proteins on partitions in aqueous two-phase systems containing thermo separating ethylene oxide–propylene oxide copolymers. Journal of Chromatography A 1995;718:67–9. [4] Johansson HO, Persson J, Tjerneld F. Thermo separating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification. Biotechnology and Bioengineering 1999;66(4):247–57. [5] Hasan A, Muallem A, Mohamed IMW, Asrof ASK. Synthesis and solution properties of a new ionic polymer and its behavior in aqueous two-phase polymer systems. Polymer 2002;43:1041–50. [6] Kumar A, Kamihira M, Galaev IYu, Mattiasson B, lijima S. Type-specific separation of animal cells in aqueous two-phase systems using antibody conjugates with temperature-sensitive polymers. Biotechnology and Bioengineering 2001;75:570–80. [7] Qui W, Cao XJ. Synthesis of a novel pH-sensitive methacrylate amphiphilic polymer and its primary application in aqueous two-phase systems. Applied Biochemistry and Biotechnology 2008;150:171–83. [8] Kong FQ, Cao XJ, Xia JA. Synthesis and application of a light-sensitive polymer forming aqueous two-phase systems. Journal of Industrial and Engineering Chemistry 2007;13(3):424–8. [9] Wang W, Wan JF, Ning B, Xia JA, Cao XJ. Preparation of a novel light-sensitive copolymer and its application in recycling aqueous two-phase systems. Journal of Chromatography A 2008;1205:171–6. [10] Hasan F, Shah AA, Hameed A. Industrial applications of microbial lipases. Enzyme and Microbial Technology 2006;39:235–51. [11] Zhu J, Zhu X, Lu J, Guo W, Yuan Y. Study on the synthesis and swelling behaviors of pH and temperature sensitive interpenetrating network material with poly(sodium acrylate) and N-isopropylacrylamide. Journal of Chemical Engineering of Chinese Universities 2002;3(16):302–5. [12] Wang J, Fan M. Protein technique handbook. Beijing: Science Press; 2002. p. 43–46. [13] Saravanan S, Rao JR, Nair BU, Ramasami T. Aqueous two-phase poly(ethylene glycol)–poly(acrylic acid) system for protein partitioning: Influence of molecular weight, pH and temperature. Process Biochemistry 2008;43:905–11.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Preliminary application of light-pH sensitive recycling aqueous two-phase systems to purification of lipase Xian Li, Junfen Wan, Xuejun Cao * State Key Laboratory of Bioreactor Engineering, Department of Biochemical Engineering, East China University of Science & Technology, 130 Meilong Road, Shanghai 200237, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 May 2009 Received in revised form 10 November 2009 Accepted 12 November 2009
One key problem of aqueous two-phase systems (ATPS) is that phase-forming polymers could not be recycled efficiently. This results in high cost and environmental pollution. In this study, we introduced novel aqueous two-phase systems which are composed by pH-sensitive polymer PADB and light-sensitive polymer PNNC. PNNC is enriched in the top phase while PADB is found in the bottom phase. And recoveries of two-phase-forming polymers can both reach over 96%. This aqueous two-phase system was used for purification of lipase from its crude material. The influences of various process parameters such as concentration of the phase-forming polymer, system pH, different types and concentrations of neutral salts on partitioning of lipase are evaluated. It has been found that partition coefficient of pure lipase could reach 0.061 under optimized conditions. Lipase from crude material was purified with 83.7% recovery and a purification factor of approximately 18 folds. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: pH-sensitive polymer Light-sensitive polymer Aqueous two-phase systems Purification Recovery Lipase
1. Introduction Aqueous two-phase systems (ATPS) were developed by Albertsson in 1950s [1,2]. Then the application study has been extended into areas of bio-separation and bio-catalysis. However, one key problem of ATPS is that most phase-forming copolymers could not be recycled effectively at low cost. Traditional ATPS are based on polyethylene glycol (PEG)/dextran, or PEG/potassium phosphate. However, high cost and environmental pollution of these systems limits their application in biotechnology industry. So few reports on the application of this technique are available. In recent years, many scientists have made their great efforts on exploring recyclable copolymers. It was reported that ethylene oxide–propylene oxide copolymers (EOPO) could be recycled by temperature [3]. Then random copolymer of EO and PO modified with a hydrophobical aliphatic C14H29-group (HM-EOPO) was synthesized by Johansson et al. [4]. Hasan et al. [5] synthesized an anion polymer with N,N-diallyl-N-carboethoxymethylammonium chloride as a monomer to form aqueous two-phase systems, which was a pH-sensitive polymer and would precipitate in 0.1 M HCl. Kumar et al. [6] synthesized a temperature-sensitive polymer, poly-N-isopropyl-acrylamide [poly(NIPAM)] to form ATPS. In our laboratory, Wei et al. [7] synthesized a novel pH-sensitive polymer (PABC) copolymerized by 2-(dimethylamino) ethyl methacrylate (DMAEMA), tert-butyl methacrylate (tBMA) and methyl methac-
* Corresponding author. Fax: +86 21 64252695. E-mail address: [email protected] (X. Cao). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.11.008
rylate (MMA) as monomers. It can form ATPS with polyethylene glycol of molecular mass 20,000 (PEG20000). PABC could be recovered by adjusting isoelectric point (pI) to 8.4, with recovery of 95%. Meanwhile, Kong et al. [8] synthesized the visible lightsensitive polymer PNBC which was copolymerized by using Nisopropylacrylamide (NIPA), n-butyl acrylate, and chlorophyllin sodium copper salt (CHL) as monomers. The ATPS were formed by the PNBC and dextran with molecular mass 20,000 (DEX20000). Over 95% of the PNBC could be recycled. Recently, Wang et al. [9] synthesized two novel polymers called PADB and PNNC which are pH-sensitive and light-sensitive, respectively. They could form novel ATPS. PADB could be recovered by adjusting pH to its isoelectric point (pI = 4.1) and PNNC by laser at 488 nm. Their recoveries can reach more than 97%. High recovery means that they could be re-utilized for several tens of times. Lipase is an important enzyme due to its wide applications in fats and oleo chemical industry, production of biodegradable polymers, textile industry, detergent industry, diagnostic tool and food industry, etc. [10]. Especially, the lipase will play a key role in bio-diesel production. Conventionally, lipase is purified by various steps such as ammonium sulphate fractionation and chromatography, etc. However, these methods could not be used in large volume and dilute crude material solution. So it is necessary to develop a purification process in large scale in industry. In our laboratory, a pH-sensitive PADB polymer and a lightsensitive PNNC polymer were prepared by co-workers, and they could form ATPS each other. In this study, the novel PADB–PNNC ATPS were attempted to purify lipase from its crude material. And recycle times of the novel PADB–PNNC ATPS were investigated after
X. Li et al. / Process Biochemistry 45 (2010) 598–601
extraction. Concentration of the phase-forming polymer, system pH, different types and concentrations of neutral salts were also investigated in the process of extraction. Some interesting results have been obtained.
599
Lipase recovery in the bottom phase y (%) was also calculated according to the given equation
yð%Þbottom ¼
100 1 þ RK
(3)
where R = VT/VB, VB and VT are the bottom and top phase volume, and K is the lipase partition coefficient.
2. Materials and methods 2.1. Materials
The purification factor (PF) was calculated as
N-(2-(dimethylamino) ethyl)-methacrylamide was purchased from Wanduofu Chemical Co. (Zibo, Shandong Province, China). Acrylic Acid (AA), n-butyl methacrylate (BMA) and 2-20 -azoisobutyronitrile (AIBN) were obtained from Ling Feng Chemical Co. (Shanghai, China). N-vinyl-2-pyrrolidone (NVP) and chlorophyllin sodium copper salt (CHL) were from Sigma. Benzene and n-hexane were from Fei Da Co. (Shanghai China). N-isopropylacrylamide was synthesized by reacting acryloyl chloride with isopropyl amine and crystallized from hexane solution [11]. Commercial porcine pancreatic lipase (lipase) was purchased from Shanghai KaiYang Biotechnology Co. Ltd. Crude enzyme material was from Suzhou Dongwu Biochemical Products Company (Jiangsu province, China). All chemicals were of analytical grade and were used without further purification.
PF ¼
½Actbottom ½Actoriginal
(4)
[Act]bottom and [Act]top are the lipase specific activity in the bottom phase and in original mixture, respectively. 2.2.3.4. Polyacrylamide gel electrophoresis. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the following method [12]. 30% polyacrylamide slab gel was prepared and electrophoresis was run at 120 V, 12.5 mA, for 2–3 h. The gel was stained with the solution containing Coomassie Brilliant Blue R250 of 0.05% (w/v), 50% (v/v) methanol and 12% (v/v) acetic acid. The gel was distained using the same buffer without Coomassie Brilliant Blue.
2.2. Methods 2.2.1. Synthesis of P (AA-co-DMAEMA-co-BMA) (PADB) and P (NIPA-co-NVP-co-CHL) (PNNC) PADB and PNNC were synthesized according to the methods described by Wang et al. [9]. 2.2.2. Recycling of copolymers 2.2.2.1. Recovery of PADB copolymer. The dissolved copolymer solution was adjusted pH to pI (4.1) to produce a precipitate and then the precipitate was separated from solution by centrifugation, and dried in the vacuum drier. 2.2.2.2. Light recovery of PNNC copolymer. The dissolved copolymer solution was recovered by irradiation with a 488 nm laser (model 5500A ion laser equipment from Ion laser Technology). And during light irradiation, the temperature of PNNC solution is controlled in 258C with water bath, and kept below the LCST (31.8 8C) of PNNC. The precipitate of PNNC was shrinked into a clot and can be taken out from the solution with a tweezers, and then dried in the vacuum drier. 2.2.3. Determination of lipase 2.2.3.1. Assay of lipase activity. The activity of lipase was determined by olive oil hydrolysis. A 100 ml olive oil emulsion was prepared by mixing 25 ml olive oil and 75 ml polyvinyl alcohol (PVA) solution. The assay mixture consisted of 4 ml emulsion, 5 ml phosphate buffer (pH 7.5) and 1 ml free enzyme (1.0 mg/ml). Oil hydrolysis was carried out at 40 8C for 15 min in a water bath. The reaction was stopped by the addition of 15 ml of ethanol solution (95%). The released fatty acid in the reaction was determined by titration with 50 mM NaOH solution. One lipase unit was expressed as release of 1 mmol fatty acid per minute in the assay conditions, and the specific activity was expressed as lipase units per mg protein. Activity was calculated by the following formula:
X¼
ðB AÞ c 50 n 0:05t
3. Results and discussion 3.1. Effect of PNNC concentration in top phase on the partitioning of lipase Concentration of PADB is fixed at 5% (w/w), and PNNC concentration range investigated was 7–10% (w/w). Fig. 1 shows the effect of PNNC concentration on lipase partition coefficient. From Fig. 1, it could be seen that partitioning of pure lipase and crude lipase can be altered by change PNNC concentration. Partition coefficient of pure lipase decreases sharply with increase of PNNC concentration from 7% (w/w) to 10%. This indicates that lipase bias to bottom with increase of PNNC concentration. The result can be explained by Donnan effect. PNNC is a polymer with negative charges in top phase and it can repulse lipase with negative charges in top phase to bottom phase. Compare with pure lipase, crude material contains many proteins. Some proteins have also negative charge as lipase. Therefore, lipase and the contaminants have same charge property. As a result, partition coefficient of lipase decreases slowly with the increase of PNNC concentration due to competition of contaminant proteins. As we can see from Fig. 1, with the increase of PNNC concentration, the partition coefficients of crude enzymes decrease slowly from 0.98 to 0.90.
(1)
where X is the enzyme activity, B the NaOH volume consumed by sample, A the NaOH volume consumed by blank sample, c the NaOH concentration, t the reaction time and n the dilution factor. 2.2.3.2. Determination of protein concentration. When proteins partition reached equilibrium in ATPS, bottom phase was taken out by a syringe. After PADB in it was recovered by adjusting pH to 4.1, protein concentration in the supernatant was determined by the absorbance at 280 nm. 2.2.3.3. Determination of partition coefficient (K). Lipase was dissolved in ATPS with 1 mg/ml total systems. Samples were taken out from two phases and Lipase activity in each phase was determined by aforementioned method. The partition coefficient was defined as
K¼
½Acttop ½Actbottom
(2)
where [Act]top and [Act]bottom are activity of partitioned lipase in the top phase and bottom phases, respectively.
Fig. 1. Partition coefficient of lipase affected by PNNC concentration.
X. Li et al. / Process Biochemistry 45 (2010) 598–601
600
3.2. Effect of pH on the partitioning of lipase Considering PADB solubility and lipase stability, pH range investigated was 7.0–8.0. pH was controlled by using phosphate buffer system. The volume change of top and bottom phases was negligible within the pH range. Fig. 2 shows the effect of pH on partition coefficient. For pure enzyme, K decreases sharply from 3.5 to 0.142 in the range of 7.0–7.5. The minimum K = 0.142 appears at pH 7.5. And then it increased slowly from 7.5 to 8.0. Contrarily, change of crude enzyme partition coefficient is not so much. Most of points distribute around 1.0. The minimum K is 0.61 at pH 7.4. This could also be attributed to charge effect (Donnan effect). Lipase was negatively charged protein (pI = 5.0) at pH 7.0–8.0. PNNC has negative charge above pH 7.0. With pH increasing, both PNNC and PADB have more and more negative charges. But this effect is more significant for PNNC. The more charges PNNC carries, the stronger repulsive force between the polymer and protein are. So much pure Lipase transfers into bottom phase which leads to the decrease of partition coefficient. At pH 7.5, apparently lipase shows lowest partition coefficient. If pH continually increases, partition coefficient slight increases. The result may be a complex balance between charges on polymers and charges on lipase. Crude material contains many impurities with unknown pI. Some are above 7.0 and some are below 7.0. At pH 7.0 partition coefficient is around 1.0. With pH increasing, contaminants, lipase, PNNC and PADB have competition balance among them. Finally, we can see the curve shown in Fig. 2.
ammonium salts. At the pH values, lipase has a negative net charge. Most of partitioning coefficient values is more than 1.0. For different positive ions, the partition coefficients decreased according to the following order: NH4+ < Na+ < K+. And these nine curves show this trend. According to Fig. 3, we choose four salts with better partitioning effect for lipase. They are listed as followed: NaBr, K2SO4, KCl and KBr. we find that all partition coefficients are less than 1.0. We chose four conditions with 50 mM NaBr, 40 mM K2SO4, 40 mM KCl and 20 mM KBr, which had brilliant effect on protein partitioning, to investigate partition of lipase due to better partition coefficients of pure lipase in these four systems. The results are listed in Table 1.
3.3. Effect of neutral salts and their concentrations on partition of lipase The addition of neutral salts to ATPS can manipulate partition of proteins. Changes of the salt type and concentration can produce an electrical potential difference between the two phases due to uneven partition of ions. The effect of nine neutral salts (KCl, KBr, KI, K2SO4, NaCl, Na2SO4, NH4Cl, (NH4)2SO4) on partitioning for pure lipase is investigated from 10 to 80 mM concentrations. The results are shown in Fig. 3a–c. Salts could lead to electric potential difference between two phases, which influences on partition of charged biological macromolecules. For pure enzyme, the effects of potassium salts on partition of lipase are much better than that of sodium salts and
Fig. 2. Partition coefficient of lipase affected by pH.
Fig. 3. Partition coefficient of lipase affected by different salts. (a) Partition coefficient of lipase affected by sodium salts; (b) partition coefficient of lipase affected by potassium salts; and (c) partition coefficient of lipase affected by ammonium salts.
X. Li et al. / Process Biochemistry 45 (2010) 598–601 Table 1 Partition coefficient and specific activity of crude enzyme in phase systems composed of four different salt (sodium bromide, potassium sulphate, potassium chloride, potassium bromide) solutions. Each experiment was performed in duplicate. The results are expressed as the mean of triplicate readings, which have an estimated error of 10%. NaBr (50 mM)
K2SO4
KCl (40 mM)
KBr (20 mM)
0.265 0.431 135
0.061 0.584 270
(40 mM) Pure enzyme Crude enzyme Specific activity (U/mg)
0.38 0.112 135
0.192 0.418 180
601
trend, lipase has the largest possibility partitioning to the bottom phase. That is why it has the best partition result in ATPS with 20 mM KBr. 3.5. Recycling of copolymers After extraction of ATPS, two copolymers PNNC and PADB were recovered by laser at 488 nm for PNNC and pH at 4.1 for PADB. 97.8% and 96.7% recovery related to initial amount of the two polymers at fifth cycles.
p.s. The original lipase specific activity is 15 U/mg.
4. Conclusions
According to Table 1, the optimal condition for partitioning lipase was observed in 20 mM potassium bromide system, which had a partition coefficient of 0.061 for pure lipase and the highest lipase specific activity of 270 U/mg for crude lipase. The results indicate that lipase has more satisfactory partition behavior at lower concentrations of potassium salt. Generally speaking, different types of salts have different ions partition in the two phases. This results in an electrostatic potential difference over the interface [13].
A novel ATPS composed by 5% (w/w) PADB pH-sensitive polymer and 10% (w/w) PNNC light-sensitive polymers were firstly applied for purification of lipase. The partition coefficient of pure lipase could reach 0.061 at the concentration of 5% (w/w) PADB, 10% (w/w) PNNC and 20 mM KBr at pH 7.5. Under the same condition, lipase was purified from crude lipase material with recovery of 83.7% and purification factor of 18. Partition coefficient of lipase could be effectively controlled by using types and concentration of some special inorganic salts. The system is composed by light-sensitive PNNC and pH-sensitive PADB. PADB could be recycled by adjusting the pH to its isoelectric point (pI = 4.1). Light-sensitive PNNC can be recycled by laser irradiation at 488 nm. This novel ATPS show potential application in biotechnology industry due to low cost and no any environmental problem.
3.4. SDS-PAGE After partition in ATPS reached equilibrium, bottom phase was taken out and PADB was recovered by adjusting pH to 4.1. The supernatant was used for SDS-PAGE analysis. The result is shown in Fig. 4. Fig. 4 showed that some contaminants are removed by ATPS partition. Especially, KBr shows obvious effect. In Fig. 4, lanes 1–5 show the partition effect after added KBr in the system. From it, we can see that the fifth lane has the best partition effect among these five lanes because of fewer impurities in electrophoresis bands. In the crude material, some proteins with pI above 7.0 such as trypsin and other proteins are influenced by the charged polymer. They transfer to the top phase and could be separated from crude material. For the other impurities such as chymotrypsin and pancreatic kininogenase, their molecular weights are lower than lipase. So lipase has larger space volume. This leads to less binding force with PNNC. To the negatively charged protein, its partition coefficient decreases according to the following order: Na+ < K+ and Cl < Br. So summarizing this
Fig. 4. SDS-PAGE analysis of purified lipase. Samples from bottom phase were procured from ATPS with 5% (w/w) PADB and 10% (w/w) PNNC. Each sample contains different salts. Lane 1: ATPS with 50 mM NaBr; lane 2: ATPS with 40 mM K2SO4; lane 3: ATPS with 40 mM KCl; lane 4: sample of crude enzyme dissolved in Na2HPO4–NaH2PO4 buffer; lane 5: ATPS with 20 mM KBr; and lane 6: Marker.
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