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Biodegradation of cellulose in novel recyclable aqueous two-phase systems with water-soluble immobilized cellulase
Process Biochemistry 47 (2012) 1998–2004
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Biodegradation of cellulose in novel recyclable aqueous two-phase systems with water-soluble immobilized cellulase Jingjing Liu, Xuejun Cao ∗ State Key Laboratory of Bioreactor Engineering, Department of Bioengineerng, East China University of Science and Technology, Shanghai 200237, China
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Article history: Received 21 May 2012 Received in revised form 16 June 2012 Accepted 10 July 2012 Available online 22 July 2012 Keywords: pH-response copolymer Cellulase Cellulose Water-soluble immobilized enzyme Recyclable aqueous two-phase systems
a b s t r a c t Aqueous two-phase systems (ATPS) are an attractive technology in bioseparation engineering. However, one key problem is that phase-forming copolymer could not be recycled efficiently. This results in high cost and environmental pollution. In this study, we have developed recyclable aqueous two-phase systems composed by pH-response copolymer PMDB and thermo-response copolymer PNB and have carried out biodegradation of cellulose in the ATPS. The phase-forming copolymers could be recycled with over 95.0% recovery. In the systems, cellulase was immobilized on pH-response copolymer PMDB by using 1ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride as cross-linker, and optimized partition coefficient of product was 3.68. Insoluble substrate and immobilized enzyme were biased in bottom phase, while product was partitioned in top phase. Microcrystalline cellulose was catalyzed into reducing sugar, then the product entering into the top phase. In the end, inhibition of product was removed, and the yield of reducing sugar in ATPS was increased 10.94% compared with the reaction in the single aqueous phase. The saccharification in ATPS could reach 40.16% when the reaction reached equilibrium. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Since Albertsson developed aqueous two-phase systems (ATPS) in 1950s [1], the systems have shown an attractive prospect in bioseparation [2] and bioconversion areas [3]. Traditional ATPS consisted of polyethylene glycol/dextran, or polyethylene glycol/salts (ammonium sulfate and potassium phosphate). Unfortunately, recoveries of phase-forming copolymers cannot be achieved to result in high cost and environmental pollution. Recently, scientists have attempted to look for new phase-forming copolymers that can be recycled by changing pH, temperature, ionic strength and so on [4,5]. In early 1990s, thermo-sensitive ethylene oxide–propylene oxide copolymers (EO–PO) were firstly used in recyclable ATPS. Johansson et al. [6] synthesized linear random copolymer of ethylene oxide (EO) and propylene oxide copolymers (PO) with aliphatic C14 H29 -groups modified at the end of the polymer chain (HMEOPO). Single copolymer HM-EOPO can form two phases with water. This copolymer can be recycled by temperature-inducing phase separation [6]. Persson et al. [7] used two thermo-response copolymers EO50 PO50 and HM-EOPO to form new ATPS. These two copolymers can be recovered by temperature-inducing. Another kind of attractive recycling copolymer is pH-response. Al-Muallem
∗ Corresponding author. Tel.: +86 21 64252695; fax: +86 21 624252695. E-mail address: [email protected] (X. Cao). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.07.016
et al. [8] synthesized an anion polymer with N, N-diallyl-Ncarboethoxymethyl ammonium chloride, which was a pH-response polymer, and could form two phase systems with PEG-35000. The recovery of the polymer can be achieved by changing pH of the solution. In our lab, Kong and Cao [9] synthesized a visible light-response copolymer PNBC , which could form aqueous two-phase systems with Dextran 20000. Over 98% of the copolymer can be recycled by using light radiation. Qin and Cao [10] synthesized a novel pHresponse polymer PABC . The ATPS were formed by 5% (w/w) PABC and 10% (w/w) PEG 20000. The recovery of copolymer was 95% when adjusting the pH to 8.4. Wang et al. [11] reported a novel light-response copolymer PNNC forming aqueous two-phase systems with a pH-response copolymer PADB . Copolymer PNNC can be recycled by light radiation at 488 nm, and copolymer PADB can be recovered by adjusting pH. Ning et al. [12] reported that the pHresponse copolymer PADB can form ATPS with the light-response copolymer PNBC . More than 97% of copolymer PADB can be recycled. Chen et al. [13] synthesized two novel light-response copolymers PNBAC and PNDBC that they could form recycling ATPS. Five batches of recoveries of two copolymers in the ATPS were 96.6% and 97.4%. Miao and Chen [14] reported that a new novel thermo-response copolymer PNB can form ATPS with the pH-response copolymer PADB . Cellulosic material is the most abundant renewable natural resource on the earth which can be converted into glucose and soluble sugars by chemical or enzymatic process. The production of
J. Liu, X. Cao / Process Biochemistry 47 (2012) 1998–2004
biofuels from cellulosic feedstocks has much more economic and environmental advantages than traditional fossil fuels, so scientists pay more and more attention on these green routes to obtain ethanol [15,16]. Cellulase catalyzing cellulosic material into soluble sugars and glucose shows a promising application [17–19]. Cellulose saccharification is catalyzed by complex cellulase systems. The complex cellulase systems include three classes of enzyme: cellobiohydrolases (EC 3.2.1.91), i.e. exozymes releasing cellobiose as main product from crystalline cellulose; endoglucanases (EC 3.2.1.4), attacking soluble cellulose derivatives by endoaction; and -glucosidases (EC 3.2.1.21), hydrolyzing cellooligosaccharides and cellobiose into glucose [20]. However, the main bottlenecks are the difficulty in the recovery of cellulase which leads to the high cost. To overcome this problem, cellulase was immobilized on water-insoluble support which can be recycled readily [21,22]. There arised another problem that both the enzyme and cellulosic material were insoluble. It was difficult to separate the enzyme from substrate in the reaction mixture. In addition, the reaction efficiency was decreased due to the limitation of diffusion. The reversibly soluble-insoluble supports are better than insoluble supports for enzyme immobilization. This kind of support is soluble state during the catalyzing reaction and insoluble by adjusting the pH or the temperature to recover the enzyme easily. Recently, covalently immobilizing enzyme by carbodiimide and reversibly soluble-insoluble carriers has been studied for bioconversion processes [23–28]. Enzymatic hydrolysis efficiency of cellulose is usually decreased due to many factors, such as substrate inhibition and product inhibition. Liaw and Penner [29] demonstrated that the saccharification yield at relative high substrate concentrations was 35% lower than that observed at lower substrate concentrations. Andric et al. [30] indicated that the existence of product inhibition decreased the efficiency of the bioconvertion of cellulose to valuable products. They designed a reactor to minimize product inhibition during cellulose hydrolysis. This inhibition could be overcome by removing the product during the hydrolysis reaction [31,32]. Nowadays, research efforts are directing to remove product inhibition. If the reaction is carried out in aqueous two-phase systems, products are partitioned to one phase, while the substrate and enzyme exist in the other phase, then, products yield will be improved. In this study, we report a novel pH-response copolymer (PMDB ) which can form two-phase systems with previous thermoresponse copolymer (PNB ) in our group. Both the two copolymers were synthesized in our laboratory. The copolymer (PMDB ) was synthesized by using methacrylic, 2-dimethylamino ethyl methacrylate and butyl methacrylate as monomers. PMDB can be recycled by adjusting pH. This novel PNB /PMDB ATPS show a promising prospect in carrying out the phase transfer bioconvertion. The novel ATPS achieve recycle of cellulase and phase-forming polymers and improvement of products yield. As we know, this is the first report that degradation of cellulose is carried out in recycling ATPS.
2. Materials and methods 2.1. Materials 2-Dimethylamino ethyl methacrylate (DMAEMA) was obtained from Wanduofu Chemical Co. (Zibo, Shandong Province, China). ␣-Methacrylic acid (MAA), butyl methacrylate (BMA), butyl acrylate (BA), ammonium persulfate (APS), sodium hydrogen sulfite (NaHSO3 ) and 2-2 -azo-bis-isobutyronitrile (AIBN) were purchased from Ling Feng Chemical Co. (Shanghai, China). N-isopropylacrylamide (NIPA) was obtained from Aladdin Co. All other chemicals were of analytical grade and were used without further purification. Celluclast 1.5L FG was obtained from Novozymes (Denmark). Microcrystalline cellulose was purchased from YuanJu Biotechnol Co. (Shanghai, China). 1-ethyl-3-(3dimethyaminopropyl)-carbodiimide hydrochloride (EDC) was obtained from sigma (Shanghai, China).
1999
2.2. Preparation of copolymer PMDB Copolymer PMDB was synthesized as illustrated in Fig. 1a. 5.1 ml of ␣-methacrylic (MAA), 0.53 ml of 2-dimethylamino ethyl methacrylate (DMAEMA) and 0.5 ml of butyl methacrylate (BMA) were added into a conical flask containing 120 ml of deionized water. Then initiator (APS–NaHSO3 ) was added into the solution by 1.3% (w/w). The polymerization reaction was carried out under nitrogen protection for 24 h at 55 ◦ C. After the reaction finished, the product was dissolved in 1 M NaOH solution, and filtrated to remove the undissolved impurities. Then the copolymer was precipitated by adjusting the solution pH to 3.1. The precipitate was washed three times by absolute ethyl alcohol, and then dried in the vacuum [27]. 2.3. Preparation of copolymer PNB The chemical structure of copolymer PNB was illustrated in Fig. 1b. Methods of preparation of copolymer PNB referred to Miao’s publication [14]. 2.4. Phase-forming test Different concentrations of PMDB were dissolved in 150 mM NaOH solution and pH was adjusted to 6.0 by gradually adding 100 mM NaOH. Thermo-response copolymer PNB was dissolved in purified water. Different concentrations of PMDB were used to test possibility of forming ATPS with different concentrations of PNB . Take out the samples from the top and bottom phases after 6 h when the two phases were formed, and then determine the concentration of PNB and PMDB in both phases to draw the phase diagram. 2.5. Recycling of copolymers The pH-response copolymer PMDB can be precipitated by adjusting the pH to the pI point, and the recovery of PMDB can be calculated by the percentage of the dry weight of recovered polymer to that of the initial polymer weight. The thermo-response copolymer PNB can be precipitated by raising the temperature above 33 ◦ C, then calculating the recovery of PNB by similar to aforementioned method. 2.6. Cellulase immobilization Cellulase was immobilized on PMDB as illustrated in Fig. 2. One gram of PMDB was dissolved in 50 ml of NaOH solution (0.5 M) in a beaker with constant stirring, and pH of the solution was adjusted to 6.0 by gradually adding 3 M HCl. 300 mg EDC was added to the copolymer solution with stirring, and then added 1.2 ml celluclast 1.5L (150 mg protein). After being stirred gently at room temperature for 4 h, pH of the mixture was adjusted to 3.5 by using 3 M acetic acid. The precipitate was collected as immobilized enzyme by centrifugation (8000 rpm for 15 min at 4 ◦ C) and was washed three times with distilled water (pH was about 3.5). The immobilized enzyme was stored at 4 ◦ C. 2.7. Enzyme activity assays The activity of cellulase was measured by filter paper activity (FPA) method using DNS as chromogenic reagent and glucose as standard. One unit of filter paper cellulase (FPU) was defined as the amount of enzyme producing 2.0 mg reducing sugar from 50.0 mg filter paper strip in 60 min [33]. The reserved enzymed activity was calculated by the activity of immobilized cellulase related to the activity of free cellulase. 2.8. Partition of reducing sugar in ATPS In this experiment, the reaction product was reducing sugar including glucose, cellobiose, and cellooligosaccharide. Different kinds of salts including KCl, KSCN, NaCl, NaSCN, NH4 Cl were added into PNB /PMDB systems to adjust the partition coefficients, respectively. The concentrations of all the salts were from 10 to 90 mM with 10 mM step. Samples taken from top and bottom phase by a syringe were determined by spectrophotometer at 540 nm after the aqueous two-phase systems reached equilibrium. The partition coefficient was determined by the concentration of glucose partitioning in the top phase related to the concentration of glucose partitioning in the bottom phase. 2.9. Biodegradation reaction of cellulose in ATPS Reducing sugar was produced by using cellulase as a catalyst and microcrystalline cellulose as substrate in PNB /PMDB ATPS and single aqueous phase system as control. The reaction was taken place under emulsion state. In this experiment, the reaction was carried out in a shaker with 150 rpm at 30 ◦ C. After the reaction, the tube was centrifuged at 4 ◦ C, 8000 × g for 20 min to accelerate phase separation. The total reaction volume was 10 ml. The effect of different concentration of microcrystalline cellulose on the yield of reducing sugar was investigated. The sample was taken out from top and bottom phase every 12 h to determine the concentration of product
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J. Liu, X. Cao / Process Biochemistry 47 (2012) 1998–2004
Fig. 1. (a) Synthesis of copolymers PMDB . Synthesis of pH-response copolymer PMDB . The functional monomers mole ratio of copolymer PMDB are MAA:DM:BMA = 19:1:1. (b) The chemical structure of thermo-response copolymer PNB .
after the ATPS was formed by using centrifuge. The degree of saccharification can be calculated by the following equation [34]: Saccharification (%) =
Reducing sugar (g) × 0.89 × 100% Carbohydrates (g)
3. Results and discussion 3.1. Preparation of PNB –PMDB aqueous two-phase systems We aim to synthesize a recyclable copolymer forming ATPS and to immobilize cellulase on it. Copolymer PMDB was synthesized by others in our lab. The reserved enzyme activity of cellulase immobilized on copolymer PMDB with different monomer ratio was shown in Table 1. It has been found that the reserved enzyme activity had correlation with the molar ratio of DMAEMA of three monomers in copolymer. MAA is an acid monomer with carboxyl groups. Cellulase has amino groups, which were suitable for covalently binding carboxyl groups on copolymer (Fig. 2). Therefore, Table 1 Cellulase immobilized on pH-response copolymer PMDB with different monomer ratio.
1 2 3 4 5 6
Copolymer PMDB with different monomer ratioa
Reserved enzyme activity (%)b
19:8:1 19:4:1 19:2:1 19:1.5:1 19:1:1 19:1:2
<10% <50% 60–70% 60–70% 80–90% 60–70%
a Monomer ratio indicates M(␣-methacrylic):M(DMAEMA):M(butyl methacrylate) (w/w). b Immobilization conditions: reaction time is 4 h; pH is 6.0; EDC amount is 300 mg/g PMDB .
copolymer with high content of MAA has more possibility to bind cellulase. When the molar ratio of DMAEMA of three monomers was high (the monomers molar ratio of MAA/DMAEMA/BMA was 19:8:1), there was not any activity after cellulase immobilized on it, while the enzyme activity in residual solution after immobilization was decreasing obviously. This indicated that cellulase was immobilized on copolymer PMDB . But the immobilized cellulase cannot show any activity after immobilization at high molar ratio of DMAEMA of three monomers. One possible explanation was that the copolymer might generate steric hindrances which blocked substrate approach to the active sites of immobilized enzyme. As a result, immobilized enzyme cannot show any activity during the reaction. In fact, the amount of cellulase immobilized on copolymer was increasing with molar ratio of DMAEMA of three monomers decreasing and ratio of carboxyl group of copolymer increasing. It could be observed from the enzyme activity in residual solution after immobilization. The reserved enzyme activity was more than 80% when the monomer molar ratio of MAA/DMAEMA/BMA was 19:1:1. As shown in Table 2, PMDB with different monomer molar ratio was synthesized to test the possibility of forming ATPS with thermo-response copolymer PNB . PMDB is a pH-response amphiphilic copolymer, which has acid group and alkaline group. Monomer MAA is an acid monomer with negative charge, while
Table 2 The ATPS phase-forming test of copolymers with different monomer ratio.
1 2 3 4
Copolymer PMDB (M ␣-methacrylic:M DMAEMA:M butyl methacrylate):copolymer PNB
Formation of ATPS
PMDB PMDB PMDB PMDB
NO NO YES NO
(19:2:1):PNB (19:1.5:1):PNB (19:1:1):PNB (19:1:2):PNB
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Fig. 2. Schematic illustration for cellulase immobilization. The functional monomers mole ratio of PMDB are: MAA:DM:BMA = 19:1:1. Immobilization conditions: reaction time: 4 h, pH: 6.0, EDC amount: 300 mg/g PMDB .
monomer DMAEMA is an alkaline monomer with a positive charge. We should prepare a copolymer with the most suitable monomer molar ratio (the monomer molar ratio of MAA/DMAEMA/BMA) to form ATPS with PNB . It can be seen that only copolymer PMDB at the special monomers ratio (the monomer molar ratio of MAA/DMAEMA/BMA was 19:1:1) can form ATPS with copolymer PNB . Eventually, we chose PMDB (the monomers molar ratio of MAA/DMAEMA/BMA was 19:1:1) as the pH response copolymer to immobilize cellulase on it. In this experiment, the novel PNB /PMDB ATPS are used.
3.2. Phase diagram The phase diagram for the PNB /PMDB ATPS is shown in Fig. 3. The phase separation is driven by repulsion between the copolymer PNB and PMDB . Thermo-response copolymer PNB is enriched in the top phase and pH-response copolymer PMDB is mainly in the bottom phase. The region above the binodal curve is two-phase region. M, T and B represent the total composition of the copolymer in the aqueous two-phase systems, the concentration of copolymers in the top phase and the concentration of copolymer in the bottom
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Fig. 3. The phase diagram of PNB /PMDB aqueous two-phase systems. All the experiments are carried out at room temperature (below LCST).
phase, respectively. The volume ratio of the phase could be calculated by the length ratio of line MB and MT on tie line TMB. In the experiment, the concentration of copolymer PNB was 2.5% (w/w) in total phase systems, and the concentration of copolymer PMDB was 3% (w/w) in total phase systems. The volume ratio of top phase to bottom phase was 2:1. 3.3. Recovery of copolymers Copolymer PMDB with acid monomer (MAA) and alkaline monomer (DM) is an amphiphilic copolymer and it could be precipitated from solution by adjusting pH to its isoelectric point (pI = 3.1) by adding HCl. Fig. 4 indicated that the recoveries of pH-response copolymer PMDB after recycled five times. Each recovery was average value of three parallel experimental points. The average recovery of PMDB was kept 97.2–97.9% after using five times. The values were high and stable. This indicated that the copolymer PMDB could still keep 50% after 40 recycling.
Fig. 4. The recycle recovery of copolymer PMDB . PMDB was precipitated by adjusting pH to 3.1, and collected by centrifugation in 8000 rpm. The precipitate was dried to a constant weight and dissolved again. The recovery of PMDB was calculated as the ratio of the dry weight and that of the initial weight.
Fig. 5. The recoveries of two copolymers in ATPS.
The recoveries of the two copolymers in PNB /PMDB ATPS were showed in Fig. 5. Each recovery was also average value of three parallel experimental points. Thermo-response copolymer was precipitated by raising the temperature above its LCST (33 ◦ C). The average recoveries of PNB and PMDB were 96.5% and 95.4% at the five cycles with relation to initial amount, respectively. 3.4. Partition of reducing sugar in ATPS The partition coefficient of reducing sugar was measured in this experiment, and different salt types with varied concentration were used to improve partition of reducing sugar. Reducing sugar was evenly partitioned to the top and bottom phases without neutral salts. The effect of different kinds of salts including KCl, KSCN, NaCl, NaSCN, NH4 Cl on partition of reducing sugar was investigated from 10 mM to 90 mM concentrations. Partition between two phases depends on many factors since interactions between the partitioned substance and the components of each phase are a complex phenomenon involving hydrogen bonds, charge interaction, Van der Waals forces, hydrophobic interactions, and steric effects. The results were shown in Fig. 6. As the results shown, reducing sugar was enriched in the top phase and the partition coefficient was 3.68, in presence of 50 mM KCl. Different species of ions have different effects on the partition coefficient in ATPS. The physicochemical interactions of copolymers with each other and with salts determine the equilibrium distribution of all the components in the system. Copolymer PMDB with acid monomer (MAA) and alkaline monomer (DMAEMA) is an amphiphilic copolymer enriched in the bottom phase. PNB enriched in the top phase. One of possible explanation for the partition coefficient of reducing sugar is that the existence of KCl could change the intermolecular attraction including Van der Waals force and hydrophobic interaction between product and copolymers, which makes reducing sugar distribute unevenly between two phases. The intermolecular attraction between reducing sugar and copolymer PNB might be bigger than that between reducing sugar and copolymer PMDB , and then produce forces to drive reducing sugar into the top phase to keep the phase system stable. For partition of substrate, it is known to all, cellulose is insoluble, which are completely partitioned in the bottom phase due to larger density of particles than that of water phase or large size of the
J. Liu, X. Cao / Process Biochemistry 47 (2012) 1998–2004
2003
Fig. 6. Effect of salts on partition coefficient of reducing sugar.
particles. The insoluble particles would be distributed in interphase between two phases at excess. Similar observation was previously reported [1].
3.5. Bioconversion reaction of microcrystalline cellulose Cellulase was immobilized on pH-response copolymer PMDB before bioconversion reaction. The hydrolysis of microcrystalline cellulose by immobilized water-soluble cellulase was carried out in a shaker with 150 rpm at 30 ◦ C. Phase transfer catalyzing reaction of cellulase with different concentrations of substrate was carried out in this experiment. When the initial concentration of substrate was 0.5% (w/v), both the reaction equilibrium time in ATPS and in single aqueous phase were 48 h. The saccharification percentage yield and reducing sugar concentration in ATPS were 40.16% and 2.23% (w/v), respectively. While the values in the single aqueous phase reaction were 29.22% and 1.62% (w/v) (Fig. 7a). It can be seen from the phase diagram that copolymer PMDB was mainly in the bottom phase. Cellulase was immobilized on PMDB before bioconversion reaction. As a result, immobilized cellulase was mainly partitioned in the bottom phase. In PNB /PMDB ATPS, product was partitioned in the top phase with adding 50 Mm KCl. With biodegradation reaction went on, the concentration of reducing sugar in the top phase increased three times more than that in the bottom phase. This indicated that microcrystalline cellulose was catalyzed into reducing sugar, which entered into top phase at the presence of 50 mM KCl. In this way, inhibition of product was removed and saccharification percentage yield was improved in ATPS compared with that in the single aqueous phase. As shown in Fig. 7b, when the concentration of substrate was increased to 1% (w/v), the reaction equilibrium time in the ATPS and in the single aqueous phase were 48 h. The saccharification percentage yield and reducing sugar concentration in ATPS were 24.23% and 2.69% (w/v), respectively. While the values in the single aqueous phase reaction were 14.09% and 1.56% (w/v), respectively. Substrate was catalyzed into reducing sugar in the bottom phase, and the product entered into the top phase. As a result, saccharification percentage yield was also improved. Although the increase of reducing sugar yield was limited because of low partition coefficient of product, it still had a large space for improvement. If the partition coefficient could be increased more, the yields of reducing
Fig. 7. (a) Bioconversion of cellulose in ATPS and in single aqueous phase. Microcrystalline cellulose hydrolysis is carried out in PNB /PMDB ATPS at 30 ◦ C. Substrate concentration was 0.5% (w/v). (b) Bioconversion of cellulose in ATPS and in single aqueous phase. Substrate concentration was 1% (w/v).
sugar were also increased much more than that is now compared with the yield in single aqueous phase. It could be seen that the concentration of substrate had not much effect on the reaction equilibrium time, but had obvious effects on the yield of reducing sugar. At low concentration of microcrystalline cellulose, yield of reducing sugar in ATPS was 10.94% higher than that in the single aqueous phase, while at high concentration of substrate, yield of reducing sugar in ATPS was 10.14% higher than that in the single aqueous phase. Substrate was not soluble in this experiment. The increase of substrate concentration in the reaction medium might limit the amount of saccharification, probably due to decrease mobility of insoluble substrate in the aqueous phase of the reaction mixture or increase the viscosity of the reaction mixture which had an adverse effect on the mobility of the reactants and the release of products. On the other hand, the concentration of the released reducing sugar in ATPS was 2.69% (w/v). Higher concentration of reducing sugar may also lead to the decrease of reaction rate because of product inhibition. Similar observations were previously reported [35,36]. After saccharification reaction, copolymer PMDB and PNB could be recovered by aforementioned method. Copolymer PMDB with immobilized cellulase could be recovered over 95%.
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4. Conclusions In this study, novel PNB /PMDB ATPS with water-soluble immobilized cellulase was used to biodegradation of cellulose. Cellulase was immobilized on PMDB , with over 80% reserved enzyme activity. The recoveries of both copolymers are over 95%. A significant advantage is that phase-forming polymers and cellulase could be recycled at a very low cost and there is not any environmental problem. On the other hand, phase transfer bioconversion of cellulose has been carried out in this novel ATPS. The saccharification in ATPS could reach 40.16% after 48 h, 10.94% higher than that in the single aqueous phase. Since the optimum temperature of cellulase hydrolyzing microcrystalline cellulose is 50 ◦ C, while our experiment can only carried out at 30 ◦ C because of limitation of thermo-response temperature of copolymer PNB . Although the reaction temperature was limited, saccharification in ATPS was absolutely improved, compared with the reaction in single aqueous phase. Though the yield of product yet is not quite satisfied, we are now looking for some new methods and have made some progress. In this work, we focus on the feasibility of biodegradation cellulose in recycling ATPS. Through the further study, the yield should achieve higher. It is believed that bioconversion reactions in the recycling ATPS are potential application value in future bioenergy and biochemicals industry not limited to cellulose biodegradation. References [1] Albertsson PA. Partition of cell particles and macromoleculars. Canada: John Wiley & Sons, Inc.; 1986. p. 1–110. [2] Benavides J, Aguilar O, Lapizco-Encinas BH, Rito-Palomares M. Extraction and purification of bioproducts and nanoparticles using aqueous two-phase systems strategies. Chem Eng Technol 2008;31:838–45. [3] Hatti-Kaul R. Extractive bioconversion in aqueous twophase systems in aqueous two-phase systems. Methods Biotechnol 2000;11:411–7. [4] Chen GH, Holman AS. Graft copolymers that exhibit temperature-induced phase transitions over a wide range of pH. Nature 1995;373:49–52. [5] Ricka J, Tanaka T. Phase transition in ionic gels induced by copper complexation. Macromolecules 1985;18:83–5. [6] Johansson HO, Persson J, Tjerneld F. Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification. Biotechnol Bioeng 1999;66:247–57. [7] Persson J, Johansson HO, Tjernekd F. Purification of protein and recycling of polymers in a new aqueous two-phase systems using two thermoseparating polymers. J Chromatogr A 1999;864:31–48. [8] Al-Muallem HA, Wazeer MI, Ali SA. Synthesis and solution properties of a new ionic polymer and its behavior in aqueous two-phase polymer systems. Polymer 2002;43:1041–50. [9] Kong FQ, Cao XJ, Xia JA, Byung KH. Synthesis and application of a light-response polymer forming aqueous two-phase systems. J Ind Eng Chem 2007;13:424–8. [10] Qin W, Cao XJ. Synthesis of a novel pH-response methacrylate amphiphilic polymer and its primary application in aqueous two-phase systems. Appl Biochem Biotechnol 2008;150:171–83. [11] Wang W, Wan JF, Ning B, Xia JN, Cao XJ. Preparation of a novel light-response copolymer and its application in recycling aqueous two-phase systems. J Chromatogr A 2008;1205:171–6.
[12] Ning B, Wan JF, Cao XJ. Preparation and recycling of aqueous two-phase systems with pH-response amphiphilic terpolymer PADB . Biotechnol Progr 2009;5:20–824. [13] Chen JP, Miao S, Wan JF, Xia JN, Cao xj. Synthesis and application of two lightresponse copolymers forming recyclable aqueous two-phase systems. Process Biochem 2010;45:1928–36. [14] Miao S, Chen JP, Cao XJ. Preparation of a novel thermo-response copolymer forming recyclable aqueous two-phase system and its application in bioconversion of Penicillin G. Sep Purif Technol 2010;75:156–64. [15] Hahn-Hagerdal B, Galbe M, Gorwa-Grauslund MF, Liden G, Zacchi G. Bioethanol-the fuel of tomorrow from the residues of today. Trends Biotechnol 2006;24:549–56. [16] Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, et al. Biomass recalcitrance:engineering plants and enzymes for biofuels production. Science 2007;315:804. [17] Bhat MK. Cellulase and related enzymes in biotechnology. Biotechnol Adv 2000;18:355–83. [18] Artur CP, Luis A. Hydrolysis of cotton cellulose by engineered cellulases from Trichoderma reesei. Text Res J 1998;68:273–80. [19] Mandels M, Hontz L, Nystrom J. Enzymatic hydrolysis of waste cellulose. Biotechnol Bioeng 1974;105:1471–93. [20] Natividad O, Maria DB, Perez-Mateos M. Kinetics of cellulose saccharification by Trichoderma reeseri cellulases. Int Biodeterior Biodegradation 2001;47:7–14. [21] Li Ch-Zh Yoshimoto M, Fukunaga K, Nakao K. Characterization and immobilization of liposome-bound cellulase for hydrolysis of insoluble cellulose. Bioresour Technol 2007;98:1366–72. [22] Simionescu CI, Popa VI, Popa M, Maxim S. On the possibilities of immobilization and utilization of some cellulase enzymes. J Appl Polym Sci 1990;39:1837–46. [23] Chen SH, Yen YH, Wang CL, Wang SL. Reversible immobilization of lysozyme via coupling to reversibly soluble polymer. Enzyme Microb Technol 2003;33:643–9. [24] Dourado F, Bastos M, Mota M, Gama FM. Studies on the properties of celluclast/Eudragit L-100 conjugate. J Biotechnol 2002;99:121–31. [25] Hoshino K, Taniguchi M. Repeated utilization of -glucosidase immobilized on a reversibly soluble-insoluble polymer for hydrolysis of Phloridzin as a model reaction producing a water-insoluble product. J Ferment Bioeng 1996;82:253–8. [26] Hoshino K, Akakabe S, Morohashi S, Sasakura T. Immobilization of enzymes on thermo-responsive polymers. Methods Biotechnol 1997;1:101–8. [27] Liang WJ, Cao XJ. Preparation of a pH-sensitive polyacrylate amphiphilic copolymer and its application in cellulase immobilization. Bioresour Technol 2012;116:140–6. [28] Zhou JQ. Immobilization of cellulase on a reversibly soluble-insoluble support:properties and application. J Agric Food Chem 2010;58:6741–6. [29] Liaw ET, Penner MH. Substrate–velocity relationships for the Trichoderma viride cellulase-catalyzed hydrolysis of cellulose. Appl Environ Microbiol 1990;56:2311–8. [30] Andric P, Meyer AS, Jensen PA, Dam-Johansen K. Reactor design for minimizing product inhibition during enzymatic lignocelluloses hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes. Biotechnol Adv 2010;28:308–24. [31] Gan Q, Allen SJ, Taylor G. Design and operation of an integrated membrane reactor for enzymatic cellulose hydrolysis. Biochem Eng J 2002;12:223–9. [32] Lopez JL, Matson SL, Stanley TJ, Quinn JA. Liquid–Liquid extractive membrane reacters. Bioprocess Technol 1991;11:27–66. [33] Ghose TK. Measurement of cellulase activities. Pure Appl Chem 1987;59:257–68. [34] Szczodrak J. The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol. Biotechnol Bioeng 1998;32:771–6. [35] Chen M, Zhao J, Xia L. Enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars. Carbohydr Polym 2008;71:411–5. [36] Abdel-Fattah AF, Abdel-Naby MA. Pretreatment and enzymic saccharification of water hyacinth cellulose. Carbohydr Polym 2012;87:2109–13.
Contents lists available at SciVerse ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Biodegradation of cellulose in novel recyclable aqueous two-phase systems with water-soluble immobilized cellulase Jingjing Liu, Xuejun Cao ∗ State Key Laboratory of Bioreactor Engineering, Department of Bioengineerng, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 21 May 2012 Received in revised form 16 June 2012 Accepted 10 July 2012 Available online 22 July 2012 Keywords: pH-response copolymer Cellulase Cellulose Water-soluble immobilized enzyme Recyclable aqueous two-phase systems
a b s t r a c t Aqueous two-phase systems (ATPS) are an attractive technology in bioseparation engineering. However, one key problem is that phase-forming copolymer could not be recycled efficiently. This results in high cost and environmental pollution. In this study, we have developed recyclable aqueous two-phase systems composed by pH-response copolymer PMDB and thermo-response copolymer PNB and have carried out biodegradation of cellulose in the ATPS. The phase-forming copolymers could be recycled with over 95.0% recovery. In the systems, cellulase was immobilized on pH-response copolymer PMDB by using 1ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride as cross-linker, and optimized partition coefficient of product was 3.68. Insoluble substrate and immobilized enzyme were biased in bottom phase, while product was partitioned in top phase. Microcrystalline cellulose was catalyzed into reducing sugar, then the product entering into the top phase. In the end, inhibition of product was removed, and the yield of reducing sugar in ATPS was increased 10.94% compared with the reaction in the single aqueous phase. The saccharification in ATPS could reach 40.16% when the reaction reached equilibrium. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Since Albertsson developed aqueous two-phase systems (ATPS) in 1950s [1], the systems have shown an attractive prospect in bioseparation [2] and bioconversion areas [3]. Traditional ATPS consisted of polyethylene glycol/dextran, or polyethylene glycol/salts (ammonium sulfate and potassium phosphate). Unfortunately, recoveries of phase-forming copolymers cannot be achieved to result in high cost and environmental pollution. Recently, scientists have attempted to look for new phase-forming copolymers that can be recycled by changing pH, temperature, ionic strength and so on [4,5]. In early 1990s, thermo-sensitive ethylene oxide–propylene oxide copolymers (EO–PO) were firstly used in recyclable ATPS. Johansson et al. [6] synthesized linear random copolymer of ethylene oxide (EO) and propylene oxide copolymers (PO) with aliphatic C14 H29 -groups modified at the end of the polymer chain (HMEOPO). Single copolymer HM-EOPO can form two phases with water. This copolymer can be recycled by temperature-inducing phase separation [6]. Persson et al. [7] used two thermo-response copolymers EO50 PO50 and HM-EOPO to form new ATPS. These two copolymers can be recovered by temperature-inducing. Another kind of attractive recycling copolymer is pH-response. Al-Muallem
∗ Corresponding author. Tel.: +86 21 64252695; fax: +86 21 624252695. E-mail address: [email protected] (X. Cao). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.07.016
et al. [8] synthesized an anion polymer with N, N-diallyl-Ncarboethoxymethyl ammonium chloride, which was a pH-response polymer, and could form two phase systems with PEG-35000. The recovery of the polymer can be achieved by changing pH of the solution. In our lab, Kong and Cao [9] synthesized a visible light-response copolymer PNBC , which could form aqueous two-phase systems with Dextran 20000. Over 98% of the copolymer can be recycled by using light radiation. Qin and Cao [10] synthesized a novel pHresponse polymer PABC . The ATPS were formed by 5% (w/w) PABC and 10% (w/w) PEG 20000. The recovery of copolymer was 95% when adjusting the pH to 8.4. Wang et al. [11] reported a novel light-response copolymer PNNC forming aqueous two-phase systems with a pH-response copolymer PADB . Copolymer PNNC can be recycled by light radiation at 488 nm, and copolymer PADB can be recovered by adjusting pH. Ning et al. [12] reported that the pHresponse copolymer PADB can form ATPS with the light-response copolymer PNBC . More than 97% of copolymer PADB can be recycled. Chen et al. [13] synthesized two novel light-response copolymers PNBAC and PNDBC that they could form recycling ATPS. Five batches of recoveries of two copolymers in the ATPS were 96.6% and 97.4%. Miao and Chen [14] reported that a new novel thermo-response copolymer PNB can form ATPS with the pH-response copolymer PADB . Cellulosic material is the most abundant renewable natural resource on the earth which can be converted into glucose and soluble sugars by chemical or enzymatic process. The production of
J. Liu, X. Cao / Process Biochemistry 47 (2012) 1998–2004
biofuels from cellulosic feedstocks has much more economic and environmental advantages than traditional fossil fuels, so scientists pay more and more attention on these green routes to obtain ethanol [15,16]. Cellulase catalyzing cellulosic material into soluble sugars and glucose shows a promising application [17–19]. Cellulose saccharification is catalyzed by complex cellulase systems. The complex cellulase systems include three classes of enzyme: cellobiohydrolases (EC 3.2.1.91), i.e. exozymes releasing cellobiose as main product from crystalline cellulose; endoglucanases (EC 3.2.1.4), attacking soluble cellulose derivatives by endoaction; and -glucosidases (EC 3.2.1.21), hydrolyzing cellooligosaccharides and cellobiose into glucose [20]. However, the main bottlenecks are the difficulty in the recovery of cellulase which leads to the high cost. To overcome this problem, cellulase was immobilized on water-insoluble support which can be recycled readily [21,22]. There arised another problem that both the enzyme and cellulosic material were insoluble. It was difficult to separate the enzyme from substrate in the reaction mixture. In addition, the reaction efficiency was decreased due to the limitation of diffusion. The reversibly soluble-insoluble supports are better than insoluble supports for enzyme immobilization. This kind of support is soluble state during the catalyzing reaction and insoluble by adjusting the pH or the temperature to recover the enzyme easily. Recently, covalently immobilizing enzyme by carbodiimide and reversibly soluble-insoluble carriers has been studied for bioconversion processes [23–28]. Enzymatic hydrolysis efficiency of cellulose is usually decreased due to many factors, such as substrate inhibition and product inhibition. Liaw and Penner [29] demonstrated that the saccharification yield at relative high substrate concentrations was 35% lower than that observed at lower substrate concentrations. Andric et al. [30] indicated that the existence of product inhibition decreased the efficiency of the bioconvertion of cellulose to valuable products. They designed a reactor to minimize product inhibition during cellulose hydrolysis. This inhibition could be overcome by removing the product during the hydrolysis reaction [31,32]. Nowadays, research efforts are directing to remove product inhibition. If the reaction is carried out in aqueous two-phase systems, products are partitioned to one phase, while the substrate and enzyme exist in the other phase, then, products yield will be improved. In this study, we report a novel pH-response copolymer (PMDB ) which can form two-phase systems with previous thermoresponse copolymer (PNB ) in our group. Both the two copolymers were synthesized in our laboratory. The copolymer (PMDB ) was synthesized by using methacrylic, 2-dimethylamino ethyl methacrylate and butyl methacrylate as monomers. PMDB can be recycled by adjusting pH. This novel PNB /PMDB ATPS show a promising prospect in carrying out the phase transfer bioconvertion. The novel ATPS achieve recycle of cellulase and phase-forming polymers and improvement of products yield. As we know, this is the first report that degradation of cellulose is carried out in recycling ATPS.
2. Materials and methods 2.1. Materials 2-Dimethylamino ethyl methacrylate (DMAEMA) was obtained from Wanduofu Chemical Co. (Zibo, Shandong Province, China). ␣-Methacrylic acid (MAA), butyl methacrylate (BMA), butyl acrylate (BA), ammonium persulfate (APS), sodium hydrogen sulfite (NaHSO3 ) and 2-2 -azo-bis-isobutyronitrile (AIBN) were purchased from Ling Feng Chemical Co. (Shanghai, China). N-isopropylacrylamide (NIPA) was obtained from Aladdin Co. All other chemicals were of analytical grade and were used without further purification. Celluclast 1.5L FG was obtained from Novozymes (Denmark). Microcrystalline cellulose was purchased from YuanJu Biotechnol Co. (Shanghai, China). 1-ethyl-3-(3dimethyaminopropyl)-carbodiimide hydrochloride (EDC) was obtained from sigma (Shanghai, China).
1999
2.2. Preparation of copolymer PMDB Copolymer PMDB was synthesized as illustrated in Fig. 1a. 5.1 ml of ␣-methacrylic (MAA), 0.53 ml of 2-dimethylamino ethyl methacrylate (DMAEMA) and 0.5 ml of butyl methacrylate (BMA) were added into a conical flask containing 120 ml of deionized water. Then initiator (APS–NaHSO3 ) was added into the solution by 1.3% (w/w). The polymerization reaction was carried out under nitrogen protection for 24 h at 55 ◦ C. After the reaction finished, the product was dissolved in 1 M NaOH solution, and filtrated to remove the undissolved impurities. Then the copolymer was precipitated by adjusting the solution pH to 3.1. The precipitate was washed three times by absolute ethyl alcohol, and then dried in the vacuum [27]. 2.3. Preparation of copolymer PNB The chemical structure of copolymer PNB was illustrated in Fig. 1b. Methods of preparation of copolymer PNB referred to Miao’s publication [14]. 2.4. Phase-forming test Different concentrations of PMDB were dissolved in 150 mM NaOH solution and pH was adjusted to 6.0 by gradually adding 100 mM NaOH. Thermo-response copolymer PNB was dissolved in purified water. Different concentrations of PMDB were used to test possibility of forming ATPS with different concentrations of PNB . Take out the samples from the top and bottom phases after 6 h when the two phases were formed, and then determine the concentration of PNB and PMDB in both phases to draw the phase diagram. 2.5. Recycling of copolymers The pH-response copolymer PMDB can be precipitated by adjusting the pH to the pI point, and the recovery of PMDB can be calculated by the percentage of the dry weight of recovered polymer to that of the initial polymer weight. The thermo-response copolymer PNB can be precipitated by raising the temperature above 33 ◦ C, then calculating the recovery of PNB by similar to aforementioned method. 2.6. Cellulase immobilization Cellulase was immobilized on PMDB as illustrated in Fig. 2. One gram of PMDB was dissolved in 50 ml of NaOH solution (0.5 M) in a beaker with constant stirring, and pH of the solution was adjusted to 6.0 by gradually adding 3 M HCl. 300 mg EDC was added to the copolymer solution with stirring, and then added 1.2 ml celluclast 1.5L (150 mg protein). After being stirred gently at room temperature for 4 h, pH of the mixture was adjusted to 3.5 by using 3 M acetic acid. The precipitate was collected as immobilized enzyme by centrifugation (8000 rpm for 15 min at 4 ◦ C) and was washed three times with distilled water (pH was about 3.5). The immobilized enzyme was stored at 4 ◦ C. 2.7. Enzyme activity assays The activity of cellulase was measured by filter paper activity (FPA) method using DNS as chromogenic reagent and glucose as standard. One unit of filter paper cellulase (FPU) was defined as the amount of enzyme producing 2.0 mg reducing sugar from 50.0 mg filter paper strip in 60 min [33]. The reserved enzymed activity was calculated by the activity of immobilized cellulase related to the activity of free cellulase. 2.8. Partition of reducing sugar in ATPS In this experiment, the reaction product was reducing sugar including glucose, cellobiose, and cellooligosaccharide. Different kinds of salts including KCl, KSCN, NaCl, NaSCN, NH4 Cl were added into PNB /PMDB systems to adjust the partition coefficients, respectively. The concentrations of all the salts were from 10 to 90 mM with 10 mM step. Samples taken from top and bottom phase by a syringe were determined by spectrophotometer at 540 nm after the aqueous two-phase systems reached equilibrium. The partition coefficient was determined by the concentration of glucose partitioning in the top phase related to the concentration of glucose partitioning in the bottom phase. 2.9. Biodegradation reaction of cellulose in ATPS Reducing sugar was produced by using cellulase as a catalyst and microcrystalline cellulose as substrate in PNB /PMDB ATPS and single aqueous phase system as control. The reaction was taken place under emulsion state. In this experiment, the reaction was carried out in a shaker with 150 rpm at 30 ◦ C. After the reaction, the tube was centrifuged at 4 ◦ C, 8000 × g for 20 min to accelerate phase separation. The total reaction volume was 10 ml. The effect of different concentration of microcrystalline cellulose on the yield of reducing sugar was investigated. The sample was taken out from top and bottom phase every 12 h to determine the concentration of product
2000
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Fig. 1. (a) Synthesis of copolymers PMDB . Synthesis of pH-response copolymer PMDB . The functional monomers mole ratio of copolymer PMDB are MAA:DM:BMA = 19:1:1. (b) The chemical structure of thermo-response copolymer PNB .
after the ATPS was formed by using centrifuge. The degree of saccharification can be calculated by the following equation [34]: Saccharification (%) =
Reducing sugar (g) × 0.89 × 100% Carbohydrates (g)
3. Results and discussion 3.1. Preparation of PNB –PMDB aqueous two-phase systems We aim to synthesize a recyclable copolymer forming ATPS and to immobilize cellulase on it. Copolymer PMDB was synthesized by others in our lab. The reserved enzyme activity of cellulase immobilized on copolymer PMDB with different monomer ratio was shown in Table 1. It has been found that the reserved enzyme activity had correlation with the molar ratio of DMAEMA of three monomers in copolymer. MAA is an acid monomer with carboxyl groups. Cellulase has amino groups, which were suitable for covalently binding carboxyl groups on copolymer (Fig. 2). Therefore, Table 1 Cellulase immobilized on pH-response copolymer PMDB with different monomer ratio.
1 2 3 4 5 6
Copolymer PMDB with different monomer ratioa
Reserved enzyme activity (%)b
19:8:1 19:4:1 19:2:1 19:1.5:1 19:1:1 19:1:2
<10% <50% 60–70% 60–70% 80–90% 60–70%
a Monomer ratio indicates M(␣-methacrylic):M(DMAEMA):M(butyl methacrylate) (w/w). b Immobilization conditions: reaction time is 4 h; pH is 6.0; EDC amount is 300 mg/g PMDB .
copolymer with high content of MAA has more possibility to bind cellulase. When the molar ratio of DMAEMA of three monomers was high (the monomers molar ratio of MAA/DMAEMA/BMA was 19:8:1), there was not any activity after cellulase immobilized on it, while the enzyme activity in residual solution after immobilization was decreasing obviously. This indicated that cellulase was immobilized on copolymer PMDB . But the immobilized cellulase cannot show any activity after immobilization at high molar ratio of DMAEMA of three monomers. One possible explanation was that the copolymer might generate steric hindrances which blocked substrate approach to the active sites of immobilized enzyme. As a result, immobilized enzyme cannot show any activity during the reaction. In fact, the amount of cellulase immobilized on copolymer was increasing with molar ratio of DMAEMA of three monomers decreasing and ratio of carboxyl group of copolymer increasing. It could be observed from the enzyme activity in residual solution after immobilization. The reserved enzyme activity was more than 80% when the monomer molar ratio of MAA/DMAEMA/BMA was 19:1:1. As shown in Table 2, PMDB with different monomer molar ratio was synthesized to test the possibility of forming ATPS with thermo-response copolymer PNB . PMDB is a pH-response amphiphilic copolymer, which has acid group and alkaline group. Monomer MAA is an acid monomer with negative charge, while
Table 2 The ATPS phase-forming test of copolymers with different monomer ratio.
1 2 3 4
Copolymer PMDB (M ␣-methacrylic:M DMAEMA:M butyl methacrylate):copolymer PNB
Formation of ATPS
PMDB PMDB PMDB PMDB
NO NO YES NO
(19:2:1):PNB (19:1.5:1):PNB (19:1:1):PNB (19:1:2):PNB
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2001
Fig. 2. Schematic illustration for cellulase immobilization. The functional monomers mole ratio of PMDB are: MAA:DM:BMA = 19:1:1. Immobilization conditions: reaction time: 4 h, pH: 6.0, EDC amount: 300 mg/g PMDB .
monomer DMAEMA is an alkaline monomer with a positive charge. We should prepare a copolymer with the most suitable monomer molar ratio (the monomer molar ratio of MAA/DMAEMA/BMA) to form ATPS with PNB . It can be seen that only copolymer PMDB at the special monomers ratio (the monomer molar ratio of MAA/DMAEMA/BMA was 19:1:1) can form ATPS with copolymer PNB . Eventually, we chose PMDB (the monomers molar ratio of MAA/DMAEMA/BMA was 19:1:1) as the pH response copolymer to immobilize cellulase on it. In this experiment, the novel PNB /PMDB ATPS are used.
3.2. Phase diagram The phase diagram for the PNB /PMDB ATPS is shown in Fig. 3. The phase separation is driven by repulsion between the copolymer PNB and PMDB . Thermo-response copolymer PNB is enriched in the top phase and pH-response copolymer PMDB is mainly in the bottom phase. The region above the binodal curve is two-phase region. M, T and B represent the total composition of the copolymer in the aqueous two-phase systems, the concentration of copolymers in the top phase and the concentration of copolymer in the bottom
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Fig. 3. The phase diagram of PNB /PMDB aqueous two-phase systems. All the experiments are carried out at room temperature (below LCST).
phase, respectively. The volume ratio of the phase could be calculated by the length ratio of line MB and MT on tie line TMB. In the experiment, the concentration of copolymer PNB was 2.5% (w/w) in total phase systems, and the concentration of copolymer PMDB was 3% (w/w) in total phase systems. The volume ratio of top phase to bottom phase was 2:1. 3.3. Recovery of copolymers Copolymer PMDB with acid monomer (MAA) and alkaline monomer (DM) is an amphiphilic copolymer and it could be precipitated from solution by adjusting pH to its isoelectric point (pI = 3.1) by adding HCl. Fig. 4 indicated that the recoveries of pH-response copolymer PMDB after recycled five times. Each recovery was average value of three parallel experimental points. The average recovery of PMDB was kept 97.2–97.9% after using five times. The values were high and stable. This indicated that the copolymer PMDB could still keep 50% after 40 recycling.
Fig. 4. The recycle recovery of copolymer PMDB . PMDB was precipitated by adjusting pH to 3.1, and collected by centrifugation in 8000 rpm. The precipitate was dried to a constant weight and dissolved again. The recovery of PMDB was calculated as the ratio of the dry weight and that of the initial weight.
Fig. 5. The recoveries of two copolymers in ATPS.
The recoveries of the two copolymers in PNB /PMDB ATPS were showed in Fig. 5. Each recovery was also average value of three parallel experimental points. Thermo-response copolymer was precipitated by raising the temperature above its LCST (33 ◦ C). The average recoveries of PNB and PMDB were 96.5% and 95.4% at the five cycles with relation to initial amount, respectively. 3.4. Partition of reducing sugar in ATPS The partition coefficient of reducing sugar was measured in this experiment, and different salt types with varied concentration were used to improve partition of reducing sugar. Reducing sugar was evenly partitioned to the top and bottom phases without neutral salts. The effect of different kinds of salts including KCl, KSCN, NaCl, NaSCN, NH4 Cl on partition of reducing sugar was investigated from 10 mM to 90 mM concentrations. Partition between two phases depends on many factors since interactions between the partitioned substance and the components of each phase are a complex phenomenon involving hydrogen bonds, charge interaction, Van der Waals forces, hydrophobic interactions, and steric effects. The results were shown in Fig. 6. As the results shown, reducing sugar was enriched in the top phase and the partition coefficient was 3.68, in presence of 50 mM KCl. Different species of ions have different effects on the partition coefficient in ATPS. The physicochemical interactions of copolymers with each other and with salts determine the equilibrium distribution of all the components in the system. Copolymer PMDB with acid monomer (MAA) and alkaline monomer (DMAEMA) is an amphiphilic copolymer enriched in the bottom phase. PNB enriched in the top phase. One of possible explanation for the partition coefficient of reducing sugar is that the existence of KCl could change the intermolecular attraction including Van der Waals force and hydrophobic interaction between product and copolymers, which makes reducing sugar distribute unevenly between two phases. The intermolecular attraction between reducing sugar and copolymer PNB might be bigger than that between reducing sugar and copolymer PMDB , and then produce forces to drive reducing sugar into the top phase to keep the phase system stable. For partition of substrate, it is known to all, cellulose is insoluble, which are completely partitioned in the bottom phase due to larger density of particles than that of water phase or large size of the
J. Liu, X. Cao / Process Biochemistry 47 (2012) 1998–2004
2003
Fig. 6. Effect of salts on partition coefficient of reducing sugar.
particles. The insoluble particles would be distributed in interphase between two phases at excess. Similar observation was previously reported [1].
3.5. Bioconversion reaction of microcrystalline cellulose Cellulase was immobilized on pH-response copolymer PMDB before bioconversion reaction. The hydrolysis of microcrystalline cellulose by immobilized water-soluble cellulase was carried out in a shaker with 150 rpm at 30 ◦ C. Phase transfer catalyzing reaction of cellulase with different concentrations of substrate was carried out in this experiment. When the initial concentration of substrate was 0.5% (w/v), both the reaction equilibrium time in ATPS and in single aqueous phase were 48 h. The saccharification percentage yield and reducing sugar concentration in ATPS were 40.16% and 2.23% (w/v), respectively. While the values in the single aqueous phase reaction were 29.22% and 1.62% (w/v) (Fig. 7a). It can be seen from the phase diagram that copolymer PMDB was mainly in the bottom phase. Cellulase was immobilized on PMDB before bioconversion reaction. As a result, immobilized cellulase was mainly partitioned in the bottom phase. In PNB /PMDB ATPS, product was partitioned in the top phase with adding 50 Mm KCl. With biodegradation reaction went on, the concentration of reducing sugar in the top phase increased three times more than that in the bottom phase. This indicated that microcrystalline cellulose was catalyzed into reducing sugar, which entered into top phase at the presence of 50 mM KCl. In this way, inhibition of product was removed and saccharification percentage yield was improved in ATPS compared with that in the single aqueous phase. As shown in Fig. 7b, when the concentration of substrate was increased to 1% (w/v), the reaction equilibrium time in the ATPS and in the single aqueous phase were 48 h. The saccharification percentage yield and reducing sugar concentration in ATPS were 24.23% and 2.69% (w/v), respectively. While the values in the single aqueous phase reaction were 14.09% and 1.56% (w/v), respectively. Substrate was catalyzed into reducing sugar in the bottom phase, and the product entered into the top phase. As a result, saccharification percentage yield was also improved. Although the increase of reducing sugar yield was limited because of low partition coefficient of product, it still had a large space for improvement. If the partition coefficient could be increased more, the yields of reducing
Fig. 7. (a) Bioconversion of cellulose in ATPS and in single aqueous phase. Microcrystalline cellulose hydrolysis is carried out in PNB /PMDB ATPS at 30 ◦ C. Substrate concentration was 0.5% (w/v). (b) Bioconversion of cellulose in ATPS and in single aqueous phase. Substrate concentration was 1% (w/v).
sugar were also increased much more than that is now compared with the yield in single aqueous phase. It could be seen that the concentration of substrate had not much effect on the reaction equilibrium time, but had obvious effects on the yield of reducing sugar. At low concentration of microcrystalline cellulose, yield of reducing sugar in ATPS was 10.94% higher than that in the single aqueous phase, while at high concentration of substrate, yield of reducing sugar in ATPS was 10.14% higher than that in the single aqueous phase. Substrate was not soluble in this experiment. The increase of substrate concentration in the reaction medium might limit the amount of saccharification, probably due to decrease mobility of insoluble substrate in the aqueous phase of the reaction mixture or increase the viscosity of the reaction mixture which had an adverse effect on the mobility of the reactants and the release of products. On the other hand, the concentration of the released reducing sugar in ATPS was 2.69% (w/v). Higher concentration of reducing sugar may also lead to the decrease of reaction rate because of product inhibition. Similar observations were previously reported [35,36]. After saccharification reaction, copolymer PMDB and PNB could be recovered by aforementioned method. Copolymer PMDB with immobilized cellulase could be recovered over 95%.
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4. Conclusions In this study, novel PNB /PMDB ATPS with water-soluble immobilized cellulase was used to biodegradation of cellulose. Cellulase was immobilized on PMDB , with over 80% reserved enzyme activity. The recoveries of both copolymers are over 95%. A significant advantage is that phase-forming polymers and cellulase could be recycled at a very low cost and there is not any environmental problem. On the other hand, phase transfer bioconversion of cellulose has been carried out in this novel ATPS. The saccharification in ATPS could reach 40.16% after 48 h, 10.94% higher than that in the single aqueous phase. Since the optimum temperature of cellulase hydrolyzing microcrystalline cellulose is 50 ◦ C, while our experiment can only carried out at 30 ◦ C because of limitation of thermo-response temperature of copolymer PNB . Although the reaction temperature was limited, saccharification in ATPS was absolutely improved, compared with the reaction in single aqueous phase. Though the yield of product yet is not quite satisfied, we are now looking for some new methods and have made some progress. In this work, we focus on the feasibility of biodegradation cellulose in recycling ATPS. Through the further study, the yield should achieve higher. It is believed that bioconversion reactions in the recycling ATPS are potential application value in future bioenergy and biochemicals industry not limited to cellulose biodegradation. References [1] Albertsson PA. Partition of cell particles and macromoleculars. Canada: John Wiley & Sons, Inc.; 1986. p. 1–110. [2] Benavides J, Aguilar O, Lapizco-Encinas BH, Rito-Palomares M. Extraction and purification of bioproducts and nanoparticles using aqueous two-phase systems strategies. Chem Eng Technol 2008;31:838–45. [3] Hatti-Kaul R. Extractive bioconversion in aqueous twophase systems in aqueous two-phase systems. Methods Biotechnol 2000;11:411–7. [4] Chen GH, Holman AS. Graft copolymers that exhibit temperature-induced phase transitions over a wide range of pH. Nature 1995;373:49–52. [5] Ricka J, Tanaka T. Phase transition in ionic gels induced by copper complexation. Macromolecules 1985;18:83–5. [6] Johansson HO, Persson J, Tjerneld F. Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification. Biotechnol Bioeng 1999;66:247–57. [7] Persson J, Johansson HO, Tjernekd F. Purification of protein and recycling of polymers in a new aqueous two-phase systems using two thermoseparating polymers. J Chromatogr A 1999;864:31–48. [8] Al-Muallem HA, Wazeer MI, Ali SA. Synthesis and solution properties of a new ionic polymer and its behavior in aqueous two-phase polymer systems. Polymer 2002;43:1041–50. [9] Kong FQ, Cao XJ, Xia JA, Byung KH. Synthesis and application of a light-response polymer forming aqueous two-phase systems. J Ind Eng Chem 2007;13:424–8. [10] Qin W, Cao XJ. Synthesis of a novel pH-response methacrylate amphiphilic polymer and its primary application in aqueous two-phase systems. Appl Biochem Biotechnol 2008;150:171–83. [11] Wang W, Wan JF, Ning B, Xia JN, Cao XJ. Preparation of a novel light-response copolymer and its application in recycling aqueous two-phase systems. J Chromatogr A 2008;1205:171–6.
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