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Process Biochemistry 47 (2012) 1998–2004

Contents lists available at SciVerse ScienceDirect

Process Biochemistry

jo u rn al hom epa ge: www .e lsev ier .com/ locate /procbio

iodegradation of cellulose in novel recyclable aqueous two-phase systems withater-soluble immobilized cellulase

ingjing Liu, Xuejun Cao ∗

tate Key Laboratory of Bioreactor Engineering, Department of Bioengineerng, East China University of Science and Technology, Shanghai 200237, China

r t i c l e i n f o

rticle history:eceived 21 May 2012eceived in revised form 16 June 2012ccepted 10 July 2012vailable online 22 July 2012

eywords:

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 highcost and environmental pollution. In this study, we have developed recyclable aqueous two-phase sys-tems composed by pH-response copolymer PMDB and thermo-response copolymer PNB and have carriedout biodegradation of cellulose in the ATPS. The phase-forming copolymers could be recycled with over95.0% recovery. In the systems, cellulase was immobilized on pH-response copolymer PMDB by using 1-

H-response copolymerellulaseelluloseater-soluble immobilized enzyme

ecyclable aqueous two-phase systems

ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride as cross-linker, and optimized partitioncoefficient of product was 3.68. Insoluble substrate and immobilized enzyme were biased in bottomphase, while product was partitioned in top phase. Microcrystalline cellulose was catalyzed into reduc-ing 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 singleaqueous phase. The saccharification in ATPS could reach 40.16% when the reaction reached equilibrium.

. Introduction

Since Albertsson developed aqueous two-phase systems (ATPS)n 1950s [1], the systems have shown an attractive prospect inioseparation [2] and bioconversion areas [3]. Traditional ATPS con-isted of polyethylene glycol/dextran, or polyethylene glycol/saltsammonium sulfate and potassium phosphate). Unfortunately,ecoveries of phase-forming copolymers cannot be achieved toesult in high cost and environmental pollution. Recently, scien-ists have attempted to look for new phase-forming copolymershat can be recycled by changing pH, temperature, ionic strengthnd so on [4,5].

In early 1990s, thermo-sensitive ethylene oxide–propylenexide copolymers (EO–PO) were firstly used in recyclable ATPS.ohansson et al. [6] synthesized linear random copolymer of ethy-ene oxide (EO) and propylene oxide copolymers (PO) with aliphatic14H29-groups modified at the end of the polymer chain (HM-OPO). Single copolymer HM-EOPO can form two phases withater. This copolymer can be recycled by temperature-inducinghase separation [6]. Persson et al. [7] used two thermo-response

opolymers EO50PO50 and HM-EOPO to form new ATPS. These twoopolymers can be recovered by temperature-inducing. Anotherind of attractive recycling copolymer is pH-response. Al-Muallem

∗ Corresponding author. Tel.: +86 21 64252695; fax: +86 21 624252695.E-mail address: caoxj@ecust.edu.cn (X. Cao).

359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.procbio.2012.07.016

© 2012 Elsevier Ltd. All rights reserved.

et al. [8] synthesized an anion polymer with N, N-diallyl-N-carboethoxymethyl ammonium chloride, which was a pH-responsepolymer, and could form two phase systems with PEG-35000. Therecovery of the polymer can be achieved by changing pH of thesolution.

In our lab, Kong and Cao [9] synthesized a visible light-responsecopolymer PNBC, which could form aqueous two-phase systemswith Dextran 20000. Over 98% of the copolymer can be recycledby using light radiation. Qin and Cao [10] synthesized a novel pH-response polymer PABC. The ATPS were formed by 5% (w/w) PABCand 10% (w/w) PEG 20000. The recovery of copolymer was 95%when adjusting the pH to 8.4. Wang et al. [11] reported a novellight-response copolymer PNNC forming aqueous two-phase sys-tems with a pH-response copolymer PADB. Copolymer PNNC can berecycled by light radiation at 488 nm, and copolymer PADB can berecovered by adjusting pH. Ning et al. [12] reported that the pH-response copolymer PADB can form ATPS with the light-responsecopolymer PNBC. More than 97% of copolymer PADB can be recycled.Chen et al. [13] synthesized two novel light-response copolymersPNBAC and PNDBC that they could form recycling ATPS. Five batchesof recoveries of two copolymers in the ATPS were 96.6% and 97.4%.Miao and Chen [14] reported that a new novel thermo-responsecopolymer PNB can form ATPS with the pH-response copolymer

PADB.

Cellulosic material is the most abundant renewable naturalresource on the earth which can be converted into glucose and sol-uble sugars by chemical or enzymatic process. The production of

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iofuels from cellulosic feedstocks has much more economic andnvironmental advantages than traditional fossil fuels, so scien-ists pay more and more attention on these green routes to obtainthanol [15,16]. Cellulase catalyzing cellulosic material into solubleugars and glucose shows a promising application [17–19]. Cel-ulose saccharification is catalyzed by complex cellulase systems.he complex cellulase systems include three classes of enzyme:ellobiohydrolases (EC 3.2.1.91), i.e. exozymes releasing cellobioses main product from crystalline cellulose; endoglucanases (EC.2.1.4), attacking soluble cellulose derivatives by endoaction; and-glucosidases (EC 3.2.1.21), hydrolyzing cellooligosaccharidesnd cellobiose into glucose [20]. However, the main bottlenecksre the difficulty in the recovery of cellulase which leads to theigh cost. To overcome this problem, cellulase was immobilizedn water-insoluble support which can be recycled readily [21,22].here arised another problem that both the enzyme and cellulosicaterial were insoluble. It was difficult to separate the enzyme

rom substrate in the reaction mixture. In addition, the reactionfficiency was decreased due to the limitation of diffusion. Theeversibly soluble-insoluble supports are better than insoluble sup-orts for enzyme immobilization. This kind of support is solubletate during the catalyzing reaction and insoluble by adjusting theH or the temperature to recover the enzyme easily. Recently,ovalently immobilizing enzyme by carbodiimide and reversiblyoluble-insoluble carriers has been studied for bioconversion pro-esses [23–28].

Enzymatic hydrolysis efficiency of cellulose is usually decreasedue to many factors, such as substrate inhibition and product inhi-ition. Liaw and Penner [29] demonstrated that the saccharificationield at relative high substrate concentrations was 35% lower thanhat observed at lower substrate concentrations. Andric et al. [30]ndicated that the existence of product inhibition decreased thefficiency of the bioconvertion of cellulose to valuable products.hey designed a reactor to minimize product inhibition duringellulose hydrolysis. This inhibition could be overcome by remov-ng the product during the hydrolysis reaction [31,32]. Nowadays,esearch efforts are directing to remove product inhibition. If theeaction is carried out in aqueous two-phase systems, products areartitioned to one phase, while the substrate and enzyme exist inhe other phase, then, products yield will be improved.

In this study, we report a novel pH-response copolymerPMDB) which can form two-phase systems with previous thermo-esponse copolymer (PNB) in our group. Both the two copolymersere synthesized in our laboratory. The copolymer (PMDB)as synthesized by using methacrylic, 2-dimethylamino ethylethacrylate and butyl methacrylate as monomers. PMDB can be

ecycled by adjusting pH. This novel PNB/PMDB ATPS show a promis-ng prospect in carrying out the phase transfer bioconvertion. Theovel ATPS achieve recycle of cellulase and phase-forming poly-ers and improvement of products yield. As we know, this is the

rst report that degradation of cellulose is carried out in recyclingTPS.

. Materials and methods

.1. Materials

2-Dimethylamino ethyl methacrylate (DMAEMA) was obtained from Wan-uofu Chemical Co. (Zibo, Shandong Province, China). �-Methacrylic acid (MAA),utyl methacrylate (BMA), butyl acrylate (BA), ammonium persulfate (APS), sodiumydrogen sulfite (NaHSO3) and 2-2′-azo-bis-isobutyronitrile (AIBN) were purchased

rom Ling Feng Chemical Co. (Shanghai, China). N-isopropylacrylamide (NIPA) was

btained from Aladdin Co. All other chemicals were of analytical grade and weresed without further purification.

Celluclast 1.5L FG was obtained from Novozymes (Denmark). Microcrystallineellulose was purchased from YuanJu Biotechnol Co. (Shanghai, China). 1-ethyl-3-(3-imethyaminopropyl)-carbodiimide hydrochloride (EDC) was obtained from sigmaShanghai, China).

try 47 (2012) 1998–2004 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 ofbutyl methacrylate (BMA) were added into a conical flask containing 120 ml ofdeionized water. Then initiator (APS–NaHSO3) was added into the solution by 1.3%(w/w). The polymerization reaction was carried out under nitrogen protection for24 h at 55 ◦C. After the reaction finished, the product was dissolved in 1 M NaOHsolution, and filtrated to remove the undissolved impurities. Then the copolymerwas precipitated by adjusting the solution pH to 3.1. The precipitate was washedthree 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 ofpreparation of copolymer PNB referred to Miao’s publication [14].

2.4. Phase-forming test

Different concentrations of PMDB were dissolved in 150 mM NaOH solutionand pH was adjusted to 6.0 by gradually adding 100 mM NaOH. Thermo-responsecopolymer 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 phaseswere formed, and then determine the concentration of PNB and PMDB in both phasesto draw the phase diagram.

2.5. Recycling of copolymers

The pH-response copolymer PMDB can be precipitated by adjusting the pH to thepI point, and the recovery of PMDB can be calculated by the percentage of the dryweight of recovered polymer to that of the initial polymer weight.

The thermo-response copolymer PNB can be precipitated by raising the temper-ature above 33 ◦C, then calculating the recovery of PNB by similar to aforementionedmethod.

2.6. Cellulase immobilization

Cellulase was immobilized on PMDB as illustrated in Fig. 2. One gram of PMDB wasdissolved in 50 ml of NaOH solution (0.5 M) in a beaker with constant stirring, andpH of the solution was adjusted to 6.0 by gradually adding 3 M HCl. 300 mg EDCwas added to the copolymer solution with stirring, and then added 1.2 ml celluclast1.5L (150 mg protein). After being stirred gently at room temperature for 4 h, pHof the mixture was adjusted to 3.5 by using 3 M acetic acid. The precipitate wascollected as immobilized enzyme by centrifugation (8000 rpm for 15 min at 4 ◦C) andwas washed three times with distilled water (pH was about 3.5). The immobilizedenzyme was stored at 4 ◦C.

2.7. Enzyme activity assays

The activity of cellulase was measured by filter paper activity (FPA) methodusing DNS as chromogenic reagent and glucose as standard. One unit of filter papercellulase (FPU) was defined as the amount of enzyme producing 2.0 mg reducingsugar from 50.0 mg filter paper strip in 60 min [33]. The reserved enzymed activitywas calculated by the activity of immobilized cellulase related to the activity of freecellulase.

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, NH4Cl were added into PNB/PMDB systems to adjust the partition coef-ficients, respectively. The concentrations of all the salts were from 10 to 90 mMwith 10 mM step. Samples taken from top and bottom phase by a syringe weredetermined by spectrophotometer at 540 nm after the aqueous two-phase systemsreached equilibrium. The partition coefficient was determined by the concentra-tion of glucose partitioning in the top phase related to the concentration of glucosepartitioning in the bottom phase.

2.9. Biodegradation reaction of cellulose in ATPS

Reducing sugar was produced by using cellulase as a catalyst and microcrys-talline cellulose as substrate in PNB/PMDB ATPS and single aqueous phase system ascontrol. 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 tubewas centrifuged at 4 ◦C, 8000 × g for 20 min to accelerate phase separation. The totalreaction volume was 10 ml. The effect of different concentration of microcrystallinecellulose on the yield of reducing sugar was investigated. The sample was taken outfrom top and bottom phase every 12 h to determine the concentration of product

2000 J. Liu, X. Cao / Process Biochemistry 47 (2012) 1998–2004

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ig. 1. (a) Synthesis of copolymers PMDB. Synthesis of pH-response copolymer PMDB

he chemical structure of thermo-response copolymer PNB.

fter the ATPS was formed by using centrifuge. The degree of saccharification cane calculated by the following equation [34]:

accharification (%) = Reducing sugar (g) × 0.89Carbohydrates (g)

× 100%

. Results and discussion

.1. Preparation of PNB–PMDB aqueous two-phase systems

We aim to synthesize a recyclable copolymer forming ATPSnd to immobilize cellulase on it. Copolymer PMDB was synthe-ized by others in our lab. The reserved enzyme activity of cellulasemmobilized on copolymer PMDB with different monomer ratio washown in Table 1. It has been found that the reserved enzyme

ctivity had correlation with the molar ratio of DMAEMA of threeonomers in copolymer. MAA is an acid monomer with carboxyl

roups. Cellulase has amino groups, which were suitable for cova-ently binding carboxyl groups on copolymer (Fig. 2). Therefore,

able 1ellulase immobilized on pH-response copolymer PMDB with different monomeratio.

Copolymer PMDB withdifferent monomer ratioa

Reserved enzymeactivity (%)b

1 19:8:1 <10%2 19:4:1 <50%3 19:2:1 60–70%4 19:1.5:1 60–70%5 19:1:1 80–90%6 19:1:2 60–70%

a Monomer ratio indicates M(�-methacrylic):M(DMAEMA):M(butyl methacry-ate) (w/w).

b Immobilization conditions: reaction time is 4 h; pH is 6.0; EDC amount is00 mg/g PMDB.

unctional monomers mole ratio of copolymer PMDB are MAA:DM:BMA = 19:1:1. (b)

copolymer with high content of MAA has more possibility to bindcellulase. When the molar ratio of DMAEMA of three monomerswas high (the monomers molar ratio of MAA/DMAEMA/BMA was19:8:1), there was not any activity after cellulase immobilized onit, while the enzyme activity in residual solution after immobiliza-tion was decreasing obviously. This indicated that cellulase wasimmobilized on copolymer PMDB. But the immobilized cellulasecannot show any activity after immobilization at high molar ratioof DMAEMA of three monomers. One possible explanation was thatthe copolymer might generate steric hindrances which blockedsubstrate approach to the active sites of immobilized enzyme. Asa result, immobilized enzyme cannot show any activity during thereaction. In fact, the amount of cellulase immobilized on copolymerwas increasing with molar ratio of DMAEMA of three monomersdecreasing and ratio of carboxyl group of copolymer increasing. Itcould be observed from the enzyme activity in residual solutionafter immobilization. The reserved enzyme activity was more than80% when the monomer molar ratio of MAA/DMAEMA/BMA was19:1:1.

As shown in Table 2, PMDB with different monomer molarratio was synthesized to test the possibility of forming ATPS

with thermo-response copolymer PNB. PMDB is a pH-responseamphiphilic copolymer, which has acid group and alkaline group.Monomer MAA is an acid monomer with negative charge, while

Table 2The ATPS phase-forming test of copolymers with different monomer ratio.

Copolymer PMDB (M �-methacrylic:M DMAEMA:Mbutyl methacrylate):copolymer PNB

Formation of ATPS

1 PMDB (19:2:1):PNB NO2 PMDB (19:1.5:1):PNB NO3 PMDB (19:1:1):PNB YES4 PMDB (19:1:2):PNB NO

J. Liu, X. Cao / Process Biochemistry 47 (2012) 1998–2004 2001

Fig. 2. Schematic illustration for cellulase immobilization. The functional monomers mole ratio of PMDB are: MAA:DM:BMA = 19:1:1. Immobilization conditions: reactiont

mWmtPomMi

ime: 4 h, pH: 6.0, EDC amount: 300 mg/g PMDB.

onomer DMAEMA is an alkaline monomer with a positive charge.e should prepare a copolymer with the most suitable monomerolar ratio (the monomer molar ratio of MAA/DMAEMA/BMA)

o form ATPS with PNB. It can be seen that only copolymerMDB at the special monomers ratio (the monomer molar ratiof MAA/DMAEMA/BMA was 19:1:1) can form ATPS with copoly-

er PNB. Eventually, we chose PMDB (the monomers molar ratio ofAA/DMAEMA/BMA was 19:1:1) as the pH response copolymer to

mmobilize 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. Thephase separation is driven by repulsion between the copolymer PNBand PMDB. Thermo-response copolymer PNB is enriched in the topphase 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 theaqueous two-phase systems, the concentration of copolymers inthe top phase and the concentration of copolymer in the bottom

2002 J. Liu, X. Cao / Process Biochemistry 47 (2012) 1998–2004

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ig. 3. The phase diagram of PNB/PMDB aqueous two-phase systems. All the experi-ents are carried out at room temperature (below LCST).

hase, respectively. The volume ratio of the phase could be calcu-ated by the length ratio of line MB and MT on tie line TMB. In thexperiment, the concentration of copolymer PNB was 2.5% (w/w) inotal phase systems, and the concentration of copolymer PMDB was% (w/w) in total phase systems. The volume ratio of top phase toottom phase was 2:1.

.3. Recovery of copolymers

Copolymer PMDB with acid monomer (MAA) and alkalineonomer (DM) is an amphiphilic copolymer and it could be precip-

tated from solution by adjusting pH to its isoelectric point (pI = 3.1)y adding HCl.

Fig. 4 indicated that the recoveries of pH-response copolymerMDB after recycled five times. Each recovery was average value of

hree parallel experimental points. The average recovery of PMDBas kept 97.2–97.9% after using five times. The values were high

nd stable. This indicated that the copolymer PMDB could still keep0% after 40 recycling.

ig. 4. The recycle recovery of copolymer PMDB. PMDB was precipitated by adjustingH to 3.1, and collected by centrifugation in 8000 rpm. The precipitate was dried to

constant weight and dissolved again. The recovery of PMDB was calculated as theatio 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 ATPSwere showed in Fig. 5. Each recovery was also average valueof three parallel experimental points. Thermo-response copoly-mer was precipitated by raising the temperature above its LCST(33 ◦C). The average recoveries of PNB and PMDB were 96.5% and95.4% at the five cycles with relation to initial amount, respec-tively.

3.4. Partition of reducing sugar in ATPS

The partition coefficient of reducing sugar was measured inthis experiment, and different salt types with varied concentra-tion were used to improve partition of reducing sugar. Reducingsugar was evenly partitioned to the top and bottom phases with-out neutral salts. The effect of different kinds of salts including KCl,KSCN, NaCl, NaSCN, NH4Cl on partition of reducing sugar was inves-tigated from 10 mM to 90 mM concentrations. Partition betweentwo phases depends on many factors since interactions betweenthe partitioned substance and the components of each phase area complex phenomenon involving hydrogen bonds, charge inter-action, Van der Waals forces, hydrophobic interactions, and stericeffects. The results were shown in Fig. 6. As the results shown,reducing sugar was enriched in the top phase and the partitioncoefficient was 3.68, in presence of 50 mM KCl. Different speciesof ions have different effects on the partition coefficient in ATPS.The physicochemical interactions of copolymers with each otherand with salts determine the equilibrium distribution of all thecomponents in the system. Copolymer PMDB with acid monomer(MAA) and alkaline monomer (DMAEMA) is an amphiphilic copoly-mer enriched in the bottom phase. PNB enriched in the top phase.One of possible explanation for the partition coefficient of reducingsugar is that the existence of KCl could change the intermolecularattraction including Van der Waals force and hydrophobic inter-action between product and copolymers, which makes reducingsugar distribute unevenly between two phases. The intermolecu-lar attraction between reducing sugar and copolymer PNB might bebigger than that between reducing sugar and copolymer PMDB, andthen 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 largerdensity of particles than that of water phase or large size of the

J. Liu, X. Cao / Process Biochemistry 47 (2012) 1998–2004 2003

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Fig. 7. (a) Bioconversion of cellulose in ATPS and in single aqueous phase. Micro-crystalline cellulose hydrolysis is carried out in PNB/PMDB ATPS at 30 ◦C. Substrate

Fig. 6. Effect of salts on partition coefficient of reducing sugar.

articles. The insoluble particles would be distributed in interphaseetween two phases at excess. Similar observation was previouslyeported [1].

.5. Bioconversion reaction of microcrystalline cellulose

Cellulase was immobilized on pH-response copolymer PMDBefore bioconversion reaction. The hydrolysis of microcrystallineellulose by immobilized water-soluble cellulase was carried out in

shaker with 150 rpm at 30 ◦C. Phase transfer catalyzing reaction ofellulase with different concentrations of substrate was carried outn this experiment. When the initial concentration of substrate was.5% (w/v), both the reaction equilibrium time in ATPS and in singlequeous phase were 48 h. The saccharification percentage yield andeducing sugar concentration in ATPS were 40.16% and 2.23% (w/v),espectively. While the values in the single aqueous phase reactionere 29.22% and 1.62% (w/v) (Fig. 7a). It can be seen from the phaseiagram that copolymer PMDB was mainly in the bottom phase. Cel-

ulase was immobilized on PMDB before bioconversion reaction. As result, immobilized cellulase was mainly partitioned in the bot-om phase. In PNB/PMDB ATPS, product was partitioned in the tophase with adding 50 Mm KCl. With biodegradation reaction wentn, the concentration of reducing sugar in the top phase increasedhree times more than that in the bottom phase. This indicated that

icrocrystalline cellulose was catalyzed into reducing sugar, whichntered into top phase at the presence of 50 mM KCl. In this way,nhibition of product was removed and saccharification percentageield was improved in ATPS compared with that in the single aque-us phase. As shown in Fig. 7b, when the concentration of substrateas increased to 1% (w/v), the reaction equilibrium time in the ATPS

nd in the single aqueous phase were 48 h. The saccharificationercentage yield and reducing sugar concentration in ATPS were4.23% and 2.69% (w/v), respectively. While the values in the singlequeous phase reaction were 14.09% and 1.56% (w/v), respectively.ubstrate was catalyzed into reducing sugar in the bottom phase,nd the product entered into the top phase. As a result, saccharifi-ation percentage yield was also improved. Although the increase

f reducing sugar yield was limited because of low partition coef-cient of product, it still had a large space for improvement. If theartition coefficient could be increased more, the yields of reducing

concentration was 0.5% (w/v). (b) Bioconversion of cellulose in ATPS and in singleaqueous phase. Substrate concentration was 1% (w/v).

sugar were also increased much more than that is now comparedwith the yield in single aqueous phase.

It could be seen that the concentration of substrate had not mucheffect on the reaction equilibrium time, but had obvious effects onthe yield of reducing sugar. At low concentration of microcrystallinecellulose, yield of reducing sugar in ATPS was 10.94% higher thanthat in the single aqueous phase, while at high concentration ofsubstrate, yield of reducing sugar in ATPS was 10.14% higher thanthat in the single aqueous phase. Substrate was not soluble in thisexperiment. The increase of substrate concentration in the reactionmedium might limit the amount of saccharification, probably dueto decrease mobility of insoluble substrate in the aqueous phaseof the reaction mixture or increase the viscosity of the reactionmixture which had an adverse effect on the mobility of the reactantsand the release of products. On the other hand, the concentrationof the released reducing sugar in ATPS was 2.69% (w/v). Higherconcentration of reducing sugar may also lead to the decrease ofreaction rate because of product inhibition. Similar observationswere previously reported [35,36].

After saccharification reaction, copolymer PMDB and PNB couldbe recovered by aforementioned method. Copolymer PMDB withimmobilized cellulase could be recovered over 95%.

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. Conclusions

In this study, novel PNB/PMDB ATPS with water-soluble immo-ilized cellulase was used to biodegradation of cellulose. Cellulaseas immobilized on PMDB, with over 80% reserved enzyme activ-

ty. The recoveries of both copolymers are over 95%. A significantdvantage is that phase-forming polymers and cellulase could beecycled at a very low cost and there is not any environmen-al problem. On the other hand, phase transfer bioconversion ofellulose has been carried out in this novel ATPS. The saccharifi-ation in ATPS could reach 40.16% after 48 h, 10.94% higher thanhat in the single aqueous phase. Since the optimum temperaturef cellulase hydrolyzing microcrystalline cellulose is 50 ◦C, whileur experiment can only carried out at 30 ◦C because of limita-ion of thermo-response temperature of copolymer PNB. Althoughhe reaction temperature was limited, saccharification in ATPS wasbsolutely improved, compared with the reaction in single aqueoushase. Though the yield of product yet is not quite satisfied, we areow looking for some new methods and have made some progress.

n this work, we focus on the feasibility of biodegradation cellu-ose in recycling ATPS. Through the further study, the yield shouldchieve higher. It is believed that bioconversion reactions in theecycling ATPS are potential application value in future bioenergynd biochemicals industry not limited to cellulose biodegradation.

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