immobilization of cellulase in nanofibrous pva membranes by electrospinning

Journal of Membrane Science 250 (2005) 167–173

Immobilization of cellulase in nanofibrous PVA membranesby electrospinning

Lili Wu, Xiaoyan Yuan∗, Jing ShengSchool of Materials Science and Engineering, Tianjin University, Tianjin 300072, PR China

Received 12 August 2004; accepted 26 October 2004Available online 28 December 2004

Abstract

Electrospinning is a nanofiber-forming process by which either polymer solution or melt is charged to high voltages. With high specificsurface area and porous structure, electrospun fibrous membranes are excellent candidates for immobilization of enzymes. In this paper,immobilization of cellulase in nanofibrous poly(vinyl alcohol) (PVA) membranes was studied by electrospinning. PVA and cellulase weredissolved together in an acetic acid buffer (pH 4.6) and electrospun into nanofibers with diameter of around 200 nm. The nanofibrousm mobilizedc olution fori sslinkingt©

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embranes were crosslinked by glutaraldehyde vapor and examined catalytic efficiency for biotransformations. The activity of imellulase in PVA nanofibers was over 65% of that of the free enzyme. Nanofibers were superior to casting films from the same smmobilization of cellulase. The activity of immobilized cellulase descended with ascending in enzyme loading efficiency and croime, which retained 36% its initial activity after six cycles of reuse.

2004 Elsevier B.V. All rights reserved.

eywords:Electrospinning; Nanofiber; Immobilization; Cellulase; Poly(vinyl alcohol)

. Introduction

Electrospinning was first reported in 1934 and has been re-eived a dramatic revival of interests in recent years becausef its potential to produce ultrafine fibers with diameters in

he range of nanometer to sub-micrometer. Electrospun fibersre excellent candidates for tissue engineering scaffolds andound dressings due to their high specific surface area andorous structure[1]. Strong electrostatic field is applied to theetal capillary of a syringe which held polymer solution dur-

ng electrospinning. The pendent droplet is deformed into aaylor cone by electrostatic field. When the voltage surpasseshe threshold value, the electrostatic force overcomes the sur-ace tension of the droplet and a charged jet of the solutions ejected from the tip of the Taylor cone. As the jet movesoward a grounded metal collector, the solvent evaporates

∗ Corresponding author.E-mail address:[email protected] (X. Yuan).

and non-woven fibrous membranes are formed[2–5]. In theprocess of electrospinning, the governing parameters suthe applied voltage, the solution flow rate, the capillarycollector distance and the polymer/solvent systems, seriaffect the electric current and charge density in the polyjet. These parameters mainly determined the spinnabilitystability of the polymer[6].

Enzymatic biotransformations have been pursued esively, largely as a result of their unparalleled selectivitymild reaction conditions. In many cases, however, lowalytic efficiency and stability of enzymes were considerebarriers for the development of large-scale operations anplications. Immobilization of enzymes on inert, insoluble mterials is currently an active research area in order to impthe functionality and performance of enzymes for biopcessing applications. Immobilized enzymes allow their repermit better control reaction and are more stable than soones[7–11]. The structure of the support materials has a gimpact on the performance of the immobilized enzyme.duction of the geometric size of the enzyme support can e

376-7388/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.memsci.2004.10.024

168 L. Wu et al. / Journal of Membrane Science 250 (2005) 167–173

tively improve the catalytic efficiency of enzymes because thediffusion resistance can be remarkably reduced. It is thoughtthat the large specific surface area and the fine porous struc-ture of electrospun fibrous membranes could greatly increasethe catalyzing ability of the immobilized enzymes. Jia et al.[8] first prepared immobilized�-chymotrypsin on the surfaceof polystyrene nanofibers produced via electrospinning. Thehydrolytic activity of immobilized enzyme was over 65% ofthat of the free enzyme. Xie and You[9] investigated thefeasibility of electrospinning in converting natural proteinsinto nanofibers and explored the immobilized lipase by elec-trospinning of enzyme-containing solutions. However, thecatalytic activities of lipase in electrospun membranes wereabout two orders of magnitude lower than that of free lipasebecause of crosslinking reaction and electrospinning. It ispresumed that the activity of enzymes immobilized in elec-trospun fibers may vary with the crosslinking method andthe enzyme species. In recent years, advantages in reducingthe geometrical size of the support materials play importantroles in enzyme immobilization. However, no further reportsabout electrospun nanofibers as the support for immobilizedenzymes were found except the above two papers. Therefore,it is necessary to further investigate this efficient method ofimmobilization.

Cellulase, with the highest activity at pH 3.0–5.5 and4 ◦ hy-d d canc tainc isl ter-s mals n-s tion[

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2.2. Formation of nanofibrous PVA/cellulase membranes

PVA and cellulase were dissolved in an acetic acid buffer(pH 4.6) to obtain PVA solution containing cellulase in 0%,2.5%, 5.0%, 7.5% and 10.0% amount, respectively. For elec-trospinning, each solution was placed in a glass syringe(50 ml) bearing a metal capillary (0.8 mm i.d.) which wasconnected with a high voltage power supply (GDW-a, Tian-jin University). Grounded counter electrode was connectedto the copper net collector. Typically, electrospinning wasperformed at 10 kV voltage, 10 cm distance between the cap-illary tip and the collector. The flow rate of the solutionwas controlled by a syringe pump (WZ-50C2, Zhejiang Uni-versity) to maintain at 0.2 ml/h from the capillary outlet. Itusually took 12–14 h to obtain sufficiently thick membrane(about 10�m) that can be detached from the copper net col-lector.

2.3. Immobilization of cellulase

Immobilization of cellulase in nanofibrous PVA mem-branes was carried out by crosslinking the electrospunPVA/cellulase membranes at 37◦C by glutaraldehyde vaporfor 1 h [18]. For the comparison purpose, casting films wereprepared from the same solution as those used for electro-s

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0–50 C, can be used as the biocatalyst for celluloserolysis. Cellulase has a wide range of substrates anatalyze various kinds of natural polymers which conellulose[12,13]. The study of immobilization of cellulase

ess reported. Poly(vinyl alcohol) (PVA) is a non-toxic, waoluble synthetic polymer with good chemical and thertability[14,15]. Because of its biocompatibility, PVA is coidered as an appropriate matrix for enzyme immobiliza16,17].

This paper will present the immobilized cellulase in Panofibers by electrospinning. Morphology of electrosVA fibers embedded with cellulase is discussed along

he effect of enzyme loading efficiency, crosslinkingeuse on the activity of immobilized cellulase.

. Experimental

.1. Materials

PVA with 1750± 50 degree of polymerization and 98f degree of hydrolysis was supplied by Experimehemical Plant of Tianjin University (Tianjin, China). C

ulase was purchased from Xinjingke Biologic Techniompany (Beijing, China). The substrate carboxymeellulose (CMC) (Beijing Chemical Agent Compaeijing, China) and the crosslinking agent glutarayde (50%, Damao Chemical Apparatus Supply Agianjin, China) were used without further treatment.ther agents used in the experiments were of anarade.

pinning and crosslinked in the same way.

.4. Analyses

The morphology of nanofibrous PVA/cellulase meranes was viewed under a Philips XL-30 scanning electicroscope (SEM) after being sputtered with gold. T

hemical composition was verified by Fourier transformrared spectroscopy (IR) (Bio-Rad FTS3000) and X-ray poelectron spectroscopy (XPS) (Perkin-Elmer PHI-1600

.5. Determination of enzyme loading efficiency

The PVA/cellulase membranes before crosslinking wncubated in 3 ml of the acetic acid buffer (pH 4.6) at 4◦Cor 12 h. It was supposed that all the cellulase could beased from the membrane and got into the buffer. Thenmount of cellulase was measured by a spectrophotoU-1800, Japan) at 280 nm. The enzyme loading efficias determined by ratioing the cellulase amount (mg) inembrane to the whole membrane mass (mg).

.6. Measurement of cellulase activities

The hydrolytic activity of cellulase was measured by u2% (w/v) CMC solution as the substrate. An acetic

uffer (pH 4.6) was used as the medium. After incubatiowater bath at 50◦C for 30 min, the reaction was stoppedddition of 1.5 M NaOH solution. The amount of generalucose was measured by the spectrophotometer at 5ith DNS agent as a color indicator[19]. An activity unit

L. Wu et al. / Journal of Membrane Science 250 (2005) 167–173 169

(U) of cellulase was defined as the amount of cellulase thatcatalyzes CMC to generate 1 mg glucose per minute under theabove assay conditions. Therefore, the activity of cellulasewas calculated according to the following equation:

activity of cellulase (U/g) = mg × 1000

t × mc

wheret is the time of reaction,mg (mg) andmc (mg) are re-ferred to the amount of glucose and cellulase, respectively.The retained activity of immobilized cellulase was deter-

mined by the percentage of the activity of immobilized cel-lulase in the activity of free cellulase.

3. Results and discussion

3.1. SEM analysis

Pure PVA solution can be electrospun into nanofibers withdiameter of around 200 nm as shown inFig. 1(a). With the

Fr

ig. 1. SEM micrographs of electrospun PVA/cellulase fibers from PVA soespectively (original magnification 5000×).

lutions with (a) 0%, (b) 2.5%, (c) 5.0%, (d) 7.5% and (e) 10.0% of cellulase,


Fig. 2. IR spectra of electrospun PVA (a) and PVA/cellulase (b) membranes.

addition of cellulase, the electrospun fibers became irregu-lar and were interspersed with shuttle-shape beads in 3�mlength and 500 nm diameter. Increasing cellulase amount inthe solution mixture resulted in more beads (Fig. 1(b)–(e)). Asa protein, it is impossible for cellulase to be electrospun aloneinto nanofibers due to its complicatedly three-dimensionalstructures as well as strong inter- and intra-molecular forces[9]. The blend of cellulase with PVA can interrupt its complexstructure and destroy molecular interactions because PVA hascapacity to form secondary bonding with proteins. Therefore,the mixture of PVA and enzyme can form nanofibers by elec-trospinning. On the other hand, the interaction of PVA andcellulase resulted in the reduction of stability of PVA solu-tion and induced the appearance of beads. It is assumed thatsmall amount of beads in the electrospun membranes havenot effect on the activity of immobilized enzyme.

3.2. IR and XPS analyses

In order to examine whether cellulase existed in theelectrospun nanofibers, IR spectra of both electrospun PVAand PVA/cellulase membranes were measured as shown inFig. 2. PVA (Fig. 2(a)) exhibited stretching vibration bandof hydrogen-bonded alcohol (OH) at 3372 cm−1 [20]. In

IR spectrum of PVA/cellulase membranes (Fig. 2(b)), theabsorption band at 3372 cm−1 was wider than that of PVAfibers due to the superposition of stretching vibration of OHand N H [20]. Besides, the characteristic absorption bandsat 1652 cm−1 in Fig. 2(b) were induced from OC NH [19].It is concluded that there were a given amount of cellulase inelectrospun fibrous membranes.

XPS was widely used in analyzing the surface structure ofsubstances. Element components and functional groups canbe detected by XPS spectra. Results of XPS analysis of elec-trospun fibers from PVA solutions containing cellulase rang-ing from 0% to 10% were shown inTable 1. It indicated thatsulfur and nitrogen element coming from cellulase existed onthe PVA/cellulase membranes, but not on the PVA membrane.Furthermore, with increasing the cellulase amount in the elec-trospun solutions, the quantity of amide group OC NH,which was identified as C 1s 288.2 eV[21,22], was alsoincreased. However, amounts of sulfur, nitrogen and amidegroup, that are assigned to the amount of enzyme, were not ashigh as that anticipated. These indicated that cellulase werenot only attached on the surface of electrospun fibers but alsoembedded within the PVA nanofibers.

Results of IR and XPS could verify that cellulase has beenimmobilized in PVA nanofibers by electrospinning.

TA

Ce

(%)

)

1

able 1nalysis by XPS

ellulase amount inlectrospun solutions (%)

O 1s (%) S 2p (%) N 1s

0 25.1 0 02.5 25.8 0.2 05.0 24.5 0 0.60.0 28.5 0.2 0.5

C 1s

– (%) 285.0 eV (%) 286.4 eV (%) 288.2 eV (%

74.9 66.61 31.02 2.3674.0 62.63 34.27 3.1074.9 72.40 23.63 3.9870.8 58.23 38.23 3.53


Table 2Activity of immobilized cellulase

Cellulase amount inelectrospun solutions (%)

Electrospun membrane Casting film

Loadingefficiency (%)

Activitya (U/g) Retainedactivity (%)

Loadingefficiency (%)

Activity (U/g) Retainedactivity (%)

2.5 3.9 149.2 99.0 2.7 121.8 80.85.0 6.2 136.3 90.4 5.0 86.7 57.57.5 8.9 107.9 71.6 7.4 65.2 43.3

10.0 11.4 98.7 65.5 9.5 51.5 34.2a The activity of free cellulase was 150.7 U/g.

3.3. Activity of immobilized cellulase

The activity and the retained activity of immobilizedcellulase with different enzyme loading efficiencies by bothelectrospinning and casting methods were given inTable 2.A comparison of the values inTable 2clearly showed thatthe activity of cellulase immobilized by electrospinning wasover 65% of that of free enzyme (150.7 U/g), and was nearlytwo times higher than that in casting films from the samesolution. This is a quite high activity as compared with otherforms of immobilization[12,13]. The apparent activities ofimmobilized enzymes are usually much lower than the homo-geneous activities of free enzymes. In the case of nanofibrousenzyme, the enzyme is more likely exposed to the surfaceand much less diffusion resistance can be expected becauseof their higher surface area and porous structure. Otherfactors, however, may become predominant in limiting theactivity of the nanofibrous cellulase. For example, enzymesin the solid state and bond to solids had much more limitedaccess to the substrate than free ones. Besides, not allcellulase incorporated in polymer blends resided on thesurface or were exposed to the substrate to participate in thereaction.

3.4. Effect of crosslinking time

tionh mesb yme.T thiss nedbc o-b s ofc berss morea therp edc tiono ree timer inkingr lesse flu-e hich

may be another factor that caused the reduction of activ-ity of cellulase. As shown inFig. 4, crosslinking of thenanofibrous PVA/cellulase membranes caused the fibers tobecome densely packed due to the conglutination betweenPVA molecules. As a result, the surface area was reduced,which made enzymes more difficult to access to the substrateand decrease of the catalytic activity.

Comparing with the results of Xie and You[9], the activityof enzyme immobilized by electrospinning was much higherin our work. This could be mainly attributed to the change ofenzyme species and crosslinking method. It is suggested thatcrosslinking by glutaraldehyde vapor could largely reducethe toxicity of the crosslinking agent.

3.5. Reuseability of immobilized cellulase

The recycling stability of immobilized cellulase was ex-amined by measuring the activity repeatedly. As seen fromFig. 5, the relative activity of the immobilized cellulase com-pared to its initial value in percentage decreased along withthe reusing times and the immobilized enzyme retained over36% of its initial activity after six cycles of reuse. Suchreusability is advantageous for the continuous use of this en-zyme in industry application. The loss of catalytic activitymay be explained by the following two reasons. Firstly, there

F es.

It has been well demonstrated that crosslinking reacas a great impact on the activity of immobilized enzyecause crosslinking could destroy the active site of enzhe effect of crosslinking by glutaraldehyde vapor intudy on the activity of immobilized cellulase was determiy changing crosslinking time. As shown inFig. 3, with in-reasing the crosslinking time, catalytic efficiency of immilized cellulase was reduced gradually. In the procesrosslinking, cellulase was further bonded to PVA nanofio that it accessed more difficultly to the substrate andctive centers of it were destroyed. However, with furrolonging of crosslinking time, the activity of immobilizellulase descended more slowly compared to the initiaf crosslinking becauseOH groups in PVA molecules weasy to react with glutaraldehyde. When crosslinkingeached a certain value as shown by the data, the crossleaction occurred only among PVA molecules and hadffect on enzymes. Besides, crosslinking would also innce the morphology of the electrospun membrane, w
ig. 3. Effect of crosslinking time on the activity of immobilized cellulas


Fig. 4. SEM micrographs of electrospun PVA/cellulase membranes after crosslinking for (a) 1 h, (b) 2 h and (c) 4 h, respectively (original magnification 3000×).

may be some cellulase molecules, which were not chemicalbonded on PVA fibers but only physical embedded, were lostduring the process of measurement. Secondly, the diameterof PVA fibers became larger and the surface area got smallerafter several arrays (Fig. 6) due to the hydrophilicity of PVA.

Fig. 5. Relative activity of immobilized cellulase after repeated assay.

Fig. 6. SEM micrograph of the electrospun PVA/cellulase membrane afterrepeated assays for six times (original magnification 5000×).

These resulted in the reduction of immobilized cellulase ac-tivity in the subsequent measurement.

4. Conclusions

Cellulase was successfully immobilized in PVAnanofibers by electrospinning under proper conditions. The


chemical composition of nanofibers was confirmed by IRand XPS. Results indicated that cellulase was embedded inPVA nanofibers. The activity of immobilized cellulase in theelectrospun PVA nanofibrous membranes after crosslinkingby glutaraldehyde vapor was over 65% of that of the freeenzyme and nanofibers were superior to casting films tobe used in immobilization of cellulase. The activity ofimmobilized cellulase descended with ascending of thecrosslinking time and retained 36% its initial activity aftersix cycles of reuse. It is implied that electrospun nanofibersare appropriate for immobilization of enzymes.

Acknowledgments

This work was funded by RFDP, Ministry of Education,PR China, No. 20020056048.

References

[1] Z.M. Huang, Y.Z. Zhang, M. Kotaki, et al., A review on polymernanofibers by electrospinning and their applications in nanocompos-ites, Comp. Sci. Technol. 63 (2003) 2223–2253.

[2] J. Zeng, X.Y. Xu, X.S. Chen, et al., Biodegradable electrospun fibersfor drug delivery, J. Control. Release 92 (2003) 227–231.

pun

kingLett.

ul-003)

n oftions,

bil-stry

[8] H.F. Jia, G.Y. Zhu, B. Vugrinovich, et al., Enzyme-carrying poly-meric nanofibers prepared via electrospinning for use as unique bio-catalysts, Biotechnol. Prog. 18 (2002) 1027–1032.

[9] J.B. Xie, Y.L. Hsieh, Ultra-high surface fibrous membranes fromelectrospinning of natural proteins: casein and lipase enzyme, J.Mater. Sci. 38 (2003) 2125–2133.

[10] Y.M. Yang, J.W. Wang, R.X. Tan, Immobilization of glucose oxidaseon chitosan–SiO2 gel, Enzyme Microb. Technol. 34 (2004) 126–131.

[11] S. Onal, A. Telefoncu, Preparation and properties of�-galactosidasechemically attached to activated chitin, Artif. Cell. Blood SubstitutesBiotechnol. 31 (2003) 339–355.

[12] X.Y. Yuan, N.X. Shen, J. Sheng, et al., Immobilization of cellu-lase using acrylamide grafted acrylonitrile copolymer membranes, J.Membr. Sci. 155 (1999) 101–106.

[13] A.C. Chakrabarti, K.B. Storey, Immobilization of cellulase usingpolyurethane foam, Appl. Biochem. Biotechnol. 19 (1988) 189–207.

[14] A. Koski, K. Yim, K. Shivkumar, Effect of molecular weight onfibrous PVA produced by electrospinning, Mater. Lett. 58 (2004)493–497.

[15] B. Ding, H.Y. Kim, S.C. Lee, et al., Preparation and characterizationof nanoscale poly(vinyl alcohol) fibers via electrospinning, FiberPolym. 3 (2002) 73–79.

[16] V.I. Lozinsky, F.M. Plieva, Poly(vinyl alcohol) cryogels employedas matrices for cell immobilization. 3. Overview of recent re-search and developments, Enzyme Microb. Technol. 23 (1998) 227–242.

[17] Y.C. Liu, X.H. Zhang, H.Y. Liu, et al., Immobilization of glucoseoxidase onto the blend membrane of poly(vinyl alcohol) and regen-erated silk fibroin: morphology and application to glucose biosensor,

[ LGArticu-Res.

[ . 59

[ an, pp.

[ Or-

[ ers,

[3] A. Pedicini, R.J. Farris, Mechanical behavior of electrospolyurethane, Polymer 44 (2003) 6857–6862.

[4] C.L. Shao, H.Y. Guan, S.B. Wen, et al., A novel method for maNiO nanofibres via an electrospinning technique, Chin. Chem.15 (2004) 365–367.

[5] C. Nah, S.H. Han, M.H. Lee, et al., Characteristics of polyimidetrafine fibers prepared through electrospinning, Polym. Int. 52 (2429–432.

[6] S.A. Theron, E. Zussman, A.L. Yarin, Experimental investigatiothe governing parameters in the electrospinning of polymer soluPolymer 45 (2004) 2017–2030.

[7] A.V. Bacheva, O.V. Baibak, A.V. Belyaeva, et al., Activity and staity of native and modified subtilisins in various media, Biochemi(Moscow) 68 (2003) 1261–1266.

J. Biotechnol. 46 (1996) 131–138.18] G.P. Chen, T. Sato, T. Ushida, et al., The use of a novel P

fiber/collagen composite web as a scaffold for engineering of alar cartilage tissue with adjustable thickness, J. Biomed. Mater.A 67A (2003) 1170–1180.

19] K. Ghose, Measurement of cellulase activities, Pure Appl. Chem(1987) 257–268.

20] A.H. Kuptsov, G.N. Zhizhin, Handbook of Fourier Transform Ramand Infrared Spectra of Polymers, Elsevier, Amsterdam, 199871–72.

21] R.M. Silverstiein, F.X. Webster, Spectrometroc Identification ofganic Compounds, Wiley, New York, 1998, pp. 71–143.

22] G. Beamson, D. Broggs, High Resolution XPS of Organic PolymWiley, New York, 1998, pp. 96–97.

immobilization of cellulase in nanofibrous pva membranes by electrospinning

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