Noncovalent immobilization of cellulases using the reversibly soluble polymers for biopolishing of cotton fabric
Yuanyuan Yu Jiugang Yuan ∗ Qiang Wang Xuerong Fan Ping Wang Li Cui
Key Laboratory of Science and Technology of Eco-Textile, Ministry of Education, Jiangnan University, Wuxi, Jiangsu, 214122, People’s Republic of China
Abstract The hydrolytic reaction of cellulases can occur in the interior of cellulosic fibers, causing tensile strength loss of the fabrics. Cellulase immobilization is an approach to solve this problem, because enlarging the molecule size of cellulases will limit the hydrolysis to the surfaces of the fibers. In this study, commercial cellulases were noncovalently immobilized onto the reversibly soluble polymers (Eudragit S-100 and Eudragit L-100). The characteristics of cellulase-Eudragit S-100 (CES) and cellulase-Eudragit L-100 (CEL) were evaluated using Fourier transform infrared spectra, circular dichroism spectra, and fluorescence spectra. The CES showed higher stability
than CEL and free cellulase, especially at higher pH and temperature. CES and CEL retained 51% and 42% of their original activities after three cycles of repeated uses, respectively. In addition, the effects of cellulase treatment on the cotton yarn and fabric have been investigated. The bending stiffness results showed that the cotton fabric samples treated with the free and immobilized cellulases were softer than untreated samples. However, less fiber damage in terms of weight loss and tensile strength of treated cotton was C 2014 International Union of Biochemistry and Molecular observed. Biology, Inc. Volume 00, Number 00, Pages 1–8, 2014
Keywords: cellulase, cotton, Eudragit, noncovalent immobilization, recycling
1. Introduction In recent years, the problem of environmental pollution has aroused extensive concern all over the world. In the textile industry, the wastewater from wet processes is one of the main industrial pollution [1, 2]. To solve the problem, many enzymes have been used in the wet processes to replace harsh chemicals used in traditional processes [3]. For example, amylases are used in desizing, cellulases are used in cotton biopolishing and
Abbreviations: CD, circular dichroism; CEL, cellulase-Eudragit L-100; CES, cellulase-Eudragit S-100; EL, Eudragit L-100; ES, Eudragit S-100; FTIR, Fourier transform infrared; SEM, scanning electron microscopy; UV, ultraviolet. ∗ Address
for correspondence: Professor Qiang Wang, Key Laboratory of Science and Technology of Eco-Textile, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China. Tel.: +86-510-85912007; Fax: +86-510-85912009; e-mail: [email protected]. Received 25 February 2014; accepted 2 September 2014 DOI: 10.1002/bab.1289 Published online in Wiley Online Library (wileyonlinelibrary.com)
denim washing, and lipases and pectinases are used in cotton biopretreatment [4–10]. Cellulases are the most successful enzymes used in the textile industry, and many valuable effects can be acquired after cellulase treatments. However, the cellulase treatments have some critical drawbacks. First, cellulases are difficult to recover and reuse after the treatments. Second, the treatments can reduce the fabric strength because cellulases can easily penetrate into cotton fiber, leading to the hydrolysis of cellulose molecule in the interior of fiber [11, 12]. Cellulase immobilization is an approach to solve the problems. Cellulase immobilization can offer several advantages over the free cellulases such as recovery from the reaction medium, reuse, and operation in continuous reactors. Cellulase immobilization on some macromolecule carriers can enlarge the sizes of cellulases, and the diffusion of an enlarged enzyme molecule is significantly inhibited in the interior of the fiber. Eudragit S-100 (ES) and Eudragit L-100 (EL), the commercial copolymers of methacrylic acid and methyl methacrylate, have been extensively used as a carrier for enzyme immobilization. Eudragit can be precipitated at low pH around 4.5 and dissolved in aqueous solutions at pH over 5.5. When enzymes are immobilized on Eudragit, the solubility of the immobilized
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Biotechnology and Applied Biochemistry enzymes becomes similar to that of Eudragit. Thus, the immobilized enzymes can be used in their soluble form during the enzymatic reaction and recovered in their insoluble form after the reaction by altering the pH of the reaction mixture [13, 14]. Silva et al. [15] had immobilized protease on ES to decrease the loss of wool fabric strength after protease treatment. Xu et al. [16] had co-immobilized cellulase and xylanase on EL using a covalent coupling method. In our previous work, cellulases had been successfully immobilized on ES by a covalent coupling method, and the application of covalent–Eudragit–cellulase on the cotton biopolishing was studied. The cotton samples treated with covalent–Eudragit–cellulase showed lower weight loss and considerably higher tensile strength than those treated with free cellulase [17, 18]. Noncovalent adsorption is another frequently used technique for enzyme immobilization. The great advantage of noncovalent immobilization is that the enzyme does not have to be pretreated or chemically modified [19]. In the present work, noncovalent immobilization of cellulases on ES or EL was carried out. The structure of the immobilized cellulases was characterized by Fourier transform infrared (FTIR) spectra, circular dichroism (CD), and fluorescence spectra. The stability and reusability of the immobilized cellulases were studied. Biopolishing of cotton yarns and fabrics using the free and immobilized cellulases were investigated to evaluate the effect of the treatments and the degradation of cotton fibers.
2. Materials and Methods 2.1. Materials Acidic cellulase Suhong 989N with a CMCase activity of 92 U/mL was supplied by Novozymes (Suzhou, People’s Repub¨ lic of China). ES and EL were kindly donated by Degussa-Huls, S.A. (Shanghai, People’s Republic of China). Both ES and EL had a molecular weight of 135,000 Da, but the ratio of the free carboxyl groups to the ester groups was 1:1 for EL and 1:2 for ES. All other chemicals were of analytical grade. Wuxi Cotton Textile Company (Wuxi, People’s Republic of China) supplied the cotton yarns (38 tex) and woven fabrics. The specifications of the fabrics were as follows: thread density 18.276 tex, warp 13 threads/cm, weft 7 threads/cm, and weight per area 150 g/m2 .
2.2. Noncovalent immobilization of cellulase onto Eudragit Noncovalent immobilization of cellulase was carried out following a protocol developed by Sardar et al. [20] with some modifications. The pI of free cellulase is 7.7, and the enzyme has positive charges at pH 7.2. Eudragit has negative charges at pH 7.2. So the strong electrostatic interaction exists between free cellulase and Eudragit. Five grams of ES or EL was completely dissolved in 100 mL phosphate buffer (pH 7.2). To prepare 2 mL polymer solutions, 0.6 mL free cellulase (17.5 mg protein/mL) was added and then the total volume was made up to 10 mL with phosphate buffer (pH 7.2). After stirring at room
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temperature for 1 H, the pH of the solutions was adjusted to 4.2 by adding 0.1 M HCl solution to precipitate the immobilized cellulases. The precipitates were separated by centrifugation (10,000g, 10 Min), and then washed twice with HCl solution (pH 4.2). The immobilized cellulases were redissolved in 0.2 M acetate buffer at pH 5.5 for further study.
2.3. Determination of amount of binding cellulases and cellulase activity The amount of cellulase proteins was determined by the Bradford method, using bovine serum albumin as the standard [21]. The amount of binding cellulases was calculated according to Eq. (1). Amount of binding cellulase protein = M1 − M2
(1)
where M1 and M2 are the amounts of cellulase proteins initially used for the reaction and the unbinding cellulase proteins collected, respectively. The hydrolytic activity of the cellulase used in this work was measured using 1% (w/v) carboxy-methylated cellulose as a substrate. The reaction mixture was incubated in a water bath at 50 ◦ C for 30 Min, and the amount of glucose generated was measured by an ultraviolet (UV)–Vis spectrophotometer at 540 nm using 3,5-dinitrosalicylic acid agent as the color indicator [22]. The activity recovery yield was calculated based on Eq. (2) as follows: Activity recovery yield % (2) = activity recovered/activity loaded × 100
2.4. Characterization of immobilized cellulases The structure of the immobilized cellulases was investigated by FTIR spectroscopy with the KBr pellet technique. CD spectra were obtained using a MOS-450 spectropolarimeter in the range of 190–250 nm. A total of 150 µL of enzyme solution was added into the cell. The spectra were run with protein concentration ranging from 0.15 to 0.25 mg/mL at 20 ◦ C with a scan rate of 100 nm/Min, response time of 1 Sec, and resolution of 1 nm [23]. Fluorescence spectra were recorded on a Hitachi F4500 spectrofluorometer (Hitachi, Tokyo, Japan) at 20 ◦ C using a slit width of 5 nm and a 1 cm path length cuvette with a scan speed of 60 nm/Min. The spectra of free and immobilized cellulases were recorded from 300 to 400 nm using an excitation wavelength of 280 nm [24].
2.5. Solubility of immobilized cellulases The solubility of Eudragit and the immobilized cellulases was determined at 470 nm by a UV–Vis spectrophotometer and expressed as a ratio of absorbance [25]. The maximum value of the absorbance was set at pH 3.5 of the systems. Zero absorbance was set at the maximum solubility of the systems.
2.6. Enzymatic treatment of cotton samples The cotton yarn and fabric samples were treated with cellulases in 0.2 M acetate buffer with a liquor-to-fiber ratio of 20:1 at
Immobilization of Cellulases for Biopolishing
pH 5.5 and 50 ◦ C for 2 H. After the cellulase treatments, the samples were rinsed with deionized water, followed by deactivation of residual cellulases on the samples in deionized water at 80 ◦ C for 10 Min. Finally, the cotton samples were dried at 60 ◦ C.
2.7. Weight loss and tensile strength The weight loss of the cotton samples was calculated according to the following equation: Weight loss % = (W1 − W2 ) /W1
(3)
where W1 is the weight of the sample before the cellulase treatment and W2 is the weight of the sample after the cellulase treatment. The tensile strength of the cotton yarn samples was determined in accordance with ASTM D 1682-64 using a YG(B)025S Tensile Strength Tester (Wenzhou Darong Company, Wenzhou, People’s Republic of China). The tensile strength of the cotton fabric samples was determined in accordance with ASTM D5035-2006(2008)e1 using a YG(B)026H Tensile Strength Tester (Wenzhou Darong Company).
2.8. Wrinkle recovery angle, bending stiffness, and scanning electron microscopy of cotton fabric A standard method (AATCC test method 66-1984) was used to measure the wrinkle recovery angles. The bending stiffness was measured using a KES-FB-AUTO-A system. The surface morphology of the cotton samples was observed using a SU1510 scanning electron microscope (Hitachi). Platinum was sputtered onto the fabric samples as a conducting material to analyze the samples.
TABLE 1
Amount of binding cellulases and activity recovery yields after immobilization reaction
Amount of binding cellulases (mg)
Activity recovery yield (%)
CellulaseEudragit S-100
2.31 ± 0.10
82.2 ± 2.8
CellulaseEudragit L-100
5.62 ± 0.32
78.2 ± 3.4
Immobilized cellulase
shown in Fig. 1. The amide I and II bands are two of the most prominent vibrational bands of the protein backbone. The amide I absorption band is located in the region between 1,700 and 1,600 cm−1 , and the amide II band is located in the region between 1,600 and 1,500 cm−1 [26]. From CES and CEL spectra, it was found that distinctive absorption bands appeared at 1,658 (amide I) and 1,534 cm−1 (amide II). The absorption intensity of CES and CEL at about 3,400 cm−1 was stronger than that of ES and EL because of the superposition of stretching vibration of O—H and N—H in the protein structure of the cellulase. In addition, the characteristic adsorption
3. Results and Discussion 3.1. Noncovalent immobilization of cellulase on ES or EL By assaying the amount of the cellulase proteins collected in the supernatant after the immobilization process, the amount of binding cellulases was determined. As shown in Table 1, the amount of binding cellulases on EL (5.62 mg) was higher than that of binding cellulases on ES (2.31 mg). In the noncovalent immobilization process, the binding force is mainly the electrostatic force between carboxyl (Eudragit) and amino (cellulase). The amount of carboxyl in EL molecule is twice that of carboxyl in ES molecule. Therefore, more cellulase molecules were bound to EL than ES. Moreover, enzymes usually lose some of their activities after immobilization because of the blocking of the catalytic active site by binding of enzymes to polymers [13]. After the immobilization, the activity recovery yields of cellulase-Eudragit S-100 (CES) and cellulase-Eudragit L-100 (CEL) were 82.2% and 78.2%, respectively. The structure characteristics of the immobilized cellulases were analyzed by FTIR to verify the binding of cellulases onto Eudragit. FTIR spectra of ES, EL, CES, and CEL are
Biotechnology and Applied Biochemistry
FTIR spectra of (a) ES and CES and (b) EL and CEL.
FIG. 1
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Biotechnology and Applied Biochemistry
FIG. 2
FIG. 3
Circular dichroism spectra of free and immobilized cellulases.
peaks appeared at 520 cm−1 in the spectra of the immobilized cellulases, which could be assigned to the disulfide bond (—S—S—) in cellulase protein molecule. The results indicated the formation of immobilized cellulases via noncovalent binding.
3.2. CD of immobilized cellulases CD has been increasingly used as a spectroscopic tool for detecting the secondary and tertiary structures of proteins in a solution [27]. As shown in Fig. 2, there was a significant change in CD spectra of CES and CEL compared with that of the free cellulase. Analysis of the CD spectrum of the free cellulase resulted in secondary structural fractions of 53.04% helix and 11.47% strand (Table 2). After immobilization, CES contained about 46.67% helix and 13.46% strand and CEL contained about 24.52% helix and 27.33% strand. ES and EL were linear macromolecules. When the enzyme was bound to Eudragit, the enzyme molecules could be stretched and unfolded, causing the decrease in helix in the structure of immobilized enzyme. Moreover, the difference in the CD spectra could be due to either the change in the secondary structure of the enzyme after immobilization or the formation of some aggregative structures (e.g., less enzyme molecules were closely bound on one polymer backbone) [25].
Fluorescence emission spectra of Eudragit, free, and immobilized cellulases.
3.3. Fluorescence spectroscopy Fluorescence emission spectra of EL, ES, CEL, CES, and free cellulase excited at 280 nm are shown in Fig. 3. The fluorescence emission characteristics can be considered as a more sensitive parameter to study the structural changes involving the microenvironment of tryptophan [25]. Fluorescence was not detected at fluorescence emission spectra of ES and EL. The fluorescence intensity for the free cellulase was lower than that for CEL and CES. The increase in fluorescence intensity indicated that the enzyme molecules were unfolded after immobilization [25]. The results also showed that the secondary structure of the cellulase was altered, which was consistent with the finding in CD spectra.
3.4. Effects of temperature and pH on enzymatic activity Figure 4 shows the effects of pH and temperature on the relative activities of the free and immobilized cellulases. The maximum activities of the free and immobilized cellulases were observed at 50 ◦ C and 60 ◦ C, respectively. Immobilization of cellulases changed its temperature profile. Moreover, CES had better temperature stability than free cellulase and CEL. The activities of free cellulase and CEL showed a significant loss above 70 ◦ C, which was lower than 30% of the initial activity
The secondary structure of free and immobilized cellulases as determined by circular dichroism spectroscopy
TABLE 2 Enzyme
Helix (%)
Strand (%)
Turns (%)
Unordered (%)
Free cellulase
53.04
11.47
14.26
22.23
CES
46.67
13.46
15.45
24.42
CEL
24.52
27.33
20.42
27.73
4
Immobilization of Cellulases for Biopolishing
FIG. 4
Effects of (a) temperature and (b) pH on the relative activities of the free and immobilized cellulases.
at 90 ◦ C. However, the activity of CES remained above 60% at 90 ◦ C. It is known that enzyme activity is dependent on the ionization state of the amino acids in the active site. Hence, pH plays a significant role in maintaining the proper conformation of an enzyme. Figure 4b shows the effect of pH on the relative activities of cellulases. The relative activities of the free and immobilized cellulases increased and reached the maximum at pH 5, and then decreased with further increase in the pH value. In addition, CES was much more stable than free cellulase and CEL when the pH was above 5.0. The relative activity of CES remained 60% of its initial activity at pH 8.0, which was higher than that of CEL and free cellulase.
3.5. Solubility and reusability of immobilized cellulases The solubility of Eudragit and immobilized cellulases can be controlled by adjusting the pH of the solutions. As shown in Fig. 5a, ES was soluble in aqueous solutions above pH 4.8 and precipitated below pH 4.6, and EL was soluble in aqueous solutions above pH 4.5 and precipitated below pH 4.0. The
Biotechnology and Applied Biochemistry
FIG. 5
(a) Solubility and (b) reusability of immobilized cellulases.
solubility of the immobilized cellulases became similar to that of Eudragit. CES and CEL were soluble in aqueous solutions above pH 5.2 and precipitated below pH 4.5. Figure 5b shows the reusability of the immobilized cellulases. The CES and CEL retained 51% and 42% of their original activities after three cycles of repeated uses, respectively. The results demonstrated that the immobilized cellulases had certain reusability characteristics, which was important for the potential industry application.
3.6. Weight loss and tensile strength of cotton yarn The cotton yarn samples were treated with free or immobilized cellulases with same enzyme activities at 50 ◦ C and pH 5.5 for 2 H. The weight loss of the samples caused by the cellulase treatments is shown in Fig. 6a. The cotton yarn samples treated with the free cellulase showed the weight loss of 3.82% and 4.84% at the cellulase concentration of 2% and 4%, respectively. However, the immobilized cellulase treatments resulted in less weight loss at the same treatment conditions. Figure 6b shows the tensile strengths of the cotton yarn samples treated with the free or immobilized cellulases. The tensile strengths of the
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Biotechnology and Applied Biochemistry cotton yarn samples decreased after the cellulase treatments. When the cellulase concentration was 4%, the tensile strengths of the cotton yarn samples treated with CES and CEL were 12.52 and 12.88 cN/tex, respectively. At same cellulase concentration, the tensile strength of the cotton yarn was 10.32 cN/tex after free cellulase treatment, which was lower than that of the samples treated with the immobilized cellulases. The findings indicated that the cotton fibers suffered less damage after the immobilized cellulase treatments than those treated with the free cellulase, because the hydrolytic reaction of the immobilized cellulases was more concentrated on the surfaces of the cotton yarn samples because of their larger molecule size and volume.
3.7. Biopolishing of cotton fabric with immobilized cellulase
FIG. 6
Effects of enzymatic treatment on (a) weight losses and (b) tensile strengths of the cotton yarns.
Cellulases are widely used as textile auxiliaries for improving handle and surface appearance of cotton fabric. Table 3 shows the effects of the enzymatic treatments using free and immobilized cellulases on cotton fabric. Bending stiffness was measured by KES-F to evaluate the handle of the cotton fabric samples. Bending rigidity (B) is defined as the ability of a fabric sample to resist the bending movement. Bending moment (2HB) is defined as the recovery ability of a fabric sample after being bent. The smaller B and 2HB values of the samples, the better handle of the samples will be [28]. The bending stiffness results revealed that cotton fabric samples treated with free and immobilized cellulases were softer than untreated samples. In addition, the cellulase treatments improved the wrinkle recovery angle of the fabrics. Weight loss and tensile strength of cotton fabrics treated with the free cellulase were 5.82% and 482 N, respectively. However, the treatments with CES and CEL, which had the same enzyme units as free cellulase, resulted in the weight loss of 3.14% and 2.88%, and the tensile strength of 538 and 542 N, respectively. Thus, less damage was inflicted to the cotton fabrics after the immobilized cellulase treatments compared with the free cellulase treated ones. Figure 7 shows scanning electron microscopy (SEM) images of the cotton fabrics. The untreated sample had many projecting
Effects of immobilized cellulase treatments on cotton fabric
TABLE 3
Type of treatment
Wrinkle Tensile strength recovery angles Weight loss (%) (N) (◦ )
B (Ncm2 /cm)
2HB (Ncm/cm)
Warp
Weft
Warp
Weft
Control
0.26 ± 0.08
556 ± 16
138 ± 6
0.0264
0.0203
0.0169
0.0098
Free cellulase
5.82 ± 0.30
482 ± 12
170 ± 8
0.0186
0.0157
0.0153
0.0084
CES
3.14 ± 0.24
538 ± 12
165 ± 5
0.0207
0.0172
0.0165
0.0093
CEL
2.88 ± 0.32
542 ± 14
162 ± 6
0.0195
0.0152
0.0159
0.0089
6
Immobilization of Cellulases for Biopolishing
FIG. 7
SEM morphology of cotton fabric treated with (a) buffer (control), (b) free cellulase, (c) CES, and (d) CEL.
fuzz, and the fabric surfaces became smoother after the cellulase treatments. The degradation of cotton fibers was quite serious after the free cellulase treatment, and many cracks appeared on the fibers. However, it can be seen from the images that the immobilized cellulase treatment caused less damage to the fibers.
4. Conclusions In this paper, cellulases were immobilized on the reversibly soluble carriers by the noncovalent method. The immobilized cellulases were successfully applied to cotton biopolishing. The cotton fabrics treated with the immobilized cellulases suffered less damage than those treated with the free cellulase and were softer and smoother than control. Moreover, the immobilized cellulases could be recycled after the enzymatic reaction. An efficient recycling bioprocess was established for cotton biopolishing, and the immobilized-cellulase treatments can overcome the strength-loss problem in free cellulase treatment and the environmental problems in the cotton chemical treatments.
Biotechnology and Applied Biochemistry
5. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (51173071 and 31201134), the Program for New Century Excellent Talents in University (NCET-12-0883), the Jiangsu Provincial Natural Science Foundation of China (BK2012112), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1135), the Fundamental Research Funds for the Central Universities (JUSRP51312B and JUSRP111A01), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors declare no conflict of interest.
6. References [1] Fu, Z., Zhang, Y., and Wang, X. (2011) Bioresour. Technol. 102, 3748–3753. [2] Santos, A. B., Cervantes, F. J., and Lier, J. B. (2007) Bioresour. Technol. 98, 2369–2385. ´ C., Lant, N., Smets, J., and Cavaco-Paulo, A. (2012) Biocatal. [3] Caparros, Biotransfor. 30, 260–271. [4] Vankar, P. S., Shanker, R., and Verma, A. (2007) J. Clean. Prod. 15, 1441–1450. [5] Aly, A. S., Moustafa, A. B., and Hebeish, A. (2004) J. Clean. Prod. 12, 697–705. [6] Anish, R., Rahman, M. S., and Rao, M. (2007) Biotechnol. Bioeng. 96, 48–56. [7] Belghiht, H., Ellouz-Chaabouni, S., and Gargouri, A. (2001) J. Biotechnol. 89, 257–262. [8] Hebeish, A., Hashem, M., Shaker, N., Ramadan, M., El-Sadek, B., and Hady, M. A. (2009) Carbohydr. Polym. 78, 953–960.
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[19] Hanefeld, U., Gardossi, L., and Magner, E. (2009) Chem. Soc. Rev. 38, 453–468. [20] Sardar, M., Roy, I., and Gupta, M. N. (2000) Enzyme Microb. Technol. 27, 672–679. [21] Bradford, M. (1976) Anal. Biochem. 72, 248–254. [22] Kondo, A., Urabe, T., and Higashitani, K. (1994) J. Ferment. Bioeng. 77, 700–703. [23] Liu, P., Xia, W., and Liu, J. (2008) Biochem. Eng. J. 41, 142–148. [24] Sharma, S., Kaur, P., Jain, A., Rajeswari, M. R., and Gupta, M. N. (2003) Biomacromolecules 4, 330–336. [25] Dourado, F., Bastos, M., Mota, M., and Gama, F. M. (2002) J. Biotechnol. 99, 121–131. [26] Kweon, H. Y., Um, I. C., and Park, Y. H. (2001) Polymer 42, 6651–6656. [27] Asuri, P., Bale, S. S., Pangule, R. C., Shah, D. A., Kane R. S., and Dordick, J. S. (2007) Langmuir 23, 12318–12321. [28] Kan, C. W., Yuen, C. W. N., Lam, Y. L., and Chan, C. K. (2009) Fiber Polym. 10, 325–332.
Immobilization of Cellulases for Biopolishing
Yuanyuan Yu Jiugang Yuan ∗ Qiang Wang Xuerong Fan Ping Wang Li Cui
Key Laboratory of Science and Technology of Eco-Textile, Ministry of Education, Jiangnan University, Wuxi, Jiangsu, 214122, People’s Republic of China
Abstract The hydrolytic reaction of cellulases can occur in the interior of cellulosic fibers, causing tensile strength loss of the fabrics. Cellulase immobilization is an approach to solve this problem, because enlarging the molecule size of cellulases will limit the hydrolysis to the surfaces of the fibers. In this study, commercial cellulases were noncovalently immobilized onto the reversibly soluble polymers (Eudragit S-100 and Eudragit L-100). The characteristics of cellulase-Eudragit S-100 (CES) and cellulase-Eudragit L-100 (CEL) were evaluated using Fourier transform infrared spectra, circular dichroism spectra, and fluorescence spectra. The CES showed higher stability
than CEL and free cellulase, especially at higher pH and temperature. CES and CEL retained 51% and 42% of their original activities after three cycles of repeated uses, respectively. In addition, the effects of cellulase treatment on the cotton yarn and fabric have been investigated. The bending stiffness results showed that the cotton fabric samples treated with the free and immobilized cellulases were softer than untreated samples. However, less fiber damage in terms of weight loss and tensile strength of treated cotton was C 2014 International Union of Biochemistry and Molecular observed. Biology, Inc. Volume 00, Number 00, Pages 1–8, 2014
Keywords: cellulase, cotton, Eudragit, noncovalent immobilization, recycling
1. Introduction In recent years, the problem of environmental pollution has aroused extensive concern all over the world. In the textile industry, the wastewater from wet processes is one of the main industrial pollution [1, 2]. To solve the problem, many enzymes have been used in the wet processes to replace harsh chemicals used in traditional processes [3]. For example, amylases are used in desizing, cellulases are used in cotton biopolishing and
Abbreviations: CD, circular dichroism; CEL, cellulase-Eudragit L-100; CES, cellulase-Eudragit S-100; EL, Eudragit L-100; ES, Eudragit S-100; FTIR, Fourier transform infrared; SEM, scanning electron microscopy; UV, ultraviolet. ∗ Address
for correspondence: Professor Qiang Wang, Key Laboratory of Science and Technology of Eco-Textile, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China. Tel.: +86-510-85912007; Fax: +86-510-85912009; e-mail: [email protected]. Received 25 February 2014; accepted 2 September 2014 DOI: 10.1002/bab.1289 Published online in Wiley Online Library (wileyonlinelibrary.com)
denim washing, and lipases and pectinases are used in cotton biopretreatment [4–10]. Cellulases are the most successful enzymes used in the textile industry, and many valuable effects can be acquired after cellulase treatments. However, the cellulase treatments have some critical drawbacks. First, cellulases are difficult to recover and reuse after the treatments. Second, the treatments can reduce the fabric strength because cellulases can easily penetrate into cotton fiber, leading to the hydrolysis of cellulose molecule in the interior of fiber [11, 12]. Cellulase immobilization is an approach to solve the problems. Cellulase immobilization can offer several advantages over the free cellulases such as recovery from the reaction medium, reuse, and operation in continuous reactors. Cellulase immobilization on some macromolecule carriers can enlarge the sizes of cellulases, and the diffusion of an enlarged enzyme molecule is significantly inhibited in the interior of the fiber. Eudragit S-100 (ES) and Eudragit L-100 (EL), the commercial copolymers of methacrylic acid and methyl methacrylate, have been extensively used as a carrier for enzyme immobilization. Eudragit can be precipitated at low pH around 4.5 and dissolved in aqueous solutions at pH over 5.5. When enzymes are immobilized on Eudragit, the solubility of the immobilized
1
Biotechnology and Applied Biochemistry enzymes becomes similar to that of Eudragit. Thus, the immobilized enzymes can be used in their soluble form during the enzymatic reaction and recovered in their insoluble form after the reaction by altering the pH of the reaction mixture [13, 14]. Silva et al. [15] had immobilized protease on ES to decrease the loss of wool fabric strength after protease treatment. Xu et al. [16] had co-immobilized cellulase and xylanase on EL using a covalent coupling method. In our previous work, cellulases had been successfully immobilized on ES by a covalent coupling method, and the application of covalent–Eudragit–cellulase on the cotton biopolishing was studied. The cotton samples treated with covalent–Eudragit–cellulase showed lower weight loss and considerably higher tensile strength than those treated with free cellulase [17, 18]. Noncovalent adsorption is another frequently used technique for enzyme immobilization. The great advantage of noncovalent immobilization is that the enzyme does not have to be pretreated or chemically modified [19]. In the present work, noncovalent immobilization of cellulases on ES or EL was carried out. The structure of the immobilized cellulases was characterized by Fourier transform infrared (FTIR) spectra, circular dichroism (CD), and fluorescence spectra. The stability and reusability of the immobilized cellulases were studied. Biopolishing of cotton yarns and fabrics using the free and immobilized cellulases were investigated to evaluate the effect of the treatments and the degradation of cotton fibers.
2. Materials and Methods 2.1. Materials Acidic cellulase Suhong 989N with a CMCase activity of 92 U/mL was supplied by Novozymes (Suzhou, People’s Repub¨ lic of China). ES and EL were kindly donated by Degussa-Huls, S.A. (Shanghai, People’s Republic of China). Both ES and EL had a molecular weight of 135,000 Da, but the ratio of the free carboxyl groups to the ester groups was 1:1 for EL and 1:2 for ES. All other chemicals were of analytical grade. Wuxi Cotton Textile Company (Wuxi, People’s Republic of China) supplied the cotton yarns (38 tex) and woven fabrics. The specifications of the fabrics were as follows: thread density 18.276 tex, warp 13 threads/cm, weft 7 threads/cm, and weight per area 150 g/m2 .
2.2. Noncovalent immobilization of cellulase onto Eudragit Noncovalent immobilization of cellulase was carried out following a protocol developed by Sardar et al. [20] with some modifications. The pI of free cellulase is 7.7, and the enzyme has positive charges at pH 7.2. Eudragit has negative charges at pH 7.2. So the strong electrostatic interaction exists between free cellulase and Eudragit. Five grams of ES or EL was completely dissolved in 100 mL phosphate buffer (pH 7.2). To prepare 2 mL polymer solutions, 0.6 mL free cellulase (17.5 mg protein/mL) was added and then the total volume was made up to 10 mL with phosphate buffer (pH 7.2). After stirring at room
2
temperature for 1 H, the pH of the solutions was adjusted to 4.2 by adding 0.1 M HCl solution to precipitate the immobilized cellulases. The precipitates were separated by centrifugation (10,000g, 10 Min), and then washed twice with HCl solution (pH 4.2). The immobilized cellulases were redissolved in 0.2 M acetate buffer at pH 5.5 for further study.
2.3. Determination of amount of binding cellulases and cellulase activity The amount of cellulase proteins was determined by the Bradford method, using bovine serum albumin as the standard [21]. The amount of binding cellulases was calculated according to Eq. (1). Amount of binding cellulase protein = M1 − M2
(1)
where M1 and M2 are the amounts of cellulase proteins initially used for the reaction and the unbinding cellulase proteins collected, respectively. The hydrolytic activity of the cellulase used in this work was measured using 1% (w/v) carboxy-methylated cellulose as a substrate. The reaction mixture was incubated in a water bath at 50 ◦ C for 30 Min, and the amount of glucose generated was measured by an ultraviolet (UV)–Vis spectrophotometer at 540 nm using 3,5-dinitrosalicylic acid agent as the color indicator [22]. The activity recovery yield was calculated based on Eq. (2) as follows: Activity recovery yield % (2) = activity recovered/activity loaded × 100
2.4. Characterization of immobilized cellulases The structure of the immobilized cellulases was investigated by FTIR spectroscopy with the KBr pellet technique. CD spectra were obtained using a MOS-450 spectropolarimeter in the range of 190–250 nm. A total of 150 µL of enzyme solution was added into the cell. The spectra were run with protein concentration ranging from 0.15 to 0.25 mg/mL at 20 ◦ C with a scan rate of 100 nm/Min, response time of 1 Sec, and resolution of 1 nm [23]. Fluorescence spectra were recorded on a Hitachi F4500 spectrofluorometer (Hitachi, Tokyo, Japan) at 20 ◦ C using a slit width of 5 nm and a 1 cm path length cuvette with a scan speed of 60 nm/Min. The spectra of free and immobilized cellulases were recorded from 300 to 400 nm using an excitation wavelength of 280 nm [24].
2.5. Solubility of immobilized cellulases The solubility of Eudragit and the immobilized cellulases was determined at 470 nm by a UV–Vis spectrophotometer and expressed as a ratio of absorbance [25]. The maximum value of the absorbance was set at pH 3.5 of the systems. Zero absorbance was set at the maximum solubility of the systems.
2.6. Enzymatic treatment of cotton samples The cotton yarn and fabric samples were treated with cellulases in 0.2 M acetate buffer with a liquor-to-fiber ratio of 20:1 at
Immobilization of Cellulases for Biopolishing
pH 5.5 and 50 ◦ C for 2 H. After the cellulase treatments, the samples were rinsed with deionized water, followed by deactivation of residual cellulases on the samples in deionized water at 80 ◦ C for 10 Min. Finally, the cotton samples were dried at 60 ◦ C.
2.7. Weight loss and tensile strength The weight loss of the cotton samples was calculated according to the following equation: Weight loss % = (W1 − W2 ) /W1
(3)
where W1 is the weight of the sample before the cellulase treatment and W2 is the weight of the sample after the cellulase treatment. The tensile strength of the cotton yarn samples was determined in accordance with ASTM D 1682-64 using a YG(B)025S Tensile Strength Tester (Wenzhou Darong Company, Wenzhou, People’s Republic of China). The tensile strength of the cotton fabric samples was determined in accordance with ASTM D5035-2006(2008)e1 using a YG(B)026H Tensile Strength Tester (Wenzhou Darong Company).
2.8. Wrinkle recovery angle, bending stiffness, and scanning electron microscopy of cotton fabric A standard method (AATCC test method 66-1984) was used to measure the wrinkle recovery angles. The bending stiffness was measured using a KES-FB-AUTO-A system. The surface morphology of the cotton samples was observed using a SU1510 scanning electron microscope (Hitachi). Platinum was sputtered onto the fabric samples as a conducting material to analyze the samples.
TABLE 1
Amount of binding cellulases and activity recovery yields after immobilization reaction
Amount of binding cellulases (mg)
Activity recovery yield (%)
CellulaseEudragit S-100
2.31 ± 0.10
82.2 ± 2.8
CellulaseEudragit L-100
5.62 ± 0.32
78.2 ± 3.4
Immobilized cellulase
shown in Fig. 1. The amide I and II bands are two of the most prominent vibrational bands of the protein backbone. The amide I absorption band is located in the region between 1,700 and 1,600 cm−1 , and the amide II band is located in the region between 1,600 and 1,500 cm−1 [26]. From CES and CEL spectra, it was found that distinctive absorption bands appeared at 1,658 (amide I) and 1,534 cm−1 (amide II). The absorption intensity of CES and CEL at about 3,400 cm−1 was stronger than that of ES and EL because of the superposition of stretching vibration of O—H and N—H in the protein structure of the cellulase. In addition, the characteristic adsorption
3. Results and Discussion 3.1. Noncovalent immobilization of cellulase on ES or EL By assaying the amount of the cellulase proteins collected in the supernatant after the immobilization process, the amount of binding cellulases was determined. As shown in Table 1, the amount of binding cellulases on EL (5.62 mg) was higher than that of binding cellulases on ES (2.31 mg). In the noncovalent immobilization process, the binding force is mainly the electrostatic force between carboxyl (Eudragit) and amino (cellulase). The amount of carboxyl in EL molecule is twice that of carboxyl in ES molecule. Therefore, more cellulase molecules were bound to EL than ES. Moreover, enzymes usually lose some of their activities after immobilization because of the blocking of the catalytic active site by binding of enzymes to polymers [13]. After the immobilization, the activity recovery yields of cellulase-Eudragit S-100 (CES) and cellulase-Eudragit L-100 (CEL) were 82.2% and 78.2%, respectively. The structure characteristics of the immobilized cellulases were analyzed by FTIR to verify the binding of cellulases onto Eudragit. FTIR spectra of ES, EL, CES, and CEL are
Biotechnology and Applied Biochemistry
FTIR spectra of (a) ES and CES and (b) EL and CEL.
FIG. 1
3
Biotechnology and Applied Biochemistry
FIG. 2
FIG. 3
Circular dichroism spectra of free and immobilized cellulases.
peaks appeared at 520 cm−1 in the spectra of the immobilized cellulases, which could be assigned to the disulfide bond (—S—S—) in cellulase protein molecule. The results indicated the formation of immobilized cellulases via noncovalent binding.
3.2. CD of immobilized cellulases CD has been increasingly used as a spectroscopic tool for detecting the secondary and tertiary structures of proteins in a solution [27]. As shown in Fig. 2, there was a significant change in CD spectra of CES and CEL compared with that of the free cellulase. Analysis of the CD spectrum of the free cellulase resulted in secondary structural fractions of 53.04% helix and 11.47% strand (Table 2). After immobilization, CES contained about 46.67% helix and 13.46% strand and CEL contained about 24.52% helix and 27.33% strand. ES and EL were linear macromolecules. When the enzyme was bound to Eudragit, the enzyme molecules could be stretched and unfolded, causing the decrease in helix in the structure of immobilized enzyme. Moreover, the difference in the CD spectra could be due to either the change in the secondary structure of the enzyme after immobilization or the formation of some aggregative structures (e.g., less enzyme molecules were closely bound on one polymer backbone) [25].
Fluorescence emission spectra of Eudragit, free, and immobilized cellulases.
3.3. Fluorescence spectroscopy Fluorescence emission spectra of EL, ES, CEL, CES, and free cellulase excited at 280 nm are shown in Fig. 3. The fluorescence emission characteristics can be considered as a more sensitive parameter to study the structural changes involving the microenvironment of tryptophan [25]. Fluorescence was not detected at fluorescence emission spectra of ES and EL. The fluorescence intensity for the free cellulase was lower than that for CEL and CES. The increase in fluorescence intensity indicated that the enzyme molecules were unfolded after immobilization [25]. The results also showed that the secondary structure of the cellulase was altered, which was consistent with the finding in CD spectra.
3.4. Effects of temperature and pH on enzymatic activity Figure 4 shows the effects of pH and temperature on the relative activities of the free and immobilized cellulases. The maximum activities of the free and immobilized cellulases were observed at 50 ◦ C and 60 ◦ C, respectively. Immobilization of cellulases changed its temperature profile. Moreover, CES had better temperature stability than free cellulase and CEL. The activities of free cellulase and CEL showed a significant loss above 70 ◦ C, which was lower than 30% of the initial activity
The secondary structure of free and immobilized cellulases as determined by circular dichroism spectroscopy
TABLE 2 Enzyme
Helix (%)
Strand (%)
Turns (%)
Unordered (%)
Free cellulase
53.04
11.47
14.26
22.23
CES
46.67
13.46
15.45
24.42
CEL
24.52
27.33
20.42
27.73
4
Immobilization of Cellulases for Biopolishing
FIG. 4
Effects of (a) temperature and (b) pH on the relative activities of the free and immobilized cellulases.
at 90 ◦ C. However, the activity of CES remained above 60% at 90 ◦ C. It is known that enzyme activity is dependent on the ionization state of the amino acids in the active site. Hence, pH plays a significant role in maintaining the proper conformation of an enzyme. Figure 4b shows the effect of pH on the relative activities of cellulases. The relative activities of the free and immobilized cellulases increased and reached the maximum at pH 5, and then decreased with further increase in the pH value. In addition, CES was much more stable than free cellulase and CEL when the pH was above 5.0. The relative activity of CES remained 60% of its initial activity at pH 8.0, which was higher than that of CEL and free cellulase.
3.5. Solubility and reusability of immobilized cellulases The solubility of Eudragit and immobilized cellulases can be controlled by adjusting the pH of the solutions. As shown in Fig. 5a, ES was soluble in aqueous solutions above pH 4.8 and precipitated below pH 4.6, and EL was soluble in aqueous solutions above pH 4.5 and precipitated below pH 4.0. The
Biotechnology and Applied Biochemistry
FIG. 5
(a) Solubility and (b) reusability of immobilized cellulases.
solubility of the immobilized cellulases became similar to that of Eudragit. CES and CEL were soluble in aqueous solutions above pH 5.2 and precipitated below pH 4.5. Figure 5b shows the reusability of the immobilized cellulases. The CES and CEL retained 51% and 42% of their original activities after three cycles of repeated uses, respectively. The results demonstrated that the immobilized cellulases had certain reusability characteristics, which was important for the potential industry application.
3.6. Weight loss and tensile strength of cotton yarn The cotton yarn samples were treated with free or immobilized cellulases with same enzyme activities at 50 ◦ C and pH 5.5 for 2 H. The weight loss of the samples caused by the cellulase treatments is shown in Fig. 6a. The cotton yarn samples treated with the free cellulase showed the weight loss of 3.82% and 4.84% at the cellulase concentration of 2% and 4%, respectively. However, the immobilized cellulase treatments resulted in less weight loss at the same treatment conditions. Figure 6b shows the tensile strengths of the cotton yarn samples treated with the free or immobilized cellulases. The tensile strengths of the
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Biotechnology and Applied Biochemistry cotton yarn samples decreased after the cellulase treatments. When the cellulase concentration was 4%, the tensile strengths of the cotton yarn samples treated with CES and CEL were 12.52 and 12.88 cN/tex, respectively. At same cellulase concentration, the tensile strength of the cotton yarn was 10.32 cN/tex after free cellulase treatment, which was lower than that of the samples treated with the immobilized cellulases. The findings indicated that the cotton fibers suffered less damage after the immobilized cellulase treatments than those treated with the free cellulase, because the hydrolytic reaction of the immobilized cellulases was more concentrated on the surfaces of the cotton yarn samples because of their larger molecule size and volume.
3.7. Biopolishing of cotton fabric with immobilized cellulase
FIG. 6
Effects of enzymatic treatment on (a) weight losses and (b) tensile strengths of the cotton yarns.
Cellulases are widely used as textile auxiliaries for improving handle and surface appearance of cotton fabric. Table 3 shows the effects of the enzymatic treatments using free and immobilized cellulases on cotton fabric. Bending stiffness was measured by KES-F to evaluate the handle of the cotton fabric samples. Bending rigidity (B) is defined as the ability of a fabric sample to resist the bending movement. Bending moment (2HB) is defined as the recovery ability of a fabric sample after being bent. The smaller B and 2HB values of the samples, the better handle of the samples will be [28]. The bending stiffness results revealed that cotton fabric samples treated with free and immobilized cellulases were softer than untreated samples. In addition, the cellulase treatments improved the wrinkle recovery angle of the fabrics. Weight loss and tensile strength of cotton fabrics treated with the free cellulase were 5.82% and 482 N, respectively. However, the treatments with CES and CEL, which had the same enzyme units as free cellulase, resulted in the weight loss of 3.14% and 2.88%, and the tensile strength of 538 and 542 N, respectively. Thus, less damage was inflicted to the cotton fabrics after the immobilized cellulase treatments compared with the free cellulase treated ones. Figure 7 shows scanning electron microscopy (SEM) images of the cotton fabrics. The untreated sample had many projecting
Effects of immobilized cellulase treatments on cotton fabric
TABLE 3
Type of treatment
Wrinkle Tensile strength recovery angles Weight loss (%) (N) (◦ )
B (Ncm2 /cm)
2HB (Ncm/cm)
Warp
Weft
Warp
Weft
Control
0.26 ± 0.08
556 ± 16
138 ± 6
0.0264
0.0203
0.0169
0.0098
Free cellulase
5.82 ± 0.30
482 ± 12
170 ± 8
0.0186
0.0157
0.0153
0.0084
CES
3.14 ± 0.24
538 ± 12
165 ± 5
0.0207
0.0172
0.0165
0.0093
CEL
2.88 ± 0.32
542 ± 14
162 ± 6
0.0195
0.0152
0.0159
0.0089
6
Immobilization of Cellulases for Biopolishing
FIG. 7
SEM morphology of cotton fabric treated with (a) buffer (control), (b) free cellulase, (c) CES, and (d) CEL.
fuzz, and the fabric surfaces became smoother after the cellulase treatments. The degradation of cotton fibers was quite serious after the free cellulase treatment, and many cracks appeared on the fibers. However, it can be seen from the images that the immobilized cellulase treatment caused less damage to the fibers.
4. Conclusions In this paper, cellulases were immobilized on the reversibly soluble carriers by the noncovalent method. The immobilized cellulases were successfully applied to cotton biopolishing. The cotton fabrics treated with the immobilized cellulases suffered less damage than those treated with the free cellulase and were softer and smoother than control. Moreover, the immobilized cellulases could be recycled after the enzymatic reaction. An efficient recycling bioprocess was established for cotton biopolishing, and the immobilized-cellulase treatments can overcome the strength-loss problem in free cellulase treatment and the environmental problems in the cotton chemical treatments.
Biotechnology and Applied Biochemistry
5. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (51173071 and 31201134), the Program for New Century Excellent Talents in University (NCET-12-0883), the Jiangsu Provincial Natural Science Foundation of China (BK2012112), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1135), the Fundamental Research Funds for the Central Universities (JUSRP51312B and JUSRP111A01), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors declare no conflict of interest.
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[19] Hanefeld, U., Gardossi, L., and Magner, E. (2009) Chem. Soc. Rev. 38, 453–468. [20] Sardar, M., Roy, I., and Gupta, M. N. (2000) Enzyme Microb. Technol. 27, 672–679. [21] Bradford, M. (1976) Anal. Biochem. 72, 248–254. [22] Kondo, A., Urabe, T., and Higashitani, K. (1994) J. Ferment. Bioeng. 77, 700–703. [23] Liu, P., Xia, W., and Liu, J. (2008) Biochem. Eng. J. 41, 142–148. [24] Sharma, S., Kaur, P., Jain, A., Rajeswari, M. R., and Gupta, M. N. (2003) Biomacromolecules 4, 330–336. [25] Dourado, F., Bastos, M., Mota, M., and Gama, F. M. (2002) J. Biotechnol. 99, 121–131. [26] Kweon, H. Y., Um, I. C., and Park, Y. H. (2001) Polymer 42, 6651–6656. [27] Asuri, P., Bale, S. S., Pangule, R. C., Shah, D. A., Kane R. S., and Dordick, J. S. (2007) Langmuir 23, 12318–12321. [28] Kan, C. W., Yuen, C. W. N., Lam, Y. L., and Chan, C. K. (2009) Fiber Polym. 10, 325–332.
Immobilization of Cellulases for Biopolishing
