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1、Structural changes of lignin in the jute fiber treated by laccase and mediator systemYongbing Zhang, Qiang Wang*, Xuerong Fan, Jiugang YuanKey Laboratory of Science and Technology of Eco-Textile, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, PR China*Corresponding author. Tel.: +
2、86-510-85912007; fax: 86-510-855912009.E-mail address: qiang_.AbstractTo study the structural changes of lignin in the jute fiber treated with laccase and mediator system (LMS), lignins from the control and LMS-treated jute fiber were isolated and characterized by gel permeation chromatography (GPC)
3、, elemental analysis, measurement of phenolic hydroxyl group content, FTIR and 1H NMR. The results showed that the molecular weights of the lignin from LMS-treated jute fiber were lower than those of the lignin from the control jute fiber. The contents of phenolic hydroxyl group, aliphatic hydroxyl
4、group and methoxy group of the lignin from LMS-treated jute fiber decreased, while the content of carboxyl group increased.Keywords: laccase, jute fiber, lignin, degradation, mediator1. IntroductionThe jute fiber lignin is composed of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units with a H
5、/G/S composition of 2:32:66 and a S/G ration of 2.1 1. The lignin content in jute fiber is up to ca. 16%, which resulted in the coarseness and rigidity of the fiber. Therefore, jute fibers are mainly used to make the low-grade goods such as package fabrics and bags 2-3. The removal of lignin from ju
6、te fiber is proved to be a key step in the manufacturing of high-value textile products. In the traditional process, the lignins in jute fibers are eliminated mainly in degumming using some chemical products, which often cause severe environmental pollution. In order to overcome the disadvantages of
7、 chemical degumming, enzymatic degumming has been attracted a great deal of attention.Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are a widespread group of multi-copper enzymes 4. It has been reported by many researchers that laccase can degrade or polymerize the phenolic compounds in
8、 lignin 5-6. Furthermore, when laccase is used in the presence of a mediator, such as 2,2-azonabis(3-ethylbenzthiazoline-6sulfonate) (ABTS) 6, 1-hydroxybenzotriazole (HOBT) 7 and 2,2,6,6-tetramethyl-piperidine-N-oxyl (TEMPO) 8, it can further degrade the nonphenolic subunits of lignin. The mediators
9、 have high redox potential values and can produce radicals which transfer electrons from lignin to the enzyme, which finally reduces oxygen to water 9-11. The use of a laccase-mediator system (LMS) is one of the promising possibilities as environmentally benign processes for pulp biobleaching 12-13,
10、 enzymatic pulping 14 and old newspaper deinking 15 because of its ability to delignify.Bio-degumming refers to the enzymatic removal of the non-cellulosic matters such as waxes, pectins and lignins from the surface of bast fiber, which endows the fiber with better hydrophilicity in favor of subsequ
11、ent processes. Laccase is also a promising enzyme for the degumming of bast fibers to remove lignins because of its ability of delignification. Ren et al. reported that the lignin content of linen fibers treated by laccase was decreased from 4.4% to 2.3% 16. Liu et al. investigated the degumming of
12、jute fibers with laccase and pectinase 17. They found that the complex enzyme showed better removing effect for lignin. In our previous work, the degumming of linen/cotton fabric with pectinase, cellulose, xylanase and laccase were investigated 18. The results showed that laccase treatment was the b
13、est way to remove lignins from linen/cotton fabric, but it still had a big gap compared to the traditional process. These results indicated that degumming of bast fibers with laccase is a feasible method. Nevertheless, the mechanism of lignin oxidation during bast fiber degumming with LMS is not wel
14、l established. Degumming of bast fibers with LMS may be improved if the fundamental chemical reactions contributing to this process are well understood. In this study, the jute fibers were treated by LMS, and then the lignins were extracted from it with dioxane/water solution. The structure changes
15、of lignins from the control and LMS-treated jute fibers were characterized by GPC, elemental analysis, FTIR and 1H NMR. We hope the results will provide useful references to the degumming of jute fibers with LMS.2. Materials and Methods2.1 MaterialsJute fiber was supplied by Changshu Aocun Longtai w
16、eaving Co., Ltd. Laccase from Trametes Versicolor with an activity of 5.43 U/mg was supplied by Sigma. One unit of laccase activity was defined as the amount of enzyme converting 1 mole of catechol per minute in 50 mM sodium citrate buffer (pH 6) at 25oC using catechol as substrate. 2,2-azinobis(3-e
17、thylbenzthiazoline-6-sulphonate) (ABTS) provided by Sigma was used as a mediator.2.2 Treatment of the jute fibers with LMSThe reaction solution (300 mL) contained jute fibers (13 g), laccase (total activity 160 U), ABTS (10 mg), sodium acetate buffer (0.05M, pH 6). The mixtures were shaken and bubbl
18、ed air at 25oC for 6 h. After the enzymatic reaction, the jute fibers were washed several times with water, and air-dried.2.3 Isolation of lignins from the jute fibersThe corresponding residual lignins in the control and LMS-treated jute fibers, Lc and Lt, were isolated using a method as described b
19、y Evtuguin et al. with slight modification 19.The control and LMS-treated jute fibers were ground to 40 mesh fractions, refluxed with ethanol-benzene (1:2, v/v) solvent for 6 h, and then dried at room temperature. These fractions were refluxed with dioxane-water (9:1, v/v) solution containing 0.2 M
20、HCl at 90oC for 60 min. The liquid phase was decanted after the mixture was cooled to room temperature. The solid residue was subjected to the next extraction as described above, and then decanted the liquid phase. The two portions of the liquid phases were mixed, and concentrated to around 60 mL by
21、 vacuum evaporation at 40oC. The lignins were precipitated from dioxane solution by dilution into cold water (about 800 mL). The precipitate was separated by centrifugation, followed by being washed with water and freeze-dried. The crude lignins were further purified according to the method of Lundq
22、uist et al. 20.2.4 Acetylation of lignins from the jute fibersThe Lc and Lt were acetylated using a method proposed by Jahan et al. 21. Lignin of 100 mg was added in 9 mL of pyridine-acetic anhydride solution（1:2, v/v）and kept for 72 h in dark. The solution was poured into a 10-fold volume of an ice
23、-water bath where the acetylated lignins were recovered as a precipitate, which were further purified by successive washing with water and dried under vacuum. The acetylated lignins were used for 1H NMR analysis.2.5 Estimation of molecular weightThe number-average molecular weight (Mn) and weight-av
24、erage molecular weight (Mw) of Lc and Lt were determined by GPC. The GPC equipment used was a Waters 1515 Isocratic HPLC Pump (Waters Corporation, Milford, USA), with a Waters 2414 Refractive Index Detector (Waters Corporation, Milford, USA) and a GPC KD-802 Packed Column (Shodex, Japan).The lignin
25、samples were dissolved in N,N-dimethylformamide (DMF) and 20 L solution was injected into the HPLC column. The test was operated at 35oC and eluted with DMF at a flow rate of 1.5 mL/min. The molecular weight was calibrated with a polystyrene standard.2.6 Chemical analysisC, H and N elements of Lc an
26、d Lt were determined using a Vario ZL elemental analyzer. The percentage of oxygen was calculated by subtracting the C, H and N contents from 100%. Methoxyl group contents were calculated according to 1H NMR spectra. Phenolic hydroxyl group contents were determined by an ultraviolet spectrophotomete
27、r 22.2.7 Analysis by FTIR FTIR spectra were recorded on a Nicolet iS10 FTIR spectrometer. The lignin samples were embedded in KBr pellets in the concentration of ca. 1 mg/200 mg KBr. The spectra were recorded in the absorption band mode in the range from 4000 to 500 cm-1.2.8 Analysis by 1H NMRThe 1H
28、 NMR spectra of 20 mg acetylated lignins solved in 0.5 mL chloroform (CDCl3) were recorded, using tetramethylsilane (TMS) as the internal standard in a Bruker Avance 400 spectrometer with an operating frequency at 400 MHz.3. Results and Discussion3.1 Molecular weight distribution of the jute fiber l
29、igninsThe molecular weight distribution curves of Lc and Lt were shown in Fig. 1. The values of the weight-average (Mw) and number-average (Mn) molecular weights of Lc and Lt were calculated from the curves, and the polydisperisty (Mw/Mn) was given in Table 1. As can be seen from Table 1, the Mw and
30、 Mn were 34130 and 24177 for Lt, respectively, and the polydispersity value was 1.412. Comparing with Lc, Mw, Mn and polydispersity of Lt were decreased. The similar outcome was also obtained by Fu et al. in studies on the degradation of residual lignin in kraft pulp by laccase and mediator system 2
31、3. This result meant that the lignins in jute fibers could be degraded by laccase and mediator system into smaller fragments.3.2 Chemical analysis Table 2 summarized the results from C, H, N, O, methoxyl and phenolic hydroxyl analyses of Lc and Lt, together with the approximate C9 formula calculated
32、 therefrom 24. The Lt contained a high percentage of oxygen. It is in agreement with the change of oxygen content of residual lignin in the biobleaching of pulp with LMS reported by Balakshin et al. 25. It may be the result of the oxidation of the LMS treatment. The methoxyl content was calculated a
33、ccording to 1H NMR spectra. Although it is an approximate calculation method, the variation tendency of methoxy group content in lignin before and after LMS treatment could be observed through this result. As can be seen from Table 2, the methoxyl content in Lt was lower than that in Lc, which sugge
34、sted that lignin demethylation took place during the LMS treatment. This result was compatible to earlier report of Bourbonnais and Paice 26. In addition, compared to Lc, Lt presented a decrease in the amount of phenolic hydroxyl group, indicating that the phenolic hydroxyl group participated in the
35、 degradation reaction during the LMS treatment.3.3 FTIR analysis of the jute fiber ligninsFTIR spectra of Lc and Lt were shown in Fig. 2, and the assignments of the observed bands 27-28 and their relative transmittance were listed in Table 3. As can be seen from Fig. 2, Lc and Lt were structurally s
36、imilar, but the contents of functional groups were different according to Table 3. The band at 3443 cm-1 was assigned to O-H stretching vibration in aromatic and aliphatic OH groups. Its relative transmittance increased after LMS treatment, showing that the LMS treatment caused a decrease in the hyd
37、roxyl group contents. It was agreement with the result of 1H NMR analysis. The absorption at 1717 cm-1 was attributed to carboxylic acid and unconjugated carbonyl group. Its relative transmittance decreased after LMS treatment, which may be ascribed to the enzymatic oxidation of lignin and generated
38、 new unconjugated carbonyl groups. This is in agreement with the result reported by Sealey et al. in lignin studies of laccase-delignified kraft pulps 29. The band at 1423 cm-1 originated from aromatic skeletal vibrations together with OCH3 in-plane deformations. The increase in its relative transmi
39、ttance illustrated that some methoxyl groups were removed during the LMS treatment, which was consistent with the result of the chemical analysis.3.4 1H NMR analysis of the jute fiber ligninsThe 1H NMR spectra obtained for acetylated lignins were shown in Fig. 3, and Table 4 listed the position of s
40、ignals assigned by Jahan et al. 21,27 and numbers for each protons type per C9 unit. The numbers of phenolic and aliphatic hydroxyl groups per C9 unit for each lignin, as calculated from the above proton numbers, were listed in Table 5.The proton of phenolic hydroxyl group of Lc was 0.27/C9, while t
41、hat of the Lt decreased to 0.22/C9. The variation tendency was consistent with the result measured by the UV spectrophotometry. The proton of aliphatic hydroxyl group was 1.02/C9 for Lc, whereas it reduced to 0.98/C9 for Lt, which could be attributed to the fact that laccase can oxidize aliphatic hy
42、droxyl group of lignin in the presence of ABTS. The similar change was observed by Xu et al. from the deinking of old newsprint with laccase-violuric acid system 30. Silva et al. considered that the oxidation was probably associated with the cleavage of arylether bonds and the modification of alipha
43、tic side chains 31.4. ConclusionsThe lignins from the jute fibers as the main substrates of laccases would inevitably produce some structural changes. Compared to the control, the results obtained from the lignins from LMS-treated jute fibers led to the following conclusions:(a) The weight-average a
44、nd number-average molecular weights of the lignin from LMS-treated jute fiber were lower than those of the lignin from the control jute fiber, which showed the lignin in the jute fiber was degraded during the LMS treatment. The phenolic hydroxyl content of the lignin from LMS-treated jute fiber decr
45、eased, which indicated that the phenolic compounds in lignin participated in the degradation reaction.(b) The methoxyl content of the lignin from LMS-treated jute fiber decreased, while the content of carboxyl group increased. The fomer suggested that the LMS treatment had the role of demethylation
46、and the latter might be the result of the oxidative action of the LMS treatment.The conclusions in this paper may provide useful references to reveal the mechanism of lignin degradation of jute fiber during LMS treatment, which could guide the enzymatic processes of lignocellulose materials in texti
47、le, pulping and papermaking, as well as biofuel industries.AcknowledgementsThis work was nancially supported by National Natural Science Foundation of China (51173071, 21274055), Program for New Century Excellent Talents in University (NCET-12-0883), The natural science foundation of Jiangsu Provinc
48、e (BK2011157), Ph.D. Programs Foundation of Ministry of Education of China (20110093110003), the Fundamental Research Funds for the Central Universities (JUSRP51312B), Program for Changjiang Scholars and Innovative Research Team in University (IRT1135).References1 J.C. del Ro, J. Rencoret, G. Marque
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