- Email: [email protected]
Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw
Bioresource Technology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw Yanfei Li, Xiaoyan Ge, Zongping Sun, Junhua Zhang ⇑ College of Forestry, Northwest A&F University, 3 Taicheng Road, Yangling 712100, China
h i g h l i g h t s Additives affected the adsorption and desorption behavior of xylanase on lignin. The adsorption of xylanase onto lignin could be alleviated by additives. The additives could enhance desorption of xylanase from lignin. More additives could adsorb onto WS-lignin compared to CS-lignin. The released xylanase from lignin still exhibited hydrolytic activity.
a r t i c l e
i n f o
Article history: Received 28 January 2015 Received in revised form 9 March 2015 Accepted 10 March 2015 Available online xxxx Keywords: Xylanase Acid-insoluble lignin Additives Adsorption Desorption
a b s t r a c t The competitive adsorption between cellulases and additives on lignin in the hydrolysis of lignocelluloses has been confirmed, whereas the effect of additives on the interaction between xylanase and lignin is not clear. In this work, the effects of additives, poly(ethylene glycol) 2000, poly(ethylene glycol) 6000, Tween 20, and Tween 80, on the xylanase adsorption/desorption onto/from acid-insoluble lignin from corn stover (CS-lignin) and wheat straw (WS-lignin) were investigated. The results indicated that the additives could adsorb onto isolated lignin and reduce the xylanase adsorption onto lignin. Compared to CS-lignin, more additives could adsorb onto WS-lignin, making less xylanase adsorbed onto WS-lignin. In addition, the additives could enhance desorption of xylanase from lignin, which might be due to the competitive adsorption between xylanase and additives on lignin. The released xylanase from lignin still exhibited hydrolytic capacity in the hydrolysis of isolated xylan and xylan in corn stover. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Many previous studies have proved that the bioconversion of cellulosic biomass is mainly limited by the recalcitrance and complex structure of lignocellulosic material (Zhu et al., 2006). Although pretreatment has been found to be one technical approach to improve cellulose accessibility to cellulases in enzymatic hydrolysis by removing lignin and hemicelluloses, low amounts of residual lignin and xylan after pretreatment still limit cellulose hydrolysis by cellulases (Yang and Wyman, 2008). The negative effects of lignin in biomass hydrolysis have been confirmed by many authors. The close association of lignin with
Abbreviations: BWX, beechwood xylan; CS, corn stover; CS-lignin, lignin from corn stover; PEG, poly(ethylene glycol); WS, wheat straw; WS-lignin, lignin from wheat straw. ⇑ Corresponding author. Tel.: +86 13892883052; fax: +86 29 87082892. E-mail address: [email protected] (J. Zhang).
cellulose microfibrils prevents swelling of fibers, thus limits the accessibility of cellulases to cellulose (Zhang et al., 2006). Except for the effect of lignin on fiber swelling, steric hindrance from the residual lignin is a significant effect on enzyme digestibility (Mooney et al., 1998). Furthermore, the hydrophobic surface of lignin can easily induce the binding to the hydrophobic residues (i.e. Tryptophan, Phenylalanine and Tyrosine) on the surface of enzymes (Palonen et al., 2004). The unproductive adsorption and inactivation of enzymes by lignin are important factors that inhibit the effectiveness of enzymatic hydrolysis (Rahikainen et al., 2011). In addition, the irreversible adsorption of cellulases and xylanases by lignin reduces the recyclability after enzymatic hydrolysis (Lu et al., 2002). In the past three decades, numerous studies have investigated the effect of surfactants and other additives on the hydrolysis of lignocellulosic substrates. Non-ionic surfactants (Eriksson et al., 2002) and polymers (Börjesson et al., 2007a) have been found to be effective additives. The major explanations are as follows: (1)
http://dx.doi.org/10.1016/j.biortech.2015.03.058 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Li, Y., et al. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.058
2
Y. Li et al. / Bioresource Technology xxx (2015) xxx–xxx
the interaction between additives and lignin which could reduce the unproductive adsorption of enzymes on lignin (Eriksson et al., 2002; Börjesson et al., 2007b). (2) The additives protect enzymes from denaturing by heat, solvents and shear force (Kaar and Holtzapple, 1998; Okino et al., 2013). (3) Additives could alter substrate structure and increasing cellulose accessibility (Seo et al., 2011). In the previous studies, effects of additives on cellulases adsorption onto lignin and Avicel have been extensively investigated (Eriksson et al., 2002; Börjesson et al., 2007b). Xylanase as one of the major hydrolytic enzymes has been reported by many authors and its supplementation can clearly increase cellulose hydrolysis in xylan-containing lignocellulosic materials (Öhgren et al., 2007). However, the xylanase would be inhibited by the residual lignin (Berlin et al., 2006). However, there is a lack of information on whether this inhibition could be relieved by additives. In addition, the effects of additives on xylanase adsorption onto lignin and desorption from lignin has not been clearly elucidated. In this work, the effects of additives (PEG 2000, PEG 6000, Tween 20, and Tween 80) on xylanase adsorption and desorption onto/from isolated lignin preparations from corn stover and wheat straw were investigated. The objective of this work is to explore the role of additives in the reduction of unproductive adsorption of xylanase onto lignin.
2. Methods 2.1. Substrates Beechwood xylan (BWX) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Corn stover (CS) and wheat straw (WS) were both harvested from a local farm in Yangling, Shaanxi Province, China. They were dried at 60 °C for 10 h, and then milled to give a size about 1 mm and stored at 4 °C for further use. The corn stover was pretreated with 21% ammonia at a solid-to-liquid ratio of 1:6 and 70 °C for 20 h. The chemical compositions of pretreated corn stover were determined according to Laboratory Analytical Procedures established by the National Renewable Energy Laboratory (Sluiter et al., 2008). The compositions of pretreated corn stover were 60.4% cellulose, 20.8% xylan, and 10.8% lignin.
2.2. Lignin preparation The lignin from corn stover (CS-lignin) and wheat straw (WSLignin) were isolated from the milled materials by a two-step sulfuric acid hydrolysis according to the method of National Renewable Energy Laboratory (Sluiter et al., 2008). The lignin obtained by this method was acid-insoluble lignin, which was washed by deionized water to neutral and was lyophilized later to used as adsorbed substrates.
2.3. Enzymes The Thermomyces lanuginosus xylanase, Pentopan Mono BG, was purchased from Sigma Chemical Co., USA. The amount of protein in enzyme preparation was determined by BCA Assay Reagent Kit (Beyotime, Shanghai, China).
2.4. Additives PEG 2000, PEG 6000, Tween 20, and Tween 80 were purchased from Tianjin Kermel Reagent Co. Ltd, China.
2.5. Xylanase adsorption Xylanase adsorption experiment was conducted in a system of 50 mM sodium citrate buffer (pH 5.0) with 1% (w/v) substrate (CS-lignin or WS-lignin) with a working volume of 1.2 ml. The system was incubated with 0 or 2.5 mg/ml additives in 50 mM sodium citrate buffer (pH 5.0) at 4 °C with magnetic stirring for 30 min. The solid and liquid in the system were separated by centrifugation (10,000g, 10 min), and the solid was washed with 5 ml 50 mM sodium citrate buffer (pH 5.0) for 3 times. After that, the solid was incubated with xylanase (0.20 mg/ml) in 1.2 ml 50 mM sodium citrate buffer (pH 5.0). After incubated at 4 °C with magnetic stirring for 1 h, the solid and liquid were separated by centrifugation (10,000g, 10 min). The protein in the liquid was determined and the solid was washed with 5 ml 50 mM sodium citrate buffer (pH 5.0) to remove the unbound xylanase and additives. After wash, the residual solid was used as lignin–xylanase complex for hydrolysis experiment.
2.6. Xylanase desorption Xylanase desorption experiment was also carried out in a system as described earlier. Xylanase preparation (0.20 mg/ml) was incubated with 1% (w/v) substrate (CS-lignin, WS-lignin) at 4 °C with magnetic stirring for 1 h. The solid and liquid were separated by centrifugation (10,000g, 10 min), then the solid was washed as described earlier. Next, the solid was incubated with 0 or 2.5 mg/ ml additives in the 50 mM sodium citrate buffer (pH 5.0). After incubated at 4 °C with magnetic stirring for 1 h, the solid and liquid was separated by centrifugation (10,000g, 10 min). The solid was washed as described earlier, and was used as lignin–xylanase complex for hydrolysis experiments.
2.7. Enzymatic hydrolysis The hydrolysis of BWX (1%, w/v) and CS (2%, w/v) by lignin–xylanase complex was performed in screw-capped 10 ml tubes with a working volume of 3 ml in 50 mM sodium citrate buffer (pH 5.0) containing 0.02% NaN3. The reaction mixture was incubated at 50 °C on an orbital shaker at 200 rpm. Samples were withdrawn at 4 h (BWX) or 48 h (CS) and boiled for 10 min to stop the enzymatic hydrolysis. After cooling to room temperature, the samples were centrifuged at 10,000g for 10 min, and the supernatants were collected for further analysis.
2.8. Carbohydrate analysis The reducing sugars were measured using the dinitrosalicylic acid method with xylose as the standards (Miller, 1959). Two replicate tests were carried out in all hydrolysis experiments and average values are presented.
2.9. Fourier Transform Infrared Spectroscopy (FTIR) analysis of lignins The functional groups of the isolated lignins (CS-lignin and WSlignin) were analyzed using a Thermo Nicolet 470 Fourier Transform Infrared Spectrometer (FTIR, Nexus., Thermo Nicolet Corporation., VA, USA) with KBr pellets over the range of 400–4 000 cm 1.
Please cite this article in press as: Li, Y., et al. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.058
Y. Li et al. / Bioresource Technology xxx (2015) xxx–xxx
3. Results and discussion 3.1. Effect of additives on xylanase adsorption To investigate the effect of additives on adsorption of xylanase onto isolated acid-insoluble lignin (CS-lignin and WS-lignin), the hydrolysis yields of BWX by lignin–xylanase complex obtained from the adsorption experiments were determined (Fig. 1A). In this adsorption experiments, higher adsorption capacity of additive onto lignin corresponded to lower xylanase adsorption onto lignin and lower hydrolytic capacity of lignin–xylanase complex. With the addition of Tween and PEG, higher amounts of protein in supernatants were observed (data not shown) and the hydrolysis yields of BWX by lignin–xylanase complex were significantly decreased (Fig. 1A). With the addition of Tween 80, the hydrolysis yields of BWX by CS- and WS-lignin–xylanase complex decreased from 11.2% and 6.1% to 1.3% and 0.8%, respectively. The results here confirmed the strong adsorption of additives onto lignin and the reduction of xylanase adsorption onto lignin by additives. In the present work, there was no free xylanase released from the solid after washing several times with buffer (data not shown), indicating the result of xylan hydrolysis was due to the xylanase adsorbed on lignin. The hydrolytic capacity of lignin–xylanase complex could be explained by the adsorbed xylanase released to the supernatant, exhibiting xylanase activity in the hydrolysis process. It was reported by Kumar and Wyman (2009) that when the buffer containing unbound proteins was replaced by an equal amount of fresh buffer, the desorption of cellulase from various pretreated poplar enzyme lignin was noticed. However, in this work, the desorption of xylanase from lignin after the addition of buffer was limited and the addition of surfactants could increase such desorption by competitive adsorption between xylanase and additives on
A
14 CS-Lignin
Hydrolysis yield(%)
12
WS-lignin
10 8 6 4 2 Control
PEG 2000 PEG 6000 Tween 20 Tween 80
30 25
Hydrolysis yield (%)
lignin. Alternatively, the xylanase bound to lignin might be maintain its enzymatic activity and functioned as an immobilized xylanase in hydrolysis process, which was reported in the experiment of cellulase adsorbed onto lignophenol that lignophenol–cellulase complex maintains about half of the enzymatic activity of free cellulase (Nonaka et al., 2013). The hydrolysis yields of BWX by lignin–xylanase complex with PEG 6000 were less than those with PEG 2000 (Fig. 1A). It suggested the adsorption affinity of PEG 6000 on lignin was larger than that of PEG 2000, which resulted in a lower amount of xylanase adsorbed on lignin. That was conformed to the previous observation that with longer ethylene oxide chains on the PEG, the adsorption of cellobiohydrolase I on lignin was decreased (Börjesson et al., 2007a). It was noticed that the CS-lignin–xylanase complex had higher hydrolytic capacity than WS-lignin–xylanase complex (Fig. 1A), which could be due to more adsorption of additives onto WS-lignin, making less xylanase adsorbed onto lignin, and lower hydrolysis yield of BWX. Structure analysis of two lignin preparations by FTIR was performed to elucidate the cause of the difference in adsorption capacity (Fig. S1 and Table S1). The FTIR analysis suggested that both CS-lignin and WS-lignin were GSH-type lignin (G, guaiacyl; S, syringyl; H, p-hydroxyphenyl). Furthermore, the CS-lignin contained higher S unit, much lower G and H units leading to a higher S/G ratio compared to WS-lignin. According to the conclusion of Guo et al. (2014), G unit has a higher adsorption capacity on enzymes than S unit. In addition, the low S/G ratio and higher H unit content have good correlations with high adsorption capacity. The results supported that WS-lignin was more effective to bind enzymes or additives than CS-lignin. However, the isolation method, molecular weight, and functional groups of lignin would also affect the adsorption capacity of enzymes and additives toward lignin. Hydrolysis of BWX with CS-lignin–xylanase complex, which was obtained by incubating lignin with PEG 6000 followed by different concentrations of xylanase, was carried out (Fig. 1B). In the case of xylanase dosages of 100 and 500 mg/g DM, the hydrolysis yields of BWX with CS-lignin–xylanase complex decreased from 24.0% and 29.3% to 11.2% and 21.9%, respectively, with the presence of PEG 6000. The results indicated that the xylanase adsorbed on CS-lignin was reduced by PEG 6000, which was in good line with the data of Fig. 1A. With the increase of xylanase dosage, more xylanase adsorbed onto the hydrophobic surface of lignin and exhibited higher hydrolytic capacity. 3.2. Effect of additives on the xylanase desorption
0
B
3
20 15 10 Control
5
PEG 6000 0 0
100
200
300
400
500
XYL dosage (mg/g DM) Fig. 1. Hydrolysis of BWX (1 %) by lignin–xylanase complex obtained from incubating lignin with additives followed by xylanase in 50 mM sodium citrate buffer (pH 5.0) at 50 °C for 4 h. (A) Effect of different additives (2.5 mg/ml) on xylanase (0.2 mg/ml) adsorption; (B) Effect of PEG 6000 (2.5 mg/ml) on xylanase (0, 20, 30, 50, 100, 200, 500 mg/g DM) adsorption. Error bars represent the standard errors.
To investigate the role of additives in xylanase desorption from acid-insoluble lignin, the hydrolysis yields of BWX and CS with lignin–xylanase complex after desorption by additives (2.5 mg/ml) were determined (Fig. 2). Aqueous ammonia pretreated CS was used as the substrate because high xylan content (20.8%) existed and xylanase played an important role in the enzymatic hydrolysis. High xylan content (17.5%) in CS after aqueous ammonia pretreatment was also reported by other authors (Kim and Lee, 2007). In this work, higher contents of cellulose and lignin were noticed, which was due to the differences of the sources of raw materials and the conditions of pretreatment. In this desorption experiments, higher adsorption capacity of additives onto lignin resulted in higher xylanase desorption from lignin and lower hydrolytic capacity of lignin–xylanase complex. The hydrolysis yields of BWX and CS by CS- and WS-lignin–xylanase complex decreased after the addition of additives (Fig. 2). With the addition of Tween 80, the hydrolysis yields of BWX by CS- and WS-lignin–xylanase complex decreased from 2.8% and 5.4% to 1.1% and 2.3%, respectively. In the hydrolysis of CS, after the addition Tween 80
Please cite this article in press as: Li, Y., et al. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.058
4
Y. Li et al. / Bioresource Technology xxx (2015) xxx–xxx
A
4. Conclusions
6 CS-Lignin
Hydrolysis yield (%)
5
WS-Lignin
4 3 2 1 0 Control
PEG 2000 PEG 6000 Tween 20 Tween 80
The adsorption of xylanase onto lignin could be alleviated by additives (PEG and Tween) due to the strong adsorption of additives onto lignin, occupying partial hydrophobic surface of lignin. In addition, the presence of additives did great benefit to the xylanase desorption from lignin. The xylanase adsorbed on both CSand WS-lignin could be released to supernatant, exhibiting hydrolytic capacity toward isolated xylan and xylan in corn stover. The conclusions of this work help us further understanding the role of additives in the reduction of non-productive adsorption of xylanase on lignin.
B 15 Hydrolysis yield (%)
CS-Lignin
12
WS-Lignin
Acknowledgement This work was supported by Natural Science Foundation of China (31270622).
9 6
Appendix A. Supplementary data
3 0 Control
PEG 2000 PEG 6000 Tween 20 Tween 80
Fig. 2. Effect of additives on the hydrolytic capacity of lignin–xylanase complex obtained from incubating lignin with xylanase (0.2 mg/ml) followed by additives (2.5 mg/ml). The hydrolysis of 1% BWX (A) and 2% CS (B) were performed in 50 mM sodium citrate buffer (pH 5.0) at 50 °C for 4 and 48 h, respectively. Error bars represent the standard errors.
the hydrolysis yields by CS-and WS-lignin–xylanase complex decreased from 12.0% and 13.8% to 6.7% and 6.1%, respectively. Such decreases in hydrolysis yield were due to the desorption of xylanase from lignin–xylanase complex and the decrease of the amount of xylanase available for hydrolysis. The desorption of xylanase from lignin might be due to the competitive adsorption between xylanase and additives on lignin. The results indicated that the presence of additives did great benefit to the xylanase desorbed from the lignin, which conformed with the results of Park et al. (1992) that surfactants help the cellulases to desorb from the binding sites on the surface of used newspaper. The lowest hydrolysis yields were observed in the hydrolysis of BWX and CS by lignin–xylanase complex after desorption of xylanase by Tween 80. The results suggested that Tween 80 had the highest affinity with lignin and occupied the hydrophobic surface of lignin strongly. The stronger affinity of Tween 80 with lignin compared to PEG was also reported by Yao et al. (2007). In addition, the effect of PEG 6000 concentration (0–5.0 mg/ml) on the desorption of xylanase from CS-lignin was also investigated. The results showed that higher concentration of PEG 6000 could increase the desorption of xylanase from CS-lignin (data not shown), indicating higher concentration of additives, to some extent, exhibited stronger reduction of the non-productive adsorption of xylanase onto lignin. Higher hydrolysis yields of BWX and CS by lignin–xylanase complex after desorption by additives were noticed with lignin from wheat straw (Fig. 2B). That was because WS-lignin was more effective to bind xylanase than CS-lignin, and resulted in more amounts of xylanase adsorbed on WS-lignin. For WS-lignin–xylanase complex, after desorption of lignin-bound xylanase by additives, more xylanase bound on WS-lignin and exhibited higher hydrolytic capacity. However, the difference of adsorption capacity of additives onto two lignin preparations were unknown and further work was still ongoing.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.03. 058.
References Berlin, A., Balakshin, M., Gilkes, N., Kadla, J., Maximenko, V., Kubo, S., Saddler, J., 2006. Inhibition of cellulase, xylanase and b-glucosidase activities by softwood lignin preparations. J. Biotechnol. 125, 198–209. Börjesson, J., Peterson, R., Tjerneld, F., 2007a. Enhanced enzymatic conversion of softwood lignocellulose by poly(ethylene glycol) addition. Enzyme Microb. Technol. 40, 754–762. Börjesson, J., Engqvist, M., Sipos, B., 2007b. Effect of poly(ethylene glycol) on enzymatic hydrolysis and adsorption of cellulase enzymes to pretreated lignocellulose. Enzyme Microb. Technol. 41, 186–195. Eriksson, T., Börjesson, J., Tjerneld, F., 2002. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme Microb. Technol. 31, 353–364. Guo, F., Shi, W., Sun, W., Li, X., Wang, F., Zhao, J., Qu, Y., 2014. Differences in the adsorption of enzymes onto lignins from diverse types of lignocellulosic biomass and the underlying mechanism. Biotechnol. Biofuels 7, 38. Kaar, W.E., Holtzapple, M.T., 1998. Benefits from Tween during enzymic hydrolysis of corn stover. Biotechnol. Bioeng. 59, 419–427. Kim, T.H., Lee, Y.Y., 2007. Pretreatment of corn stover by soaking in aqueous ammonia at moderate temperatures. Appl. Biochem. Biotech. 137, 81–92. Kumar, R., Wyman, C.E., 2009. Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies. Biotechnol. Prog. 25, 807–819. Lu, Y., Yang, B., Gregg, D., Saddler, J.N., Mansfield, S.D., 2002. Cellulase adsorption and an evaluation of enzyme recycle during hydrolysis of steam-exploded softwood residues. Appl. Biochem. Biotechnol. 98, 641–654. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determinantion of reducing sugars. Anal. Chem. 31, 426–428. Mooney, C.A., Mansfield, S.D., Touhy, M.G., Saddler, J.N., 1998. The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods. Bioresour. Technol. 64, 113–119. Nonaka, H., Kobayashi, A., Funaoka, M., 2013. Behavior of lignin-binding cellulase in the presence of fresh cellulosic substrate. Bioresour. Technol. 135, 53–57. Öhgren, K., Bura, R., Saddler, J., Zacchi, G., 2007. Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover. Bioresour. Technol. 98, 2503–2510. Okino, S., Ikeo, M., Ueno, Y., Taneda, D., 2013. Effects of Tween 80 on cellulase stability under agitated conditions. Bioresour. Technol. 142, 535–539. Palonen, H., Tjerneld, F., Zacchi, G., Tenkanen, M., 2004. Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin. J. Biotechnol. 107, 65–72. Park, J.W., Takahata, Y., Kajiuchi, T., Akehata, T., 1992. Effects of nonionic surfactant on enzymatic hydrolysis of used newspaper. Biotechnol. Bioeng. 39, 117–120. Rahikainen, J., Mikander, S., Marjamaa, K., Tamminen, T., Lappas, A., Viikari, L., Kruus, K., 2011. Inhibition of enzymatic hydrolysis by residual lignins from softwood-study of enzyme binding and inactivation on lignin-rich surface. Biotechnol. Bioeng. 108, 2823–2834. Seo, D.J., Fujita, H., Sakoda, A., 2011. Effects of a non-ionic surfactant, Tween 20, on adsorption/desorption of saccharification enzymes onto/from lignocelluloses and saccharification rate. Adsorption 17, 813–822.
Please cite this article in press as: Li, Y., et al. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.058
Y. Li et al. / Bioresource Technology xxx (2015) xxx–xxx Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Determination of structural carbohydrates and lignin in biomass. National Renewable Energy Laboratory (NREL), Golden, CO. Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels. Bioprod. Bioref. 2, 26–40. Yao, R., Qi, B., Deng, S., Liu, N., Peng, S., Cui, Q., 2007. Use of surfactants in enzymatic hydrolysis of rice straw and lactic acid production from rice straw by simultaneous saccharification and fermentation. Biorsources. 2, 389–398.
5
Zhu, S.D., Wu, Y.X., Chen, Q.M., Yu, Z.N., Wang, C.W., Jin, S.W., Ding, Y.G., Wu, G., 2006. Dissolution of cellulose with ionic liquids and its application: a minireview. Green Chem. 8, 325–327. Zhang, Y.H., Cui, J., Lynd, L.R., Kuang, L.R., 2006. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 7, 644–648.
Please cite this article in press as: Li, Y., et al. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.058
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw Yanfei Li, Xiaoyan Ge, Zongping Sun, Junhua Zhang ⇑ College of Forestry, Northwest A&F University, 3 Taicheng Road, Yangling 712100, China
h i g h l i g h t s Additives affected the adsorption and desorption behavior of xylanase on lignin. The adsorption of xylanase onto lignin could be alleviated by additives. The additives could enhance desorption of xylanase from lignin. More additives could adsorb onto WS-lignin compared to CS-lignin. The released xylanase from lignin still exhibited hydrolytic activity.
a r t i c l e
i n f o
Article history: Received 28 January 2015 Received in revised form 9 March 2015 Accepted 10 March 2015 Available online xxxx Keywords: Xylanase Acid-insoluble lignin Additives Adsorption Desorption
a b s t r a c t The competitive adsorption between cellulases and additives on lignin in the hydrolysis of lignocelluloses has been confirmed, whereas the effect of additives on the interaction between xylanase and lignin is not clear. In this work, the effects of additives, poly(ethylene glycol) 2000, poly(ethylene glycol) 6000, Tween 20, and Tween 80, on the xylanase adsorption/desorption onto/from acid-insoluble lignin from corn stover (CS-lignin) and wheat straw (WS-lignin) were investigated. The results indicated that the additives could adsorb onto isolated lignin and reduce the xylanase adsorption onto lignin. Compared to CS-lignin, more additives could adsorb onto WS-lignin, making less xylanase adsorbed onto WS-lignin. In addition, the additives could enhance desorption of xylanase from lignin, which might be due to the competitive adsorption between xylanase and additives on lignin. The released xylanase from lignin still exhibited hydrolytic capacity in the hydrolysis of isolated xylan and xylan in corn stover. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Many previous studies have proved that the bioconversion of cellulosic biomass is mainly limited by the recalcitrance and complex structure of lignocellulosic material (Zhu et al., 2006). Although pretreatment has been found to be one technical approach to improve cellulose accessibility to cellulases in enzymatic hydrolysis by removing lignin and hemicelluloses, low amounts of residual lignin and xylan after pretreatment still limit cellulose hydrolysis by cellulases (Yang and Wyman, 2008). The negative effects of lignin in biomass hydrolysis have been confirmed by many authors. The close association of lignin with
Abbreviations: BWX, beechwood xylan; CS, corn stover; CS-lignin, lignin from corn stover; PEG, poly(ethylene glycol); WS, wheat straw; WS-lignin, lignin from wheat straw. ⇑ Corresponding author. Tel.: +86 13892883052; fax: +86 29 87082892. E-mail address: [email protected] (J. Zhang).
cellulose microfibrils prevents swelling of fibers, thus limits the accessibility of cellulases to cellulose (Zhang et al., 2006). Except for the effect of lignin on fiber swelling, steric hindrance from the residual lignin is a significant effect on enzyme digestibility (Mooney et al., 1998). Furthermore, the hydrophobic surface of lignin can easily induce the binding to the hydrophobic residues (i.e. Tryptophan, Phenylalanine and Tyrosine) on the surface of enzymes (Palonen et al., 2004). The unproductive adsorption and inactivation of enzymes by lignin are important factors that inhibit the effectiveness of enzymatic hydrolysis (Rahikainen et al., 2011). In addition, the irreversible adsorption of cellulases and xylanases by lignin reduces the recyclability after enzymatic hydrolysis (Lu et al., 2002). In the past three decades, numerous studies have investigated the effect of surfactants and other additives on the hydrolysis of lignocellulosic substrates. Non-ionic surfactants (Eriksson et al., 2002) and polymers (Börjesson et al., 2007a) have been found to be effective additives. The major explanations are as follows: (1)
http://dx.doi.org/10.1016/j.biortech.2015.03.058 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Li, Y., et al. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.058
2
Y. Li et al. / Bioresource Technology xxx (2015) xxx–xxx
the interaction between additives and lignin which could reduce the unproductive adsorption of enzymes on lignin (Eriksson et al., 2002; Börjesson et al., 2007b). (2) The additives protect enzymes from denaturing by heat, solvents and shear force (Kaar and Holtzapple, 1998; Okino et al., 2013). (3) Additives could alter substrate structure and increasing cellulose accessibility (Seo et al., 2011). In the previous studies, effects of additives on cellulases adsorption onto lignin and Avicel have been extensively investigated (Eriksson et al., 2002; Börjesson et al., 2007b). Xylanase as one of the major hydrolytic enzymes has been reported by many authors and its supplementation can clearly increase cellulose hydrolysis in xylan-containing lignocellulosic materials (Öhgren et al., 2007). However, the xylanase would be inhibited by the residual lignin (Berlin et al., 2006). However, there is a lack of information on whether this inhibition could be relieved by additives. In addition, the effects of additives on xylanase adsorption onto lignin and desorption from lignin has not been clearly elucidated. In this work, the effects of additives (PEG 2000, PEG 6000, Tween 20, and Tween 80) on xylanase adsorption and desorption onto/from isolated lignin preparations from corn stover and wheat straw were investigated. The objective of this work is to explore the role of additives in the reduction of unproductive adsorption of xylanase onto lignin.
2. Methods 2.1. Substrates Beechwood xylan (BWX) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Corn stover (CS) and wheat straw (WS) were both harvested from a local farm in Yangling, Shaanxi Province, China. They were dried at 60 °C for 10 h, and then milled to give a size about 1 mm and stored at 4 °C for further use. The corn stover was pretreated with 21% ammonia at a solid-to-liquid ratio of 1:6 and 70 °C for 20 h. The chemical compositions of pretreated corn stover were determined according to Laboratory Analytical Procedures established by the National Renewable Energy Laboratory (Sluiter et al., 2008). The compositions of pretreated corn stover were 60.4% cellulose, 20.8% xylan, and 10.8% lignin.
2.2. Lignin preparation The lignin from corn stover (CS-lignin) and wheat straw (WSLignin) were isolated from the milled materials by a two-step sulfuric acid hydrolysis according to the method of National Renewable Energy Laboratory (Sluiter et al., 2008). The lignin obtained by this method was acid-insoluble lignin, which was washed by deionized water to neutral and was lyophilized later to used as adsorbed substrates.
2.3. Enzymes The Thermomyces lanuginosus xylanase, Pentopan Mono BG, was purchased from Sigma Chemical Co., USA. The amount of protein in enzyme preparation was determined by BCA Assay Reagent Kit (Beyotime, Shanghai, China).
2.4. Additives PEG 2000, PEG 6000, Tween 20, and Tween 80 were purchased from Tianjin Kermel Reagent Co. Ltd, China.
2.5. Xylanase adsorption Xylanase adsorption experiment was conducted in a system of 50 mM sodium citrate buffer (pH 5.0) with 1% (w/v) substrate (CS-lignin or WS-lignin) with a working volume of 1.2 ml. The system was incubated with 0 or 2.5 mg/ml additives in 50 mM sodium citrate buffer (pH 5.0) at 4 °C with magnetic stirring for 30 min. The solid and liquid in the system were separated by centrifugation (10,000g, 10 min), and the solid was washed with 5 ml 50 mM sodium citrate buffer (pH 5.0) for 3 times. After that, the solid was incubated with xylanase (0.20 mg/ml) in 1.2 ml 50 mM sodium citrate buffer (pH 5.0). After incubated at 4 °C with magnetic stirring for 1 h, the solid and liquid were separated by centrifugation (10,000g, 10 min). The protein in the liquid was determined and the solid was washed with 5 ml 50 mM sodium citrate buffer (pH 5.0) to remove the unbound xylanase and additives. After wash, the residual solid was used as lignin–xylanase complex for hydrolysis experiment.
2.6. Xylanase desorption Xylanase desorption experiment was also carried out in a system as described earlier. Xylanase preparation (0.20 mg/ml) was incubated with 1% (w/v) substrate (CS-lignin, WS-lignin) at 4 °C with magnetic stirring for 1 h. The solid and liquid were separated by centrifugation (10,000g, 10 min), then the solid was washed as described earlier. Next, the solid was incubated with 0 or 2.5 mg/ ml additives in the 50 mM sodium citrate buffer (pH 5.0). After incubated at 4 °C with magnetic stirring for 1 h, the solid and liquid was separated by centrifugation (10,000g, 10 min). The solid was washed as described earlier, and was used as lignin–xylanase complex for hydrolysis experiments.
2.7. Enzymatic hydrolysis The hydrolysis of BWX (1%, w/v) and CS (2%, w/v) by lignin–xylanase complex was performed in screw-capped 10 ml tubes with a working volume of 3 ml in 50 mM sodium citrate buffer (pH 5.0) containing 0.02% NaN3. The reaction mixture was incubated at 50 °C on an orbital shaker at 200 rpm. Samples were withdrawn at 4 h (BWX) or 48 h (CS) and boiled for 10 min to stop the enzymatic hydrolysis. After cooling to room temperature, the samples were centrifuged at 10,000g for 10 min, and the supernatants were collected for further analysis.
2.8. Carbohydrate analysis The reducing sugars were measured using the dinitrosalicylic acid method with xylose as the standards (Miller, 1959). Two replicate tests were carried out in all hydrolysis experiments and average values are presented.
2.9. Fourier Transform Infrared Spectroscopy (FTIR) analysis of lignins The functional groups of the isolated lignins (CS-lignin and WSlignin) were analyzed using a Thermo Nicolet 470 Fourier Transform Infrared Spectrometer (FTIR, Nexus., Thermo Nicolet Corporation., VA, USA) with KBr pellets over the range of 400–4 000 cm 1.
Please cite this article in press as: Li, Y., et al. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.058
Y. Li et al. / Bioresource Technology xxx (2015) xxx–xxx
3. Results and discussion 3.1. Effect of additives on xylanase adsorption To investigate the effect of additives on adsorption of xylanase onto isolated acid-insoluble lignin (CS-lignin and WS-lignin), the hydrolysis yields of BWX by lignin–xylanase complex obtained from the adsorption experiments were determined (Fig. 1A). In this adsorption experiments, higher adsorption capacity of additive onto lignin corresponded to lower xylanase adsorption onto lignin and lower hydrolytic capacity of lignin–xylanase complex. With the addition of Tween and PEG, higher amounts of protein in supernatants were observed (data not shown) and the hydrolysis yields of BWX by lignin–xylanase complex were significantly decreased (Fig. 1A). With the addition of Tween 80, the hydrolysis yields of BWX by CS- and WS-lignin–xylanase complex decreased from 11.2% and 6.1% to 1.3% and 0.8%, respectively. The results here confirmed the strong adsorption of additives onto lignin and the reduction of xylanase adsorption onto lignin by additives. In the present work, there was no free xylanase released from the solid after washing several times with buffer (data not shown), indicating the result of xylan hydrolysis was due to the xylanase adsorbed on lignin. The hydrolytic capacity of lignin–xylanase complex could be explained by the adsorbed xylanase released to the supernatant, exhibiting xylanase activity in the hydrolysis process. It was reported by Kumar and Wyman (2009) that when the buffer containing unbound proteins was replaced by an equal amount of fresh buffer, the desorption of cellulase from various pretreated poplar enzyme lignin was noticed. However, in this work, the desorption of xylanase from lignin after the addition of buffer was limited and the addition of surfactants could increase such desorption by competitive adsorption between xylanase and additives on
A
14 CS-Lignin
Hydrolysis yield(%)
12
WS-lignin
10 8 6 4 2 Control
PEG 2000 PEG 6000 Tween 20 Tween 80
30 25
Hydrolysis yield (%)
lignin. Alternatively, the xylanase bound to lignin might be maintain its enzymatic activity and functioned as an immobilized xylanase in hydrolysis process, which was reported in the experiment of cellulase adsorbed onto lignophenol that lignophenol–cellulase complex maintains about half of the enzymatic activity of free cellulase (Nonaka et al., 2013). The hydrolysis yields of BWX by lignin–xylanase complex with PEG 6000 were less than those with PEG 2000 (Fig. 1A). It suggested the adsorption affinity of PEG 6000 on lignin was larger than that of PEG 2000, which resulted in a lower amount of xylanase adsorbed on lignin. That was conformed to the previous observation that with longer ethylene oxide chains on the PEG, the adsorption of cellobiohydrolase I on lignin was decreased (Börjesson et al., 2007a). It was noticed that the CS-lignin–xylanase complex had higher hydrolytic capacity than WS-lignin–xylanase complex (Fig. 1A), which could be due to more adsorption of additives onto WS-lignin, making less xylanase adsorbed onto lignin, and lower hydrolysis yield of BWX. Structure analysis of two lignin preparations by FTIR was performed to elucidate the cause of the difference in adsorption capacity (Fig. S1 and Table S1). The FTIR analysis suggested that both CS-lignin and WS-lignin were GSH-type lignin (G, guaiacyl; S, syringyl; H, p-hydroxyphenyl). Furthermore, the CS-lignin contained higher S unit, much lower G and H units leading to a higher S/G ratio compared to WS-lignin. According to the conclusion of Guo et al. (2014), G unit has a higher adsorption capacity on enzymes than S unit. In addition, the low S/G ratio and higher H unit content have good correlations with high adsorption capacity. The results supported that WS-lignin was more effective to bind enzymes or additives than CS-lignin. However, the isolation method, molecular weight, and functional groups of lignin would also affect the adsorption capacity of enzymes and additives toward lignin. Hydrolysis of BWX with CS-lignin–xylanase complex, which was obtained by incubating lignin with PEG 6000 followed by different concentrations of xylanase, was carried out (Fig. 1B). In the case of xylanase dosages of 100 and 500 mg/g DM, the hydrolysis yields of BWX with CS-lignin–xylanase complex decreased from 24.0% and 29.3% to 11.2% and 21.9%, respectively, with the presence of PEG 6000. The results indicated that the xylanase adsorbed on CS-lignin was reduced by PEG 6000, which was in good line with the data of Fig. 1A. With the increase of xylanase dosage, more xylanase adsorbed onto the hydrophobic surface of lignin and exhibited higher hydrolytic capacity. 3.2. Effect of additives on the xylanase desorption
0
B
3
20 15 10 Control
5
PEG 6000 0 0
100
200
300
400
500
XYL dosage (mg/g DM) Fig. 1. Hydrolysis of BWX (1 %) by lignin–xylanase complex obtained from incubating lignin with additives followed by xylanase in 50 mM sodium citrate buffer (pH 5.0) at 50 °C for 4 h. (A) Effect of different additives (2.5 mg/ml) on xylanase (0.2 mg/ml) adsorption; (B) Effect of PEG 6000 (2.5 mg/ml) on xylanase (0, 20, 30, 50, 100, 200, 500 mg/g DM) adsorption. Error bars represent the standard errors.
To investigate the role of additives in xylanase desorption from acid-insoluble lignin, the hydrolysis yields of BWX and CS with lignin–xylanase complex after desorption by additives (2.5 mg/ml) were determined (Fig. 2). Aqueous ammonia pretreated CS was used as the substrate because high xylan content (20.8%) existed and xylanase played an important role in the enzymatic hydrolysis. High xylan content (17.5%) in CS after aqueous ammonia pretreatment was also reported by other authors (Kim and Lee, 2007). In this work, higher contents of cellulose and lignin were noticed, which was due to the differences of the sources of raw materials and the conditions of pretreatment. In this desorption experiments, higher adsorption capacity of additives onto lignin resulted in higher xylanase desorption from lignin and lower hydrolytic capacity of lignin–xylanase complex. The hydrolysis yields of BWX and CS by CS- and WS-lignin–xylanase complex decreased after the addition of additives (Fig. 2). With the addition of Tween 80, the hydrolysis yields of BWX by CS- and WS-lignin–xylanase complex decreased from 2.8% and 5.4% to 1.1% and 2.3%, respectively. In the hydrolysis of CS, after the addition Tween 80
Please cite this article in press as: Li, Y., et al. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.058
4
Y. Li et al. / Bioresource Technology xxx (2015) xxx–xxx
A
4. Conclusions
6 CS-Lignin
Hydrolysis yield (%)
5
WS-Lignin
4 3 2 1 0 Control
PEG 2000 PEG 6000 Tween 20 Tween 80
The adsorption of xylanase onto lignin could be alleviated by additives (PEG and Tween) due to the strong adsorption of additives onto lignin, occupying partial hydrophobic surface of lignin. In addition, the presence of additives did great benefit to the xylanase desorption from lignin. The xylanase adsorbed on both CSand WS-lignin could be released to supernatant, exhibiting hydrolytic capacity toward isolated xylan and xylan in corn stover. The conclusions of this work help us further understanding the role of additives in the reduction of non-productive adsorption of xylanase on lignin.
B 15 Hydrolysis yield (%)
CS-Lignin
12
WS-Lignin
Acknowledgement This work was supported by Natural Science Foundation of China (31270622).
9 6
Appendix A. Supplementary data
3 0 Control
PEG 2000 PEG 6000 Tween 20 Tween 80
Fig. 2. Effect of additives on the hydrolytic capacity of lignin–xylanase complex obtained from incubating lignin with xylanase (0.2 mg/ml) followed by additives (2.5 mg/ml). The hydrolysis of 1% BWX (A) and 2% CS (B) were performed in 50 mM sodium citrate buffer (pH 5.0) at 50 °C for 4 and 48 h, respectively. Error bars represent the standard errors.
the hydrolysis yields by CS-and WS-lignin–xylanase complex decreased from 12.0% and 13.8% to 6.7% and 6.1%, respectively. Such decreases in hydrolysis yield were due to the desorption of xylanase from lignin–xylanase complex and the decrease of the amount of xylanase available for hydrolysis. The desorption of xylanase from lignin might be due to the competitive adsorption between xylanase and additives on lignin. The results indicated that the presence of additives did great benefit to the xylanase desorbed from the lignin, which conformed with the results of Park et al. (1992) that surfactants help the cellulases to desorb from the binding sites on the surface of used newspaper. The lowest hydrolysis yields were observed in the hydrolysis of BWX and CS by lignin–xylanase complex after desorption of xylanase by Tween 80. The results suggested that Tween 80 had the highest affinity with lignin and occupied the hydrophobic surface of lignin strongly. The stronger affinity of Tween 80 with lignin compared to PEG was also reported by Yao et al. (2007). In addition, the effect of PEG 6000 concentration (0–5.0 mg/ml) on the desorption of xylanase from CS-lignin was also investigated. The results showed that higher concentration of PEG 6000 could increase the desorption of xylanase from CS-lignin (data not shown), indicating higher concentration of additives, to some extent, exhibited stronger reduction of the non-productive adsorption of xylanase onto lignin. Higher hydrolysis yields of BWX and CS by lignin–xylanase complex after desorption by additives were noticed with lignin from wheat straw (Fig. 2B). That was because WS-lignin was more effective to bind xylanase than CS-lignin, and resulted in more amounts of xylanase adsorbed on WS-lignin. For WS-lignin–xylanase complex, after desorption of lignin-bound xylanase by additives, more xylanase bound on WS-lignin and exhibited higher hydrolytic capacity. However, the difference of adsorption capacity of additives onto two lignin preparations were unknown and further work was still ongoing.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.03. 058.
References Berlin, A., Balakshin, M., Gilkes, N., Kadla, J., Maximenko, V., Kubo, S., Saddler, J., 2006. Inhibition of cellulase, xylanase and b-glucosidase activities by softwood lignin preparations. J. Biotechnol. 125, 198–209. Börjesson, J., Peterson, R., Tjerneld, F., 2007a. Enhanced enzymatic conversion of softwood lignocellulose by poly(ethylene glycol) addition. Enzyme Microb. Technol. 40, 754–762. Börjesson, J., Engqvist, M., Sipos, B., 2007b. Effect of poly(ethylene glycol) on enzymatic hydrolysis and adsorption of cellulase enzymes to pretreated lignocellulose. Enzyme Microb. Technol. 41, 186–195. Eriksson, T., Börjesson, J., Tjerneld, F., 2002. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme Microb. Technol. 31, 353–364. Guo, F., Shi, W., Sun, W., Li, X., Wang, F., Zhao, J., Qu, Y., 2014. Differences in the adsorption of enzymes onto lignins from diverse types of lignocellulosic biomass and the underlying mechanism. Biotechnol. Biofuels 7, 38. Kaar, W.E., Holtzapple, M.T., 1998. Benefits from Tween during enzymic hydrolysis of corn stover. Biotechnol. Bioeng. 59, 419–427. Kim, T.H., Lee, Y.Y., 2007. Pretreatment of corn stover by soaking in aqueous ammonia at moderate temperatures. Appl. Biochem. Biotech. 137, 81–92. Kumar, R., Wyman, C.E., 2009. Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies. Biotechnol. Prog. 25, 807–819. Lu, Y., Yang, B., Gregg, D., Saddler, J.N., Mansfield, S.D., 2002. Cellulase adsorption and an evaluation of enzyme recycle during hydrolysis of steam-exploded softwood residues. Appl. Biochem. Biotechnol. 98, 641–654. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determinantion of reducing sugars. Anal. Chem. 31, 426–428. Mooney, C.A., Mansfield, S.D., Touhy, M.G., Saddler, J.N., 1998. The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods. Bioresour. Technol. 64, 113–119. Nonaka, H., Kobayashi, A., Funaoka, M., 2013. Behavior of lignin-binding cellulase in the presence of fresh cellulosic substrate. Bioresour. Technol. 135, 53–57. Öhgren, K., Bura, R., Saddler, J., Zacchi, G., 2007. Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover. Bioresour. Technol. 98, 2503–2510. Okino, S., Ikeo, M., Ueno, Y., Taneda, D., 2013. Effects of Tween 80 on cellulase stability under agitated conditions. Bioresour. Technol. 142, 535–539. Palonen, H., Tjerneld, F., Zacchi, G., Tenkanen, M., 2004. Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin. J. Biotechnol. 107, 65–72. Park, J.W., Takahata, Y., Kajiuchi, T., Akehata, T., 1992. Effects of nonionic surfactant on enzymatic hydrolysis of used newspaper. Biotechnol. Bioeng. 39, 117–120. Rahikainen, J., Mikander, S., Marjamaa, K., Tamminen, T., Lappas, A., Viikari, L., Kruus, K., 2011. Inhibition of enzymatic hydrolysis by residual lignins from softwood-study of enzyme binding and inactivation on lignin-rich surface. Biotechnol. Bioeng. 108, 2823–2834. Seo, D.J., Fujita, H., Sakoda, A., 2011. Effects of a non-ionic surfactant, Tween 20, on adsorption/desorption of saccharification enzymes onto/from lignocelluloses and saccharification rate. Adsorption 17, 813–822.
Please cite this article in press as: Li, Y., et al. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.058
Y. Li et al. / Bioresource Technology xxx (2015) xxx–xxx Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Determination of structural carbohydrates and lignin in biomass. National Renewable Energy Laboratory (NREL), Golden, CO. Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels. Bioprod. Bioref. 2, 26–40. Yao, R., Qi, B., Deng, S., Liu, N., Peng, S., Cui, Q., 2007. Use of surfactants in enzymatic hydrolysis of rice straw and lactic acid production from rice straw by simultaneous saccharification and fermentation. Biorsources. 2, 389–398.
5
Zhu, S.D., Wu, Y.X., Chen, Q.M., Yu, Z.N., Wang, C.W., Jin, S.W., Ding, Y.G., Wu, G., 2006. Dissolution of cellulose with ionic liquids and its application: a minireview. Green Chem. 8, 325–327. Zhang, Y.H., Cui, J., Lynd, L.R., Kuang, L.R., 2006. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 7, 644–648.
Please cite this article in press as: Li, Y., et al. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.03.058