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Quantitative analysis of adsorption and desorption behavior of individual cellulase components during the hydrolysis of lignocellulosic biomass with the addition of lysozyme
Bioresource Technology 234 (2017) 150–157
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Quantitative analysis of adsorption and desorption behavior of individual cellulase components during the hydrolysis of lignocellulosic biomass with the addition of lysozyme Yoshiko Toyosawa, Makoto Ikeo, Daisuke Taneda, Shohei Okino ⇑ JGC Corporation, 2205, Narita-cho, Oarai-machi, Higashiibaraki-gun, Ibaraki Pref. 311-1313, Japan
h i g h l i g h t s Effect of non-catalytic protein addition on enzymatic hydrolysis was investigated. Lysozyme was used as a model non-catalytic protein. Lysozyme adsorbed only to lignin containing substrate. Hydrolysis and desorption of cellulase components were enhanced by lysozyme. Lysozyme is likely to reduce non-specific adsorption of cellulase to lignin.
a r t i c l e
i n f o
Article history: Received 14 December 2016 Received in revised form 27 February 2017 Accepted 28 February 2017
Keywords: Lignocellulosic bioethanol Lysozyme Non-specific adsorption Enzyme adsorption/desorption Sugarcane bagasse
a b s t r a c t The effect of non-catalytic protein addition on the adsorption/desorption behavior of individual cellulase components on/from substrates during the hydrolysis of microcrystalline cellulose and steam exploded sugarcane bagasse (SEB) were investigated. The addition of non-catalytic protein enhanced the enzymatic hydrolysis of SEB, but did not enhance the hydrolysis of microcrystalline cellulose. During the hydrolysis of SEB, adsorption of beta-glucosidase (BGL) was prevented in the presence of non-catalytic protein. Cellobiohydrolase I (CBH I) and endoglucanase I (EG I) desorbed from the substrate after temporary adsorption in the presence of non-catalytic protein during SEB hydrolysis. This suggested that reduction of the non-specific adsorption of cellulase components, CBH I, EG I, and BGL, on lignin in SEB led to the improving of enzymatic hydrolysis. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulosic biomass has a potential to produce a clean and renewable biofuel that does not compete with food resources (Limayem and Ricke, 2012). However, its commercialization is still hampered by economic obstacles, one of them being high enzyme costs (Johnson, 2016; Limayem and Ricke, 2012). Various studies have been conducted to determine how to reduce the usage of the cellulolytic enzyme, cellulase, in the hydrolysis of biomass (Gao et al., 2014; Kumar et al., 2012; Nakagame et al., 2011; Pareek et al., 2013). Lignocellulosic biomass is mainly composed of cellulose, a linear glucose polymer, hemicellulose, a highly branched heteropolymer, and lignin, a very high molecular weight and cross-linked aromatic macromolecular (Himmel et al., 2007). ⇑ Corresponding author. E-mail address: [email protected] (S. Okino). http://dx.doi.org/10.1016/j.biortech.2017.02.132 0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
In the enzymatic hydrolysis of pretreated lignocellulosic materials, cellulases tend to adsorb to lignin-rich surfaces through what is called non-specific adsorption (Pan, 2008; Rahikainen et al., 2013). It has been proposed that non-specific adsorption of cellulase on lignin surfaces is one of the factors that decreases the effectiveness of hydrolysis (Gao et al., 2014; Nakagame et al., 2011; Pareek et al., 2013; Rahikainen et al., 2013; Yang and Wyman, 2006). One of the methods to overcome the non-specific adsorption is the use of additives, such as non-catalytic proteins (e.g. bovine serum albumin: BSA), which enhances enzymatic hydrolysis (Brethauer et al., 2011; Yang and Wyman, 2006). The effect of the addition of non-catalytic protein on the enzymatic hydrolysis of lignocellulosic substrates has been ascertained by many studies (Florencio et al., 2016; Wang et al., 2015; Yang and Wyman, 2006), and it is believed that this type of protein adsorbs to the surface of lignin and prevents the non-specific adsorption of cellulase (Brethauer et al., 2011; Yang and Wyman, 2006).
Y. Toyosawa et al. / Bioresource Technology 234 (2017) 150–157
151
In this study, the non-catalytic protein addition effect for non-specific adsorption of individual cellulase components such as CBH I, CBH II, EG I, and BGL during enzymatic hydrolysis was investigated utilizing lysozyme as the non-catalytic protein.
from Nagase Chemtex was used as cellulase. Lysozyme from Wako Pure Chemical Co. was used as the non-catalytic protein additive.
2. Materials and methods
Enzymatic hydrolysis experiments were conducted using 200 ml-baffled Erlenmeyer flasks with a final reaction volume of 50 ml and 25 ml for microcrystalline cellulose (17 days) and SEB (14 days), respectively. The commercial enzyme solutions, lysozyme, and 0.02% (w/v) NaN3 were mixed and diluted to the desired concentrations using 50 mM sodium acetate buffer (pH 5.0). The prepared solution was added to each flask containing substrates. The concentration of SEB and microcrystalline cellulose were set to 20 w/v% and 10 w/v%, respectively. Enzymatic hydrolysis was carried out at 50 °C under agitation using a rotary shaker set at
2.1. Substrates and enzymes Commercial microcrystalline cellulose (FUNACEL II, Funakoshi Co. Ltd., Japan) and steam explosion pretreated sugarcane bagasse (SEB) were used as substrate in these experiments. The experimental condition of the steam explosion pretreatment was a temperature of 230 °C and a hold time of 7 min, and the solid fraction was washed with hot water. For hydrolysis, Cellulase SS (cellulase)
2.2. Enzymatic hydrolysis conditions
Fig. 1. Changes in glucose concentrations during hydrolysis of microcrystalline cellulose and SEB. (a): Microcrystalline cellulose. (b): SEB. Open and closed symbols show glucose concentration in the presence and absence of lysozyme, respectively. Square and circle symbols show glucose concentration on cellulase loadings of 8 and 20 mgprotein/g-dry substrate, respectively.
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120 rpm. Liquid loss from evaporation of the buffer solution was prevented by tightly sealing the flasks. 2.3. Analytical methods Sample slurries were centrifuged (16,000g at 4 °C for 15 min) and filtered (Millex-LH 0.45 lm PVDF, Millipore). The glucose concentration was quantified by high-performance liquid chromatography (LC20, Shimadzu) equipped with an RI detector, and ULTRON PS-80P (Shinwa chemical industries) operating at 80 °C with ultrapure water as a mobile phase at a flow rate of 1.0 ml/min. The glucose yield of reaction was evaluated by setting the final sugar concentration yielded during hydrolysis when using excess
enzyme loading (60 mg-protein/g-dry substrate) as 100%. Protein concentrations were measured by the Bradford method using bovine gamma globulin (Bio-Rad) as a standard. The gel electrophoresis of hydrolyzates was performed on 10% and 15% polyacrylamide gels when analyzing individual components of the cellulase and lysozyme concentrations, respectively. The gels were stained with Coomassie Brilliant Blue R-250 (BioRad Laboratories). The identities of the bands in the cellulase solution, BGL (78 kDa), CBH I (63 kDa), and EG I (55 kDa) were deduced by comparing the molecular masses with those cited in a previous report (Herpoel-Gimbert et al., 2008). CBH II (58 kDa) was identified by LC-MS analysis in our previous report (Taneda et al., 2012). For quantitative analysis of the intensity of each protein
Fig. 2. Adsorption/desorption behavior of lysozyme. (a): SDS-PAGE of lysozyme in the presence of microcrystalline cellulose. The marker is in Lane 1, and 0, 1, 2, 3, 4, 6, 9, 11, and 17th day are Lanes 2 to 10, respectively. (b): SDS-PAGE of lysozyme in the presence of SEB. The marker is in Lane 1, and 0, 1, 2, 3, 4, 6, 9, 11, and 14th day are Lanes 2 to 10, respectively. (c): Changes in the relative amount of lysozyme by densitograph analysis from results of SDS-PAGE ((a) and (b)).
Y. Toyosawa et al. / Bioresource Technology 234 (2017) 150–157
band in the hydrolysis supernatants, image analysis was conducted with densitograph software (CS Analyzer ver. 2.0, ATTO Co.). Concentrations of each component were standardized so that the initial concentration of each component became 100. The initial adsorption amount was calculated by the difference between the initial band intensity and the band intensity after 24 h. The maximum desorption amount was calculated when an increase in concentration was observed in the component. The difference between the maximum band intensity after the decrease in the concentration and the minimum band intensity is defined as the maximum desorption amount.
3. Results and discussion 3.1. Effect of non-catalytic protein addition on the enzymatic hydrolysis of microcrystalline cellulose and SEB The effect of non-catalytic protein on the hydrolysis of microcrystalline cellulose and SEB was investigated (Fig. 1a, b). Lysozyme was used as a non-catalytic protein in this study. During the hydrolysis of microcrystalline cellulose, no significant differences in glucose production rate and final yield were observed between the conditions with and without lysozyme (Fig. 1a). On the other hand, the glucose concentration in SEB hydrolysis with lysozyme was higher than without lysozyme (Fig. 1b). This result indicates that lysozyme was especially effective for the
153
enzymatic hydrolysis of lignin-containing substrate. Hydrolysis was not observed upon incubation of the substrate with lysozyme alone (Fig. 1a, b). Bovine serum albumin (BSA) is well known as a protein which enhances the hydrolysis of lignocellulosic biomass due to its adsorption on the surface of lignin leading to the prevention of non-specific adsorption of cellulase on lignin (Brethauer et al., 2011; Yang and Wyman, 2006). The result of enhancement of SEB hydrolysis in this study is consistent with previous studies.
3.2. Effect of non-catalytic protein addition on adsorption/desorption of individual cellulase components on/from substrates during hydrolysis To investigate the enhancement of SEB hydrolysis by means of the addition of non-catalytic protein, the adsorption/desorption behavior of CBH I, CBH II, EG 1, and BGL on/from SEB and microcrystalline cellulose was analyzed by SDS-PAGE analysis of hydrolysate supernatant (Fig. 3; Fig. 5). Bovine serum albumin (BSA) commonly used as a non-catalytic protein in cellulase hydrolysis is unsuitable for SDS-PAGE analysis due to the fact that the molecular weight is too close to CBH I, one of the most important components in cellulase. Lysozyme, which has different molecular weight (14.0 kDa) from the main components of cellulase, CBH I, CBH II, EG I, and BGL, was used as a non-catalytic protein in this study.
Fig. 3. SDS-PAGE analysis of hydrolysate supernatant during microcrystalline cellulose hydrolysis. (a): Cellulase loading is 8 mg-protein/g-dry substrate. (b): Cellulase loading is 8 mg-protein/g-dry substrate in the presence of lysozyme. (c): Cellulase loading is 20 mg-protein/g-dry substrate. (d): Cellulase loading is 20 mg-protein/g-dry substrate in the presence of lysozyme. The marker is in Lane 1, and 0, 1, 2, 3, 4, 6, 9, 11, and 17th day are analyzed from Lanes 2 to10.
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3.2.1. Adsorption/desorption behavior of non-catalytic protein, CBH1, and EG1 No adsorption of lysozyme onto the microcrystalline cellulose was observed upon incubation with lysozyme alone (Fig. 2a, c). On the other hand, when lysozyme was incubated with SEB, lysozyme disappeared from supernatants within 24 h (Fig. 2b, c). These results indicate that lysozyme adsorbed to the lignin and not the cellulose. In the hydrolysis of microcrystalline cellulose, the concentrations of CBH I and EG I in the supernatants decreased within 24 h, indicating these two enzymes adsorbed to microcrystalline cellulose (Fig. 3; Fig. 4 a, b). Increasing CBH I and EG I in the super-
natant after 24 h indicates that these two enzymes were desorbed from the substrate (Fig. 4a, b). In the hydrolysis of SEB, more than 90% of CBH I and EG I disappeared from the supernatant within 24 h under all conditions (Fig. 6; Table 1). Under the lysozyme added condition on loading 20 mg-protein/g-dry substrate of cellulase (Fig. 5d; Fig. 6a), the amount of CBH I in the supernatant increased further as the hydrolysis proceeded (Fig. 5c; Fig. 6a). There was the same trend under the without lysozyme condition (Fig. 5d; Fig. 6a), but the maximum desorption amount of CBH I was 1.6 times lower than that of the lysozyme added condition (Table 1).
Fig. 4. Changes in the relative amount of individual cellulase components and lysozyme during microcrystalline cellulose hydrolysis. Data were calculated by densitograph analysis from SDS-PAGE of Fig. 3(a) to (d). Open and closed symbols show the presence and absence of lysozyme, respectively. Triangle and square symbols show loadings of 8 and 20 mg-protein/g-dry substrate of cellulase, respectively.
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Fig. 5. SDS-PAGE analysis of hydrolysate supernatant during SEB hydrolysis. (a): Cellulase loading is 8 mg-protein/g-dry substrate. (b): Cellulase loading is 8 mg-protein/gdry substrate in the presence of lysozyme. (c): Cellulase loading is 20 mg-protein/g-dry substrate. (d): Cellulase loading is 20 mg-protein/g-dry substrate in the presence of lysozyme. The Marker is in Lane 1, and 0, 1, 2, 3, 4, 5, 6, 8, 10, and 14th day are analyzed from Lanes 2 to11.
In the case of EG1 on loading 20 mg-protein/g-dry substrate of cellulase with lysozyme, the maximum desorption amount was 3.9 times higher than the without lysozyme condition (Table 1; Fig. 6b). Under the lysozyme added condition on loading 8 mgprotein/g-dry of cellulase for SEB, most of the cellulase did not desorb except CBH I (Fig. 6a to d), most probably due to the lower enzyme loading. These results suggested that adding non-catalytic protein on SEB hydrolysis influences the desorption behavior of CBH I and EG I. CBH I and EG I once adsorbed on the substrate temporarily, and thereafter both enzymes desorbed to the supernatant as hydrolysis proceeded, which might be due to both enzymes not being adsorbed on the lignin but on the cellulose. The desorption amounts of CBH 1 and EG I were higher in the non-catalytic protein added condition. These results indicate that the presence of noncatalytic protein prevents re-adsorption of CBH I and EG I to the substrate. 3.2.2. Adsorption /desorption behavior of CBH II In the case of CBH II, during the hydrolysis of microcrystalline cellulose, the SDS-PAGE analysis indicated that almost all of the CBH II disappeared from the supernatants within 24 h, irrespective of whether lysozyme was absent or present in both loading amounts of cellulase (Fig. 3; Fig. 4c). The amount of CBH II in the supernatant under all conditions did not increase during hydrolysis (Fig. 4c). During hydrolysis of SEB, the results for all conditions were similar to those for microcrystalline cellulose (Fig. 5; Fig. 6c). The amount of CBH II in the supernatant did not increase until the end of hydrolysis under all conditions (Fig. 6c). Change in the concentration of CBH II in the supernatant is likely to indicate precipitation of CBH II rather than non-specific adsorption. Taneda et al. reported that CBH II is weak against the agitation stress and precip-
itates rapidly under the agitated condition (Taneda et al., 2012). Thus, even if lysozyme prevented non-specific adsorption of CBH II, CBH II is likely to precipitate rapidly, therefore resulting in no observation of CBH II in the supernatant. Other additives such as Tween80 stabilize CBH II under the agitated condition, therefore enhancing the hydrolysis of biomass (Okino et al., 2013). This result indicates that lysozyme does not stabilize CBH II, and the enhancement of enzymatic hydrolysis is solely caused by the prevention of non-specific adsorption. 3.2.3. Adsorption behavior of BGL During the hydrolysis of microcrystalline cellulose, the BGL concentration gradually decreases as the reaction progresses (Fig. 3; Fig. 4d). This tendency was the same regardless of whether it was with or without the addition of lysozyme and the loading amount of cellulase (Fig. 3; Fig. 4d). An even more significant difference was observed in SEB. On loading 20 mg-protein/g-dry substrate of cellulase under no lysozyme condition, almost all BGL disappeared from the supernatant within 24 h. Thereafter, the amount of BGL in supernatant did not increase by the end of the hydrolysis (Fig. 5c; Fig. 6d). Although on loading 20 mg-protein/g-dry substrate of cellulase under lysozyme added condition, 80.9% of BGL in the supernatant still remained after 24 h (Fig. 5d; Fig. 6d). Thereafter, the concentration of BGL in the supernatant decreased faster than in the case of microcrystalline cellulose hydrolysis, although 39.7% of BGL still remained in the supernatant by the end of hydrolysis (Fig. 5d; Fig. 6d). These results indicate that the addition of non-catalytic protein suppresses non-specific adsorption of BGL for lignin-containing substrate. It is generally known that the substrate of BGL is cellobiose, and BGL from A. niger was less bound onto surfaces of the pretreated biomasses than that from T. reesei (Haven and
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Fig. 6. Changes in the relative amounts of individual cellulase components and lysozyme during SEB hydrolysis. Data were calculated by densitograph analysis from SDSPAGE of Fig. 5(a) to (d). Open and closed symbols show the presence and absence of lysozyme, respectively. Triangle and square symbols show loadings of 8 and 20 mgprotein/g-dry substrate of cellulase.
Jorgensen, 2013; Varnai et al., 2011; Weiss et al., 2013). Visser et al. reported that BGL from Chrysoporthe cubensis and Penicillium pinophilum was potentially bound to the solid fraction (Visser et al., 2015). In the present experiment, BGL in Cellulase SS (T. reesei derived cellulase) did not adsorb to microcrystalline cellulose, but adsorbed to SEB. While lysozyme also prevents BGL from adsorbing to the lignin (Fig. 6d), the effect of this prevention contributing to the enhancement of hydrolysis is debatable (Fig. 1b).
If the BGL adsorbed to the substrate is completely inactivated, no glucose production should be observed in the without lysozyme condition. However, glucose is still produced in significant amounts under the without lysozyme condition. Moreover, Haven & Jorgensen reported that the BGL (T. reesei derived cellulase) adsorbed strongly to the residual solids but was still catalytically active (Haven and Jorgensen, 2013), indicating that BGL binding to the lignin is still active.
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Y. Toyosawa et al. / Bioresource Technology 234 (2017) 150–157 Table 1 Summary of adsorption and desorption amounts of individual cellulase components during hydrolysis of microcrystalline cellulose and SEB. Initial Adsorption amounta Cellulase 8 mg Lysozyme
a b *
*
0 mg
*
Maximum Desorption amountb Cellulase 20 mg
*
20 mg
0 mg
Microcrystalline cellulose CBH I 87.5 EG I 66.7 CBH II 86.0 BGL 20.2
89.7 82.5 82.7 7.3
50.5 62.4 84.4 18.6
SEB CBH I EG I CBH II BGL
98.8 93.6 92.5 85.3
98.0 98.8 90.3 86.6
99.0 93.7 91.4 98.3
*
*
Cellulase 8 mg* *
Cellulase 20 mg*
0 mg
20 mg
0 mg*
20 mg*
54.6 60.2 86.2 18.0
26.2 6.1 3.4 1.9
25.2 0 0.7 7.9
28.54 11.1 2.0 0.0
32.2 9.3 0.7 1.1
97.8 86.7 87.8 19.1
1.4 0.0 0.0 0.0
1.9 3.7 1.8 0.1
43.6 11.3 4.2 1.0
68.5 44.3 1.1 0.0
20 mg
*
*
The difference between the initial band intensity and the band intensity after 24 h. The difference between the maximum band intensity after the decrease in the concentration and the minimum band intensity. mg-protein/g-dry substrate.
4. Conclusion The lysozyme addition to the SEB hydrolysis had a positive effect on the glucose yield while it had no positive effect on microcrystalline cellulose hydrolysis. During the hydrolysis of two types of substrates, the quantitative SDS-PAGE analysis reveals that the lysozyme addition enhancement of enzymatic hydrolysis is solely caused by the prevention of non-specific adsorption of cellulase components, which might be caused by the masking of lignin by lysozyme. In the hydrolysis of SEB, lysozyme addition prevented re-adsorption of CBH I and EG I to the substrate, but CBH II did not stabilize. Additionally, adsorbed BGL might be catalytically active. Acknowledgements Funding for this project was provided from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. References Brethauer, S., Studer, M.H., Yang, B., Wyman, C.E., 2011. The effect of bovine serum albumin on batch and continuous enzymatic cellulose hydrolysis mixed by stirring or shaking. Bioresour. Technol. 102, 6295–6298. Florencio, C., Badino, A.C., Farinas, C.S., 2016. Soybean protein as a cost-effective lignin-blocking additive for the saccharification of sugarcane bagasse. Bioresour. Technol. 221, 172–180. Gao, D., Haarmeyer, C., Balan, V., Whitehead, T.A., Dale, B.E., Chundawat, S.P., 2014. Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification. Biotechnol. Biofuels 7, 175. Haven, M.O., Jorgensen, H., 2013. Adsorption of beta-glucosidases in two commercial preparations onto pretreated biomass and lignin. Biotechnol. Biofuels 6, 165. Herpoel-Gimbert, I., Margeot, A., Dolla, A., Jan, G., Molle, D., Lignon, S., Mathis, H., Sigoillot, J.C., Monot, F., Asther, M., 2008. Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains. Biotechnol. Biofuels 1, 18.
Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W., Foust, T.D., 2007. Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315, 804–807. Johnson, E., 2016. Integrated enzyme production lowers the cost of cellulosic ethanol. Biofuels Bioproud. Biorefin. 10, 164–174. Kumar, L., Arantes, V., Chandra, R., Saddler, J., 2012. The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility. Bioresour. Technol. 103, 201–208. Limayem, A., Ricke, S.C., 2012. Lignocellulosic biomass for bioethanol production: Current perspectives, potential issues and future prospects. Prog. Energy Combust. Sci. 38, 449–467. Nakagame, S., Chandra, R.P., Kadla, J.F., Saddler, J.N., 2011. The isolation, characterization and effect of lignin isolated from steam pretreated Douglasfir on the enzymatic hydrolysis of cellulose. Bioresour. Technol. 102, 4507– 4517. Okino, S., Ikeo, M., Ueno, Y., Taneda, D., 2013. Effects of Tween 80 on cellulase stability under agitated conditions. Bioresour. Technol. 142, 535–539. Pan, X., 2008. Role of functional groups in lignin inhibition of enzymatic hydrolysis of cellulose to glucose. J. Biobased Mater. Bioenergy 2, 25–32. Pareek, N., Gillgren, T., Jonsson, L.J., 2013. Adsorption of proteins involved in hydrolysis of lignocellulose on lignins and hemicelluloses. Bioresour. Technol. 148, 70–77. Rahikainen, J.L., Martin-Sampedro, R., Heikkinen, H., Rovio, S., Marjamaa, K., Tamminen, T., Rojas, O.J., Kruus, K., 2013. Inhibitory effect of lignin during cellulose bioconversion: the effect of lignin chemistry on non-productive enzyme adsorption. Bioresour. Technol. 133, 270–278. Taneda, D., Ueno, Y., Ikeo, M., Okino, S., 2012. Characteristics of enzyme hydrolysis of cellulose under static condition. Bioresour. Technol. 121, 154–160. Varnai, A., Viikari, L., Marjamaa, K., Siika-aho, M., 2011. Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates. Bioresour. Technol. 102, 1220–1227. Visser, E.M., Leal, T.F., de Almeida, M.N., Guimaraes, V.M., 2015. Increased enzymatic hydrolysis of sugarcane bagasse from enzyme recycling. Biotechnol. Biofuels 8, 5. Wang, H., Kobayashi, S., Mochidzuki, K., 2015. Effect of non-enzymatic proteins on enzymatic hydrolysis and simultaneous saccharification and fermentation of different lignocellulosic materials. Bioresour. Technol. 190, 373–380. Weiss, N., Borjesson, J., Pedersen, L.S., Meyer, A.S., 2013. Enzymatic lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling. Biotechnol. Biofuels 6, 5. Yang, B., Wyman, C.E., 2006. BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechnol. Bioeng. 94, 611–617.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Quantitative analysis of adsorption and desorption behavior of individual cellulase components during the hydrolysis of lignocellulosic biomass with the addition of lysozyme Yoshiko Toyosawa, Makoto Ikeo, Daisuke Taneda, Shohei Okino ⇑ JGC Corporation, 2205, Narita-cho, Oarai-machi, Higashiibaraki-gun, Ibaraki Pref. 311-1313, Japan
h i g h l i g h t s Effect of non-catalytic protein addition on enzymatic hydrolysis was investigated. Lysozyme was used as a model non-catalytic protein. Lysozyme adsorbed only to lignin containing substrate. Hydrolysis and desorption of cellulase components were enhanced by lysozyme. Lysozyme is likely to reduce non-specific adsorption of cellulase to lignin.
a r t i c l e
i n f o
Article history: Received 14 December 2016 Received in revised form 27 February 2017 Accepted 28 February 2017
Keywords: Lignocellulosic bioethanol Lysozyme Non-specific adsorption Enzyme adsorption/desorption Sugarcane bagasse
a b s t r a c t The effect of non-catalytic protein addition on the adsorption/desorption behavior of individual cellulase components on/from substrates during the hydrolysis of microcrystalline cellulose and steam exploded sugarcane bagasse (SEB) were investigated. The addition of non-catalytic protein enhanced the enzymatic hydrolysis of SEB, but did not enhance the hydrolysis of microcrystalline cellulose. During the hydrolysis of SEB, adsorption of beta-glucosidase (BGL) was prevented in the presence of non-catalytic protein. Cellobiohydrolase I (CBH I) and endoglucanase I (EG I) desorbed from the substrate after temporary adsorption in the presence of non-catalytic protein during SEB hydrolysis. This suggested that reduction of the non-specific adsorption of cellulase components, CBH I, EG I, and BGL, on lignin in SEB led to the improving of enzymatic hydrolysis. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulosic biomass has a potential to produce a clean and renewable biofuel that does not compete with food resources (Limayem and Ricke, 2012). However, its commercialization is still hampered by economic obstacles, one of them being high enzyme costs (Johnson, 2016; Limayem and Ricke, 2012). Various studies have been conducted to determine how to reduce the usage of the cellulolytic enzyme, cellulase, in the hydrolysis of biomass (Gao et al., 2014; Kumar et al., 2012; Nakagame et al., 2011; Pareek et al., 2013). Lignocellulosic biomass is mainly composed of cellulose, a linear glucose polymer, hemicellulose, a highly branched heteropolymer, and lignin, a very high molecular weight and cross-linked aromatic macromolecular (Himmel et al., 2007). ⇑ Corresponding author. E-mail address: [email protected] (S. Okino). http://dx.doi.org/10.1016/j.biortech.2017.02.132 0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
In the enzymatic hydrolysis of pretreated lignocellulosic materials, cellulases tend to adsorb to lignin-rich surfaces through what is called non-specific adsorption (Pan, 2008; Rahikainen et al., 2013). It has been proposed that non-specific adsorption of cellulase on lignin surfaces is one of the factors that decreases the effectiveness of hydrolysis (Gao et al., 2014; Nakagame et al., 2011; Pareek et al., 2013; Rahikainen et al., 2013; Yang and Wyman, 2006). One of the methods to overcome the non-specific adsorption is the use of additives, such as non-catalytic proteins (e.g. bovine serum albumin: BSA), which enhances enzymatic hydrolysis (Brethauer et al., 2011; Yang and Wyman, 2006). The effect of the addition of non-catalytic protein on the enzymatic hydrolysis of lignocellulosic substrates has been ascertained by many studies (Florencio et al., 2016; Wang et al., 2015; Yang and Wyman, 2006), and it is believed that this type of protein adsorbs to the surface of lignin and prevents the non-specific adsorption of cellulase (Brethauer et al., 2011; Yang and Wyman, 2006).
Y. Toyosawa et al. / Bioresource Technology 234 (2017) 150–157
151
In this study, the non-catalytic protein addition effect for non-specific adsorption of individual cellulase components such as CBH I, CBH II, EG I, and BGL during enzymatic hydrolysis was investigated utilizing lysozyme as the non-catalytic protein.
from Nagase Chemtex was used as cellulase. Lysozyme from Wako Pure Chemical Co. was used as the non-catalytic protein additive.
2. Materials and methods
Enzymatic hydrolysis experiments were conducted using 200 ml-baffled Erlenmeyer flasks with a final reaction volume of 50 ml and 25 ml for microcrystalline cellulose (17 days) and SEB (14 days), respectively. The commercial enzyme solutions, lysozyme, and 0.02% (w/v) NaN3 were mixed and diluted to the desired concentrations using 50 mM sodium acetate buffer (pH 5.0). The prepared solution was added to each flask containing substrates. The concentration of SEB and microcrystalline cellulose were set to 20 w/v% and 10 w/v%, respectively. Enzymatic hydrolysis was carried out at 50 °C under agitation using a rotary shaker set at
2.1. Substrates and enzymes Commercial microcrystalline cellulose (FUNACEL II, Funakoshi Co. Ltd., Japan) and steam explosion pretreated sugarcane bagasse (SEB) were used as substrate in these experiments. The experimental condition of the steam explosion pretreatment was a temperature of 230 °C and a hold time of 7 min, and the solid fraction was washed with hot water. For hydrolysis, Cellulase SS (cellulase)
2.2. Enzymatic hydrolysis conditions
Fig. 1. Changes in glucose concentrations during hydrolysis of microcrystalline cellulose and SEB. (a): Microcrystalline cellulose. (b): SEB. Open and closed symbols show glucose concentration in the presence and absence of lysozyme, respectively. Square and circle symbols show glucose concentration on cellulase loadings of 8 and 20 mgprotein/g-dry substrate, respectively.
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120 rpm. Liquid loss from evaporation of the buffer solution was prevented by tightly sealing the flasks. 2.3. Analytical methods Sample slurries were centrifuged (16,000g at 4 °C for 15 min) and filtered (Millex-LH 0.45 lm PVDF, Millipore). The glucose concentration was quantified by high-performance liquid chromatography (LC20, Shimadzu) equipped with an RI detector, and ULTRON PS-80P (Shinwa chemical industries) operating at 80 °C with ultrapure water as a mobile phase at a flow rate of 1.0 ml/min. The glucose yield of reaction was evaluated by setting the final sugar concentration yielded during hydrolysis when using excess
enzyme loading (60 mg-protein/g-dry substrate) as 100%. Protein concentrations were measured by the Bradford method using bovine gamma globulin (Bio-Rad) as a standard. The gel electrophoresis of hydrolyzates was performed on 10% and 15% polyacrylamide gels when analyzing individual components of the cellulase and lysozyme concentrations, respectively. The gels were stained with Coomassie Brilliant Blue R-250 (BioRad Laboratories). The identities of the bands in the cellulase solution, BGL (78 kDa), CBH I (63 kDa), and EG I (55 kDa) were deduced by comparing the molecular masses with those cited in a previous report (Herpoel-Gimbert et al., 2008). CBH II (58 kDa) was identified by LC-MS analysis in our previous report (Taneda et al., 2012). For quantitative analysis of the intensity of each protein
Fig. 2. Adsorption/desorption behavior of lysozyme. (a): SDS-PAGE of lysozyme in the presence of microcrystalline cellulose. The marker is in Lane 1, and 0, 1, 2, 3, 4, 6, 9, 11, and 17th day are Lanes 2 to 10, respectively. (b): SDS-PAGE of lysozyme in the presence of SEB. The marker is in Lane 1, and 0, 1, 2, 3, 4, 6, 9, 11, and 14th day are Lanes 2 to 10, respectively. (c): Changes in the relative amount of lysozyme by densitograph analysis from results of SDS-PAGE ((a) and (b)).
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band in the hydrolysis supernatants, image analysis was conducted with densitograph software (CS Analyzer ver. 2.0, ATTO Co.). Concentrations of each component were standardized so that the initial concentration of each component became 100. The initial adsorption amount was calculated by the difference between the initial band intensity and the band intensity after 24 h. The maximum desorption amount was calculated when an increase in concentration was observed in the component. The difference between the maximum band intensity after the decrease in the concentration and the minimum band intensity is defined as the maximum desorption amount.
3. Results and discussion 3.1. Effect of non-catalytic protein addition on the enzymatic hydrolysis of microcrystalline cellulose and SEB The effect of non-catalytic protein on the hydrolysis of microcrystalline cellulose and SEB was investigated (Fig. 1a, b). Lysozyme was used as a non-catalytic protein in this study. During the hydrolysis of microcrystalline cellulose, no significant differences in glucose production rate and final yield were observed between the conditions with and without lysozyme (Fig. 1a). On the other hand, the glucose concentration in SEB hydrolysis with lysozyme was higher than without lysozyme (Fig. 1b). This result indicates that lysozyme was especially effective for the
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enzymatic hydrolysis of lignin-containing substrate. Hydrolysis was not observed upon incubation of the substrate with lysozyme alone (Fig. 1a, b). Bovine serum albumin (BSA) is well known as a protein which enhances the hydrolysis of lignocellulosic biomass due to its adsorption on the surface of lignin leading to the prevention of non-specific adsorption of cellulase on lignin (Brethauer et al., 2011; Yang and Wyman, 2006). The result of enhancement of SEB hydrolysis in this study is consistent with previous studies.
3.2. Effect of non-catalytic protein addition on adsorption/desorption of individual cellulase components on/from substrates during hydrolysis To investigate the enhancement of SEB hydrolysis by means of the addition of non-catalytic protein, the adsorption/desorption behavior of CBH I, CBH II, EG 1, and BGL on/from SEB and microcrystalline cellulose was analyzed by SDS-PAGE analysis of hydrolysate supernatant (Fig. 3; Fig. 5). Bovine serum albumin (BSA) commonly used as a non-catalytic protein in cellulase hydrolysis is unsuitable for SDS-PAGE analysis due to the fact that the molecular weight is too close to CBH I, one of the most important components in cellulase. Lysozyme, which has different molecular weight (14.0 kDa) from the main components of cellulase, CBH I, CBH II, EG I, and BGL, was used as a non-catalytic protein in this study.
Fig. 3. SDS-PAGE analysis of hydrolysate supernatant during microcrystalline cellulose hydrolysis. (a): Cellulase loading is 8 mg-protein/g-dry substrate. (b): Cellulase loading is 8 mg-protein/g-dry substrate in the presence of lysozyme. (c): Cellulase loading is 20 mg-protein/g-dry substrate. (d): Cellulase loading is 20 mg-protein/g-dry substrate in the presence of lysozyme. The marker is in Lane 1, and 0, 1, 2, 3, 4, 6, 9, 11, and 17th day are analyzed from Lanes 2 to10.
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3.2.1. Adsorption/desorption behavior of non-catalytic protein, CBH1, and EG1 No adsorption of lysozyme onto the microcrystalline cellulose was observed upon incubation with lysozyme alone (Fig. 2a, c). On the other hand, when lysozyme was incubated with SEB, lysozyme disappeared from supernatants within 24 h (Fig. 2b, c). These results indicate that lysozyme adsorbed to the lignin and not the cellulose. In the hydrolysis of microcrystalline cellulose, the concentrations of CBH I and EG I in the supernatants decreased within 24 h, indicating these two enzymes adsorbed to microcrystalline cellulose (Fig. 3; Fig. 4 a, b). Increasing CBH I and EG I in the super-
natant after 24 h indicates that these two enzymes were desorbed from the substrate (Fig. 4a, b). In the hydrolysis of SEB, more than 90% of CBH I and EG I disappeared from the supernatant within 24 h under all conditions (Fig. 6; Table 1). Under the lysozyme added condition on loading 20 mg-protein/g-dry substrate of cellulase (Fig. 5d; Fig. 6a), the amount of CBH I in the supernatant increased further as the hydrolysis proceeded (Fig. 5c; Fig. 6a). There was the same trend under the without lysozyme condition (Fig. 5d; Fig. 6a), but the maximum desorption amount of CBH I was 1.6 times lower than that of the lysozyme added condition (Table 1).
Fig. 4. Changes in the relative amount of individual cellulase components and lysozyme during microcrystalline cellulose hydrolysis. Data were calculated by densitograph analysis from SDS-PAGE of Fig. 3(a) to (d). Open and closed symbols show the presence and absence of lysozyme, respectively. Triangle and square symbols show loadings of 8 and 20 mg-protein/g-dry substrate of cellulase, respectively.
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Fig. 5. SDS-PAGE analysis of hydrolysate supernatant during SEB hydrolysis. (a): Cellulase loading is 8 mg-protein/g-dry substrate. (b): Cellulase loading is 8 mg-protein/gdry substrate in the presence of lysozyme. (c): Cellulase loading is 20 mg-protein/g-dry substrate. (d): Cellulase loading is 20 mg-protein/g-dry substrate in the presence of lysozyme. The Marker is in Lane 1, and 0, 1, 2, 3, 4, 5, 6, 8, 10, and 14th day are analyzed from Lanes 2 to11.
In the case of EG1 on loading 20 mg-protein/g-dry substrate of cellulase with lysozyme, the maximum desorption amount was 3.9 times higher than the without lysozyme condition (Table 1; Fig. 6b). Under the lysozyme added condition on loading 8 mgprotein/g-dry of cellulase for SEB, most of the cellulase did not desorb except CBH I (Fig. 6a to d), most probably due to the lower enzyme loading. These results suggested that adding non-catalytic protein on SEB hydrolysis influences the desorption behavior of CBH I and EG I. CBH I and EG I once adsorbed on the substrate temporarily, and thereafter both enzymes desorbed to the supernatant as hydrolysis proceeded, which might be due to both enzymes not being adsorbed on the lignin but on the cellulose. The desorption amounts of CBH 1 and EG I were higher in the non-catalytic protein added condition. These results indicate that the presence of noncatalytic protein prevents re-adsorption of CBH I and EG I to the substrate. 3.2.2. Adsorption /desorption behavior of CBH II In the case of CBH II, during the hydrolysis of microcrystalline cellulose, the SDS-PAGE analysis indicated that almost all of the CBH II disappeared from the supernatants within 24 h, irrespective of whether lysozyme was absent or present in both loading amounts of cellulase (Fig. 3; Fig. 4c). The amount of CBH II in the supernatant under all conditions did not increase during hydrolysis (Fig. 4c). During hydrolysis of SEB, the results for all conditions were similar to those for microcrystalline cellulose (Fig. 5; Fig. 6c). The amount of CBH II in the supernatant did not increase until the end of hydrolysis under all conditions (Fig. 6c). Change in the concentration of CBH II in the supernatant is likely to indicate precipitation of CBH II rather than non-specific adsorption. Taneda et al. reported that CBH II is weak against the agitation stress and precip-
itates rapidly under the agitated condition (Taneda et al., 2012). Thus, even if lysozyme prevented non-specific adsorption of CBH II, CBH II is likely to precipitate rapidly, therefore resulting in no observation of CBH II in the supernatant. Other additives such as Tween80 stabilize CBH II under the agitated condition, therefore enhancing the hydrolysis of biomass (Okino et al., 2013). This result indicates that lysozyme does not stabilize CBH II, and the enhancement of enzymatic hydrolysis is solely caused by the prevention of non-specific adsorption. 3.2.3. Adsorption behavior of BGL During the hydrolysis of microcrystalline cellulose, the BGL concentration gradually decreases as the reaction progresses (Fig. 3; Fig. 4d). This tendency was the same regardless of whether it was with or without the addition of lysozyme and the loading amount of cellulase (Fig. 3; Fig. 4d). An even more significant difference was observed in SEB. On loading 20 mg-protein/g-dry substrate of cellulase under no lysozyme condition, almost all BGL disappeared from the supernatant within 24 h. Thereafter, the amount of BGL in supernatant did not increase by the end of the hydrolysis (Fig. 5c; Fig. 6d). Although on loading 20 mg-protein/g-dry substrate of cellulase under lysozyme added condition, 80.9% of BGL in the supernatant still remained after 24 h (Fig. 5d; Fig. 6d). Thereafter, the concentration of BGL in the supernatant decreased faster than in the case of microcrystalline cellulose hydrolysis, although 39.7% of BGL still remained in the supernatant by the end of hydrolysis (Fig. 5d; Fig. 6d). These results indicate that the addition of non-catalytic protein suppresses non-specific adsorption of BGL for lignin-containing substrate. It is generally known that the substrate of BGL is cellobiose, and BGL from A. niger was less bound onto surfaces of the pretreated biomasses than that from T. reesei (Haven and
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Fig. 6. Changes in the relative amounts of individual cellulase components and lysozyme during SEB hydrolysis. Data were calculated by densitograph analysis from SDSPAGE of Fig. 5(a) to (d). Open and closed symbols show the presence and absence of lysozyme, respectively. Triangle and square symbols show loadings of 8 and 20 mgprotein/g-dry substrate of cellulase.
Jorgensen, 2013; Varnai et al., 2011; Weiss et al., 2013). Visser et al. reported that BGL from Chrysoporthe cubensis and Penicillium pinophilum was potentially bound to the solid fraction (Visser et al., 2015). In the present experiment, BGL in Cellulase SS (T. reesei derived cellulase) did not adsorb to microcrystalline cellulose, but adsorbed to SEB. While lysozyme also prevents BGL from adsorbing to the lignin (Fig. 6d), the effect of this prevention contributing to the enhancement of hydrolysis is debatable (Fig. 1b).
If the BGL adsorbed to the substrate is completely inactivated, no glucose production should be observed in the without lysozyme condition. However, glucose is still produced in significant amounts under the without lysozyme condition. Moreover, Haven & Jorgensen reported that the BGL (T. reesei derived cellulase) adsorbed strongly to the residual solids but was still catalytically active (Haven and Jorgensen, 2013), indicating that BGL binding to the lignin is still active.
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Y. Toyosawa et al. / Bioresource Technology 234 (2017) 150–157 Table 1 Summary of adsorption and desorption amounts of individual cellulase components during hydrolysis of microcrystalline cellulose and SEB. Initial Adsorption amounta Cellulase 8 mg Lysozyme
a b *
*
0 mg
*
Maximum Desorption amountb Cellulase 20 mg
*
20 mg
0 mg
Microcrystalline cellulose CBH I 87.5 EG I 66.7 CBH II 86.0 BGL 20.2
89.7 82.5 82.7 7.3
50.5 62.4 84.4 18.6
SEB CBH I EG I CBH II BGL
98.8 93.6 92.5 85.3
98.0 98.8 90.3 86.6
99.0 93.7 91.4 98.3
*
*
Cellulase 8 mg* *
Cellulase 20 mg*
0 mg
20 mg
0 mg*
20 mg*
54.6 60.2 86.2 18.0
26.2 6.1 3.4 1.9
25.2 0 0.7 7.9
28.54 11.1 2.0 0.0
32.2 9.3 0.7 1.1
97.8 86.7 87.8 19.1
1.4 0.0 0.0 0.0
1.9 3.7 1.8 0.1
43.6 11.3 4.2 1.0
68.5 44.3 1.1 0.0
20 mg
*
*
The difference between the initial band intensity and the band intensity after 24 h. The difference between the maximum band intensity after the decrease in the concentration and the minimum band intensity. mg-protein/g-dry substrate.
4. Conclusion The lysozyme addition to the SEB hydrolysis had a positive effect on the glucose yield while it had no positive effect on microcrystalline cellulose hydrolysis. During the hydrolysis of two types of substrates, the quantitative SDS-PAGE analysis reveals that the lysozyme addition enhancement of enzymatic hydrolysis is solely caused by the prevention of non-specific adsorption of cellulase components, which might be caused by the masking of lignin by lysozyme. In the hydrolysis of SEB, lysozyme addition prevented re-adsorption of CBH I and EG I to the substrate, but CBH II did not stabilize. Additionally, adsorbed BGL might be catalytically active. Acknowledgements Funding for this project was provided from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. References Brethauer, S., Studer, M.H., Yang, B., Wyman, C.E., 2011. The effect of bovine serum albumin on batch and continuous enzymatic cellulose hydrolysis mixed by stirring or shaking. Bioresour. Technol. 102, 6295–6298. Florencio, C., Badino, A.C., Farinas, C.S., 2016. Soybean protein as a cost-effective lignin-blocking additive for the saccharification of sugarcane bagasse. Bioresour. Technol. 221, 172–180. Gao, D., Haarmeyer, C., Balan, V., Whitehead, T.A., Dale, B.E., Chundawat, S.P., 2014. Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification. Biotechnol. Biofuels 7, 175. Haven, M.O., Jorgensen, H., 2013. Adsorption of beta-glucosidases in two commercial preparations onto pretreated biomass and lignin. Biotechnol. Biofuels 6, 165. Herpoel-Gimbert, I., Margeot, A., Dolla, A., Jan, G., Molle, D., Lignon, S., Mathis, H., Sigoillot, J.C., Monot, F., Asther, M., 2008. Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains. Biotechnol. Biofuels 1, 18.
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