Improving the Hydrolytic Action of Cellulases by Tween 80 - American

Research Article pubs.acs.org/journal/ascecg

Improving the Hydrolytic Action of Cellulases by Tween 80: Offsetting the Lost Activity of Cellobiohydrolase Cel7A Donglin Xin, Ming Yang, Xiang Chen, Ying Zhang, Li Ma, and Junhua Zhang*

ACS Sustainable Chem. Eng. 2017.5:11339-11345. Downloaded from pubs.acs.org by TULANE UNIV on 01/23/19. For personal use only.

College of Forestry, Northwest A&F University, 3 Taicheng Road, Yangling 712100, Shaanxi, China ABSTRACT: Deactivation of cellulase components has been shown to play a key role in restricting the efficient conversion of biomass to fermentable sugars and other chemicals. A potential strategy to increase the cellulases’ hydrolytic efficiency could be the development of costeffective technologies to offset the easily inactivated components in commercial cellulase preparations. In this work, a potential strategy to address this issue is reported. During the hydrolysis of Avicel and aqueous ammonia-pretreated corn stover and spruce, the deactivation of cellobiohydrolase was found to be the primary reason for the loss of total cellulase activities. Kinetic data indicated that Tween 80 was a specific activator of cellobiohydrolase, but not of endoglucanase and βglucosidase. The activation effect of Tween 80 showed a specific positive role in suppressing inhibition of cellobiohydrolase by lignin, hemicelluloses, and their derivatives and thus maintained the enzyme in high activity during the enzymatic hydrolysis process. When cellobiohydrolase was supplemented with a mixture of these inhibitors (0.5 mg mL−1 lignin, 0.5 mg mL−1 hemicelluloses, and 0.5 mmol L−1 hemicellulose oligomers), 60% of the original cellobiohydrolase activity was lost, while approximately 40% of the lost activity was restored by Tween 80. These results are expected to be significant for future research concerning the beneficial action of surfactants, improvement of cellulase activities, and recycling of enzymes during the industrial cellulose conversion process. KEYWORDS: Enzymatic hydrolysis, Cellobiohydrolases, Tween 80, Kinetic, Activator

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INTRODUCTION The shortage of fossil fuels has motivated investment in the production of alternative renewable fuels by biotechnical routes, which involves exploiting enzymatic hydrolysis of lignocellulosic biomass to fermentable sugars, followed by fermentation to bioethanol. Enzymatic hydrolysis is the key step in all conversion processes aiming at production of fermentable sugars from lignocellulosic biomass. However, enzymatic hydrolysis efficiency is limited by the complex structure of the biomass, and a large amount of enzyme is required to make the conversion efficient. Therefore, the cost of enzyme is a limiting factor in all conversion processes. In the cellulase systems of Trichoderma reesei, cellobiohydrolases (Cel6A and Cel7A) regularly comprise approximately 75% of the total protein and are considered key enzymes due to their significant roles in the conversion of insoluble cellulose to soluble sugars.1 However, in previous studies, it was found that the activity of cellobiohydrolases was low because they were susceptible to inhibition by other compounds in the hydrolysates. Aside from their end products cellobiose and glucose, other compounds such as xylan, xylan oligomers (XOS), mannan, mannan oligomers (MOS), and phenolic compounds were all found to be inhibitors of cellobiohydrolases, especially Cel7A.2−8 We believe that the inhibition of cellobiohydrolases by those compounds could be part of the key reason for the low hydrolytic efficiency of cellulases. However, little attention has © 2017 American Chemical Society

been focused on offsetting the lost cellobiohydrolase activity and thus improving the hydrolytic action of cellulases. There is considerable evidence indicating the beneficial role of surfactants, such as Tween or polyethylene glycol, in the hydrolysis of pretreated biomass materials.9−12 Explanations of how these additives boosted the hydrolysis efficiency mainly focused on the following aspects: (1) The surfactant could increase positive interactions between substrates and enzymes by interacting with the hydrophobic lignin and forming a coating on the lignin surface, and thus reducing nonproductive adsorption of cellulases on lignin.13,14 (2) The surfactant could increase the thermal stability of the enzyme and prevent thermal deactivation of enzymes during the hydrolysis.15,16 However, these explanations could not consistently explain how surfactants improve enzymatic digestion, such as, although the amount of lignin in Avicel is negligible, addition of surfactants could significantly increase its hydrolysis yield;11 although enzymes are thermostable, surfactants could also enhance their hydrolytic action.17 It is thus necessary to develop a mechanism that can make up these gaps. To our best knowledge, evidence of whether the surfactants could offset the lost cellobiohydrolase activity is currently lacking; this may be another Received: July 14, 2017 Revised: September 29, 2017 Published: October 19, 2017 11339

DOI: 10.1021/acssuschemeng.7b02361 ACS Sustainable Chem. Eng. 2017, 5, 11339−11345

Research Article

ACS Sustainable Chemistry & Engineering

Figure 1. Activities of cellulase during the hydrolysis of Avicel, spruce, and corn stover. Filter paper activity (FPA) (A), Cel5A activity (B), Cel7A activity (C), and Cel3A activity (D) in the hydrolysates of Avicel, spruce, and corn stover by cellulases (10 FPU g−1 DM Celluclast 1.5 L and 500 nkat g−1 DM Novozyme 188) and Tween 80 (2.5 mg mL−1). Incubation of CEL in the absence of material and Tween 80 was performed as a control. The error bars represent the standard errors of three experiments. ANOVA was performed to compare the statistical significance of the observed differences in cellulase activity before and after addition of Tween 80. The p value is indicated in the text at the appropriate place. The pretreatment conditions were as follows: the corn stover was pretreated by 21% (W/V) aqueous ammonia with a solid to liquid ratio of 1:10 at 50 °C for 12 h and the spruce was pretreated by 21% (W/V) aqueous ammonia with a solid to liquid ratio of 1:10 at 70 °C for 72 h. The pretreated materials were washed to neutral with pure water and then stored at −20 °C for further use. The chemical compositions of the pretreated materials were determined by the National Renewable Energy Laboratory Analytical Procedure.20 The contents of cellulose, xylan, and lignin in the pretreated corn stover were 58.0%, 21.8%, and 6.2%, respectively. The contents of cellulose, xylan, mannan, and lignin in the pretreated spruce were 46.8%, 2.7%, 13.9%, and 18.7%, respectively. Enzymes. Glycosyl hydrolase (GH) 5 family endoglucanase Ta Cel5A, GH 7 family cellobiohydrolase Ta Cel7A, and GH 3 family βglucosidase At Cel3A21 were produced in a genetically modified Trichoderma reesei strain where the genes coding for the major cellulases Tr Cel7A, Tr Cel6A, Tr Cel7B, and Tr Cel5A had been deleted.22−24 The enzyme preparations were adjusted to pH 6.0 and treated at 60 °C for 2 h to inactivate the background T. reesei enzymes. The commercial enzyme preparations Celluclast 1.5L and Novozyme 188 (Novozymes A/S, Bagsværd, Denmark) were used as the reference cellulases (CEL). Celluclast 1.5L had an activity of 74.7 FPU mL−1 (169.6 mg protein mL−1) measured according to the IUPAC standard assay.25 The βG activity of Novozyme 188 was determined to be 5121 nkat mL−1 (187.9 mg protein mL−1) using the method described by Bailey and Nevalainen.26 The protein contents of these enzymes were quantified by the Lowry method27 using bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) as a standard. Enzymatic Hydrolysis. The hydrolysis of Avicel, aqueous ammonia-pretreated corn stover, and spruce by the cellulase preparations was performed in 50 mmol L−1 sodium citrate buffer (pH 5.0) in tubes with a working volume of 3 mL at 50 °C. The dry matter (DM) content of the substrate was 10%. 0.02% NaN3 was added to the hydrolysis broth to prevent bacterial contamination.28

potential mechanism for the positive effect of surfactant on biomass hydrolysis. To fill this gap, we investigated the effect of the surfactant Tween 80 on changes in cellulase activities (including total filter paper activity (FPA), endoglucanase (Cel5A) activity, Cel7A activity, and β-glucosidase (Cel3A) activity) during biomass hydrolysis. The negative effects of xylan, mannan, XOS, MOS, and lignin on Cel7A activity and the role of Tween 80 in offsetting the lost activity of Cel7A were investigated. In addition, the kinetics of the promotion of Cel7A by Tween 80 was analyzed to elucidate the mechanisms involved.

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EXPERIMENTAL SECTION

Materials. Microcrystalline cellulose (Avicel PH-101), Beechwood xylan, hydroxyethyl cellulose (HEC), p-nitrophenyl-β-D-glucoside (pNPG), and p-nitrophenol-D-cellobioside (pNPC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Carob galactomannan (mannose to galactose ratio: 3.76:1, Lot10501b), xylobiose (X2), and mannobiose (M2) were purchased from Megazyme (Bray, Wicklow, Ireland). Enzymatic hydrolysis lignin was purchased from Shandong Nongli Biological Technology Co., Ltd. (Shandong, China). The purity of the lignin was >95%. Corn stover was collected from a local farm in Yangling, China. Spruce was purchased from Shanghai Tingzhong Industrial Co., Ltd. (Shanghai, China). These materials were milled, sieved through a 60 mesh screen scale and pretreated by aqueous ammonia, because this pretreatment approach was found to be a promising method for improving enzymatic saccharification of lignocellulosic biomass and showed strong ability to remove lignin but maintain most hemicelluloses.18,19 The purpose of this work is to evaluate the effects of Tween 80 on cellulase activity in the presence of inhibitors (hemicelluloses, oligosaccharides, and lignin) and it is necessary to maintain some hemicelluloses in the pretreated materials. 11340


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ACS Sustainable Chemistry & Engineering The CEL was dosed at 10 FPU/g DM Celluclast 1.5 L and 500 nkat g−1 DM Novozyme 188. The hydrolysis experiments were performed in a shaking incubator (200 rpm) in triplicate. Tween 80 (2.5 mg mL−1) was added into the reaction system at the beginning of the enzymatic hydrolysis. Samples were withdrawn and centrifuged at 10 000 g for 10 min, and the supernatants were analyzed for glucose and cellulase activities. Activity of Cellulase in the Hydrolysates. The activities of Ta Cel7A and Ta Cel5A were determined using pNPC and HEC as substrate, respectively, as previously described.25,29 When measuring the activities of cellulase (including FPA, Cel5A activity, Cel7A activity, and Cel3A activity) in the hydrolysates, sugars were removed using Amicon Ultra centrifuge tubes (Millipore) by centrifuging at 16 280g, 4 °C for 15 min. The remaining supernatant was brought up to its original volume with citrate buffer. Kinetic Analysis on Ta Cel5A, Ta Cel7A, and At Cel3A. Kinetic of analysis of Ta Cel5A (HEC as substrate), Ta Cel7A (pNPC as substrate), and At Cel3A (pNPG as substrate) was performed, and the corresponding kinetic parameters were determined as previously described.2 Carbohydrate Analysis. The concentration of glucose in the supernatants was determined using an HPLC system as previously described.4 The glucose yield in the hydrolysis of biomass materials was calculated according to NREL LAP-009.30 The degree of promoting was evaluated using eq 1 below:

degree of promoting =

V0 − Vi × 100% Vi

The nonproductive adsorption of cellulases onto lignin and the inhibition of products in the supernatant on cellulase activities could be the major reason for this phenomenon.35,36 After 48 h of hydrolysis of ammonia-pretreated corn stover, the activities of Cel5A, Cel7A, and Cel3A clearly decreased by 38.3%, 45.5%, and 22.1%, respectively. A similar phenomenon was also observed in the hydrolysis of ammonia-pretreated spruce and Avicel. Thus, it could be concluded that Cel7A was the most easily inactivated constituent and bore primary responsibility for the loss of total cellulase activities among the investigated pure enzymes, followed by Cel5A and Cel3A. After supplementation with Tween 80, the activities of cellulase in the supernatant dramatically increased (Figure 1A). As reported previously, Tween 80 shows the ability to prevent the denaturation of cellulases and reduce the nonproductive adsorption of cellulases onto material,13,15,37 which can partly explain the increases in cellulase activities in the hydrolysis supernatant of Avicel, corn stover, and spruce. However, somewhat surprisingly, the activities of cellulase were also enhanced in the absence of substrate. The results indicated that, aside from reducing the nonproductive adsorption of cellulases onto material, Tween 80 had significant (P < 0.05) positive effect on cellulase activities. The phenomenon could be mainly attributed to the fact that Tween 80 could prevent the thermal deactivation of enzyme15,16 and may also have an activating effect on cellulase activity. It is worth noting that the beneficial effect on cellulase activities mainly focused on the promotion of Cel7A activity (P < 0.05), but not Cel5A and Cel3A activity (Figure 1B−D). Mechanism Behind the Specific Positive Effect of Tween 80 on Cel7A. Kinetic experiments were performed to investigate the possible mechanism behind the specific beneficial effect of Tween 80 on Cel7A, but not Cel5A and Cel3A (Figure 2). In order to rule out the effect of Tween 80 on preventing the thermal deactivation of enzymes, three thermostable cellulases (Thermoascus aurantiacus cellobiohydrolase (Ta Cel7A), T. aurantiacus endoglucanase (Ta Cel5A), and Acremonium thermophilum β-glucosidase (At Cel3A)), identified as promising enzymes in their categories (cellobiohydrolase, endoglucanase, and β-glucosidase), were cloned and produced in Trichoderma reesei and used as pure enzyme preparations in this work.1,38 In our previous results, activities of the three pure enzyme preparations (including endoglucanase activity, cellobiohydrolase activity, β-glucosidase activity, and xylanase activity) were measured and the results indicated that the target proteins were the main components in these enzyme preparations and most background cellulases were inactivated.39 Therefore, they are adequately suitable for subsequent experiments as pure enzymes. The results of the kinetic experiments were presented as Lineweaver−Burk plots of 1 V−1 versus 1 S−1. The results show that Tween 80 was an activator of Ta Cel7A based on the intersection of the lines on the Y-axis (Figure 2A) and the reduction in Km values with increasing concentrations of Tween 80 (Table 1). However, Lineweaver−Burk plots of Ta Cel5A and At Cel3A activities remained almost unchanged, showing that Tween 80 had no activation effect on Ta Cel5A and At Cel3A (Figure 2B and C). Therefore, the specific activation effect on Cel7A activity could be another potential reason for the promotion of cellulase activities by Tween 80 in the hydrolysis supernatants of Avicel, corn stover, and spruce (Figure 1).

(1)

Where, Vi is the glucose yield without the addition of Tween 80 and V0 is the glucose yield with the addition of Tween 80. Statistical Analysis. Analysis of variance (ANOVA) was performed at 95% confidence level to compare group means of experimental data in triplicate using Microsoft Excel 2007 and Graphpad Prism. Differences with P values of 0.05 or less were considered significant.

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RESULTS Behavior of Cellulase Activity in the Hydrolysis of Biomass. It is known that the activities of cellulase decrease with increasing reaction time.31,32 The decrease in cellulase activities directly results in a low efficiency of biomass degradation. Commercial cellulases, such as cellulases from T. reesei, are composed of endoglucanases, cellobiohydrolases, and β-glucosidase. The loss of total cellulase activity could be caused by the deactivation of these individual cellulase components. However, which one of these components is most prone to deactivation and should take major responsibility for the decrease in total cellulase activity is currently unclear. Therefore, in this work, we investigated the changes in cellulase activities (including the total FPA activity and the activities of Cel5A, Cel7A, and Cel3A) in the hydrolysis of Avicel, aqueous ammonia-pretreated corn stover, and spruce. The incubation of cellulases in the absence of substrate was performed as a control. In addition, the effect of Tween 80, a common nonionic surfactant that was found to play an efficient role in enhancing the hydrolysis of biomass in our previous results,33 on the change in cellulase activities was also investigated. In the absence of biomass materials, the total FPA activity of cellulase decreased by 30.2% after 48 h of incubation (Figure 1A). This loss of activity could result from the thermal deactivation of Cel5A (by 10.1%), Cel7A (by 6.3%), and Cel3A (by 4.2%) activity (Figure 1B−D) because the commercial cellulase preparations were found to be not thermostable at 50 °C.11,34 When there is a substrate, the total FPA activities of cellulase in the supernatant decreased by 35.9% (Avicel), 52.8% (corn stover), and 58.4% (spruce), respectively (Figure 1A). 11341


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Figure 3. Activity of Ta Cel7A in the presence of Tween 80 and/or inhibitors. Effects of X2 (0.5 mmol L−1), M2 (0.5 mmol L−1), and Tween 80 (1 mg mL−1) on Ta Cel7A activity at different substrate concentrations (A). Effect of xylan (0.5 mg mL−1), mannan (0.5 mg mL−1), and Tween 80 (1 mg mL−1) on Ta Cel7A activity (B). Effect of lignin (0.5 mg mL−1), a mixture of the inhibitors (0.5 mg mL−1 mannan, 0.5 mg mL−1 xylan, 0.5 mg mL−1 lignin, 0.5 mmol L−1 M2, and 0.5 mmol L−1 X2) and Tween 80 (1 mg mL−1) on Ta Cel7A activity (C). All determinations were performed in 50 mmol L−1 sodium citrate buffer (pH 5.0) at 50 °C using p-nitrophenol-Dcellobioside as substrate. The error bars represent the standard errors of three experiments.

Figure 2. Lineweaver−Burk plots of Ta Cel7A (A), Ta Cel5A (B), and At Cel3A (C) activity with the addition of Tween 80. The determinations were performed in 50 mmol L−1 sodium citrate buffer (pH 5.0) at 50 °C using p-nitrophenol-D-cellobioside (A), hydroxyl ethyl cellulose (B), and p-nitrophenyl-β-D-glucopyranoside (C) as the substrate. The error bars represent the standard errors of three experiments. a Table 1. Effect of Tween 80 on the Kapp m values of Ta Cel7A

activators

concentrations (mg mL−1)

−1 Km or Kapp m (mmol L )

none Tween 80

0 0.05 0.5 1.0

0.81 0.69 0.65 0.44

lignin all reduced Ta Cel7A activity to some extent, and under the investigated conditions, xylan showed the strongest inhibitory effect on Ta Cel7A activity, followed by lignin, mannan, M2, and X2. It seems that polysaccharides showed a stronger inhibitory effect on Cel7A activity as compared with oligosaccharides, which could be partly attributed to the binding of enzyme on these polysaccharides. When supplemented with a mixture of the inhibitors, the activity of Ta Cel7A clearly decreased by approximately 60%. After the addition of Tween 80, approximately 40% of the inactivated enzyme could be recovered (Figure 3C). However, it seems that Tween 80 exhibited a specific recovery effect on the Ta Cel7A activity that was inactivated by lignin, mannan and hemicelluloses oligomers, but not by xylan. It was observed that xylan dramatically decreased the activity of Ta Cel7A from 4.8 to 2.6 nkat mL−1, and further addition of Tween 80 slightly increased the activity to 2.82 nkat mL−1, far below the control. In the conversion of stover and spruce to glucose, a greater

a

The value of Km was obtained for the reaction without additives. The determinations were carried out at 50 °C and pH 5.0 using pnitrophenol-D-cellobioside as the substrate.

The composition of a hydrolysis supernatant of biomass is complex. As the hydrolysis reaction proceeds, more and more products will accumulate in the supernatant. Among these products, many have been found to be inhibitors of Cel7A.2,3,5,7,8 When these inhibitors are mixed with Tween 80, it is unclear which shows a stronger impact on Cel7A activity. Therefore, we further investigated the behavior of Ta Cel7A in the presence of Tween 80 and the inhibitors (including xylan, mannan, xylan oligomers, mannan oligomers and lignin) (Figure 3). As expected, xylan, mannan, X2, M2, and 11342


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addition of BSA prior to cellulase could also enhance the hydrolysis yield of cellulose in dilute acid and ammonia fiber explosion pretreated corn stover by 11.4% and 7.7%. In addition, Kumar and Wyman43 reported that BSA, Tween 20, and PEG 6000 could increase digestibility of Avicel and corn stover after pretreatment with ammonia fiber expansion, ammonia recycled percolation, dilute acid, lime, controlled pH, and sulfur dioxide. They speculated that these additives may accelerate cellobiohydrolase action and/or reduce inhibition of cellulase by oligomers, our results here confirmed the positive effect of Tween 80 on Cel7A activity and supported their speculation. Hsieh et al.44 previously reported a specific positive effect of PEGs on Cel7A’s hydrolytic action from the view of increased water availability due to PEGs. The Tween 80-induced specific boosting of Cel7A activity presented here clearly revealed another potential explanation of surfactants on biomass hydrolysis. During the hydrolysis of lignocellulosic biomass, both the accumulated soluble fraction and enzymatic residual fraction (main composed of lignin) were found to be inhibitors of cellulases.45 Luckily, for all inhibitors (except xylan) tested in this work, addition of Tween 80 suppressed their inhibition effect on Cel7A (Figure 3). When the inhibitors (including xylan, mannan, xylan oligomers, and mannan oligomers) were incubated with Cel7A and Tween 80, the effect of nonproductive adsorption of enzyme on substrate and thermal deactivation of the enzyme did not exist because the substrates used in this experiment were soluble and the Cel7A was thermostable below 60 °C as shown in our previous results.17 Under this condition, the positive effect of Tween 80 on Ta Cel7A activity could be possible by suppressing the enzyme deactivation caused by these inhibitors. However, it should be noted that the positive effect of Tween 80 varied with the inhibitors. In our previous results, we found that all the soluble inhibitors tested were competitive inhibitors of Ta Cel7A except for xylan.2,3,5,46 The results in Figure 3 showed that Tween 80 could completely suppress the inhibition caused by these competitive inhibitors, but not by xylan. Based on these results, we suggest that Tween 80 and these inhibitors may competitively bind with Cel7A, thus reducing the binding of these inhibitors into the active site of Cel7A, preventing the deactivation of enzyme caused by these competitive inhibitors, and therefore suppressing the competitive inhibition effect on the enzyme. The detailed mechanism describing the process should be further investigated. When insoluble lignin was added, enzyme would nonproductively adsorb onto the surface of it as previously reported,47,48 thus resulting in a decrease in Cel7A activity (Figure 3C). After addition of Tween 80, the lost activity was completely recovered, indicating that, under the investigated condition, Tween 80 could efficiently eliminate the inhibition of Cel7A caused by nonproductive adsorption on lignin. Previously published results also proved the positive role of Tween 80 in recovering the enzyme that bound onto the enzymatic residual fraction.49 Based on above results, we concluded that Tween 80 could significantly recover the lost activity of Cel7A that was not only caused by competitive inhibition but also by the accumulated enzymatic residual fraction. Aside from enzyme activity, other factors, such as enzyme accessibility, have vital roles in efficient conversion of lignocellulosic biomass to fermentable sugars. Jeoh et al.50 reported that conversion yield of cellulose showed a close

promoting effect of Tween 80 on spruce was observed (Figure 4). Previously, this phenomenon was mainly explained by the

Figure 4. Effect of Tween 80 on spruce and corn stover hydrolysis. Hydrolysis of spruce and corn stover (100 mg mL−1) by Celluclast 1.5 L (10 FPU g−1 DM) and Novozyme 188 (500 nkat g−1 DM) in the presence of Tween 80 (2.5 mg mL−1) at 50 °C and pH 5.0 for 2, 24, 48 h (A). Degree of promotion of Tween 80 on spruce and corn stover hydrolysis (B). The error bars represent the standard errors of three experiments.

stronger role of Tween 80 in reducing the nonproductive adsorption of cellulases on lignin in softwood.40 In previous results, bovine serum albumin (BSA) was also believed to mainly reduce the unproductive adsorption of enzyme to lignin and thus it could significantly enhance the hydrolysis yield of pretreated corn stover, but not pure cellulose (Avicel).41 However, aside from lignin, hemicellulose was another limiting factor in the efficient cellulose conversion by cellulases. Our results here indicated that, aside from reducing the nonproductive adsorption of cellulases on lignin, the specific effect of Tween 80 on recovery of the Cel7A inactivated by mannan (the major hemicellulose in softwood), but not xylan (the major hemicellulose in hardwood and gramineae biomasses), could be another potential mechanism behind the varying degree of promotion of Tween 80 on the hydrolysis of softwood and gramineae biomasses.

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DISCUSSION In the conversion of ammonia-pretreated corn stover and spruce to glucose, addition of Tween 80 clearly increased the conversion yield by approximately 10% and 25%, respectively (Figure 4). These results were consistent with those of Gupta et al.42 in which Tween 60 enhanced the reducing sugar yield of dilute sulfuric acid pretreated Prosopis juliflora by approximately 20%. Aside from Tween, Yang and Wyman41 found that 11343


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(4) Xin, D.; Yang, M.; Chen, X.; Zhang, J. The access of Trichoderma reesei 6A to cellulose is blocked by isolated hemicelluloses and their derivatives in biomass hydrolysis. RSC Adv. 2016, 6, 73859−73868. (5) Zhang, J.; Viikari, L. Xylo-oligosaccharides are competitive inhibitors of cellobiohydrolase I from Thermoascus aurantiacus. Bioresour. Technol. 2012, 117, 286−291. (6) Rajan, K.; Carrier, D. J. Insights into exo-cellulase inhibition by the hot water hydrolyzates of rice straw. ACS Sustainable Chem. Eng. 2016, 4, 3627−3633. (7) Kumar, R.; Wyman, C. Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies. Biotechnol. Bioeng. 2009, 102, 457−467. (8) Ximenes, E.; Kim, Y.; Mosier, N.; Dien, B.; Ladisch, M. Inhibition of cellulases by phenols. Enzyme Microb. Technol. 2010, 46, 170−176. (9) Jin, W.; Chen, L.; Hu, M.; Sun, D.; Li, A.; Li, Y.; Hu, Z.; Zhou, S.; Tu, Y.; Xia, T.; et al. Tween-80 is effective for enhancing steamexploded biomass enzymatic saccharification and ethanol production by specifically lessening cellulase absorption with lignin in common reed. Appl. Energy 2016, 175, 82−90. (10) Mesquita, J. F.; Ferraz, A.; Aguiar, A. Alkaline-sulfite pretreatment and use of surfactants during enzymatic hydrolysis to enhance ethanol production from sugarcane bagasse. Bioprocess Biosyst. Eng. 2016, 39, 441−448. (11) Ouyang, J.; Dong, Z.; Song, X.; Lee, X.; Chen, M.; Yong, Q. Improved enzymatic hydrolysis of microcrystalline cellulose (Avicel PH101) by polyethylene glycol addition. Bioresour. Technol. 2010, 101, 6685−6691. (12) Liu, H.; Sun, J.; Leu, S. Y.; Chen, S. Toward a fundamental understanding of cellulase-lignin interactions in the whole slurry enzymatic saccharification process. Biofuels, Bioprod. Biorefin. 2016, 10, 648−663. (13) Eriksson, T.; Börjesson, J.; Tjerneld, F. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme Microb. Technol. 2002, 31, 353−364. (14) Malmsten, M.; Van Alstine, J. M. Adsorption of poly (ethylene glycol) amphiphiles to form coatings which inhibit protein adsorption. J. Colloid Interface Sci. 1996, 177, 502−512. (15) Kaar, W. E.; Holtzapple, M. T. Benefits from Tween during enzymic hydrolysis of corn stover. Biotechnol. Bioeng. 1998, 59, 419− 427. (16) Kim, M.; Lee, S.; Ryu, D. D.; Reese, E. Surface deactivation of cellulase and its prevention. Enzyme Microb. Technol. 1982, 4, 99−103. (17) Xin, D.; Yang, M.; Zhang, J. Mechanism of improving hydrolytic capacity of cellobiohydrolase I by PEG 6000 and BSA. J. Forest. Eng. 2016, 1, 88−95 (in Chinese). (18) Alvira, P.; Tomás-Pejó, E.; Ballesteros, M.; Negro, M. J. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review. Bioresour. Technol. 2010, 101, 4851−4861. (19) Jia, L.; Sun, Z.; Ge, X.; Xin, D.; Zhang, J. Comparison of the delignifiability and hydrolysability of wheat straw and corn stover in aqueous ammonia pretreatment. BioResources 2013, 8, 4505−4517. (20) Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D. Determination of structural carbohydrates and lignin in biomass; Lab. Anal. Proced. 1617; National Renewable Energy Laboratory: Golden, CO, 2011. (21) Lombard, V.; Ramulu, H. G.; Drula, E.; Coutinho, P. M.; Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 2014, 42, D490−D495. (22) Suominen, P. L.; Mäntylä, A. L.; Karhunen, T.; Hakola, S.; Nevalainen, H. High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes. Mol. Gen. Genet. 1993, 241, 523−530. (23) Leskinen, S.; Mäntylä, A.; Fagerström, R.; Vehmaanperä, J.; Lantto, R.; Paloheimo, M.; Suominen, P. Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea f lexuosa: isolation of the genes and characterization of recombinant Xyn11A

relationship with the concentrations of Cel7A bound to the substrate, indicating the potential of enzyme accessibility for affecting cellulose digestibility. The positive effect of pretreatment on subsequent hydrolysis of biomass was also largely contributed to the increase of cellulase accessibility to cellulose.51,52 Cellulase binding to cellulose is a prerequisite of cellulose hydrolysis. Without binding, there is no hydrolysis. However, efficient binding of cellulase requires high active enzymes. The results presented reveal that Tween 80 could significantly suppress the deactivation of Cel7A induced by mannan, hemicellulose oligomers, and lignin and maintain its high activity during enzymatic hydrolysis. The findings of this work not only offer new insights into the relationship between surfactant and cellulase but pave an efficient way for recycling of highly active cellulase during sustainable bioethanolproducing processes.

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CONCLUSIONS

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AUTHOR INFORMATION

Here, we investigated the behaviors of cellulase activities, including Cel5A, Cel7A, and Cel3A activity, during the hydrolysis of corn stover and spruce and the positive effect of Tween 80 on these activities. It was found that the activation effect of Tween 80 significantly suppressed the inhibition of the accumulated soluble fraction (especially competitive inhibitors of Cel7A in soluble fractions) and enzymatic residual fraction on Cel7A activity. The results in this work offer a new perspective toward understanding the positive effect of Tween 80 on cellulose hydrolysis and provide a novel concept of avoiding the deactivation of cellobiohydrolase in the cellulasecatalyzed hydrolysis of lignocellulosic biomass. In addition, activators of Cel6A, endoglucanases, and β-glucosidase should be further investigated to recover inactivated enzymes, optimize the cellulase cocktail and improve the conversion efficiency.

Corresponding Author

*E-mail: [email protected]. ORCID

Donglin Xin: 0000-0003-0983-6595 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Prof. Liisa Viikari (University of Helsinki, Finland) and Roal Oy (Rajamäki, Finland) for providing Ta Cel5A, Ta Cel7A, and At Cel3A. This work was supported by Natural Science Foundation of China (31670598 and 31270622) and the Science Foundation for Distinguished Young Scholars of Northwest A&F University (2452015098).

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REFERENCES

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Improving the Hydrolytic Action of Cellulases by Tween 80 - American

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