Improving the Hydrolytic Action of Cellulases by Tween 80: Offsetting

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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., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02361 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 25, 2017

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ACS Sustainable Chemistry & Engineering

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, Junhua Zhang*

College of Forestry, Northwest A&F University, 3 Taicheng Road, Yangling 712100, Shaanxi, China.

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Corresponding author: Email: [email protected].

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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 cost-effective 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 β-glucosidase. 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 active during 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 high 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 been focused on offsetting the lost cellobiohydrolases activity and thus improving the hydrolytic action of cellulases. There is considerable evidence indicating a beneficial role of surfactants, such as Tween or polyethylene glycol, in the hydrolysis of pretreated biomass materials 9-12. Explanations how these additives boosted the hydrolysis efficiency mainly focused on

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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 lignin surface, and thus reducing non-productive 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, whether the surfactants could offset the lost cellobiohydrolases activity is currently lacking, which may be another 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 behind the promoting.

EXPERIMENTAL SECTION Materials Microcrystalline cellulose (Avicel PH-101), Beechwood xylan, hydroxyethyl cellulose (HEC), p-nitrophenyl-β-D-glucoside (pNPG), and p-nitrophenol-D-cellobioside 4


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(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. 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 ºC 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 ºC for 72 h. The pretreated materials were washed to neutral with pure water and then stored at -20 ºC 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 5



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cellobiohydrolase Ta Cel7A, and GH 3 family β-glucosidase 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 method 27 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. The CEL was dosed at 10 FPU/g DM Celluclast 1.5 L and 500 nkat/g 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. 6


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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 centrifugating at 16280 g, 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 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 equation 1 below: Degree of promoting =

× 100%

(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 97 and Graphpad 7



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Prism. Differences with P values of 0.05 or less were considered significant. Results Behavior of cellulases 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 non-ionic 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). The non-productive adsorption of cellulases onto

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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 non-productive 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 non-productive adsorption of cellulases onto material, Tween 80 had significant (P

Improving the Hydrolytic Action of Cellulases by Tween 80: Offsetting

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