Strong cellulase inhibitors from the hydrothermal pretreatment of wheat straw
© Kont et al.; licensee BioMed Central Ltd. 2013
Received: 3 June 2013
Accepted: 13 September 2013
Published: 21 September 2013
The use of the enzymatic hydrolysis of lignocellulose with subsequent fermentation to ethanol provides a green alternative for the production of transportation fuels. Because of its recalcitrant nature, the lignocellulosic biomass must be pretreated before enzymatic hydrolysis. However, the pretreatment often results in the formation of compounds that are inhibitory for the enzymes or fermenting organism. Although well recognized, little quantitative information on the inhibition of individual cellulase components by identified inhibitors is available.
Strong cellulase inhibitors were separated from the liquid fraction of the hydrothermal pretreatment of wheat straw. HPLC and mass-spectroscopy analyses confirmed that the inhibitors were oligosaccharides (inhibitory oligosaccharides, IOS) with a degree of polymerization from 7 to 16. The IOS are composed of a mixture of xylo- (XOS) and gluco-oligosaccharides (GOS). We propose that XOS and GOS are the fragments of the xylan backbone and mixed-linkage β-glucans, respectively. The IOS were approximately 100 times stronger inhibitors for Trichoderma reesei cellobiohydrolases (CBHs) than cellobiose, which is one of the strongest inhibitors of these enzymes reported to date. Inhibition of endoglucanases (EGs) by IOS was weaker than that of CBHs. Most of the tested cellulases and hemicellulases were able to slowly degrade IOS and reduce the inhibitory power of the liquid fraction to some extent. The most efficient single enzyme component here was T. reesei EG Tr Cel7B. Although reduced by the enzyme treatment, the residual inhibitory power of IOS and the liquid fraction was strong enough to silence the major component of the T. reesei cellulase system, CBH Tr Cel7A.
The cellulase inhibitors described here may be responsible for the poor yields from the enzymatic conversion of the whole slurries from lignocellulose pretreatment under conditions that do not favor complete degradation of hemicellulose. Identification of the inhibitory compounds helps to design better enzyme mixtures for their degradation and to optimize the pretreatment regimes to minimize their formation.
KeywordsCellulase Cellulose Lignocellulose Hydrothermal pretreatment Hemicellulose Xylooligosaccharides Inhibition Cellobiohydrolase Bioethanol Trichoderma reesei
Lignocellulose is the most abundant biopolymer on the Earth and has a significant potential as a renewable energy source. Therefore, the use of cellulosic biomass for the production of ethanol that can replace oil-based transportation fuels is currently being researched intensively . The complex structure of lignocellulose consists of three primary components: cellulose, hemicellulose, and lignin [2, 3]. The main component of plant cell walls, cellulose, consists of linear β-1,4-glucan chains that adhere to each other, forming crystalline higher-order fibrous structures. Hemicellulose includes a number of polysaccharides that vary in sugar composition, types of linkages, branching, and substitutions. Different plants, such as woody plants and grasses, have different hemicellulose compositions, and therefore, different classifications of hemicelluloses have been used . Hemicelluloses in cereals are often divided into four groups: (i) xylans, (ii) mannans, (iii) xyloglucans, and (iv) mixed-linkage β-glucans . Xylan, the main hemicellulose in hardwoods and annual plants, consists of a linear backbone of β-1,4-linked xylopyranose (Xyl) residues. The latter are often substituted at its 2-O and/or 3-O with arabinose (Ara), glucuronic acid, and acetic acid . Glucomannan, the most abundant hemicellulose in softwoods, consists of a β-1,4-linked mannose and glucose backbone that is substituted with α-galactose. The backbone of xyloglucan consists of β-1,4-linked glucose residues, over half of which are substituted with α-linked Xyl residues. Mixed-linkage β-glucans consist of β-1,3-linked segments of β-1,4-linked glucose residues and are characteristic of the Poales, including cereals. Glucose residues in mixed-linkage β-glucans are not substituted [5, 7]. In plant cell walls, cellulose elementary fibrils are associated with hemicellulose, forming a complex network of polysaccharides, which is in turn embedded in the matrix of lignin .
The most efficient lignocellulose degraders in nature are fungi. They secrete a number of enzymes involved in cellulose, hemicellulose, and lignin breakdown. These enzymes are collectively referred to as the lignocellulolytic system . The best-characterized cellulolytic system is that of the soft rot fungus Trichoderma reesei. The most abundant cellulase of T. reesei is cellobiohydrolase (CBH), Tr Cel7A, which constitutes approximately 60% of the secreted enzymes. Tr Cel7A is also a major component of many commercial cellulase preparations. Another CBH, Tr Cel6A, constitutes approximately 20% of the enzymes secreted by T. reesei. Beside two CBHs, T.reesei also secretes a number of endoglucanases (EGs), including Tr Cel7B, Tr Cel5A, and Tr Cel12A, and enzymes involved in hemicellulose degradation. The main product of cellulose hydrolysis is cellobiose, which is also a strong inhibitor for CBHs. Therefore, the cellulolytic systems also contain β-glucosidase, an enzyme that hydrolyses cellobiose into two molecules of glucose.
Owing to its function in plant cell walls, lignocellulose has evolved into a structure that makes it recalcitrant toward chemical and enzymatic breakdown . Therefore, a physicochemical pretreatment of biomass is necessary before enzymatic hydrolysis [10, 11]. Pretreatment opens up the plant cell wall structure and improves the access of enzymes to cellulose. In the lignocellulose-to-ethanol process, the pretreated biomass is subjected to enzymatic hydrolysis, followed by fermentation of the resulting soluble sugars to ethanol. Depending on the conditions used, the pretreatments can be broadly divided into alkali, acid, organosolv, and hydrothermal pretreatments. Alkali and organosolv pretreatments are effective in removing lignin, whereas the hemicellulose is not degraded. Acid and hydrothermal pretreatments result in alteration of the structure of lignin and its relocation. Depending on the severity of the pretreatment (pH, temperature, and residence time), acid and hydrothermal pretreatments result in the partial or complete hydrolysis of hemicellulose . Because there is no addition of chemicals, hydrothermal pretreatments provide a green route for the pretreatment of biomass and are employed in many operational lignocellulose-to-ethanol pilot units around the world [12–14]. During hydrothermal pretreatment, most of the hemicellulose is solubilized through the fragmentation to oligosaccharides and ends up in the liquid fraction (LF) [11, 15]. Although it has an altered structure, most of the lignin remains associated with cellulose and stays in the solid fraction [11, 16]. Various low-molecular-weight degradation products of hemicellulose and lignin that have been shown to be inhibitory for yeast fermentation also concentrate in the LF . Therefore, the LF is usually separated before the solid fraction is added to the hydrolysis and fermentation tanks. The separated LF can be used in different ways, e.g., in the Inbicon process, the oligosaccharide-rich LF is used for the production of animal feed. However, to maximize ethanol yields from biomass, there is a strong interest in using whole slurries from pretreatment rather than separated solid fractions. This has led to an intensive search for inhibitor-tolerant microorganisms and to the engineering of microorganisms to have a better tolerance for biomass-derived inhibitors [17–19]. Besides inhibitors for fermentation, the pretreatment can also result in the formation of compounds that are inhibitory for the enzymatic hydrolysis of pretreated biomass [20–25]. However, quantitative studies of the inhibition of cellulases by biomass-derived isolated inhibitors are scarce . Previously, we developed a 14C-labeled cellulose-based method to characterize the product inhibition of cellulases . In this study, we employ these methods to characterize strong cellulase inhibitors from the LF of the hydrothermal pretreatment of wheat straw. The inhibitors were oligosaccharides, and they were approximately 100 times stronger inhibitors for T. reesei cellulases than cellobiose, one of the most potent cellulase inhibitors described to date.
Results and discussion
CBH Tr Cel7A is strongly inhibited by the liquid fraction from the hydrothermal pretreatment of wheat straw
Composition of wheat straw and solid fraction from the pretreatment a
Raw wheat straw
Pretreated wheat straw
Composition (g l -1 ) of the liquid fraction from the pretreatment of wheat straw a
Free monomeric sugars
Sugars in oligosaccharidesb
Identification of the inhibitors from the liquid fraction
Inhibition of cellulases by IOS
Inhibition of cellulases on 14 C-cellulose substrates by IOS and cellobiose
0.0082 ± 0.0018c
0.68 ± 0.24
0.0126 ± 0.0026d
0.076 ± 0.034c
16 ± 0.5
0.093 ± 0.033d
168 ± 2
For more details of the calculation of IC50 values, see . As in the case of the MUL substrate, the IOS inhibition of the hydrolysis of 14C-BC by Tr Cel7A was approximately 100 times stronger than the cellobiose inhibition (Table 3). IOS inhibition of another T. reesei CBH, Tr Cel6A, was also assessed on the 14C-BC substrate (Figure 7C). Here, the inhibition by cellobiose released during cellulose hydrolysis was not significant, and the term [CB]/IC50(CB) was omitted from Equation 3 in the analysis of the data. Although Tr Cel6A was more resistant to IOS inhibition than Tr Cel7A, the IOS were also approximately 100 times stronger inhibitors than cellobiose for Tr Cel6A (Table 3). For both Tr Cel7A and Tr Cel6A, the inhibitory strength of IOS appeared somewhat weaker if the total concentration of IOS was used in analyses rather than the [IOS]free (Table 3). Inhibition of EGs, Tr Cel7B, Tr Cel5A, and Tr Cel12A was assessed on 14C-amorphous cellulose (Figure 7D). For the time courses of the hydrolysis of 14C-celluoses in the presence and absence of IOS, see Additional file 1: Figure S1. The inhibition of EGs was much weaker than that of CBHs. The availability of IOS limited the highest concentration of IOS used, and this did not permit the calculation of IC50 values for EGs. For Tr Cel7B, one can estimate, using long extrapolation, an apparent IC50 value in the sub-millimolar range (Table 3). For Tr Cel5A and Tr Cel12A, it was not possible to say whether the enzymes were inhibited or not (Additional file 1: Figure S1). Similarly to cellobiose inhibition of these EGs [26, 28], the most sensitive to IOS inhibition appeared to be Tr Cel7B.
Inhibition of cellulose hydrolysis by polymeric xylans and by XOS is well known. For the mechanistic interpretation, at least two scenarios have been proposed: (i) by binding to the cellulose surface, xylans restrict the accessibility of cellulose to cellulases, and (ii) by binding to the active sites of cellulases, xylans compete with the binding of the cellulose chain. The first scenario is a plausible way to explain the mechanism of inhibition by polymeric xylans. Clear correlations between the cellulose digestibility and the amount of residual xylan on cellulose or between the degree of conversion of cellulose and xylan have been reported [20, 42–46]. The second scenario has been primarily used to explain the inhibition of cellulases by XOS [21, 22, 29, 30, 47]. Because IOS were able to bind to cellulose (Figure 7A), the contribution of cellulose-bound IOS in inhibition cannot be excluded. The binding affinity of XOS to Tr Cel7A has been shown to increase with increasing DP of XOS . The strongest binding to Tr Cel7A, with a Kd value of 3.4 μM, reported to date is for the binding of a mixture XOS ((Xyl)8/(Xyl)9/(Xyl)10 in a 1/1/1 ratio) . Because the active site of Tr Cel7A contains 10 glucose unit binding sites, the stronger binding of IOS observed here may be due to the higher DP of IOS. The mechanistic interpretation of the strong inhibitory power of IOS may be that, by mimicking the structure of the cellulose chain, XOS and GOS span the active site tunnel of Tr Cel7A. By doing so, they can use the cumulative binding energy of all 10 glucose unit binding sites, whereas the binding of cellobiose relies primarily on interactions with the product binding sites (+1/+2) (Figure 5).
Enzymatic degradation of IOS
Residual inhibitory power of IOS and the LF after enzymatic treatment
Residual inhibitory powera(%)
3.0 ± 0.8
45 ± 10
3.8 ± 0.3
16 ± 2
9.9 ± 1.6
85 ± 4
13.1 ± 2.1
43 ± 1.4
15.6 ± 2.2
55 ± 9
24.9 ± 3.6
49 ± 8
26.0 ± 2.4
49 ± 9
6.9 ± 0.6
2.8 mg/ml (1.8 FPU/ml)
3.1 ± 0.3
96.8 ± 0.1
29.0 ± 9.4
45.0 ± 4.2
Reducing the inhibitory power of the entire LF against Tr Cel7A by enzymatic treatment was also studied. As in the case of IOS, the most efficient individual enzyme component here was Tr Cel7B (Table 4). In contrast, whereas Ta Xyn10A was efficient in reducing the inhibitory power of IOS, the enzyme was rather inefficient in doing so with the LF. With all enzymes tested, the efficiency of reducing the inhibitory power of the LF was worse than that of IOS. This apparently reflects the more complex nature of inhibitory compounds in the LF. In addition to IOS, the LF may contain other inhibitors of Tr Cel7A that cannot be degraded by the enzymes. It may also be that the LF contains inhibitors for the enzymes used for its treatment so that the degradation of IOS in the LF is hampered. Two cellulase mixtures, Celluclast/Novozymes®188 and the mixture of cellulases referred to as Thermomix  that was developed during the EU FP7 funded project HYPE, were also used for the treatment of the LF. Both mixtures were better than any individual enzyme components, but Thermomix outperformed the conventional Celluclast/Novozymes®188 mixture in reducing the inhibitory strength of the LF against Tr Cel7A (Table 4). It must be noted, however, that although the inhibitory power of the LF was greatly reduced by enzyme treatment, the remaining inhibitory power was still strong enough to silence Tr Cel7A. Recall that whereas treatment of the LF reduces its inhibitory power by a factor of approximately 100, the 10,000-fold diluted LF halved the activity of Tr Cel7A on MUL (Figure 1B). Thus, the strong inhibition of cellulases by IOS reported here may be responsible for the poor enzymatic conversion of the whole slurries from the hydrothermal pretreatment of lignocellulose compared with that of the separated solid fractions [23, 56–58]. However, poor conversion of whole slurries has also been observed for lignocelluloses pretreated using an acid catalyst, conditions that favor the degradation of hemicellulose [58–61]. Apparently, the oligosaccharides are not the sole determinants of the poor conversion of whole slurries, and the subject requires more study. The washing of solids after pretreatment increases the water consumption and is not economically feasible. Furthermore, a portion of the LF that is entrapped in the pores in pretreated solids will be transferred to the hydrolysis tank even after the washing of solids . Although there may be other inhibitors beside IOS, our results suggest that the optimization of enzyme mixtures for better alleviation of the inhibition by IOS or pretreatment regimes that minimize the production of IOS may lead to better economics for the lignocellulose-to-ethanol process.
Here, we separated and identified strong cellulase inhibitors from the liquid fraction of the hydrothermal pretreatment of wheat straw. The inhibitors were confirmed to be oligosaccharides (IOS) with a DP ranging from 7 to 16. The IOS were composed of a mixture of XOS and GOS. We propose that XOS and GOS are fragments of the xylan backbone and mixed-linkage β-glucans, respectively. The IOS were approximately 100 times stronger inhibitors for T. reesei CBHs than cellobiose. The mechanistic interpretation of the strong inhibitory power of IOS may be that, by mimicking the structure of the cellulose chain, XOS and GOS bind to the active site of CBHs through all glucose unit binding sites. Most of the tested cellulases and hemicellulases were able to slowly degrade IOS and reduce the inhibitory power of IOS and the liquid fraction to some extent. Although reduced by the enzyme treatment, the residual inhibitory power of IOS and the liquid fraction was strong enough to silence the major component of the T. reesei cellulase system, CBH Tr Cel7A.
The LF was kindly provided by Jan Larsen from Inbicon (Fredericia, Denmark). Glucose, MUL, MUG, pNPL, Novozyme®188, Celluclast®, and BSA were purchased from Sigma-Aldrich. The lichenase was from Megazyme (Bray, Ireland). Cellobiose (≥ 99%) was from Fluka. D-[U-14C] glucose with a specific activity of 262 mCi mmol-1 was from Hartmann Analytic GmbH. The scintillation cocktail was from Merck.
14C-BC was prepared by laboratory fermentation of the Gluconobacter xylinum strain ATCC 53582 in the presence of a [U-14C] glucose carbon source [63, 64]. 14C-BC had a specific activity of 450,000 DPM mg-1. 14C-amorphous cellulose was prepared from 14C-bacterial microcrystalline cellulose by dissolution and regeneration from phosphoric acid . The total concentration of cellulose was determined by the anthrone-sulfuric acid method.
Tr Cel7A, Tr Cel6A, Tr Cel7B, Tr Cel5A, and Tr Cel12A were purified from the culture filtrate of T. reesei QM 9414 as described previously [65–68]. N188 BG was purified from Novozyme®188 according to . The culture filtrate containing Ta Xyn10A heterologously expressed in the T. reesei strain lacking the genes of four major cellulases was kindly provided by Terhi Puranen from Roal Oy (Rajamäki, Finland). For purification of Ta Xyn10A, the above culture filtrate was heat treated in 50 mM sodium phosphate buffer with a pH of 6.0 for 2 h at 60°C to sediment the background T. reesei enzymes . Thermomix was also kindly provided by Terhi Puranen from Roal Oy (Rajamäki, Finland). The purified Tr XG (Tr Cel74A) and Tr AXE were gifts from Matti Siika-aho from VTT (Espoo, Finland). The lichenase (Megazyme) was used as purchased.
Separation and purification of IOS from the LF
Before its application to the SEC column (Toyopearl HW40-F), the LF was centrifuged (10,000 × g) and pressed through a 0.2 μm PVDF filter. SEC was performed using the ÄKTA Explorer chromatography system (GE Healthcare) at 4°C. The column was equilibrated and eluted with water at a flow rate of 0.5 ml min-1. The fractions (2.5 ml) were analyzed for the concentration of reducing groups using the modified BCA method [33, 63] and for the inhibitory strength against Tr Cel7A on MUL. The fractions from SEC were also analyzed by HPLC. HPLC was performed using a Prominex HPLC system (Shimadzu) equipped with an Aminex HPX-87P (BioRad, 5 μm, 250 mm × 7.8 mm) column and a refractive index detector RID-10A (Shimadzu). The column temperature was kept at 80°C, the flow rate was 0.6 ml min-1, and the eluent was water. The fractions from HPLC (0.3 ml) were also collected and analyzed for the reducing groups and for the inhibitory strength against Tr Cel7A on MUL. Selected fractions from SEC were pooled, concentrated under reduced pressure, and purified on HPLC using the above-described conditions. HPLC fractions with retention times between 8–10 min were pooled, concentrated under reduced pressure, and stored at −18°C before use. This HPLC purified material is referred to as IOS throughout the study.
Characterization of IOS
Determination of the monosaccharide composition of IOS was performed essentially as described in . IOS were autoclaved in 4% sulfuric acid (1 atm, 121°C) for 3 × 20 min. Autoclaved samples were neutralized to pH 5 – 6 by the addition of CaCO3. Precipitate was separated by centrifugation, and aliquots of supernatant were analyzed by HPLC. Monosaccharide standards were treated similarly to account for the sugar recovery . The recovery of monosaccharide standards was above 90%.
Offline ESI-MS measurements were performed on an LTQ-Orbitrap classic mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a nanoelectrospray ion source (Proxeon, Odense, Denmark) using Proxeon medium nanospray needles. A 5 μl sample of IOS (100 μM) in 10 mM ammonium acetate (pH 5) was introduced into the LTQ Orbitrap mass spectrometer operating at a 180°C capillary temperature, a 105.0 V tube lens voltage, and a 1.0 kV needle voltage. Spectra (10 scans) were acquired in positive ion mode in profile (m/z 500 – 2000) with a resolution of 100000 FWHM.
For deacetylation, IOS were incubated in 50 mM NaOH at 4°C overnight. The pH was adjusted to 6.0 by adding 0.5 M acetic acid. Deacetylated and neutralized IOS were purified by HPLC (see purification of IOS) before ESI-MS analysis.
Inhibition of Tr Cel7A and Tr Cel7B on MUL
All experiments were performed in 1.5 ml microcentrifuge tubes in 50 mM sodium acetate (containing BSA, 0.1 g l-1) at pH 5 and 35°C. The concentration of MUL was 5 μM and 20 μM in the case of experiments with Tr Cel7A and Tr Cel7B, respectively, and that of the inhibitor was varied as appropriate. Reactions were initiated by the addition of the enzyme to a final concentration of 10 nM and stopped by the addition of 1.0 M ammonium hydroxide (10% of the total volume). The released MU was quantified by the fluorescence using a Hitachi F-4500 fluorimeter with excitation and emission wavelengths set to 360 nm and 450 nm, respectively. The hydrolysis time was selected so that all rates of MU liberation correspond to the initial rates.
Binding of IOS to cellulose
IOS (0–100 μM on a reducing groups basis) were incubated with 14C-cellulose in 50 mM sodium acetate buffer (containing BSA, 0.1 g l-1) at pH 5 and 35°C for 30 min. The concentration of 14C-BC and 14C-amorphous cellulose was 0.25 g l-1 and 0.5 g l-1, respectively. Cellulose was separated by centrifugation, and supernatants were analyzed for their inhibitory strength against Tr Cel7A on MUL, as described above. The concentration of IOS in the supernatant was calculated from the inhibitory strength and the IC50 value of 0.31 μM for IOS inhibition of Tr Cel7A using Equation 2. The concentration of IOS bound to cellulose was found as the difference between the total concentration of IOS and that in the supernatant.
Inhibition of cellulases on 14C-cellulose
All experiments were performed in 50 mM sodium acetate buffer, pH 5.0, containing BSA (0.1 g l-1) at 35°C. Inhibition of Tr Cel7A was assessed by following the synergistic hydrolysis of 14C-BC. For that, 14C-BC (0.25 g l-1) was pre-incubated (without stirring) with IOS at selected concentrations at 35°C for 30 min. Hydrolysis was initiated by the addition of the mixture of Tr Cel7A and Tr Cel5A to the final concentrations of 0.25 μM and 0.025 μM, respectively. In the case of experiments with no added IOS, the reaction mixtures were supplied with N188 BG (0.06 μM). At selected times, 0.2 ml aliquots were withdrawn and added to 20 μl 1 M NaOH to stop the reaction. Residual cellulose was separated by centrifugation (2 min, 104 × g), and radioactivity in the supernatant was quantified using a liquid scintillation counter. The degree of cellulose degradation was found from the ratio of radioactivity in the supernatant to the total radioactivity in the hydrolysis mixture. In the case of the inhibition of Tr Cel6A, the same procedure and conditions were followed, except that Tr Cel5A was omitted.
IOS inhibition of EGs was assessed on 14C-amorphous cellulose. 14C-amorphous cellulose (0.5 g l-1) was pre-incubated (with shaking at 700 rpm) with IOS at selected concentrations at 35°C for 30 min. Hydrolysis was initiated by the addition of EG to a final concentration of 2.5 nM, 5.0 nM, and 50 nM for Tr Cel7B, Tr Cel5A, and Tr Cel12A, respectively. The rest of the procedure was identical to that described above for CBHs.
Treatment of IOS and LF with enzymes
All of the experiments were performed in 50 mM sodium acetate buffer, pH 5.0, containing BSA (0.1 g l-1) at 35°C. IOS (100 μM on a reducing groups basis) were treated with different enzymes for 2 h. Reactions were stopped by heating at 100°C for 20 min. Heat-inactivated enzymes were pelleted by centrifugation (3 min, 104 × g), and aliquots of supernatants were used to quantify the residual inhibitory power against Tr Cel7A on MUL (see inhibition of Tr Cel7A on MUL). Enzymes treated identically but without the presence of IOS were used for background measurements in the determination of the activity of Tr Cel7A on MUL. In the case of the treatment of the LF, the remaining solids in the LF were separated by centrifugation (10,000 × g) and filtration through a 0.2 μm PVDF filter. The concentration of the LF in the enzymatic treatment was as provided (Table 2). To maintain its original concentration, the volume of the LF was reduced using a vacuum concentrator followed by the addition of enzymes to restore the original volume of the LF. The following enzymes were used in the treatment of IOS and/or the LF: Tr Cel7A (21 μM), Tr Cel6A (7 μM), Tr Cel7B (3.5 μM), Tr Cel5A (3.5 μM), Tr Cel12A (3.5 μM), N188 BG (1.75 μM), Ta Xyn10A (1.75 μM), Tr AXE (0.1 μM), Tr XG (0.1 μM), and lichenase (0.1 μM). Cellulase mixtures were loaded on an activity (FPU/CBU, for Celluclast/Novozyme®188) or on a mg protein (for Thermomix) basis. In the case of the treatment of IOS with N188 BG and Ta Xyn10A, a series with varying enzyme concentrations (between 1.0 nM and 1.75 μM) was also made.
In the case of the initial assessment of the inhibitory power of the LF (data in Figure 1), the LF (with the pH adjusted to pH 5 by the addition of 0.5 M sodium acetate buffer) was treated with 0.1 μM N188 BG for 48 h before the inhibition studies. For the acid treatment, the LF was incubated with 2% sulfuric acid at 121°C for 20 min, followed by neutralization with NaOH before inhibition studies on 14C-BC (Figure 1A).
Acetyl-xylan esterase, BSA, Bovine serum albumin
14C-labeled bacterial cellulose
Degree of conversion in the presence of IOS
Degree of conversion in the absence of IOS
Dry matter, DP, Degree of polymerization
Electrospray ionization mass spectroscopy
Liquid fraction from hydrothermal pretreatment of wheat straw
β-glucosidase purified from Novozyme®188, pNPL, para-nitrophenole-β-lactoside
Size exclusion chromatography
Xylanase from Thermoascus aurantiacus
This work was funded by the EU Commission (FP7/2007-2013, grant agreement no. 213139) and the Estonian Science Foundation (grant no 9227). Jan Larsen from Inbicon (Fredericia, Denmark) is acknowledged for the LF. We are grateful to Terhi Puranen from Roal Oy (Rajamäki, Finland) for the Ta Xyn10A and Thermomix and Matti Siika-Aho from VTT (Espoo, Finland) for Tr XG and Tr AXE. Lauri Peil from the University of Edinburgh (UK) and Triin Tammsalu and Liisa Arike from the University of Tartu are acknowledged for the help with ESI-MS. Silja Kuusk from the University of Tartu is acknowledged for critical reading.
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