Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover
© Qing and Wyman; licensee BioMed Central Ltd. 2011
Received: 10 August 2010
Accepted: 24 June 2011
Published: 24 June 2011
Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses.
In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX) and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan.
This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy.
The abundance and diversity of feedstocks and the potential to prevent changes of the thermal equilibrium of the atmosphere caused mostly by carbon dioxide makes conversion of cellulosic biomass to ethanol and other fuels more advantageous than fossil fuels or first-generation corn ethanol . However, the inherent recalcitrance of cellulosic materials means that they require more severe processing than do corn or sugarcane. In addition, even for biomass pretreated under optimum conditions by leading pretreatment technologies, very high enzyme doses are still required to achieve high-yield conversion of polymeric cellulose and hemicellulose into sugar monomers that can be utilized by fermentative microorganisms . Thus, the costs of enzymes and pretreatment are the major barriers to low costs by biological processing of cellulosic biomass, and must be lowered substantially to make the cost of cellulosic ethanol competitive with that of fossil fuels or corn ethanol . Advances in current pretreatment technologies to generate more digestible substrates and modifying hydrolytic enzyme cocktails to improve enzyme efficacy could benefit from more in-depth and comprehensive understanding of the interaction between pretreated biomass and enzymes, and the synergism operating between different enzyme components in producing fermentable sugars.
Enzymatic digestion of cellulosic materials involves synergic action of a group of different functional enzymes [4, 5]. In general, endoglucanases (EC 184.108.40.206) and exoglucanases (cellobiohydrolases; CBHs) break down cellulose at the solid-liquid interface, whereas accessory enzymes such as hemicellulases, acetyl xylan esterase , α-L-arabinofuranosidase, feruloyl esterase and p-coumaroyl esterase help cleave physical shields that cover cellulose microfibrils [7, 8]. Therefore, the accessibility of the cellulose surface to cellulases and the subsequent efficacy of these enzymes have been identified as important factors that dominate cellulose hydrolysis yield [9–12]. However, the heterogeneous nature of cellulosic materials makes enzyme access to the cellulose surface very difficult, and the presence of lignin and hemicelluloses in pretreated biomass have been suggested to cause major obstacles to enzymatic digestion of cellulosic materials, by physically blocking the access of cellulase and non-productively binding with enzymes [13–17]. Our recent research suggests that xylose, and particularly soluble xylo-oligomers released from hemicelluloses during enzymatic hydrolysis, could present an additional important barrier to enzyme action by competitively inhibiting cellulase activity . Furthermore, it is difficult to totally hydrolyze these xylo-oligomers and reduce their effect within a typical hydrolysis span of 72 hours using moderate doses of commonly used and commercially available enzymes such as cellulase preparations (Spezyme CP) and β-glucosidase (Novozyme 188). Thus, there is a need to understand the mechanisms of cellulase inhibition by solid xylan and soluble xylo-oligomers and reduce their negative effects on cellulose hydrolysis.
In this study, xylanase (Multifect; Genencor) and/or β-xylosidase were added to cellulase to evaluate the synergism of hemicellulases and cellulases in hydrolyzing pure cellulose and the cellulose in pretreated biomass. For comparison, corn-stover solids were used, with two different pretreatments: 1) ammonia fiber expansion (AFEX), which allows the stover to retain almost all of the xylan and other hemicellulose, cellulose and lignin fractions, and 2) dilute acid, which results in a relatively low amount of xylan left in the solid residue. Because enzymes must adsorb to the solid substrate surface before the hydrolyzing reaction occurs, adsorption of cellulase and hemicellulase on different substrates was measured to understand the interactions of the different enzymes and the substrates. In addition, the effects of xylanase and β-xylosidase in reducing inhibition by xylo-oligomers were evaluated, and different cellulase and hemicellulase supplementation sequences were used to test our hypothesis that hydrolyzing xylans and xylo-oligomers before adding cellulase could reduce loss of enzyme activity.
Compostion of corn stover and washed solids from its pretreatment by dilute acid and AFEX technologies.
Untreated corn stover
39.1 ± 0.4
23.7 ± 0.3
19.3 ± 0.5
140°C, 1% sulfuric acid, 40 minutes
57.6 ± 0.2
5.7 ± 0.7
22.1 ± 0.4
AFEX 3 -pretreated
90°C, 220 psi, 1:1 NH3 to Biomass, 5 minutes
39.6 ± 0.5
24.5 ± 0.4
18.2 ± 0.9
Cellulase (Spezyme® CP, lot number 301-05330-205, cellulase activity 59 ± 5 filter paper units (FPU)/ml, xylanase activity 2622 OSX (oat spelt xylan)/ml, protein content 123 ± 10 mg protein/ml), xylanase (Multifect®, lot number 301-04021-015; xylanase activity 25203 OSX/ml, protein content 42 ± 5 mg protein/ml) and β-xylosidase (lot number 20050881-0882, protein content 75 ± 5 mg protein/ml) enzyme preparations were all supplied by Genencor (a Danisco Division, Rochester, NY, USA). The β-glucosidase (Novozyme 188; 18066K0676, 665 CBU/ml, protein content 140 ± 5 mg protein/ml) was purchased from Sigma-Aldrich. The β-xylosidase preparation used in this study was a non-commercial enzyme expressed in a Trichoderma strain in which the major cellulase genes were deleted, specifically CBH1, CBH2, EG1 and EG2.
Dilute acid pretreatment
The dilute acid-pretreated corn stover used in this study was pretreated at 140°C for 40 minutes with 1% by weight (wt%) sulfuric acid in a 1 litre stainless-steel batch reactor from Parr instruments (Moline, IL, USA) with a solids loading of 5 wt%, these conditions having been previously determined to maximize recovery of total glucose plus xylose . The reactor vessel was sealed and placed in a sand bath (model SBL-2D; Techne Co., Princeton, NJ, USA) set at 320°C for fast heating, and transferred to a sand bath at 140°C to maintain the target temperature. Mechanical agitation at 200 rpm by a turbine impeller was applied during heating, reaction and cooling. After 40 minutes, the reactor was quenched quickly in iced water to room temperature, and after the solution cooled down to room temperature, the solids were separated from the liquid hydrolyzate by centrifugation (model 5424; Eppendorf, Hauppauge, NY) at 18895 g for 5 minutes. The solids were collected and washed at least three times with 1 litre deionized water to neutralize and remove possible degradation products.
A xylo-oligomer rich solution was produced by pretreatment of birchwood xylan (lot number 038K0751, Sigma Chemicals) with water only at 200°C for 15 minutes at 10 wt% solids loading in the same 1 litre stainless-steel Parr reactor and using the same procedure described above. After separation of the liquid hydrolyzate from the solid residue, the liquid portion was collected for further use and stored at 50°C in an incubation shaker (Infors HT, Bottmingen, Switzerland) to prevent precipitation of high degree of polymerization (DP) xylo-oligomers . To avoid possible degradation or precipitation of xylo-oligomers, this solution was usually generated the day before use.
All monomeric sugars were quantified by high-performance liquid chromatography (HPLC) (Waters Alliance HPLC System, model 2695; Waters Corporation, Milford, MA, USA) with an Aminex column (HPX-87P; Bio-Rad Laboratories, Hercules, CA, USA) and a refractive index detector (2414; Waters). Liquid samples were neutralized when appropriate and filtered through 0.2 μm nylon filter vials (Alltech Associates Inc., Deerfield, IL, USA), transferred by pipette into 500 μl polyethylene HPLC vials (Alltech Associates Inc.), and kept refrigerated at 4°C until analyzed. A series of sugar standards with different concentrations were run together with the liquid samples, and used as the calibration basis.
To determine the structural carbohydrates and lignin in raw and pretreated corn stover, all samples were dried to constant weight by placing in a convection oven (series 0504-6593; Barnstead Lab-Line, Melrose Park, IL, USA) at temperature of less than 45°C. These constituents were measured using a two-step acid hydrolysis method to fractionate the biomass into forms that are more easily quantified. The acid-insoluble lignin including ash was quantified by gravimetric analysis. During acid hydrolysis, the polymeric carbohydrates were hydrolyzed into monomers that are soluble in the hydrolysis liquid and could be measured by HPLC. A series of sugar-recovery standards were run in parallel to correct for sugar degradation during this process .
All enzymatic hydrolysis experiments were performed according to the National Renewable Energy Laboratory analytical procedures at 2% (w/v) solids loading with 0.05 mol/l citrate buffer (pH 4.8) in a thermostatically controlled shaker at 50°C . To prevent possible microbial growth on the sugars generated, 100 μL of 2% sodium azide was added before enzymes. Spezyme CP cellulase (16.1 mg protein/g glucan), Novozyme 188 β-glucosidase (3.16 mg protein/g glucan), Multifect xylanase (16.1 mg protein/g glucan) and the non-commercial β-xylosidase (32.2 mg protein/g glucan) were added to the hydrolysis broth with different loadings and in different combinations. Hydrolysis samples taken after 4, 24, 48 and 72 hours of hydrolysis were analyzed according to the sugar-quantification method described above to follow the reaction course and determine final yields.
Protein-adsorption experiments were carried out at 4°C to prevent hydrolysis of the substrate at a concentration of 1% solids in a total volume of 1.1 ml in 1.5 ml microcentrifuge tubes (Lobind; Eppendorf) (protein loss <3%). Cellulase, β-glucosidase, xylanase and β-xylosidase were added separately to bring the final protein concentration to a range from 0 to 15 mg/ml. The samples were then rotated slowly at 4°C in a refrigerator for 4 hours to allow equilibration, followed by separation in a centrifuge (model 5415D; Eppendorf) at 4°C at a maximum speed of 2897968 g for a minimum of 10 minutes. The resulting solids were dried in a convection oven at 105°C overnight. Following a protocol developed previously, the protein adsorbed on solids was directly determined by measuring the nitrogen content of the samples using an elemental analyzer (Flash 1112 CHNOS Analyzer, CE Elantech, Lakewood, NJ, USA). For nitrogen-content analysis, the samples were weighed (~ 6 mg) into a tin capsule, which was then sealed, and were run on the analyzer along with appropriate nitrogen standards such as aspartic acid or BBOT (2,5-Bis-(5-tert.-butyl-benzoxazol-2-yl)-thiophen) . Protein adsorption could then be calculated based on a mass balance for nitrogen, with a nitrogen factor (NF) of 8.40 for Spezyme CP cellulase, 8.28 for Multifect xylanase and 3.25 for Novozyme 188 β-glucosidase [23, 24]. The NF for Genencor β-xylosidase was determined to be 7.85 ± 0.3 in this study, using the same method described by Kumar and Wyman 
where Ebound and Efree represent the amount of enzyme adsorbed to the solids (mg/g substrate) and the enzyme remaining in the solution (mg/ml), respectively; S is the substrate concentration in mg/ml; σ is the maximum adsorption capacity in mg/g substrate; and Kd (L/g) is the equilibrium constant.
To elucidate the importance of removing or totally hydrolyzing xylo-oligomers on cellulose hydrolysis, hemicellulases (xylanase and β-xylosidase) were added several hours before or after addition of cellulase. To eliminate the effects of lignin, pure Avicel cellulose was combined with mixed DP xylo-oligomers at a weight ratio of 8:5 cellulose to the equivalent amount of xylose in the xylo-oligomers, to simulate the glucan to xylan ratio for solids produced by AFEX pretreatment. One part of this mixture was incubated at 50°C with xylanase and β-xylosidase only, at a loading of 30 mg protein/g equivalent xylose (xylanase: β-xylosidase 1:1) for 2, 4, 24 and 72 hours. After incubation, cellulase was added at a loading of 10.7 mg/g glucan (about 5 FPU/g glucan) with β-glucosidase supplementation at a cellobiase units (CBU): FPU ratio of 2:1. As a comparison, xylanase and β-xylosidase were also added to another portion of samples that were initially incubated with cellulase and β-glucosidase for 2, 4 or 24 hours. Samples of cellulose and a mixture of cellulose and xylo-oligomers were run with cellulase and β-glucosidase only as controls. The time of the samples taken before cellulase addition was designated as time 0 even if they were incubated with hemicellulase.
Results and Discussion
Composition of solids after dilute acid and AFEX pretreatments
The disruption of the lignin-hemicellulose matrix enhances the susceptibility of cellulosic biomass solids to attack by enzymes. Low and neutral pH pretreatment technologies such as dilute acid for the former and liquid hot water for the latter usually remove a large fraction of the hemicellulose from biomass, but remove very little lignin. Furthermore, pretreatments at low pH hydrolyze most of the hemicellulose to monomers, whereas pretreatments at near neutral pH produce mostly oligosaccharides with some monomers. Depending upon the pretreatment severity, some degradation products are released over this range of pH conditions. By contrast, applying a high pH pretreatment, such as lime, soaking in aqueous ammonia (SAA) or ammonia recycle percolation (ARP), leaves the majority of the hemicellulose in the solids while releasing large amounts of lignin [26, 27]. An anomaly at high pH is that treating cellulosic biomass with anhydrous ammonia in the AFEX method at relatively mild temperatures (60-100°C) and high pressures (250-300 psi) for short durations, followed by rapid decompression, breaks up the recalcitrant barriers to increase the exposed surface area without much apparent change in the solids composition [28, 29]. Therefore, solids from dilute acid and AFEX pretreatments were chosen to explore how xylan-hydrolyzing enzymes could influence the enzymatic hydrolysis of cellulose in the presence of the lowest to highest possible xylan contents while retaining most of the lignin from the original corn-stover solids.
The glucan and xylan contents in corn- stover solids before pretreatment and after application of dilute acid and AFEX are summarized in Table 1. As expected, and shown in earlier studies, AFEX did not substantially change the carbohydrate or lignin contents, but still enabled high enzymatic conversion through the combined chemical and physical effects of lignin relocation, cellulose decrystallization and increased surface area [26, 27, 29]. By contrast, dilute acid pretreatment performed at 140°C with 1% sulfuric acid for 40 minutes hydrolyzed a large portion of the hemicelluloses into sugars in solution. As measured in this study, the AFEX-pretreated corn-stover solids contained 39.6% glucan and 24.5% xylan, virtually the same as in the raw corn stover, whereas the solids after dilute acid pretreatment contained 57.6% glucan and only 5.7% xylan. The lignin content for the non-pretreated feedstock and the AFEX-pretreated solids were very similar, whereas that of the dilute acid-pretreated solids was slightly higher, because removal of so much xylan overwhelmed the effect of the limited solubilization of lignin.
The effects of xylanase and β-xylosidase on enzymatic hydrolysis of cellulose and pretreated corn stover
To overcome the negative effects of residual hemicellulose on enzymatic hydrolysis of pretreated biomass [30–34], some recent research added xylanase to cellulase as an accessory activity to reduce the physical barrier of hemicelluloses . Their study also found a strong linear relationship between release of xylobiose and cellobiose, which reinforced their hypothesis that the polysaccharide network in the cell-wall matrix was strongly interconnected . The substantial inhibition of cellulase by xylo-oligomers revealed by our recent research emphasized the benefits of removing or hydrolyzing xylan and xylo-oligomers in enhancing cellulose digestibility, and prompted our addition of xylanase and β-xylosidase to cellulase to determine the synergic effects of these two classes of enzymes on hydrolyisis of different substrates 
Protein adsorption to different substrates
Maximum protein-adsorption capacity, affinity constants and correlation coefficients of different enzymes with Avicel, birchwood xylan and solids from corn stover (CS) pretreated with dilute acid and ammonia fiber expansion (AFEX).
σ, mg/g substrate1
Spezyme CP cellulase
Dilute acid CS
Novozyme 188 β-glucosidase
Dilute acid CS
Dilute acid CS
Dilute acid CS
The two hemicellulase preparations had a much stronger adsorption preference for birchwood xylan than Avicel glucan. For Multifect xylanase on birchwood xylan, the adsorption capacity was 114.1 mg/g and the affinity was 2.7 L/g, compared with just 60.8 mg/g adsorption capacity and 1.9 L/g adsorption affinity for Avicel cellulose. Similarly, β-xylosidase found stronger binding to birchwood xylan than Avicel cellulose but had even higher adsorption parameters than xylanase. In addition, β-xylosidase adsorption was stronger on AFEX-pretreated corn-stover solids than on dilute acid-pretreated corn-stover solids, possibly as a result of the higher xylan content of the AFEX processed material. Overall, both hemicellulase activities displayed greater adsorption on higher xylan content substrates whereas higher glucan content did not necessarily result in stronger cellulase binding. These observations suggest that cellulase competitively binds to xylan and thereby could provide a possible mechanism for xylan or xylo-oligomer inhibition [5, 18, 37, 38]. However, the hypothesis of protein adsorption to soluble/insoluble xylo-oligomers is still to be proved.
The effects of enzyme-supplementation sequence
Although the mechanism is still subject to debate, cellulose digestion generally improves with xylan removal by chemical or enzymatic treatment, and it is widely believed that both lignin and hemicelluloses form a physical barrier that hinders access by cellulase [33, 39]. In addition, enzymatic removal of xylan and glucomannan were shown to enlarge the pore size of pine or birch Kraft pulps . Thus, studies of this nature attribute the key benefit of xylan removal to enhancement of substrate accessibility to enzymes through exposing more crystalline cellulose surface. However, because our recent research suggested that soluble xylo-oligomers released from xylan during enzymatic hydrolysis could be powerful inhibitors of cellulase activity, removing xylan before adding cellulase could therefore improve enzyme effectiveness. The protein-adsorption data of this study, which show a stronger binding of cellulase to birchwood xylan than to Avicel cellulose, and the possibility of competitive inhibition of cellulase by xylan and xylan derivatives, are consistent with the latter reasoning.
Implications for possible mechanisms
The key findings of this research suggest competitive binding of cellulase and hemicellulase on cellulose reactive sites (Figure 1), and as a result, the efficacy of cellulase is reduced by comparable amounts of hemicellulase protein. However, if the substrate has relatively high xylan content, hemicellulases preferably bind to hemicelluloses and also hydrolyze inhibitory xylo-oligomers to enhance the overall glucose and xylose yields (Figure 2, Figure 3). In addition, Multifect xylanase and Genencor β-xylosidase have a higher binding affinity to xylan than to glucan, thereby reducing the possibility of steric hindrance of one by the other when used with substrates of high xylan content. By contrast, cellulase binds more strongly to xylan than glucan, whereas β-glucosidase binds primarily to higher lignin content substrates (Table 2). Therefore, xylan, xylo-oligomers and lignin seem to reduce cellulase availability to react with cellulose by undesirable binding with these proteins. As supported by other research, supplementation with xylanase and β-xylosdiase could reduce this negative effect by hydrolyzing xylan and its oligomers to xylose (unpublished data). IN addition, this research further indicated that adding hemicellulase before cellulase could enhance the enzymatic benefits by reducing competitive binding of cellulases to xylan and xylo-oligomers instead of to cellulose. These findings highlight the need to modify enzyme-cocktail compositions and to have rational strategies for advancing enzyme efficacy.
Adding xylanase or β-xylosidase improved the enzymatic hydrolysis of cellulose and hemicellulose in solids after AFEX and dilute acid pretreatment of corn stover. Xylan removal has been widely believed to disrupt barriers that hinder enzyme access to cellulose, but this research suggests that cellulase binding to xylan and, possibly, xylo-oligomers strongly reduces cellulase activity, and that xylan and xylo-oligomer removal or conversion to xylose can greatly reduce their inhibition. Chemically or enzymatically removing xylan and xylo-oligomers before adding cellulase seems to provide a particularly important method of enhancing cellulase effectiveness and thereby reducing the doses needed to achieve a given performance.
Major portions of this study were funded by Mascoma Corporation headquartered in Lebanon, NH, and we are grateful for their support and helpful discussions. In addition, the UCR Bourns College of Engineering through the Center for Environmental Research and Technology (CE-CERT) and the Chemical and Environmental Engineering Department provided funding and vital equipment and facilities. Genencor, a Danisco Division, generously provided the Spezyme CP cellulase, Multifect xylanase and β-xylosidase used in this research. We are grateful to Dr Bruce Dale's group at Michigan State University for providing the AFEX-pretreated corn stover. CEW is particularly grateful to the Ford Motor Company for funding the Chair in Environmental Engineering at the Center for Environmental Research and Technology of the Bourns College of Engineering at UCR, which augments support for many projects such as this.
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