Article pubs.acs.org/JAFC
Cellulase Inhibition by High Concentrations of Monosaccharides Chia-wen C. Hsieh,* David Cannella, Henning Jørgensen,‡ Claus Felby, and Lisbeth G. Thygesen Department of Geosciences and Natural Resource Management, Faculty of Science, University of Copenhagen, Rolighedsvej 23, DK-1958 Frederiksberg C, Denmark ABSTRACT: Biological degradation of biomass on an industrial scale culminates in high concentrations of end products. It is known that the accumulation of glucose and cellobiose, end products of hydrolysis, inhibit cellulases and decrease glucose yields. Aside from these end products, however, other monosaccharides such as mannose and galactose (stereoisomers of glucose) decrease glucose yields as well. NMR relaxometry measurements showed direct correlations between the initial T2 of the liquid phase in which hydrolysis takes place and the total glucose production during cellulose hydrolysis, indicating that low free water availability contributes to cellulase inhibition. Of the hydrolytic enzymes involved, those acting on the cellulose substrate, that is, exo- and endoglucanases, were the most inhibited. The β-glucosidases were shown to be less sensitive to high monosaccharide concentrations except glucose. Protein adsorption studies showed that this inhibition effect was most likely due to catalytic, and not binding, inhibition of the cellulases. KEYWORDS: water availability, enzymatic hydrolysis, T2 relaxation, cellulase adsorption, cellulase inhibition
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INTRODUCTION Biological conversion of biomass to ethanol requires highperforming enzymes to break down the biomass insoluble polymers to soluble molecules.1 The main substrate, cellulose, degrades slowly in nature, and many efforts have been made to understand the cellulase mode of action to optimize its efficiency.2,3 Despite the fact that cellulases hydrolyze an insoluble substrate and can degrade cellulose at very low water content,4 both laboratory- and industrial-scale reactions have shown that the hydrolysis yield of the enzyme decreases as the dry matter content increases.5,6 This apparent substrate inhibition7,8 is now also known as the “high solids effect”.9 The advantage of performing hydrolysis at high dry matter content is that the final product concentration is higher, which, from a commercial point of view, decreases costs as there is less water to remove during the downstream product separation process.10,11 Thus, it is of particular interest to investigate the reasons behind the decrease in enzyme performance at high dry matter content. Many factors have been proposed to explain the high solids effect9 besides the well-known product inhibition.12 One factor that has been suggested is mass transfer limitations,13,14 the idea being that high amounts of soluble molecules in the hydrolysate increase its viscosity and thereby affect the way the cellulases diffuse to reaction sites within the substrate.14 More recently, Selig et al.15 proposed that the decreasing yields during hydrolysis could also be related to the decreased availability of water brought on by significant increases in the concentration of soluble molecules at high solids loadings. In light of this study, it is possible that the decreasing yields are due to properties not only of the substrate (biomass, cellulose) but also of the liquid fraction (water, buffer) in which the hydrolysis takes place. We propose that water-related factors could also affect enzymatic hydrolysis in addition to substrate and enzyme-related factors. Water is involved in many life processes and as such contributes to the proper function of proteins.16 As water is a reactant and the medium in which cellulose © 2014 American Chemical Society
hydrolysis takes place, it is an important but often overlooked factor in optimization of the catalytic rate. Factors that affect the liquid phase during saccarification include pH, presence of dissolved salts and minerals, dissolved organic molecules, and surfactants.17 Here we explore the impacts of individual monosaccharides (glucose, galactose, mannose, xylose, and fructose) at high concentrations on the hydrolysis yield with a commercial cellulase cocktail (Novozymes Cellic CTec2). It was previously shown by Xiao et al.12 that monosaccharides other than glucose (mannose, galactose, xylose) were strong inhibitors of cellulases, but the mechanism behind the cellulase inhibition was not fully understood. In this paper, we aim to correlate these findings with new data and potentially attribute this behavior to the lower free water availability caused by the presence of monosaccharides during hydrolysis. Low-field nuclear magnetic resonance (LF-NMR) has been used as a way to observe differences in the hydration behavior by measuring the proton spin−spin relaxation times (T2) of different soluble molecules in solution. Mora-Gutierrez and Baianu18 stressed the importance of the stereochemistry of the solute in determining the extent of hydration. In other words, the hydration sphere is unique to all small molecules in solution as they form different interactions (H-bonding and van der Waals) with water. When the concentration of solutes in water increases, the molecular mobility of “free” water decreases as its interaction with the solute becomes stronger. Thus, as more “bound” water is present, the amount of free water decreases. In this study, different concentrations of sugars in solution were used to change the free water availability to observe the influence of this change on the performance of cellulases. It is hypothesized that Received: Revised: Accepted: Published: 3800
January 7, 2014 April 10, 2014 April 14, 2014 April 14, 2014 dx.doi.org/10.1021/jf5012962 | J. Agric. Food Chem. 2014, 62, 3800−3805
Journal of Agricultural and Food Chemistry
Article
Enzymatic Hydrolysis of Avicel and Cellobiose with Novozymes Cellic CTec2. Avicel hydrolysis samples were prepared as described above (5% water insoluble solids, 13 mg/g enzyme loading) in 50 mM acetate buffer at pH 4.8 and additional 50, 270, 540, and 810 mM glucose or galactose. At the end of 96 h, 200 μL of hydrolysate was removed from the sample and 200 μL of cellobiose (125 g/L in 50 mM acetate buffer) was introduced. The hydrolysate aliquots were centrifuged, filtered, and frozen as described above, whereas the original samples with freshly added cellobiose were incubated for 1 h further before termination of the hydrolysis and processed as described above, followed by HPLC quantification. HPLC Sugar Quantification. D-Cellobiose, D-glucose, D-mannose, D-fructose, D-galactose, and D-xylose (standards obtained from SigmaAldrich, USA) were quantified with UltiMate 3000 HPLC (Dionex, Germany) equipped with a refractive index detector (Shodex, Japan). The separation was performed in a Phenomenex Resex ROA column at 80 °C with 5 mM H2SO4 as eluent at a flow rate of 0.6 mL/min. As mannose and xylose, plus fructose and galactose, have the same retention time of 11 min, with this column, their concentrations were separately quantified using their respective calibration standard curves. The results were analyzed using Chromeleon software from Dionex. LF-NMR T2 Relaxation. NMR measurements were performed on a Bruker mq20 minispec with a 0.47 T permanent magnet (equivalent to 20 MHz proton resonance frequency). The internal magnet temperature was 40 °C. T2 values of hydrolysis solutions (before Avicel and cellulase addition) were measured using the CPMG pulse sequence. Each sample was run with 32 scans and a 5 s recycle delay containing 8000 points and a pulse separation of 1.2 ms between each scan. The obtained relaxation curves were analyzed using the inverse Laplace transformation method CONTIN,22 where all solutions gave a single T2 relaxation peak. The T2 reported are thus the peak maxima positions of this peak. Protein Adsorption Measurements. Fifty milligrams of Avicel was weighed in 2 mL screw-cap tubes and filled with diluted Novozymes Cellic CTec2 (10 FPU) for a total water-insoluble solids content of 5% w/w. The liquid fraction contained 50, 270, 540, or 810 mM glucose, galactose, mannose, or xylose. The samples were prepared in triplicates and incubated at 50 °C for 30 min to allow the protein to bind to the substrate. Avicel blanks without enzyme were run in parallel. To determine the free protein content in solution, diluted enzyme solutions without Avicel were also run in parallel. After incubation, the samples were centrifuged at 850g and 5 °C for 2 min, from which 400 μL of the supernatant was sampled. Two microliters of this supernatant was then analyzed for protein content on a Thermo Scientific NanoDrop 2000 spectrophotometer (Wilmington, DE, USA) at 280 nm UV wavelength absorption.
just like the high solids effect, higher concentrations of dissolved solids lead to a decrease in the product yield. We used a pure microcrystalline cellulose substrate, Avicel, for the hydrolysis experiments to rule out other potential substraterelated inhibiting factors (lignin, hemicellulose) and focus on the liquid phase. The hydrolysis was furthermore run at 5% water insoluble solids to observe the impact of soluble compounds and not of a high solids content. The findings generated from this study remind us that enzymatic hydrolysis remains a biological process; enzyme performance is very sensitive to small changes in the external environment, which have to be accounted for during process design. This study is particularly relevant in industrial settings where hydrolysis is to be performed at high dry matter contents as soluble product accumulation is high in the liquid phase during hydrolysis11 under these circumstances.
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MATERIALS AND METHODS
Enzymatic Hydrolysis of Avicel with Novozymes Cellic CTec2. Fifty milligrams of Avicel was weighed in 2 mL screw-cap tubes and filled with diluted enzyme solution for a total waterinsoluble solids loading of 5% w/w. The commercial cellulolytic enzyme mixture Cellic CTec2 from Novozymes A/S, Bagsværd, Denmark, was used in all studies. The mixture had a protein content of 161 mg/g, a filter paper activity of 120 FPU/g, and a β-glucosidase activity of 2731 U/g. The protein content was measured using the ninhydrin assay with BSA as the protein standard,19 the filter paper activity was determined according to the method of Ghose,20 and the β-glucosidase activity was measured using 5 mM p-nitrophenyl-β-Dglucopyranoside (Sigma-Aldrich) as substrate.21 The enzymatic loading for all studies was 13 mg enzyme protein/g dry matter (approximately 10 FPU/g Avicel). The hydrolysis of Avicel was performed in pure water as well as 50 mM citrate buffer at pH 4.8. Additionally, five soluble monosaccharides (glucose, galactose, mannose, fructose, and xylose) at 50, 270, 540, and 810 mM were used (in separate experiments) to spike the samples prior to hydrolysis. For the first four monosaccharides, the molar concentrations are equivalent to approximately 10, 50, 100, and 150 g/L mass concentration, respectively, that is, in or above the range of soluble glucose found during lignocellulose hydrolysis at high dry matter for an industrial setup.11 The samples were spiked with glucose to study its effect as a hydrolysis end product; that is, product inhibition was expected. Galactose and mannose were chosen as they are stereoisomers of glucose but are not end products of hydrolysis. Xylose was used as it is one of the major monosaccharides found in biomass, and fructose, even though not a major component in biomass, is a structural isomer of glucose and is the most water-soluble of all monosaccharides. Samples were prepared in triplicates, and the hydrolysis was carried out using an incubator at 50 °C with an orbital shaker set at 150 rpm. Hydrolysis was run for 96 h. Control samples (without enzyme) were prepared and analyzed for their sugar content at 15 min and at the end of hydrolysis to verify whether the sugars had degraded over time. The hydrolyses were terminated by centrifuging each sample at 13200g and 5 °C for 10 min and filtering through a 0.45 μm syringe filter (Millipore, Bedford, MA, USA). Filtrates were stored at −20 °C for further HPLC quantification. Enzymatic Hydrolysis of Cellobiose with Novozymes Cellic CTec2. Fifty milligrams of D-cellobiose (Sigma-Aldrich, USA) was weighed in 2 mL screw-cap tubes and filled with diluted enzyme solution in 50 mM acetate buffer at pH 4.8 for a total dry matter content of 5% w/w. The enzymatic loading was 13 mg/g of cellobiose. Furthermore, 50, 270, 540, or 810 mM glucose or galactose was added. Samples were prepared in triplicates and incubated at 50 °C with an orbital shaker set at 150 rpm for 1 h. The hydrolysis was terminated by centrifugation and filtration as described above, followed by HPLC quantification.
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RESULTS AND DISCUSSION Hydrolysis of Avicel at High Monosaccharide Concentrations. Solutions containing high concentrations of monosaccharides can decrease the mobility of free water and inhibit hydrolysis. Previous studies have indicated that high concentrations of glucose have an inhibitory effect on cellulase activity.12,23,24 End products of hydrolysis, glucose and cellobiose, inhibit cellulases based on the classical definition of end-product inhibition.25 However, as Xiao et al.12 and Roberts et al. 14 have shown, the presence of other monosaccharides that are not end products of hydrolysis also decrease glucose yields. Figure 1 shows the glucose yield after 96 h for samples that were spiked with different monosaccharides. As expected, the yield decreased for all sugars studied as the initial monosaccharide concentration increased. Sugars at low concentration (50 mM) slightly boosted glucose production (especially galactose), but at higher concentrations (>270 mM) the yield decreased considerably compared to pure water. 3801
dx.doi.org/10.1021/jf5012962 | J. Agric. Food Chem. 2014, 62, 3800−3805
Journal of Agricultural and Food Chemistry
Article
Figure 2. Glucose production after 96 h of Avicel hydrolysis in water (solid symbols) versus 50 mM acetate buffer (open symbols) with initial glucose or galactose spikes to determine additive effects between acetate and monosaccharide. Despite a more stable pH with a small added concentration of buffer, the additional presence of soluble salts at higher concentrations has a detrimental effect on the glucose yield.
Figure 1. Hydrolysis of Avicel at 5% dry matter after 96 h spiked with glucose, xylose, galactose, fructose, or mannose from low to high concentrations of monosaccharides. Monosaccharides present at low concentrations seem to be beneficial to hydrolysis as there is a small boost in the product yield in some cases. At higher concentrations of sugars, however, they are detrimental to the cellulases as the yield keeps decreasing.
sugars, for example, galactose and fructose give higher glucose hydrolysis yields than when having additional mannose in the hydrolysates. Thus, the saccharification rates must be limited by other factors aside from mass transfer and end-product (glucose) inhibition, which we suggest may have to do with the way each monosaccharide interacts with water. In this section, we look at three possibilities related to monosaccharide solutions: solubility, partial molar volume, and T2 relaxation time. Selig et al.15 suggested that an increase in the amount of water-soluble species in the system can significantly inhibit cellulases. One can speculate that when soluble species form strong interactions with water to maintain their solubility, water has lower mobility and hydrolysis slows. In this case, it would mean that sugars which interact more with water in the solution should inhibit hydrolysis to a greater extent than those with weaker interactions. Therefore, we tested if the decrease in glucose yield could be linked to the water solubility of the monosaccharides, as more soluble monosaccharides would be expected to interact with fewer water molecules and hence produce higher glucose yields than the less soluble monosaccharides. Table 1 shows reported literature values29,30 for the
Some of the sugar concentrations tested in Figure 1 are unrealistically high when compared to realistic biorefinery scenarios. For example, for hydrolysis of pretreated wheat straw at 30% dry matter content using Cellic CTec2, 95 g/L (approximately 527 mM) was the highest glucose concentration reported for a separate hydrolysis and fermentation process.11 In a simultaneous hydrolysis and fermentation process, however, the glucose concentration will remain at
