Product inhibition of cellulases studied with 14C-labeled cellulose substrates
© Teugjas and Väljamäe; licensee BioMed Central Ltd. 2013
Received: 20 May 2013
Accepted: 11 July 2013
Published: 24 July 2013
As a green alternative for the production of transportation fuels, the enzymatic hydrolysis of lignocellulose and subsequent fermentation to ethanol are being intensively researched. To be economically feasible, the hydrolysis of lignocellulose must be conducted at a high concentration of solids, which results in high concentrations of hydrolysis end-products, cellobiose and glucose, making the relief of product inhibition of cellulases a major challenge in the process. However, little quantitative information on the product inhibition of individual cellulases acting on cellulose substrates is available because it is experimentally difficult to assess the hydrolysis of the heterogeneous polymeric substrate in the high background of added products.
The cellobiose and glucose inhibition of thermostable cellulases from Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum acting on uniformly 14C-labeled bacterial cellulose and its derivatives, 14C-bacterial microcrystalline cellulose and 14C-amorphous cellulose, was studied. Cellulases from Trichoderma reesei were used for comparison. The enzymes most sensitive to cellobiose inhibition were glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs), followed by family 6 CBHs and endoglucanases (EGs). The strength of glucose inhibition followed the same order. The product inhibition of all enzymes was relieved at higher temperatures. The inhibition strength measured for GH7 CBHs with low molecular-weight model substrates did not correlate with that measured with 14C-cellulose substrates.
GH7 CBHs are the primary targets for product inhibition of the synergistic hydrolysis of cellulose. The inhibition must be studied on cellulose substrates instead of on low molecular-weight model substrates when selecting enzymes for lignocellulose hydrolysis. The advantages of using higher temperatures are an increase in the catalytic efficiency of enzymes and the relief of product inhibition.
KeywordsCellulase Cellulose Cellobiose Glucose Inhibition Acremonium thermophilum Thermoascus aurantiacus Chaetomium thermophilum Trichoderma reesei
Cellulose is the most abundant biopolymer on Earth and has great potential as a renewable energy source. In nature, cellulose is degraded mainly by fungi and bacteria, which secrete cellulolytic enzymes . These enzymes include cellulases, hemicellulases, and enzymes involved in lignin breakdown. Cellulases are divided into cellobiohydrolases (CBHs), endoglucanases (EGs) and β-glucosidases (BGs). CBHs are processive enzymes that liberate consecutive cellobiose units from cellulose chain ends, whereas EGs non-processively attack cellulose chains at random positions. β-Glucosidases hydrolyze cellobiose to glucose, thus relieving the product inhibition of CBHs . One of the most efficient and best-characterized cellulolytic systems is that of the soft rot fungus Tricoderma reesei (Tr). The major component of the Tr cellulolytic system is the glycoside hydrolase (GH) family 7 [3, 4] CBH, TrCel7A (formerly CBH I). Tr also secretes a less abundant CBH, TrCel6A (CBH II), and a number of EGs, including TrCel7B, TrCel5A and TrCel12A (EG I, EG II and EG III, respectively).
Cellulases are used in many biotechnological applications, such as fiber modification in the paper and textile industries, but they also have great potential in the emerging industry of ethanol production from lignocellulose. To decrease the water consumption and reduce the costs of equipment and distillation, the hydrolysis of lignocellulose must be conducted at a high concentration of solids. This approach inevitably results in high concentrations of the hydrolysis end-products cellobiose and glucose, and it has been proposed that the end-product inhibition of cellulases is rate limiting for lignocellulose hydrolysis in high-solid conditions . Thus, relieving the product inhibition is a major challenge in the process, as well as in enzyme engineering . The end-product inhibition can be relieved in a simultaneous saccharification and fermentation process, where the fermenting organism is added in parallel with hydrolytic enzymes, but one drawback is the need for different conditions for optimal hydrolysis and fermentation. The optimal temperature for yeast fermentation is approximately 35°C, whereas temperatures near 50°C are optimal for the performance of cellulases. A process concept using high temperature liquefaction with thermostable enzymes preceding simultaneous saccharification and fermentation has been developed , and this has triggered the search for novel thermostable enzymes [8, 9].
Despite intensive efforts, little quantitative information about the end-product inhibition of cellulases is available. Many of the studies can be classified as “semi-quantitative”. Most often, the rates of cellulose hydrolysis measured in the presence and absence of β-glucosidase are compared [10–13]. In some studies, the experimental setup enabling the continuous elimination of end-products has been used . The numerical values of inhibition constants have been obtained by the fitting of hydrolysis data to the complex equations derived for the full time-course [14–20]. The validity of these figures depends on the validity of the model . Another problem lies in the possible interplay between parameters in trials, where values of multiple parameters are approximated by a single fit. The inhibition types reported include competitive, non-competitive, uncompetitive and mixed inhibition, whereas the values of inhibition constants vary over several orders of magnitude. One reason for the variation of reported inhibition types and the values of inhibition constants is that complex cellulase mixtures are often used instead of purified cellulase components in experiments. Different cellulase components may be inhibited to different extents and by different mechanisms, which clearly complicates the interpretation of the data. For literature reviews of earlier and more recent studies, see  and , respectively.
An inherent problem in measuring the strength of product inhibition is associated with difficulties in measuring the initial rates of product formation in the high background of the product added as an inhibitor. Three approaches can be used to overcome this: (i) measurement of the initial rates of substrate consumption instead of product formation ; (ii) measurement of the hydrolysis rate with a method that does not rely on measuring the concentration of the substrate or product; and (iii) the use of model substrates, whose conversion can be followed independently of the added products. Although emerging new methods, such as flow ellipsometry  and quartz crystal microbalance , enable the monitoring of changes in cellulose concentration in real time, these methods have not yet been applied to quantification of the inhibition of cellulases. The second approach has been applied for cellulases by following the rate of cellulose hydrolysis using isothermal titration calorimetry [27, 28]. Because of the moderate standard enthalpy change of glycosidic bond hydrolysis, the low sensitivity is a drawback of calorimetry. While signal amplification systems can be used to measure cellulose hydrolysis, these systems are not applicable in studies of inhibition . The third approach has been most widely used in studies of the inhibition of cellulases. The model substrates used can be divided into two classes, low-Mw and polymeric model substrates. Among low-Mw model substrates, the chromo- or fluorogenic derivatives of lactose or cellobiose are most often used . However, these derivatives are not generally applicable. As an example, para-nitrophenyl-β-lactoside (pNPL) and 4-methylumbelliferyl-β-lactoside (MUL) are good substrates for GH7 CBHs such as TrCel7A and some EGs such as TrCel7B, but they are not hydrolyzed by GH6 CBHs such as TrCel6A. Another drawback of using low-Mw model substrates is that cellobiose inhibition appears to be much stronger with these substrates than with cellulose . The reason for this may lie in different modes of action of cellulases on low-Mw model substrates and on cellulose  and in the experimental conditions used to measure enzyme inhibition . Therefore, it is not possible to determine whether and to what extent the inhibition strength measured with low-Mw substrates reflects the inhibition strength with the real substrate, cellulose. Among polymeric model substrates, cellulose derivatives, in which hydroxyls are randomly substituted with chromo- or fluorophores (dyed cellulose), can be used [22, 23]. The drawback of their use is that the tunnel-shaped active sites of CBHs cannot accommodate the bulky substitutes, and the application of these substrates is limited with EGs. Derivatives in which the reducing ends of cellulose are 3H-reduced to corresponding alditols have also been used . The disadvantage of these substrates is that only the cleavage of reducing-end terminal glycosidic bonds can be measured. Therefore, these substrates are not applicable with non-reducing-end active CBHs such as TrCel6A. To overcome these limitations, we prepared uniformly 14C-labeled bacterial cellulose (14C-BC) by cultivating Gluconobacterium xylinum in the presence of 14C-glucose. 14C-BC and its derivatives, 14C-bacterial microcrystalline cellulose (14C-BMCC) and 14C-amorphous cellulose, were used to study the cellobiose and glucose inhibition of thermostable cellulases from Acremonium thermophilum (At), Thermoascus aurantiacus (Ta), and Chaetomium thermophilum (Ct). Cellulases from these organisms have great potential in biotechnological applications [34–39]. Well-characterized cellulases from Tr were used for comparison.
Results and discussion
Measuring the strength of inhibition
The best parameter for describing the inhibitory strength of an inhibitor is Ki, the equilibrium dissociation constant of an enzyme-inhibitor complex. Ki is a fundamental parameter of enzyme kinetics that is directly related to the thermodynamic stability of the enzyme-inhibitor complex. The conventional approach for the measurement of Ki involves the measurement of kcat and KM values for the substrate at different concentrations of an inhibitor. The plotting of kcat and KM or their combination as a function of inhibitor concentration allows the determination of both the type of inhibition and the Ki value. However, this approach is not applicable to cellulases acting on cellulose. The complex, multiple-mode binding of cellulases to the solid substrate obeys the so-called double-saturation character . KM values measured for cellulose depend on the enzyme concentration, and therefore, KM has not its usual meaning. Because of the non-productive binding and strong time dependency, the measurement of the kcat value is also not straightforward [40–42].
In the case of mixed inhibition, the interplay among IC50, Ki (there are two different Kis now) and [S]/KM is more complicated, and whether the inhibition appears to be stronger at low or high [S]/KM ratio depends on which type of inhibition (competitive or un-competitive) is dominating. However, in the case of pure non-competitive inhibition, IC50 = Ki, so IC50 represents the value of the true Ki at any substrate concentration used for its measurement.
GH family 7 cellobiohydrolases
GH7 CBHs are major components of efficient fungal cellulase systems. They are processive enzymes that are responsible for the degradation of crystalline cellulose . Because of their central role in cellulose degradation, the inhibition of GH7 CBHs is of utmost importance. Here, we undertook a study of the inhibition of GH7 CBHs acting on 14C-BC. Thermostable GH7 CBHs AtCel7, TaCel7A, and CtCel7A , along with TrCel7A, were characterized in terms of cellobiose and glucose inhibition. Tm values of 75°C, 69°C, 75°C and 65°C have been reported for TaCel7A, AtCel7A, CtCel7A and TrCel7A, respectively . Although highly crystalline, the BC fiber contains a small fraction of heterogeneities [45, 46]. These heterogeneities are preferentially degraded by cellulases, and their depletion is thought to be responsible for rate retardation of cellulose hydrolysis . Thus, interpretation of the results of product inhibition is more straightforward if measured at a higher degree of substrate conversion. A very high degree of synergy between TrCel7A and EG has been reported with BC substrates [32, 48, 49]. To reach a higher degree of conversion and characterize the hydrolysis of bulk cellulose, the GH7 CBHs were thus provided with the EG, TrCel5A (10% on a molar basis).
Inhibition of GH7 CBHs by cellobiose and glucose studied with 14 C-BC substrate
IC50for cellobiose (mM)
IC50for glucose (mM)
0.38 ± 0.03a
0.68 ± 0.24b
2.61 ± 0.10
420 ± 230c
0.19 ± 0.10c
0.44 ± 0.10
2.12 ± 1.40b
420 ± 180c
0.41 ± 0.06
1.08 ± 0.22
2.48 ± 0.91b
360 ± 170c
0.58 ± 0.35
0.93 ± 0.10
Inhibition of GH7 CBHs by cellobiose studied with MUL substrate
Kifor cellobiose (mM)
DGlc and DGlc=0 represent the degree of conversion of 14C-BC in the presence and absence of added glucose, respectively; [Glc] is the concentration of added glucose; [14CBC] is the 14C-BC concentration used in the experiment; and C1, C2 and H are empirical constants. The values of C1, C2 and H obtained by the fitting of the data to Equation 7 were used to calculate the IC50 for glucose according to Equation 6. The glucose inhibition of GH7 CBHs was more than two orders of magnitude weaker than cellobiose inhibition (Table 1). Although relatively weak, glucose inhibition may become significant in the separate hydrolysis and fermentation of lignocellulose at a high dry matter consistency, where glucose may accumulate to well above 50 g/l (0.28 M) [5, 23].
GH family 6 cellobiohydrolases
GH6 CBHs are the second most abundant components of fungal cellulase systems. They are inverting CBHs that preferentially attack cellulose chains from non-reducing ends. To date, there are no good chromo- or fluorogenic model substrates for GH6 CBHs . Because of the different chain-end preferences, inhibition studies on reducing-end-labeled cellulose substrates are also not applicable . Therefore, little is known about the strength of the product inhibition of GH6 CBHs. From the reported binding constants measured using fluorophore competition experiments [60, 61] and analysis of the progress curves of cellotriose hydrolysis [51, 62], Ki values in a sub- to low-millimolar range can be calculated for the interaction of TrCel6A with cellobiose and glucose.
Inhibition of GH6 CBHs by cellobiose and glucose studied with 14 C-BC and 14 C-BMCC substrates
16 ± 0.5
20 ± 1.4
240 ± 26
28 ± 4.5
301 ± 30
Our data presented here, together with those from the literature, strongly suggest that the inhibition of cellulases must be studied on cellulose substrates instead of on low-Mw model substrates. The enzymes most sensitive to cellobiose inhibition were GH7 CBHs, followed by GH6 CBHs and EGs. The strength of glucose inhibition followed the same order. Thus, the GH7 CBHs are primary targets for product inhibition of the synergistic hydrolysis of cellulose. With all enzymes, the strength of the product inhibition decreased with increasing temperature.
Glucose, MUL, pNPL, Novozyme®188, and BSA were purchased from Sigma-Aldrich. Cellobiose (≥ 99%) was from Fluka. D-[U-14C] glucose with a specific activity of 262 mCi mmol-1 was from Hartmann Analytic GmbH. Scintillation cocktail was from Merck. All chemicals were used as purchased.
14C-BC was prepared by laboratory fermentation of the Gluconobacter xylinum strain ATCC 53582  in the presence of [U-14C] glucose carbon source . 14C-BC had a specific activity 450,000 DPM mg-1. 14C-BMCC was prepared by the limited acid hydrolysis of 14C-BC, and 14C-amorphous cellulose was prepared from 14C-BMCC by dissolution and regeneration from phosphoric acid . The total concentration of cellulose was determined by the anthrone sulfuric acid method.
TrCel7A was purified from the culture filtrate of Tr QM 9414 as described previously . Culture filtrates containing AtCel7A, CtCel7A or TaCel7A were kindly provided by Terhi Puranen from Roal Oy (Rajamäki, Finland). CBHs were heterologously expressed in a Tr strain lacking the genes of four major cellulases [34, 44]. The natively carbohydrate-binding module-less TaCel7A was provided with the carbohydrate binding module of TrCel7A [34, 44]. CBHs were purified on a Q-Sepharose column after buffer exchange on a Toyopearl HW-40 column. For ion-exchange chromatography on Q-Sepharose, the column was equilibrated with 20 mM sodium phosphate, pH 6.0 (in the case of AtCel7A and TaCel7A) or with 20 mM sodium phosphate, pH 6.5 (in the case of CtCel7A). CBHs were eluted with a linear gradient of 0 – 0.3 M NaCl in equilibration buffer.
TrCel6A was purified from the culture filtrate of Tr QM 9414 as described previously [72, 73]. The culture filtrate of CtCel6A heterologously expressed in Tr originated from Roal OY (Rajamäki, Finland) and was kindly provided by Matti Siika-Aho from VTT (Espoo, Finland). CtCel6A was purified on a DEAE-Sepharose column after buffer exchange on a Toyopearl HW-40 column. For ion-exchange chromatography on DEAE-Sepharose, the column was equilibrated with 20 mM sodium phosphate (pH 7.0), and CtCel6A was eluted with a linear gradient of 0 – 0.5 M NaCl in equilibration buffer.
The concentration of the enzymes was measured from the absorbance at 280 nm using theoretical ϵ280 values.
Activity and inhibition of GH7 CBHs
The activity and inhibition of GH7 CBHs were assessed by following the synergistic hydrolysis of 14C-BC. For that, 14C-BC (0.25 g l-1) was incubated (without stirring) with a mixture of CBH (0.25 μM), TrCel5A (0.025 μM) and N188BG (0.06 μM) in 50 mM sodium acetate buffer pH 5.0 containing BSA (0.1 g l-1). 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 x g), and radioactivity in the supernatant was quantified using liquid scintillation counting. The degree of cellulose degradation was calculated from the ratio of radioactivity in the supernatant to the total radioactivity in the hydrolysis mixture. In the case of inhibition studies, the reactions were supplied with cellobiose and glucose at different concentrations, and N188BG was omitted.
For the inhibition of enzyme acting on the low-Mw substrate, the initial rates of the hydrolysis of MUL in the presence and absence of added cellobiose were followed. MUL (5 μM) was incubated with CBH (10 nM) in 50 mM sodium acetate buffer, pH 5.0, containing BSA (0.1 g l-1). Reactions were stopped by the addition of NH3 (final concentration 0.1 M), and the released 4-methylumbelliferone was quantified by fluorescence using excitation and emission wavelengths of 360 nm and 450 nm, respectively.
Activity and inhibition of GH6 CBHs
GH6 CBHs were assessed by observing the hydrolysis of 14C-BMCC. 14C-BMCC (0.25 g l-1) was incubated (with shaking at 350 rpm) with CBH (0.25 μM) and N188BG (0.06 μM) in 50 mM sodium acetate buffer, pH 5.0, containing BSA (0.1 g l-1). The remainder of the procedure was identical to that described for GH7 CBHs. In the case of inhibition studies, the reactions were supplied with cellobiose and glucose at different concentrations, and N188BG was omitted.
The cellobiose inhibition of the synergistic hydrolysis of 14C-BC was performed identically to the procedure described for GH7 CBHs, but the CBH component was 0.25 μM TrCel6A.
Activity and inhibition of EGs
EGs were assessed on 14C-amorphous cellulose. 14C-amorphous cellulose (0.5 g l-1) was incubated (with shaking at 700 rpm) with EG in 50 mM sodium acetate buffer, pH 5.0, containing BSA (0.1 g l-1) in the presence and absence of added cellobiose. The concentration of EG was 2.5 nM, 5.0 nM, and 50 nM for TrCel7B, TrCel5A, and TrCel12A, respectively. The remainder of the procedure was identical to that described for GH7 CBHs.
Bovine serum albumin
14C-labeled bacterial cellulose
14C-labeled bacterial microcrystalline cellulose
Degree of conversion in the presence of inhibitor i
Degree of conversion in the absence of inhibitor
Degree of polymerization
This work was funded by the EU Commission (FP7/2007-2013, grant agreement no. 213139). Dr. Terhi Puranen from Roal Oy (Rajamäki, Finland) and Dr. Matti Siika-Aho from VTT (Espoo, Finland) are acknowledged for crude preparations of TaCel7A, AtCel7A, CtCel7A, and CtCel6A. Among our colleagues from the University of Tartu, we thank Jürgen Jalak and Mihhail Kurašin for their assistance in protein purification and in preparing figures, and we thank Dr. Silja Kuusk for critical reading.
a This work is dedicated to lecturer Hele Teugjas, who passed away during the preparation of this paper.
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