Product inhibition of cellulases studied with ¹⁴C-labeled cellulose substrates

Measuring the strength of inhibition

The best parameter for describing the inhibitory strength of an inhibitor is K_i, the equilibrium dissociation constant of an enzyme-inhibitor complex. K_i 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 K_i involves the measurement of k_cat and K_M values for the substrate at different concentrations of an inhibitor. The plotting of k_cat and K_M or their combination as a function of inhibitor concentration allows the determination of both the type of inhibition and the K_i 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 [1]. K_M values measured for cellulose depend on the enzyme concentration, and therefore, K_M has not its usual meaning. Because of the non-productive binding and strong time dependency, the measurement of the k_cat value is also not straightforward [40–42].

In the case of mixed inhibition, the interplay among IC₅₀, K_i (there are two different K_is now) and [S]/K_M is more complicated, and whether the inhibition appears to be stronger at low or high [S]/K_M ratio depends on which type of inhibition (competitive or un-competitive) is dominating. However, in the case of pure non-competitive inhibition, IC₅₀ = K_i, so IC₅₀ represents the value of the true K_i 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 [43]. 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 [44], along with TrCel7A, were characterized in terms of cellobiose and glucose inhibition. T_m values of 75°C, 69°C, 75°C and 65°C have been reported for TaCel7A, AtCel7A, CtCel7A and TrCel7A, respectively [44]. 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 [47]. 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).

Figure 1 shows the time courses for the synergistic hydrolysis of ¹⁴C-BC by CBHs (supplemented with TrCel5A and β-glucosidase, N188BG) at different temperatures. With all CBHs, the time courses of ¹⁴C-BC degradation measured at lower temperatures (25°C – 35°C) were nearly linear, whereas the time courses measured at higher temperatures gradually deviated from linearity. With TaCel7A as an exception, the degree of conversion after 30 min of hydrolysis measured at 60°C was less than that measured at 50°C (Figure 1). However, such a decrease in the degree of conversion with increasing temperature was not observed after 5 min of hydrolysis. Similar observations have also been made for the hydrolysis of pre-treated lignocellulose [36], suggesting that this phenomenon is not ¹⁴C-BC specific. The simplest explanation would be the thermal inactivation of enzymes that progresses with time. We tested the possible thermal inactivation of enzymes in an experiment where the hydrolysis began at 55°C, and after 30 min, the temperature was decreased to 40°C. TrCel7A was used as the CBH because of its lowest T_m value among the CBHs studied. Figure 2 demonstrates that despite a 15°C drop in temperature, the rate of cellulose hydrolysis actually increased. This finding rules out the irreversible inactivation of enzymes as the primary cause of the non-linearity in time curves observed at higher temperatures. However, the contribution of the reversible denaturation of enzymes cannot be ruled out. The hydrolysis of cellulose by CBH is a multi-step process including binding to cellulose, the capture of the cellulose chain-end, processive degradation, and dissociation [32, 50]. Therefore, another possibility is that some kinetic property of CBHs is negatively affected by temperature. Whatever the underlying mechanisms, the change in the linearity of time curves depending on temperature may also result in a change in the apparent inhibition strength with hydrolysis time.

where [CB] is the concentration of cellobiose; [¹⁴CBC] is the concentration of ¹⁴C-BC used in the experiment; and C₁, C₂ and H are empirical constants. Equation 5 differs from Equation 1 by the presence of constant H. H was included to improve the fit and is a constant that accounts for the background radioactivity (the degree of conversion that is independent of CBH). The degree of conversion resulting from the activity of the EG was measured in a separate experiment (Additional file 1: Figure S4) and was subtracted from the degree of conversion resulting from the synergistic hydrolysis. Thus, in the case of complete inhibition, H should have a value of zero. Non-zero H values are indicative of partial inhibition. However, because the H values remained between 0 and 0.2 and were even negative in some cases, they may also be a result of experimental uncertainty. Provided with the values of C₁, C₂ and H, the value of IC₅₀ was calculated as follows:

$I{C}_{50}=\frac{\left[{}^{14}C\mathit{BC}\right]+C_1}{\frac{C_1}{C_2}\left(1-2H\right)}$

(6)

The cellobiose inhibition of GH7 CBHs is most often studied on low-Mw model substrates. However, it has been shown that the inhibition of CBHs acting on low-Mw substrates appears to be much stronger than that on cellulose substrates [31, 33]. The K_i values for cellobiose inhibition of GH7 CBHs measured on low-Mw substrates are in the micromolar range [44, 51, 52], whereas those measured on cellulose are in the low- to high-millimolar range [28, 31, 32]. An interesting exception is Cel7A from Trichoderma harzianum, which shows a 7.2 mM K_i value for the cellobiose inhibition of the hydrolysis of chloro-nitrophenyl lactoside [53]. Unfortunately, the inhibition of this enzyme on cellulose has not been studied. We also studied the cellobiose inhibition of GH7 CBHs acting on MUL. The initial rates of MUL hydrolysis measured in the presence and absence of added cellobiose were first analyzed according to Equation 1, and the IC₅₀ values were found using Equation 2. As cellobiose was shown to be a competitive inhibitor for these CBHs acting on MUL [44] and the concentration of MUL used in the experiments (5 μM) was far below its K_M value (approximately 300 μM [44]), the measured IC₅₀ value represents the true K_i (see Equation 3). The resulting K_i values are listed in Table 2. Van´t Hoff analysis of the temperature dependency of the IC₅₀ and K_i values of TrCel7A resulted in standard enthalpy changes of 63.6 ± 2.6 kJ mol^-1 (for IC₅₀ values on ¹⁴C-BC, Table 1) and approximately 63 kJ mol^-1 (for K_i values on MUL, Table 2). The inhibition of MUL hydrolysis is attributable to the binding of cellobiose to the product sites (+1/+2) of TrCel7A [52]. Similar standard enthalpy changes thus suggest that the cellobiose inhibition of the synergistic hydrolysis of ¹⁴C-BC is also attributable to the binding of cellobiose to sites +1/+2. Nonetheless, for all CBHs, the K_i values found for the cellobiose inhibition of MUL hydrolysis (Table 2) were smaller than the corresponding IC₅₀ values for the inhibition of ¹⁴C-BC hydrolysis (Table 1). The reason for this difference may lie in the different modes of action used by CBHs with low-M_w model substrates and cellulose and therefore the different types of inhibition [32]. Another possible explanation is that the cellobiose inhibition of CBHs on cellulose is competitive and that the concentration of cellulose chain ends used in the measurement of the IC₅₀ value is higher than the corresponding K_M value. In this case, the observed IC₅₀ is expected to be higher than the K_i (see Equation 3), and the inhibition of cellulose hydrolysis appears to be weak. This scenario has been proposed to explain the differences in the inhibitory strength of xylo-oligosaccharides toward CBHs acting on MUL and cellulose [33]. The binding of xylo-oligosaccharides with DP 8 – 10 is expected to mimic the binding of the cellulose chain to the active site of TrCel7A, resulting in competitive inhibition. In contrast, despite the strong binding of cellobiose to the product sites (+1/+2) of TrCel7A [52, 54, 55], the cellulose chain can still bind to the substrate sites (from −7 to −1), and this predicts non-competitive inhibition [23, 31, 32]. The results of our previous studies of the inhibition of TrCel7A under single-turnover and steady-state conditions suggested that cellobiose might be a mixed-type inhibitor of TrCel7A acting on cellulose. The binding of cellobiose to the product and substrate binding sites was proposed to be responsible for the non-competitive and competitive components of inhibition, respectively [32]. Observations that the binding affinity of TrCel7A and TrCel6A towards cellulose increased in the presence of cellobiose also suggest an inhibition mode that is not competitive [56, 57]. From the practical point of view, it is important to note that for different CBHs, the differences in inhibition strength observed on MUL and cellulose were not of the same magnitude (Figure 5). This result can be exemplified best by TaCel7A, which appeared to be most resistant to cellobiose inhibition on MUL substrate (Table 2) but was most sensitive to cellobiose inhibition on cellulose (Table 1). This finding stresses the importance of the use of “as native as possible” screening systems for selecting cellulases [58].

The glucose inhibition of CBHs with ¹⁴C-BC as the substrate was also studied. CBHs were provided with EG TrCel5A (TrCel5A is not significantly inhibited by glucose [28]) and also with BG in the experiments without added glucose. The time courses of ¹⁴C-BC hydrolysis in the presence and absence of added glucose are shown in Figure 6. As revealed by the scattering of data points in the plot of (D_Glc/D_Glc=0) versus [glucose] (Figure 6D), the inhibition by the cellobiose released during hydrolysis was significant. This result was accounted for by adding the term [CB]/IC_50(CB) ([CB] is the concentration of the released cellobiose, and IC_50(CB) is the IC₅₀ for cellobiose previously determined) to Equation 5 to create Equation 7:

$\frac{D_{\mathit{Glc}}}{D_{\mathit{Glc}=0}}=\frac{\left(\left[{}^{14}C\mathit{BC}\right]+C_1\right)\left(1-H\right)}{\left[{}^{14}C\mathit{BC}\right]+C_1\left(1+\frac{\left[\mathit{CB}\right]}{I{C}_{50\left(\mathit{CB}\right)}}+\frac{\left[\mathit{Glc}\right]}{C_2}\right)}+H$

(7)

D_Glc and D_Glc=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 [59]. Because of the different chain-end preferences, inhibition studies on reducing-end-labeled cellulose substrates are also not applicable [31]. 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], K_i values in a sub- to low-millimolar range can be calculated for the interaction of TrCel6A with cellobiose and glucose.

Here, we characterized the cellobiose and glucose inhibition of TrCel6A and its thermophilic counterpart, CtCel6A [9, 63]. First, the cellobiose inhibition of the synergistic hydrolysis of ¹⁴C-BC by TrCel6A and TrCel5A was studied (Figure 7A). As TrCel6A was less sensitive to cellobiose inhibition than TrCel7A, the contribution of the cellobiose released during hydrolysis was not significant, and an average (D_CB/D_CB=0) over all time points was used in plotting (D_CB/D_CB=0) versus [cellobiose] (Figure 7D). No significant systematic variation of D_CB/D_CB=0 depending on hydrolysis time was observed. As in the case of GH7 CBHs, the IC₅₀ value was found using Equations 5 and 6. Because the IC₅₀ value for synergistic hydrolysis (Table 3) was of the same order as the apparent K_i value reported for TrCel5A [31], we further tested the inhibition of individual TrCel6A. BC is not a good substrate for TrCel6A, but its acid-treated derivative, BMCC, is readily degraded by the enzyme. Therefore, we prepared ¹⁴C-BMCC by the heterogeneous acid hydrolysis of ¹⁴C-BC. The time courses of ¹⁴C-BMCC hydrolysis by TrCel6A and CtCel6A are shown in Figures 7B and 7C. Without supplied cellobiose, both enzymes had similar activity with the ¹⁴C-BMCC substrate, but CtCel6A was somewhat more resistant to cellobiose inhibition (Figure 7D, Table 3). The IC₅₀ value for TrCel6A by itself was similar to that found for the synergistic hydrolysis. This result suggests that the inhibition of TrCel6A was responsible for the cellobiose inhibition of the synergistic hydrolysis of ¹⁴C-BC. The glucose inhibition of TrCel6A and CtCel6A with ¹⁴C-BMCC as a substrate was also studied (Figure 8). Because the inhibition by cellobiose released during hydrolysis was not significant, a simpler equation, Equation 5 (the terms referring to cellobiose were replaced with corresponding terms for glucose), was used instead of Equation 7 to analyze the glucose inhibition of GH6 CBHs. Glucose appeared to be an approximately 10 times weaker inhibitor of TrCel6A and CtCel6A than cellobiose (Table 3), but comparison with corresponding figures for GH7 CBHs (Table 1) reveals that glucose is a relatively stronger inhibitor of GH6 than GH7 CBHs. The same result was also observed in a recent calorimetry study of the inhibition of Tr cellulases acting on amorphous cellulose [28]. However, the IC₅₀ values found by Murphy et al. [28] for the cellobiose inhibition of TrCel7A and TrCel6A were approximately one order of magnitude higher than ours. Whether the differences in the strengths of cellobiose inhibition reflect the differences in substrates or the methods used for the measurement of inhibition is not known. Comparison of the IC₅₀ values measured here with K_i values derived from binding constants measured using low-Mw substrates and ligands as competitors [60–62] reveals the same trend as in the case of GH7 CBHs: the binding of cellobiose and glucose appears to be weaker when assessed on polymeric substrates.

Endoglucanases

EGs are a diverse group of enzymes present in all efficient cellulase systems. Their best recognized function is their synergism with CBHs. Depending on the conditions, the degree of synergistic effect may be more than 10-fold [32, 64]. Therefore, the inhibition of the EG component may result in a drastic decrease in the rate of the synergistic hydrolysis of cellulose. The main soluble product of the EG-catalyzed cellulose hydrolysis is cellobiose, but some glucose and higher-order oligosaccharides are also produced [65]. Here, we studied the cellobiose inhibition of the EGs TrCel7B, TrCel5A and TrCel12A with ¹⁴C-amorphous cellulose substrate. The enzyme concentrations and hydrolysis times were adjusted so that the linear region of the time course was studied. The time courses for the hydrolysis of ¹⁴C-amorphous cellulose by TrCel7B in the presence and absence of added cellobiose are shown in Figure 9A. For the results with TrCel5A and TrCel12A, see Additional file 1: Figure S5. The “conventional” inhibition pattern was observed only in the case of TrCel7B, with an IC₅₀ value of 168 ± 2 mM. This figure is reasonably well in line with that measured for TrCel7B on amorphous cellulose using isothermal titration calorimetry [28]. Calorimetry measures the amount of glycosidic bonds that are cleaved irrespective of the solubility of the products [41]. Thus, the agreement between the IC₅₀ values from calorimetric measurements and those reported here suggests that the inhibition of the release of soluble products represents the inhibition of the total activity of TrCel7B. However, we have previously reported an apparent K_i value of 11 ± 3 mM for TrCel7B with a ³H-reduced amorphous cellulose substrate [31]. Thus, the cellobiose inhibition of TrCel7B on uniformly ¹⁴C-labeled amorphous cellulose was much weaker. The same was also true for TrCel5A. The inhibition of TrCel5A and TrCel12A was not accountable by Equation 5 (Figure 9B). In the case of TrCel5A, the initial drop in activity was followed by a slight increase at the highest cellobiose concentration tested. In the case of TrCel12A, there was an apparent activation at a lower cellobiose concentration of 75 mM, followed by a decrease in activity with increasing cellobiose concentration (Figure 9B). We previously observed the apparent activation of TrCel12A in the cellobiose concentration range of 1 mM – 100 mM acting on a ³H-reduced amorphous cellulose substrate [31]. Glucose concentration dependent apparent activation or inhibition of pNPG-ase activity of BGs has also been observed [66–70]. The concentration-dependent apparent activation or inhibition most likely reflects the complex kinetics with competing hydrolytic and transglycosylation reactions [28, 31]. Whether the sugar appears to be an inhibitor or an activator may depend on the rate-limiting step, which may also change depending on the sugar concentration and the experiment conditions, e.g., the method used for rate measurement. Although the IC₅₀ values cannot be calculated for TrCel5A and TrCel12A, approximate figures in a few hundred millimolar range can be estimated by visual inspection of the data in Figure 9B. The K_i value of 424 μM has been reported for the cellobiose inhibition of TrCel5A acting on cellohexaose [27]. Thus, the strong dependence of inhibition strength on the type of substrate used seems to also be true for EGs. Despite some discrepancies in IC₅₀ values, the inhibition of EGs is far weaker than that of CBHs and is not responsible for the cellobiose inhibition of synergistic hydrolysis.

Materials

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-cellulose substrates

¹⁴C-BC was prepared by laboratory fermentation of the Gluconobacter xylinum strain ATCC 53582 [71] in the presence of [U-¹⁴C] glucose carbon source [32]. 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 [71]. The total concentration of cellulose was determined by the anthrone sulfuric acid method.

Enzymes

TrCel7A was purified from the culture filtrate of Tr QM 9414 as described previously [72]. 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.

TrCel7B, TrCel5A, and TrCel12A were purified from the culture filtrate of Tr QM 9414 as described previously [72, 74, 75]. N188BG was purified from Novozyme®188 as described in [76].

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.