Practical screening of purified cellobiohydrolases and endoglucanases with α-cellulose and specification of hydrodynamics
© Jäger et al; licensee BioMed Central Ltd. 2010
Received: 11 June 2010
Accepted: 18 August 2010
Published: 18 August 2010
It is important to generate biofuels and society must be weaned from its dependency on fossil fuels. In order to produce biofuels, lignocellulose is pretreated and the resulting cellulose is hydrolyzed by cellulases such as cellobiohydrolases (CBH) and endoglucanases (EG). Until now, the biofuel industry has usually applied impractical celluloses to screen for cellulases capable of degrading naturally occurring, insoluble cellulose. This study investigates how these cellulases adsorb and hydrolyze insoluble α-cellulose − considered to be a more practical substrate which mimics the alkaline-pretreated biomass used in biorefineries. Moreover, this study investigates how hydrodynamics affects cellulase adsorption and activity onto α-cellulose.
First, the cellulases CBH I, CBH II, EG I and EG II were purified from Trichoderma reesei and CBH I and EG I were utilized in order to study and model the adsorption isotherms (Langmuir) and kinetics (pseudo-first-order). Second, the adsorption kinetics and cellulase activities were studied under different hydrodynamic conditions, including liquid mixing and particle suspension. Third, in order to compare α-cellulose with three typically used celluloses, the exact cellulase activities towards all four substrates were measured.
It was found that, using α-cellulose, the adsorption models fitted to the experimental data and yielded parameters comparable to those for filter paper. Moreover, it was determined that higher shaking frequencies clearly improved the adsorption of cellulases onto α-cellulose and thus bolstered their activity. Complete suspension of α-cellulose particles was the optimal operating condition in order to ensure efficient cellulase adsorption and activity. Finally, all four purified cellulases displayed comparable activities only on insoluble α-cellulose.
α-Cellulose is an excellent substrate to screen for CBHs and EGs. This current investigation shows in detail, for the first time, the adsorption of purified cellulases onto α-cellulose, the effect of hydrodynamics on cellulase adsorption and the correlation between the adsorption and the activity of cellulases at different hydrodynamic conditions. Complete suspension of the substrate has to be ensured in order to optimize the cellulase attack. In the future, screenings should be conducted with α-cellulose so that proper cellulases are selected to best hydrolyze the real alkaline-pretreated biomass used in biorefineries.
Lignocellulose is a renewable resource that can be used for the sustainable production of platform chemicals or fuels [1, 2]. Essential for its industrial use is the hydrolysis of its main component cellulose to glucose involving at least three different types of cellulases [3–6]: cellobiohydrolase (CBH, EC 18.104.22.168), endoglucanase (EG, EC 22.214.171.124) and β-glucosidase (EC 126.96.36.199). As the cellulose depolymerization performed by CBHs and EGs is the rate-limiting step for the whole hydrolysis , screening for CBHs and EGs is important. However, CBHs and EGs are often characterized with different impractical model substrates that do not mimic the real biomass in biorefineries . Thus, screening experiments need to be conducted with a more practical substrate such as α-cellulose so that proper cellulases are selected which best hydrolyze the biomass actually used in biorefineries.
Physical properties and product information of applied cellulosic substrates
Solubility in water
DP w [AGU]
d p [μm]
200 - 240
1590 - 1960
2140 - 2420
Since α-cellulose is insoluble, the adsorption of cellulases onto α-cellulose is a prerequisite for hydrolysis [4, 12, 13]. Cellulase adsorption is usually analysed using the Langmuir isotherm . It assumes a single, reversible adsorption step to uniform cellulose binding sites without interactions among cellulases. However, according to various authors, the cellulase adsorption onto the respective cellulose was found to be irreversible [15–17]. In addition, cellulase interactions [18, 19], cellulose heterogeneity and porosity were also cited [20–22]. Consequently, several alternative adsorption models were developed [23–26]. Nevertheless, the Langmuir isotherm is the most common mechanistic model for cellulase adsorption [4, 12, 27, 28] and is easily interpretable. Besides the applied cellulases and substrates, temperature is especially important as it affects cellulase adsorption. The amount of adsorbed cellulase is decreased with increasing temperature [16, 29–31].
Few cellulase adsorption studies have been performed using α-cellulose or other fibrous substrates , and these studies utilized complex cellulase systems [32–35]; as yet, no purified cellulases have been analysed. As insoluble substrates are applied, attention has to be paid to hydrodynamics. Until now, cellulase adsorption and activity have not been investigated systematically by considering liquid mixing and particle suspension.
In this study, insoluble α-cellulose is proposed as a more practical substrate to screen for purified CBHs and EGs. Moreover, this study investigates and correlates in detail cellulase adsorption and activity under different hydrodynamic conditions.
The cellulosic substrates carboxymethyl cellulose (CMC), Avicel PH101, Sigmacell 101 and α-cellulose (Figure 1) were purchased from Sigma-Aldrich (MO, USA). The physical properties and product information are presented in Table 1. The crystallinity index (CrI) was determined by powder X-ray diffraction (XRD) (STOE & Cie GmbH, Darmstadt, Germany). XRD patterns were obtained using CuKα radiation, a diffraction angle 2θ from 10° to 30° (step size of 0.02°) and a counting time of 2 s. The CrI was calculated using the equation CrI [%] = (I 002 -I AM )/I 002 × 100, where I 002 is the maximum intensity of the crystalline plane (002) reflection (2θ = 22.5°) and I AM is the intensity of the scattering for the amorphous component at about 18° in cellulose-I . Different CrI values for Sigmacell 101 can be found in the literature [37, 38]. This may be explained by the varying quality of cellulose depending on batches and production location . Nevertheless, Sigmacell 101 is typically chosen as a more amorphous cellulose . The weight-average degree of polymerization (DP w ) was determined by gel permeation chromatography . The geometric mean particle size (d P ) was analysed by laser diffraction  using a LS13320 (Beckman Coulter, CA, USA).
Purification of cellulases
The commercial cellulase preparation Celluclast® 1.5L (Novozymes, Bagsværd, Denmark) was applied to purify the cellulases CBH I, CBH II, EG I and EG II by using column chromatography, with an Äkta FPLC (GE Healthcare, Buckinghamshire, UK) which automatically measures conductivity and ultraviolet absorbance at 280 nm. All the columns were purchased from GE Healthcare. In addition, chromatographic experiments were carried out at room temperature and the automatically collected fractions were directly cooled at 4°C. For anion exchange chromatography, 7.5 mL Celluclast® was previously rebuffered using 0.05 M Tris-HCl buffer (pH 7) and Sephadex G-25 Fine (dimensions: 2.6 cm × 10 cm) at 110 cm/h. The rebuffered sample was loaded on DEAE-Sepharose (dimensions: 1.6 cm × 10 cm) at 60 cm/h using 0.05 M Tris-HCl (pH 7) as a running buffer. The bound proteins were eluted stepwise (35% v/v, 100% v/v) with 0.2 M sodium chloride in 0.05 M Tris-HCl buffer (pH 7). Furthermore, hydrophobic interaction chromatography was performed with 1 M ammonium acetate buffer (pH 5.5) and phenyl-Sepharose (dimensions: 1.6 cm × 2.5 cm) at 30 cm/h. No additional salts had to be added as ammonium effectively promotes ligand-protein interactions in hydrophobic interaction chromatography [42, 43]. After loading of a rebuffered sample, the bound proteins were eluted with 0.05 M ammonium acetate buffer (pH 5.5). Moreover, cation exchange chromatography was performed with 0.02 M sodium acetate buffer (pH 3.6) and SP-Sepharose (dimensions: 1.6 cm × 2.5 cm) at 60 cm/h. The rebuffered sample was loaded and bound proteins were eluted stepwise (15% v/v, 100% v/v) with 1 M sodium chloride in 0.02 M sodium acetate buffer (pH 3.6). Finally, when size exclusion chromatography (SEC; dimensions: 1.6 cm × 60 cm) was applied, a 0.6 mL sample was directly injected using 0.01 M sodium acetate buffer (pH 4.8) and Sephacryl S-200 HR at 15 cm/h.
Measurement of protein concentration
After cellulase purification, the protein concentrations of the final samples were analysed by the bicinchoninic acid assay  using the BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA) and bovine serum albumin as a standard. The absorbance at 562 nm was measured with a Synergy 4 microtitre plate reader (BioTek Inst, VT, USA).
SDS-polyacrylamide gel electrophoresis
SDS-polyacrylamide gel electrophoresis  was applied to analyse the identity and purity of single cellulases. Novex® 12% polyacrylamide Tris-Glycine gels (Invitrogen, CA, USA) and samples were prepared according to the manufacturer's protocol. A prestained protein marker (New England Biolabs, MA, USA) was used as a molecular mass marker. Finally, the proteins were stained with coomassie brilliant blue  and analysed densitometrically using the scanner Perfection V700 (Epson, Suwa, Japan).
Adsorption experiments were performed in 0.1 M sodium acetate buffer (pH 4.8) using 10 g/L α-cellulose and various amounts (see below) of the particular cellulases CBH I and EG I. Solutions with α-cellulose and solutions with cellulases were preincubated separately at 45°C for 10 min and experiments were started by mixing both solutions. The final mixtures were incubated as duplicates in 2 mL Eppendorf tubes with a filling volume V L = 1 mL on a thermo mixer MHR23 (HLC Biotech, Bovenden, Germany) with a shaking diameter d 0 = 3 mm. Blanks, either without cellulase, neither substrate nor cellulase or without substrate were incubated similarly. The reaction was stopped by centrifugation (8000 g, 1 min), and the supernatants were immediately analysed for unbound cellulase using the bicinchoninic acid assay. As single cellulases and short incubation times were applied, only small amounts of reducing sugars were produced and, therefore, cellulase adsorption could be analysed by the bicinchoninic acid assay . The adsorbed cellulase concentration was calculated as the difference between initial (blanks) and unbound cellulase concentration.
in which: A denotes the amount of adsorbed cellulase (μmolcellulase/gcellulose); A max , the maximum cellulase adsorption at equilibrium (μmolcellulase/gcellulose); E, the free cellulase concentration (μmolcellulase/L); and K D , the dissociation constant (μmolcellulase/L).
in which A(t) is the amount of adsorbed cellulase (μmolcellulase/gcellulose) at time t (s), and k ad reflects the pseudo-first-order adsorption rate constant for approaching equilibrium (s-1).
Activity experiments and sugar analysis
Cellulase activity assays with a final concentration of 10 g/L cellulose and 0.1 g/L enzyme were performed in 0.1 M sodium acetate buffer (pH 4.8). The mixtures were incubated as triplicates in 2 mL Eppendorf tubes with V L = 1 mL on a thermo mixer MHR23 at 45°C, n = 0-1000 rpm and d 0 = 3 mm. Depending on the substrate applied, the incubation time was: 10 min for CMC; 120 min for Avicel PH101; 30 min for Sigmacell 101; and 60 min for α-cellulose. The cellulase activities on Avicel and CMC were used to differentiate CBHs and EGs, respectively . Blanks, either without cellulase - neither substrate nor cellulase - or without substrate, were incubated similarly. Preliminary kinetic experiments showed that inhibiting product concentrations were not reached during the applied incubation times. In addition, low enzyme concentrations were applied, so jamming of cellulases could be neglected . After incubation, the reaction was stopped by boiling for 10 min. The amount of released reducing sugars was determined with the dinitrosalicylic acid assay . For accurate determination of low reducing sugar concentrations, 1.25 g/L glucose was added to each sample . The absorbencies were measured at 540 nm in a Synergy 4 microtitre plate reader. Product concentrations were calculated using glucose as a standard and activities were expressed as the unit U (μmolglucose equivalents/min). As CBHs and EGs show different product profiles , cellulase activities may be underestimated when glucose is used as a standard in reducing sugar assays . Nevertheless, glucose is often applied  when analyzing relative changes in single cellulase activities.
Determination of hydrodynamics
The critical shaking frequency (n crit ) is reached when the labour delivered by the centrifugal force is equal to the surface tension of the liquid. Since sodium acetate is capillary-inactive and cellulose loading was low, both their impacts were negligible.
After SDS-polyacrylamide gel electrophoresis, the molecular mass and purity of each cellulase were analysed using the software TotalLab TL100 (Nonlinear Dynamics, Newcastle, UK). Parameters of the mathematical adsorption models were calculated by nonlinear, least squares regression analysis using MATLAB version R2008b (The MathWorks, MA, USA).
Results and discussion
Purification of cellulases
Langmuir and kinetic adsorption parameters of purified cellulases using α-cellulose at n = 1000 rpm
Langmuir adsorption parameters*
Kinetic adsorption parameters†
A max [μmol/g]
K D [μmol/L]
A max [μmol/g]
k ad [s -1 ]
0.155‡ ± 0.003
0.433 ± 0.039
0.170‡ ± 0.003
0.0031 ± 0.0002
0.212 ± 0.010
2.146 ± 0.216
0.213 ± 0.007
0.0019 ± 0.0002
Influence of hydrodynamics
As seen in Figure 6B, the activities of CBH I and EG I were also investigated at different shaking frequencies using α-cellulose as substrate. For both cellulases, the same trend in activity was observed, whereby a sharp increase occurred between 400 rpm and 800 rpm as in the adsorption kinetic experiments (Figure 6A). Consequently, higher shaking frequencies clearly improved the adsorption of cellulases, thereby bolstering their respective activity, because adsorption is a prerequisite for cellulose hydrolysis [4, 12, 13]. Thus, when short incubation times of cellulases are applied (for example, washing agent), catalyst optimization should also be focused on improving the cellulase binding properties. In other studies, the effect of agitation on cellulose hydrolysis was investigated without considering adsorption. These studies showed that enhanced agitation increases initial cellulose hydrolysis rates [85–87]. However, attention has to be paid to cellulase inactivation reducing the final yield of cellulose hydrolysis . This is especially important when using high solid concentrations  and shear-force sensitive cellulases . In this current study, however, low solid concentrations, short incubation times and a shaken system were applied, so cellulase inactivation could be neglected. Moreover, upon using immobilized or displayed cellulases [91–93], lower shaking frequencies are beneficial to ensure sufficient surface contact between cellulase and solid substrate . In comparison to the adsorption parameters, a disproportionate increase in cellulase activity was observed with enhanced agitation (Figure 6A and B). As CBHs and EGs are inhibited by soluble hydrolysis products, such as glucose and cellobiose [95–97], agitation may transport these inhibiting products away from the cellulases, thus decreasing the local concentration of inhibiting products and improving the cellulase activity.
In order to understand the influence of shaking frequency on the adsorption and cellulase activity, the hydrodynamics inside the respective reaction tube were investigated in detail. Pictures of the liquid phase with immersed α-cellulose particles were taken at different shaking frequencies (Figure 6C). For n ≤ 200 rpm, the liquid surface remained horizontal and no liquid mixing was observed. Once n ≥ 400 rpm, liquid mixing started (white arrow; Figure 6C). According to Hermann et al. , a critical shaking frequency (n crit ) is necessary for liquid mixing and can be calculated according to Eqn (3). In this current investigation, n crit was 260 rpm (black arrows; Figure 6A and B), which fitted well to the start of liquid mixing between 200 rpm and 400 rpm. The boundary layer at the cellulose-liquid interface is relatively thick without mixing  and can decrease the rate of cellulase adsorption. However, mixing was increased by exceeding n crit , leading to a decrease in the width of the boundary layer at the cellulose-liquid interface. Since adsorption kinetics and cellulase activity did not significantly change between 0 rpm and 400 rpm, liquid mixing was not the rate limiting step.
A sharp increase in adsorption parameter values and cellulase activities was observed once suspension of cellulose particles began (n = 600 rpm). As soon as the particles were completely suspended (n = 800 rpm), their whole surface was exposed to the liquid and all external cellulose binding sites were accessible to the cellulases. Hence, an optimal particle-liquid mass transfer was achieved  and the parameters A max , k ad as well as cellulase activities reached their maximum values. Complete suspension is defined as the point when no particles are deposited on the tank bottom for longer than one second . This criterion is designated as the just suspending speed or off bottom speed. A correlation for calculating the just suspending speed in shaking vessels can be found in the literature . However, it can not be applied to cellulose particles because of their fibrous structure and wide particle size distribution. Complete suspension is known to be required for high cellulase activity . However, this current paper shows for the first time the effect of suspension on cellulase adsorption as well as the correlation between the adsorption and the activity of cellulases at different hydrodynamic conditions.
Cellulase activity with conventional model substrates and α-cellulose
In this study, insoluble α-cellulose was found to be an excellent practical substrate to characterize and screen for purified CBHs and EGs. First, a novel and reproducible purification method was established to prepare the major cellulases from T. reesei with high purity. Second, the adsorption isotherms and kinetics of the purified cellulases were analysed for the first time using α-cellulose as a cellulosic substrate. Here, the calculated adsorption parameters (A max , K D , k ad ) of the studied cellulases were comparable to those for filter paper as an established model substrate. In addition, this investigation shows in detail, for the first time, the effect of hydrodynamics on cellulase adsorption as well as the correlation between the adsorption and the activity of cellulases at different hydrodynamic conditions. Complete suspension of α-cellulose particles clearly enhanced the adsorption of cellulases thereby augmenting cellulase activity. By comparing conventional model substrates with α-cellulose, CBHs and EGs showed similar cellulase activities only on insoluble α-cellulose.
Even though other researchers use conventional pure cellulosic substrates, these are not suitable for characterizing purified CBHs and EGs. Instead, α-cellulose is ideal when alkaline pretreatment is considered as a previous pretreatment step. In the future, screening experiments should be conducted with α-cellulose so that proper cellulases are selected to best hydrolyze the real alkaline-pretreated biomass used in biorefineries. In addition, α-cellulose can be used in automated screening platforms  as suspensions of α-cellulose particles can be handled by pipetting. Since lignocellulose pretreatment in biorefineries significantly alters the structure of cellulose, cellulases should be characterized with other practical cellulosic substrates that represent other pretreatment techniques [105, 106] (such as ammonia fibre explosion, ionic liquid process, organosolv process and steam explosion).
size exclusion chromatography
- A :
adsorbed cellulase (μmolcellulase/gcellulose)
- A max :
maximum cellulase adsorption at equilibrium (μmolcellulase/gcellulose)
- CrI :
crystallinity index (%)
- d 0 :
shaking diameter (mm)
- d p :
geometric mean particle size (μm)
- DP w :
weight-average apparent degree of polymerization (AGU)
- D t :
inner tube diameter (mm)
- E :
free cellulase concentration (μmolcellulase/L)
- I 002 :
maximum intensity of the crystalline plane (002) reflection (s-1)
- I AM :
XRD scattering for the amorphous component at 18° in cellulose-I (s-1)
- k ad :
pseudo-first-order adsorption rate constant (s-1)
- K D :
dissociation constant (μmolcellulase/L)
- n :
shaking frequency (rpm)
- n crit :
critical shaking frequency for liquid mixing (rpm)
- t :
- T :
- V L :
filling volume (mL)
- θ :
diffraction angle (°)
- ρ L :
liquid density (kg/L)
- σ :
surface tension (N/m)
This work was performed as part of the Cluster of Excellence 'Tailor-Made Fuels from Biomass', which is funded by the Excellence Initiative by the German federal and state governments to promote science and research at German universities.
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