Practical screening of purified cellobiohydrolases and endoglucanases with a-cellulose and specification of hydrodynamics

Background: 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 a-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 a-cellulose. Results: 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 a-cellulose with three typically used celluloses, the exact cellulase activities towards all four substrates were measured. It was found that, using a-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 a-cellulose and thus bolstered their activity. Complete suspension of a-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 a-cellulose. Conclusions: a-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 a-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 a-cellulose so that proper cellulases are selected to best hydrolyze the real alkaline-pretreated biomass used in biorefineries. Background 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 3.2.1.91), endoglucanase (EG, EC 3.2.1.4) and b-glucosidase (EC 3.2.1.21). As the cellulose depolymerization performed by CBHs and EGs is the rate-limiting step for the whole hydrolysis [7], 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 [7]. Thus, screening experiments need to be conducted with a more practical substrate such as a-cellulose so that proper cellulases are * Correspondence: [email protected] AVT-Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany Full list of author information is available at the end of the article Jäger et al. Biotechnology for Biofuels 2010, 3:18 http://www.biotechnologyforbiofuels.com/content/3/1/18 © 2010 Jäger et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. selected which best hydrolyze the biomass actually used in biorefineries. a-Cellulose is a solid residue of lignocellulose after extraction with strong alkali [8-10] and mainly consists of cellulose and a small amount of hemicellulose (Table 1) [11]. a-Cellulose exhibits similar crystallinity and porosity to wood biomass [12] and shows the natural structure of cellulose fibres (Figure 1). Up to now, it has just been used for assaying total cellulase activity [7]. In contrast, conventional model substrates, further processed from a-cellulose, are more artificial [8], because they are dyed, derivatized or water-soluble and show unnatural physical properties (such as crystallinity, degree of polymerization, porosity) [7]. Consequently, a-cellulose is more natural and most similar to alkaline-pretreated cellulosic biomass used in biorefineries [7]. Since a-cellulose is insoluble, the adsorption of cellulases onto a-cellulose is a prerequisite for hydrolysis [4,12,13]. Cellulase adsorption is usually analysed using the Langmuir isotherm [14]. 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 a-cellulose or other fibrous substrates [14], 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 a-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.