Biological pretreatment of cellulose: Enhancing enzymatic hydrolysis rate using cellulose-binding domains from cellulases Mélanie Hall a , Prabuddha Bansal b , Jay H. Lee c , Matthew J. Realff b , Andreas S. Bommarius b,⇑ a Department of Chemistry, Organic and Bioorganic Chemistry, Heinrichstraße 28, 8010 Graz, Austria b School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive, N.W., Atlanta, GA 30332-0100, USA c Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Korea article info Article history: Received 13 August 2010 Received in revised form 27 October 2010 Accepted 1 November 2010 Available online 9 November 2010 Keywords: Cellulose-binding domains Biofuel Cellulose hydrolysis Pretreatment Crystallinity abstract In this study, cellulose-binding domains (CBDs) of cellulases from Trichoderma reesei were used in a pre- treatment step and were found to effectively reduce the crystallinity of cellulose (both Avicel and fibrous cellulose). This, in turn, led to higher glucose concentrations (up to 25% increase) in subsequent hydro- lysis of cellulose using a mixture of cellulases and without the need for any intermediate purification step. CBDs were shown to be active in a range of temperatures (up to 50 °C), while cellulase hydrolytic activity was greatly reduced after incubation at 50 °C. This was explained by retention of full binding capacity after incubation at 50 °C for 15 h. Our findings suggest that CBDs may be a valuable tool in pre- treating cellulose and eventually afford faster enzymatic conversion of cellulose to glucose, thus contrib- uting to more affordable processes in the production of biofuels. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction The current and urgent need for alternative renewable energies to supplement petroleum-based fuels has led to the development of various technologies based on wind, solar, tidal energy or bio- mass. The use of biomass as a source for biofuels relies on the great abundance of cellulose, as it is one of the most abundant sources of organic material on our planet. Currently, widespread production of biofuels from lignocellulosic materials is prevented by high processing costs. To render lignocellulosic biofuels more competi- tive, improvements in the enzymatic degradation of cellulose are crucial (Himmel et al., 2007; Lynd et al., 2008). The enzymatic hydrolysis of cellulose, a polysaccharide consisting of repeated cellobiose units (glucose dimers), requires the synergistic action of various cellulases (endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91) and b-glucosidase (EC 3.2.1.21)) to eventually pro- duce glucose, a valuable key intermediate that can be fermented to bioalcohols or transformed into various other chemicals (Davies and Henrissat, 1995; Lynd et al., 2002). Various process limitations account for overall slow reaction and decreasing rates as the con- version proceeds, and can be directly related to cellulose properties (insoluble nature, crystallinity, surface area, porosity, degree of polymerization) and enzymatic features (deactivation, inhibition, jamming, clogging, adsorption, processivity) (Bansal et al., 2009; Hall et al., 2010; Zhang and Lynd, 2004). Cellulases are glycosyl hydrolases of greatly varying structure, though they all catalyze the hydrolysis of 1,4-b-D-glycosidic bonds (Davies and Henrissat, 1995). Most cellulases consist of three do- mains: a catalytic domain (the locus of hydrolysis), a cellulose- binding domain (that anchors the whole enzyme onto the cellulose surface and orients the cellulose fiber towards the tunnel contain- ing the active site) and a glycosylated flexible linker connecting the two other domains (and providing sufficient spatial separation re- quired between them; it allows processive motion and likely also plays a role in energy storage) (Gilkes et al., 1991; Zhao et al., 2008). Cellulose-binding domains (CBDs) of cellulases from Tricho- derma reesei (belonging to family I CBD) form a wedge-like fold, where a flat face provides key (aromatic) residues strongly inter- acting with crystalline cellulose (Linder et al., 1995; Takashima et al., 2007). Recent investigation of the effect of CBDs (without the catalytic unit) on cellulose provides evidence that family I CBD may play a role in the hydrolysis of crystalline cellulose by weakening and splitting off the hydrogen bond network in the cel- lulose crystal (Gao et al., 2001; Wang et al., 2008; Xiao et al., 2001). Given our latest study on the role of cellulose crystallinity in deter- mining hydrolysis rate (Hall et al., 2010), and given the processive nature of cellulases (Beckham et al., 2010), we hypothesized that CBDs could be used in a separate step prior to the hydrolysis, and without the requirement for change of solvent, such as in the case of pretreatment with ionic liquids (Brennan et al., 2010; 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.010 ⇑ Corresponding author. Tel.: +1 404 385 1334; fax: +1 404 894 2295. E-mail address: Andreas.Bommarius@chbe.gatech.edu (A.S. Bommarius). Bioresource Technology 102 (2011) 2910–2915 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech