Carbohydrate Polymers 97 (2013) 581–586 Contents lists available at SciVerse ScienceDirect Carbohydrate Polymers jo ur nal homep age: www.elsevier.com/locate/carbpol Short communication Assessing cellulose microfibrillar structure changes due to cellulase action Tina Jeoh ∗ , Monica C. Santa-Maria 1 , Patrick J. O’Dell Department of Biological and Agricultural Engineering, University of California at Davis, Davis, CA 95616, United States a r t i c l e i n f o Article history: Received 21 December 2012 Received in revised form 13 May 2013 Accepted 14 May 2013 Available online 21 May 2013 Keywords: Trichoderma reesei Cel7A Cellobiohydrolase Cellulose microfibrils Fibrillation AFM a b s t r a c t There is a need to understand how cellulose structural properties impact productive cellulase–cellulose interactions toward solving the mechanisms of the heterogeneous reaction. We coupled biochemical studies of cellulose hydrolysis by a purified Trichoderma reesei Cel7A (TrCel7A) cellobiohydrolase with atomic force microscopy (AFM) to study the impact of the cellulolytic activity on the fibrillar structure of cellulose. Bacterial cellulose (BC) fibrils were hydrolyzed by TrCel7A then immobilized by hydrophobic interactions on glass for AFM imaging. Commonly used methods to culture and isolate cellulose fibrils resulted in significant oxidation of the reducing-ends but minimal oxidation along the fibrils. We observed extensive fibrillation of BC fibrils to ∼3 nm microfibrils during the course of hydrolysis by TrCel7A, leaving thinned un-fibrillated recalcitrant fibrils at >80% hydrolysis extents. Additionally, this remaining fraction appeared to be segmented along the fibril length. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Cellulose is of industrial importance as a renewable resource for conversion to biofuels, biochemicals (Foust, Aden, Dutta, & Phillips, 2009; Himmel et al., 2007) and as functional components in advanced materials (Lahiji, Boluk, & McDermott, 2012; Lu & Hsieh, 2010). Due to its natural recalcitrance, one of the biggest technological challenges to realizing a cellulosic bioproducts indus- try lies in engineering inexpensive strategies to depolymerize cellulose. A persistent mystery surrounding cellulase hydrolysis of cellu- lose is the characteristic decline in hydrolysis rates over the course of the reaction. In studies using purified Trichoderma reesei Cel7A (TrCel7A), a reducing-end specific cellobiohydrolase, it was shown that the hydrolysis rate decline tracks with a decline in the appar- ent catalytic rate constant (Jalak, Kuraˇ shin, Teugjas, & Väljamäe, 2012; Kurasin & Valjamae, 2011). Substrate properties have been speculated to be the limiting factor (Cruys-Bagger, Elmerdahl, & Praestgaard, 2012; Kurasin & Valjamae, 2011; Zhang, Wolfgang, & Wilson, 1999), but understanding is limited. Native cellulose microfibrils are linear cellulose polymers associated via extensive hydrogen bonding and hydrophobic stacking interactions to orga- nize into crystalline lattices that exclude water (Nishiyama, Langan, & Chanzy, 2002; Nishiyama, Sugiyama, Chanzy, & Langan, 2003). ∗ Corresponding author. Tel.: +1 530 752 1020; fax: +1 530 752 2460. E-mail address: tjeoh@ucdavis.edu (T. Jeoh). 1 Present address: Global Alimentos S.A.C., Sevilla 244, Lima 18, Peru. Microfibrils associate into larger fibrils (White & Brown, 1981) resulting in insoluble fibrillar structures where only celluloses on the outer surfaces are accessible to hydrolysis at the solid/aqueous interface by soluble cellulolytic enzymes. Cellulose fibrils undergo macroscopic changes in the supramolecular organization (Chanzy, Henrissat, Vuong, & Schulein, 1983; Santa-Maria & Jeoh, 2010; White & Brown, 1981) and nano-scale changes in surface properties (Wang et al., 2012) due to cellulolytic action. Here we present details of methods to characterize and immo- bilize cellulose fibrils to facilitate biochemical and imaging studies of cellulose fibrillar properties. With these methods, we present results showing changes in the microstructure of cellulose fibrils due to the action of a purified TrCel7A. 2. Materials and methods 2.1. Preparation of cellulose fibrils Pellicles from Gluconacetobacter xylinus (ATCC 700178) cul- tures (Santa-Maria & Jeoh, 2010) were rinsed, then washed in 1% sodium hydroxide (4 ◦ C, 12 h, shaking at 60 rpm) and 0.3% sodium hypochlorite (pH 4.9 adjusted with glacial acetic acid, 2 h, 70 ◦ C, gentle agitation) and repeated as necessary. The pellicles were rinsed until the conductivity reached 0.5–5 S/cm and stored with 0.02% sodium azide at 4 ◦ C. Microfibrils (4 mL of ∼0.5 mg/mL) were dispersed by ultra- sonication with a 1/8 in microtip (Misonix Ultrasonicator S-4000, Qsonica, LLC, Newtown, CT) at 30% amplitude and 2× 15-s pulses (220–283 J). 0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carbpol.2013.05.027