Simulation studies of the insolubility of cellulose Malin Bergenstråhle a , Jakob Wohlert a,  , Michael E. Himmel b , John W. Brady a, * a Department of Food Science, Cornell University, Ithaca, NY 14853, United States b National Renewable Energy Laboratory, 1617 Cole Boulevard, Golden, CO 80401-3393, United States article info Article history: Received 4 February 2010 Received in revised form 5 June 2010 Accepted 25 June 2010 Available online 6 July 2010 Keywords: Cellulase Cellobiohydrolase I Cellulose Computer modeling Molecular dynamics abstract Molecular dynamics simulations have been used to calculate the potentials of mean force for separating short cellooligomers in aqueous solution as a means of estimating the contributions of hydrophobic stacking and hydrogen bonding to the insolubility of crystalline cellulose. A series of four potential of mean force (pmf) calculations for glucose, cellobiose, cellotriose, and cellotetraose in aqueous solution were performed for situations in which the molecules were initially placed with their hydrophobic faces stacked against one another, and another for the cases where the molecules were initially placed adjacent to one another in a co-planar, hydrogen-bonded arrangement, as they would be in cellulose Ib. From these calculations, it was found that hydrophobic association does indeed favor a crystal-like structure over solution, as might be expected. Somewhat more surprisingly, hydrogen bonding also favored the crystal packing, possibly in part because of the high entropic cost for hydrating glucose hydroxyl groups, which significantly restricts the configurational freedom of the hydrogen-bonded waters. The crystal was also favored by the observation that there was no increase in chain configurational entropy upon disso- lution, because the free chain adopts only one conformation, as previously observed, but against intuitive expectations, apparently due to the persistence of the intramolecular O3–O5 hydrogen bond. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Cellulose, the b-(1?4)-linked polymer of D-glucose that is the primary structural component of plant cell walls, has been one of the most studied of all biopolymer molecules. In the plant cell wall, cellulose acts as the load-bearing component, and co-exists with other cell wall polymers, such as hemicelluloses and lignin, as well as with water. 1 As the principal cell wall component, it is the single most abundant biological molecule in the biosphere, and thus rep- resents the most important feedstock for the industrial production of liquid fuels using biomass conversion technologies, a topic of considerable current interest. 2 Its biosynthesis takes place on the surfaces of the plasma membranes of plant cell walls, where newly formed cellulose chains are organized (coalesced) into elementary fibrils with lateral dimensions of 3–5 nm. 3 The nature of the aggre- gated structure of these microfibrils is complex and still an open question. Cellulose may aggregate in at least six different crystal polymorphs, and in addition exists in less-ordered structures, sometimes referred to as amorphous or paracrystalline cellulose. 1,4 Native crystalline cellulose is found in a metastable form called cellulose I, with two polymorphs, cellulose Ib and Ia, differing slightly in their unit cells. 5 These crystalline polymorphs are found to co-exist in nature with source-dependent ratios. 6,7 When native cellulose is processed through regeneration or mercerization, it will irreversibly restructure to form so-called cellulose II. The unit cell of cellulose II differs from that of cellulose I in that neighboring chains are probably oriented in an antiparallel fashion, instead of parallel as they are in the native crystals. 1,8,9 The most significant limitation for the use of cellulosic material for fuel production is its insolubility in water, which hinders the action of cellulase enzymes. Cellulose remains completely insolu- ble below temperatures of about 300 °C, where it rapidly decom- poses. 10 This insolubility is a strong function of the chain length; as the degree of polymerization (DP) of cellooligomers increases, their solubility rapidly drops to zero beyond celloheptaose. 11 Cellu- lose insolubility makes evolutionary sense in terms of its biological role. As the structural framework of plant cell walls, it would be highly disadvantageous if cellulose dissolved on contact with water. However, while the biological advantages arising from insolubility are clear, the physical reasons for this insolubility of cellulose are not obvious. The fact of cellulose insolubility has been so well-known for so long that rarely are the features of this poly- mer that contribute to its insolubility examined. When attempting to understand the solubility of a molecule, it is necessary to compare the many contributions to the free energy of both the solid and solution states. For example, cellulose in solution would be expected to have much larger translational and rotational entropy than when confined in the crystal, and the solution of course is also favored by the entropy of mixing. These 0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2010.06.017 * Corresponding author. Tel.: +1 (607) 255 2897. E-mail address: jwb7@cornell.edu (J.W. Brady).   Present address: Wallenberg Wood Science Center, Teknikringen 56-58, Royal Institute of Technology, SE-10044 Stockholm, Sweden. Carbohydrate Research 345 (2010) 2060–2066 Contents lists available at ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres