Biofuel generation from sources such as cornstarch, sugarcane or sweet sorghum syrups, produces large amounts of biomass waste products. For example, over 90% of the plant is unused in the case of ethanol production from cornstarch [1–3]. Current commercial enterprises produce ethanol from cornstarch, sugar cane or sweet sorghum syrups and large amounts of waste biomass accumulate alongside refineries and most of them are not recycled. Biofuels production would therefore be significantly more efficiently utilized if this biomass could be converted into fermentable sugars.
Biomass polysaccharides consist of cellulose, hemicellulose and pectin. Cellulose is a linear, crystalline self-assembled nanofiber formed from the linear polymer containing exclusively glucose monomers linked through β-1,4-glycosydic bonds [1–4]. Recalcitrance of cellulose towards enzymatic degradation is a widely reported phenomenon [1, 5, 6] and is the result of the wide range of possible intra- and inter-molecular hydrogen bonds within and among linear cellulose molecules assuming crystalline or amorphous cellulosic nanofiber structures. Cell walls from different plants contain various amounts of crystalline/amorphous cellulose fibers , reflected by the relative crystalinity index (RCI).
Because cellulose is structurally complex and crystalline in nature, it is recalcitrant towards microbial or enzymatic attack. Unfortunately, current pretreatment methods – e.g., acid hydrolysis [8, 9] or pyrolysis [10, 11], generate compounds that inhibit subsequent fermentation. Enzymatic hydrolysis of cellulose results in glucose the universal carbon source for all organisms to drive oxidative metabolism including the production of biofuels, chemicals and pharmaceuticals.
A complete enzymatic cellulose degrading system consists of at least three related partially redundant biochemical reactions. Endo-glucanases (EC 22.214.171.124) randomly hydrolyze internal glycosydic bonds to decrease the length of the cellulose chain and multiply polymer ends [12–14]. Exo-glucanases (EC 126.96.36.199) split-off cellobiose from cellulose termini [15–17] and β-glucosidases (EC 188.8.131.52) hydrolyze cellobiose and oligomers to render glucose [18–20]. All three types of enzymes have similar catalytic domains all splitting the β-1,4-glycosidc bond between glucose molecules, however they differ in their binding and substrate interaction domains resulting in cooperation and synergism in releasing glucose [21–23].
Exo-glucanases hydrolyze cellulose chains by removing cellobiose either from the non-reducing end (GH6, EC 184.108.40.206) or reducing end (GH7, EC 220.127.116.11), which in both cases results in the release of reducing sugars (cellobiose) but little polymer length reduction . In fungi, exo-glucanases are commonly known as 1,4-β-D-glucan cellobiohydrolases . The main characteristic of a cellobiohydrolase is its processive action on individual cellulose chains by reiterated release of cellobiose [24, 26–32].
The catalytic domain (CD) of a typical cellobiohydrolase forms a channel-shaped cavity topped by several flexible loops resulting in a tunnel like structure [33–39], which is frequently connected through a linker to a carbohydrate binding domain (CBD) [40, 41]. CBDs are thought to bind to the crystalline cellulosic fiber sliding down the cellulosic surface [6, 42] and carrying the CD with it. Three tyrosine residues on the CBD hydrophobic surface align with the cellulose chain adjacent to the reducing end and a fourth tyrosine residue moves from its internal position to form van der Waals interactions with the cellulose surface resulting in an induced change near the surface allowing the CBD to progress [16, 40].
From a comprehensive bioinformatics survey of seven Aspergillii completely sequenced genomes, we found between three and five genes encoding cellobiohydrolases, typically two in each of the CAZy families GH6 and GH7, respectively. Remarkably, for each GH family 6 or 7 fungal cellobiohydrolase examined, one enzyme was linked to a CBD and a second was not.
Here we describe two GH7-cellobiohydrolases, Cbh1 and CelD from Aspergillus niveus. The structural differences between these two enzymes were the lack of a CBD in CelD and a conserved loop that obstructed the catalytic tunnel in enzymes that were linked to a CBD, but missing in enzymes that did not link to a CBD, ensuring that the tunnel was always open. These structural differences reflected directly on kinetic properties, thermostability, inhibition by glucose and cellobiose and the way these proteins interacted with crystalline substrate molecules. We hypothesize that the cellobiohydrolase linked to a CBD is competent to release cellulosic chains from the crystalline nanofiber and the cellobiohydrolase without a CBD can only use loose ends, without the aid of a CBD.