The use of renewable resources for the production of fuels and chemicals has been a continuing topic of interest. The bioconversion process involves the use of enzymes to convert cellulose into fermentable sugars, which are then substrate for further processing into mainly ethanol. The core pool of enzymes, known generally as cellulases, makes up a well established system of action divided in two groups: cellobiohydrolases and endoglucanases, plus a third component known as the β-glucosidase [1
Despite significant progress in this field, the enzymatic deconstruction of the lignocellulosic biomass is not yet fully understood, especially regarding the action of non-hydrolytic enzymatic activities [2
]. Cellulose-degrading microorganisms also produce accessory proteins that are co-regulated and co-expressed with the cellulase enzymes. These auxiliary proteins do not hydrolyze cellulosic material per se
, but play a significant role in enhancing the yield by increasing the access of cellulases to the substrate and opening the crystalline structure: such enzymes are the swollenins and expansins [3
]. A novel auxiliary enzyme activity capable of an oxidative cleavage of the glycosidic bond is currently classified in the Glycoside Hydrolase family 61 (GH61) [4
]. Since Vaaje-Kolstad et al.
] identified the oxidative process as a result of enzymatic activity, a variety of GH61-like proteins from different fungi as well as bacteria (GH61D from P. chrysosporium
], GH61A from T. auranticus
], several from N. crassa
] and CelS2 (CBM33) from S. coelicolor
]) have been isolated and studied. Notwithstanding the key role of GH61, the correct placing on the lignocellulosic degradation scenario, especially in relation to the classical cellulases, still remains ambiguous. Although a final model mechanism of action has not yet been found, some common features can be generalized: i) GH61s are metallo-enzymes that need a bivalent metal ion to act, and copper seems to be the metal ion coordinated in the active site; ii) since the oxidation of the glycosidic bond is the main activity, all GH61s need a reductant cofactor that works as an external electron donor: gallate, ascorbate, and the enzyme CDH (often up regulated and expressed together with GH61s [8
]) are indicated to enhance the GH61s activity; iii) as substrate, aggregated cellulose is preferred: no activity was detected on soluble cellodextrines; iv) finally but most important, mass spectrometry and HPAEC analysis of reaction products of GH61s show a variety (different DP) of native as well as oxidized cellodextrines as a result of the glycosidic bond cleavage. Even though the oxidation may take place at several carbons in the glucose ring structure (C1, C4 or C6), the C1 oxidized (aldonic) cellodextrines are the most represented [5
During enzymatic deconstruction of lignocelluloses, the presence of exocellulase and β-glucosidase enzymes rapidly degrade native as well as oxidized cellodextrines into di- and monosaccharides. We suppose that cellobiohydrolases also hydrolyze those cellodextrines carrying a C1 oxidized glucose thereby releasing cellobiose and cellobionic acid as products. Similarly, we suppose that cellobionic acid is also one of the substrates for β-glucosidase. Thus the final products expected in a hydrolysate are native glucose and intermediate cellobiose, as well as their oxidized forms glucono-δ-lactone/gluconic acid and cellobio-δ-lactone/cellobionic acid respectively as shown in Figure . Once C1 oxidized cello-oligosaccharides are produced in solution, there exist a chemical equilibrium between their lactone and aldonic acid forms which is dependent on pH, temperature, and concentration [11
]. The lactone form can hydrolyze non-enzymatically to the aldonic acid form. The rate of aldonic acid formation can be increased by lowering the pH, but lactonization as well as its reverse reaction does not alter the acidity of the solution [12
]. During the enzymatic hydrolysis process at a pH of about 5, equilibrium tends to shift toward the aldonic acids. The presence of gluconic acid is relevant from an industrial bioconversion point of view because it has been proven to be a β-glucosidase inhibitor [13
] and is also a non-fermentable sugar for S. cerevisiae
], which in turn means that part of the potential glucose is lost as gluconic acid, that cannot be fermented into ethanol.
Oxidized C1 products from glucose.
The boosting and synergetic effect of oxidative enzymes such as GH61 on lignocellulosic hydrolysis is well recognized [5
] and oxidative enzymes are now present in commercially available cellulase preparations to improve the conversion yields [16
]. An example is Novozymes Cellic CTec2 (used in this work) as opposed to its predecessor, the combined Celluclast 1.5 L/Novozym 188 mixture (Novozymes A/S, Bagsværd, Denmark). Despite the presence of two genes encoding for the GH61 family enzymes in Trichoderma reesei
], oxidized products are generally not found in cellulose and lignocellulosic hydrolysate using commercial T. reesei
cellulolytic systems [19
Most of the papers cited above show the production of oxidized products by GH61 activity under ideal conditions using pure cellulose or PASC (phosphoric acid swollen cellulose) as substrate at low dry matter concentration and boosted by an externally added electron donor. In this work we wanted to study the action of oxidative enzymes (GH61) in commercial enzyme preparations during hydrolysis of an industrially relevant substrate at conditions as close as possible to a setup for bioethanol production. The substrate used was hydrothermally pretreated wheat straw at very high dry matter concentration (30% water insoluble solids, WIS), with lignin present and without the addition of electron donors as normally used in studies of oxidative enzymes. The work focused on the production of oxidized products at various process conditions, especially the impact on the β-glucosidase activity.