Valdeir Arantes

The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility.

The influence of cellulose accessibility and protein loading on the efficiency of enzymatic hydrolysis of steam pretreated Douglas-fir was assessed. It was apparent that the lignin component significantly influences the swelling/accessibility of cellulose as at low protein loadings (5FPU/g cellulose), only 16% of the cellulose present in the steam pretreated softwood was hydrolyzed while almost complete hydrolysis was achieved with the delignified substrate. When lignin (isolated from steam pretreated Douglas-fir) was added back in the same proportions it was originally found to the highly accessible and swollen, delignified steam pretreated softwood and to a cellulose control such as Avicel, the hydrolysis yields decreased by 9 and 46%, respectively. However, when higher enzyme loadings were employed, the greater availability of the enzyme could overcome the limitations imposed by both the lignins restrictions on cellulose accessibility and direct binding of the enzymes, resulting in a near complete hydrolysis of the cellulose.

Peculiarities of brown-rot fungi and biochemical Fenton reaction with regard to their potential as a model for bioprocessing biomass

This work reviews the brown-rot fungal biochemical mechanism involved in the biodegradation of lignified plant cell walls. This mechanism has been acquired as an apparent alternative to the energetically expensive apparatus of lignocellulose breakdown employed by white-rot fungi. The mechanism relies, at least in the incipient stage of decay, on the oxidative cleavage of glycosidic bonds in cellulose and hemicellulose and the oxidative modification and arrangement of lignin upon attack by highly destructive oxygen reactive species such as the hydroxyl radical generated non-enzymatically via Fenton chemistry

The adsorption and enzyme activity profiles of specific Trichoderma reesei cellulase/xylanase components when hydrolyzing steam pretreated corn stover.

({\text{F}}{{\text{e}}^{{{3} + }}} + {{\text{H}}_{{2}}}{{\text{O}}_{{2}}} \to {\text{F}}{{\text{e}}^{{{2} + }}} + \cdot {\text{OH}}{{ + }^{ - }}{\text{OH}})

Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis

. Modifications in the lignocellulose macrocomponents associated with this non-enzymatic attack are believed to aid in the selective, near-complete removal of polysaccharides by an incomplete cellulase suite and without causing substantial lignin removal. Utilization of this process could provide the key to making the production of biofuel and renewable chemicals from lignocellulose biomass more cost-effective and energy efficient. This review highlights the unique features of the brown-rot fungal non-enzymatic, mediated Fenton reaction mechanism, the modifications to the major plant cell wall macrocomponents, and the implications and opportunities for biomass processing for biofuels and chemicals.

Steam pretreatment of agricultural residues facilitates hemicellulose recovery while enhancing enzyme accessibility to cellulose.

The polysaccharide monooxygenase enzyme AA9 (formerly known as GH61) was shown to interact synergistically with cellulases to enhance the enzymatic hydrolysis of a range of “commercially-relevant” pretreated and “model” cellulosic substrates. Although an exogenous source of reducing power was required when AA9 was added with cellulases to a “pure” cellulosic substrate, it was not required when added to pretreated lignocellulosic substrates. It appears that the non-cellulosic components such as soluble components, lignin, and possibly hemicellulose, can all act as AA9 reducing cofactor. Of the various substrate characteristics that influenced the efficacy of the enzyme mixture, the relative amount of accessible crystalline cellulose, assessed by the specific cellulose binding module (CBM), appeared to be the most critical. Cellulases and AA9 acted synergistically when hydrolysing cellulose I but it did not occur during the hydrolysis of cellulose II and III.

The Use of Carbohydrate Binding Modules (CBMs) to Monitor Changes in Fragmentation and Cellulose Fiber Surface Morphology during Cellulase- and Swollenin-induced Deconstruction of Lignocellulosic Substrates

Recycling of enzymes during biomass conversion is one potential strategy to reduce the cost of the hydrolysis step of cellulosic ethanol production. Devising an efficient enzyme recycling strategy requires a good understanding of how the enzymes adsorb, distribute, and interact with the substrate during hydrolysis. We investigated the interaction of individual Trichoderma reesei enzymes present in a commercial cellulase mixture during the hydrolysis of steam-pretreated corn stover (SPCS). The enzyme profiles were followed using zymograms, gel electrophoresis, enzyme activity assays and mass spectrometry. The adsorption and activity profiles of 6 specific enzymes Cel7A (CBH I), Cel7B (EG I), Cel5A (EG II), Xyn 10 (endo-1,4-β-xylanase III), Xyn 11 (endo-xylanase II), and β-glucosidase were characterized. Initially, each of the enzymes rapidly adsorbed onto the SPCS. However, this was followed by partial desorption to an adsorption equilibrium where the Cel7A, Cel7B, Xyn 10, and β-glucosidase were partially adsorbed to the SPCS and also found free in solution throughout the course of hydrolysis. In contrast, the Cel5A and Xyn 11 components remained primarily free in the supernatant. The Cel7A component also exhibited a partial desorption when the rate of hydrolysis leveled off as evidenced by MUC zymogram and SDS-PAGE. Those cellulase components that did not bind to the substrate were generally less stable and lost their activities within the first 24h when compared to enzymes that were distributed in both the liquid and solid phases. Therefore, to ensure maximum enzyme activity recovery, enzyme recycling seems to be most effective when short-term rounds of hydrolysis are combined with the recovery of enzymes from both the liquid and the solid phases and potentially enzyme supplementation to replenish lost activity.

Biomimetic oxidative treatment of spruce wood studied by pyrolysis-molecular beam mass spectrometry coupled with multivariate analysis and 13C-labeled tetramethylammonium hydroxide thermochemolysis: implications for fungal degradation of wood.

A key limitation in the overall hydrolysis process is the restricted access that the hydrolytic enzymes have due to the macro-and-micro structure of cellulose and its association with hemicellulose and lignin. Previous work has shown that several non-hydrolytic proteins can disrupt cellulose structure and boost the activity of hydrolytic enzymes when purer forms of cellulose are used. In the work reported here, Swollenin primarily disrupted the hemicellulosic fraction of pretreated corn stover, resulting in the solubilisation of monomeric and oligomeric sugars. Although Swollenin showed little synergism when combined with the cellulase monocomponents exoglucanase (CEL7A) and endoglucanase (CEL5A), it showed pronounced synergism with xylanase monocomponents Xylanase GH10 and Xylanase GH11, resulting in the release of significantly more xylose (>300%). It appears that Swollenin plays a role in amorphogenesis and that its primary action is enhancing access to the hemicellulose fraction that limits or masks accessibility to the cellulose component of lignocellulosic substrates.

The Accessible Cellulose Surface Influences Cellulase Synergism during the Hydrolysis of Lignocellulosic Substrates

BackgroundCellulose amorphogenesis, described as the non-hydrolytic “opening up” or disruption of a cellulosic substrate, is becoming increasingly recognized as one of the key steps in the enzymatic deconstruction of cellulosic biomass when used as a feedstock for fuels and chemicals production. Although this process is thought to play a major role in facilitating hydrolysis, the lack of quantitative techniques capable of accurately describing the molecular-level changes occurring in the substrate during amorphogenesis has hindered our understanding of this process.ResultsIn this work, techniques for measuring changes in cellulose accessibility are reviewed and a new quantitative assay method is described. Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibres during amorphogenesis. The extents of phosphoric acid-induced and Swollenin-induced changes to cellulose accessibility were successfully quantified using this technique.ConclusionsThe adsorption of substructure-specific CBMs can be used to accurately quantify the extent of changes to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick, accurate and quantitative measure of the accessibility of cellulosic substrates. Expanding the range of CBMs used for adsorption studies to include those specific for such compounds as xylan or mannan should also allow for the accurate quantitative tracking of the accessibility of these and other polymers within the lignocellulosic biomass matrix.