BackgroundThe efficiency of enzymatic hydrolysis is reduced by the structural properties of cellulose. Although efforts have been made to explain the mechanism of enzymatic hydrolysis of cellulose by considering the interaction of cellulolytic enzymes with cellulose or the changes in the structure of cellulose during enzymatic hydrolysis, the process of cellulose hydrolysis is not yet fully understood. We have analysed the characteristics of the complex supramolecular structure of cellulose on the nanometre scale in terms of the spatial distribution of fibrils and fibril aggregates, the accessible surface area and the crystallinity during enzymatic hydrolysis. Influence of the porosity of the substrates and the hydrolysability was also investigated. All cellulosic substrates used in this study contained more than 96% cellulose.ResultsConversion yields of six cellulosic substrates were as follows, in descending order: nano-crystalline cellulose produced from never-dried soda pulp (NCC-OPHS-ND) > never-dried soda pulp (OPHS-ND) > dried soda pulp (OPHS-D) > Avicel > cotton treated with sodium hydroxide (cotton + NaOH) > cotton.ConclusionsNo significant correlations were observed between the yield of conversion and supramolecular characteristics, such as specific surface area (SSA) and lateral fibril dimensions (LFD). A strong correlation was found between the average pore size of the starting material and the enzymatic conversion yield. The degree of crystallinity was maintained during enzymatic hydrolysis of the cellulosic substrates, contradicting previous explanations of the increasing crystallinity of cellulose during enzymatic hydrolysis. Both acid and enzymatic hydrolysis can increase the LFD, but no plausible mechanisms could be identified. The sample with the highest initial degree of crystallinity, NCC-OPHS-ND, exhibited the highest conversion yield, but this was not accompanied by any change in LFD, indicating that the hydrolysis mechanism is not based on lateral erosion.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0236-9) contains supplementary material, which is available to authorized users.

Lytic polysaccharide monooxygenase (LPMO) and copper binding protein CopC share a similar mononuclear copper site. This site is defined by an N-terminal histidine and a second internal histidine side chain in a configuration called the histidine brace. To understand better the determinants of reactivity, the biochemical and structural properties of a well-described cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A) is compared with that of CopC from Pseudomonas fluorescens (PfCopC) and with the LPMO-like protein Bim1 from Cryptococcus neoformans. PfCopC is not reduced by ascorbate but is a very strong Cu(II) chelator due to residues that interacts with the N-terminus. This first biochemical characterization of Bim1 shows that it is not redox active, but very sensitive to H2O2, which accelerates the release of Cu ions from the protein. TaAA9A oxidizes ascorbate at a rate similar to free copper but through a mechanism that produce fewer reactive oxygen species. These three biologically relevant examples emphasize the diversity in how the proteinaceous environment control reactivity of Cu with O2.

The industrial production of cellulolytic enzymes is dominated by the filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina). In order to develop optimal enzymatic cocktail, it is of importance to understand the natural regulation of the enzyme profile as response to the growth substrate. The influence of the complexity of cellulose on enzyme production by the microorganisms is not understood. In the present study we attempted to understand how different physical and structural properties of cellulose-rich substrates affected the levels and profiles of extracellular enzymes produced by T. reesei. Enzyme production by T. reesei Rut C-30 was studied in submerged cultures on five different cellulose-rich substrates, namely, commercial cellulose Avicel® and industrial-like cellulosic pulp substrates which consist mainly of cellulose, but also contain residual hemicellulose and lignin. In order to evaluate the hydrolysis of the substrates by the fungal enzymes, the spatial polymer distributions were characterised by cross-polarisation magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS (13)C-NMR) in combination with spectral fitting. Proteins in culture supernatants at early and late stages of enzyme production were labeled by Tandem Mass Tags (TMT) and protein profiles were analysed by liquid chromatography-tandem mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD001304. In total 124 proteins were identified and quantified in the culture supernatants, including cellulases, hemicellulases, other glycoside hydrolases, lignin-degrading enzymes, auxiliary activity 9 (AA9) family (formerly GH61), supporting activities of proteins and enzymes acting on cellulose, proteases, intracellular proteins and several hypothetical proteins. Surprisingly, substantial differences in the enzyme profiles were found even though there were minor differences in the chemical composition between the cellulose-rich substrates.

BackgroundThe bioconversion of lignocellulosic feedstocks to ethanol is being commercialised, but further process development is required to improve their economic feasibility. Efficient saccharification of lignocellulose to fermentable sugars requires oxidative cleavage of glycosidic linkages by lytic polysaccharide monooxygenases (LPMOs). However, a proper understanding of the catalytic mechanism of this enzyme class and the interaction with other redox processes associated with the saccharification of lignocellulose is still lacking. The in-use stability of LPMO-containing enzyme cocktails is increased by the addition of catalase implying that hydrogen peroxide (H2O2) is generated in the slurry during incubation. Therefore, we sought to characterize the effects of enzymatic and abiotic sources of H2O2 on lignocellulose hydrolysis to identify parameters that could improve this process. Moreover, we studied the abiotic redox reactions of steam-pretreated wheat straw as a function of temperature and dry-matter (DM) content.ResultsAbiotic reactions in pretreated wheat straw consume oxygen, release carbon dioxide (CO2) to the slurry, and decrease the pH. The magnitude of these reactions increased with temperature and with DM content. The presence of LPMO during saccharification reduced the amount of CO2 liberated, while the effect on pH was insignificant. Catalase led to increased decarboxylation through an unknown mechanism. Both in situ-generated and added H2O2 caused a decrease in pH.ConclusionsAbiotic redox processes similar to those that occur in natural water-logged environments also affect the saccharification of pretreated lignocellulose. Heating of the lignocellulosic material and adjustment of pH trigger rapid oxygen consumption and acidification of the slurry. In industrial settings, it will be of utmost importance to control these processes. LPMOs interact with the surrounding redox compounds and redirect abiotic electron flow from decarboxylating reactions to fuel the oxidative cleavage of glycosidic bonds in cellulose.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1159-z) contains supplementary material, which is available to authorized users.

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