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Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61-3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end. [ABSTRACT FROM AUTHOR]

Abstract: In steam pretreatment the defibration is usually achieved by an explosion at the end of the treatment, but can also be carried out in a subsequent refiner step. A steam explosion and a steam refining unit were compared by using the same raw material and pretreatment conditions, i.e. temperature and time. Smaller particle size was needed for the steam explosion unit to obtain homogenous slurries without considerable amounts of solid chips. A higher amount of volatiles could be condensed from the vapour phase after steam refining. The results from enzymatic hydrolysis showed no significant differences. It could be shown that, beside the chemical changes in the cell wall, the decrease of the particle size is the decisive factor to enhance the enzymatic accessibility while the explosion effect is not required. [Copyright &y& Elsevier]

Products containing the perennial grain intermediate wheatgrass (IWG) have recently entered the market, but little is known about the effect of processing operations on its constituents. This study investigated the effect of steam explosion (SE) conditions, i.e., temperature (130, 150 or 170 °C) and residence time (5 or 10 min), spanning severity factors from 1.6 to 3.1, on physicochemical properties of IWG bran. Even the lowest temperature and shortest time combination (130 °C, 5 min), was sufficient to inactivate peroxidase, generally regarded as the most heat-stable enzyme in grains. Contents of free phenolic acids as well as water-extractable arabinoxylans significantly increased while phytic acid was reduced after SE. However, starch damage, free fatty acids as well as browning (all negatively affecting quality) increased with treatment severity. Thus, SE is a promising technology to reduce enzymatic activity, enhance phenolic and soluble fiber contents in IWG bran, but severity factors >2.5 may negatively affect quality. • Steam explosion (SE) altered characteristics of bran from intermediate wheatgrass. • SE increased extractability of antioxidants, including hydroxycinnamic acids. • SE partially converted arabinoxylans from water-unextractable to extractable. • SE decreased phytic acid contents and inactivated peroxidase. • Treatment severity affected color, free fatty acids, and water retention capacity. [ABSTRACT FROM AUTHOR]

Brown macroalgae synthesize large amounts of fucoidans, sulfated fucose-containing polysaccharides, in the ocean. Fucoidans are of importance for their recently discovered contribution to marine carbon dioxide sequestration and due to their potential applications in biotechnology and biomedicine. However, fucoidans have high intra- and intermolecular diversity that challenges assignment of structure to biological function and the development of applications. Fucoidan-active enzymes may be used to simplify this diversity by producing defined oligosaccharides more applicable for structural refinement, characterization, and structure to function assignment for example via bioassays. In this study, we combined MALDI mass spectrometry with biocatalysis to show that the endo -fucoidanases P5AFcnA and Wv323 can produce defined oligosaccharide structures directly from unrefined macroalgal biomass. P5AFcnA released oligosaccharides from seven commercial fucoidan extracts in addition to unrefined biomass of three macroalgae species indicating a broadly applicable approach reproducible across 10 species. Both MALDI-TOF/TOF and AP-MALDI-Orbitrap systems were used, demonstrating that the approach is not instrument-specific and exploiting their combined high-throughput and high-resolution capabilities. Overall, the combination of MALDI-MS and endo -fucoidanase assays offers high-throughput evaluation of fucoidan samples and also enables extraction of defined oligosaccharides of known structure from unrefined seaweed biomass. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: The conversion of wheat straw to fermentable sugar for bioethanol production typically involves a thermal pretreatment step, followed by enzymatic hydrolysis. In this study we have investigated the effect of steam explosion parameters on wheat straw digestibility using a newly designed steam explosion unit and a process without acid impregnation. The wheat straw was steam pretreated using 18 different conditions in the temperature range of 170–220 °C and the resulting material was used directly (i.e. without washing) for enzymatic hydrolysis and fermentation in either a separate hydrolysis and fermentation (SHF)-type or a simultaneous saccharification and fermentation (SSF)-type set-up. Maximum glucose yields upon enzymatic hydrolysis were obtained after pretreatment at 210 °C for 10 min and yields were similar at harsher conditions. Xylose yields increased with temperature and residence time up to 190 °C, but decreased at harsher pretreatment conditions since these led to xylan degradation and concomitant production of furfural. In an SHF-type set-up ethanol formation did not follow enzymatic glucose release and was inversely correlated with furfural levels. An SFF-type set-up displayed a straightforward correlation between the expected amount of released glucose and the ethanol yields. The highest saccharification yields corresponded to about 90% of the cellulose in the substrate. Overall, this study shows that steam explosion without an acid catalyst is a good pretreatment method for saccharification of wheat straw. Optimal steam explosion conditions need to be a compromise between sugar accessibility and sugar degradation. [Copyright &y& Elsevier]

Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown. To address this question, here we carried out comparative structural and functional characterizations of three cellulose-active C4-oxidizing family AA9 LPMOs from the fungus Neurospora crassa, NcLPMO9A (NCU02240), NcLPMO9C (NCU02916), and NcLPMO9D (NCU01050). We solved the three-dimensional structure of copper-bound NcLPMO9A at 1.6-Å resolution and found that NcLPMO9A and NcLPMO9C, containing a CBM1 carbohydrate-binding module, bind cellulose more strongly and were less susceptible to inactivation than NcLPMO9D, which lacks a CBM. All three LPMOs were active on tamarind xyloglucan and konjac glucomannan, generating similar products but clearly differing in activity levels. Importantly, in some cases, the addition of phosphoric acid-swollen cellulose (PASC) had a major effect on activity: NcLPMO9A was active on xyloglucan only in the presence of PASC, and PASC enhanced NcLPMO9D activity on glucomannan. Interestingly, the three enzymes also exhibited large differences in their interactions with enzymatic electron donors, which could reflect that they are optimized to act with different reducing partners. All three enzymes efficiently used H2O2 as a cosubstrate, yielding product profiles identical to those obtained in O2-driven reactions with PASC, xyloglucan, or glucomannan. Our results indicate that seemingly similar LPMOs act preferentially on different types of copolymeric substructures in the plant cell wall, possibly because these LPMOs are functionally adapted to distinct niches differing in the types of available reductants. [ABSTRACT FROM AUTHOR]

Efficient digestate dewatering is crucial to reduce the volume and transportation cost of solid residues from anaerobic digestion (AD) plants. Large variations in dewatered cake solids have been reported and predictive models are therefore important in design and operation of such plants. However, current predictive models lack validation across several digestion substrates, pre-treatments and full-scale plants. In this study, we showed that thermogravimetric analysis is a reliable prediction model for dewatered cake solids using digestates from 15 commercial full-scale plants. The tested digestates originated from different substrates, with and without the pre-AD thermal hydrolysis process (THP). Moreover, a novel combined physicochemical parameter (C/N•ash) characterizing different digestate blends was identified by multiplying the C/N ratio with ash content of the dried solids. Using samples from 22 full-scale wastewater, food waste and co-waste plants, a linear relationship was found between C/N•ash and predicted cake solids for digestates with and without pre-AD THP. Pre-AD THP improved predicted cake solids by increasing the amount of free water. However, solids characteristics like C/N ratio and ash content had a more profound influence on the predicted cake solids than pre-AD THP and type of dewatering device. Finally, C/N•ash was shown to have a linear relationship to cake solids and reported polymer dose from eight full-scale pre-AD THP plants. In conclusion, we identified the novel parameter C/N•ash which can be used to predict dewatered cake solids regardless of dewatering device and sludge origin. Image 1 • Full-scale dewatered cake solids were predicted by thermogravimetric analysis. • Digestates were described by the C/N ratio and ash content (C/N.•ash) • Dewatered cake solids can be predicted from C/N.•ash • Thermal hydrolysis increased predicted cake solids. [ABSTRACT FROM AUTHOR]

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. [ABSTRACT FROM AUTHOR]

Highlights: [•] Xylo-oligos’ released during pretreatment have DP≤20 (most 3–8); more acetylated in stems. [•] AX and MLG released during pretreatment; xylan, XG, glucan and mannan remain only alkali-extractable. [•] All polysaccharides partly digestible after pretreatment, regardless extractability. [•] Digestibility correlates with polysaccharides’ structural organisation. [•] Results correlate with previous models of the primary type II cell wall. [Copyright &y& Elsevier]

Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain. [ABSTRACT FROM AUTHOR]

Abstract: Even if carbohydrate preparations from plant/fungal sources have a high degree of purity, observed immune-stimulation may be caused by minute sample contaminations. Using the example of different β-glucans we present a range of analytical tools crucial for validation of possible immune-stimulatory effects. Two yeast (MacroGard and Zymosan) and one cereal β-glucan (CBG40) increased IL-8 secretion by HT-29 cells considerably. Degradation of the β-glucan samples with β-glucan specific enzymes did hardly influence the effect of Zymosan and CBG40 but significantly decreased the effect of MacroGard. Stimulation of IL-8 secretion by CBG40 and Zymosan was hence not due to their β-glucan content. Instead, the effect of the CBG40 sample was due to low levels of LPS despite the inability of the known LPS inhibitor Polymyxin B to supress its stimulatory effect. We conclude that targeted enzymatic degradation of samples is a powerful validation tool to investigate carbohydrate specific immune-modulation. [Copyright &y& Elsevier]

We describe enzymological and structural analyses of the interaction between the family 18 chitinase ChiB from Serratia marcescens and the designed inhibitor N,N'-diacetylchitobionoxime-N-phenylcarbamate (HM508). HM508 acts as a competitive inhibitor of this enzyme with a K[sub i] in the 50 µM range. Active site mutants of ChiB show K[sub i] values ranging from 1 to 200 µM, providing insight into some of the interactions that determine inhibitor affinity. Interestingly, the wild type enzyme slowly degrades HM508, but the inhibitor is essentially stable in the presence of the moderately active D142N mutant of ChiB. The crystal structure of the D142N-HM508 complex revealed that the two sugar moieties bind to the -2 and -1 subsites, whereas the phenyl group interacts with aromatic side chains that line the +1 and +2 subsites. Enzymatic degradation of HM508, as well as a Trp → Ala mutation in the +2 subsite of ChiB, led to reduced affinity for the inhibitor, showing that interactions between the phenyl group and the enzyme contribute to binding. Interestingly, a complex of enzymatically degraded HM508 with the wild type enzyme showed a chitobiono-δ-lactone bound in the -2 and -1 subsites, despite the fact that the equilibrium between the lactone and the hydroxy acid forms in solution lies far toward the latter. This shows that the active site preferentially binds the [sup 4]E conformation of the -1 sugar, which resembles the proposed transition state of the reaction. [ABSTRACT FROM AUTHOR]