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Polysaccharides from plant biomass are the most abundant renewable chemicals on Earth and can potentially be converted to a wide variety of useful glycoconjugates. Potential applications of glycoconjugates include therapeutics and drug delivery, vaccine development and as fine chemicals. While anomeric hydroxyl groups of carbohydrates are amenable to a variety of useful chemical modifications, selective cross-coupling to non-reducing ends has remained challenging. Several lytic polysaccharide monooxygenases (LPMOs), powerful enzymes known for their application in cellulose degradation, specifically oxidize non-reducing ends, introducing carbonyl groups that can be utilized for chemical coupling. This study provides a simple and highly specific approach to produce oxime-based glycoconjugates from LPMO-functionalized oligosaccharides. The products are evaluated by HPLC, mass spectrometry and NMR. Furthermore, we demonstrate potential biodegradability of these glycoconjugates using selective enzymes. [ABSTRACT FROM AUTHOR]

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: We present an evaluation of HPLC-based analytical tools for the simultaneous analysis of native and oxidized cello-oligosaccharides, which are products of enzymatic cellulose degradation. Whereas cello-oligosaccharides arise from cellulose depolymerization by glycoside hydrolases, oxidized cello-oligosaccharides are produced by cellobiose dehydrogenase and the recently identified copper dependent lytic polysaccharide monooxygenases (LPMOs) currently classified as CBM33 and GH61. The latter enzymes are wide-spread and expected to play crucial roles in further development of efficient enzyme technology for biomass conversion. Three HPLC approaches with well documented performance in the field of oligosaccharide analysis have been investigated: high-performance anion-exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC) and porous graphitized carbon liquid chromatography (PGC-LC). HPAEC with pulsed amperometric detection (PAD) was superior for analysis of oxidized oligosaccharides, combining the best separation with superior sensitivity for oligosaccharide species with a degree of polymerization (DP) ranging from 1 to 10. Furthermore, the HPAEC method can be optimized for operation in a high-throughput manner (run time 10min). Both PGC-LC and HILIC allow reasonable run times (41 and 25min, respectively), with acceptable separation, but suffer from poor sensitivity compared to HPAEC-PAD. On the other hand, PGC-LC and HILIC benefit from being fully compatible with online mass spectrometry. Using an LC–MS setup, these methods will deliver much better sensitivity than what can be obtained with conventional detectors such as ultraviolet-, charged aerosol-, or evaporative light scattering and may reach sensitivities similar to or even better than what is obtained in HPAEC-PAD. Pure oxidized cello-oligosaccharide standards, ranging from DP2 to DP5, were obtained by semi-preparative PGC and characterized by MS and NMR analysis. [Copyright &y& Elsevier]

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]

Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency. [ABSTRACT FROM AUTHOR]

Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy but remains unfavorably inefficient. We describe an enzyme that acts on the surface of crystalline chitin, where it introduces chain breaks and generates oxidized chain ends, thus promoting further degradation by chitinases. This enzymatic activity was discovered and further characterized by using mass spectrometry and chromatographic separation methods to detect oxidized products generated in the absence or presence of H218O or 18O2. There are strong indications that similar enzymes exist that work on cellulose. Our findings not only demonstrate the existence of a hitherto unknown enzyme activity but also provide new avenues toward more efficient enzymatic conversion of biomass. [ABSTRACT FROM AUTHOR]

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]

Summary: The Bacteroidetes phylum is renowned for its ability to degrade a wide range of complex carbohydrates, a trait that has enabled its dominance in many diverse environments. The best studied species inhabit the human gut microbiome and use polysaccharide utilization loci (PULs), discrete genetic structures that encode proteins involved in the sensing, binding, deconstruction, and import of target glycans. In many environmental species, polysaccharide degradation is tightly coupled to the phylum‐exclusive type IX secretion system (T9SS), which is used for the secretion of certain enzymes and is linked to gliding motility. In addition, within specific species these two adaptive systems (PULs and T9SS) are intertwined, with PUL‐encoded enzymes being secreted by the T9SS. Here, we discuss the most noteworthy PUL and non‐PUL mechanisms that confer specific and rapid polysaccharide degradation capabilities to the Bacteroidetes in a range of environments. We also acknowledge that the literature showcasing examples of PULs is rapidly expanding and developing a set of assumptions that can be hard to track back to original findings. Therefore, we present a simple universal description of conserved PUL functions and how they are determined, while proposing a common nomenclature describing PULs and their components, to simplify discussion and understanding of PUL systems. [ABSTRACT FROM AUTHOR]

The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most land plants and was originally thought to be absent in CGA. This study presents evidence that XyG evolved in the CGA. This is based on a) the identification of orthologs of the genetic machinery to produce XyG, b) the identification of XyG in a range of CGA and, c) the structural elucidation of XyG, including uronic acid-containing XyG, in selected CGA. Most notably, XyG fucosylation, a feature considered as a late evolutionary elaboration of the basic XyG structure and orthologs to the corresponding biosynthetic enzymes are shown to be present in Mesotaenium caldariorum. Mikkelsen et al. demonstrate molecular and cellular evidence of the evolution of xyloglucan (XyG), a structural component of the cell walls of most land plants, in charophyte algae. This study describes the structure of XyG in charophyte algae, including identification of fucosylated XyG, and furthermore identifies orthologs required to produce XyG. [ABSTRACT FROM AUTHOR]

Background: Utilization of commensal bacteria for delivery of medicinal proteins, such as vaccine antigens, is an emerging strategy. Here, we describe two novel food-grade strains of lactic acid bacteria, Lactiplantibacillus pentosus KW1 and KW2, as well as newly developed tools for using this relatively unexplored but promising bacterial species for production and surface-display of heterologous proteins. Results: Whole genome sequencing was performed to investigate genomic features of both strains and to identify native proteins enabling surface display of heterologous proteins. Basic characterization of the strains revealed the optimum growth temperatures for both strains to be 35–37 °C, with peak heterologous protein production at 33 °C (KW1) and 37 °C (KW2). Negative staining revealed that only KW1 produces closely bound exopolysaccharides. Production of heterologous proteins with the inducible pSIP-expression system enabled high expression in both strains. Exposure to KW1 and KW2 skewed macrophages toward the antigen presenting state, indicating potential adjuvant properties. To develop these strains as delivery vehicles, expression of the mycobacterial H56 antigen was fused to four different strain-specific surface-anchoring sequences. Conclusion: All experiments that enabled comparison of heterologous protein production revealed KW1 to be the better recombinant protein production host. Use of the pSIP expression system enabled successful construction of L. pentosus strains for production and surface display of an antigen, underpinning the potential of these strains as novel delivery vehicles. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases ( LPMOs) are important for the enzymatic conversion of biomass and seem to play a key role in degradation of the plant cell wall. In this study, we characterize an LPMO from the fungal plant pathogen Fusarium graminearum ( Fg LPMO9A) that catalyzes the mixed C1/C4 oxidative cleavage of cellulose and xyloglucan, but is inactive toward other (1,4)-linked β-glucans. Our findings indicate that Fg LPMO9A has unprecedented broad specificity on xyloglucan, cleaving any glycosidic bond in the β-glucan main chain, regardless of xylosyl substitutions. Interestingly, we found that when incubated with a mixture of xyloglucan and cellulose, Fg LPMO9A efficiently attacks the xyloglucan, whereas cellulose conversion is inhibited. This suggests that removal of hemicellulose may be the true function of this LPMO during biomass conversion. [ABSTRACT FROM AUTHOR]

The study of specific glycan uptake and metabolism is an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards this target. Here, we successfully used the fluorescent label 2-aminobenzamide (2-AB) to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, are able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it is possible to better understand the metabolism of labelled glycans in both supernatants and at a single cell level. We envisage this combination of complementary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities. A reductive amination-based fluorophore labelling of complex wood-derived glycans provides a proof-of-principle multi-modal platform for monitoring glycan uptake by bacteria. [ABSTRACT FROM AUTHOR]

Background: Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes. Results: We have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates of biomass materials in which the complexity of plant cell walls is partially maintained. Conclusions: We show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems. [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]

The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases) by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first "extracting" these chains from their crystalline matrix. [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]

Solid residues such as primary sludge (PS), waste activated sludge (WAS), and food waste (FW) can be stabilized through anaerobic digestion (AD). Application of the thermal hydrolysis process (THP) prior to AD results in several benefits in AD and dewatering. However, soluble recalcitrant compounds associated with Maillard reactions have been identified after THP which can impact downstream processes and water discharge limits. In this study, the soluble colloidal chemical oxygen demand, color, ultraviolet absorbance at 254 nm and dissolved organic nitrogen in seven full‐scale THP facilities were quantified and compared. The THP substrate influenced the concentration of soluble melanoidin‐associated compounds in the digestates. THP implementation in five water resource recovery facilities (WRRFs) was modeled and found to give a 3–8 mg/L increase on the water effluent COD concentration depending on the PS/WAS ratio. The results provide novel information useful in planning new WRRFs and optimization of existing facilities. Practitioner points: High amounts of WAS in substrate resulted in higher concentrations of CODsc, color and dissolved organic nitrogen in the digestate.Food waste treated at 145°C showed equal or lower concentrations of all components compared with sludge operated at 165°C.Installation of THP will increase the COD concentration in the water effluent of a water resource recovery facility.The characteristics of the THP substrate are important to consider in cost/benefit analysis when planning the installation of THP. [ABSTRACT FROM AUTHOR]

Traditional sour beers are produced by spontaneous fermentations involving numerous yeast and bacterial species. One of the traits that separates sour beers from ales and lagers is the high concentration of organic acids such as lactic acid and acetic acid, which results in reduced pH and increased acidic taste. Several challenges complicate the production of sour beers through traditional methods. These include poor process control, lack of consistency in product quality, and lengthy fermentation times. This review summarizes the methods for traditional sour beer production with a focus on the use of lactobacilli to generate this beverage. In addition, the review describes the use of selected pure cultures of microorganisms with desirable properties in conjunction with careful application of processing steps. Together, this facilitates the production of sour beer with a higher level of process control and more rapid fermentation compared to traditional methods. [ABSTRACT FROM AUTHOR]

ß-mannans and xylans are important components of the plant cell wall and they are acetylated to be protected from degradation by glycoside hydrolases. ß-mannans are widely present in human and animal diets as fiber from leguminous plants and as thickeners and stabilizers in processed foods. There are many fully characterized acetylxylan esterases (AcXEs); however, the enzymes deacetylating mannans are less understood. Here we present two carbohydrate esterases, RiCE2 and RiCE17, from the Firmicute Roseburia intestinalis, which together deacetylate complex galactoglucomannan (GGM). The three-dimensional (3D) structure of RiCE17 with a mannopentaose in the active site shows that the CBM35 domain of RiCE17 forms a confined complex, where the axially oriented C2-hydroxyl of a mannose residue points toward the Ser41 of the catalytic triad. Cavities on the RiCE17 surface may accept galactosylations at the C6 positions of mannose adjacent to the mannose residue being deacetylated (subsite -1 and +1). In-depth characterization of the two enzymes using time-resolved NMR, high-performance liquid chromatography (HPLC), and mass spectrometry demonstrates that they work in a complementary manner. RiCE17 exclusively removes the axially oriented 2-O-acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the RiCE2 is active on 3-O-, 4-O-, and 6-O-acetylations. Activity of RiCE2 is dependent on RiCE17 removing 2-O-acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2-O-specific RiCE17 provided insight into how temperature and pH affects acetyl migration on manno-oligosaccharides. [ABSTRACT FROM AUTHOR]

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]

There is a general agreement that fish meal (FM) and fish oil (FO) are valuable resources for aquafeed, but that the production cannot keep the same pace as the current growth of the aquaculture industry. Therefore, there is a need to find alternative sources for lipids and protein. This study examines the possibility of using proteins recovered from seafood industry side stream waters as a complement to FM in feed for Atlantic salmon. To recover the proteins, herring and shrimp process side streams waters were flocculated then treated with dissolved air flotation (DAF), which is considered a gentle technology. Shrimp steam water was used to obtain shrimp protein (SP) and herring salt brine to obtain herring protein (HP). The recovered semi-solid protein fraction was spray dried and formulated into experimental diets at inclusion levels of 1.9–10% (dw/dw). Two feeding trials were conducted to investigate; 1) the properties of different flocculants to recover SP from shrimp steam water, alginate (Alg; 1.9% inclusion), carrageenan (Carr; 2.0% inclusion) and a synthetic flocculant from Kemira (Kem; 3.8% inclusion). 2) total or partial replacement of FM with SP-Alg (10% and no FM) and HP-Alg (3% and 8% FM). For both feeding trials each diet was provided to triplicate tanks (n = 31 and 30/tank) of Atlantic salmon with a start weight of 193 g (duration 10 weeks) and 304 g (duration 7 weeks) respectively. All fish showed similar feed intake, feed conversion ratio (on tank basis) and weight gain. The inclusion of SP or HP as total or partial replacement of FM did neither influence adiposity of the fish, as measured by condition factor, nor heposomatic index (HSI). No negative effect of the alternative protein could be found through histological examination of the intestine. In feeding trial two, diets did not affect the adaptive immune indicators CD8α and MHC II. The SP-Alg diet did not affect intestinal barrier and transporting functions, assessed using Ussing-chamber technology. However, HP-Alg affected the trans-epithelial resistance, which indicate that the intestinal barrier function could be affected by low inclusions. We conclude that from a biological perspective, SP recovered from shrimp steaming waters using Alg and DAF technology represents a new marine biomass with potential as a replacement for FM in Atlantic salmon feed. To diversify the possibility of using flocculants to retrieve proteins we also suggest further investigation of the potential to use Carr in larger inclusions. • Alginate can work as a natural flocculant to aggregate proteins from industry side-stream waters for feed production. • Shrimp protein extracted from industry side-stream water show great potential as replacement for fishmeal. • Dietary shrimp protein performed equally to fishmeal in terms of growth and welfare indicators. • Low Inclusion of herring protein affected intestinal barrier function and reduced IGF-1 conc. and need further assessment. [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]

Sour beer is beer with an intentionally sour taste. In traditionally produced sour beer, the acidic character results from spontaneous, mixed fermentation where different bacteria and yeast species participate. These complex fermentations take years to complete and can be difficult to control. Owing to the increasing interest in sour beer and challenges related to commercial sour beer brewing, alternative production methods are being explored. In the current paper, pre‐fermentation with lactic acid bacteria (LAB) was investigated as a timesaving and controllable strategy for the production of sour beer. Four beers were produced with either biological or chemical acidification of wort, and a reference beer was produced with no acidification. Volatile compounds and organic acids were analysed by headspace gas chromatography and high‐performance liquid chromatography to explore any contribution from LAB to the composition of beer. Finally, descriptive sensory analysis was performed to evaluate the sensory contribution from LAB. A significant effect was observed from LAB pre‐fermentation, with respect to both volatile compounds (e.g. reduced production of 2‐methyl‐1‐butanol) and organic acids (e.g. production of acetic acid). Biological acidification by LAB pre‐fermentation had a significant impact on the sensory character of beer. This sensory impact did, however, not surpass that obtained by chemical acidification, as few significant differences were found between biologically and chemically acidified beers. © 2019 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are a group of recently discovered enzymes that play important roles in the decomposition of recalcitrant polysaccharides. Here, we report the biochemical, structural, and computational characterization of an LPMO from the white‐rot fungus Heterobasidion irregulare (HiLPMO9B). This enzyme oxidizes cellulose at the C1 carbon of glycosidic linkages. The crystal structure of HiLPMO9B was determined at 2.1 Å resolution using X‐ray crystallography. Unlike the majority of the currently available C1‐specific LPMO structures, the HiLPMO9B structure contains an extended L2 loop, connecting β‐strands β2 and β3 of the β‐sandwich structure. Molecular dynamics (MD) simulations suggest roles for both aromatic and acidic residues in the substrate binding of HiLPMO9B, with the main contribution from the residues located on the extended region of the L2 loop (Tyr20) and the LC loop (Asp205, Tyr207, and Glu210). Asp205 and Glu210 were found to be involved in the hydrogen bonding with the hydroxyl group of the C6 carbon of glucose moieties directly or via a water molecule. Two different binding orientations were observed over the course of the MD simulations. In each orientation, the active‐site copper of this LPMO preferentially skewed toward the pyranose C1 of the glycosidic linkage over the targeted glycosidic bond. This study provides additional insight into cellulose binding by C1‐specific LPMOs, giving a molecular‐level picture of active site substrate interactions. Database: The atomic coordinates and structure factors for HiLPMO9B have been deposited in the Protein Data Bank with accession code 5NNS. [ABSTRACT FROM AUTHOR]

Pectic homogalacturonan ( HG) is one of the main constituents of plant cell walls. When processed to low degrees of esterification, HG can form complexes with divalent calcium ions. These macromolecular structures (also called egg boxes) play an important role in determining the biomechanics of cell walls and in mediating cell-to-cell adhesion. Current immunological methods enable only steady-state detection of egg box formation in situ. Here we present a tool for efficient real-time visualisation of available sites for HG crosslinking within cell wall microdomains. Our approach is based on calcium-mediated binding of fluorescently tagged long oligogalacturonides ( OGs) with endogenous de-esterified HG. We established that more than seven galacturonic acid residues in the HG chain are required to form a stable complex with endogenous HG through calcium complexation in situ, confirming a recently suggested thermodynamic model. Using defined carbohydrate microarrays, we show that the long OG probe binds exclusively to HG that has a very low degree of esterification and in the presence of divalent ions. We used this probe to study real-time dynamics of HG during elongation of Arabidopsis pollen tubes and root hairs. Our results suggest a different spatial organisation of incorporation and processing of HG in the cell walls of these two tip-growing structures. [ABSTRACT FROM AUTHOR]

Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD andMSand that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolyticLPMOactivity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. [ABSTRACT FROM AUTHOR]

The enzymatic conversion of acetylated hardwood glucuronoxylan to functional food oligomers, biochemicals or fermentable monomers requires besides glycoside hydrolases enzymes liberating acetic acid esterifying position 2 and/or 3 in xylopyranosyl (Xyl p ) residues. The 3- O -acetyl group at internal Xyl p residues substituted by MeGlcA is the only acetyl group of hardwood acetylglucuronoxylan and its fragments not attacked by acetylxylan esterases of carbohydrate esterase (CE) families 1, 4, 5 and 6 and by hemicellulolytic acetyl esterases classified in CE family 16. Monoacetylated aldotetraouronic acid 3″-Ac 3 MeGlcA 3 Xyl 3 , generated from the polysaccharide by GH10 endoxylanases, appears to be one of the most resistant fragments. The presence of the two substituents on the non-reducing-end Xyl p residue prevents liberation of MeGlcA by α-glucuronidase of family GH67 and blocks the action of acetylxylan esterases. The Ac 3 MeGlcA 3 Xyl 3 was isolated from an enzymatic hydrolysate of birchwood acetylglucuronoxylan and characterized by 1 H NMR spectroscopy as a mixture of two positional isomers, 3″-Ac 3 MeGlcA 3 Xyl 3 and 4″-Ac 3 MeGlcA 3 Xyl 3 , the latter being the result of acetyl group migration. The mixture was used as a substrate for three members of CE16 family of fungal origin. Trichoderma reesei CE16 esterase, inactive on polymeric substrate, deacetylated both isomers. Podospora anserina and Aspergillus niger esterases, active on acetylglucuronoxylan, deesterified effectively only the 4″-isomer. The results indicate catalytic diversity among CE16 enzymes, but also their common and unifying catalytic ability to exo-deacetylate positions 3 and 4 on non-reducing-end Xyl p residues, which is an important step in plant hemicellulose saccharification. [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]

Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems.We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons. [ABSTRACT FROM AUTHOR]

A xyloglucan-specific endo-1,4β-glucanase (XcXGHA) from Xanthomonas citri pv. mangiferaeindicae has been cloned, expressed in Escherichia coli, purified and characterised. The XcXGHA enzyme belongs to CAZy family GH74 and has catalytic site residues conserved with other xyloglucanases in this family. At its optimal reaction conditions, pH 7.0 and 40 °C, the enzyme has a k/ K value of 2.2 × 10 min M on a tamarind seed xyloglucan substrate. XcXGHA is relatively stable within a broad pH range (pH 4-9) and up to 50 °C ( t of 74 min). XcXGHA is proven to be xyloglucan-specific, and a glycan microarray study verifies that XcXGHA catalyses cleavage of xyloglucan extracted from both monocot and dicot plant species. The enzyme catalyses hydrolysis of tamarind xyloglucan in a unique way by cleaving XXXG into XX and XG (X is xylosyl-substituted glucose; G is unsubstituted glucose), is able to degrade more complex xyloglucans and notably is able to cleave near more substituted xyloglucan motifs such as L [i.e. α- l-Fuc p-(1 → 2)-β- d-Gal p-(1 → 2)-α- d-Xyl p-(1 → 6)-β- d-Glc p]. LC-MS/MS analysis of product profiles of tamarind xyloglucan which had been catalytically degraded by XcXGHA revealed that XcXGHA has specificity for X in subsite −1. The 3D model suggests that XcXGHA consists of two seven-bladed β-propeller domains with the catalytic center formed by the interface of these two domains, which is conserved in xyloglucanases in the GH74 family. However, the XcXGHA has two amino acids (D264 and R472) that differ from the conserved residues of other GH74 xyloglucanases. These two amino acids were predicted to be located on the opposite side of the active site pocket, facing each other and forming a closing surface above the active site pocket. These two amino acids may contribute to the unique substrate specificity of the XcXGHA enzyme. [ABSTRACT FROM AUTHOR]

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures. [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]