Xylose Residues Research Articles

Crystal structure of reducing-end xylose-releasing exoxylanase in subfamily 7 of glycoside hydrolase family 30.

TcXyn30A from Talaromyces cellulolyticus, which belongs to subfamily 7 of the glycoside hydrolase family 30 (GH30-7), releases xylose from the reducing end of xylan and xylooligosaccharides (XOSs), the so-called reducing-end xylose-releasing exoxylanase (ReX). In this study, the crystal structures of TcXyn30A with and without xylose at subsite +1 (the binding site of the xylose residue at the reducing end) were determined. This is the first report on the structure of ReX in the family GH30-7. TcXyn30A forms a dimer. The complex structure of TcXyn30A with xylose revealed that subsite +1 is located at the dimer interface. TcXyn30A recognizes xylose at subsite +1 composed of amino acid residues from each monomer and blocks substrate binding to subsite +2 by dimer formation. Thus, the dimeric conformation is responsible for ReX activity. The structural comparison between TcXyn30A and the homologous enzyme indicated that subsite -2 is composed of assembled three stacked Trp residues, Trp49, Trp333, and Trp334, allowing TcXyn30A to accommodate xylan and any branched XOSs decorated with a substitution such as α-1,2-linked 4-O-methyl-d-glucuronic acid or α-1,2- and/or -1,3-linked L-arabinofuranose. These findings provide an insight into the structural determinants for ReX activity of TcXyn30A.

Optimization and Physicochemical Characterization of Polysaccharide Purified from Sonneratia caseolaris Mangrove Leaves: a Potential Antioxidant and Antibiofilm Agent.

The Box-Behnken design was applied to determine the optimal parameters of the extraction condition by using the response surface methodology (RSM) from the leaves of Sonneratia caseolaris L. The result indicates the best-optimized conditions used for the extraction of polysaccharides at 84.02°C temperature, 3.12h time, and 27.31mL/g for the water-to-material ratio. The maximum experimental yield of 8.81 ± 0.09% was obtained which is in agreement with the predicted value of 8.79%. Thereafter, low molecular weight polysaccharide (SCLP) was separated after sequentially being purified through column chromatography with a relative molecular weight of 3.74kDa. The physicochemical properties were evaluated by characterization techniques such as FT-IR spectra, NMR spectrum, and SEM analysis. RP-HPLC analysis confirmed that SCLP was a heteropolysaccharide, majorly comprising rhamnose (28.25%), and xylose (27.17%) residues, followed by mannose (18.90%), and galactose (17.17%), respectively. Thermal analysis (TGA-DSC) results showed that SCLP is a highly thermostable polymer with a degradation temperature of 361.63°C. X-ray diffraction patterns and tertiary structure analyses indicate that SCLP had a semi-crystalline polymer having a triple-helical configuration. Moreover, SCLP displayed potential antibiofilm ability for all the tested pathogens while stronger activity against Klebsiella pneumoniae and Pseudomonas aeruginosa. In addition, SCLP has potential in vitro antioxidant activity on DPPH, ABTS radical, superoxide, and Fe2+ chelating. These findings indicate that the polysaccharide has potentially been used in functional food, cosmetics, and pharmacological industries.

Chemo-Enzymatic Synthesis of Long-Chain Oligosaccharides for Studying Xylan-Modifying Enzymes.

Plant research is hampered in several aspects by a lack of pure oligosaccharide samples that closely represent structural features of cell wall glycans. An alternative to purely chemical synthesis to access these oligosaccharides is chemo-enzymatic synthesis using glycosynthases. These enzymes enable the ligation of oligosaccharide donors, when activated for example as α-glycosyl fluorides, with suitable acceptor oligosaccharides. Herein, the synthesis of xylan oligosaccharides up to dodecasaccharides is reported, with glycosynthase-mediated coupling reactions as key steps. The xylo-oligosaccharide donors were protected at the non-reducing end with a 4-O-tetrahydropyranyl (THP) group to prevent polymerization. Installation of an unnatural 3-O-methylether substituent at the reducing end xylose of the oligosaccharides ensured good water solubility. Biochemical assays demonstrated enzymatic activity for the xylan acetyltransferase XOAT1 from Arabidopsis thaliana, xylan arabinofuranosyl-transferase XAT3 enzymes from rice and switchgrass, and the xylan glucuronosyltransferase GUX3 from Arabidopsis thaliana. In case of the glucuronosyltransferase GUX3, MALDI-MS/MS analysis of the reaction product suggested that a single glucuronosyl substituent was installed primarily at the central xylose residues of the dodecasaccharide acceptor, demonstrating the value of long-chain acceptors for assaying biosynthetic glycosyltransferases.

Nitrogen doped carbon solid acid for improving its catalytic transformation of xylose and agricultural biomass residues to furfural

Nitrogen doped carbon solid acid catalyst was prepared by sulfonation of the nitrogen doped carbon derived from polyaniline. The structure and morphology of the as-prepared catalyst was investigated and the acidity was tested by the titration method. The catalytic performances were evaluated by conversion of xylose to furfural as well as one-pot transformation of biomass to furfural. Moreover, influence of the reaction conditions including reaction temperature, time, solvents, and catalyst dosage were investigated. It can yield 88.2% furfural from xylose in γ-valerolactone at 170 °C for 45 min. Moreover, biomass of agricultural biomass residues were used to test the performance for one-pot transformation of biomass to furfural. Satisfied yield of 95% and 77.4% furfural was respectively obtained from corn cob and rice husk.

Efficient fractionation and targeted valorization of industrial xylose residue by synergistic and mild alkaline deep eutectic solvent‑hydrogen peroxide pretreatment

Achieving efficient fractionation and tailored downstream high value-added applications of bulk industrial waste biomass could guarantee the sustainability of biorefinery strategies. A synergistic and mild alkaline deep eutectic solvent‑hydrogen peroxide (DES-HP) pretreatment was designed in this study to significantly reduce pretreatment costs while achieving efficient fractionation of industrial xylose residues for preparing high antioxidative lignin and high enzymatic digestion of cellulose substrates for subsequent bioenergy and biochemicals production. The results demonstrated that this DES-HP pretreatment had admirable biomass deconstruction ability and the obtained DES-HP lignin displayed well-preserved structures and excellent antioxidative properties, which facilitated downstream catalytic degradation as well as the production of lignin-based materials and chemicals. Under the optimal condition (60 °C, 2 h, 10 FPU/g), the fermentable glucose yield of the DES-HP pretreated substrate could achieve 95.89%. In conclusion, this synergistic DES-HP is an ideal pretreatment strategy for the current tailored biorefinery scenario of industrial lignocellulosic biomass.

Extension of O-Linked Mannosylation in the Golgi Apparatus Is Critical for Cell Wall Integrity Signaling and Interaction with Host Cells in Cryptococcus neoformans Pathogenesis.

The human-pathogenic yeast Cryptococcus neoformans assembles two types of O-linked glycans on its proteins. In this study, we identified and functionally characterized the C. neoformans CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to minor O-glycans containing xylose in the Golgi apparatus. Two cell surface sensor proteins, Wml1 (WSC/Mid2-like) and Wml2, were found to be independent substrates of Cap6-mediated minor or Ktr3-mediated major O-mannosylation, respectively. The double deletion of KTR3 and CAP6 (ktr3Δ cap6Δ) completely blocked the mannose addition at the second position of O-glycans, resulting in the accumulation of proteins with O-glycans carrying only a single mannose. Tunicamycin (TM)-induced phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) was greatly decreased in both ktr3Δ cap6Δ and wml1Δ wml2Δ strains. Transcriptome profiling of the ktr3Δ cap6Δ strain upon TM treatment revealed decreased expression of genes involved in the Mpk1-dependent cell wall integrity (CWI) pathway. Consistent with its defective growth under several stress conditions, the ktr3Δ cap6Δ strain was avirulent in a mouse model of cryptococcosis. Associated with this virulence defect, the ktr3Δ cap6Δ strain showed decreased adhesion to lung epithelial cells, decreased proliferation within macrophages, and reduced transcytosis of the blood-brain barrier (BBB). Notably, the ktr3Δ cap6Δ strain showed reduced induction of the host immune response and defective trafficking of ergosterol, an immunoreactive fungal molecule. In conclusion, O-glycan extension in the Golgi apparatus plays critical roles in various pathobiological processes, such as CWI signaling and stress resistance and interaction with host cells in C. neoformans. IMPORTANCE Cryptococcus neoformans assembles two types of O-linked glycans on its surface proteins, the more abundant major O-glycans that do not contain xylose residues and minor O-glycans containing xylose. Here, we demonstrate the role of the Cap6 α1,3-mannosyltransferase in the synthesis of minor O-glycans. Previously proposed to be involved in capsule biosynthesis, Cap6 works with the related Ktr3 α1,2-mannosyltransferase to synthesize O-glycans on their target proteins. We also identified two novel C. neoformans stress sensors that require Ktr3- and Cap6-mediated posttranslational modification for full function. Accordingly, the ktr3Δ cap6Δ double O-glycan mutant strain displays defects in stress signaling pathways, CWI, and ergosterol trafficking. Furthermore, the ktr3Δ cap6Δ strain is completely avirulent in a mouse infection model. Together, these results demonstrate critical roles for O-glycosylation in fungal pathogenesis. As there are no human homologs for Cap6 or Ktr3, these fungus-specific mannosyltransferases are novel targets for antifungal therapy.

Post-extrusion physical properties, techno-functionality and microbiota-modulating potential of hempseed (Cannabis sativa L.) hull fiber

One-third of hempseed is composed of fiber, yet this major nutrient is far understudied than the seed's oil, protein, polyphenol or cannabinoid fractions. Here we provided a comprehensive and novel study covering the monosaccharide-linkage composition, physical, functional and microbiota-shifting properties of the fiber-rich (>77%) hempseed hull, which is a major industrial processing by-product. Xylose and cellulose-derived 4-linked glucopyranosyl linkage residue dominated the monosaccharide profile and 1D/2D NMR molecular assignments of hempseed hull. Processing with extrusion technology altered the particle size distribution, FTIR structural characteristics, X-ray diffraction crystallinity and thermal stability of hempseed fiber. Extruded hempseed fiber showed significantly greater soluble fiber content, water and oil binding capacity, dynamic modulus and flow viscosity values in oil and oil-water dispersions, delayed starch gelatinization and inhibited its retrogradation. Integration of 16S rRNA gene sequencing and the rapidly emerging metaproteomics revealed a higher population of Megasphaera elsdenii and Lactobacillus spp., along with their expressed ferredoxin and enolases respectively, in porcine faecal samples after fermentation with hempseed hull fiber. Higher production of propionic, butyric and valeric acid from the microbiota was further observed in fiber-treated faecal inoculum. Finally, the possible microbiota modulating-effect of fiber-bounded unique phenylpropionamides ( N - trans -caffeoyltyramine and lignanamides) from hempseed hull was assessed for the first time. • The effects of extrusion on the fiber profile of hempseed hull were investigated. • Linkage and NMR analysis revealed the molecular composition of hempseed fiber. • Functionality of hempseed fiber was tested in oil, water and starch matrices. • Hempseed fiber may alter gut microbial populations and their expressed proteins. • Microbiota and phenolic amides interaction were explored.

Hydration of Wheat Flour Water-Unextractable Cell Wall Material Enables Structural Analysis of Its Arabinoxylan by High-Resolution Solid-State 13C MAS NMR Spectroscopy.

To enable its structural characterization by nuclear magnetic resonance (NMR) spectroscopy, the native structure of cereal water-unextractable arabinoxylan (WU-AX) is typically disrupted by alkali or enzymatic treatments. Here, WU-AX in the wheat flour unextractable cell wall material (UCWM) containing 40.9% ± 1.5 arabinoxylan with an arabinose-to-xylose ratio of 0.62 ± 0.04 was characterized by high-resolution solid-state NMR without disrupting its native structure. Hydration of the UCWM (1.7 mg H2O/mg UCWM) in combination with specific optimizations in the NMR methodology enabled analysis by solid-state 13C NMR with magic angle spinning and 1H high-power decoupling (13C HPDEC MAS NMR) which provided sufficiently high resolution to allow for carbon atom assignments. Spectral resonances of C-1 from arabinose and xylose residues of WU-AX were here assigned to the solid state. The proportions of un-, mono-, and di-substituted xyloses were 59.2, 19.5, and 21.2%, respectively. 13C HPDEC MAS NMR showed the presence of solid-state fractions with different mobilities in the UCWM. This study presents the first solid-state NMR spectrum of wheat WU-AX with sufficient resolution to enable assignment without prior WU-AX solubilization.

Phosphorylation and sulfation share a common biosynthetic pathway, but extend biochemical and evolutionary diversity of biological macromolecules in distinct ways.

Phosphate and sulfate groups are integral to energy metabolism and introduce negative charges into biological macromolecules. One purpose of such modifications is to elicit precise binding/activation of protein partners. The physico-chemical properties of the two groups, while superficially similar, differ in one important respect—the valency of the central (phosphorus or sulfur) atom. This dictates the distinct properties of their respective esters, di-esters and hence their charges, interactions with metal ions and their solubility. These, in turn, determine the contrasting roles for which each group has evolved in biological systems. Biosynthetic links exist between the two modifications; the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate being formed from adenosine triphosphate (ATP) and adenosine phosphosulfate, while the latter is generated from sulfate anions and ATP. Furthermore, phosphorylation, by a xylosyl kinase (Fam20B, glycosaminoglycan xylosylkinase) of the xylose residue of the tetrasaccharide linker region that connects nascent glycosaminoglycan (GAG) chains to their parent proteoglycans, substantially accelerates their biosynthesis. Following observations that GAG chains can enter the cell nucleus, it is hypothesized that sulfated GAGs could influence events in the nucleus, which would complete a feedback loop uniting the complementary anionic modifications of phosphorylation and sulfation through complex, inter-connected signalling networks and warrants further exploration.

Turning lignin into treasure: An innovative filler comparable to commercial carbon black for the green development of the rubber industry

Driven by the global carbon neutrality action, biomass-derived functional materials have been applied in many fields to alleviate the pressure brought by the depletion of fossil energy. However, due to the complex structure, lignin faces many difficulties in its high-value utilization. The second largest biomass in the world has become the largest “natural waste”. In this paper, the lignin-based biochar-silica (LB-S) hybrid nanoparticles were prepared via a combination of two-step acid precipitation and carbonization using lignin black liquor extracted from xylose residue and sodium silicate as raw materials. The effects of carbonization temperature and lignin-based biochar (LB) content on the reinforcing properties of LB-S were studied. The results show that the particle size, specific surface area, pore characteristics, and surface polarity of LB-S all affect the mechanical properties of the final vulcanizates. The reinforcement performance of the best sample (LMB500-S) with “high structure” characteristics can be comparable to that of commercial carbon black (CB) N550. This study shows that LMB500-S hybrid nanoparticles with economic benefits possess the potential to completely replace commercial CB, which can turn lignin waste into treasure and promote the green development of traditional rubber industry in the context of carbon neutrality.

Xyloglucan Biosynthesis: From Genes to Proteins and Their Functions.

The plant’s recalcitrant cell wall is composed of numerous polysaccharides, including cellulose, hemicellulose, and pectin. The most abundant hemicellulose in dicot cell walls is xyloglucan, which consists of a β-(1- > 4) glucan backbone with α-(1- > 6) xylosylation producing an XXGG or XXXG pattern. Xylose residues of xyloglucan are branched further with different patterns of arabinose, fucose, galactose, and acetylation that varies between species. Although xyloglucan research in other species lag behind Arabidopsis thaliana, significant advances have been made into the agriculturally relevant species Oryza sativa and Solanum lycopersicum, which can be considered model organisms for XXGG type xyloglucan. In this review, we will present what is currently known about xyloglucan biosynthesis in A. thaliana, O. sativa, and S. lycopersicum and discuss the recent advances in the characterization of the glycosyltransferases involved in this complex process and their organization in the Golgi.

Structural characterization and antagonistic effect against P-selectin-mediated function of SFF-32, a fucoidan fraction from Sargassum fusiforme

Ethnopharmacological relevanceSargassum fusiforme (Harvey) Setchell, or Haizao, has been used in traditional Chinese medicine (TCM) since at least the eighth century a.d. S. fusiforme is an essential component of several Chinese formulas, including Haizao Yuhu Decoction, used to treat goiter, and Neixiao Lei Li Wan used to treat scrofuloderma. The pharmacological efficacy of S. fusiforme may be related to its anti-inflammatory effect. Aim of the studyTo determine the structural characteristics of SFF-32, a fucoidan fraction from S. fusiforme, and its antagonistic effect against P-selectin mediated function. Materials and methodsThe primary structure of SFF-32 was determined using methylation/GC–MS and NMR analysis. Surface morphology and solution conformation of SFF-32 were determined by scanning electron microscopy (SEM), Congo red test, and circular dichroic (CD) chromatography, respectively. The inhibitory effects of SFF-32 against the binding of P-selectin to HL-60 cells were evaluated using flow cytometry, static adhesion assay, and parallel-plate flow chamber assay. Furthermore, the blocking effect of SFF-32 on the interaction between P-selectin and PSGL-1 was evaluated using an in vitro protein binding assay. ResultsThe main linkage types of SFF-32 were proven to →[3)-α-l-Fucp-(1→3,4)-α-l-Fucp-(1]2→[4)-β-d-Manp-(1→3)-d-GlcAp-(1]2→4)-β-d-Manp-(1→3)-β-d-Glcp-(1→4)-β-d-Manp-(1→2,3)-β-d-Galp-(1→4)-β-d-Manp-(1→[4)-α-l-Rhap-(1]3→. The sulfated unit or terminal xylose residues were attached to the backbone through the C-3 of some fucose residues and terminal xylose residues were attached to C-3 of galactose residues. Moreover, SFF-32 disrupted P-selectin-mediated cell adhesion and rolling as well as blocked the interaction between P-selectin and its physiological ligand PSGL-1 in a dose-dependent manner. ConclusionsBlocking the binding between P-selectin and PSGL-1 is the possible underlying mechanism by which SFF-32 inhibits P-selectin-mediated function, which demonstrated that SFF-32 may be a potential anti-inflammatory lead compound.

Characterisation, rheological properties and immunomodulatory efficiency of corn silk polysaccharides

SummaryCorn silk polysaccharide (CSP) was isolated by heated 80 °C ethanol 70%. The extracted light (83.70 L*) with a yellowish (13.09 b*) reddish (1.98 a*) CSP had an ash content of 2.53 ± 0.03%. It is mainly composed of seven monosaccharides (xylose 23.64, glucose 16.65, ribose 17.35, galactose 13.64, rhamnose 11.93, arabinose 11.87 and mannose 4.85 mol%) and showed four peaks in 13C NMR correlated to the anomeric carbon of arabinose and/or xylose residues. The thermal characteristics exhibited the breakdown of CSP glycosidic bonds at 321.16 °C. The rheological properties showed that the raising of share rate (γ) caused a reduction in apparent viscosity for all tested concentrations, whilst the elastic G′ and viscous G″ storage moduli of CSP were enhanced by increasing the oscillation frequency. The immunomodulatory effects of CSP on RAW264.7 cells showed a significant (P < 0.05) increase in NO secretion up to a concentration of 80 μg mL−1 from CSP. Additionally, the phagocytosis index and acid phosphatase activity were enhanced (P < 0.05) in a dose‐dependent manner by increasing CSP concentrations.

Production of Deglucose-Apiose-Xylosylated Platycosides from Glycosylated Platycosides by Crude Enzyme from Aspergillus tubingensis.

Platycosides, Platycodi radix (Platycodon grandiflorus root) saponins, are used as food supplements and exert diverse pharmacological activities. Deglycosylation of saponins enhances their biological efficacy, and deglycosylated platycosides are produced mainly through enzymatic hydrolysis. However, the types of available deglycosylated platycosides remain limited because of a lack of hydrolyzing enzymes that can act on specific glycosides in glycosylated platycosides. In this study, a crude enzyme from Aspergillus tubingensis converted platycoside E (PE) and polygalacin D3 (PGD3) into deglucose-apiose-xylosylated (deGAX)-platycodin D (PD) and deGAX-polygalacin D (PGD), respectively. The products were identified through LC/MS analysis by specifically hydrolyzing all glucose residues at C-3, and apiose and xylose residues at C-28 of platycoside. The hydrolytic activity of the crude enzyme obtained after the cultivation of the fungus using citrus pectin and corn steep solid as carbon and nitrogen sources, respectively, in culture medium was increased compared with those using other carbon and nitrogen sources. The crude enzyme from A. tubingensis was the most effective in producing deGAX platycoside at pH 5.0 and 60°C. The crude enzyme produced 0.32 mg/ml deGAX-PD and 0.34 mg/ml deGAX-PGD from 1 mg/ml PE and 1 mg/ml PGD3 (at pH 5.0 and 60°C) for 12 and 10 h, with productivities of 32.0 and 42.5 mg/l/h and molar yields of 62.1 and 59.6%, respectively. To the best of our knowledge, this is the first study to produce deGAX platycosides from glycosylated platycosides.

Ultrafast alkaline deep eutectic solvent pretreatment for enhancing enzymatic saccharification and lignin fractionation from industrial xylose residue

An aspirational pretreatment method for efficient fractionation and tailored valorization of large industrial biomass can ensure the realizability of sustainable biorefinery strategies. In this study, an ultrafast alkaline deep eutectic solvents (DES) pretreatment strategy was developed to efficiently extract the lignin nanoparticles and retain cellulose residues that could be readily enzymatic saccharified to obtain fermentative glucose for the bioenergy production from industrial xylose residue. Results showed that the DES pretreatment had excellent delignification performance and the regenerated DES lignin nanoparticles exhibited well-preserved structures and excellent antioxidant activity, as well as low molecular weights and relatively uniform size distribution, which could facilitate downstream catalytic degradation for production of chemicals and preparation of lignin-based materials. Under the optimal condition (DES pretreatment: 80 °C, 10 min; saccharification: 10 FPU/g, 5 wt%, 100 mg/g Tween 80), the glucose yield of 90.12% could be achieved, which was dramatically increased compared to raw materials.

High performance removal of sulfamethoxazole using large specific area of biochar derived from corncob xylose residue

To remove antibiotics from waste water, an alkali active porous biochar, 850BC, was prepared from corncob xylose residue. In preparation, NaOH dipping was used for silicon removal and KOH activation was operated at 850℃. Further characterization containing BET, SEM, and FTIR were confirmed. 850BC possessed a huge specific surface area of 3043 m2·g−1, developed pore structure and abundant oxygen functional groups. The adsorption performance of sulfamethoxazole on 850BC was quick and efficient, and the adsorption capacity reached 1429 mg·g−1, which was significantly higher than other adsorbents reported previously. While pseudo-second-order kinetic model and Langmuir model could better describe the adsorption, chemisorption dominated the SMX adsorption onto 850BC. In virtue of pore-filling and π–π interaction as major mechanism, a large surface area and rich oxygen-containing functional groups led to an excellent adsorption performance. Thus, this preparation method provided a biochar-based adsorbent with enhanced specific surface for efficient removal of antibiotic pollutants.Graphical

Comparative studies on the structure, biological activity and molecular mechanisms of polysaccharides from Boletus aereus (BA-T) and Pleurotus cornucopiae (PC-1)

In this study, two new polysaccharides were extracted from the fruiting bodies of Boletus aereus and Pleurotus cornucopiae, respectively. The results of structure identification showed that BA-T was composed of xylose residues, glucose residues and galactose residues, and the ratio is 3:6:6. The BA-T takes 1,6-glucose and 1,6-galactose as skeletons, extends a branched chain from galactose 2-O to connect 1,4-xylose, and connects β-4-glucose terminal monosaccharide to xylose. PC-1 was composed of of xylose, glucose and galactose in the ratio of 2:8:2 and takes 1,6-glucose as skeletons, extends a branched chain from galactose 4-O to connect (1→4,,6)-α-D-Glcp and (1→2,6)-α-D-galactose, and →4)-β-D-Glcp and →1)-α-D-Xylp as terminal group. The results of immunoactivity showed that BA-T and PC-1 have the proliferation activity of B cells, T cells and RAW264.7 cells in vitro, and the effect of BA-T on the proliferation of T cells was the greatest. The results of RNA-sequencing showed that the protein synthesis and metabolism of T cells are more vigorous under the stimulation of BA-T, and oxidative phosphorylation is the main energy source in the process of T cell proliferation.

Functional characterization of a GH62 family α-L-arabinofuranosidase from Eupenicillium parvum suitable for monosaccharification of corncob arabinoxylan in combination with key enzymes

Corncob rich in arabinoxylan is an important raw material widely used in bio-refinery. Complete saccharification of arabinoxylan depends on the synergism of different enzymes including α-L-arabinofuranosidase (ABF). This study aimed to investigate the functional characteristics of a new ABF EpABF62A belonging to glycoside hydrolase (GH) 62 family from the fungus Eupenicillium parvum, and to explore its potential in the saccharification of corncob arabinoxylan. The recombinant EpABF62A showed high activity against wheat arabinoxylan and rye arabinoxylan, with the optimal temperature of 55 °C and pH of 4.5. The protein contains an N-terminal cellulose-binding domain family 1 (CBM_1) domain, and displayed a 59.5% absorption rate to phosphoric acid swollen cellulose. Regioselectivity analysis indicated that the enzyme selectively removed α-1,2 or α-1,3 linked arabinofuranosyl residues on mono-substituted xylose residues on arabinoxylan. Corncob arabinoxylans (CAX1 or CAX2) with different (low or high) branching degrees were extracted from the raw material by alkaline hydrogen peroxide pretreatment and graded ethanol precipitation. Single EpABF62A removed 69.5% or 67.1% arabinose from CAX1 or CAX2, respectively. EpABF62A combined with a GH10 xylanase, a GH43 β-D-xylosidase and a GH67 α-glucuronidase released 75.0% or 64.5% xylose from CAX1 or CAX2, respectively. The addition of the four hemicellulases enhanced the saccharification the solid fraction of the pretreated corncob by the commercial cellulase Cellic® CTec2, and the conversion ratios of glucose, xylose and arabinose were up to 94.0%, 91.8% and 82.6%, respectively.

Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo.

Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the "Y"- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues.

Structure of heteroxylans from vitreous and floury endosperms of maize grain and impact on the enzymatic degradation

Heteroxylans (HX) from vitreous and floury parts of maize endosperm were isolated. Structural analysis showed a xylan backbone with few unsubstituted xylose residues (<9%) demonstrating the high content in side chains in both fractions. HX from floury endosperm contained more arabinose and galactose than vitreous HX. The mono-substitution rate was 15% higher in the vitreous endosperm HX. Similar amounts of uronic acids were present in both fractions (~7% DM). Galactose in the floury endosperm HX was present exclusively in terminal position. A xylanase preparation solubilized more material from floury (40.5%) than from vitreous endosperm cell walls (15%). This could be a consequence of the structural differences between the two fractions and/or of the impact of structure on the interaction abilities of these fractions with other cell wall polysaccharides.Our study advances the understanding of cell wall polysaccharides in maize endosperm and their role in enzymatic susceptibility of maize grain.