Laminaribiose

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Marine macroalgae and aquaculture organisms have in common to form problematic biomass, either when washed ashore after extensive blooms or as processing remains, which accumulate in aquaculture facilities. Both sources of biomass are commonly regarded as waste. This study aimed to investigate whether both sources of waste can be combined in a beneficial way to yield value-added products. Crude extracts of shrimp (Penaeus vannamei) and crayfish (Cherax quadricarinatus) remains were analyzed for their catalytic potential and functional properties. Shrimp extracts showed a high potential for degrading β-1,3-glycosidic bonds (laminarin), while crayfish extracts showed a high potential for degrading β-1,4-glycosidic bonds (cellulose). The highest activities were observed at pH 4 to pH 6 and at 50 to 60°C, with an optimum range between 30 and 40°C. Pre-treated brown algae, Sargassum horridum, were incubated with the crude crustacean extracts. The extracts were capable of hydrolyzing brown algae biomass, thereby liberating glucose. Blends of shrimp and crayfish extracts were more efficient than shrimp extracts alone. The produced glucose was fermented by common yeast to bio-ethanol. This “proof of concept” showed that putative bio-waste can be utilized to extract active enzymes and suitable substrates for the production of value-added products such as bio-ethanol. This approach of combining two different sources of waste in a complementary process may contribute to the mitigation of marine bio-waste and be considered a valuable feedstock for biotechnological applications.

The glycoside hydrolase (GH) 64 β-1,3-glucanase Bgl64Fj was cloned from the gram-negative bacterium Flavobacterium johnsoniae. Bgl64Fj includes a signal sequence, catalytic domain, βγ-crystallin domain (βγ-Cry), ricin B-like lectin domain (RicinB), carbohydrate-binding module family 6 (CBM6), and carboxyl-terminal domain. Bgl64Fj without the signal sequence and carboxyl-terminal domain hydrolyzed zymosan A, homogenized curdlan-gel, and pachyman, and Bgl64Fj released laminaripentaose from homogenized curdlan-gel. Bgl64Fj independently inhibited hyphal extension in Trichoderma reesei, and Bgl64Fj contributed to enhancing the mycelial extension inhibitory activity of GH19 chitinase. To clarify the domain function, green fluorescent protein-fused βγ-Cry, RicinB, and CBM6 were constructed. As a result, RicinB is bound to β-glucans, such as zymosan A, homogenized curdlan gel, and lichenan. In contrast, the deletion enzyme Bgl64FjCat, consisting only of the catalytic domain, showed hydrolytic activity and mycelial extension inhibitory activity similar to those of Bgl64Fj. This result suggests that Bgl64Fj exhibits sufficient activity in the absence of a substrate-binding domain.

Background: Aspergillus niger is an important lignocellulose-degrading enzyme-producing strain. Multiple regulatory factors regulate the synthesis of lignocellulose-degrading enzymes in A. niger. We previously found that A. niger possessed an intracellular β-glucosidase BGL1B, and the intracellular localization of BGL1B and its active transglycosylation action prompted us to explore whether BGL1B was involved in the regulation of the synthesis of lignocellulose-degrading enzymes in A. niger. Results: In this study, by investigating the production of lignocellulose-degrading enzymes of bgl1B knockout strain (Δbgl1B) and overexpression strain (OE::bgl1B), it was found that BGL1B exhibited a repressive role on the expression of lignocellulose-degrading enzyme genes through carbon catabolite repression (CCR) way. On the other hand, BGL1B’s transglycosylation products sophorose and laminaribiose were proved to be able to induce the expression of lignocellulose-degrading enzyme genes, which explained why OE::bgl1B showed the same enhanced enzyme activity and gene expression as Δbgl1B strain compared to the starting strain (WT). Conclusions: The present study demonstrates that BGL1B plays dual regulatory roles in the regulation of the synthesis of lignocellulose-degrading enzymes in A. niger: the repressive role caused by BGL1B’s hydrolysis product glucose and the induction role caused by BGL1B’s transglycosylation products sophorose and laminaribiose. This study broadens the understanding of the regulatory network of the synthesis of lignocellulose-degrading enzymes in A. niger. Also, it provides a strategy to create an engineered strain with high production of lignocellulose-degrading enzymes.

β-1,3-glucanases can degrade β-1,3-glucoside bonds in β-glucan which is the main cell-wall component of most of fungi, and have the crucial application potential in plant protection and food processing. Herein, a β-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659 composed of 333 amino acids with a predicted molecular mass of 36.6 kDa was expressed in Escherichia coli BL21, purified and characterized. The deduced amino acid sequence of FlGluA showed the high identity with the β-1,3-glucanase belonging to glycoside hydrolase (GH) family 16. Enzymological characterization indicated FlGluA had the highest activity on zymosan A, with a specific activity of 3.87 U/mg, followed by curdlan (1.16 U/mg) and pachymaran (0.88 U/mg). It exhibited optimal catalytic activity at the pH 5.0 and 40°C, and was stable when placed at 4°C for 12 h in the range of pH 3.0-8.0 or at a temperature below 50°C for 3 h. Its catalytic activity was enhanced by approximately 36 % in the presence of 1 mM Cr³⁺. The detection of thin-layer chromatography and mass spectrometry showed FlGluA hydrolyzed zymosan A mainly to glucose and disaccharide, and trace amounts of tetrasaccharide and pentasaccharide, however, it had no action on laminaribiose, indicating its endo-β-1,3-glucanase activity. The mycelium growth of F. oxysporum treated by FlGluA was inhibited, with approximately 37% of inhibition rate, revealing the potential antifungal activity of the enzyme. These results revealed the hydrolytic properties and biocontrol activity of FlGluA, laying a crucial foundation for its potential application in agriculture and industry.

Laminarin, a β(1,3)-glucan, serves as a storage polysaccharide in marine microalgae such as diatoms. Its abundance, water solubility and simple structure make it an appealing substrate for marine bacteria. Consequently, many marine bacteria have evolved strategies to scavenge and decompose laminarin, employing carbohydrate-binding modules (CBMs) as crucial components. In this study, we characterized two previously unassigned domains as laminarin-binding CBMs in multimodular proteins from the marine bacterium Christiangramia forsetii KT0803^T, thereby introducing the new laminarin-binding CBM families CBM102 and CBM103. We identified four CBM102s in a surface glycan-binding protein (SGBP) and a single CBM103 linked to a glycoside hydrolase module from family 16 (GH16_3). Our analysis revealed that both modular proteins have an elongated shape, with GH16_3 exhibiting greater flexibility than SGBP. This flexibility may aid in the recognition and/or degradation of laminarin, while the constraints in SGBP could facilitate the docking of laminarin onto the bacterial surface. Exploration of bacterial metagenome-assembled genomes (MAGs) from phytoplankton blooms in the North Sea showed that both laminarin-binding CBM families are widespread among marine Bacteroidota. The high protein abundance of CBM102- and CBM103-containing proteins during phytoplankton blooms further emphasizes their significance in marine laminarin utilization.

Penicillium roqueforti is used worldwide in the production of blue-veined cheese. The blue-green colour derives from pigmented spores formed by fungal growth. Using a combination of bioinformatics, targeted gene deletions, and heterologous gene expression we discovered that pigment formation was due to a DHN-melanin biosynthesis pathway. Systematic deletion of pathway genes altered the arising spore colour, yielding white to yellow-green to red-pink-brown phenotypes, demonstrating the potential to generate new coloured strains. There was no consistent impact on mycophenolic acid production as a result of pathway interruption although levels of roquefortine C were altered in some deletants. Importantly, levels of methyl-ketones associated with blue-cheese flavour were not impacted. UV-induced colour mutants, allowed in food production, were then generated. A range of colours were obtained and certain phenotypes were successfully mapped to pathway gene mutations. Selected colour mutants were subsequently used in cheese production and generated expected new colourations with no elevated mycotoxins, offering the exciting prospect of use in future cheese manufacture.

Fungal cell walls undergo continual remodeling that generates β-1,3-glucan fragments as products of endo-glycosyl hydrolases (GHs), which can be recognized as pathogen-associated molecular patterns (PAMPs) and trigger plant immune responses. How fungal pathogens suppress those responses is often poorly understood. Here, we study mechanisms underlying the suppression of β-1,3-glucan-triggered plant immunity by the blast fungus Magnaporthe oryzae. We show that an exo-β-1,3-glucanase of the GH17 family, named Ebg1, is important for fungal cell wall integrity and virulence of M. oryzae. Ebg1 can hydrolyze β-1,3-glucan and laminarin into glucose, thus suppressing β-1,3-glucan-triggered plant immunity. However, in addition, Ebg1 seems to act as a PAMP, independent of its hydrolase activity. This Ebg1-induced immunity appears to be dampened by the secretion of an elongation factor 1 alpha protein (EF1α), which interacts and co-localizes with Ebg1 in the apoplast. Future work is needed to understand the mechanisms behind Ebg1-induced immunity and its suppression by EF1α.

Background: Grifola frondosa is a Basidiomycete fungus belonging to the family of Grifolaceae and the order of Polyporales. β-Glucans are the main polymers in G. frondosa, playing a crucial role in the physiology and representing the healthy benefits for humans. The membrane-integrated β-1, 3-glucan synthase (GLS) is responsible for glucan synthesis, cell wall assembly, differentiation and growth of the edible fungi. However, the structural/catalytic characteristics and mechanisms of β-1, 3-glucan synthases in G. frondosa are still unknown due to their extremely complex structures with multi-transmembranes and large molecular masses. Results: Herein, a β-1, 3-glucan synthase (GFGLS2) was purified and identified from the cultured mycelia with a specific activity of 60.01 pmol min⁻¹ μg⁻¹ for the first time. The GFGLS2 showed a strict specificity to UDP-glucose with a V_max value of 1.29 ± 0.04 µM min⁻¹ at pH 7.0 and synthesized β-1, 3-glucan with a maximum degree of polymerization (DP) of 62. Sequence Similarity Network (SSN) analysis revealed that GFGLS2 has a close relationship with others in Ganoderma sinense, Trametes coccinea, Polyporus brumalis, and Trametes pubescens. With the assistance of 3D structure modelling by AlphaFold 2, molecular docking and molecular dynamics simulations, the central hydrophilic domain (Class III) in GFGLS2 was the main active sites through binding the substrate UDP–glucose to 11 amino acid residues via hydrogen bonds, π-stacking and salt bridges. Conclusions: The biochemical, 3D structural characterization and potential catalytic mechanism of a membrane-bound β-1, 3-glucan synthase GFGLS2 from cultured mycelia of G. frondosa were well investigated and would provide a reasonable full picture of β-1, 3-glucan synthesis in fungi.

The β-glucans are structurally varied, naturally occurring components of the cell walls, and storage materials of a variety of plant and microbial species. In the human diet, mixed-linkage glucans [MLG - β-(1,3/4)-glucans] influence the gut microbiome and the host immune system. Although consumed daily, the molecular mechanism by which human gut Gram-positive bacteria utilize MLG largely remains unknown. In this study, we used Blautia producta ATCC 27340 as a model organism to develop an understanding of MLG utilization. B. producta encodes a gene locus comprising a multi-modular cell-anchored endo-glucanase (BpGH16_MLG), an ABC transporter, and a glycoside phosphorylase (BpGH94_MLG) for utilizing MLG, as evidenced by the upregulation of expression of the enzyme- and solute binding protein (SBP)-encoding genes in this cluster when the organism is grown on MLG. We determined that recombinant BpGH16_MLG cleaved various types of β-glucan, generating oligosaccharides suitable for cellular uptake by B. producta. Cytoplasmic digestion of these oligosaccharides is then performed by recombinant BpGH94_MLG and β-glucosidases (BpGH3-AR8_MLG and BpGH3-X62_MLG). Using targeted deletion, we demonstrated BpSBP_MLG is essential for B. producta growth on barley β-glucan. Furthermore, we revealed that beneficial bacteria, such as Roseburia faecis JCM 17581^T, Bifidobacterium pseudocatenulatum JCM 1200T, Bifidobacterium adolescentis JCM 1275T, and Bifidobacterium bifidum JCM 1254, can also utilize oligosaccharides resulting from the action of BpGH16_MLG. Disentangling the β-glucan utilizing the capability of B. producta provides a rational basis on which to consider the probiotic potential of this class of organism.