Glycoside hydrolase subfamily GH5_57 features a highly redesigned catalytic interface to process complex hetero-ß-mannans.

Glycoside hydrolase family 5 (GH5) harbors diverse substrate specificities and modes of action, exhibiting notable molecular adaptations to cope with the stereochemical complexity imposed by glycosides and carbohydrates such as cellulose, xyloglucan, mixed-linkage ß-glucan, laminarin, (hetero)xylan, (hetero)mannan, galactan, chitosan, N-glycan, rutin and hesperidin. GH5 has been divided into subfamilies, many with higher functional specificity, several of which have not been characterized to date and some that have yet to be discovered with the exploration of sequence/taxonomic diversity. In this work, the current GH5 subfamily inventory is expanded with the discovery of the GH5_57 subfamily by describing an endo-ß-mannanase (CapGH5_57) from an uncultured Bacteroidales bacterium recovered from the capybara gut microbiota. Biochemical characterization showed that CapGH5_57 is active on glucomannan, releasing oligosaccharides with a degree of polymerization from 2 to 6, indicating it to be an endo-ß-mannanase. The crystal structure, which was solved using single-wavelength anomalous diffraction, revealed a massively redesigned catalytic interface compared with GH5 mannanases. The typical aromatic platforms and the characteristic α-helix-containing ß6-α6 loop in the positive-subsite region of GH5_7 mannanases are absent in CapGH5_57, generating a large and open catalytic interface that might favor the binding of branched substrates. Supporting this, CapGH5_57 contains a tryptophan residue adjacent and perpendicular to the cleavage site, indicative of an anchoring site for a substrate with a substitution at the -1 glycosyl moiety. Taken together, these results suggest that despite presenting endo activity on glucomannan, CapGH5_57 may have a new type of substituted heteromannan as its natural substrate. This work demonstrates the still great potential for discoveries regarding the mechanistic and functional diversity of this large and polyspecific GH family by unveiling a novel catalytic interface sculpted to recognize complex heteromannans, which led to the establishment of the GH5_57 subfamily.

Glucogalactan: a polysaccharide isolated from the cell-wall of Verticillium lecanii.

A water-soluble polysaccharide was extracted with alkali from the cell wall of Verticillium lecanii (also called Lecanicillium lecanii). After freezing and thawing, the water-soluble fraction was purified by gel filtration chromatography on Sepharose CL-6B and eluted as one peak by HPSEC/RID. Monosaccharide analysis showed galactose and glucose (1.1:1), with traces of mannose (<1%). The structural characteristics were determined by spectroscopic analysis, FT-IR and 1D and 2D (1)H and (13)C NMR, and methylation results. On the basis of the data obtained, the following structure of the polysaccharide (E3SIV fraction) was established: [Formula: see text] where n≈22 and m≈22.

Sulfonation and anticoagulant activity of fungal exocellular ß-(1→6)-D-glucan (lasiodiplodan).

An exocellular ß-(1→6)-D-glucan (lasiodiplodan) produced by a strain of Lasiodiplodia theobromae (MMLR) grown on sucrose was derivatized by sulfonation to promote anticoagulant activity. The structural features of the sulfonated ß-(1→6)-D-glucan were investigated by UV-vis, FT-IR and (13)C NMR spectroscopy, and the anticoagulant activity was investigated by the classical coagulation assays APTT, PT and TT using heparin as standard. The content of sulfur and degree of substitution of the sulfonated glucan was 11.73% and 0.95, respectively. UV spectroscopy showed a band at 261 nm due to the unsaturated bond formed in the sulfonation reaction. Results of FT-IR and (13)C NMR indicated that sulfonyl groups were inserted on the polysaccharide. The sulfonated ß-(1→6)-D-glucan presented anticoagulant activity as demonstrated by the increase in dose dependence of APTT and TT, and these actions most likely occurred because of the inserted sulfonate groups on the polysaccharide. The lasiodiplodan did not inhibit the coagulation tests.

Rheological characterization of a xanthan-galactomannan hydrogel loaded with lipophilic substances.

We compared the structures and rheology of xanthan-galactomannan (X:G) hydrogels with the addition of curcumin in microemulsion (X:GMC) and ethanol (X:GEC). X:GMC hydrogels have gel characteristics and exhibited a significantly higher elastic response than the X:GEC and X:G hydrogels at room temperature, but after heating, an increase in the elastic modulus was observed for the last two systems. The visualization of the hydrogel microstructures by cryo-scanning electronic microscopy revealed pores within the lamellar structure only for X:GMC. In vitro skin permeation tests showed a more pronounced lag time for X:GMC; however, a more efficient permeation from X:GMC than from X:GEC. This study demonstrates that the X:G system is an alternative to traditional gels for the topical applications of hydrophobic drugs.

A galactomannan-driven enhancement of the in vitro multiplication rate for the Marubakaido apple rootstock (Malus prunifolia (Willd.) Borkh) is not related to the degradation of the exogenous galactomannan.

Agar is a complex mixture of gel-forming polysaccharides. Gelling agents are very often used to provide proper support for plants grown in semisolid culture media. And agar is the most frequently used gelling agent in plant tissue culture media. Galactomannans, another group of gel-forming polysaccharides, consists of a (1 → 4)-linked ß-D: -mannopyranosyl backbone partially substituted at O-6 with D: -galactopyranosyl side groups. In this work, we demonstrate that a statistically significant 2.7-fold increase on the multiplication rate (MR) for in vitro-grown Marubakaido (Malus prunifolia) shoots was associated with a 12.5% replacement of agar in the semi-solid culture media for a galactomannan obtained from seeds of Schizolobium paraybae. This increase on MR was due mainly to a 1.9-fold increase in the number of main branches and an 8.6-fold increase in the number of primary lateral branches. Gas liquid chromatography and thin layer chromatography analyzes demonstrated that the galactomannan-driven enhancement of the in vitro multiplication rate of the Marubakaido apple rootstock was not related to the galactomannan degradation. To the best of our knowledge, this is the first report on the successful use of partial replacement of high quality agar by a galactomannan from S. paraybae in a micropropagation system for a tree species.

Chemical modification of botryosphaeran: structural characterization and anticoagulant activity of a water-soluble sulfonated (1-->3)(1-->6)-ß-D-glucan.

The exopolysaccharide botryosphaeran (EPS(GLC); a (1--> 3)(1-->6)-ß-D-glucan from Botryosphaeria rhodina MAMB- 05) was sulfonated to produce a water-soluble fraction (EPS(GLC)-S) using pyridine and chlorosulfonic acid in formamid. This procedure was then repeated twice to produce another fraction (EPSGLC-RS) with a higher degree of substitution (DS, 1.64). The purity of each botryosphaeran sample (unsulfonated and sulfonated) was assessed by gel filtration chromatography (Sepharose CL-4B), where each polysaccharide was eluted as a single symmetrical peak. The structures of the sulfonated and re-sulfonated botryosphaerans were investigated using ultraviolet-visible (UV-Vis), Fourier-transform infrared (FT-IR), and (13)C nuclear magnetic resonance ((13)C NMR) spectroscopies. EPS(GLC) and EPS(GLC)-RS were also assayed for anticoagulation activity, and EPS(GLC)-RS was identified as an anticoagulant.

Thermal and rheological properties of a family of botryosphaerans produced by Botryosphaeria rhodina MAMB-05.

Differential scanning calorimetry (DSC), thermogravimetry (TG) and Fourier-transform infra-red spectroscopy (FT-IR) analyses were performed to investigate changes in the physico-chemical properties of botryosphaerans, a family of exopolysaccharides (EPS) produced by the fungus Botryosphaeria rhodina MAMB-05 grown on glucose (EPS(GLC)), sucrose (EPS(SUC)) and fructose (EPS(FRU)). A slight endothermic transition and small mass loss attributable to the removal of water of hydration were observed in the DSC and TG analyses, respectively, for the three EPS samples. The FT-IR spectra confirmed no structural changes occurred during thermal treatment. Viscometry was utilized to obtain information on the rheological behaviour of the EPS in aqueous solutions. The Power Law and Cross Equations determined the natural pseudoplastic characteristics of the EPS. Comparatively, results obtained for EPS produced when B. rhodina MAMB-05 was grown on each of the three carbohydrate sources demonstrated similar apparent viscosity values for EPS(GLC) and EPS(SUC), while EPS(FRU) displayed the lowest apparent viscosity of the three botryosphaerans, suggesting a higher degree of ramification and lower Mw. EPS(GLC) and EPS(SUC) possessed similar degrees of ramification. The slight differences found in their viscosities can be explained by the differences in the type of branching among the three botryosphaerans, thus varying the strength of intermolecular interactions and consequently, consistency and viscosity. The physico-chemical studies of botryosphaerans represent the originality of this work, and the knowledge of these properties is an important criterion for potential applications.

Three exopolysaccharides of the beta-(1-->6)-D-glucan type and a beta-(1-->3;1-->6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit.

Four exopolysaccharides (EPS) obtained from Botryosphaeria rhodina strains isolated from rotting tropical fruit (graviola, mango, pinha, and orange) grown on sucrose were purified on Sepharose CL-4B. Total acid hydrolysis of each EPS yielded only glucose. Data from methylation analysis and (13)C NMR spectroscopy indicated that the EPS from the graviola isolate consisted of a main chain of glucopyranosyl (1-->3) linkages substituted at O-6 as shown in the putative structure below: [carbohydrate structure: see text]. The EPS of the other fungal isolates consisted of a linear chain of (1-->6)-linked glucopyranosyl residues of the following structure: [carbohydrate structure: see text]. FTIR spectra showed one band at 891 cm(-1), and (13)C NMR spectroscopy showed that all glucosidic linkages were of the beta-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. beta-(1-->6)-d-Glucans produced as exocellular polysaccharides by fungi are uncommon.

Structural characterization of the cell wall D-glucans isolated from the mycelium of Botryosphaeria rhodina MAMB-05.

Three D-glucans were isolated from the mycelium of the fungus Botryosphaeria rhodina MAMB-05 by sequential extraction with hot-water and hot aqueous KOH (2% w/v) followed by ethanol precipitation. Following their purification by gel permeation chromatography on Sepharose CL-4B, the structural characteristics of the D-glucans were determined by FT-IR and 13C NMR spectroscopy and, after methylation, by GC-MS. The hot-water extract produced a fraction designated Q1A that was a beta-(1-->6)-D-glucan with the following structure: [Formula: see text] The alkaline extract, when subjected to repeated freeze-thawing, yielded two fractions: K1P (insoluble) that comprised a beta-(1-->3)-D-glucan with beta-D-glucose branches at C-6 with the structure: [Formula: see text] and K1SA (soluble) consisting of a backbone chain of alpha-(1-->4)-linked D-glucopyranosyl residues substituted at O-6 with alpha-D-glucopyranosyl residues: [Formula: see text]