Extração, purificação e caracterização de polissacarídeos da biomassa do fungo ascomiceto Botryosphaeria rhodina MAMB-05

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

The composition of the polysaccharides constituting the biomass of the ascomyceteous fungus, Botryosphaeria rhodina MAMB-05, cultivated on glucose as carbon source was investigated following separation of the exopolysaccharides (EPS). Fungal mycelial biomass was exhaustingly washed with water at room temperature until no residual sugars could be detected (phenol-sulfuric acid method), indicating total removal of the EPS, botryosphaeran. The mycelium was then lyophilised, followed by homogenisation and consecutively submitted to the following extraction procedures to extract the biomass polysaccharides: cold water (3x), boiling water (4x) and 2 % KOH/100 °C (2x), which separated the biomacromolecules into groups according to their solubilities in these solvents. Fractions Q1 (hot water extract) and K1 (alkaline extract) were selected for further studies: quantification and monosaccharide constitution. Fraction Q1 was fractionated by Sepharose CL-4B gel filtration chromatography into four peaks: Q1A, Q1B, Q1C and Q1D. Sub-fraction Q1A with an apparent large molecular mass as indicated by its elution volume on a chromatographic run, presented glucose as the major component. Spectroscopy studies (Fourier Transformation-Infra-Red (FT-IR), and 13Carbon-nuclear magnetic resonance, 13C-NMR) demonstrated that all of the glycosidic linkages were of the b-configuration. The absence of a signal in the region d 60.0 ppm and the presence of an intense signal at 69.01, as well as the results arising from methylation, characterized Q1A as a ß-(1?6)-D-glucan. Sub-fraction Q1C consisting mainly of galactose residues had a structure characterised as galactofuranan with substitutions at 5-O and 6-O as indicated by spectroscopy and methylation analyses. Sub-fractions Q1B and Q1D were not further investigated in this work because they required detailed purification. The fraction from alkaline extraction (K1) followed by neutralization, dialysis, ethanol precipitation and solubilisation in water, presented an insoluble residue (K1P) which could be separated from its soluble counterpart (K1S) by successive treatments of freezing and thawing. K1P consisted mainly of glucose (93 %) and was characterized through spectroscopy and methylation analyses as a ß-(1?3)-glucan with ramifications at C-6. K1S fractionated by Sepharose CL-4B gel filtration chromatography resolved into three peaks: K1SA, K1SB and K1SC. Sub-fraction K1SA, after acid hydrolysis presented mainly glucose (92 %), and analysis by gas chromatography of the alditol acetate derivatives presented three peaks identified as: 2,3,4,6-tetra-O-methyl-, 2,3,6-tri-O-methyl- and 2,3-di-O-methyl-glucitol. FT-IR signals at 1550 and 920 cm-1 were characteristic of glucans, and 850 cm-1 of a-glucans. 13C-NMR spectra showed signals corresponding to a-(1?4)-D-glucan with a low degree of substitution at C-6. The results found in this study indicate that the composition of the biomass polysaccharides of the ascomycete Botryosphaeria rhodina MAMB-05 was similar to the biomass of other fungi reported in the literature, and the differences in the degree of substitutions of the polysaccharides probably reflects variations in times of cultivation, and/or the relative degrees of purity.

ASSUNTO(S)

biotechnology fungi - biotechnology polysaccharides biotecnologia fungos - biotecnologia polissacarídeos

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