Purification and Structural Determination of a Native Biofilm Obtained from Copper Pipes of a Hospital - Chemical Sequence and Partial Folding

摘要

For the first time the chemical structures of the matrix of a native biofilmisolated from a microbially induced copper corrosion process has been analysed quantitatively by means of MALDI-TOF-mass-spectroscopy techniques and partially sequenced, revealing a polysaccharide backbone of cross-linked oligosaccharides, mainly hexoses, (95%) and approx. 5% of a linkage between serine or threonine to the polysaccharide chain. In addition some pyruvyl residues have also been determined including the total content of α-oxo acids being present in the matrix of the biofilm through covalent linkages. The matrix of the biofilm investigated is an ionic polysaccharide consisting (tentatively) partly of linear β-glucosidic and mannosidic like backbone (1 → 4) cross-linked to β-D-mannosidic residues, and galactose-or mannosidic residues resulting in a three-sugar side chain linkage attached to every third or fourth linkage of mannose (galactose) of the polysaccharide backbone. The O-glycosidic nature of the linkages between the prosthetic disaccharide and hydroxyl groups of L-(+)-serine and L-(+)-threonine residues has been established according to the chemical sequence. The strong alkali-lability of the sugar component and peptide linkages was attributed to a β-elimination mechanism on part of the sugar component, and the stoichiometric conversion of serine to dehydro-alanine and of threonine to α-crotonic acid was shown for the alkali-treated matrix of the biofilm. Serine and threonine residues of the peptide are most likely involved within these O-glycosidic linkages in equimolar proportion. Preliminary light scattering experiments of the native matrix of the biofilm and cleaved forms of this exopolymer yielded weight average molecular weights of 0.70 x 10~5 to 23.0 x 10~5, depending on chemical and enzymatic treatment of the matrix. The radii of gyration and second virial coefficients were found to be dependent on molecular weights, but show a behaviour which is consistent with that of semiflexible chains of the matrix of the biofilm. Some of the physical data obtained, particularly those of the high molecular fractions are also consistent with the view that this matrix can exist in aqueous solutions at low ionic strengths as double-stranded helical chains, which is also supported by optical rotary measurements as a function of temperature. On the basis of the molecular weight distribution and the chemical analysis a tentative model was proposed to encompass all data obtained by physical and MALDI-TOF-MS experiments.