Ex situ and in situ investigation of protein/exopolysaccharide complex in Porphyridium cruentum biomass resuspension
Résumé
During the extraction processes of B-phycoerythrin from P. cruentum, the possibility of the occurrence of extraction-inhibiting B-phycoerythrin/exopolysaccharide (EPS) complexes was highlighted. The use of confocal laser scanning microscopy allowed the observation of the cells including their sheath and their chloroplast, the bound EPS matrix, and extracellular vesicles involving phycobiliproteins and EPS in P. cruentum biomass resuspensions. The observations were performed using autofluorescence and fluorescence marking.pH variations showed more impact than ionic strength increase induced by NaCl addition on the B-phycoerythrin content and the organization of vesicles. Namely, agglomeration of the vesicles could be observed at pH 8.5 without NaCl addition. With addition of 1 M NaCl, this phenomenon was prevented but the vesicles were not visibly dissociated for any pH value. These results point towards a precipitate rather than a coacervate, which are expected to have narrow stability conditions regarding pH and ionic strength.In an attempt to dissolve or break the vesicles and the extracellular matrix, successive macerations in water and use of high pressure homogenizer process in one-step or two-step treatments were applied to the resuspensions. The successive macerations could free the cells and the vesicles from the extracellular matrix, whereas the homogenizer treatment disrupted the extracellular matrix, the vesicle and a part of the cells. In our case, only the successive macerations showed a positive extraction since the high pressure treatment caused unwanted denaturation.It is concluded that more experiments in model conditions could help understand the nature of the interactions between phycobiliproteins and EPS leading to the formation of the vesicles.
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