Integrated bioethanol and protein production from brown seaweed Laminaria digitata.

A wild-growing glucose-rich (i.e. 56.7% glucose content) brown seaweed species Laminaria digitata, collected from the North Coast of Denmark in August 2012, was used as the feedstock for an integrated bioethanol and protein production. Glutamic acid and aspartic acid are the two most abundant amino acids in the algal protein, both with proportional content of 10% in crude protein. Only minor pretreatment of milling was used on the biomass to facilitate the subsequent enzymatic hydrolysis and fermentation. The Separate Hydrolysis and Fermentation (SHF) resulted in obviously higher ethanol yield than the Simultaneous Saccharification and Fermentation (SSF). High conversion rate at maximum of 84.1% glucose recovery by enzymatic hydrolysis and overall ethanol yield at maximum of 77.7% theoretical were achieved. Protein content in the solid residues after fermentation was enriched by 2.7 fold, with similar distributions of amino acids, due to the hydrolysis of polymers in the seaweed cell wall matrix.

Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants.

Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant-robust broad-specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS-PAGE and N-terminal sequencing. We observe well-defined cleavage fragments, which suggest that flexibility is limited to certain regions of the protein. Cleavage sites for alpha-lactalbumin and myoglobin correspond to regions identified in other studies as partially unfolded at low pH or in the presence of organic solvents. For Tnfn3, which does not form partially folded structures under other conditions, cleavage sites can be rationalized from the structure of the protein's folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded structures. Thus, for proteins accumulating stable intermediates on the folding pathway, surfactants encourage the formation of these states, while the situation is more complex for proteins that do not form these intermediates.

Aggregation of S6 in a quasi-native state by sub-micellar SDS.

Anionic surfaces promote protein fibrillation in vitro and in vivo. Monomeric SDS has also been shown to stimulate this process. We describe the dynamics of conformational changes and aggregative properties of the model protein S6 at sub-micellar SDS concentrations. S6 exhibits a rich and pH-sensitive diversity in conformational changes around 0.2-2 mM SDS, in which several transitions occur over time scales spanning milliseconds to hours. Monomeric SDS readily precipitates S6 within minutes at pH-values of 5 and below to form states able to bind the fibril-specific dye thioflavin T. At pH 5.5, the process is much slower and shows a mutagenesis-sensitive lag, leading to different forms of organized but not classically fibrillar aggregates with native-like levels of secondary structure, although the tertiary structure is significantly rearranged. The slow aggregation process may be linked to conformational changes that occur at the second-time scale in the same SDS concentration range, leading to an altered structure, possibly with unfolding around the C-terminal helix. The S6 aggregates may be differently trapped states, equivalent to pre-fibrillar structures seen at early stages in the fibrillation process for other proteins. The low quantities of anionic species required suggest that the aggregates may have parallels in vivo.