Surfactants find wide commercial use as foaming agents, emulsifiers, and dispersants. Currently, surfactants are produced from petroleum, or from seed oils such as palm or coconut oil. Due to concerns with CO(2) emissions and the need to protect rainforests, there is a growing necessity to manufacture these chemicals using sustainable resources In this report, we describe the engineering of a native nonribosomal peptide synthetase pathway (i.e., surfactin synthetase), to generate a Bacillus strain that synthesizes a highly water-soluble acyl amino acid surfactant, rather than the water insoluble lipopeptide surfactin. This novel product has a lower CMC and higher water solubility than myristoyl glutamate, a commercial surfactant. This surfactant is produced by fermentation of cellulosic carbohydrate as feedstock. This method of surfactant production provides an approach to sustainable manufacturing of new surfactants.
Natural tetrameric streptavidin has two subunit interfaces; one is a strong interface between subunits in a tightly associated dimer, and the other is a weak interface between a pair of such dimers (dimer-dimer interface). To test whether strengthening the weak dimer-dimer interface could provide streptavidin with additional structural stability, covalent crosslinks were introduced between adjacent subunits through the dimer-dimer interface. Specific crosslinking sites were designed by site-directed mutations of His-127 residues that are in close proximity in natural streptavidin. The first and second streptavidin constructs have a disulfide bond and an irreversible covalent bond, respectively, between two Cys-127 residues across the dimer-dimer interface. The third variant is a hybrid tetramer consisting of two different streptavidin species, one having lysine and the other aspartic acid at position 127, which are covalently crosslinked. All streptavidin constructs with intersubunit crosslinks showed higher biotin-binding ability than natural core streptavidin after heat treatment. All of these crosslinked streptavidins retained bound biotin more stably than natural core streptavidin in guanidine hydrochloride at very acidic pH. These results suggest that the introduction of covalent bonds across the dimer-dimer interface enhances the overall stability of streptavidin.
A streptavidin mutant has been designed and produced that allows the specific, covalent immobilization of streptavidin on solid surfaces. This streptavidin mutant was constructed by fusing a six-residue sequence, containing a single cysteine, to the carboxyl terminus of streptavidin. Because this mutant has no other cysteine residues, the reactive sulfhydryl group of the cysteine residue serves as a unique immobilization site for conjugation using sulfhydryl chemistry. This streptavidin mutant was efficiently immobilized on maleimide-coated solid surfaces via its unique immobilization site. Characterization of the immobilized streptavidin mutant for the ability to bind to biotinylated macromolecules and the dissociation rates of bound biotin showed that the biotin-binding properties of this mutant were minimally affected by immobilization on solid surfaces. This streptavidin could be readily incorporated into a wide variety of solid-phase diagnostic tests and biomedical assays. This could enhance the performance of streptavidin-based solid-phase assay systems.
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