Modulation of kernel storage proteins in grain sorghum (Sorghum bicolor (L.) Moench).

Sorghum prolamins, termed kafirins, are categorized into subgroups α, ß, and γ. The kafirins are co-translationally translocated to the endoplasmic reticulum (ER) where they are assembled into discrete protein bodies that tend to be poorly digestible with low functionality in food and feed applications. As a means to address the issues surrounding functionality and digestibility in sorghum, we employed a biotechnology approach that is designed to alter protein body structure, with the concomitant synthesis of a co-protein in the endosperm fraction of the grain. Wherein perturbation of protein body architecture may provide a route to impact digestibility by reducing disulphide bonds about the periphery of the body, while synthesis of a co-protein, with known functionality attributes, theoretically could impact structure of the protein body through direct association and/or augment end-use applications of sorghum flour by stabilizing ß-sheet formation of the kafirins in sorghum dough preparations. This in turn may improve viscoelasticity of sorghum dough. To this end, we report here on the molecular and phenotypic characterizations of transgenic sorghum events that are down-regulated in γ- and the 29-kDa α-kafirins and the expression of a wheat Dy10/Dx 5 hybrid high-molecular weight glutenin protein. The results demonstrate that down-regulation of γ-kafirin alone does not alter protein body formation or impacts protein digestibility of cooked flour samples. However, reduction in accumulation of a predicted 29-kDa α-kafirin alters the morphology of protein body and enhances protein digestibility in both raw and cooked samples.

Large-scale cultivation of acidophilic hyperthermophiles for recovery of secreted proteins.

An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins. Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system. These alterations provided accurate temperature and pH control. The bioreactor was used to cultivate selected strains of the archaeon Sulfolobus solfataricus and other species within this genus. Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium components. Substrate induction of synthesis of the S. solfataricus-secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes. An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant. These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins.