DESCRIPTION

Soy leghemoglobin protein is produced by controlled submerged fed-batch fermentation of a non-pathogenic, non-toxigenic, genetically engineered strain of the yeast Pichia pastoris. The production strain is constructed to overexpress the P. pastoris enzymes responsible for catalyzing heme B biosynthesis and leghemoglobin C2 from the root nodules of the soybean plant Glycine max. At the end of the fermentation, bead mill mechanical shearing is used to lyse the P. pastoris cells in the fermentation broth. Centrifugation and microfiltration are then performed to remove insoluble materials in the lysate, followed by ultrafiltration of the fermentation broth to concentrate the soy leghemoglobin protein. Sodium chloride and sodium ascorbate are added to the ultrafiltered fermentation broth to create the liquid concentrate, which is frozen until use. The liquid concentrate soy leghemoglobin is referred to as LegH Prep. LegH Prep is a mixture containing the soy leghemoglobin protein, residual P. pastoris proteins and added stabilizers (e.g., sodium ascorbate and sodium chloride). The terms soy leghemoglobin and soy leghemoglobin protein are used interchangeably to refer to the protein itself and the coloring component of LegH Prep.