Biochemical characterization and optimized production of Vigna angularis hemoglobin in Pichia pastoris

Hemoglobin (HB) is a valuable additive for enhancing the taste, flavor, and nutrition of plant-based protein products. With rising demand for sustainable and health-conscious food options, the exploration of novel HB sources has become increasingly important to meet consumer needs. In this study, a plant-derived HB from Vigna angularis (VaHB) was heterologously expressed and characterized in Pichia pastoris . The data indicated that VaHB exhibited prominent absorption peaks at 415 and 540 nm, a secondary structure comparable to reference standards, high peroxidase activity (123.6 U/mg), and remarkable thermal stability (Tm = 79.6 °C). These attributes were found to differ from those of Glycine a HB (GmHB), an approved color additive in plant-based protein products. To optimize its production, the copy number of the globin-coding gene was enhanced and the entire heme biosynthesis pathway was reengineered, achieving a titer of 117.7 mg/L. These findings indicate that VaHB could be considered a comparable alternative to GmHB.