Recombinant napin expression in Pichia pastoris: Structural and functional attributes in comparison with...

The increasing demand for alternative proteins necessitates efficient production platforms. This study establishes a scalable method for producing recombinant napin (rNapin) using Pichia pastoris and compares it to canola-derived napin (cNapin). While cNapin is processed into a two-chain protein, rNapin remains a single polypeptide, allowing for structural and functional comparisons. Fermentation optimization showed that the GAP promoter increased rNapin yield by 2.4-fold compared to the AOX1 promoter in haploid strains, while further strain engineering using a diploid GAP strain led to an additional 1.5-fold increase. Circular dichroism and nanoDSF analysis confirmed that rNapin retains key secondary structure elements similar to cNapin, though it exhibited greater aggregation and lower thermal stability. In terms of functionality, rNapin demonstrated high solubility across all pH values and superior emulsifying activity under acidic conditions, though it lacked foamability. These findings demonstrate rNapin's potential for food applications and the benefits of P. pastoris optimization for production.