Nie_2025_Food.Chem_482_144155

Patatin, a multifunctional glycoprotein from potatoes, is a promising lipase for industrial applications due to its emulsifying, antioxidant, and lipid-modifying properties. However, its low expression efficiency and preference for short-chain substrates limit practical utility. Here, we addressed these challenges by heterologously expressing patatin in Pichia pastoris X-33, achieving a yield of 121 mg/L through optimized fermentation. The enzyme showed optimal activity at 35 degreesC and pH 10.0, with methanol enhancing activity, while Fe(2+)/Fe(3+) inhibited it. Rational design of the D286A mutant significantly improved long-chain substrate specificity (3.2-fold higher activity for pNP-C16) and thermal stability (deltaTm = +5.4 degreesC). Molecular dynamics revealed that the mutation disrupted an alpha-helix (residues 280-286), forming a flexible loop to accommodate long-chain substrates via hydrogen bonding and Pi-alkyl interactions. Structural integrity was confirmed by circular dichroism. This work provides a scalable platform for engineering patatin, with future studies targeting industrial-scale production and applications in functional lipids and biocatalysis.