Codon-optimized production of Cu/Zn superoxide dismutase (SOD1) in Komagataella phaffii: Functional characterization...

Cu/Zn superoxide dismutase (SOD1) is a vital enzyme that catalyzes the dismutation of superoxide radicals into oxygen and hydrogen peroxide. The large-scale production of recombinant eukaryotic SOD1 remains limited by challenges in achieving high yields and functional stability. In this study, the SOD1 gene from Saccharomyces bayanus was cloned, characterized, and overexpressed in Komagataella phaffii. The SOD1 gene (465 bp, encoding 154 residues) was first codon-optimized and cloned into the pPIC9K vector, yielding pPIC9K-SOD1. The SOD was expressed in a shake flask, reaching a maximum activity of 2074.9 U/mg after 96 h of induction. The protein was further analyzed for its enzymatic properties. Enzymatic studies revealed stability within the physiologically relevant pH range (6.0-8.0), with an optimal pH of 6.0 and an optimal temperature of 40 °C. The enzyme maintained over 70 % of its initial activity after incubation for 2 h at 60 °C, demonstrating high thermostability. Molecular docking further supported the enzyme's functional integrity, revealing conserved interactions with key Cu²⁺-binding residues His47, His49, His64, and His121. High-cell-density cultivation in a 5-L fermenter yielded a secretory SOD1 activity of 15,120 U/mg. This study presents an efficient, scalable strategy for recombinant SOD1 production in K. phaffii, enabling broader applications in industrial and therapeutic antioxidant formulations.