Study on the High-Efficiency Expression of Horseradish Peroxidase in Pichia pastoris

Horseradish peroxidase (HRP) is a heme-containing oxidoreductase with extensive applications in biotechnology, medical diagnostics, and environmental protection. In this study, <i>Pichia pastoris</i> was utilized to produce HRP. Successfully, expression strains with 1-5 copies of HRP-C were constructed, and the strain with the highest expression level and activity of HRP-C was obtained. Different molecular chaperones (PDI1, HAC1, BIP1) were selected, and co-expression was carried out through co-induction and separate induction methods. The results showed that the yield of HRP increased approximately 1.4 times with the assistance of PDI1 and HAC1 molecular chaperones in the 3-copy <i>Pichia pastoris</i> expression strain, with enzyme activities increasing by 1.2-fold and 1.3-fold, respectively. High-density fermentation of the recombinant strain transformed with BDM-PDI1-HRP-C-3C was carried out in a 50 L fermenter, and after methanol induction for 72 h, a target protein expression level of up to 200 mg/L was achieved. The enzyme activity reached 1796 U/mL, which is nearly three times higher than that of shake-flask fermentation and is the highest reported in the literature to date.