Rational Engineering and Preparation of Horseradish Peroxidase C

To address the persistent challenges of low yield and significant batch-to-batch variability inherent in the traditional plant-based extraction of horseradish peroxidase (HRP), this study aimed to develop an efficient production strategy for HRP-C through rational molecular design and process optimization. A recombinant engineering strain was constructed in Pichia pastoris GS115, and site-directed mutagenesis was performed based on FoldX virtual screening to evaluate mutations affecting enzyme stability and catalytic properties. A comprehensive 50 L scale process was established, integrating high-density fermentation, purification, and lyophilized formulation. Results indicated that exogenous supplementation with 5 μM hemin significantly enhanced enzymatic activity. The HRP-C-D282I variant exhibited a 17.7% increase in specific activity and a 2.14-fold extension of half-life at 60 °C compared with the wild-type enzyme. Furthermore, Endo H-mediated deglycosylation was associated with a dose-dependent decrease in HRP-C-D282I activity, suggesting that N-glycosylation contributes to enzyme performance under the conditions tested. The optimized process achieved a recombinant protein concentration of 216 mg/L during fermentation and a purified enzyme specific activity of 2600 U/mg, with a lyophilized powder reconstitution time of less than 60 s. This study provides a proof-of-concept basis for the potential industrial production and application of recombinant HRP.