Rational Engineering and Preparation of Horseradish Peroxidase C
- 1National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan 430064, China
- 2State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences of Hubei University, Hubei University, Wuhan 430062, China
Abstract
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.
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BibTeX
@article{cui2026rational,
title = {{Rational Engineering and Preparation of Horseradish Peroxidase C}},
author = {Juran Cui and Qifeng Yu and Sen Yang and Quanxi Wang and Yaping Wang and Ben Rao},
journal = {Molecules},
year = {2026},
month = oct,
doi = {10.3390/molecules31203730},
url = {https://www.mdpi.com/1420-3049/31/20/3730},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Rational Engineering and Preparation of Horseradish Peroxidase C
AU - Juran Cui
AU - Qifeng Yu
AU - Sen Yang
AU - Quanxi Wang
AU - Yaping Wang
AU - Ben Rao
PY - 2026
DA - 2026/10/01
JO - Molecules
DO - 10.3390/molecules31203730
UR - https://www.mdpi.com/1420-3049/31/20/3730
AB - 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.
SN - 1420-3049
ER -