High-level expression of active horseradish peroxidase in methyltrophic yeast.

Expression of active horseradish (Armoracia rusticana) peroxidase (HRP) in microbes is necessary not only to gain an insight into its mechanisms, structure and function, and physiological role in plants, but also to supply large quantities of this useful enzyme. Exploiting new approach to express HRP in methyltrophic yeast, Pichia pastoris, the cDNA coding for mature HRP isozyme C was subcloned into the vector pPIC9. After transformation; the recombinant P. pastoris strain which secreted nonglycosylated HRP (about 30 ku) and hyperglycosylated HRP ( about 100 ku) was obtained. Through optimization of the growth conditions, the hyperglycosylated HRP as the sole product was secreted into fermentation broth and reached a level of approximately 4 similar to 6 mg per mi of culture medium. Remarkably, a certain activity of 2.24 U/ml was determined in the fermentation broth and showed the maximum absorbance at 403 nm.