Cholesterol oxidase (ChO) belongs to the family of flavin-containing enzymes that catalyze the oxidation of 3β-hydroxy-5-ene steroids with the formation of hydrogen peroxide and the products with a 3‑keto-4-ene structure. This study dealt with obtaining a secreted mature form of heterologous ChO from the actinobacterium Nocardioides simplex (ChONs) in the cells of a recombinant methylotrophic yeast Pichia pastoris GS115. Based on the plasmid vector pPICZαA, genetic constructs were designed for heterologous expression of the target gene choN (KR76₀9550) from N. simplex in recombinant yeast. We compared the activity of the cell-free culture supernatant of recombinant P. pastoris strains containing secreted heterologous ChONs with respect to the conversion of cholesterol to cholestenone, depending on the presence and position of a 6 × His tag in the ChONs molecule. The conditions for synthesis of the most active heterologous secreted ChONs in the selected recombinant yeast strain were optimized.
Kollerov et al. (Mon,) studied this question.