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Recombinant Extracellular Cholesterol Oxidase from Nocardioides simplex

Victoria V. Fokina1,a*, Mikhail V. Karpov1,b, Vyacheslav V. Kollerov1,c, Eugeny Yu. Bragin1,d, Dmitry O. Epiktetov1,e, Alexey V. Sviridov1,f, Alexey V. Kazantsev2,g, Andrey A. Shutov1,h, and Marina V. Donova1,i

1Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Center for Biological Research of the Russian Academy of Sciences”, 142290 Pushchino, Moscow Region, Russia

2Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

* To whom correspondence should be addressed.

Received July 5, 2022; Revised August 1, 2022; Accepted August 1, 2022
Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3β-hydroxy-5-ene- to 3-keto-4-ene- steroids with the formation of hydrogen peroxide. Here, we expressed 6xHis-tagged mature form of the extracellular cholesterol oxidase (ChO) from the actinobacterium Nocardioides simplex VKM Ac-2033D (55.6 kDa) in Escherichia coli cells. The recombinant enzyme (ChONs) was purified using affinity chromatography. ChONs proved to be functional towards cholesterol, cholestanol, phytosterol, pregnenolone, and dehydroepiandrosterone. Its activity depended on the structure and length of the aliphatic side chain at C17 atom of the steroid nucleus and was lower with pregnenolone and dehydroepiandrosterone. The enzyme was active in a pH range of 5.25÷6.5 with the pH optimum at 6.0. Kinetic assays and storage stability tests demonstrated that the characteristics of ChONs were generally comparable with or superior to those of commercial ChO from Streptomyces hygroscopicus (ChOSh). The results contribute to the knowledge on microbial ChOs and evidence that ChO from N. simplex VKM Ac-2033D is a promising agent for further applications.
KEY WORDS: cholesterol, cholesterol oxidase, actinobacteria, recombinant enzyme

DOI: 10.1134/S0006297922090048