Recombinant Extracellular Cholesterol Oxidase from Nocardioides simplex

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 chole...

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Bibliographic Details
Published in:Biochemistry (Moscow) 2022-09, Vol.87 (9), p.903-915
Main Authors: Fokina, Victoria V., Karpov, Mikhail V., Kollerov, Vyacheslav V., Bragin, Eugeny Yu, Epiktetov, Dmitry O., Sviridov, Alexey V., Kazantsev, Alexey V., Shutov, Andrey A., Donova, Marina V.
Format: Article
Language:English
Subjects:
Actinomycetes
Affinity chromatography
Biochemistry
Biomedical and Life Sciences
Biomedicine
Bioorganic Chemistry
Biotechnology
Cholesterol
Cholesterol oxidase
Dehydroepiandrosterone
E coli
Enzymes
Escherichia coli
Hydrogen peroxide
Identification and classification
Life Sciences
Microbiology
Microorganisms
Nocardioides
Oxidases
Oxidation
pH effects
Pregnenolone
Properties
Shelf life
Stability tests
Steroid hormones
Steroids
Storage stability
Streptomyces hygroscopicus
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Summary: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 (ChO Ns ) was purified using affinity chromatography. ChO Ns 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 ChO Ns were generally comparable with or superior to those of commercial ChO from Streptomyces hygroscopicus (ChO Sh ). 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.
ISSN:0006-2979
1608-3040
DOI:10.1134/S0006297922090048