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Extraction, Purification and Characterization of Cholesterol Oxidase Enzyme Biosynthesized by Probiotic Lactiplantibacillus plantarum MF1
  • Mohamed farouk,
  • samar mahmoud,
  • Mohamed Abdel razik
Mohamed farouk
Zagazig University Faculty of Science
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samar mahmoud
Suez Canal University

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Mohamed Abdel razik
Suez Canal University
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This study aims to purify and characterize the cholesterol oxidase enzyme extracted from probiotic bacteria isolated from raw cow milk. The bacterial isolate was identified genotypically by 16S rRNA such as Lactiplantibacillus plantarum MF1 and then deposited in GenBank under the accession number MW242720. Cholesterol oxidase from Lactiplantibacillus plantarum MF1 was purified using acetone and Sephadex G 100 column chromatography, yielding enzyme recovery and purification folds of 19.3% and 4.2, respectively. The purity of purified cholesterol oxidase was confirmed using SDS-PAGE, which revealed a single protein band at 51 kDa. The optimal conditions for maximum activity of the purified CHO enzyme produced by Lactiplantibacillus plantarum MF1 were a 30-minute incubation period at 30°C, a substrate concentration of 150 μL in the reaction mixture, and an enzyme concentration of 100 μL in the reaction mixture. The purified cholesterol oxidase was activated by metal ions (CuSO4), whereas it was inhibited by CaCl2, MgSO4, ZnSO4, and MnCl2. After four days of storage at -20 °C, the purified enzyme retained 57% of its initial enzyme activity. The purified enzyme demonstrated an active effect on serum human cholesterol samples, resulting in a 29% reduction.