| Summary: | Cholesterol oxidases (CHOXs) are flavin‐adenine dinucleotide‐dependent oxidoreductases with a range of biotechnological applications. There remains an urgent need to identify novel CHOX family members to meet the demands of enzyme markets worldwide. Here, we report the cloning, heterologous expression, and structural modeling of the cholesterol oxidase of Acinetobacter sp. strain RAMD. The cholesterol oxidase gene was cloned and expressed in pGEM®‐T and pET‐28a(+) vectors, respectively, using a gene‐specific primer based on the putative cholesterol oxidase ORF of Acinetobacter baumannii strain AB030 (GenBank [gb] locus tag: IX87_05230). The obtained nucleotide sequence (1671 bp, gb: MK575469.2), translated to a protein designated choxAB (556 amino acids), was overexpressed as inclusion bodies (IBs) (MW ˜ 62 kDa) in 1 mm IPTG‐induced Escherichia coli BL21 (DE3) Rosetta cells. The optimized expression conditions (1 mm IPTG with 2% [v/v] glycerol and at room temperature) yielded soluble active choxAB of 0.45 U·mL−1, with 56.25‐fold enhancement. The recombinant choxAB was purified to homogeneity using Ni2+‐affinity agarose column with specific activity (0.054 U·mg−1), yield (8.1%), and fold purification (11.69). Capillary isoelectric‐focusing indicated pI of 8.77 for choxAB. LC‐MS/MS confirmed the IBs (62 kDa), with 82.6% of the covered sequence being exclusive to A. baumannii cholesterol oxidase (UniProtKB: A0A0E1FG24). The 3D structure of choxAB was predicted using the LOMETS webtool with the cholesterol oxidase template of Streptomyces sp. SA‐COO (PDB: 2GEW). The predicted secondary structure included 18 α‐helices and 12 β‐strands, a predicted catalytic triad (E220, H380, and N514), and a conserved FAD‐binding sequence (GSGFGGSVSACRLTEKG). Future studies should consider fusion to solubilization tags and switching to the expression host Pichia pastoris to reduce IB formation.
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