| Summary: | Cholest-4-en-3-one Δ<sup>1</sup>-dehydrogenase (AcmB) from <i>Sterolibacterium denitrificans</i> was successfully immobilized on 3-aminopropyltrimethoysilane functionalized mesoporous cellular foam (MCF) and Santa Barbara Amorphous (SBA-15) silica supports using adsorption or covalently with glutaraldehyde or divinyl sulfone linkers. The best catalyst, AcmB on MCF linked covalently with glutaraldehyde, retained the specific activity of the homogenous enzyme while exhibiting a substantial increase of the operational stability. The immobilized enzyme was used continuously in the fed-batch reactor for 27 days, catalyzing 1,2-dehydrogenation of androst-4-en-3-one to androst-1,4-dien-3-one with a final yield of 29.9 mM (8.56 g/L) and 99% conversion. The possibility of reuse of the immobilized catalyst was also demonstrated and resulted in a doubling of the product amount compared to that in the reference homogenous reactor. Finally, it was shown that molecular oxygen from the air can efficiently be used as an electron acceptor either reoxidizing directly the enzyme or the reduced 2,4-dichlorophenolindophenol (DCPIPH<sub>2</sub>).
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