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Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6-9 % (w/v) 2-hydroxypropyl-β-cyclodextrin as a solubilizer and 1.25-5 % (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8-15 mM K[Fe(CN)] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8 g/L for 25-hydroxycholesterol, 1.4 g/L for calcifediol and 2.2 g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3 mg of pure S25DH from 30 g of wet cell mass (specific activity of 14 nmol min mg), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.

An efficient stereoselective syntheses of a series of functionalized optically active γ‐aryl‐γ‐butyrolactones is achieved by enzymatic asymmetric reduction of the corresponding sterically demanding γ‐keto esters employing wild‐type and recombinant alcohol dehydrogenases. The best stereoselectivities for the reduction via hydrogen transfer was obtained with two short chain dehydrogenases (SDRs) of complementary stereospecificity from Aromatoleum aromaticum, namely the Prelog‐specific NADH‐dependent (S)‐1‐phenylethanol dehydrogenase [(S)‐PED] and the anti‐Prelog‐specific (R)‐1‐(4‐hydroxyphenyl)‐ethanol dehydrogenase [(R)‐HPED], respectively. Biotransformations catalyzed by both enzymes, followed by TFA‐catalyzed cyclization of the resulting γ‐hydroxy esters, furnished the respective (S)‐ and (R)‐configured products with exquisite optical purity (up to >99% ee). The synthetic value was demonstrated on preparative scale for the asymmetric bioreduction of the model compound, methyl 4‐oxo‐4‐phenylbutanoate, affording optically pure (S)‐γ‐phenyl‐γ‐butyrolactone (>99% ee) in 67–74% isolated yield at 89–95% conversion depending on the applied scale.magnified image

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