Abstract
3-Ketosteroid-Δ1-dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ1-dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, nine putative kstD genes from different origins were selected and overexpressed in Escherichia coli BL21(DE3). These recombinant enzymes catalyzed the Δ1-desaturation of a variety of steroidal compounds. Among them, the KstD from Propionibacterium sp. (PrKstD) displayed the highest specific activity and broad substrate spectrum. The detailed catalytic characterization of PrKstD showed that it can convert a wide range of 3-ketosteroid compounds with diverse substituents, ranging from substituents at the C9, C10, C11 and C17 position through substrates without C4-C5 double bond, to previously inactive C6-substituted ones such as 11β,17-dihydroxy-6α-methyl-pregn-4-ene-3,20-dione. Reaction conditions were optimized for the biotransformation of hydrocortisone in terms of pH, temperature, co-solvent and electron acceptor. By using 50 g/L wet resting E. coli cells harboring PrKstD enzyme, the conversion of hydrocortisone was about 92.5% within 6 h at the substrate concentration of 80 g/L, much higher than the previously reported results, demonstrating the application potential of this new KstD.
Highlights
IntroductionPublisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations
These enzymes play functions in steroid degradation andthe show plication implementation is prevented by the limited availability of
Ketosteroid-∆1 -dehydrogenase (KstD) with high acapplication implementation is prevented by the limited availability of
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. ∆1 -dehydrogenation of steroid compounds is an important reaction in the synthesis of medically useful steroidal molecules [3–6] Such a transformation may be achieved by chemical or biocatalytic methods. ∆1 -KSTDs from several microorganisms were overexpressed in Bacillus species [23–25], Corynebacterium crenatum [26], Escherichia coli [21,27–32], or Pichia pastoris [27] to enable ∆1 -dehydrogenation of various valuable steroid intermediates. This approach has drawn increasing attention because it avoids the by-product accumulation in the microbial transformation using the wild-type microorganisms as the biocatalyst due to other active enzymes in the microorganisms. A new KstD enzyme from Propionibacterium sp (PrKstD) was purified and characterized, and its substrate scope and application in the synthesis of prednisolone were explored
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