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Characterization of new recombinant 3-ketosteroid-Δ 1 -dehydrogenases for the biotransformation of steroids.

Authors :
Wang X
Feng J
Zhang D
Wu Q
Zhu D
Ma Y
Source :
Applied microbiology and biotechnology [Appl Microbiol Biotechnol] 2017 Aug; Vol. 101 (15), pp. 6049-6060. Date of Electronic Publication: 2017 Jun 20.
Publication Year :
2017

Abstract

3-Ketosteroid-Δ <superscript>1</superscript> -dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ <superscript>1</superscript> -dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, we cloned 12 putative KstD-encoding (kstd) genes from both fungal and Gram-positive microorganisms and attempted to overproduce the recombinant proteins in E. coli BL21(DE3). Five successful recombinant enzymes catalyzed the Δ <superscript>1</superscript> -desaturation of a variety of steroidal compounds such as 4-androstene-3,17-dione (AD), 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), hydrocortisone, cortisone, and cortexolone. However, the substrate specificity and catalytic efficiency of the enzymes differ depending on their sources. The purified KstD from Mycobacterium smegmatis mc <superscript>2</superscript> 155 (MsKstD1) displayed high catalytic efficiency toward hydrocortisone, progesterone, and 9-OH-AD, where it had the highest affinity (K <subscript>m</subscript> 36.9 ± 4.6 μM) toward 9-OH-AD. On the other hand, the KstD from Rhodococcus erythropolis WY 1406 (ReKstD) exhibited high catalytic efficiency toward androst-4,9(11)-diene-3,17-dione (Diene), 21-acetoxy-pregna-4,9(11),16-triene-3,20-dione (Triene), and cortexolone, where in all three cases the K <subscript>m</subscript> values (12.3 to 17.8 μM) were 2.5-4-fold lower than that toward hydrocortisone (46.3 μM). For both enzymes, AD was a good substrate although ReKstD had a 3-fold higher affinity than MsKstD1. Reaction conditions were optimized for the biotransformation of AD or hydrocortisone in terms of pH, temperature, and effects of hydrogen peroxide, solvent, and electron acceptor. For the biotransformation of hydrocortisone with 20 g/L wet resting E. coli cells harboring MsKstD1 enzyme, the yield of prednisolone was about 90% within 3 h at the substrate concentration of 6 g/L, demonstrating the application potential of the newly cloned KstDs.

Details

Language :
English
ISSN :
1432-0614
Volume :
101
Issue :
15
Database :
MEDLINE
Journal :
Applied microbiology and biotechnology
Publication Type :
Academic Journal
Accession number :
28634849
Full Text :
https://doi.org/10.1007/s00253-017-8378-2