Journal: Environmental Microbiology

Article Title: Ring A Cleaving Beta‐Diketone Hydrolase Is a Key Enzyme of Steroid Degradation in Anaerobic Bacteria

doi: 10.1111/1462-2920.70034

Figure Lengend Snippet: Activity assay and enrichment of steroid ring A hydrolase (SRAH). (A) Time‐dependent conversion of androsta‐1‐en‐3,17‐dione (1,3,17‐ATO) to 1,17‐dioxo‐2,3‐ seco‐ androstan‐3‐carboxylate (DSAO). UPLC chromatograms at 0 and 30 min using 1 mg mL −1 SRAH enriched from crude extracts of Stl. denitrificans . (B) SDS‐PAGE analysis of active pools obtained during the enrichment of SRAH from crude extracts of Stl. denitrificans . Extract: 20 μg crude extracts, (NH 4 ) 2 SO 4 : 15 μg protein after (NH 4 ) 2 SO 4 precipitation, Butyl: 10 μg protein after Butyl‐Sepharose chromatography, CaptoQ: 7.5 μg protein after Capto Q ion exchange chromatography, Superdex 200: 5 μg protein after size‐exclusion chromatography using a Superdex 200 Increase 10/300 GL. (C) SDS‐PAGE analysis of active pools during the enrichment of SRAH after heterologous expression of the encoding gene by StrepTactin affinity chromatography: 20 μg SRAH producing E. coli BL21 soluble proteins, flow through: unbound protein fraction, StrepTacin XT: 3 μg SRAH after StrepTactin XT affinity chromatography. (D) Blue Native PAGE analysis of enriched SRAH after heterologous production in E. coli .

Article Snippet: The resulting soluble fraction was then precipitated with 1.5 M (NH 4 ) 2 SO 4 , centrifuged at 10,000 rpm for 20 min, and the resulting supernatant was applied to a 30 mL Butyl‐Sepharose column (30 mL, GE Healthcare) equilibrated with buffer A1 (1.5 M (NH 4 ) 2 SO 4 , 20 mM MOPS/KOH pH 7.0) at a flow rate of 5 mL min −1 .

Techniques: Activity Assay, SDS Page, Chromatography, Ion Exchange Chromatography, Size-exclusion Chromatography, Expressing, Affinity Chromatography, Blue Native PAGE