Androstenedione ad

M.smegmatis MC² 155 was pre-cultured in 2 mL Luria-Bertan (LB) to OD₆₀₀ = 1.0, then the cells were centrifuged down, collected and washed twice with minimal medium (1.5 g CH₃COONH₄, 0.2 g MgSO₄·7H₂O, 0.4 g K₂HPO₄, 0.8 g KH₂PO₄, 5.0 × 10⁻⁴ FeSO₄·7H₂O, 2.0 × 10⁻⁴ ZnSO₄·7H₂O, 5.0 × 10⁻⁵ MnCl₂·4H₂O). The cultures were subsequently divided into three groups: one was grown in 20 mL minimal medium plus with 1.75 mM (0.05%) androstenedione (AD) (Sigma, St. Louis, MO, USA), one in 20 mL minimal medium plus with 1.29 mM (0.05%) cholesterol (CHOL) (Sigma, St. Louis, MO, USA), and one in 20 mL minimal medium plus with 5.47 mM (0.05%) glycerol (GL) (Sigma, St. Louis, MO, USA). For optimum mixing of cholesterol and androstenedione in water, a certain amount of the steroid substrates were firstly mixed with cyclodextrin, and added to 5 mL of solution containing 50% Tween-80, sonicated for 30 min, then added to the minimal medium, and sonicated for a further 30 min. The final medium had a concentration of 0.9 mM cyclodextrin and 0.25% (v/v) Tween-80. The control sample containing glycerol also contained 0.9 mM of cyclodextrin and 0.25% of Tween-80. All of these M.smegmatis MC² 155 were cultivated at 30 °C in an orbital shaker at 180 rpm for 48 h. Three cultures were marked as AD, CHOL and GL.