Strain and media
The strain of M. neoaurum ATCC 25795 and its mutants used in this study are shown in Table 1. Engineered strains were constructed according to the procedure of reference as previously stated [17].
Table 1 Strains used in this study
M. neoaurum
|
Description
|
References
|
NwIB-R10
|
NwIB-R10-ΔkshA, AD producer.
|
[17]
|
NwIB-R10-kshA
|
NwIB-R10-ΔkshA::kshA
|
This study
|
NwIB-yV
|
NwIB-yV-pMV261-kshA; 9-OHAD producer.
|
[17]
|
Preculture medium consisted (per liter) of 0.5 g K2HPO4, 0.5 g MgSO4, 2.68 g NH4Cl, 2.0 g citric acid, 0.05 g ammonium ferric citrate, 20.0 g glycerol. Growth medium consisted (per liter) of 2.0 g citric acid, 0.05 g ammonium ferric citrate, 15.0 g glucose, 0.5 g K2HPO4, 0.5 g MgSO4, 3.5 g (NH4)2HPO4, 1 g corn steep powder, 0.1 g phytosterol. The initial pH was adjusted to 8.0 using 2 mol L− 1 NaOH. Media were sterilized at 115°C for 20 min.
Reagents
Phytosterol (purity 95.2%) was purchased from Davi Biochemistry Inc. (Huzhou, China), containing 47.5% β-sitosterol, 17.7% stigmasterol, 26.4% campesterol, 3.6% brassicasterol. AD, ADD and related steroids were purchased from Sigma (St. Louis, USA). HP-β-CD was purchased from RSC Chemical Industries Co. Ltd. (Kunshan, China). Corn steep powder was purchased from Xiwang Group Co. Ltd. (Binzhou, China). Other chemicals were analytic grade reagents from Sinochem. Co. Ltd (Shanghai, China).
Preparation Of Resting Cells And Biotransformation
The seed was incubated in preculture medium at 30°C, 200 rpm for 2 days. Then, 5% (v/v) preculture broth was transferred to growth medium for cell expansion at 30°C, 200 rpm for 3 days. M. neoaurum cells were harvested by centrifugation (8000 g) at 4°C for 15 min and the cell pellets were washed twice with 20 mmol L− 1 phosphate buffer (PB, pH 8.0) and finally resuspended in the same buffer for further use.
All the bioconversion process with resting M. neoaurum cells was performed in 20 mmol L− 1 PB (pH 8.0) under non-sterile conditions in a 250-mL flask at 30°C, 200 rpm, containing a mixture of 1:1 molar ratio of HP-β-CD to phytosterol (approximately 4:1 by weight). The concentration of wet cell pellets (WCW) and phytosterol was based on the requirement of each test. The bioconversion volume (20 mL) was maintained constant by the addition of distilled water each day to offset the evaporation. All the bioconversion of phytosterol was performed in triplicate.
Effect Of Hp-β-cd On Oxygen Availability
Sulfite could react with the oxygen in aqueous reaction system to form sulfate. Effect of HP-β-CD on oxygen availability was determined in a reaction system containing different concentration of HP-β-CD without M. neoaurum cells and substrate. The reactions were performed in a 3.7-L bioreactor with 2 L working volume at 30 °C, constant agitation and aeration rate and initiated by adding sodium sulfite (final concentration, 0.5 mol L-1). The oxygen utility rate was detected using exhaust gas analysis apparatus (Shandong Academy of Science, Jinan, China) when the oxygen utility rate was constant in each reaction with different concentration of HP-β-CD.
Analytical methods
The procedure of sample processing and steroids analysis by HPLC were described as reference [18]. The phytosterol was analyzed by HPLC with a Agilent Zorbax SB-C18 (5 μm, 4.6×250 mm, Shanghai, China) at 40 °C and a UV detector at 210 nm. The mobile phase was composed of acetonitrile/isopropanol (70: 30, v/v), and a flow rate of 1 mL min-1 was used.
According to the instructions, the level of NADH/NAD+ at one-day bioconversion was detected by EnzyFluoTM NAD+/NADH Assay Kit (EFND-100) based on an enzyme-catalyzed kinetic reaction (Beijing Lablead Biotech Co. Ltd., Beijing, China).