Production Of 4-androstene-3,17-dione Via Side-chain Cleavage Of Phytosterols By Mycobacterium Sp.MB 3683 In Biphasic Systems

4-Androstene-3,17-dione?AD?is an important precursor for the synthesis of steroid drugs in the pharmaceutical industry,which can be obtained via selectively side-chain cleavage of phytosterols by Mycobacterium sp.cells.However,low product concentration was obtained in bioconversion due to the poor solubility of substrate and possibly inhibitory?or even toxic?effects of product on microbial cells.Biphasic system is considered to be an efficient strategy to solve the above problems as the second phase can be a "reservoir" for both substrate and product.The present work focused on the side-chain cleavage of phytosterols by Mycobacterium sp.MB 3683 in biphasic system to increase solubility of substrate,shorten the reaction time and reuse cells.Firstly,the microbial transformation of phytosterols by Mycobacterium sp.growing cells to produce AD was conducted in aqueous two-phase system?ATPS?to design an extractive fermentation procedure.Partition coefficients of cells and AD in different polymer/polymer and polymer/salt systems were determined.Then,the toxicity of polyethylene glycol?PEG?with different molecular weights on Mycobacterium sp.cells,and effects of surfactants and dissolved oxygen on AD production were investigated in detail.Finally,the bioconversion at high substrate feeding concentration was studied and the system was tentatively scaled up in the lab.The results indicated that cells were preferred to the dextran-rich bottom phase,while product of AD was preferred to the PEG-rich top phase in the ATPS system composed of PEG 6000?7%?wt??and dextran 70000?8%?wt??.For a scaled-up biphasic system of 200 g,a higher yield of AD(1.1 g L^-1)was achieved after 96 h bioconversion at optimized conditions of 1%?V/V?tween 40 and rotational speed of 240 r min^-1 when 10 g L^-1 phytosterols feeding was applied.Aqueous two-phase system with good biocompatibility is considered to be an efficient strategy to alleviate the inhibitory?or even toxic?effect of AD on microbial cells,and basic data are provided for the application of developing an extractive fermentation for the production of AD.Secondly,nine kinds of natural oils were employed in the biphasic system,where Mycobacterium sp.resting cells were used as catalysts for the bioconversion of phytosterols to AD.Specifically,soybean oil/aqueous biphasic system was used to explore optimal bioconversion conditions,and cells culture time and biotransformation time were determined to be 72 h and 24 h respectively.Important parameters such as phase ratio?PR,oil to aqueous,V to V?,rotational speeds,concentrations of resting cells and substrate were studied.The result indicated that the highest AD concentration of 2.7 g L^-1 and a space-time yield above 0.1 g L^-1 h-1 were obtained at PR of 2:8,rotational speed of 240 r min^-1,resting cells concentration of 200 g L^-1 and substrate feeding concentration of 10 g L^-1.The bioconversion time was greatly shorted due to the resting cells with high concentrations,which fully demonstrates the feasibility and advantage of using resting cells as a catalyst for degradation of phytosterols in natural oil/aqueous two-phase system.Finally,soybean oil/aqueous biphasic system was used to study the degradation of phytosterols by immobilized cells.Living cells of Mycobacterium sp.MB 3683 were immobilized by entrapment onto calcium alginate,adsorption on kieselguhr and loofa sponge,in order to produce AD from phytosterols,with soybean oil/aqueous biphasic system as the bioconversion medium.Higher activity of cells was observed when loofa sponge was used as the matrix,as compared to the free cells.Scanning electron microscope?SEM?and fourier transformation infrared spectroscopy?FT-IR?were applied to analyze the efficacy of loofa sponge used as a carrier to immobilize Mycobacterium sp.growing cells.Later,cells adsorbed on loofa sponge were used to explore optimal bioconversion conditions such as quantity of immobilization carrier and inoculum.AD concentration of 2.0 g L^-1 was obtained at loofa sponge of 0.3 g in 30 mL medium,inoculum of 8%?V/V?,substrate feeding concentration of 5 g L^-1.Based on that,the operation stability and storage stability of immobilized cells were studied.The reuse of immobilized cells in ten batches biotransformation proved effectively and the storage activity retained for 80 days.This work clearly highlights the efficacy of Mycobacterium sp.MB 3683 cells immobilized on loofa sponge to be used as biocatalyst in natural oils/aqueous two-phase bioconversion systems.