| Biphasic processes are usually applied in the whole-cell biotransformation to overcome low water solubility of substrate and/or product and their inhibitory effect of substrate and/or product on the biocatalyst. However, commonly used conventional organic solvents often damage the microbial cells. Furthermore, these solvents are volatile and enviromentally hazardous. Ionic liquids (ILs) as green solvents can be used as replacements for organic solvents in biphasic system, because of their unique physical and chemical properties. In this paper, hydroxylation and dehydrogenation of 16α,17α-epoxyprogesterone by Aspergillus ochraceus and Arthrobacter simplex were used as models to evaluate the toxity of ILs on the biocatalysts and the efficiency of ILs on microbial transformation of steroid. These studies will be very important for the potential industrial application of ILs-based biphasic systems for steroid biotransformation.Firstly, the influence of fermentation condition on the stain growth and substrate biotransformation was studied. The optical fermentation condition of A. ochraceus was that the inoculums concentration was 106/mL; the initial culture pH was 5.8; 16a, 17a-epoxyprogesterone was added after 26 h cultivation and the conversion was performed for 48 h. The conversion rate came to 88% at 5 g/L substrate concentration. The optimal medium composition of A. simplex was as follows:10 g/L glucose,1.2% corn steep liquor,8 g/L yeast extract,2.5 g/L K2HPO4. The conversion rate came to 85% at 10 g/L substrate concentration.Secondly, substrate uptake and cell growth were used as measure of biocompatibility of ILs to A. ochraceus and A. simplex, respectively. The results showed that in the presence of the same cation, the [PF6]-based ILs displayed higher cell viability than those containing [NTf2]. Moreover, cell viability decreased with elongation of the alkyl side chain of ILs containing the same anions.Thirdly, effects of ILs on the efficiency of 11α hydroxylation of 16α, 17a-epoxyprogesterone by A. ochraceus in different ILs/buffer biphasic systems were investigated. And the result indicated that, of the seven ILs tested, [C3mim][PF6] exhibited the best biocompatibility, with markedly improved biotransformation efficiency. In the [C3mim][PF6]-based biphasic system, substrate conversion reached 90% under the condition in which buffer pH, volume ratio of buffer to ILs, cell concentration, and substrate concentration were 4.8,10/1,165 g L-1 and 20 g L-1, respectively. This is more efficient than that of the monophasic aqueous system. ILs were also used in biphasic systems for dehydrogenation of 11α hydroxylation of 16α,17α-epoxyprogesterone by A. simplex. [C3mim][PF6] has the same potential for promoting the efficiency of dehydrogenation mediated by A. simplex in IL/aqueous biphasic system.Finally, A. ochraceus and A. simplex were used to conduct the sequential fermeniation in [Csmim] [PF6]/buffer biphasic system, converting epoxyprogesterone to 11a-hydroxy-16a,17a epoxypregenene-1,4-diene-3,20-dion. While the substrate conversion is very low, possibly resulting from the increase in water phase viscosity with many A. ochraceus cells, which may lead to a decrease in mass transfer between the two phases. |
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