Microbial Hydroxylation Of Steroid Hormones In Ionic Liquid Aqueous Two Phase System

Glucocorticoid drugs belong to the steroid hormone family, which have been widely used in clinical therapy. Among them, it is shown that the C11-hydroxy is a necessary functional group for the biological activity of glucocorticoids. In industry, this group was usually introduced into the steroid ring system via biocatalysis and biotransformation considering its advantages in product conversion and purity. However, the extremely low solubility of steroid substrate in water is the bottleneck that restricted the wide application of bioconversion. Ionic liquid, as a new type of green solvents, shows good biocompatibility and dissolving capacity. In this dissertation, ionic liquids were first introduced into the bioconversion catalyzed by fungus, and C11hydroxylation in ionic liquid aqueous biphasic system was studied in detail.Firstly, the solubility data of16a,17-epoxyprogesterone and17a-acetoxy progesterone in ionic liquids of [BMIm][PF6] and [BMIm][Tf2N] were determined. Their distribution coefficients in [BMIm][PF6]-aqueous (phase ratio5) biphasic system were calculated to be about2, and the values were about3in [BMIm][Tf2N]-aqueous (phase ratio5) biphasic system, which indicated that the two ionic liquids would be competent nonaqueous media in "Extractive Fermentation’". Besides, the biocompatibility of the two ionic liquids with the microorganisms was also detected. The results provided fundamental data for the following further study.Secondly, the11β-hydroxylation of17a-acetoxy progesterone catalyzed by Curvularia lunata (C.lunata) cells after ultraviolet mutagenesis was optimized. Using a pretreatment method, the substrate was firstly prepared into sub-micron particles of450nm suspension, then the conversion of the11β-hydroxy products increased to85%at0.6g/L feeding substrate, and70%at higher substrate concentration of1.2g/L. Then, the bioconversion was conducted in six different ionic liquid aqueous biphasic systems. The results were contrasted with those in six organic aqueous biphasic systems accordingly. It was found that the conversions in [BMIm][PF6] and [BMIm][Tf2N] were better, however the adverse effect brought by ionic liquids was still obvious.Finally,11α-hydroxylation of16a,17-epoxyprogesterone catalyzed by Rhizopus nigricans (R. nigricans) in ionic liquid aqueous biphasic system was studied. Two ionic liquids of [BMIm][PF6] and [BMIm][NTf2] were used in the biotransformation of16a,17-epoxyprogesterone by R. nigricans. The results indicated that the conversion carried out in [BMIm][PF6]-aqueous biphasic system was greatly increased to above90%at18g/L feeding concentration. A simplified mechanism was proposed to explain the improvement of the bioconversion in a biphasic ionic liquid aqueous system. Besides, successive three batches of bioconversion were carried out in the biphasic system with a total conversion of87%at phase ratio10and75%at phase ratio5respectively. Since recycling of the [BMIm][PF6] is quite easy, there is a great potential for the application of ionic liquids in fungi biotransformation to implement green production.