Study On The Construction Of A Highly Efficient Hydroxylation System Of Steroidal Drugs Based On DES

The microbial transformation of steroid compounds plays an important role in the production of steroid drugs due to its mild reaction conditions,high stereoselectivity and regioselectivity,and environmental friendliness.In this study,the transformation of 13-ethyl-4-gonene-3,17-dione to 15a-hydrogen-13-ethyl-4-gonene-3,17-dione catalyzed by Penicillium raistrickii cells was taken as the model reaction to examine the impacts of DESs as cosolvents on whole-cell biocatalysis.In this work,13 kinds of natural eueutic solvents(NADES)with choline chloride as hydrogen bond receptor and organic acid,alcohol and sugar as hydrogen bond donors were selected to study the effect of different types of hydrogen donor DES on substrate conversion,and the sequence of its effect on substrate conversion efficiency was found to be organic acid<sugar<alcohol.Among them,The DES choline chloride/glycerol(ChCl:Gly(1:2))showed the best substrate conversion rate,which can reach 82%is 49%higher than the control.To further understand the effect of ChCl:Gly on the reaction,a comparison of the catalyst performance was carried out by the addition of ChCl:Gly and its components separately.The study found that the conversion rate is fairly independent of choline choride and only moderately increased with addition of glycerol,but upon addition of DES,a much more dramatic enhancement in conversion rate.The cytotoxicity of DES against Penicillium raistrickii was systematically investigated by sugar metabolic activity retention,extracellular OD260 and OD280 detection and substrate accumulation in cells.The results showed that when the concentration of ChCl:Gly ChCl:Eg was lower than 8%,it had almost no effect on the metabolic activity of Penicillium raistrickii cells with low toxicity,while the concentration of ChCl:MA,ChCl:OA and ChCl:IM containing 2%seriously affected the metabolic activity of Penicillium raistrickii cells with high toxicity,and the toxicity of DES gradually increased with the increase of concentration.By measuring the amount of substrates accumulated in Penicillium raistrickii cells,it found that the amount of substrates accumulated in the ChCl:Gly-buffer system is twice higher than that in the aqueous phase system.Meanwhile,the detection results of OD260 and OD280 in the extracellular solution showed that the values of OD280 and OD260 in the system containing ChCl:Gly were significantly higher than those in the control group and other DES,suggesting that addition of ChCl:Gly may be beneficial to whole-cell biocatalysis in enhancing the cellular membrane permeability.In addition,at the end of biocatalysis,the mycelial morphology of Penicillium raistrickii disappeared in the aqueous phase system,while keep complete in the DES system.This result was consistent with the results of higher bioconversion and better biocompatibility in the system containing ChCl:Gly.