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The Research of Adsorption Resins in the Application of Epothilone Fermentation

Author: LiCan
Tutor: LiuXinLi
School:
Course: Biochemical Engineering
Keywords: Sorangium cellulosum epothilones resin oxoglutarate dehydrogenasecomplex regeneration
CLC: TQ463
Type: Master's thesis
Year: 2014
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Abstract


Epothilones that act on cancer cells by stabilizing microtubules are focused on asresearch hotspot. Because of its chemical synthesis process is too complicated, thebiological synthesis research trend has been the mainstream. The XAD-16resin,which can capture the epothilones in situ and remove them from the culture broth,could isolate the epothilones from other impurities and reduce the feedback inhibition.The XAD-16resins play an important role in absorbing epothilones during microbialfermentation of Sorangium cellulosum. However, high price and large demand ofXAD-16resin significantly increase the production costs of epothilones.In order to reduce the cost, we found two types of resins from30differentdomestic resins; the mix of two resins (the mix of resin XD-5and D202with the ratioof7to3) could increase epothilone A yield by252%and epothilone B yield by262%respectively to single resin XAD-16. With our further study, we found that themacroporous resin has stronger adsorption ability of epothilones and anion exchangeresin D202could promote the growth of Sorangium cellulosum. Oxoglutaratedehydrogenase complex (OGDHC), a key enzyme in the cycle TCA, plays animportant role in epothilones biosynthesis. Its activity is affected by chloride ionobviously. In this paper, in order to determine the mechanism of action of resin D202,we studied from the level of enzyme activity and gene expression levels of enzyme,compared the curves of the enzyme activity with the epothilone biosynthesis curve,and the genetic level detection under the different condition, we found that resin D202could be used as a promoter of OGDHC enzyme activity. We further surveyed theeffects of5different organic solvents (methanol, alcohol, acetone, ethyl acetate,carbon tetrachloride) on the epothilone extraction and regeneration of macroporousadsorption resin XAD-16. Of5organic solvents, methanol showed a much bettereffect on epothilone yield than the others. Then we observed the differences instructure between the fresh-resin and the regenerative-resin with methanol by atomicforce microscope. We found that pore size of methanol-resin was larger thanfresh-resin’s, which lead it more conducive to the adsorption of epothilones. Also, wegot that regenerative-resin treated with methanol was more sturdy. Result of theimpact experiment showed the macroporous adsorption resin XAD-16could be usedfor at least five times under methanol treatment.

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