Study On The Biotransformation Of 7-xylose-10-deacetylpaclitaxel

Paclitaxel(Taxol,Paclitaxel)is a kind of anti-cancer drug extracted from Taxus species.Because of its significant anti-tumor activity and unique anti-tumor mechanisms which surprised the world,paclitaxel is thought as one of the important achievements among the anti-cancer drugs in the 1990s[1].In clinical,paclitaxel is widely used in therapy of breast,ovarian and non-small cell lung cancer,etc.[2].It also has a good effect on the treatment head and neck cancer,melanoma,colon cancer,and Kaposi's sarcoma caused by HIV[3].The content of paclitaxel in yew plants is very low,even in the highest content of bark parts only about two per millions.The low contents of paclitaxel is unable meet the massive demand in clinical[4].Currently,shrub nursery cultivation of Taxus hybrids is considered to the easiest,resource-renewable and the least expensive method to solve the problem.In addition,in the 1988 French Denis extracted paclitaxel precursor compounds 10-deacetyl baccatin ?(10-deacetylbaccatin III)with 0.1%content from the needles of the European yew,and developed it to docetaxel(Taxotere),which has stronger anti-tumor activity,better water-soluble than paclitaxel[5].Despite the levels of paclitaxel is very low in yew plants,while the contents of 7-xylose taxanes with the structure of paclitaxel nucleus is abundent,which includes 7-xylose-l 0-deacetylpaclitaxel(7-?-xylosyl-1O-deacety ltaxol 10-DAXT),7-xylose-10-deacetylbaccatin?(7-beta-xy losy l-10-deacety lbaccatin?),7-xylose-10-deacety laseCephalomannine(7-?-xylosyl-10-deacetylcepholomanine).For example,the content of 7-xylose-10-deacetyl paclitaxel(10-DAXT)is 10 times of that of paclitaxel.Among Taxus bark grown in Fujian Taining,the yield of DAXT is even high as 0.5%[6].Taxol analogues of the presence of a large number of yew material taken by chemical or biological means Removing the xylose group from 7-xylose taxanes through chemical or biological means,a large number of C-7 position hydroxylated product can be obtained.The method 7-xylose taxanes are used for the preparation of paclitaxel or Taxol Greek or an intermediate thereof,can improve utilization efficiency of yew resource.In our previous research,the content of the taxanes and the dynamic changes of them during one year were detected on the barks,branchs,leaves of yew planted in Fujian Taining,which included paclitaxel,7-xylose-10-deacetyl paclitaxel and 10-deacetyl baccatin ?.At the same time,the taxanes extraction has been studied and enlarged.Paclitaxel,7-xylose-10-deacetyl taxol and other taxanes had been successfully extracted with high purity and yield.Especially the purity of paclitaxel was more than 99%.However,in the traditional extraction process,7-xylose-10-deacetylase paclitaxel was processed as an impurity of yew resources,which is a tremendous waste.Using the basic principles of ecology,we screened micro-organisms with 7-xylose-10-deacety l paclitaxel biotransformation activity from the Taxus Chinensis origin soil.Here bacterial culture and screening techniques were applied to obtain biotransformation activity micro-organisms.[7].At the same time,the use of modern microbial classification techniques[8],such as microbial morphology,Gram dye,16SrDNA sequencing,physiological and biochemical characteristics,GC content and fatty acid composition were systematic investigated to identify and determine the growth,metabolism of the active microbial species.Results showed that 6 bacteria strains had been screened,among which ITM-1512 strain had applied for patent protection with National Culture Collection number CGMCC No.7092.16S rDNA sequence of the screened bacteriahad been submitted to the Genebank with the serial number KC568139-568144[9].Furthermore,modern analytical techniques HPLC/MS/MS was used to determine structure information of biotransformation metabolites.the establishment of the The system,complete,and reliable HPLC,UPLC analytical methods were established for the quantitation of biotransformation activity[10].The medium carbon source,pH,induction time,the addition time of the substrate and biological conversion time were controlled and optimized to fulfil the highest biothansformation.Research work was done to stabilize the activity of ITM-1512 for hydrolyzing C-7 xylose in 7-xylose-10deacetyl-taxol.The results showed in semi-solid medium which agar concentration was 0.7%,when concentration of 7-xylose-lOdeacetyl-taxol was Smg/mL join volume of 100 ul,reaction system 50 mL,inoculation ratio 1:10,28 ?,biotransformation 4 days,reaction product 10-deacetyl paclitaxel maximum conversion rate of 56.77%,10-deacetyl baccatin III maximum conversion rate of 74.58%,the average conversionefficiency of 7-xylose-10-deacetyl paclitaxel to 10-deacetyl paclitaxel was 40.66%± 13.22,to 10-deacetyl baccatin ? was 50.600%±35.71.Studying the specific impact on microbial biotransformation,such as carbon source,pH,substrate-induced factors,accumulated valuable practical experience for biotransformation preparation of compounds,improved the preparation of biological pathways paclitaxel,laid a solid foundation.At the same time research riched source of paclitaxel and made of rational use of Taxus chinensis resources,improved resource utilization,made a good attempt.