| Paclitaxel is a natural diterpenoid initially isolated from the stem bark of Taxus brevifolia Nutt.Studies on paclitaxel are still a focus in the scientific world bacause it has been effectively used for treatment for various cancers,such as ovarian cancer, breast cancer,etc.Lots of paclitaxel analogues had been found in Taxus species with less cytotoxic activity,and scientists have been searching for the approach to utilizing the useful analogues.Potential alternative is the approach through biotransformation procedure by living organisms.In this paper,two strains with the function of selective hydrolysing the C-13 side chain and C-7 xylose of taxanes were obtain.1) A total of 197 microorganisms were screened for their ability to achieve the efficient biotransformation of cephalomannine(1).Of the five positive cultures, Luteibacter sp.was selected for a two-stage fermentation process scale-up.After six days' incubation,8 products were separated by silica gel chromatography,following by semi-preparative HPLC.On the basis of their 1H-NMR,13C-NMR and MS spectroscopic data,their structures were identified as baccatinⅢ(2),baccatinⅤ(3), 10-deacetyl baccatinⅢ(4),10-deacetyl-10-oxobaccatinⅤ(5),7-epicephalomannine (6),10-deacetylcephalomannine(7),7-epi-10-deacetylcephalomannine(8), N-debenzoyl-N-(2-methylbutyryl)-7-epitaxol(9).The metabolites 2-8 were known compounds,while metabolite 9 was a new compound.In addition,the metabolites 5-9 and 11 were preliminarily evaluated for in vitro cell proliferation inhibition activities against five human tumor cell lines(HCT-8,Bel-7402,BGC-823,A549,A2780).The IC50 of metabolites 6 towards BGC-823,A549 and A2780 was 0.09,0.09,0.07μM, respectively.Although all of them did not show comparable activity as paclitaxel,the results were still useful for the studies of antitumor structure-activity relationships of taxanes.We obtained the strain with the function of reduction,oxidation, epimerization,hydrolysis at C-13 and C-10.Epimerization of 7β-hyodroxyl group and hydrolysis of C-13 side chain were the two major reactions involved in this bioprocess.This bacterium was also selectively hydrolyzed C-13 side chain when 7-xylosyl-10-deacetyltaxol as substrate,which indicated that a bulky substituent at C-7 site can not only eliminate the retro-Aldol reaction but block the effect of the combination of substrate and the isomerase.2) 4-methylumbelliferyl[β-D-xyloside was used as a fluoroprobe in high throughput screening with modem analytical techniques such as TLC,LC-UV,LC-MSn,which led to obtain a bacterial strain jh-21 which can selectively remove the xylosyl group from 7-xylosyl-10-deacetyltaxol(10).After preparative biotransformation of 7-xylosyl- 10-deacetyltaxol(10) by the strain jh-21,one major product 10-deacetaxol (12) and other two minor products 7-xylsoyl-10-deacetylbaccatinⅢ(11), 10-deacetylbaccatinⅢ(4) were obtained.And(11) was also used as a substrate for the bioconversion by jh-21 and the obtained product was 4.We include that the strain jh-21 may can selective hydrolysis C-7 xylose of several taxanes.Our further studies showed that ammonium sulfate can enhance the activity of xylosidase and xylan can induce the expression of the relative gene.7-xylosyl-10-deacetyltaxol were conversed at room temperature for 7 days,the yield of 10-deacetyltaxol was 12.69 mg/L,and the ratio reached 73.7%,when in the oat hull medium containing 0.4%ammonium sulfate and 0.06%xylan.The temperature at 37℃is the optimum temperature for this transformation.The fact that 7-xylosyl-10-deacetyltaxol in oat hull medium removed the xylosyl group but removed the C-13 side chain in medium of beef and peptone implied the xylosidase is an induced-enzyme.Meanwhile,the basic characteristic of xylosidase was also examined preliminarily,and the result showed that the xylosidase was a membrane protein.
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