| In recent years, it is becoming hot in using endophytic fungi of yew to ferment taxol. Many scholars,at home and abroad, have made a lot of research in this field.More than 200 strains endophytic fungi producing taxol were isolated and identified, but the results showed that the yields of taxol were very low. There are three efficient ways,looking for high yield strains, optimizing cultivation condition and researching metabolic regulation, to increase the yields of bioactive substances.Those can solve the problem of the shortage of taxol resource.We analyze the metabolites of endophytic fungi isolated in Taxus chinensis from the Qin-Ba mountain and screens the strains that producing taxol, BaccatinⅢand 10-DAB in this paper. Furthermore,we analyze the influence of the culture medium, fermentation conditions and growth regulators on the producing taxol. Mainly research methods and results are as follows:1.Using HPLC and HPLC-MS method to detect the extractive of zymotic fluid of separated 82 endophytic fungi. The results have showed that 11 strains endophytic fungi produced taxol, 13.4% in total taxol endophytic fungus; 19 strains endophytic fungus produced BaccatinⅢ, 23.2% in total taxol endophytic fungus; 9 strains endophytic fungus produced 10-DAB, 10.9% in total endophytic fungus.2. It is found that LB-5 producted three main taxanes compounds, including taxol, BaccatinⅢand 10-DAB,the yield was 314.29μg/L, 565.72μg/L and 573.94μg/L respectively. Combining morphological properties and ITS sequence analysis, identified the strains Rhizopus oryzae (Rhizopus oryzae.sp).3. The carbon source and nitrogen source are the main nutrients for mycelium growth and secondary metabolites accumulation in the medium, using HPLC detection methods trace and detected taxol production,the results showed that the best source of carbon is screening glucose, the best nitrogen source is ammonium nitrate. By single factor experiment, determine the glucose, the optimal concentration of ammonium nitrate, namely glucose 40g/L, ammonium nitrate 6 g/L. So as to obtain the best fermentation composition (g/L) : glucose 40.0, NH4NO3 6.0, MgSO4 0.3, KH2PO4 0.5, VB1 10 x 10-2.4. According to the structural characteristics of paclitaxel and taxane synthesis mechanism, gibberellin(GA3), Methyl Jasmonate (MeJA), cycocel(CCC), 6-amino purine benzyl(6-BA) and naphthylacetic acid(NAA) are selected to study the effects on the producting Taxol of LB-5 and study the influence on mycelium growth of LB-5 by the Dry Weight Rank Method, using HPLC detection methods traced and detected taxol production. Experimental results showed that GA3, MeJA, CCC, 6-BA and NAA all can improve taxol production, but GA3, MeJA and CCC inhibited the growth of mycelia obviously; 6-BA and NAA can stimulate the growth of mycelia.5. GA3, MeJA, CCC, 6 - BA and NAA all can improve the yield of taxol, different concentration of these materials are selected to anylyses the influence on the yield of paclitaxel, due to the physiological functions of GA3, MeJA, CCC, 6 - BA and NAA. The results showed that: as addition of 4 mg/L GA3, taxol production reaches the highest, the yield of taxol , BaccatinⅢand 10–DAB are 670.49μg/L, 413.55μg/L and 410.94μg/L; addition of 60μmol/L MeJA, taxol production reaches the highest, the yield of taxol BaccatinⅢand 10-DAB are 838.11μg/L, 179.36μg/L and 165.31μg/L; addition of 25 mg/L CCC, taxol production reaches the highest, the yield of taxol BaccatinⅢand 10-DAB are 942.57μg/L, 102.35μg/L and 98.59μg/L, addition of 4 mg/L 6-BA, taxol production reaches the highest, the yield of taxol BaccatinⅢand 10-DAB are 455.72μg/L, 402.63μg/L and 403.91μg/L, addition of 4 mg/L NAA, taxol production reaches the highest, the yield of taxol BaccatinⅢand 10-DAB reached 450.46μg/L,408.31μg/L and 410.12μg/L.6. With the increase of regulators concentration,the synthesis reaction process was advantageous to produce taxol, but BaccatinⅢa nd 10-DAB yield decreased. At one point,the concentration of each regulator was high enough to influence the yield of taxol farthest. When the yield of taxol reach max, the concentrations of the BaccatinⅢand 10-DAB decrease to mix. With the keep on increase of those regulators,the yields of BaccatinⅢand 10-DAB began to improve and the concentration of taxol started decreasing. It suggested that these regulators influence the yield of taxol is inversely proportional to the yields of BaccatinⅢand 10-DAB. Thereby we can confirm that BaccatinⅢand 10-DAB are precursors of taxol in the synthesis reaction process. |
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