Isolation And Identification Of Taxol-producing Endophytic Fungi In Taxus Media

In the experiment, Taxus media as the experimental material, the separation of Taxusendophytic fungi, through microscopic and TLC identification, capable of producing taxol ofTaxus endophytic fungi. The ultraviolet spectrophotometric determination of EndophyticFungi of paclitaxel yield, choose the highest yield strain. Screening of the highest yield strain,through the optimization of medium and cultivation conditions, add the precursor materialand inducing factors to improve the paclitaxel yield. Through the experiment, the purpose isto establish the best of Taxus endophytic fungus synthesizing taxol separation methods,identification method and training method for industrialized production of taxol, providecertain data and technical support.The experimental results are as follows.1. In Taxus media roots, stems, leaves, bark were isolated from54strains of endophyticfungi were cultured by microscopy, and UV spectrophotometry for the determination, throughthe observation of bacterium colony and mycelial morphological characteristic, and then theTLC method for the detection of bacteria in liquid culture extracts, obtained6strains are ableto produce taxol of Taxus endophytic fungi, they are Nigrospora sp., Penicillium sp.,Aspergillums sp, Cephalosporium sp.,Cladosporium sp.,Phomopsis sp..2. Using UV spectrophotometer method was used to measure the6strains of EndophyticFungi of the paclitaxel yield, respectively: Nigrospora24.17μg/L, Penicillium15.64g/L,Aspergillus20.73μg/L, cephalosporins29.45μg/L, Cladosporium34.28g/L, Phomopsis18.56μg/L. The six strains of endophytic fungi, the highest yield is Cladosporium.3. In order to culture Cladosporium, through the optimization of the fermentationconditions of Cladosporium, improves the paclitaxel yield of cladosporium. Get the bestmedium composition for: potato200g, glucose20g, agar powder15g (liquid medium withoutagar powder), NH4NO37g/L, MgSO4,7H2O0.6g/L, water1000mL, pH7.0; the optimalculture conditions for each triangle bottle: the inoculation amount4%, liquid volume was200mL/500mL, shaker culture, the culture temperature of27℃, speed is180r/min, incultured8d added precursors and inducing factor, Phe8mg/L, arachidonic acid0.4mg/L,days of cultivation for13d. Using the optimized conditions of Cladosporium in culture, paclitaxel yield of71.34μg/L, is the original culture under the conditions of the paclitaxelyield is more than two times.