红豆杉内生真菌发酵培养基和原生质体制备酶系统的筛选

Selection of Optimal Fermentative Medium and Enzyme System for Isolating Protoplasts from Endophytic Fungus of Taxus mairei

在对红豆杉内生真菌(Ozoniumsp.)适生碳源、氮源和生长情况研究的基础上,通过正交试验筛选了其发酵培养基和原生质体制备的酶系统;用L9(34)安排了四因素三水平并考虑交互作用的正交试验,对实验结果进行了分析.结果表明,最优的发酵培养基为果糖1%、蔗糖1%、蛋白胨0.2%、酵母粉0.5%、KH2PO40.5%、MgSO4·7H2O0.3%、VB10.001%;分离原生质体的最优酶系统为1.5%溶壁酶+0.5%蜗牛酶+1.5%纤维素酶+1.0%溶菌酶;用此酶系统在30℃条件下酶解3h,原生质体的产量达6.55×107个/mL酶液;经荧光素二醋酸酯(FDA)染色评估原生质体活力,表明该条件下分离的原生质体活力较高,原生质体的再生率为2.56%.该研究为利用生物技术手段改良紫杉醇生产菌奠定了基础.

The optimal culture medium and enzyme system for isolating protoplasts from an endophytic fungus from Chinese southern yew （ Taxus mairei） were studied by orthogonal test. Table L9 （ 3^4 ） was used to test four factors with three levels, and the interaction among the factors was also studied. The results showed that the optimal culture medium for the endophytic fungus consisted of 1% fructose, 1% sucrose, 0.2% peptone, 0.5% yeast powder, KH2PO4 0.5%, MgSO4 · 7H2O 0.3% and VB1 0 . 001% , while the optimal enzyme system for isolating protoplasts was the enzyme mixture containing 1.5% lywallzyme, 0.5% snailase, 1.5% cellulase and 1.0% lysozyme. The production of protoplast was 6.55 × 10^7 with selection enzyme system at 30 ℃ for 3 h. The obtained protoplasts were treated with fluorescein diacetate, and observed by phase contrast microscopy and then by fluorescence microscopy. By superimposing these images, it appeared that all protoplasts were viable based on their absorption of fluorescein. The frequency of regeneration of fungal protoplasts was 2.56%. This study provides a basis for improving the taxol - producing endophytic fungus by biotechnological method. Fig 6, Tab 4, Ref 32