红豆杉内生真菌代谢产紫杉醇发酵工艺条件研究
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摘要
紫杉醇是一种二萜类衍生化合物,最早分离自红豆杉属植物。1992年得到美国食品与药物管理局(FDA)批准作为抗晚期癌症的新药上市。近年来利用植物内生真菌发酵法生产紫杉醇成为研究热点,寻找高产菌株、优化培养条件及研究代谢调控将是提高紫杉醇产量的有效途径。
本研究以提高微生物发酵法生产紫杉醇的产量为目标,从秦巴山区野生红豆杉中分离筛选出一株紫杉醇高产内生真菌Metarhizium anisopliae LB-10。在此基础上,对LB-10培养基配方、发酵工艺条件和生长调节剂做了进一步研究。主要研究方法和结果如下:
1.从秦巴山区野生红豆杉中分离得到109株内生真菌,经纯化获得82株内生真菌。采用UV、HPLC和HPLC-MS检测方法,对82株内生真菌的发酵液中代谢产物进行检测,并通过形态学特性研究和ITS序列分析,鉴定物种。结果表明,有8株内生真菌代谢产生紫杉醇,其中有1株高产紫杉醇内生真菌LB-10,鉴定该菌株为绿僵菌(Metarhizium anisopliae),其紫杉醇产量为846.1μg/L。
2.以紫杉醇产量为指标,利用HPLC进行检测,筛选确定了发酵产紫杉醇的最佳碳源是葡萄糖,最佳氮源是硝酸铵。通过单因素实验,确定了葡萄糖、硝酸铵和无水硫酸镁的最适浓度,进一步采用L_9(3~4)正交试验,得到了三者在培养基中的最佳组合:A_1B_2C_1,即葡萄糖50.0 g/L,硝酸铵6.0 g/L,无水硫酸镁0.3 g/L。从而得到最佳发酵培养基组成:葡萄糖50.0 g/L,NH4NO_3 6.0 g/L,无水MgSO_4 0.3 g/L,KH_2PO_4 0.5 g/L,VB_1 5.0×10~(-2) g/L,其紫杉醇平均含量达到938.6μg/L。
3.采用HPLC检测方法,对内生真菌Metarhizium anisopliae LB-10发酵产紫杉醇的工艺条件进行了研究。通过单因素实验,确定了温度、pH、接种量的最适条件,进一步采用L_9(3~4)正交试验,得到了三者在发酵过程中的最佳组合:A_2B_2C_3,即温度27℃,pH 7.0,接种量5%,其紫杉醇平均含量达到949.6μg/L。同时,制作了菌体生长曲线和代谢产紫杉醇含量变化曲线,选取发酵时间为14 d并在第10 d处于对数生长末期加入调节物质为最优条件。
4.根据紫杉醇的结构特点和红豆杉中紫杉醇的合成机制,选取了部分前体物质、诱导子和抑制剂,研究其对Metarhizium anisopliae LB-10合成紫杉醇的影响。结果表明:在发酵过程的第10 d,补加下列任一生长调节剂,使发酵液中初始浓度分别达到:苯甲酸钠30.0 mg/L,酪氨酸20.0 mg/L,苯丙氨酸3.0 mg/L,乙酸钠3.0g/L,水杨酸20.0 mg/L,茉莉酸甲酯100.0μmol/L,赤霉素2.0 mg/L,矮壮素2.0 mg/L,均能提高紫杉醇产量。进一步采用L_(16)(4~5)正交试验分析,得到了4种前体物质在培养基中的最佳组合:A_3B_3C_3D_1,即苯丙氨酸3.0 mg/L,酪氨酸40.0 mg/L,苯甲酸钠30.0 mg/L,乙酸钠1.0 g/L,其紫杉醇含量达到987.3μg/L。同时,在发酵过程的第10 d补加蔗糖,在含量不高于15.0 g/L时,对紫杉醇积累和菌体生长具有促进作用,但是高于此浓度时,菌体生长受抑制,紫杉醇含量降低。
Taxol, a highly derivative diterpenoid, was firstly found in some species of Taxus. In 1992, Taxol was marketed as an anticancer drug approved by FDA. Taxol produced by plant endophytic fungus fermentation has come up to hotspot in recent years. Finding out high yield strains, screening optimal cultivation conditions and controlling metabolism will be effective ways to increase the yield of taxol.
With the goal of increasing taxol yield by microorganism fermentation, the author isolated an endophytic fungus, Metarhizium anisopliae LB-10, from Qin-Ba mountainous areas in China, and further conducted the research on culture media, fermentation conditions and growth regulators. Chief experimental methods and results are as follows:
1. One hundred and nine endophytic fungi were isolated from wild Taxus chinensis originated in Qin-Ba mountainous areas, and 82 strains were purified, which, in the fermentations, were examined by the method of UV, HPLC and HPLC-MS. The strains were identified by morphological characteristics and ITS sequence analysis. The results showed that 8 strains of endophytic fungi could produce taxol in their metabolites, including a high taxol-producing fungus, LB-10, whose yield of taxol was 846.1μg/L, and the strain was identified fungus Metarhizium anisopliae.
2. The yield of taxol was set as an index, the analytical results by HPLC showed that the best conben and nitrogen sources for producing higher yield were glucose and ammonia nitrate respectively. The optimum concentration of glucose, ammonium nitrate and anhydrous magnesium sulfate were determined by single factor esperiments. The experiment in the orthogonal design L_9(3~4) indicated that the three best combination in the medium was A_1B_2C_1, or glucose 50.0 g/L, nitrate bromide 6.0 g/L, anhydrous magnesium sulfate 0.3 g/L. Consequently, the optimal fermentation medium was obtained that glucose 50.0 g/L, NH_4NO_3 6.0 g/L, anhydrous MgSO_4 0.3 g/L, KH_2PO_4 0.5 g/L, VB1 5.0×10~(-2) g/L. Their yield of taxol was 938.6μg/L.
3. Using HPLC detection method, the fermentation conditions of Metarhizium anisopliae LB-10 was studied. The optimum conditions of temperature, pH and inoculum were determined by single factor esperiments. The experiment in the orthogonal design L_9(3~4) indicated that the three best combination in the conditions was A_2B_2C_3, or temperature was 27 oC, initial fermentation pH was 7.0, inoculum was 5%. Their yield of taxol was 949.6μg/L. At the same time, the curve of taxol accumulation and mycelium growth were drawn. The optimal fermentation period was 14 d, with the growth regulators added on the 10th day, the end of logarithmic growth period.
4. According to the structural characteristics and synthesis mechanism of taxol, parts of the precursor, elicitor and inhibitors were selected to study its effect on yield of taxol and mycelium growth. The results indicated that when each of the following growth regulators was added to the fermention process on the 10th day, and allowed its initial concentration to be corresponding value, the yield of taxol could be enhanced: sodium benzoate, 30.0 mg/L; tyrosine, 20.0 mg/L; phenylalanine, 3.0 mg/L; sodium acetate, 30.0 mg/L; salicylic acid, 20.0 mg/L; methyl jasmonate, 100.0μmol/L; GA3, 2.0 mg/L; CCC, 2.0 mg/L. The experiment in the orthogonal design L_(16)(4~5) indicated that the four best combination of precursors in the medium was A_3B_3C_3D_1, or phenylalanine 3.0 mg/L, tyrosine 40.0 mg/L, sodium acetate 30.0 mg/L, sodium acetate 1.0 g/L. Their yield of taxol was 987.3μg/L. Simultaneously, the addition of sugar to fermentation was researched. The results indicated that taxol accumulation and mycelium growth was stimulated when appending sugar no higher than 15.0 g/L at 10th day in the fermentation process. But above this concentration, mycelium growth was inhibited and yield of taxol was decreased.
本研究以提高微生物发酵法生产紫杉醇的产量为目标,从秦巴山区野生红豆杉中分离筛选出一株紫杉醇高产内生真菌Metarhizium anisopliae LB-10。在此基础上,对LB-10培养基配方、发酵工艺条件和生长调节剂做了进一步研究。主要研究方法和结果如下:
1.从秦巴山区野生红豆杉中分离得到109株内生真菌,经纯化获得82株内生真菌。采用UV、HPLC和HPLC-MS检测方法,对82株内生真菌的发酵液中代谢产物进行检测,并通过形态学特性研究和ITS序列分析,鉴定物种。结果表明,有8株内生真菌代谢产生紫杉醇,其中有1株高产紫杉醇内生真菌LB-10,鉴定该菌株为绿僵菌(Metarhizium anisopliae),其紫杉醇产量为846.1μg/L。
2.以紫杉醇产量为指标,利用HPLC进行检测,筛选确定了发酵产紫杉醇的最佳碳源是葡萄糖,最佳氮源是硝酸铵。通过单因素实验,确定了葡萄糖、硝酸铵和无水硫酸镁的最适浓度,进一步采用L_9(3~4)正交试验,得到了三者在培养基中的最佳组合:A_1B_2C_1,即葡萄糖50.0 g/L,硝酸铵6.0 g/L,无水硫酸镁0.3 g/L。从而得到最佳发酵培养基组成:葡萄糖50.0 g/L,NH4NO_3 6.0 g/L,无水MgSO_4 0.3 g/L,KH_2PO_4 0.5 g/L,VB_1 5.0×10~(-2) g/L,其紫杉醇平均含量达到938.6μg/L。
3.采用HPLC检测方法,对内生真菌Metarhizium anisopliae LB-10发酵产紫杉醇的工艺条件进行了研究。通过单因素实验,确定了温度、pH、接种量的最适条件,进一步采用L_9(3~4)正交试验,得到了三者在发酵过程中的最佳组合:A_2B_2C_3,即温度27℃,pH 7.0,接种量5%,其紫杉醇平均含量达到949.6μg/L。同时,制作了菌体生长曲线和代谢产紫杉醇含量变化曲线,选取发酵时间为14 d并在第10 d处于对数生长末期加入调节物质为最优条件。
4.根据紫杉醇的结构特点和红豆杉中紫杉醇的合成机制,选取了部分前体物质、诱导子和抑制剂,研究其对Metarhizium anisopliae LB-10合成紫杉醇的影响。结果表明:在发酵过程的第10 d,补加下列任一生长调节剂,使发酵液中初始浓度分别达到:苯甲酸钠30.0 mg/L,酪氨酸20.0 mg/L,苯丙氨酸3.0 mg/L,乙酸钠3.0g/L,水杨酸20.0 mg/L,茉莉酸甲酯100.0μmol/L,赤霉素2.0 mg/L,矮壮素2.0 mg/L,均能提高紫杉醇产量。进一步采用L_(16)(4~5)正交试验分析,得到了4种前体物质在培养基中的最佳组合:A_3B_3C_3D_1,即苯丙氨酸3.0 mg/L,酪氨酸40.0 mg/L,苯甲酸钠30.0 mg/L,乙酸钠1.0 g/L,其紫杉醇含量达到987.3μg/L。同时,在发酵过程的第10 d补加蔗糖,在含量不高于15.0 g/L时,对紫杉醇积累和菌体生长具有促进作用,但是高于此浓度时,菌体生长受抑制,紫杉醇含量降低。
Taxol, a highly derivative diterpenoid, was firstly found in some species of Taxus. In 1992, Taxol was marketed as an anticancer drug approved by FDA. Taxol produced by plant endophytic fungus fermentation has come up to hotspot in recent years. Finding out high yield strains, screening optimal cultivation conditions and controlling metabolism will be effective ways to increase the yield of taxol.
With the goal of increasing taxol yield by microorganism fermentation, the author isolated an endophytic fungus, Metarhizium anisopliae LB-10, from Qin-Ba mountainous areas in China, and further conducted the research on culture media, fermentation conditions and growth regulators. Chief experimental methods and results are as follows:
1. One hundred and nine endophytic fungi were isolated from wild Taxus chinensis originated in Qin-Ba mountainous areas, and 82 strains were purified, which, in the fermentations, were examined by the method of UV, HPLC and HPLC-MS. The strains were identified by morphological characteristics and ITS sequence analysis. The results showed that 8 strains of endophytic fungi could produce taxol in their metabolites, including a high taxol-producing fungus, LB-10, whose yield of taxol was 846.1μg/L, and the strain was identified fungus Metarhizium anisopliae.
2. The yield of taxol was set as an index, the analytical results by HPLC showed that the best conben and nitrogen sources for producing higher yield were glucose and ammonia nitrate respectively. The optimum concentration of glucose, ammonium nitrate and anhydrous magnesium sulfate were determined by single factor esperiments. The experiment in the orthogonal design L_9(3~4) indicated that the three best combination in the medium was A_1B_2C_1, or glucose 50.0 g/L, nitrate bromide 6.0 g/L, anhydrous magnesium sulfate 0.3 g/L. Consequently, the optimal fermentation medium was obtained that glucose 50.0 g/L, NH_4NO_3 6.0 g/L, anhydrous MgSO_4 0.3 g/L, KH_2PO_4 0.5 g/L, VB1 5.0×10~(-2) g/L. Their yield of taxol was 938.6μg/L.
3. Using HPLC detection method, the fermentation conditions of Metarhizium anisopliae LB-10 was studied. The optimum conditions of temperature, pH and inoculum were determined by single factor esperiments. The experiment in the orthogonal design L_9(3~4) indicated that the three best combination in the conditions was A_2B_2C_3, or temperature was 27 oC, initial fermentation pH was 7.0, inoculum was 5%. Their yield of taxol was 949.6μg/L. At the same time, the curve of taxol accumulation and mycelium growth were drawn. The optimal fermentation period was 14 d, with the growth regulators added on the 10th day, the end of logarithmic growth period.
4. According to the structural characteristics and synthesis mechanism of taxol, parts of the precursor, elicitor and inhibitors were selected to study its effect on yield of taxol and mycelium growth. The results indicated that when each of the following growth regulators was added to the fermention process on the 10th day, and allowed its initial concentration to be corresponding value, the yield of taxol could be enhanced: sodium benzoate, 30.0 mg/L; tyrosine, 20.0 mg/L; phenylalanine, 3.0 mg/L; sodium acetate, 30.0 mg/L; salicylic acid, 20.0 mg/L; methyl jasmonate, 100.0μmol/L; GA3, 2.0 mg/L; CCC, 2.0 mg/L. The experiment in the orthogonal design L_(16)(4~5) indicated that the four best combination of precursors in the medium was A_3B_3C_3D_1, or phenylalanine 3.0 mg/L, tyrosine 40.0 mg/L, sodium acetate 30.0 mg/L, sodium acetate 1.0 g/L. Their yield of taxol was 987.3μg/L. Simultaneously, the addition of sugar to fermentation was researched. The results indicated that taxol accumulation and mycelium growth was stimulated when appending sugar no higher than 15.0 g/L at 10th day in the fermentation process. But above this concentration, mycelium growth was inhibited and yield of taxol was decreased.
引文
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