CN1932024B - 紫杉醇的生物合成方法及其专用培养基 - Google Patents
紫杉醇的生物合成方法及其专用培养基 Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种紫杉醇的生物合成方法及其专用培养基。该培养基的配方为:MgSO4·7H2O 200-300mg,KNO3 200-300mg,KH2PO4 350-400mg,Ca(NO3)2·4H2O450-550mg,FeSO4 27-29mg,C10H14N2O8Na2·2H2O 36-38mg,MnSO4·H2O 0.8-1.2mg,ZnSO4·7H2O 0.4-0.6mg,KI 0.2-0.3mg,CoCl2·6H2O 0.02—0.03mg,H3BO3 0.2-0.3mg,盐酸噻胺8-12mg,Choline 0.4-0.6mg,L-半胱氨酸8-12mg,H2NCONH2 180-220mg,Inositol 80-120mg,6-苄基腺嘌呤0.1-1mg/L,α-萘乙酸0.5-1.5mg/L,蔗糖或葡萄糖20-50g/L,麦芽糖20-50g/L,组合前体物质,组合代谢调节物质和组合诱导子,用水定容至1L,pH 5.6-5.8。本发明将在紫杉醇的工业化生产中发挥重要作用,应用前景广阔。
Description
技术领域
本发明涉及生物工程领域中的天然产物的生物合成方法及其专用培养基,特别是涉及紫杉醇的生物合成方法及其专用培养基。
背景技术
紫杉醇(pacilitaxel,商品名taxol),是1967年美国化学家Wall和Wani等首先从太平洋紫杉(短叶红豆杉,Taxus.brevifolia)树皮中提取出来的具有独特抗癌活性的二萜类化合物。用该化合物制备的药物于1990年进入III期临床试验,1992年底获美国FDA批准上市,用于治疗对常规化疗无效的卵巢癌和乳腺癌。1995年,我国北京协和药厂与海口制药厂分获二类新药批准文号,成为世界上第二个生产紫杉醇及其注射液的国家。由于紫杉醇具有独特的抗癌机理和广谱高效的抗癌活性,现已成为继阿霉素和顺铂后最常用的抗癌药物。
紫杉醇是一种高效的细胞毒素,具有独特的抗癌机理。研究表明,紫杉醇作用于微管蛋白。微管是真核细胞的一种纤维蛋白,与细胞的有丝分裂紧密相关,紫杉醇可诱导和促进微管蛋白迅速聚合形成对热与钙稳定的微管,并以非共价键化学计量地与聚合微管结合。这样,对于迅速分裂的肿瘤细胞,紫杉醇可“冻结”有丝分裂纺锤体,从而使肿瘤细胞停止在G2期和M期,直至死亡。此外,紫杉醇还表现出广谱而高效的抗癌活性,对人的卵巢癌、乳腺癌、宫颈癌、肺癌、CNS癌、黑色素瘤、肝癌和白血病细胞系等均可产生细胞毒作用;其抗癌活性高于噻唑呋啉、顺铂、依托泊甙、阿霉素和氟尿嘧啶等常用抗癌药物。
紫杉又名红豆杉,是红豆杉属植物,据统计,全世界有11种红豆杉属植物,且生长缓慢,种群密度小,自身繁殖率低。紫杉醇多提取自紫杉的树皮或树叶,且天然的植物中紫杉醇的含量极低,只有0.003-0.006%,其中,含量最高的曼地亚红豆杉(杂交种)也仅为约0.01%左右。目前,全世界紫杉醇的产量只有350公斤左右,受产量限制,该药只能用于其它药物无效的癌症治疗。据估计,全世界每年的紫杉醇需求量至少约为1000kg,而目前紫杉醇的产量远远不能满足市场需求。因此,采用各种手段寻找紫杉醇及其类似物的替代资源已成为近年来全世界的研究热点。美国农业部于1991年就批准了Christen和Gibson等用细胞培养法生产紫杉醇及其类似物的专利(United States Patent 5,019,504.),但是因其存在细胞增殖缓慢,紫杉醇含量低以及大规模培养工艺复杂等问题,一直没有实现产业化生产。因此该领域的研究仍方兴未艾,相关的研究机构和研究人员一直有增无减。
发明内容
本发明的目的是一种用于生物合成紫杉醇的培养基。
本发明所提供的培养基,命名为紫杉醇合成培养基,其配方为:MgSO4·7H2O200-300mg,KNO3 200-300mg,KH2PO4 350-400mg,Ca(NO3)2·4H2O 450-550mg,FeSO427-29mg,C10H14N2O8Na2·2H2O 36-38mg,MnSO4·H2O 0.8-1.2mg,ZnSO4·7H2O 0.4-0.6mg,KI 0.2-0.3mg,CoCl2·6H2O 0.02-0.03mg,H3BO3 0.2-0.3mg,盐酸噻胺(Aneurinehydrochloride)8-12mg,氯化胆碱(Choline)0.4-0.6mg,L-半胱氨酸(L-Cysteine)8-12mg,H2NCONH2 180-220mg,肌醇(Inositol)80-120mg,6-苄基腺嘌呤(6-BA)0.1-1mg/L,α-萘乙酸(NAA)0.5-1.5mg/L,蔗糖或葡萄糖20-50g/L,麦芽糖20-50g/L,组合前体物质,组合代谢调节物质和组合诱导子,用水定容至1L,pH5.6-5.8;所述组合前体物质由100-150mL/L番茄汁,10-30mL/L丙酮酸,20-40mL/L苯甲酸,40-50mL/L β-苯丙氨酸和30-50μl/L乙酸酐组成;组合代谢调节物质由5-20mL/L鸟氨酸,20-40mL/L色氨酸,100-150mL/L谷氨酰胺和1-3mL/L辅酶A组成;组合诱导子由0.02-0.04M紫杉真菌诱导子F3,50-80μg/L水杨酸,30-50μl/L过氧化氢和80-100μmol/L甲基茉莉酮酸组成。
上述紫杉醇合成培养基的配方优选为:MgSO4·7H2O 250mg,KNO3 250mg,KH2PO4375mg,Ca(NO3)2·4H2O 500mg,FeSO427.8mg,C10H14N2O8Na2·2H2O 37.2mg,MnSO4·H2O1mg,ZnSO4·7H2O 0.5mg,KI 0.25mg,CoCl2·6H2O 0.025mg,H3BO3 0.25mg,盐酸噻胺10mg,氯化胆碱0.5mg,L-半胱氨酸10mg,H2NCONH2200mg,肌醇100mg,6-苄基腺嘌呤0.1-1mg/L,α-萘乙酸0.5-1.5mg/L,蔗糖或葡萄糖20-50g/L,麦芽糖20-50g/L,组合前体物质,组合代谢调节物质和组合诱导子,用水定容至1L,pH5.6-5.8;所述组合前体物质由100-150mL/L番茄汁,10-30mL/L丙酮酸,20-40mL/L苯甲酸,40-50mL/L β-苯丙氨酸和30-50μl/L乙酸酐组成;组合代谢调节物质由5-20mL/L鸟氨酸,20-40mL/L色氨酸,100-150mL/L谷氨酰胺和1-3mL/L辅酶A组成;组合诱导子由0.02-0.04M紫杉真菌诱导子F3,50-80μg/L水杨酸,30-50μl/L过氧化氢和80-100μmol/L甲基茉莉酮酸组成。
本发明的第二个目的是提供一种产量较高,成本较低廉的紫杉醇的生物合成方法。
本发明所提供的紫杉醇的生物合成方法,是将紫杉细胞接种于上述紫杉醇合成培养基中,在25-30℃、黑暗条件下振荡培养,收获细胞,再经萃取、纯化后得到紫杉醇。
在上述紫杉醇的生物合成方法中,所述接种的紫杉细胞与紫杉醇合成培养基的重量份数比可为1-4∶10,振荡培养时的振速为80-120rpm,合成培养时间可为2-4天。
所述紫杉细胞既可为东北红豆杉(Taxus cuspidata Sieb et Zucc)、中国红豆杉(Taxus chinensis)、南方红豆杉(T.mairei S.Y.Hu)、曼地亚红豆杉(T.media)或云南红豆杉(T.yunnanenisis Cheng et Fu)等任意品种的紫杉细胞,也可以用人工培养或商业途径购买的等任意来源的紫杉细胞,如用具有以下步骤的培养方法获得的高活性紫杉细胞:
1)以经表面杀菌的幼嫩枝条或叶片为外植体,切成小段后,将其接种于愈伤组织诱导培养基上在20-25℃、1500-3000LX、14-16小时光照/天条件下诱导愈伤组织;所述愈伤组织诱导培养基是在上述紫杉基本培养基的基础上添加了6-苄基腺嘌呤0.1-5mg/L,α-萘乙酸0.1-5mg/L,蔗糖或葡萄糖20-50g/L,琼脂5-8g/L或冷凝胶(Gellan Gum)1.8-2g/L,pH 5.6-5.8;
2)将愈伤组织接种于继代培养基上在20-25℃、避光条件下进行继代培养,并从中筛选出紫杉醇含量达到0.006%以上的初级高产细胞株;所述继代培养基是在紫杉基本培养基的基础上添加了6-苄基腺嘌呤0.1-4mg/L,α-萘乙酸0.1-4mg/L,蔗糖或葡萄糖20-30g/L,琼脂5-8g/L或冷凝胶1.8-2g/L,pH 5.6-5.8;
3)先将初级高产细胞株接种于添加有10-70mg/L的秋水仙碱的紫杉基本培养基中在20-25℃、避光条件下振荡培养,再将所述经过不同浓度秋水仙碱处理的初级高产细胞株再次接种于继代培养基上进行继代培养,并从中筛选出紫杉醇含量达到0.01%以上的多倍体高产细胞株;
4)将多倍体高产细胞株接种于扩增培养基上在20-25℃、避光条件下进行扩增培养,可得到大量高活力紫杉细胞;所述扩增培养基是在紫杉基本培养基的基础上添加了6-苄基腺嘌呤0.5-2mg/L,α-萘乙酸0.5-2mg/L,蔗糖或葡萄糖30-50g/L,琼脂5-8g/L或冷凝胶1.8-2g/L,pH 5.6-5.8。
在上述培养方法中,紫杉基本培养基的配方为:MgSO4·7H2O 200-300mg,KNO3200-300mg,KH2PO4 350-400mg,Ca(NO3)2·4H2O 450-550mg,FeSO4 27-29mg,C10H14N2O8Na2·2H2O 36-38mg,MnSO4·H2O 0.8-1.2mg,ZnSO4·7H2O 0.4-0.6mg,KI0.2-0.3mg,CoCl2·6H2O 0.02-0.03mg,H3BO3 0.2-0.3mg,盐酸噻胺(Aneurinhydrochloride)8-12mg,氯化胆碱(Choline)0.4-0.6mg,L-半胱氨酸(L-Cysteine)8-12mg,H2NCONH2 180-220mg,肌醇(Inositol)80-120mg,用水定容至1L,pH 5.6-5.8。
步骤1)中对外植体进行表面杀菌的方法可为:在无菌条件下,将紫杉的幼嫩枝条或叶片用体积百分浓度为70%的乙醇消毒30-50s,再将其置于质量百分浓度为10%的次氯酸钠溶液中杀菌15-20分钟,最后用无菌水冲洗2-4次,每次15-30s,以将表面残留的次氯酸钠溶液冲洗干净;此外,为获得较好的诱导效果,接种前将外值体切成长度约为1-3mm的小段;愈伤组织的诱导时间可为3-5周。
步骤2)中的继代培养周期可为3-4周,至少继代3次。
步骤3)中用含10-70mg/L不同浓度秋水仙碱的紫杉基本培养基对初级高产细胞株进行振荡培养时的振速可为80-120rpm,培养时间可为6-8天;经过不同浓度秋水仙碱处理的初级高产细胞的继代培养条件与步骤2)中的继代培养条件相同,周期可为3-4周,共继代3-4次。
步骤4)中的扩增培养时间可为3-4周。
本发明提供了一种紫杉醇的生物合成方法及其专用培养基。利用本发明的方法及培养基可在不破坏自然环境的条件下,将紫杉细胞用于紫杉醇的生物转化合成中,据统计,用本发明的方法可以在2-4天内将细胞内紫杉醇的含量提高到细胞干重的0.1%左右。本发明合理地解决了紫杉细胞生长与紫杉醇合成的相互矛盾,并利用活细胞作为微反应器,采取代谢调控技术快速促进了紫杉醇的合成,且成本低廉,将在紫杉醇的工业化生产中发挥重要作用,应用前景广阔。
下面结合具体实施例对本发明做进一步详细说明。
具体实施方式
下述实施例中所用方法如无特别说明均为常规方法,所有培养基的溶剂均为蒸馏水或去离子水。
实施例1、高活力紫杉细胞的获得及紫杉醇的生物合成
一、高活力紫杉细胞的获得
紫杉基本培养基:MgSO4·7H2O 300mg,KNO3 200mg,KH2PO4 400mg,Ca(NO3)2·4H2O450mg,FeSO4 27mg,C10H14N2O8Na2·2H2O 38mg,MnSO4·H2O 1.2mg,ZnSO4·7H2O 0.6mg,KI 0.2mg,CoCl2·6H2O 0.03mg,H3BO3 0.3mg,盐酸噻胺8mg,Choline 0.4mg,L-半胱氨酸12mg,H2NCONH2 220mg,Inositol 80mg,用水定容至1L,pH 5.6。
愈伤组织诱导培养基:在紫杉基本培养基的基础上添加了6-苄基腺嘌呤0.5mg/L,α-萘乙酸(NAA)0.5mg/L,葡萄糖20g/L,琼脂5g/L,pH 5.6。
继代培养基:在紫杉基本培养基的基础上添加了6-苄基腺嘌呤0.5mg/L,α-萘乙酸0.5mg/L,葡萄糖20g/L,琼脂5g/L,pH 5.6。
扩增培养基:在紫杉基本培养基的基础上添加了6-苄基腺嘌呤0.5mg/L,α-萘乙酸0.5mg/L,葡萄糖30g/L,琼脂5g/L,pH 5.6。
上述培养基的灭菌条件均为:在115-120℃、0.1-0.15MPa压力下灭菌15-20min。
现用对本发明的方法对东北红豆杉(Taxus cuspidata Sieb et Zucc)进行体外培养,并从中筛选出高活力细胞,具体方法包括以下步骤:
1)外植体的表面杀菌
以东北红豆杉的幼嫩枝条为外植体,在无菌条件下,将其用体积百分浓度为70%的乙醇消毒30-50s,再将其置于质量百分浓度为10%的次氯酸钠溶液中杀菌15-20分钟,最后用无菌水冲洗3次,每次30s,以将表面残留的次氯酸钠溶液冲洗干净。
2)诱导愈伤组织
在无菌条件下,将步骤1)经消毒灭菌的幼茎段切成长度约为1mm的小段后接种于愈伤组织诱导培养基上,在20℃、1500LX、16小时光照/天条件下诱导愈伤组织,约3-5周后白色或绿色愈伤组织长出。
3)继代培养及初级高产细胞株的获得
将愈伤组织接种于继代培养基上在25℃、避光条件下进行继代培养,培养周期为3-4周,继代5次后,当愈伤组织变得结构疏松,生长速度趋于稳定以后,即可根据愈伤组织的株系不同,分别用干物质称重和高效液相色谱(HPLC)法进行生物量和紫杉醇含量检测,并根据检测结果筛选出生长速度快,紫杉醇含量在0.006%以上的初级高产细胞株。
4)多倍体高产细胞株的筛选
先将初级高产细胞株接种于添加有10-70mg/L的秋水仙碱的紫杉基本培养基中在22℃、避光、80rpm下振荡培养7天,再将所述经过不同浓度秋水仙碱处理的初级高产细胞株再次接种于继代培养基上继代培养3次,再经生物量和紫杉醇含量检测筛选出生长速度快,生物量积累稳定,紫杉醇含量在0.01%以上的高产细胞株,该细胞系为混合多倍体细胞系,可通过染色体染色进行鉴定。
5)扩增培养
将多倍体高产细胞株接种于扩增培养基上在20-25℃、避光条件下培养4周,得到大量高活力紫杉细胞,细胞量达300g/L。
6)紫杉醇的生物合成及其鉴定
紫杉醇合成培养基:MgSO4·7H2O 300mg,KNO3 200mg,KH2PO4 400mg,Ca(NO3)2·4H2O450mg,FeSO4 27mg,C10H14N2O8Na2·2H2O 38mg,MnSO4·H2O 1.2mg,ZnSO4·7H2O 0.6mg,KI 0.2mg,CoCl2·6H2O 0.03mg,H3BO3 0.3mg,盐酸噻胺8mg,Choline 0.4mg,L-半胱氨酸12mg,H2NCONH2 220mg,Inositol 80mg,6-苄基腺嘌呤1mg/L,α-萘乙酸1.5mg/L,蔗糖30g/L,麦芽糖20g/L,组合前体物质,组合代谢调节物质和组合诱导子,用水定容至1L,pH 5.8;
组合前体物质:由150mL/L番茄汁,30mL/L丙酮酸,40mL/L苯甲酸,50mL/Lβ-苯丙氨酸和50μl/L乙酸酐组成;
组合代谢调节物质:由20mL/L鸟氨酸,40mL/L色氨酸,150mL/L谷氨酰胺和3mL/L辅酶A组成;
组合诱导子:由0.04M紫杉真菌诱导子F3,80μg/L水杨酸,50μl/L过氧化氢和100μmol/L甲基茉莉酮酸组成。
将步骤一获得的高活力紫杉细胞与紫杉醇合成培养基按重量份数比为1∶5混合,在30℃、黑暗、90rpm下振荡培养3天,收获细胞。经检测,细胞中的紫杉醇含量达到0.1%。将收获的细胞转移到搅拌式提取罐中,将萃取剂(乙醇)与细胞按重量比为10∶1加入,在300rpm下抽提6小时,然后收集抽提液,反复3次。在50℃下减压浓缩后,再用二氯甲烷等进行反萃,再将其收集液进行减压浓缩后即可获得紫杉醇浸膏,可再用凝胶分离或制备色谱(HPLC)纯化后得到紫杉醇。经质谱和核磁共振鉴定,证明所得紫杉醇的分子结构正确。此外,对紫杉醇浸膏进行纯化过程中还可得到紫杉烷类化合物中的紫杉醇的合成前体化合物10-去乙酰浆果赤霉素III、浆果赤霉素III和三尖杉碱等。
实施例2、紫杉醇的生物合成及其鉴定
紫杉醇合成培养基:MgSO4·7H2O 250mg,KNO3 250mg,KH2PO4 375mg,Ca(NO3)2·4H2O500mg,FeSO4 27.8mg,C10H14N2O8Na2·2H2O 37.2mg,MnSO4·H2O 1mg,ZnSO4·7H2O 0.5mg,KI 0.25mg,CoCl2·6H2O 0.025mg,H3BO3 0.25mg,盐酸噻胺10mg,Choline 0.5mg,L-Cysteine 10mg,H2NCONH2 200mg,Inositol 100mg,6-苄基腺嘌呤0.1mg/L,α-萘乙酸0.5mg/L,蔗糖40g/L,麦芽糖20g/L,组合前体物质,组合代谢调节物质和组合诱导子,用水定容至1L,pH 5.6;
组合前体物质:100mL/L番茄汁,10mL/L丙酮酸,20mL/L苯甲酸,40mL/L β-苯丙氨酸和30μl/L乙酸酐组成;
组合代谢调节物质:由5mL/L鸟氨酸,20mL/L色氨酸,100mL/L谷氨酰胺和1mL/L辅酶A组成;
组合诱导子:由0.02M紫杉真菌诱导子F3,50μg/L水杨酸,30μl/L过氧化氢和80μmol/L甲基茉莉酮酸组成。
将用与实施例1中相同方法经筛选获得的中国红豆杉(Taxus chinensis)多倍体高产细胞系接种在扩增培养基上扩增进行扩增培养4周,然后将扩增的中国红豆杉细胞按重量比1.5∶5接种在紫杉醇合成培养基内,在25℃、黑暗、110rpm下振荡培养4天,收获细胞。经检测,细胞中的紫杉醇含量达到0.12%。将收获的细胞转移到搅拌式提取罐中,将萃取剂(乙醇)与细胞按重量比为10∶1加入,在400rpm下抽提5小时,然后收集抽提液,反复3次。在50℃下减压浓缩后,再用二氯甲烷等进行反萃,再将其收集液进行减压浓缩后即可获得紫杉醇浸膏,可再用凝胶分离或制备色谱(HPLC)纯化后得到紫杉醇。经质谱和核磁共振鉴定,证明所得紫杉醇的分子结构正确。此外,对紫杉醇浸膏进行纯化过程中还可得到紫杉烷类化合物中的紫杉醇的合成前体化合物10-去乙酰浆果赤霉素III、浆果赤霉素III和三尖杉碱等。
实施例3、紫杉醇的生物合成及其鉴定
紫杉醇合成培养基:MgSO4·7H2O 200mg,KNO3 300mg,KH2PO4 350mg,Ca(NO3)2·4H2O550mg,FeSO4 29mg,C10H14N2O8Na2·2H2O 36mg,MnSO4·H2O 0.8mg,ZnSO4·7H2O 0.4mg,KI 0.3mg,CoCl2·6H2O 0.02mg,H3BO3 0.2mg,盐酸噻胺12mg,Choline 0.6mg,L-半胱氨酸8mg,H2NCONH2 180mg,Inositol 120mg,6-苄基腺嘌呤0.1mg/L,α-萘乙酸0.5mg/L,蔗糖50g/L,麦芽糖50g/L,组合前体物质,组合代谢调节物质和组合诱导子,用水定容至1L,pH 5.7;
组合前体物质:由100mL/L番茄汁,10mL/L丙酮酸,20mL/L苯甲酸,40mL/L β-苯丙氨酸和30μl/L乙酸酐组成;
组合代谢调节物质:由5mL/L鸟氨酸,20mL/L色氨酸,100mL/L谷氨酰胺和1mL/L辅酶A组成;
组合诱导子:由0.02M紫杉真菌诱导子F3,50μg/L水杨酸,30μl/L过氧化氢和80μmol/L甲基茉莉酮酸组成。
将用与实施例1中相同方法经筛选获得的云南红豆杉(T.yunnanenisis Chenget Fu)多倍体高产细胞系接种在扩增培养基上进行扩增培养4周,然后将扩增的云南红豆杉细胞按重量比2∶5接种在紫杉醇合成培养基内,在28℃、黑暗、100rpm下振荡培养3.5天,收获细胞。经检测,细胞中的紫杉醇含量达到0.11%。将收获的细胞转移到搅拌式提取罐中,将萃取剂(乙醇)与细胞按重量比为10∶1混合,在350rpm下抽提6小时,然后收集抽提液,反复3次。在50℃下减压浓缩后,再用二氯甲烷等进行反萃,再将其收集液进行减压浓缩后即可获得紫杉醇浸膏,可再用凝胶分离或制备色谱(HPLC)纯化后得到紫杉醇。经质谱和核磁共振鉴定,证明所得紫杉醇的分子结构正确。此外,对紫杉醇浸膏进行纯化过程中还可得到紫杉烷类化合物中的紫杉醇的合成前体化合物10-去乙酰浆果赤霉素III、浆果赤霉素III和三尖杉碱等。
Claims (4)
1.用于生物合成紫杉醇的培养基,其配方为:MgSO4·7H2O 200-300mg,KNO3200-300mg,KH2PO4 350-400mg,Ca(NO3)2·4H2O 450-550mg,FeSO4 27-29mg,C10H14N2O8Na2·2H2O 36-38mg,MnSO4·H2O 0.8-1.2mg,ZnSO4·7H2O 0.4-0.6mg,KI0.2-0.3mg,CoCl2·6H2O 0.02-0.03mg,H3BO3 0.2-0.3mg,盐酸噻胺8-12mg,氯化胆碱0.4-0.6mg,L-半胱氨酸8-12mg,H2NCONH2 180-220mg,肌醇80-120mg,6-苄基腺嘌呤0.1-1mg/L,α-萘乙酸0.5-1.5mg/L,蔗糖或葡萄糖20-50g/L,麦芽糖20-50g/L,组合前体物质,组合代谢调节物质和组合诱导子,用水定容至1L,pH5.6-5.8;所述组合前体物质由100-150mL/L番茄汁,10-30mL/L丙酮酸,20-40mL/L苯甲酸,40-50mL/L β-苯丙氨酸和30-50μl/L乙酸酐组成;组合代谢调节物质由5-20mL/L鸟氨酸,20-40mL/L色氨酸,100-150mL/L谷氨酰胺和1-3mL/L辅酶A组成;组合诱导子由0.02-0.04M紫杉真菌诱导子F3,50-80μg/L水杨酸,30-50μl/L过氧化氢和80-100μmol/L甲基茉莉酮酸组成。
2.根据权利要求1所述的培养基,其特征在于:所述培养基的配方为:MgSO4·7H2O250mg,KNO3 250mg,KH2PO4 375mg,Ca(NO3)2·4H2O 500mg,FeSO4 27.8mg,C10H14N2O8Na2·2H2O 37.2mg,MnSO4·H2O 1mg,ZnSO4·7H2O 0.5mg,KI 0.25mg,CoCl2·6H2O 0.025mg,H3BO3 0.25mg,盐酸噻胺10mg,氯化胆碱0.5mg,L-半胱氨酸10mg,H2NCONH2 200mg,肌醇100mg,6-苄基腺嘌呤0.1-1mg/L,α-萘乙酸0.5-1.5mg/L,蔗糖或葡萄糖20-50g/L,麦芽糖20-50g/L,组合前体物质,组合代谢调节物质和组合诱导子,用水定容至1L,pH 5.6-5.8;所述组合前体物质由100-150mL/L番茄汁,10-30mL/L丙酮酸,20-40mL/L苯甲酸,40-50mL/L β-苯丙氨酸和30-50μl/L乙酸酐组成;组合代谢调节物质由5-20mL/L鸟氨酸,20-40mL/L色氨酸,100-150mL/L谷氨酰胺和1-3mL/L辅酶A组成;组合诱导子由0.02-0.04M紫杉真菌诱导子F3,50-80μg/L水杨酸,30-50μl/L过氧化氢和80-100μmol/L甲基茉莉酮酸组成。
3.一种紫杉醇的生物合成方法,是将紫杉细胞接种于权利要求1或2所述的培养基中,在25-30℃、黑暗条件下振荡培养,收获细胞,再经萃取、纯化后得到紫杉醇。
4.根据权利要求3所述的方法,其特征在于:所述接种的紫杉细胞与紫杉醇合成培养基的重量比为1-4∶10,振荡培养时的振速为80-120rpm,培养时间为2-4天。
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