CN107058137B - 一种渥曼青霉及其生产渥曼青霉素的方法 - Google Patents
一种渥曼青霉及其生产渥曼青霉素的方法 Download PDFInfo
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- CN107058137B CN107058137B CN201710447621.8A CN201710447621A CN107058137B CN 107058137 B CN107058137 B CN 107058137B CN 201710447621 A CN201710447621 A CN 201710447621A CN 107058137 B CN107058137 B CN 107058137B
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- wortmannin
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- QDLHCMPXEPAAMD-QAIWCSMKSA-N wortmannin Chemical compound C1([C@]2(C)C3=C(C4=O)OC=C3C(=O)O[C@@H]2COC)=C4[C@@H]2CCC(=O)[C@@]2(C)C[C@H]1OC(C)=O QDLHCMPXEPAAMD-QAIWCSMKSA-N 0.000 title claims abstract description 60
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Abstract
本发明提供了一种渥曼青霉(Penicillium wortmannii)1507‑N13，以及通过培养它制备渥曼青霉素的方法。渥曼青霉(Penicillium wortmannii)1507‑N13于2017年01月09日保藏于“中国微生物菌种保藏管理委员会普通微生物中心”，保藏编号为CGMCC NO.13366。
Description
技术领域
本发明涉及一种新的渥曼青霉，通过发酵培养该菌产生渥曼青霉素的方法。
背景技术
渥曼青霉素(Wortmannin)的分子式为C23H24O8，分子量为428.43，其结构如下式所示：
渥曼青霉素是一类甾醇类抗生素，它主要对磷脂酰肌醇-3激酶(PI3K)有抑制作用，因此关于它治疗磷脂酰肌醇-3激酶依赖性疾病的研究很多，特别是对肿瘤的治疗作用。
Wortmannin可以通透细胞，和PI3K的110kD催化亚基相结合，特异性抑制PI3K，抑制PI3K/Akt信号途径，包括常见的抑制Akt磷酸化等。同时Wortmannin也可以抑制myosinlight chain kinase(肌球蛋白轻链激酶，MLCK)等。Wortmannin无明显的抗细菌作用，但具有很强的抑制真菌活性。但因其对细胞的毒性作用目前还都限于体外研究，尚未发展到临床应用。
现有技术中，P.W.Brian等报道了渥曼青霉素最初是从Penicillium wormanninKlocker的发酵培养物中分离得到的天然产物，并对渥曼青霉素生产菌的形态进行了详细的描述(Trans.Brit.mycol.Soc.40(3),365-368(1957))；US5378725和CN1111127A公开了渥曼青霉素生产菌A24603.1的发酵培养和渥曼青霉素的分离和纯化，A24603.1产渥曼青霉素的效价为151μg/mL，由于其效价低、渥曼青霉素产量小，现有的渥曼青霉素产生菌存在不适合工业化生产的问题；一些文献中例如WO2007120364和WO2003024183仅报道了渥曼青霉素是从真菌Penicillium wormannin的发酵培养基中分离得到的，而对于产生菌的生理生化特征、培养条件、固体、种子及发酵培养基的具体组成及含量并未有新的报道。由此可见，公开的专利及文献中渥曼青霉素的生产菌比较原始，发酵水平低，那么开发新菌株及发酵技术很有必要，因此寻找一种新的高效的渥曼青霉素生产菌的工作仍然在继续进行中。
发明内容
本发明的目的之在于提供一种新的高效的渥曼青霉素生产菌，该菌为渥曼青霉(Penicillium wortmannii)1507-N13，其保藏编号为CGMCC No.13366。
本发明渥曼青霉1507-N13的微生物菌种于2017年01月09日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院，中国科学院微生物研究所)，保藏编号为CGMCC No.13366，分类命名为渥曼青霉Penicillium wortmannii，并登记入册，证明存活。
本发明的目的还在于提供了一种渥曼青霉1507-N13(CGMCC No.13366)在制备渥曼青霉素或者含有渥曼青霉素的药物组合物中的应用。
本发明还提供了一种渥曼青霉素的制备方法，该方法包括采用渥曼青霉1507-N13(CGMCC No.13366)在含有可同化的碳源和/或氮源的营养培养基里，进行发酵培养的步骤。
优选的实施方案中，上述可同化的碳源选自葡萄糖、乳糖、甘露醇、山梨醇、蔗糖、麦芽糊精、玉米淀粉、甘油、豆油、油酸甲酯之一或上述物质的组合。
优选的实施方案中，上述可同化的氮源选自酵母抽提粉、酵母粉、牛肉浸膏、大豆蛋白胨、蛋白胨、黄豆饼粉、棉籽饼粉、花生饼粉、麸质粉、玉米浆干粉、麸皮之一或上述物质的组合。
优选的实施方案中，所述营养培养基还包括无机盐，所述无机盐选自氯化铵、硝酸铵，柠檬酸三铵、磷酸二氢钾、磷酸氢二钾、硫酸铵、碳酸钙、硫酸亚铁、硫酸锌、硫酸铜、氯化钠、氯化钾、氯化钙、硫酸镁、氯化铁、硫酸锰之一或上述物质的组合。
优选的实施方案中，所述营养培养基含有甘油10.0～50.0g/L、乳糖5.0～50.0g/L、蔗糖5.0～50.0g/L、黄豆饼粉5.0～20.0g/L、酵母粉5.0～20.0g/L、棉籽饼粉5.0～30.0g/L、磷酸二氢钾0.5～5.0g/L、硫酸铵1.0～5.0g/L、碳酸钙1.0～5.0g/L、硫酸锰0.01～1.0g/L。
优选的实施方案中，所述发酵培养的温度为20℃-30℃，优选24℃-26℃；培养基pH为5.0-8.0，优选6.0-7.0；培养时间为24-240小时，优选72-192小时。
优选的实施方案中，所述渥曼青霉1507-N13(CGMCC No.13366)是通过种子液接种至所述培养基中进行所述发酵培养的；
其中，所述种子液是将渥曼青霉1507-N13(CGMCC No.13366)进行种子培养得到的；所述种子培养的条件为：种子培养的温度为20℃-30℃，优选24℃-26℃；培养基pH为5.0-8.0，优选5.0-7.0；培养时间为24-80小时，优选24-60小时。
优选的实施方案中，所述的种子培养基含有蔗糖0～20.0g/L、葡萄糖10.0～50.0g/L、棉籽饼粉10.0～30.0g/L、酵母粉5.0～15.0g/L、磷酸二氢钾1.0～10.0g/L、硫酸铵0.5～1.5g/L、碳酸钙0.5～2.0g/L。
本发明渥曼青霉素通过以下方法进行检测：
色谱柱：C18分析柱(5μm,4.6×150mm).
进样量：10μL
流动相：甲醇：水：甲酸＝50：50：0.1(v/v/v)
流速：1mL/min
检测波长：254nm
本发明渥曼青霉(Penicillium wortmannii)1507-N13(CGMCC No.13366)生产能力高；且，其产生渥曼青霉素的能力比现有技术中其他菌种有了大幅度的提高，有利于实现工业化生产。
本发明渥曼青霉(Penicillium wortmannii)1507-N13(CGMCC No.13366)的主要生物学特征为：孢子呈椭圆形，菌落颜色是黄色，边缘呈白色，反面白色，平铺，无褶皱，有水珠产生，直径1.0～2.0cm，不易挑取。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
实施例1：菌株来源
本发明渥曼青霉1507-N13(CGMCC No.13366)系从中国东南沿海的浙江省台州市九峰山土壤中分离得到的原始菌株，再通过诱变得到的突变菌株。
原始菌株在PDA斜面培养基中30℃培养12天后，在无菌条件下用接种铲将菌丝体刮下，在磨口试管中磨碎菌丝体并悬浮于无菌水中，制得菌悬液，供NTG(亚硝基胍)诱变处理。
称取NTG晶体2mg，溶解在2mL无菌Tris缓冲液(pH6.0)中。用移液管吸取1ml NTG溶液加入到1ml菌悬液，于30℃振荡处理40min。其余具体步骤如下：
(1)菌丝体的制备与培养
固体培养基配方：PDA，经121℃灭菌30分钟后，冷却到50-60℃倒入平板，将处理过的菌悬液经适当稀释，吸取0.1mL菌悬液涂布于PDA平板上，未作诱变处理的菌悬液亦经适当稀释涂布于PDA平板作为对照，置于30℃恒温培养箱培养，菌落培养7天，菌丝成熟。
(2)种子液的制备与培养
种子培养基配方(g/L)：蔗糖10.0g/L、葡萄糖25.0g/L、棉籽饼粉10.0g/L、酵母粉15.0g/L、磷酸二氢钾5.0g/L、硫酸铵1.0g/L、碳酸钙1.0g/L，pH6.0。摇瓶装液量为20mL/250mL，121℃灭菌30分钟。每个种子摇瓶接入0.5个菌落左右，培养温度20～30℃，摇瓶机振幅5cm，转速250rpm，培养36小时。
(3)渥曼青霉素发酵培养基的制备与培养
甘油20.0g/L、乳糖40.0g/L、蔗糖30.0g/L、黄豆饼粉10.0g/L、酵母粉15.0g/L、棉籽饼粉10.0g/L、磷酸二氢钾0.5g/L、硫酸铵2.0g/L、碳酸钙2.0g/L、硫酸锰0.05g/L，pH6.5，摇瓶装液量为25mL/250mL，121℃灭菌30分钟。将种子液以10％(体积百分比)的接种量接入发酵培养基，培养温度25℃，摇瓶机振幅5cm，转速250rpm，培养98小时。
挑选经过NTG诱变后的单菌落1000株，进行摇瓶发酵，HPLC检测渥曼青霉素的产量。筛选出产渥曼青霉素最高产的突变菌株即渥曼青霉1507-N13(CGMCC No.13366)。其主要生物学特征为：孢子呈椭圆形，菌落颜色是黄色，边缘呈白色，反面白色，平铺，无褶皱，有水珠产生，直径1.0～2.0cm，不易挑取。
实施例2：菌种鉴定
参照《普通真菌学》、《常见细菌系统鉴定手册》、《分子克隆实验指南》及《中国药典》(2015版)等书中的有关内容进行实验；颜色判断参照RAL K7色卡系列的颜色进行对照。
1.菌种编号：1507-N13(CGMCC No.13366)。
2.培养特征：采用查氏、PDA、SDA、MEA和MEPG 5种培养基，28℃培养5～7天后，观察菌丝体的颜色及色素情况。菌株的培养特征见表1。
3.生理生化试验：除温度实验外，均为28℃培养5～7天。
a)碳源的利用：采用ISP9作为基础培养基，各种碳源的终浓度均为1.0％，见表2。
b)无机氮源的利用：采用ISP9作为基础培养基，硝酸钾和硫酸铵的浓度均为0.1％，见表2。
c)降解试验和NaCl耐受实验采用基础培养基为GYEA(pH6.8)，各种降解物的浓度及降解试验结果见表3；NaCl耐受实验结果见表7。
d)氧化酶和过氧化氢酶试验、pH试验和温度试验均采用PDA培养基。氧化酶和过氧化氢酶试验结果见表4，pH试验结果见表5，温度试验结果见表6。
e)M.R、V-P和脲酶实验采用《常见细菌系统鉴定手册》方法，其结果见表4。
4.28S rDNA序列分析：收集PDA培养的新鲜菌体，采用液氮破壁方法提取总DNA模板，采用Fungi Identification PCR Kit(TaKaRa)进行28S rDNA基因ITS区域扩增，PCR产物经琼脂糖电泳检测后，直接进行序列测定，PCR产物的纯化与测序由南京金斯瑞生物科技有限公司进行。菌株1507-N13所测的28S rDNA基因ITS区域序列经校对后，经BLAST比较确定其同源性。
实验结果
1.培养特征：菌株1507-N13(CGMCC No.13366)的培养特征见表1。
表1菌株1507-N13(CGMCC No.13366)在5种培养基上的培养特征
2.生理生化特征：见表2～表7。
表2菌株1507-N13(CGMCC No.13366)的碳源和氮源的利用情况
表3菌株1507-N13(CGMCC No.13366)的降解试验结果
| 降解物 | 降解物浓度 | 结果* | 降解物 | 降解物浓度 | 结果 |
| 腺嘌呤 | 0.5％ | 4，- | 酪蛋白 | 1.0％ | 4，- |
| 鸟嘌呤 | 0.5％ | 4，- | 酪氨酸 | 1.0％ | 4，- |
| 黄嘌呤 | 0.4％ | 4，- | Tween-40 | 1.0％ | 3，- |
| 木聚糖 | 0.4％ | 4，- | Tween-60 | 1.0％ | 3，- |
| 次黄嘌呤 | 0.4％ | 4，- | Tween-80 | 1.0％ | 3，- |
表4菌株1507-N13(CGMCC No.13366)主要的生理生化特征
| 试验项目 | 结果 | 试验项目 | 结果 | 试验项目 | 结果 |
| 明胶液化 | + | 牛奶胨化 | - | 纤维素利用 | - |
| 淀粉水解 | - | 硝酸盐还原 | - | 过氧化氢酶 | - |
| 牛奶凝固 | - | 硫化氢产生 | - | ||
| V.P实验 | - | M.R实验 | + |
表5菌株1507-N13(CGMCC No.13366)生长的pH试验
| pH | 3.5 | 4.0 | 4.5 | 5.0 | 5.5 | 6.0 | 6.5 | 7.0 | 7.5 |
| 生长情况 | 3 | 3 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
表6菌株1507-N13(CGMCC No.13366)生长的温度试验
| 温度(℃) | 7 | 14 | 28 | 37 | 45 |
| 生长情况 | 0 | 4 | 4 | 0 | 0 |
表7菌株1507-N13(CGMCC No.13366)对NaCl的耐受性
| NaCl浓度 | 1％ | 4％ | 7％ | 10％ |
| 菌株生长情况 | 4 | 0 | 0 | 0 |
*注：表2-7中：0，无生长；1，生长很弱；2，能生长，有少量孢子；3，生长良好，有大量孢子；4，生长最好，有丰富孢子；+，阳性；-，阴性。
3.28S rDNA序列分析：菌株1507-N13(CGMCC No.13366)的28S ITS序列与GenBank中相关序列进行BLAST比较，结果见表8(表中只列出同源性较高的菌株)。
表8菌株1507-N13(CGMCC No.13366)和相关菌株的同源性
菌种鉴定结果
通过对菌株1507-N13(CGMCC No.13366)的28S rDNA ITS区域测序，与GenBank数据库中相关种、属的序列进行同源序列BLAST比较，结果表明：菌株1507-N13(CGMCCNo.13366)和青霉属(Penicillium sp.SGE10)有很高同源性，最高的达99.6％，同时对菌株1507-N13(CGMCC No.13366)的表观特征的试验，发现该菌株和青霉属(Penicillium sp.)分类相关参数非常接近，故将菌株1507-N13鉴定为青霉属(Penicillium sp.)菌株。
本发明渥曼青霉1507-N13(CGMCC No.13366)和其他渥曼青霉素产生菌的比较如下：
P.W.Brian等报道(Trans.Brit.mycol.Soc.40(3),365-368(1957)Penicilliumwormannin Klocker(1952年以Penicillium sp.保藏)在査氏培养基上菌落相当小，菌丝体主要是浅黄色的，菌落反面呈橙红色或者浅棕色，有褶皱，微微高耸；而本发明渥曼青霉1507-N13(CGMCC No.13366)在査氏培养基上菌落直径10mm，菌落呈亮黄色的，菌落反面呈白色，无褶皱，平铺。
US5378725和CN1111127A的渥曼青霉素生产菌A24603.1产渥曼青霉素的效价为151μg/mL；而本发明渥曼青霉1507-N13(CGMCC No.13366)产渥曼青霉素的效价高达3500μg/mL。
结合前述本发明的渥曼青霉1507-N13(CGMCC No.13366)的形态学、培养特征、生理生化特征和DNA序列鉴定结果可知，本发明的渥曼青霉1507-N13(CGMCC No.13366)属于渥曼青霉Penicillium wortmannii，且它不同于已知的渥曼青霉素产生菌，故本发明渥曼青霉1507-N13(CGMCC No.13366)是一株全新的菌种。
实施例3：制备渥曼青霉素
(1)固体培养基配方及培养
固体培养基配方：PDA，经121℃灭菌30分钟后，冷却待用，接入0.1mL菌悬液培养7天。
(2)种子培养基配方及培养
种子培养基配方(g/L)：蔗糖10.0g/L、葡萄糖25.0g/L、棉籽饼粉10.0g/L、酵母粉15.0g/L、磷酸二氢钾5.0g/L、硫酸铵1.0g/L、碳酸钙1.0g/L，pH6.0。摇瓶装液量为20mL/250mL，121℃灭菌30分钟。每个种子摇瓶接入0.5个菌落左右，培养温度20～30℃，摇瓶机振幅5cm，转速250rpm，培养36小时。
(3)渥曼青霉素发酵培养基的制备与培养
甘油40.0g/L、乳糖50.0g/L、酵母粉25g/L、棉籽饼粉10.0g/L、磷酸二氢钾0.5g/L、硫酸铵2.0g/L、碳酸钙2.0g/L、硫酸锰0.05g/L，pH6.5，摇瓶装液量为25mL/250mL，121℃灭菌30分钟。将种子液以10％(体积百分比)的接种量接入发酵培养基，培养温度25℃，摇瓶机振幅5cm，转速250rpm，培养96小时。经HPLC检测，渥曼青霉素效价为3156μg/mL。
实施例4：制备渥曼青霉素
(1)固体培养基配方及培养条件等同实施例3中步骤(1)
(2)种子培养基配方及培养条件等同实施例3中步骤(2)
(3)渥曼青霉素发酵培养基的制备与培养
甘油20.0g/L、乳糖50.0g/L、蔗糖30.0g/L、黄豆饼粉15.0g/L、酵母粉15.0g/L、棉籽饼粉10.0g/L、磷酸二氢钾0.8g/L、硫酸铵1.5g/L、碳酸钙1.5g/L、硫酸锰0.05g/L，pH6.5，摇瓶装液量为25mL/250mL，121℃灭菌30分钟。将种子液以10％(体积百分比)的接种量接入发酵培养基，培养温度25℃，摇瓶机振幅5cm，转速250rpm，培养100小时。经HPLC检测，渥曼青霉素效价为3344μg/mL。
实施例5制备渥曼青霉素
(1)固体培养基配方及培养条件等同实施例3中步骤(1)
(2)种子液的制备与培养
种子培养基配方：葡萄糖35.0g/L、棉籽饼粉10.0g/L、酵母粉15.0g/L、磷酸二氢钾3.0g/L、硫酸铵1.0g/L、碳酸钙1.0g/L，pH6.0。摇瓶装液量为20mL/250mL，121℃灭菌30分钟。培养温度20～30℃，摇瓶机振幅5cm，转速250rpm，培养36小时。
(3)发酵培养基配方及培养条件等同实施例3中步骤(3)
经HPLC检测，渥曼青霉素效价为3271μg/mL。
实施例6：制备和分离纯化渥曼青霉素
(1)种子罐种子液的制备
在15L种子罐中投入10L的种子培养基(种子培养基的配方同实施例3中步骤(2))，同时添加0.05％的PPG作为消泡剂)，灭菌采用蒸汽灭菌，121℃条件下30分钟，待冷却后，接入200ml摇瓶种子液，培养温度25±1℃，搅拌转速100～500rpm，通气量0.5～1.0vvm，溶氧20～70％，培养36小时。
(2)发酵罐培养基的配制与培养
发酵培养基的配方与前述实施例3中步骤(3)发酵配方相同，但要添加0.05％PPG作为消泡剂，发酵罐装量为30L/50L，pH7.0，于121℃下蒸汽灭菌30分钟，冷却后，接入约3.5L种子罐培养液，发酵温度25±1℃，搅拌转速200～500rpm，通气量为0.5～1.0vvm，发酵培养103小时。经HPLC检测，渥曼青霉素效价为3510μg/mL。
(3)发酵液萃取
发酵得到30L发酵液，用等体积乙酸乙酯萃取2次，将所得乙酸乙酯萃取液在45℃条件下减压浓缩去除乙酸乙酯直至干，得到76g油状物质。
(4)硅胶柱柱层析纯化
将所得的油状物用5L氯仿溶解上硅胶柱(粒径100-200目)进行柱层析，用氯仿:乙酸乙酯＝85:15(V/V)洗脱，并收集含渥曼青霉素的洗脱液。
(5)粗品的纯化和纯品的结构鉴定
将含渥曼青霉素的洗脱液浓缩至干，然后用乙酸乙酯500mL溶解，滤纸过滤后加入1000mL正己烷，混合均匀后转入4℃冰箱中冷藏。2小时后，将冷藏液过滤，得到的针状晶体转入真空干燥箱，50℃真空干燥5小时。
上述晶体通过NMR、MS等波谱分析，确定其为渥曼青霉素，其结构如下：
渥曼青霉素的理化性质如下：
性状:白色针状晶体
溶解性:易溶解于氯仿、丙酮、甲醇，不溶于水
分子式:C23H24O8
ESIMS m/z:427.12[M-H]-
表9渥曼青霉素在CDCl3中的核磁数据(氢谱，400MHz；碳谱，100MHz)
上述结果表明，本发明渥曼青霉1507-N13(CGMCC No.13366)产渥曼青霉素的效价高达3500μg/mL(CGMCC No.13366)，本发明渥曼青霉1507-N13(CGMCC No.13366)产渥曼青霉素的能力较其他菌种有了大幅度的提高，有利于实现渥曼青霉素工业化生产。
Claims (17)
1.一种渥曼青霉(Penicillium wortmannii)1507-N13，其保藏编号为CGMCCNo.13366。
2.根据权利要求1所述的渥曼青霉1507-N13在制备渥曼青霉素或者含有渥曼青霉素的药物组合物中的应用。
3.一种渥曼青霉素的制备方法，其特征在于：包括采用如权利要求1所述的渥曼青霉1507-N13在含有可同化的碳源和/或氮源的营养培养基里，进行发酵培养的步骤。
4.根据权利要求3所述的方法，其特征在于：所述可同化的碳源选自葡萄糖、乳糖、甘露醇、山梨醇、蔗糖、麦芽糊精、玉米淀粉、甘油、豆油、油酸甲酯之一或上述物质的组合。
5.根据权利要求3所述的方法，其特征在于：所述可同化的氮源选自酵母抽提粉、酵母粉、牛肉浸膏、蛋白胨、黄豆饼粉、棉籽饼粉、花生饼粉、麸质粉、玉米浆干粉、麸皮之一或上述物质的组合。
6.根据权利要求5所述的方法，其特征在于：所述蛋白胨为大豆蛋白胨。
7.根据权利要求3-6任一项所述的方法，其特征在于：所述营养培养基还包括无机盐，所述无机盐选自氯化铵、硝酸铵，柠檬酸三铵、磷酸二氢钾、磷酸氢二钾、硫酸铵、碳酸钙、硫酸亚铁、硫酸锌、硫酸铜、氯化钠、氯化钾、氯化钙、硫酸镁、氯化铁、硫酸锰之一或上述物质的组合。
8.根据权利要求3-6任一项所述的方法，其特征在于：所述营养培养基含有甘油10.0～50.0g/L、乳糖5.0～50.0g/L、蔗糖5.0～50.0g/L、黄豆饼粉5.0～20.0g/L、酵母粉5.0～20.0g/L、棉籽饼粉5.0～30.0g/L、磷酸二氢钾0.5～5.0g/L、硫酸铵1.0～5.0g/L、碳酸钙1.0～5.0g/L、硫酸锰0.01～1.0g/L。
9.根据权利要求3-6任一项所述的方法，其特征在于：所述发酵培养的温度为20℃-30℃；培养基pH为5.0-8.0；培养时间为24-240小时。
10.根据权利要求9所述的方法，其特征在于：所述发酵培养的温度为24℃-26℃。
11.根据权利要求9所述的方法，其特征在于：所述培养基pH为6.0-7.0。
12.根据权利要求9所述的方法，其特征在于：所述培养时间为72-192小时。
13.根据权利要求3-6任一项所述的方法，其特征在于：所述渥曼青霉1507-N13是通过种子液接种至所述营养培养基中进行所述发酵培养的；
其中，所述种子液是将权利要求1所述的渥曼青霉1507-N13在种子培养基里进行种子培养得到的；所述种子培养的条件为：种子培养的温度为20℃-30℃；培养基pH为5.0-8.0；培养时间为24-80小时。
14.根据权利要求13所述的方法，其特征在于：所述种子培养的温度为24℃-26℃。
15.根据权利要求13所述的方法，其特征在于：所述培养基pH为5.0-7.0。
16.根据权利要求13所述的方法，其特征在于：所述培养时间为24-60小时。
17.根据权利要求13所述的方法，其特征在于：所述的种子培养基含有蔗糖0～20.0g/L、葡萄糖10.0～50.0g/L、棉籽饼粉10.0～30.0g/L、酵母粉5.0～15.0g/L、磷酸二氢钾1.0～10.0g/L、硫酸铵0.5～1.5g/L、碳酸钙0.5～2.0g/L。
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| US5378725A (en) * | 1993-07-19 | 1995-01-03 | The Arizona Board Of Regents | Inhibition of phosphatidylinositol 3-kinase with wortmannin and analogs thereof |
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