CN106636259B - 一种通过固体发酵微生物Clonostachys rogersoniana产生抗生素TMC-154的方法 - Google Patents
一种通过固体发酵微生物Clonostachys rogersoniana产生抗生素TMC-154的方法 Download PDFInfo
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Abstract
本发明公开了一种通过微生物固体发酵产生抗生素TMC‑154的方法。即:微生物Clonostachys rogersoniana 828H2(CGMCC No.12073)在合适的培养条件下固体发酵,经过合适的溶剂超声提取,过滤,浓缩后,经硅胶和凝胶柱层析分离得到抗生素TMC‑154。结合TLC薄层层析法以及高效液相色谱法,可检测到大量的TMC‑154的产生。TMC‑154对人体白血病早幼粒细胞株HL‑60,人体肝癌细胞株SMMC‑7721,人体非小细胞肺癌细胞株A‑549,人体乳腺癌细胞株MCF‑7及人体结肠癌细胞株SW‑480都表现出明显的抑制活性,其半数抑制率(IC50值)分别为2.13μM,15.38μM,17.49μM,15.80μM和14.54μM,具有开发为抗癌药物的潜质。该方法提供了抗生素TMC‑154的一个新的生产方法。
Description
技术领域
本发明涉及微生物代谢产物分离分析领域,具体来说建立了一种通过固态发酵微生物Clonostachys rogersoniana产生抗生素TMC-154的方法。
本发明使用的微生物Clonostachys rogersoniana:[Clonostachys sp 828H2(保藏号:CGMCC No.12073)]保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏单位地址:北京市朝阳区北辰西路1号院3号;保藏时间:2016年1月15日。
背景技术
近年来,因微生物具有代谢周期短、转化反应条件温和、副产物少、立体选择性强等特点,通过微生物(真菌、细菌以及藻类)发酵来制备特定化合物的方法越来越受到人们的重视。在实际生产中,已经有大量的应用实例通过微生物发酵来生产一些新颖的、活性较高的以及对人体健康有益的药物。
TMC-154是聚酮苷类化合物,其结构特征在于具有一个独特的高度甲基化的erythromycins类型聚酮母核、一个阿拉伯醇侧链和一个甲基甘露糖取代。文献报道该类化合物对多种肿瘤细胞具有生长抑制活性,且对二酰甘油乙酰转移酶(DGAT)具有抑制活性。在体外细胞毒活性筛选实验中,TMC-154对人体白血病早幼粒细胞株HL-60,人体肝癌细胞株SMMC-7721,人体非小细胞肺癌细胞株A-549,人体乳腺癌细胞株MCF-7及人体结肠癌细胞株SW-480均表现出明显的抑制活性,其半数抑制率(IC50值)分别为2.13μM,15.38μM,17.49μM,15.80μM和14.54μM。而阳性对照顺铂对五株人类肿瘤细胞HL-60,A-549,SMMC-7721,MCF-7和SW-480的IC50值依次为1.72μM,6.82μM,12.19μM,17.40μM和16.35μM。TMC-154对人体乳腺癌细胞株MCF-7和人体结肠癌细胞株SW-480的生长抑制活性强于阳性对照顺铂。因此,TMC-154具有作为先导化合物开发抗肿瘤药物的研究价值。而基于微生物发酵的大量而简单的产生抗生素TMC-154的方法,不仅可以现代环境保护和低碳经济的需求,而且为后期工业化量产的进一步研究和开发打下了坚实的基础,具有显著的应用价值。
发明内容
本发明旨在通过Clonostachys rogersoniana固体发酵生产抗生素TMC-154。本发明提供了一种转化率高,设备要求低,简便易操作,绿色无污染,适合产业化生产TMC-154的方法。
本发明的目的是通过以下具体技术方案实现的:
(1)将拟用微生物C.rogersoniana 828H2(菌种保藏号CGMCC No.12073)首先进行活化,即将C.rogersoniana 828H2接种到经过120℃高温灭菌处理的PDA斜面培养基后,于恒温培养箱中培养3-7d后,置于4℃冰箱中备用;
(2)将活化的菌种接到PDB种子培养基中,于恒温摇床中培养3-7d;
(3)取洗净的土豆切成丁后,取适量置于组织培养瓶中,加入阿拉伯糖(以质量百分比计所述发酵培养基成分:25g马铃薯,0.5g阿拉伯糖);将装有培养基的组织培养瓶加盖包装后置于120℃高温灭菌箱中灭菌30min,取出后将组织培养瓶冷却;
(4)将步骤(2)的种子培养基在无菌环境下接种到经过步骤(3)前处理的培养基上,加盖后置于培养箱中28℃下培养30d后取出;
(5)经微生物C.rogersoniana 828H2发酵后的培养基经过甲醇超声提取、过滤和浓缩得到粗提物;分离粗提物得到的TMC-154的结构式如下:
(6)经TLC薄层层析法检测,然后经过硅胶柱进行分离,分离过程采用梯度洗脱,洗脱剂为氯仿:甲醇(10:1—3:1),然后经分离得到的化合物上凝胶柱(甲醇)纯化,通过1D/2DNMR以及HR-ESIMS鉴定得到TMC-154结构;
(7)采用如下HPLC色谱条件测定TMC-154的产量。
①色谱柱:ZORBAX SB-C18(4.6×250mm,5μm);
②梯度洗脱程序:0–10min,20–40%乙腈;10–20min,40-60%乙腈;20–45min,60%乙腈;
③检测波长:235nm;
④流速:1.0mL/min;
⑤柱温:20℃。
附图说明
图1为本发明中目标化合物的1H-NMR;
图2为本发明中目标化合物的13C-NMR;
图3为本发明中目标化合物的HSQC;
图4为本发明中目标化合物的HMBC;
图5为本发明中目标化合物的HR-ESI-MS。
具体实施方式
下面结合实施例进一步阐述本发明,但本发明不局限于该具体实施例,本领域技术人员应该认识到,本发明涵盖了权利要求范围内的所有可能的备选方案、改进方案和等效方案。
实施例1
(1)将拟用微生物Clonostachys rogersoniana 828H2首先进行活化,即将C.rogersoniana接种到经过120℃高温灭菌处理的PDA斜面培养基后,于恒温培养箱中培养3-7d后,置于4℃冰箱中备用;
(2)将活化的菌种接到PDB种子培养基中,于恒温摇床中培养3-7d;
(3)取洗净的土豆切成丁后,取适量置于组织培养瓶中,加入阿拉伯糖(以质量百分比计所述发酵培养基成分:25g马铃薯,0.5g阿拉伯糖);将装有培养基的组织培养瓶加盖包装后置于120℃高温灭菌箱中灭菌30min,取出后将组织培养瓶冷却;
(4)将步骤(2)的种子培养基在无菌环境下接种到经过步骤(3)前处理的培养基上,加盖后置于培养箱中28℃下培养30d后取出;
(5)经微生物C.rogersoniana 828H2发酵后的培养基经过甲醇超声提取、过滤和浓缩得到粗提物;分离粗提物得到的TMC-154的结构式如下:
(6)经TLC薄层层析法检测,然后经过硅胶柱进行分离,分离过程采用梯度洗脱,洗脱剂为氯仿:甲醇(10:1—3:1),然后经分离得到的化合物上凝胶柱(甲醇)纯化,通过1D/2DNMR以及HR-ESIMS鉴定得到TMC-154结构;
(7)采用如下HPLC色谱条件测定TMC-154的产量为3.78g/Kg。
①色谱柱:ZORBAX SB-C18(4.6×250mm,5μm);
②梯度洗脱程序:0–10min,20–40%乙腈;10–20min,40-60%乙腈;20–45min,60%乙腈;
③检测波长:235nm;
④流速:1.0mL/min;
⑤柱温:20℃。
该方法是一种高效的,具有创新性的,反应条件温和的,绿色无污染的,易扩大化生产的,操作设备简单的大量产生抗生素TMC-154的方法,不仅满足了现代环境保护和低碳经济的需求,而且为后期工业化量产的进一步研究和开发打下了坚实的基础。
实施例2
操作同实施例1,把发酵时间改为35d后,TMC-154的产量为3.76g/Kg。
实施例3
操作同实施例1,把发酵时间改为40d后,TMC-154的产量为3.69g/Kg。
Claims (7)
1.一种通过固体发酵微生物Clonostachys rogersoniana产生抗生素TMC-154的方法,其特征在于按如下步骤进行固体发酵:
(1)将拟用微生物Clonostachys rogersoniana 828H2首先进行活化,即将C.rogersoniana 828H2接种到经过120℃高温灭菌处理的PDA斜面培养基后,于恒温培养箱中培养3-7d后,置于4℃冰箱中备用;所述微生物C.rogersoniana 828H2的保藏编号为CGMCC No.12073;
(2)将活化的菌种接到PDB种子培养基中,恒温摇床中培养3-7d;
(3)取洗净的土豆切成丁后,取适量置于组织培养瓶中,加入阿拉伯糖,以质量百分比计,所述发酵培养基成分:25g马铃薯,0.5g阿拉伯糖;将装有培养基的组织培养瓶加盖包装后置于120℃高温灭菌箱中灭菌30min,取出后将组织培养瓶冷却;
(4)将步骤(2)的种子培养基在无菌环境下接种到经过步骤(3)前处理的培养基上,加盖后置于培养箱中28℃下培养30d后取出;
(5)经微生物C.rogersoniana 828H2发酵后的培养基经过甲醇超声提取、过滤和浓缩得到粗提物;分离粗提物得到的TMC-154的结构式如下:
(6)经TLC薄层层析法检测,然后经过硅胶柱进行分离,分离过程采用梯度洗脱,洗脱剂为氯仿:甲醇=10:1—3:1,然后经分离得到的化合物上凝胶柱/甲醇纯化,通过1D/2D NMR以及HR-ESIMS鉴定得到TMC-154结构;
(7)采用HPLC测定TMC-154的产量为3.78g/kg。
3.根据权利要求1所述通过固体发酵C.rogersoniana 828H2产生抗生素TMC-154的方法,其特征在于所述发酵方法为固态发酵。
4.根据权利要求1所述通过固体发酵C.rogersoniana 828H2产生抗生素TMC-154的方法,其特征在于以质量百分比计所述发酵培养基成分:25g马铃薯,0.5g阿拉伯糖。
5.根据权利要求1所述通过固体发酵C.rogersoniana 828H2产生抗生素TMC-154的方法,其特征在于所述发酵时间为30d。
6.根据权利要求1所述通过固体发酵C.rogersoniana 828H2产生抗生素TMC-154的方法,其特征在于所述发酵温度为28℃。
7.根据权利要求1所述通过固体发酵C.rogersoniana 828H2产生抗生素TMC-154的方法,其特征在于按如下HPLC条件进行含量测定,产量为3.78g/kg;
(1)色谱柱:ZORBAX SB-C18;4.6×250mm, 5μm;
(2)梯度洗脱程序:0–10min,20–40%乙腈;10–20min,40-60%乙腈;20–45min,60%乙腈;
(3)检测波长:235nm;
(4)流速:1.0mL/min;
(5)柱温:20℃。
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