CN1108694A - 一种淡紫灰叶链霉菌dkrs菌株及用它制备阿克拉希霉素a，b，y和糖苷配基的方法 - Google Patents
一种淡紫灰叶链霉菌dkrs菌株及用它制备阿克拉希霉素a，b，y和糖苷配基的方法 Download PDFInfo
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- CN1108694A CN1108694A CN 94105525 CN94105525D CN1108694A CN 1108694 A CN1108694 A CN 1108694A CN 94105525 CN94105525 CN 94105525 CN 94105525 D CN94105525 D CN 94105525D CN 1108694 A CN1108694 A CN 1108694A
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- aclacinomycin
- streptomycete
- dkrs
- aglycone
- pale purple
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Abstract
本文公开了链霉菌的一种新菌株和一种通过培养它制造阿克拉希霉素A，B，Y和其糖苷配基的方法。
本发明的淡紫灰叶链霉菌DKRS(KCTC8539P)与仅能生产阿克拉希霉素A，B的亲菌株12/3A不同，能够以高产率产生阿克拉希霉素A，B，Y和糖苷配基。
此外，通过分别用乙酸缓冲液或盐酸将淡紫灰叶链霉素DKRS的已培养过的培养基之pH中调节到4、4或4、6而选择性地制造阿克拉希霉素A或Y是可能的。
Description
本发明涉及链霉菌属的新菌株及通过培养该菌株制备抗癌阿克拉希霉素(aclacinornycin)A，B，Y和糖苷配基的方法。
阿克拉希霉素是含有吡咯基的蒽环系抗癌药并由三个脱氧吡喃糖残基构成。它通过被插入DNA的碱基对中以抑制核酸的合成而表现出细胞毒素活性。特别是不同于其他蒽环系药如道诺红菌素(daunorubicin)，亚德里亚霉素(doxorubicin)和洋红霉素(carmi-nornycin)，阿克拉希霉素选择性地抑制RNA的合成。此外，它具有抵抗急性白血病和恶性淋巴瘤的活性，同时它对心脏毒性是低的。
阿克拉希霉素可分成三类：阿克拉希霉素A，B和Y。它们基本上具有糖苷配基，称作阿克拉菌素(aklavine)。它们当中，阿克拉希霉素B在临床中很少采用，这是因为它显示出很强的副作用和表现出较低的抗肿癌活性。阿克拉希霉素A因其高抗癌或肿瘤活性和低副作用及高产率而已经在实践上采用了。另外，预想到阿克拉希霉素Y是最有用的抗癌药，但是，与阿克拉希霉素A或B相比，它显示出非带高的抗肿瘤活性和低的副作用。
通过加利利链霉菌(Streptomyces galilaeus)的发酵制备阿克拉希霉素的方法是已知的(授权于Hamao Umezawa的US专利No.3,988,315)。这篇专利报道了加利利链霉菌制备包括阿克拉希霉素A，B和Y的约18种类似物。但是产率是非常低的：阿克拉希霉素A(主要产物)制得的量为46毫克/升培养基，阿克拉希霉素R制得的量为23毫克/升，而其它产物(包括阿克拉希霉素Y)制得的量是极低。因此，使用上述链霉菌株很难以工业规模制造阿克拉希霉素，从而需要提供能够以高产率制造阿克拉希霉素的新菌株。
本发明人已作过广泛的研究，以提供一种通过发酵以工业规模制造阿克拉希霉素的方法。结果是提供了淡紫灰叶链霉菌12/3A，它能够制造阿克拉希霉素A和B。本发明人基于提供改进了的菌株的目的，作了进一步研究，该菌株能够产生较大量的阿克拉希霉素，而这一目的可通过从菌株12/3A衍生的突变体淡紫灰叶链霉菌DKRS来实现。
因而，本发明的目的是提供淡紫灰叶链霉菌DKRS的新菌株，它能够产生大量的阿克拉希霉素A，B，Y和糖苷配基。
本发明另一个目的是提供一种通过培养淡紫灰叶链霉菌DKRS的菌株而制造阿克拉希霉素的方法并从培养基和细胞中回收阿克拉希霉素的方法。
本发明还有一个目的是提供一种通过在培养淡紫灰叶链霉菌DKRS的菌株之后用乙酸缓冲液或盐酸将培养基的pH值调到某数值来选择性制造阿克拉希霉素A或Y的方法。
根据本发明，淡紫灰叶链霉菌12/3A(淡紫灰叶链霉菌DKRS的亲株)在培养基中可制得60毫克/升的阿克霉素A和10毫克/升的阿克拉希霉素B。
本发明的淡紫灰叶链霉菌DKRS可通过用化学诱变剂N-亚硝基甲基缩二脲(NMB)让亲株淡紫灰叶链霉菌12/3A变异而获得。该突变体能够产生阿克拉希霉素A，B，Y和糖苷配基，特别能够产生大量的阿克拉希霉素Y。
通过以下方法选择本发明的突变体，在完全固定体培养基中于30℃下培养淡紫灰叶链霉菌12/3A达6-7天之后，加入杀菌后的蒸馏水，通过玻璃棉过滤器过滤收集孢子，用0.05M三苹果酸缓冲液(pH6.5)洗涤三次并用同样的缓冲液稀释到106-108孢子/毫升的数目。在向孢子悬浮液添加NMB至最终浓度为500μg/ml之后，让该混合物在30℃下存放20分钟。反应完成之后，通过离心或过滤分离孢子，用无菌生理盐水洗涤3次，然后成条状植在完全琼脂介质中。在30℃下培养7-10天之后，在琼脂盘上出现突变体的菌落并分离之。
本发明的突变体淡紫灰叶链霉菌DKRS已经在1993年8月26日由韩国标准培养物收藏中心(韩国大田市)收藏，并收到入藏登记号KCTC 8539P。在1993年8月26日转送布达佩斯条约(Bu-dapest Treaty)收藏，并收到入藏登记号KCTC 0092BP。
本发明所采用的完全培养基具有以下组成：葡萄糖10.0％，酪素水解物0.2％，肉汁萃取物0.1％，酵母萃提物0.1％和琼脂1.5％，pH7.0-7.2。
根据本发明的菌株淡紫灰叶链霉菌DKRS(KCTC 8539P)具有以下微生物学特性：
1.形态学的特性
在显微镜下观察在固体培养基中培养的菌株时，基质菌丝没有分节，孢子呈椭球形并适当地形成不太长的链形。这些孢子测得大小为2.3μm×1.4μm并形成粉孢子，它们的表面是光滑的。
2.培养学的特性
当在28℃下、在以下各种固体培养基中培养时，本发明的菌株具有以下特性：
(1)在Waksman培养基中培养，菌落带有绿色生长物；出现粉红色-褐色可溶性色素。
(2)在Gaus-I培养基中培养，菌落生成粉红色-白色生长物，基质菌丝带有红色-褐色；没有可溶性色素。
(3)在燕麦粉培养基中培养，菌落生成灰色生长物；基质菌丝带有红色-褐色；没有可溶性色素。
(4)在玉米粉培养基中培养，菌落带有暗褐色生长物；暗褐色基质菌丝。
(5)在GanS-II培养基中培养，菌落带有绿褐色生长物；暗褐色基质菌丝；生成暗褐色可溶性色素。
(6)在大豆粉培养基中培养，菌落带有淡绿色生长物；褐绿色基质菌丝；生成红褐色可溶性色素。
(7)在RieStrick培养基中培养，菌落带有褐色生长物，生成暗褐色可溶性色素。
(8)在Czapek-Dock培养基中培养，菌落是带有白色-粉红色生长物；红褐色基质菌丝；没有可溶性色素。
(9)在Bennet培养基中培养，菌落带有浅砂色生长物；没有可溶性色素。
(10)在肉胨琼脂培养基中培养，菌落带有浅砂色生长物；生成褐色可溶性色素。
(11)在葡萄糖-天门冬酰胺培养基中培养，菌落带有绿色生长物；没有可溶性色素。
3.生理特性：
本发明的菌株是一种需氧菌，在28℃显示出最佳的生长能力。它在含有玉米粉的培养基中有红桔色生长物，显示了胨化、淀粉水解和凝胶液化能力。作为碳源用麦芽糖和乳糖时菌株生长快；用鼠李糖、木糖、阿拉伯糖和半乳糖等时生长缓慢；而用蔗糖、甘露醇和山梨糖醇时没有生长。
在亲株(12/3A)和本发明菌株淡紫灰叶链霉菌DKRS(KCTC8539P)之间的形态学特性和生理特性方面的差异示于表1。
表1
| 淡紫灰叶链霉菌 | ||
| DKRS(J本发明菌株) | 12/3A(亲菌株) | |
| I.形态学特性 | ||
| 孢子囊柄 | 垂直于基础菌丝，并且是单纯的螺旋形状大小88-32μm | 垂直于基础菌丝，并且是单纯的螺旋形状大小：10-12μm |
| 孢子 | 矩形的椭球形大小2.3×1.4μm | 接近椭球形大小0.7×1.4μm |
| 孢子化的程度 | +++ | |
| 核的形态 | 圆型或接近椭球形 | 球形或接近椭球形 |
| 表面 | 光滑 | 光滑 |
| 粉孢子 | 基质菌丝和气生菌丝体的粉孢子发展快，且尺寸易变化 | 没有 |
| 核的形态 | 非常大而且填充在整个细胞中 | |
| II.生理学特性 | ||
| 胨化淀粉水解凝胶液化糖类利用麦芽糖乳糖鼠李糖葡萄糖果糖木糖阿拉伯糖丰乳糖蔗糖肌醇甘露糖醇山梨糖醇 | +++++++++++ND+++-ND-- | +++NDND++++++++++++++++-+++-ND |
a)+++：快速生长 +：一般生长 -：没有生长 ND：没有检测
使用本发明的菌株制造阿克拉希霉素的方法包括在含有碳源、氮源、无机物和其它营养物的培养基中培养淡紫灰叶链霉菌DKRS的步骤和从培养基和细胞中回收阿克拉希霉素的步骤，而该培养基通常用于链霉菌株的发酵以在培养基和细胞中聚集阿克拉希霉素。
在本发明的培养中所使用的主要碳源可包括但不限于淀粉和大豆粉。在本发明的培养过程中所使用的氮源可包括但不限于大豆粉和硫酸铵。根据本发明在发酵中使用的无机组分可包括但不限于碳酸钙、硫酸镁、硫酸锌和氯化钠等。
发酵可在28-30℃和pH6.8-7.5下，在需氧条件下进行4-5天。可用有机或无机碱性物质，氨水和碳酸钙等在发酵过程中调节pH值。
由菌株产生的产物，主要贮藏于细胞中，一部分分泌于细胞外，贮藏于培养基中。通过使用常规分离低分子物质的方法从培养基或细胞中分离出阿克拉希霉素、例如，使用有机溶剂如丙酮或氯仿或甲苯和异丙醇萃取和使用硅酸的柱色谱法。优选使用甲苯和异丙醇(30∶1-35∶1，按体积)的混合溶剂来分离或提纯阿克拉希霉素和糖苷配基。
在本发明中，在细胞内部聚集的阿克拉希霉素可通过使用甲醇和氯仿(20∶1)混合物来萃取并可用HPLC来分析。对于HPLC，硅石和氯仿∶甲醇∶乙酸∶水∶三乙胺(68∶20∶10∶2∶0.01(V/V))混合物可分别用来作静止和移动相。洗脱液以1.0毫升/分钟的流速通过色谱柱，收集的各流分测定它们在432nm处的吸光度。通过对比标准试样的吸收停留时间和细胞内部的那些试样的吸收停留时间，可以计算出细胞内阿克拉希霉素A，B，Y和糖苷配基的浓度。
根据本发明，发酵后，通过改变分离条件和提纯步骤可将阿克拉希霉素A转变为阿克拉希霉素Y，反之亦然。这种转变不仅取决于分离和提纯条件，而且与细胞的酶活性有关。如果用乙酸缓冲液将培养基调节到pH4.4，那么在培养基中和细胞内部所含的绝大部分阿克拉希霉素A被转变成Y。另一方面，如果用盐酸将培养基调节到pH4.6，那么阿克拉希霉素Y被转变成A。因此，通过调节培养基的pH而选择性地从培养基中得到阿克拉希霉素A或Y是可能的。
已经被本发明人证实，高纯度分离出的阿克拉希霉素Y比阿克拉希霉素A或B表现出更高的抵抗结肠癌的抗癌活性，即使在较高的剂量时，毒性也很低。
通过借助以下实施例将更详细说明本发明。以下实施例仅是说明性的，应该明白，本发明并不限制于这些实施例。
实施例1
用一铂环的淡紫灰叶链霉菌DKRS(KCTC 8539P)给斜面培养基(Note2)接种。在28℃下培养5天。这样获得的培养物被接种到500ml三角烧瓶内的50ml种子培养基(Note3)中，该培养基预先已调节到pH7.8并在121℃下消毒15分钟。消毒后，在回转式震荡培养机中在250转/分钟下培养2天。生产培养基(Note4)被调节到pH7.5，并用上述种子培养物接种至浓度为10％。在通气量2.0vvm，搅拌速度500转/分，温度28℃的条件下发酵4天。
在本发明中所使用的培养基具有以下组成：
Note2：斜面培养基：淀粉2％，K2HPO40.05％，MgSO40.05％，KNO30.1％，盐0.05％，Fe2(SO4)30.001％，琼脂1.5-2％(pH7.2)
Note3：种子培养基；葡萄糖2％，淀粉1.5％，大豆粉1％，酵母萃提物1％，CaCO30.3％，MgSO40.2％，NaCl0.3％(pH7.8)
Note4：生产培养基；葡萄糖3％，淀粉3.5％，大豆粉1％，Ca-CO30.5％，MgSO40.2％，FeSO40.001％，ZnSO40.001％(pH7.5)
通过按照与以上所述的同样步骤培养本发明的亲株和突变体，并测定在每一种培养基中产生的阿克拉希霉素A，B及Y的量。结果示于表2
表2
| 菌株 | 阿克拉希霉素的量(mg/l) | ||
| A | B | Y | |
| 亲菌株RS本发明菌株DKRS | 6060 | 1070 | -90 |
从表2可看出，与亲菌株RS相比较，本发明的菌DKRS产生了大量的阿克拉希霉素Y并显示阿克拉希霉素B的产率提高了，因而可以认为本发明的菌株DKRS是一种突变体，其中作为二次代谢产物的阿克拉希霉素的部分合成途径改变了。
实施例2
在本实施例中，从淡紫灰叶链霉菌DKRS的培养基中分离出阿克拉希霉素。取实施例1中得到的培养基700毫升用盐酸调节至pH4.5，并经过过滤得到菌丝体。将氯仿加入到该菌丝体中至浓度为2ml/克菌丝体，然后该混合物充分混合1小时并过滤。这一操作步骤重复两次。
收集氯仿滤液(120毫升)并通过真空蒸发进行浓缩。向该浓缩物添加15毫升乙酸丁酯和丙酮的混合溶剂(4∶1)并完全溶解。用乙酸缓冲液(pH3.4)处理所得溶液以萃取产物，然后再用氯仿再萃取。在氯仿蒸发和萃提物浓缩之后，加入正己烷或石油醚，以沉淀出阿克拉希霉素A(27mg)，B(35mg)和Y(41mg)。
实施例3
取实施例1中得到的培养基700ml用盐酸调节到pH4.6，并在28℃下充分混合3小时。然后重复实施例2中的操作步骤得到阿克拉希霉素A(50mg)，B(20mg)和Y(13mg)。
实施例4
取实施例1中得到的培养基(700ml)用乙酸缓冲液调节pH到4.4，并在28℃下充分混合2小时，然后重复实施例2的步骤，得到阿克拉希霉素A(3mg)，B(40mg)和Y(50mg)。
实施例5
将具有以下组成(Note5)的发酵培养基(5升)置于10升发酵室中并被调节到pH7.5。将在实施例中得到的种子培养物接种到浓度为10％，然后在搅拌(350转/分)和通气(1.0vvm)条件下在28℃进行发酵4天。
Note5：发酵培养基：葡萄糖3％，淀粉5.5％，大豆粉1.5％，CaCO30.9％，MgSO40.25％，ZnSO40.002％，FeSO40.002％。
这样得到的培养基(3.5升)含有阿克拉希霉素A(245mg)，B(315mg)和Y(560mg)。将该培养基用盐酸调至pH4.6，离心后得到菌丝体。向该菌丝体添加1.5升氯仿并充分混合1.5小时。重复该步骤两次。
另一方面，将600毫升氯仿添加到滤液中萃取阿克拉希霉素。
这样得到的氯仿萃取物合并到一起，并在其空下浓缩。将沉淀物溶于乙酸丁酯和丙酮(4∶1按体积)的混合溶剂中。得到的溶液与硫酸钠混合，然后过滤该混合物并浓缩。最后，添加正己烷得到沉淀物，然后蒸发后得到1.5g混合阿克拉希霉素。
实施例6
将实施例5中得到的混合阿克拉希霉素(1.5g)溶于最低量的甲苯和氯仿(8∶2按体积)混合溶剂中，并在填充有硅酸的柱上进行色谱分离。甲苯用来作为洗脱液。在含有糖苷配基的流分被洗脱之后，洗脱液转换成甲苯和异丙醇(35∶1按体积)混合溶剂，得到含有阿克拉希霉素B的流分。然后采用甲苯和异丙醇(30∶1按体积)混合溶剂作为洗脱液，得到含有阿克拉希霉素Y的流分。
向含有每一种产物的流分中添加正己烷或石油醚以沉淀产物，然后将沉淀物干燥得到糖苷配基(10mg)，阿克拉希霉素B(154mg)，Y(182mg)和A(75mg)，它们全部呈黄色粉末状。
Claims (7)
1.一种淡紫灰叶链霉菌DKRS(KCTC 8539P)，其具有产生阿克拉希霉素A，B，Y和其糖苷配基的能力。
2.权利要求1所述的淡紫灰叶链霉菌DKRS(KCTC8539P)，其特征在于当用乙酸缓冲液调节培养基的pH值为4.4时，它能够将阿克拉希霉素A转变为Y，当用盐酸调节培养基的pH值为4.6时，它能将阿克拉希霉毒Y转变为A。
3.一种由发酵制造阿克拉希霉素A，B，Y和其糖苷配基的方法，其特征在于：在培养基中培养淡紫灰叶链霉菌DKRS(KCTC8539P)以在细菌内和培养基中聚集阿克拉希霉素A、B、Y和其糖苷配基后，从培养基中和细菌内回收并提纯。
4.权利要求3的方法，其特征在培养是在需氧培养条件下在28-30℃和pH6.8-7.5下进行的。
5.权利要求3的方法，其特征在于使用甲苯和异丙醇的30∶1～35∶1体积比的混合溶剂，回收且提纯阿克拉希霉素A、B、Y及糖苷配基。
6.阿克拉希霉素Y的制造方法，其特征在于在培养基中培养淡紫灰叶链霉菌DKRS(KCTC 8539P)，将培养液用乙酸缓冲溶液调整至pH4.4，由细菌和培养液中分离阿克拉希霉素Y。
7.阿克拉希霉素A的制造方法，其特征在于在培养基中培养淡紫灰叶链霉菌DKRS(KCTC 8539P)，用盐酸调整培养液至pH4.6后，由细菌和培养液中分离阿克拉希霉素A。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019930017589A KR970000591B1 (ko) | 1993-09-03 | 1993-09-03 | 신균주 스트렙토마이세스 라벤도폴리아 DKRS 및 이를 이용한 아클라시노마이신 A, B, Y 및 아글리콘(Aglycone)의 제조방법 |
| KR17589/93 | 1993-09-03 |
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| CN94105525A Pending CN1104682A (zh) | 1993-09-03 | 1994-05-17 | 一种淡紫灰叶链霉菌dkrs菌株及用它制备阿克拉希霉素a，b，y和糖苷配基的方法 |
| CN 94105525 Pending CN1108694A (zh) | 1993-09-03 | 1994-05-17 | 一种淡紫灰叶链霉菌dkrs菌株及用它制备阿克拉希霉素a，b，y和糖苷配基的方法 |
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| JP (1) | JP2718002B2 (zh) |
| KR (1) | KR970000591B1 (zh) |
| CN (2) | CN1104682A (zh) |
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| US4071411A (en) * | 1974-07-27 | 1978-01-31 | Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai | Antitumor antibiotics aclacinomycins A and B [RD-9187A] |
| JPS5134915B2 (zh) * | 1974-07-27 | 1976-09-29 | ||
| US4204038A (en) * | 1976-04-07 | 1980-05-20 | Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai | Process for preparing antibiotics MA 144-M1 and MA 144-M2 |
| CH637160A5 (fr) * | 1976-10-05 | 1983-07-15 | Microbial Chem Res Found | Procede de fabrication de glucosides anthracyclines. |
| US4209588A (en) * | 1976-10-05 | 1980-06-24 | Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai | Process for producing new antitumor anthracycline antibiotics |
| JPS5648893A (en) * | 1979-09-07 | 1981-05-02 | Sanraku Inc | Preparation of anthracycline glycoside |
| EP0050725B1 (en) * | 1980-10-27 | 1984-07-04 | F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft | Process for aclacinomycins and microorganism used therein |
| EP0050724B1 (en) * | 1980-10-27 | 1985-01-09 | F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft | Process for anthracycline glycosides |
| US4405713A (en) * | 1981-12-28 | 1983-09-20 | Hoffmann-La Roche Inc. | Process for the preparation of optically active anthracycline glycosides A and B |
| US4471052A (en) * | 1982-01-18 | 1984-09-11 | Adria Laboratories, Inc. | Biosynthesis of simplified anthracyclines |
| SU1069433A1 (ru) * | 1982-06-29 | 1984-11-23 | Всесоюзный научно-исследовательский институт антибиотиков | Штамм @ @ 12/3а-продуцент аклациномицинов @ и @ |
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| JP2718002B2 (ja) | 1998-02-25 |
| US5484712A (en) | 1996-01-16 |
| IT1273049B (it) | 1997-07-01 |
| ES2078875B1 (es) | 1996-08-16 |
| DE4401546C2 (de) | 1997-09-25 |
| CH688151A5 (fr) | 1997-05-30 |
| FR2709497B1 (fr) | 1996-11-15 |
| GB9400513D0 (en) | 1994-03-09 |
| ITMI940035A0 (it) | 1994-01-14 |
| CN1104682A (zh) | 1995-07-05 |
| KR970000591B1 (ko) | 1997-01-14 |
| GB2281563B (en) | 1998-01-28 |
| NL9400096A (nl) | 1995-04-03 |
| ITMI940035A1 (it) | 1995-07-14 |
| TW258754B (zh) | 1995-10-01 |
| JPH0775575A (ja) | 1995-03-20 |
| GB2281563A (en) | 1995-03-08 |
| DE4401546A1 (de) | 1995-03-30 |
| CA2113871C (en) | 2000-01-18 |
| ES2078875A1 (es) | 1995-12-16 |
| US5468637A (en) | 1995-11-21 |
| FR2709497A1 (fr) | 1995-03-10 |
| KR950008679A (ko) | 1995-04-19 |
| CA2113871A1 (en) | 1995-03-04 |
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