CN104232498B - 一种纤维化纤维微细菌菌株及其应用 - Google Patents
一种纤维化纤维微细菌菌株及其应用 Download PDFInfo
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- CN104232498B CN104232498B CN201310245307.3A CN201310245307A CN104232498B CN 104232498 B CN104232498 B CN 104232498B CN 201310245307 A CN201310245307 A CN 201310245307A CN 104232498 B CN104232498 B CN 104232498B
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Abstract
一种纤维化纤维微细菌菌株及其应用,该菌株为纤维化纤维微细菌(Cellulosimicrobium cellulans)菌株F16,保藏号为CCTCC M2013201,保藏日期为2013年5月;该菌株F16或其发酵产生的水解酶,将含木糖苷键和葡糖苷键的化合物水解,得到的产物是相应的苷元,也可以是在制备相应苷元的过程中所产生的中间体化合物。
Description
技术领域
本发明属于生物化工领域,具体涉及一种纤维化纤维微细菌菌株及其应用。
背景技术
许多天然具有活性化合物往往以糖苷的形式存在于自然界中,将其糖基去除后,便可释放出有活性的化合物。如抗癌药物紫杉醇,其主要存在于红豆杉植物的树皮中。而在红豆杉的枝叶中则大量存在着木糖苷化的紫杉醇前体或其相同母核的类似物(称之为紫杉烷类化合物),如7-木糖紫杉醇和7-木糖-10去乙酰紫杉醇(10DAXT),其在如中华、云南及南方红豆杉的枝叶中的累积量可高出紫杉醇10倍以上,将7-位木糖基水解后再经一步乙酰化即可转化为紫杉醇。
另外一种典型的天然产物如来自名贵中草药人参的人参皂苷。人参皂苷是公认人参中的主要活性成分,其基本骨架主要是原人参二醇(PPD)和原人参三醇(PPT)。其中含量高的如Rb1、Rb2、Rb3、Rc、Rd、Re、Rg1等,都是基本骨架被高度“糖基化”了的化合物,其所含糖基从一个到五个不等,而具有很好抗肿瘤和抑菌活性的却是人参中一些含量很少的稀有人参皂苷如Rh1、Rh2、Rg3、Rg5、CK、PPT、PPD等,这些化合物往往含糖基较少,或直接是不带有任何糖基的苷元,是上述高含量人参皂苷去糖基化后的产物(J PharmPharmacol.1998Oct;50(10):1155-60.)。日本公开特许58-131999(JPO)58-131999公布了20(S)-原人参二醇、20(R)-原人参二醇、20(S)-原人参三醇、20(R)-原人参三醇都具有抑制肿瘤生长的活性,其中20(S)-原人参二醇(PPD)的活性最强。进一步的研究表明20(S)-PPD可以通过增强机体免疫和直接的细胞毒作用杀死肿瘤细胞,还可以抑制肿瘤间质血管生成,阻止肿瘤细胞的生长;在神经系统方面,20(S)-PPD可以抗癫痫、抗抑郁、增强学习能力等,是一个非常具有开发价值的化合物(Front Pharmacol.2012;3:25.ChineseMedicine2010,5:20)。
此外,还有很多天然化合物都存在类似情况,如黄芪甲苷,红链霉素糖苷,异槲皮苷,大豆皂苷,红景天苷,天麻苷,山慈菇苷A,薯蓣皂苷,亚麻木酚素,哈巴俄苷及哈巴苷等等。
由于人们往往对其苷元更感兴趣,但这类化合物一般骨架复杂,化学方法断裂糖苷键的选择性差,副产物多,环境污染等原因,制备相应苷元十分困难。相比之下生物转化方法由于具有高度的选择性和专一性,几乎没有副产物,反应条件温和,环境友好,成为制备此类化合物相应苷元的首选。目前已有很多关于酶法水解糖苷类化合物的报道,如韩国研究者公布了利用针尾曲霉水解红参中人参皂苷以提高其中人参皂苷苷元的含量(J.Korean Soc.Appl.Biol.Chem.53(5),553-558(2010)),Kohada报道以G-Rb1、Rb2、Rb3、Rc和Rd为原料,用醋酸水解脱去C-20位上的糖,再用粗橙皮苷酶等水解脱去3位上的糖,制得20(S)-PPD(JPO58-131999),中国专利CN03101549.2公开了利用乳酸菌或肠道细菌生物转化制备人参组合物的方法,中国专利CN102703329公开了一株能够高效转化黄姜中皂苷生产薯蓣皂苷元的菌株,Hanson RL(1997)利用细菌Moraxella sp.、Bacillus macerans、Bacillus circulans和Micrococcus sp.将C-7木糖紫杉烷转化为C-7羟基紫杉烷,中国专利CN102296053公开了一种来自真菌的β-木糖苷酶也具有水解C-7木糖紫杉烷的能力,并实现其在酵母中的胞内表达等,但往往存在使用菌体转化、产酶及转化效率低的情况,尤其对于一些水溶性差、空间位阻大的化合物,如10DAXT,普通糖苷酶往往难以水解。
发明内容
本发明的目的在于提供一种纤维化纤维微细菌菌株及其应用,该菌株可将含木糖苷键或葡糖苷键的化合物水解而获得相应的苷元。本发明使用一种纤维化纤维微细菌菌株或该菌株所产生的酶接触含有至少一种木糖苷键或葡糖苷键的黄芪皂苷类、人参皂苷类、紫杉烷木糖苷类、红链霉素糖苷、异槲皮苷、大豆皂苷类化合物,并水解相应的糖苷键。该方法可以制备至少一种上述糖苷化合物的苷元,也可以制备在转化上述苷元的过程中所产生的具有药理活性的中间产物。
本发明提供了一种纤维化纤维微细菌菌株,该菌株为纤维化纤维微细菌(Cellulosimicrobium cellulans)菌株F16,菌种保藏号为CCTCC M2013201,保藏日期为2013年5月14日,保藏单位为中国典型培养物保藏中心,保藏地址为中国武汉大学。
本发明还提供了所述纤维化纤维微细菌菌株的应用,该菌株应用于糖苷化合物苷元的制备。
本发明提供的所述纤维化纤维微细菌菌株的应用,该菌株或其发酵产生的水解酶,接触含糖苷键的糖苷化合物,将糖苷键水解,得到的产物是相应的苷元或中间体。
本发明提供的所述纤维化纤维微细菌菌株的应用,所述糖苷键为葡萄糖苷键和/或木糖苷键。
本发明提供的所述纤维化纤维微细菌菌株的应用,所述糖苷化合物结构如下:
本发明菌株制备的相应苷元结构是:
本发明提供的菌株的水解方法已考虑到具有上述分子结构的化合物的手性中心的所有立体构型,这些立体异构体或单独被水解,或与其它立体异构体混合在一起被水解。
本发明提供的菌株实用价值在于:环黄芪醇,原人参醇,紫杉烷类化合物,红链霉素,异槲皮素和大豆皂苷苷元,在药物制备领域有重要的应用价值。从植物中提取出的上述化合物通常是连接有大量糖基的糖苷化合物,且多为混合物,其中大部分糖苷化合物带有的是木糖基或葡糖基,而去糖基后的苷元才是最终想要的产品。一个或多个含有至少一种木糖苷键或葡糖苷键的黄芪甲苷、人参皂苷、紫杉烷木糖苷、红链霉素糖苷、异槲皮苷或大豆皂苷,在菌株F16或其发酵产生的水解酶的作用下被水解,得到相应苷元;所得化合物可以是具有药理活性的苷元,如环黄芪醇,原人参醇,紫杉烷类化合物,红链霉素,异槲皮素或大豆皂苷苷元,或其类似物,也可以是制备上述苷元的过程中所产生的未完全水解的中间体。
本发明提供的菌株使得被水解的黄芪甲苷、人参皂苷、紫杉烷木糖苷、红链霉素糖苷、异槲皮苷和大豆皂苷的立体构型优先保留于产品中,所得相应苷元或其水解中间产物的糖苷键的绝对立体构型与原糖苷化合物的糖苷键绝对立体构型相同。
本发明提供的菌株用于从带有木糖苷键或葡糖苷键的黄芪甲苷、人参皂苷、紫杉烷木糖苷、红链霉素糖苷、异槲皮苷或大豆皂苷制备相应的苷元或其水解中间产物时十分有效。用本发明提供的方法可以水解一种带有木糖苷键或葡糖苷键的上述糖苷化合物,也可以连续性地或同时水解不同上述糖苷化合物的混合物。由植物材料提取的人参皂苷混合物、紫杉烷木糖苷和大豆皂苷混合物用本发明提供的方法水解,效果很好。
具体实施方式
下面的实施例将对本发明予以进一步的说明,但并不因此而限制本发明。
起始材料:
本发明所采用的起始材料可以是任何带有木糖苷键或葡糖苷键的黄芪甲苷、人参皂苷、紫杉烷木糖苷、红链霉素糖苷、异槲皮苷或大豆皂苷类化合物。带有木糖苷键或葡糖苷键的上述糖苷化合物可以是自然形成的,也可以是化学半合成或全合成所得。
酶与微生物:
本发明水解方法中使用的酶或微生物可以是下面描述的任何能催化酶水解反应的酶或微生物。这些酶或微生物材料,不管其来源或纯度,都可在游离状态下使用,或者将其用物理吸附或诱捕方法固定在支持物上使用。生物学纯的纤维化纤维微细菌(Cellulosimicrobium cellulans)菌株F16是新发现的同时产β-木糖苷酶和β-葡萄糖苷酶的微生物。这种微生物的突变体,例如经化学的,物理的(如紫外辐射)或生物学方法(如分子生物学技术)改造以用于水解反应的突变菌株,也在本发明的考虑范围内。
纤维化纤维微细菌(Cellulosimicrobium cellulans)菌株F16分离自大连市甘井子区西山水库的土壤之中,其特征为:革兰氏阳性,可在好氧条件下生长,菌落呈圆形,直径0.9~2mm,黄白色突起,反光,边缘整齐。在显微镜下观察菌体为棒状,随着培养时间的延长逐渐转化成短杆,甚至球形。
本发明应用的酶是水解酶,尤其是葡萄糖苷酶和葡聚糖酶,以及木糖苷酶和木聚糖酶。本发明提供的菌株生产这些酶,可用提取和纯化的方法分离它们。本发明提供的微生物可以以完整湿细胞的形式,或者以冻干,喷雾干燥或加热干燥的形式,或者以经破碎抽提后的形式被应用于水解反应。
本发明提供的水解方法可以在微生物发酵后进行,也可以与发酵同时进行。
将菌株接种于含诱导剂的培养基中,好氧条件下发酵培养后,离心收集上清液,即为粗酶液。培养基pH在4-7之间,培养温度20-40℃之间。水解反应进行0.5-72小时,直至目的产品的产量达最大。水解反应时,pH保持在6-9,其中pH7-8效果最好。
产物的分离:
应用本发明提供的方法所产生的六类苷元或其未完全水解的中间产物,可被分离纯化,根据水解产物的特征,可用提取,蒸馏,结晶,柱层析或多种分离技术结合的方法进行纯化。
应用:
应用本发明提供的水解方法所获得的化合物如紫杉醇,是很好的抗癌药。7-木糖-10-去乙酰紫杉醇经本发明提供的方法水解后获得10-去乙酰紫杉醇,此化合物再经10位乙酰化,即可得到紫杉醇。
应用本发明提供的水解方法所获得的化合物如人参二醇,具有非常好的抗肿瘤活性,同时还可以抗癫痫、抗抑郁、增强学习能力等,是一个非常具有开发价值的化合物,近些年被各国研究者进行改造,希望能够将其开发为一种新的抗肿瘤药。
实施例1:粗酶液的制备
使用含1%淀粉,0.2%蛋白胨,0.2%酵母提取物,0.2%磷酸氢二钾的培养基(灭菌前pH7.0)作为种子培养基。
将上述种子培养基20ml分配到100ml锥形瓶中,在120度下灭菌15min,将纤维化纤维单胞菌F16菌株的琼脂斜面培养物接种到其中,并在30度下振荡培养2天作为种子液。
500ml Erlenmeyer培养瓶中放入100ml含1%桦木木聚糖,0.2%酵母浸膏,0.2%氯化铵,0.1%NaH2PO4和0.1%Na2HPO4的培养基,pH7,高压灭菌121度30min。将1ml种子液接种于此培养基,150rpm,30℃摇瓶培养5天。离心收集上清液,即为粗酶液。
实施例2:黄芪甲苷IV的水解粗酶液的制备
在0.9ml实施例1中的粗酶液中加入1ml50mM Tris-HCl缓冲液(pH7.5),再加入0.1ml黄芪甲苷IV的甲醇溶液(浓度为10mg/ml)。100rpm,35℃孵育8小时后,以环黄芪醇标准品作对照,TLC分析,黄芪甲苷IV几乎全部转化为环黄芪醇。
实施例3:人参皂苷Rb1水解制备化合物CK
在0.9ml实施例1中的粗酶液中加入1ml50mM Tris-HCl缓冲液(pH7.5),再加入0.1ml人参皂苷Rb1的甲醇溶液(浓度为10mg/ml)。100rpm,35℃孵育4小时。加入2ml甲醇终止反应,15000转/分离心20分钟取上清液进行UPLC分析。反应液中人参皂苷Rb1几乎完全转化,产生化合物K(即CK,产率75%),未完全水解的中间产物以及少量终产物PPD。
UPLC方法:
色谱柱:Kromasil ODS(2.1×150mm,3μm)
流动相:乙腈:水(0→30min:20:80→95:5梯度洗脱)
流速:0.4ml/min
柱温:室温
检测波长:203nm
实施例4:人参皂苷Rb1水解制备原人参二醇(PPD)
在0.9ml实施例1中的粗酶液中加入1ml50mM Tris-HCl缓冲液(pH7.5),再加入0.1ml人参皂苷Rb1的甲醇溶液(浓度为10mg/ml)。100rpm,35℃孵育12小时。加入2ml甲醇终止反应,15000转/分离心20分钟取上清液进行UPLC分析,方法同实施例3。反应液中人参皂苷Rb1几乎完全转化为终产物原人参二醇(PPD),产率90%。
实施例5:人参皂苷Re水解制备原人参三醇(PPT)
在0.9ml实施例1中的粗酶液中加入1ml50mM Tris-HCl缓冲液(pH7.5),再加入0.1ml人参皂苷Re的甲醇溶液(浓度为10mg/ml)。100rpm,35℃孵育8小时。加入2ml甲醇终止反应,15000转/分离心20分钟取上清液进行UPLC分析,方法同实施例3。反应液中人参皂苷Re几乎完全转化为终产物原人参三醇(PPT),产率93%。
实施例6:7-木糖紫杉醇水解制备紫杉醇
在0.9ml实施例1中的粗酶液中加入1ml50mM Tris-HCl缓冲液(pH7.5),再加入0.1ml7-木糖-紫杉醇的甲醇溶液(浓度为5mg/ml)。100rpm,30℃孵育5小时。加入2ml甲醇终止反应,15000转/分离心20分钟取上清液进行HPLC分析。反应液中无7-木糖-紫杉醇残留,并产生0.41mg紫杉醇(产率94%)。
HPLC方法:
色谱柱:Kromasil ODS(4.6×200mm,5μm)
流动相:甲醇:水(60:40)
流速:1ml/min
柱温:室温
检测波长:227nm
实施例7:7-木糖-10-去乙酰紫杉醇水解制备10-去乙酰紫杉醇
将10ml7-木糖-10-去乙酰紫杉醇的甲醇溶液(浓度为5mg/ml)加入到90ml实施例1所述的粗酶液中,100rpm,30℃下反应20小时后,加入20ml乙酸乙酯萃取,共萃取3次,合并上层有机相,减压蒸干,获得固体物质102mg,上硅胶柱(20g)并用氯仿:甲醇(98:2)进行洗脱,得到39mg10-去乙酰紫杉醇(总收率91%)。
实施例8:7-木糖紫杉烷混合物的水解
将200ml含有7-木糖-10-去乙酰紫杉醇,7-木糖-10-去乙酰三尖杉宁碱,7-木糖-10-去乙酰紫杉醇C的混合物(从天然红豆杉提取物中分离,含量分别为65%,9.9%,3.3%)的甲醇溶液(5mg/ml)加入到2000ml如前所述的粗酶液中,100rpm,35℃下反应8小时。反应液用乙酸乙酯萃取三次,每次200ml,合并有机相蒸干溶解于100ml甲醇中,用实施例6中HPLC方法进行定量检测,发现其中无三种带木糖基的底物残留,得到的产物中10-去乙酰紫杉醇,10-去乙酰三尖杉宁碱,10-去乙酰紫杉醇C的含量分别504mg,75mg,28mg,回收率达到90%。
实施例9:红链霉素龙胆二糖苷的水解
在0.9ml实施例1中的粗酶液中加入1ml50mM Tris-HCl缓冲液(pH7.5),再加入0.1ml红链霉素龙胆二糖苷的甲醇溶液(浓度为12mg/ml)。100rpm,35℃孵育4.5小时。加入2ml甲醇终止反应,15000转/分离心20分钟取上清液进行UPLC分析。反应液中无红链霉素龙胆二糖苷残留,并产生0.52mg红链霉素(产率95%)。
UPLC方法:
色谱柱:Kromasil ODS(2.1×150mm,3μm)
流动相:乙腈:水(0→10min:20:80→95:5梯度洗脱)
流速:0.4ml/min
柱温:室温
检测波长:278nm
实施例10:异槲皮苷的水解
在0.9ml实施例1中的粗酶液中加入1ml50mM Tris-HCl缓冲液(pH7.5),再加入0.1ml异槲皮苷的甲醇溶液(浓度为7mg/ml)。100rpm,35℃孵育4小时后,以异槲皮素标准品作对照,TLC分析,异槲皮苷几乎全部转化为异槲皮素。
实施例11:大豆皂苷的水解
在0.9ml实施例1中的粗酶液中加入1ml50mM Tris-HCl缓冲液(pH7.5),再加入0.1ml大豆皂苷的甲醇溶液(浓度为5mg/ml)。100rpm,35℃孵育12小时后,以大豆皂苷苷元标准品作对照,TLC分析,大豆皂苷几乎全部转化为其苷元。
Claims (5)
1.一种纤维化纤维微细菌菌株,其特征在于:该菌株为纤维化纤维微细菌(Cellulosimicrobium cellulans)菌株F16,保藏号为CCTCC M2013201,保藏日期为2013年5月。
2.权利要求1所述纤维化纤维微细菌菌株的应用,其特征在于:该菌株应用于糖苷化合物苷元的制备。
3.按照权利要求2所述纤维化纤维微细菌菌株的应用,其特征在于:该菌株或其发酵产生的水解酶,接触含糖苷键的糖苷化合物,将糖苷键水解,得到的产物是相应的苷元或中间体。
4.按照权利要求3所述纤维化纤维微细菌菌株的应用,其特征在于:所述糖苷键为葡萄糖苷键和/或木糖苷键。
5.按照权利要求2或3所述纤维化纤维微细菌菌株的应用,其特征在于:所述糖苷化合物结构如下:
。
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