海洋链霉菌Streptomyces sp.SCSIO 1666活性代谢产物替达霉素A和B的发酵优化及分离鉴定

目的对南海海洋沉积物中分离的海洋放线菌进行体外抗肿瘤细胞和抗菌模型筛选,选取生物活性强的海洋链霉菌菌株Streptomyces sp.SCSIO1666进行发酵条件优化和活性产物的分离及结构鉴定。方法用针对6种NCI肿瘤细胞株A549、DU145、H1299、HCT15、HEP3B和SF268的体外抗肿瘤模型及其抑菌模型对提取物进行筛选,进一步优化发酵条件,采用HPLC-UV和枯草芽孢杆菌(Bacillussubtilis)抑菌活性追踪,利用有机溶剂萃取、正相硅胶和反相硅胶等各种色谱层析分离,通过理化性质和波谱数据分析进行结构鉴定。结果与结论从一株海洋链霉菌菌株Streptomyces sp.SCSIO 1666发酵产物中分离得到替达霉素A和B(TirandamycinsA and B),替达霉素A和B的生产对海盐的依赖程度较高,在不加盐的条件下产量极低,加3%粗海盐时,其发酵产量提高250倍以上,说明盐胁迫是海洋链霉菌菌株SCSIO1666产生活性物质的重要条件。替达霉素B是终产物,加入2%大孔吸附树脂发酵时,替达霉素A的产量得到明显提高。     

海洋来源微生物的分离培养与抗肿瘤活性筛选

目的从海洋环境样品中分离纯化微生物,经发酵培养与活性筛选,获取抗肿瘤活性菌株以供筛选药源活性产物,获无活性菌株以供核糖体工程转化研究。方法通过单菌落挑选与划线培养分离纯化微生物菌株,经摇床发酵和提取操作制备活性测试样品。采用MTT法结合显微镜下细胞形态学检测的方法,测试样品的抗肿瘤活性。结果从渤海湾驴驹河潮间带海泥样品中分离得到了微生物127株,其中真菌80株、放线菌47株。在127株中100mg·L-1样品浓度下对K562细胞的抑制率大于40%的活性菌株为8株,占菌株总数的6.3%(其中放线菌4株,占放线菌数的8.5%,真菌4株,占真菌数的5%),抑制率在20%～40%的放线菌6株,占放线菌数的12.7%。结论从渤海湾海泥样品中分离得到真菌80株、放线菌47株,从中获得抗肿瘤活性真菌4株、放线菌10株,从放线菌中获得抗肿瘤活性菌株的频率远远高于真菌。活性菌株为寻找药源活性产物提供了菌株,无活性菌株则为核糖体工程拓展药源菌株来源研究提供了资源。     

红树林放线菌筛选及其抗肿瘤活性测定

目的 从我国湛江红树林采集的海泥样品中分离纯化微生物菌株并进行筛选及抗肿瘤活性测定.方法 采用形态学方法鉴定放线菌菌株;采用四氮唑盐(MTT)法测定筛选出的72株放线菌发酵液对肺癌细胞A549与人类慢性髓性白血病细胞K562两种肿瘤细胞的细胞毒活性.结果 经鉴定分离得到了72株放线菌,其中18%的放线菌发酵液具有不同程度的细胞毒活性.N2010-37和N2010-68两株放线菌发酵液对上述两种肿瘤细胞株作用较显著.结论 该研究结果为从湛江红树林放线菌中寻找抗肿瘤活性成分奠定了基础.     

土壤放线菌3423代谢产物的分离纯化及结构鉴定

目的 对土壤来源的Streptomycessp．3423代谢产物中的抗肿瘤活性成分进行分离纯化和结构鉴定。方法 采用MTT法，以对K562细胞的抑制活性为指标，筛选活性菌株。发酵培养液经各种色谱技术分离纯化，并通过对其理化性质、质谱、紫外、红外和核磁共振等图谱数据分析，确定化合物的结构。结果和结论 Streptomycessp．3423代谢产物中分离得到6个哌嗪二酮类化合物。生物学活性研究发现化合物Ⅲ和Ⅳ对K562细胞具有抑制活性，其中化合物Ⅳ活性最强，IC50为14．1μg／ml。     

海洋放线菌分离及菌株Streptomyces sp. AH17-3的次级代谢产物研究

目的对海洋放线菌进行分离及抗肿瘤活性筛选,并对一株具有抗肿瘤活性的海洋放线菌AH17-3的次级代谢产物进行研究。方法采用溶剂萃取、柱色谱层析及制备HPLC等方法对菌株AH17-3的发酵产物进行化学分离,通过理化性质及波谱学方法并参阅文献进行化合物结构鉴定,以SRB法评价化合物的抗肿瘤活性。结果从海洋样品中分离放线菌174株,从菌株AH17-3中分离得到了4个聚酮类化合物,经鉴定其结构分别为germicidin A(1)、germicidin B(2)、daidzein(3)、genistein(4)。其中化合物1具有弱的细胞毒活性,其IC50为3.5×10-7 M。结论海洋放线菌是重要的药用微生物资源,化合物1,2均为首次从海洋放线菌中分离得到。     

海洋真菌的分离、抗肿瘤活性筛选与发酵条件研究

目的 对青岛海域海水、海泥样品进行真菌选择性分离培养，并对其发酵物进行抗肿瘤活性筛选及活性菌株发酵条件的研究。方法 采用温敏型小鼠乳腺癌tsFT210细胞系，运用流式细胞术结合显微镜镜检，以细胞周期抑制和细胞凋亡诱导为活性筛选指标．并通过该活性指标对培养基、发酵条件及提取分离条件进行考查。结果 添加青霉素、链霉素构成的土豆培养基具有很好的培养真菌选择性；共分离得到真菌207株，活性筛选得到阳性菌19株，其中编号Z8 3200的真菌具有显著的细胞凋亡活性。     

海藻真菌的分离及其抗肿瘤活性筛

目的从海藻中分离海藻真菌并通过抗肿瘤活性筛选获得活性菌株。方法从样品中选择性分离得到真菌,并采用海虾生物致死法和人体慢性髓性白血病细胞K562为筛选模型对所得真菌的发酵产物进行抗肿瘤活性筛选。结果从黄海海岸潮间带采集到的28种海藻中分得221株真菌,经筛选得到具有海虾生物致死及细胞毒活性菌株各8株。结论实验结果表明,海藻真菌是寻找抗肿瘤活性物质的重要资源。     

深海来源的微生物抗肿瘤活性筛选及次级代谢产物的初步研究

目的 对来自深海的海水、海泥样品进行了微生物分离并通过抗肿瘤活性筛选获得活性菌株,并研究活性菌株c2b的次级代谢产物.方法 从样品中选择性分离得到真菌,并采用海虾生物致死法和人体慢性艇性白血病细胞(K562)为筛选模型对分离得到真菌的发酵产物进行抗肿瘤活性筛选;采用溶剂萃取、硅胶柱色谱及制备HPLC等分离手段对c2b菌株发酵产物的活性部位进行了活性追踪分离,通过理化性质及渡谱学手段进行化学结构鉴定,以SRB法评价了化合物的抗肿瘤活性.结果与结论 从深海来源的样品中共分离获得29株真菌,其中7株具有细胞毒活性;从c2b活性菌株的发酵产物中分离得到6个单体化合物(1～6),其化学结构分别鉴定为N-乙酰色氨(1),chrysogine(2),过氧化麦角甾醇(3),5,8-epidioxy-24-methylcholesta-6,22-dien-3β-ol(4),cerevisterol(5)和(4E,8E)-N-[(2'R,3'E)-2'-hydroxy-3'-hexadecenoyl]-1-O-β-D-glycopyranosyl-9-methyl-4,8-sphingadiene(6),其中化合物3,4对小鼠乳腺癌细胞(tsFT210)具有中等强度的细胞毒活性.     

海洋放线菌株H74-18抗菌活性及其发酵条件优化

目的 从海洋放线菌中筛选出对白色念珠菌有抗菌活性的菌株,预期获得新结构活性物。通过优化活性菌株的发酵条件,以期提高活性菌株产生活性物产量。方法 本研究以白色念珠菌作为靶标,采用抑菌圈法,从实验室保存的2632个放线菌株中筛选出对白色念珠菌有抗菌活性的菌株,并优化菌株的发酵条件。结果 筛选出对白色念珠菌有较强抑制作用的海洋放线菌H74-18菌株,明确了它对作物疫病菌和炭疽病菌有抑制作用活性,其优化发酵条件为：培养基为摇瓶培养基,培养温度为28℃,接种龄为48h,接种量为10％,摇床转速为300 r/min,发酵时间为72h。结论 菌株H74-18对白色念珠菌抗菌活性较高且抗菌谱较广。通过优化发酵条件可以提高其活性物产量,说明它具有进一步研究开发的应用前景。     

罗汉松及响叶杨内生放线菌的分离、活性及多样性研究

目的 积累微生物资源,研究内生放线菌的微生态特征及其次生代谢产物的生物活性.方法 依据Coombs等的分离方法并加以改进,从采集自成都的罗汉松及响叶杨枝条中分离内生放线菌,对分离菌株做168 rRNA基因部分序列扩增并测序,对其次生代谢产物采用滤纸片法测抗菌活性,以SRB法测其对HepG2细胞株的细胞毒活性.结果 分离得到13株内生放线菌,其中10株菌具有不同程度的抗肿瘤活性,占全部菌株的77%,菌株A243具有极强的广谱抗细菌活性和细胞毒活性.A245属于马杜拉菌属,A251属于珊瑚状放线菌属,A246属于诺卡氏菌属,A248,A252属于小四孢菌属,其余8株属于链霉菌属.结论 链霉菌是这两种木本植物枝条中的优势内生放线菌,从这两种植物枝条中分离的内生放线菌产生多种活性的代谢产物,可以进一步的研究与开发.     

北极放线菌Streptomyces sp. MLA-21发酵条件优化

对北极放线菌Streptomyces sp.MLA-21的发酵条件进行优化,提高发酵液中抗肿瘤活性部位的产量。以发酵液对肺癌A549细胞的抑制率（IR）作为观测指标,利用响应面分析法对通过单因素实验和Plackett-Burman设计筛选出培养基初始pH值、黄豆浸出物含量,可溶性淀粉含量等关键因素进行优化。建立二次回归模型。得到菌株MLA-21发酵的最优条件为KNO₃1.0 g/L,可溶性淀粉23.04g/L,黄豆浸出物7.1g/L,K₂HPO₄ 0.5g/L,MgSO₄ 0.5g/L,NaCl 0.5g/L,pH 7.67,接种量:7.5%,培养温度:28℃,摇床转速:150 r/min。对优化结果进行验证发现优化后的培养基能够显著提高发酵液对A549细胞的抑制率和活性部位的产量。     

The kapurimycins, new antitumor antibiotics produced by Streptomyces. Producing organism, fermentation, isolation and biological activities

As a result of screening for antitumor agents from actinomycetes, the kepurimycins were isolated from Streptomyces sp. DO-115. The antibiotics were produced in a fermentation medium supplemented with high porous polymer resin which adsorbs antibiotics and results in an increase of titer. The active complex was isolated from the polymer resin by a solvent extraction procedure and was separated by column chromatography, into two minor and one major component, named A1, A2 and A3. The kepurimycins were active against bacteria, particularly Gram-positive organisms, and were cytotoxic to HeLa S3 human cerivical cancer cells and T24 human bladder carcinoma cells in vitro. Among the individual components of the kapurimycins, kapurimycin A3 exhibited the strongest antibacterial and cytotoxic activities. It showed a potent antitumor activity against murine leukemia P388 in vivo.     

海洋放线菌细胞毒抗肿瘤活性物质的初筛

本文以改进的ＭＴＴ法为初筛模型,筛选海洋放线菌产生的细胞毒抗肿瘤活性物质,对４３４株海洋放线菌发酵液的筛选结果发现,约１６％的海洋放线菌发酵液对Ｐ３８８和ＫＢ癌细胞的ＩＤ８０等于或大于３２０,其中最高可达２０４８０。这表明海洋放线菌存在着活性高的细胞毒抗肿瘤活性物质,是潜在的抗癌药源。     

北极放线菌BF-1发酵条件优化及代谢产物抑菌活性研究

目的优化发酵工艺以提高北极放线菌BF-1发酵液中抑菌活性物质的产量;测定BF-1次级代谢产物的体外抑菌活性。方法以发酵液抑菌活性为指标,采用单因素实验和正交实验对放线菌BF-1发酵培养基和发酵条件进行优化;琼脂稀释法测定BF-1发酵液最低抑菌浓度。结果最佳发酵培养基:淀粉5g.L-1,NH4Cl 5g.L-1,黄豆15g.L-1,MgSO40.25g.L-1,海水晶30g.L-1;最佳发酵条件:28℃,起始pH7,接种量5%;BF-1发酵液对绿脓杆菌的最低抑菌浓度(MIC)为640μg.mL-1。结论北极放线菌BF-1发酵液中次级代谢产物具有显著的体外抑菌活性,优化后BF-1发酵液的抑菌活性与优化前相比提高了约2.6倍。     

含双噁唑环的大环双内酯化合物FW-04-806发酵条件优化

目的对链霉菌FIM-04-806的次级代谢产物,含双噁唑环的大环双内酯化合物FW-04-806进行发酵条件研究。方法采用单因素试验进行培养基优化,探讨装液量、初始pH值等发酵参数的影响,同时逐级放大进行中试验证。结果确定了最佳培养条件:种子培养基为葡萄糖3%,蔗糖3%,玉米浆粉2%,CaCO3 0.75%,pH7.5;发酵培养基为可溶性淀粉3%,葡萄糖2%,黄豆粉1%,玉米浆粉2%,CaCO3 0.7%,pH7.5,接种量10%。优化后的摇瓶效价较初始水平提高了7倍以上,并在100L自动发酵罐及1.5吨发酵罐上得到验证。结论 FW-04-806优化的发酵条件为其工业化生产奠定了基础。     

A new antitumor antibiotic, FR900840. III. Antitumor activity against experimental tumors

FR900840 [2S)-2-amino-2-carboxyethyl (3R)-2-diazo-3-hydroxybutyrate), a new antibiotic with antitumor activity was isolated from the fermentation broth of Streptomyces sp. No. 8727. Its antitumor activity was examined in three mouse tumor systems and ten human tumor systems. FR900840 had no clear effect on mouse ascitic tumors, P388 and L1210, and the B16 melanoma line, but had prominent antitumor effects on several human solid tumors. Its antitumor activity against A549 human lung adenocarcinoma was stronger than those of vinblastine, doxorubicin and cisplatin. These results suggest that FR900840 may become a useful prototype antitumor drug.     

高寒草甸链霉菌Qhu-M197活性次级代谢产物的研究

摘要：目的 从高寒草甸来源的链霉菌Qhu-M197中发现具有抗菌和抗肿瘤活性的次级代谢产物。方法 首先扩增菌株Qhu-M197的16S rRNA基因并基于测序结果,构建系统发育树,初步开展菌株分类鉴定;其次基于色谱分离技术,结合高分辨质谱和核磁共振波谱学数据,对活性次级代谢产物进行分离和结构鉴定。最后采用微量稀释二倍法及CCK8法对分离化合物进行抗菌和抗肿瘤活性体外评价。结果 菌株Qhu-M197为链霉菌,其16S rRNA基因序列与Streptomyces phaeoluteigriseus DSM 41896T相似度为100.00%。菌株Qhu-M197的MS固体培养基发酵产物对金黄色葡萄球菌和枯草芽胞杆菌具有显著的抑菌效果。从其发酵产物中分离得到4个单体化合物,分别是mithramycin、aerugine、aeruginol以及2, 3, 4, 5-tetrahydro-3, 5-dihydroxy-6H-1, 5-benzoxazocin-6-one,其中mithramycin的抗菌和抗肿瘤活性最强,对金黄色葡萄球菌和枯草芽胞杆菌的最小抑制浓度(MIC)分别为0.012和0.095 μg/mL,对HepG2人肝癌细胞株和MCF-7人乳腺癌细胞株的半数抑制浓度IC50分别为0.45和0.46 μmol/L。结论 以链霉菌Qhu-M197为代表的青藏高原高寒草甸放线菌是抗菌和抗肿瘤活性次级代谢产物的重要来源。     

Sapurimycin, new antitumor antibiotic produced by Streptomyces. Producing organism, fermentation, isolation and biological properties

In screening actinomycetes for antitumor compounds, Streptomyces sp. DO-116 was found to produce a new antitumor antibiotic sapurimycin. It is structurally related to, but distinct from, kapurimycins. The antibiotic was produced in a fermentation medium supplemented with high porous polymer resin which adsorbs antibiotic in the culture and results in an increase of titer. Active material was separated from the polymer resin by a solvent extraction procedure and isolated by repeated solvent extraction, adsorption chromatography and HPLC. Sapurimycin was active against bacteria, particularly Gram-positive organisms. It exhibited antitumor activity against leukemia P388 and sarcoma 180 in mice. Sapurimycin caused single strand breaks in supercoiled plasmid DNA in vitro. These results are discussed in comparison with data for kapurimycins.     

Bioactive actinomycetes from Krishna River sediments of Andhra Pradesh

Sediment samples from Krishna River at Nagayalanka of Andhra Pradesh, India were investigated as a source of actinomycetes to screen for the production of novel bioactive compounds. During our investigation on fresh water actinomycetes from 5 different river sediment samples, a total of 80 actinomycetes were isolated. Out of these 80 isolates, 30 isolates which showed distinct macromorphological characteristics were selected. The antimicrobial and enzymatic activities were studied for all the 30 isolates. The preliminary study for antimicrobial activity by cross streak method indicated that 16 isolates (53.3%) have excellent antagonistic properties. All these 16 isolates were subjected to detailed submerged fermentation studies. It was observed that 12 isolates (40.0%) exhibited antibacterial activity, 9 isolates (30.0%) showed antifungal activity while 5 isolates (16.6%) showed both antibacterial and antifungal activities. All the 30 isolates were also subjected for the determination of enzymatic activities 25 isolates (83.3%) exhibited amylolytic activity while 27 isolates (90.0%) showed proteolytic activity. Among these isolates, six promising isolates were selected for detailed morphological, cultural, physiological and biochemical studies. It was established that these isolates belong to the Streptomyces genus by virtue of their cell wall composition pattern and were identified as strains of different Streptomyces species like S. rochei, S. alanosinicus, S. erumpens, S. griseoplanus, S. gancidicus and S. nigrogriseolus.