HCCB10124抗真菌活性的初步研究
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摘要
本论文利用上海来益生物药物研发中心筛选得到的一株产广谱、高活性抗真菌产物的HCCB10124菌株,通过纸敏片法测定抗菌谱,发现其对20余种真菌,其中有些是植物和动物的病原菌,有显著拮抗作用,另外,对金黄色葡萄球菌和藤黄八叠球菌也有抑制作用;并对对菌丝体提取物进行不同浓度稀释,进行抗真菌活性的测定,发现活性很高。根据16SrDNA序列分析,该菌株与链霉菌Streptomyces albireticuli具有99%的序列同源性。
在分离纯化之前,对链霉菌HCCB10124进行发酵条件和培养基成分进行初步优化,以期获得较高产量的活性代谢产物。优化后培养基基本组成成分为:2%葡萄糖、2%可溶性淀粉、4%药媒、0.04%钼酸铵、0.4%的碳酸钙。
检测了菌丝体提取物及发酵液上清中生物活性产物对温度、pH值的稳定性;利用捷克八溶剂系统纸色谱的方法对生物活性产物进行了研究,发现其对温度、pH值较稳定。初步的研究结果为进一步分离提取打下一定的基础。
通过pH沉淀、有机溶剂萃取和树脂吸附、硅胶柱层析、中压色谱层析等实验,确定的工作流程为先将发酵上清液的pH调至6除去离心未除去的固形物,以大孔吸附树脂吸附上清液和菌丝体丙酮抽提液,除去色素,不同浓度的乙醇洗脱,洗脱液浓缩,进行硅胶柱层析,以丙酮:甲醇=10:1、5:1、3:1、2:1、1:1、100%甲醇分段洗脱,充分除去色素,对活性物质进行初步分离。最后,用中压色谱进行实验,除去部分杂质,达到对样品富集和纯化的目的。
鉴于链霉菌HCCB10124所产抗真菌物质具有广谱、高活性、高稳定性等优点,因此具有潜在的、重要的应用价值和理论意义。其所产抗菌活性物质有望开发为一种可广泛地应用于真菌病害的生物防治或人畜真菌性疾病的抗真菌抗生素。
In this research work, the strain conserved in HCCB10124 (R&D) was found to be strongly active against a series of fungi,some of which are plant or animal pathogenic , such as Candida albicans, Sarcina lutea, etc. The 16S rDNA sequence analysis combined with traditional identification methods was used to identify the strain. Based on the results, this strain was identified as Streptomyces albireticuli.
Tests on culture media and cultural conditions for HCCB10124, showed that carbon source and nitrogen source presented the most significant effect on the production of bioactivity.The optimized medium was composed of glucose 2%, soluble amylum 2%,cottonseed powder 3%,ammonium molybdate 0.04%,calcium carbonate 0.4%. The effects of heat and pH on the stability of the bioactive substance in the fermented broth were studied. The water solubility and ion character of the antibiotics were analysized by pH paper chromatography and thin-layer chromatography. The results showed that the weakly acidic antibiotics were stable to heat and pH.The physiological and biochemical characteristics of the highly activitive antibiotic substances were studied in orde to provide helpful information for the further isolation and purification. A brief process for the primary isolation of the active substance was worked out.
In all, Streptomyces albireticuli can produce fairly stable and broads pectrum,strongly active antifungi substance. So it's a hopeful antibiotics producing Streptomyces that its antifungal substance will be value of potential application in agriculture and health.
在分离纯化之前,对链霉菌HCCB10124进行发酵条件和培养基成分进行初步优化,以期获得较高产量的活性代谢产物。优化后培养基基本组成成分为:2%葡萄糖、2%可溶性淀粉、4%药媒、0.04%钼酸铵、0.4%的碳酸钙。
检测了菌丝体提取物及发酵液上清中生物活性产物对温度、pH值的稳定性;利用捷克八溶剂系统纸色谱的方法对生物活性产物进行了研究,发现其对温度、pH值较稳定。初步的研究结果为进一步分离提取打下一定的基础。
通过pH沉淀、有机溶剂萃取和树脂吸附、硅胶柱层析、中压色谱层析等实验,确定的工作流程为先将发酵上清液的pH调至6除去离心未除去的固形物,以大孔吸附树脂吸附上清液和菌丝体丙酮抽提液,除去色素,不同浓度的乙醇洗脱,洗脱液浓缩,进行硅胶柱层析,以丙酮:甲醇=10:1、5:1、3:1、2:1、1:1、100%甲醇分段洗脱,充分除去色素,对活性物质进行初步分离。最后,用中压色谱进行实验,除去部分杂质,达到对样品富集和纯化的目的。
鉴于链霉菌HCCB10124所产抗真菌物质具有广谱、高活性、高稳定性等优点,因此具有潜在的、重要的应用价值和理论意义。其所产抗菌活性物质有望开发为一种可广泛地应用于真菌病害的生物防治或人畜真菌性疾病的抗真菌抗生素。
In this research work, the strain conserved in HCCB10124 (R&D) was found to be strongly active against a series of fungi,some of which are plant or animal pathogenic , such as Candida albicans, Sarcina lutea, etc. The 16S rDNA sequence analysis combined with traditional identification methods was used to identify the strain. Based on the results, this strain was identified as Streptomyces albireticuli.
Tests on culture media and cultural conditions for HCCB10124, showed that carbon source and nitrogen source presented the most significant effect on the production of bioactivity.The optimized medium was composed of glucose 2%, soluble amylum 2%,cottonseed powder 3%,ammonium molybdate 0.04%,calcium carbonate 0.4%. The effects of heat and pH on the stability of the bioactive substance in the fermented broth were studied. The water solubility and ion character of the antibiotics were analysized by pH paper chromatography and thin-layer chromatography. The results showed that the weakly acidic antibiotics were stable to heat and pH.The physiological and biochemical characteristics of the highly activitive antibiotic substances were studied in orde to provide helpful information for the further isolation and purification. A brief process for the primary isolation of the active substance was worked out.
In all, Streptomyces albireticuli can produce fairly stable and broads pectrum,strongly active antifungi substance. So it's a hopeful antibiotics producing Streptomyces that its antifungal substance will be value of potential application in agriculture and health.
引文
[1] 陈代杰.微生物药物学.上海:华东理工大学出版社,1999
[2] 张致平.微生物药物学.北京:化学工业出版社,2003
[3] Hook V. Superbugs step up the pace. Chem Brit, 1997,26(5):34
[4] 陈万义,薛振祥,王能武.新农药研究与开发.北京:化学工业出版社,1996
[5] 刘正印,王爱霞.抗真菌药物的研究进展[J].中国抗生素杂志,2006,31(2): 69
[6] Eve Finkelstein, Boaz Amichai, Marcelo H etal. Griseofulvin and its uses. International Journal of Antimicrobial Agents, 1996,6(3):189
[7] 张致平. 抗真菌药物研究进展[J]. 中国新药杂志,2004,13(2):110
[8]张建茹,谢小梅,章洪华. 作用于真菌细胞壁的抗真菌药物研究进展[J]. 现代诊断与治疗,2004,15(6):364
[9] 王亚男.抗真菌药物研究进展[J].国外医药抗生素分册,2005,26 (2):59
[10] Tojo T, Ohki H, Shiraishi N, et al. Cyclic hexapeptides having antibiotic activity[P]. WO 0064927, 2000-11-02
[11] Aoki M, Kohchi M , Mitsubishi K, et al. Aerothicin analogs, their preparation and use[P]. WO 0005251,2000-02-03
[12] Roy K, Mukhopadhyay T, Reddy G C S, et al. Mulundocandin, a new lipopentide antibiotic. I Taxonomy, fermentation, isolation and characterization[J]. J Antibiot, 1987, 409(6): 275
[13] Espnel-ngroff A. Comparison of in vitro activities of the new triazole SCH56592 and the echinocandins MK20991(L-743,872) and LY303366 against opportunistic filamentous and dimorphic fungi and yeasts [J]. Clin M icrobiol,1998,36(10):2950
[14] Abruzzo G K, Flattery A M , Gill C J,et al. Evaluation of the echinocandin antifungal M K20991(L 2743, 872 ):efficacies in mouse models of disseminated aspergillosis, candidiasis and cryptococcosis [J]. Antimicrob Agents Chem other, 1997,41(11):2333
[15] Smith J G, A bruzzo G K, Gill C J , et al. Evaluation of pneumocandin L-743,872 in neutropenicmouse models of disseminated candidiasis and aspergillosis [A]. In: Proceedings of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy New Orleans [C]. American Society for Microbiology: Washington,DC, 1996: 107.
[16] Powles M A , Liberator P, Anderson J , et al. Efficacy of MK2991 (L 2743, 872), a semisynthetic, pneumocandin,in murine models of Pneumocystis carinii [J]. Antimicrob Agents Chem other, 1998, 42(8): 1985 [ 17 ] Gonza′lez G M , Tijerina R, Najvar L K, et al. Correlation between antifungal susceptibilities of Coccid ioides immitis in vitro and antifungal treatment with caspofungin in a mouse model [J]. Antimicrob Agents Chem other, 2001, 45(7): 1854
[18] Murdoch D, Ploseker G L. Anidulafungin[J]. D rug, 2004,64(19):2249
[19] 叶丽娟,朱辉.抗真菌药物作用机制及真菌耐药机制的研究进展IJ] 国外医药抗生素分册,206,27(5):221
[20] Cohen E. Chitin synthesis and degradation as targets for pesticide action[J]. Arch Insect Biochem Physiol, 1993,22(9): 245
[21] Hwang E I, Yun B S, Kim Y K, et al. Phellinsin A, a novel chitin synthesis inhibitor produced by Phellinus sp.[J ]. J Antibiot, 2000, 53(2): 903
[22] Cabib E.differential inhibition of chitin synthetase 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins[J].Antimicrob Agents Chemother,1991,35(1):170
[23] Iwamoto T,Fujie A,Tsurumi Y,etal.FR900403,a new antifungal antibiotic produced by a Kernia sp[J]. J Antibiot,1990,43(9):1183
[24] Vijayakumar EK,Roy K,Chatterjee S, etal.Arthrichitin, a new cell wall active metabolite from arthrinium phaeospermum[J]. J Org Chem,1996,61(19):6591
[25] Albaugh D,Cell wall active antifungal compounds produced by the marine fungus hypoxylon oceanicum LL-15G256[J]. J Antibiot,1998,51(3):317
[26] Ueki T,Numata K,Sawada Y, et al.Studies on the mode of antifungal action of pradimicin antibiotics[J]. J Antibiot,1993,46(3):455
[27] Takeuchi T,Hara T,Naganawa H,et al. A new antifungal antibiotic benanomicins A and Bfrom an actinomycete[J]. J Antibiot,1988,41(6):807
[28] 殷瑜,黄为一,陈代杰.微生物来源的抗真菌抗生素的研究进展.中国新药杂志,2004, 13(2):113
[29] Horn W S, Smith J L , Bills G F, et al. Sphingofungins E and F. Novel serine palmitoyltransferase inhibitors from Paecilomyces variotii[J]. J Antibiot, 1992, 47(3):376
[30] Mandala S M , Frommer B R, Thornton R A , et al. Inhibition of serine palmitoylt ransferase activity by lipoxamycin [J ]. J Antibiot, 1994, 47(11): 376
[31] Mandala S M, Thornton R A , Frommer B R, et al. Viridiofungins, novel inhibitors of sphingolipid synthesis [J]. J Antibiot, 1997,50(10): 339
[32] Wu W I, Donough V M , Nickels J T, et al. Regulation of lipid biosynthesis in Saccharomy cescerevisiae by fumonisin B1 [J ]. J Biol Chem , 1995, 270(12):13171.
[33] Wang E, Norred W P, Bacon C W, et al. Inhibition of sphingo lipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme[J]. J Biol Chem , 1991, 266(2): 14486.
[34] Mandala S M , Thornton R A , Frommer B R, et al. The discovery of australifungin, a novel inhibitor of sph inganine N -acyltransferase from Sporormiella australis: producing organism, fermentation, isolation and biological activity[J]. J Antibiot, 1995, 48: 349.
[35] Nagiec M M, Nagiec E E, Baltisberger J A ,et al. Sphingo lipid synthesis as a target for antifungal drugs. Complementation of the inositol phosphorylceramide synthase defect in a mutantstrain of Saccharomy cescerevisiae by the AUR1 gene[J]. J Biol Chem , 1997,272: 9809
[36] Mandala S M, Thornton R A , Rosenbach M , et al. Khafrefungin, a novel inhibitor of sphingo lipid synthesis [J]. J Biol Chem , 1997, 272: 32709
[37] Mandala S M, Thornton R A , Milligan J , et al. Rustm icin, a potent antifungal agent, inhibits sphingolipid synthesis at inositol phosphoceramide synthase [J]. J Biol Chem , 1998, 273: 14942
[38] Onishi J C, Milligan J A , Basilio A ,et al. Antimicrobial activity of viridiofungins[J]. J Antibiot, 1997, 50: 334.
[39] 张常然,唐英春,川上和义,等. 白细胞介素2及12对小鼠肺部曲霉菌感染治疗作用的实验研究[J]. 中国结核和呼吸杂志, 2004,27(4):234
[40] Takesako K, Ikai K, Haruna F,et al. Aureobasidins, new antifungal antibiotics: taxonomy, fermentation, isolation, and properties[J]. J Antibiot, 1991, 44: 919
[41] Fauth U, Zahner H, Muhlenfeld A, et al. Galbonolides A and B-two non glycosidic antifungal macrolides [J ].J Antibiot, 1986, 39: 1760
[42] Harris G H, Shafiee A, Cabello M A , et al. Inhibition of fungal sphingolipid biosynthesis by rustmicin, galbonolide B and their new 21-hydroxy analogs[J]. J Antibiot, 1998, 51: 837
[43] Takesako K, Kuroda H, Inoue T, et al. Biological properties of aureobasidin A , a cyclic depsipeptide antifungal antibiotic[J]. J Antibiot, 1993,46: 1414
[44] Ueki M,Taniguchi M.The mode of action of UK-2A and UK-3A,novel antifungal antibiotics from Streptomyces sp.517-02 [J]. J Antibiot, 1997,50(12): 1052
[45] Albengres E,Le H,Tillement JP.Systemic antifungal agents,drug interactions of clinical signficance[J]. Drug Saf, 1998,18(2): 83
[2] 张致平.微生物药物学.北京:化学工业出版社,2003
[3] Hook V. Superbugs step up the pace. Chem Brit, 1997,26(5):34
[4] 陈万义,薛振祥,王能武.新农药研究与开发.北京:化学工业出版社,1996
[5] 刘正印,王爱霞.抗真菌药物的研究进展[J].中国抗生素杂志,2006,31(2): 69
[6] Eve Finkelstein, Boaz Amichai, Marcelo H etal. Griseofulvin and its uses. International Journal of Antimicrobial Agents, 1996,6(3):189
[7] 张致平. 抗真菌药物研究进展[J]. 中国新药杂志,2004,13(2):110
[8]张建茹,谢小梅,章洪华. 作用于真菌细胞壁的抗真菌药物研究进展[J]. 现代诊断与治疗,2004,15(6):364
[9] 王亚男.抗真菌药物研究进展[J].国外医药抗生素分册,2005,26 (2):59
[10] Tojo T, Ohki H, Shiraishi N, et al. Cyclic hexapeptides having antibiotic activity[P]. WO 0064927, 2000-11-02
[11] Aoki M, Kohchi M , Mitsubishi K, et al. Aerothicin analogs, their preparation and use[P]. WO 0005251,2000-02-03
[12] Roy K, Mukhopadhyay T, Reddy G C S, et al. Mulundocandin, a new lipopentide antibiotic. I Taxonomy, fermentation, isolation and characterization[J]. J Antibiot, 1987, 409(6): 275
[13] Espnel-ngroff A. Comparison of in vitro activities of the new triazole SCH56592 and the echinocandins MK20991(L-743,872) and LY303366 against opportunistic filamentous and dimorphic fungi and yeasts [J]. Clin M icrobiol,1998,36(10):2950
[14] Abruzzo G K, Flattery A M , Gill C J,et al. Evaluation of the echinocandin antifungal M K20991(L 2743, 872 ):efficacies in mouse models of disseminated aspergillosis, candidiasis and cryptococcosis [J]. Antimicrob Agents Chem other, 1997,41(11):2333
[15] Smith J G, A bruzzo G K, Gill C J , et al. Evaluation of pneumocandin L-743,872 in neutropenicmouse models of disseminated candidiasis and aspergillosis [A]. In: Proceedings of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy New Orleans [C]. American Society for Microbiology: Washington,DC, 1996: 107.
[16] Powles M A , Liberator P, Anderson J , et al. Efficacy of MK2991 (L 2743, 872), a semisynthetic, pneumocandin,in murine models of Pneumocystis carinii [J]. Antimicrob Agents Chem other, 1998, 42(8): 1985 [ 17 ] Gonza′lez G M , Tijerina R, Najvar L K, et al. Correlation between antifungal susceptibilities of Coccid ioides immitis in vitro and antifungal treatment with caspofungin in a mouse model [J]. Antimicrob Agents Chem other, 2001, 45(7): 1854
[18] Murdoch D, Ploseker G L. Anidulafungin[J]. D rug, 2004,64(19):2249
[19] 叶丽娟,朱辉.抗真菌药物作用机制及真菌耐药机制的研究进展IJ] 国外医药抗生素分册,206,27(5):221
[20] Cohen E. Chitin synthesis and degradation as targets for pesticide action[J]. Arch Insect Biochem Physiol, 1993,22(9): 245
[21] Hwang E I, Yun B S, Kim Y K, et al. Phellinsin A, a novel chitin synthesis inhibitor produced by Phellinus sp.[J ]. J Antibiot, 2000, 53(2): 903
[22] Cabib E.differential inhibition of chitin synthetase 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins[J].Antimicrob Agents Chemother,1991,35(1):170
[23] Iwamoto T,Fujie A,Tsurumi Y,etal.FR900403,a new antifungal antibiotic produced by a Kernia sp[J]. J Antibiot,1990,43(9):1183
[24] Vijayakumar EK,Roy K,Chatterjee S, etal.Arthrichitin, a new cell wall active metabolite from arthrinium phaeospermum[J]. J Org Chem,1996,61(19):6591
[25] Albaugh D,Cell wall active antifungal compounds produced by the marine fungus hypoxylon oceanicum LL-15G256[J]. J Antibiot,1998,51(3):317
[26] Ueki T,Numata K,Sawada Y, et al.Studies on the mode of antifungal action of pradimicin antibiotics[J]. J Antibiot,1993,46(3):455
[27] Takeuchi T,Hara T,Naganawa H,et al. A new antifungal antibiotic benanomicins A and Bfrom an actinomycete[J]. J Antibiot,1988,41(6):807
[28] 殷瑜,黄为一,陈代杰.微生物来源的抗真菌抗生素的研究进展.中国新药杂志,2004, 13(2):113
[29] Horn W S, Smith J L , Bills G F, et al. Sphingofungins E and F. Novel serine palmitoyltransferase inhibitors from Paecilomyces variotii[J]. J Antibiot, 1992, 47(3):376
[30] Mandala S M , Frommer B R, Thornton R A , et al. Inhibition of serine palmitoylt ransferase activity by lipoxamycin [J ]. J Antibiot, 1994, 47(11): 376
[31] Mandala S M, Thornton R A , Frommer B R, et al. Viridiofungins, novel inhibitors of sphingolipid synthesis [J]. J Antibiot, 1997,50(10): 339
[32] Wu W I, Donough V M , Nickels J T, et al. Regulation of lipid biosynthesis in Saccharomy cescerevisiae by fumonisin B1 [J ]. J Biol Chem , 1995, 270(12):13171.
[33] Wang E, Norred W P, Bacon C W, et al. Inhibition of sphingo lipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme[J]. J Biol Chem , 1991, 266(2): 14486.
[34] Mandala S M , Thornton R A , Frommer B R, et al. The discovery of australifungin, a novel inhibitor of sph inganine N -acyltransferase from Sporormiella australis: producing organism, fermentation, isolation and biological activity[J]. J Antibiot, 1995, 48: 349.
[35] Nagiec M M, Nagiec E E, Baltisberger J A ,et al. Sphingo lipid synthesis as a target for antifungal drugs. Complementation of the inositol phosphorylceramide synthase defect in a mutantstrain of Saccharomy cescerevisiae by the AUR1 gene[J]. J Biol Chem , 1997,272: 9809
[36] Mandala S M, Thornton R A , Rosenbach M , et al. Khafrefungin, a novel inhibitor of sphingo lipid synthesis [J]. J Biol Chem , 1997, 272: 32709
[37] Mandala S M, Thornton R A , Milligan J , et al. Rustm icin, a potent antifungal agent, inhibits sphingolipid synthesis at inositol phosphoceramide synthase [J]. J Biol Chem , 1998, 273: 14942
[38] Onishi J C, Milligan J A , Basilio A ,et al. Antimicrobial activity of viridiofungins[J]. J Antibiot, 1997, 50: 334.
[39] 张常然,唐英春,川上和义,等. 白细胞介素2及12对小鼠肺部曲霉菌感染治疗作用的实验研究[J]. 中国结核和呼吸杂志, 2004,27(4):234
[40] Takesako K, Ikai K, Haruna F,et al. Aureobasidins, new antifungal antibiotics: taxonomy, fermentation, isolation, and properties[J]. J Antibiot, 1991, 44: 919
[41] Fauth U, Zahner H, Muhlenfeld A, et al. Galbonolides A and B-two non glycosidic antifungal macrolides [J ].J Antibiot, 1986, 39: 1760
[42] Harris G H, Shafiee A, Cabello M A , et al. Inhibition of fungal sphingolipid biosynthesis by rustmicin, galbonolide B and their new 21-hydroxy analogs[J]. J Antibiot, 1998, 51: 837
[43] Takesako K, Kuroda H, Inoue T, et al. Biological properties of aureobasidin A , a cyclic depsipeptide antifungal antibiotic[J]. J Antibiot, 1993,46: 1414
[44] Ueki M,Taniguchi M.The mode of action of UK-2A and UK-3A,novel antifungal antibiotics from Streptomyces sp.517-02 [J]. J Antibiot, 1997,50(12): 1052
[45] Albengres E,Le H,Tillement JP.Systemic antifungal agents,drug interactions of clinical signficance[J]. Drug Saf, 1998,18(2): 83