表面活性素、杆菌霉素L、罗克霉素和泛革素4种脂肽类抗生素高效制备方法及其生物学活性
|
摘要
【目的】建立一种从枯草芽胞杆菌中高效快速制备4种脂肽类抗生素的方法,初步阐明制备脂肽类抗生素的生物学活性。【方法】通过构建脂肽类抗生素突变株,采用酸沉淀、有机溶剂抽提和固相萃取的方法从枯草芽胞杆菌Bs916及其突变株的发酵液中提纯表面活性素、杆菌霉素L、泛革素和罗克霉素4种脂肽类抗生素;运用HPLC-MS、SDS-PAGE电泳对制备脂肽类抗生素的纯度进行检测;最后采用西瓜枯萎病菌、金黄色葡萄球菌和绵羊血红细胞测定4种脂肽类抗生素的抗真菌、抗细菌和溶血活性。【结果】蛋白凝胶电泳和液相色谱联用质谱检测结果表明,制备的脂肽类抗生素均达到了电泳纯和色谱纯;生物活性分析实验结果表明,表面活性素和杆菌霉素L具有较强的溶血活性,泛革素和罗克霉素的溶血活性较弱,杆菌霉素L和泛革素具有较强的抗真菌活性,而表面活性素和罗克霉素具有较强的抗细菌活性。【结论】建立快速从芽胞杆菌中提纯4种脂肽类抗生素色谱方法,测定了4种脂肽类化合物的生物活性,为进一步探究脂肽类抗生素的构效关系、生物合成和开发应用奠定基础。
【Objective】The efficient and simple methods for the preparation of four lipopeptide antibiotics produced by Bacillus subtilis strains were established and the biological functions of these four lipopeptide antibiotics were elucideted.【Method】The four family lipopeptide antibiotics surfactin,bacillomycin,fengycin and locillomycin were prepared by acid precipitation,organic solvent extraction and solid phase extraction from the culture broth of different Bacillus subtilis 916 and its mutants; The purities of the four lipopeptides were detected by HPLCMS and Tricine-SDS-PAGE; The antifungal,antibacterial and hemolytic activities of the four lipopeptide antibiotics were analyzed by Fusarium oxysporum,Staphylococcus aureus and sheep red blood cells.【Result】The results of HPLC-MS and Tricine-SDS-PAGE showed that all the four family lipopeptide antibiotics prepared by this study reached electrophoresis pure and chromatography pure. Investigation of the biological functions of the four class lipopeptides showed that surfactin and bacillomycin L had strong haemolytic activities,but fengycin and locillomycin only had weak haemolytic activities,while bacillomycin L and fengycin showed strong antifungal activities,but surfactin and locillomycin showed antibacterial and antiviral activities.【Conclusion】In general,the efficient methods for purification of the four lipopeptide antibiotics surfactin,bacillomycin L,fengycin and locillomycin from Bacillus subtilis was set up and their biological activities were elucideted,which laid a solid foundation to further elucidate the structure function relationship,biosynthesis pathway and application of these lipopeptide antibiotics.
【Objective】The efficient and simple methods for the preparation of four lipopeptide antibiotics produced by Bacillus subtilis strains were established and the biological functions of these four lipopeptide antibiotics were elucideted.【Method】The four family lipopeptide antibiotics surfactin,bacillomycin,fengycin and locillomycin were prepared by acid precipitation,organic solvent extraction and solid phase extraction from the culture broth of different Bacillus subtilis 916 and its mutants; The purities of the four lipopeptides were detected by HPLCMS and Tricine-SDS-PAGE; The antifungal,antibacterial and hemolytic activities of the four lipopeptide antibiotics were analyzed by Fusarium oxysporum,Staphylococcus aureus and sheep red blood cells.【Result】The results of HPLC-MS and Tricine-SDS-PAGE showed that all the four family lipopeptide antibiotics prepared by this study reached electrophoresis pure and chromatography pure. Investigation of the biological functions of the four class lipopeptides showed that surfactin and bacillomycin L had strong haemolytic activities,but fengycin and locillomycin only had weak haemolytic activities,while bacillomycin L and fengycin showed strong antifungal activities,but surfactin and locillomycin showed antibacterial and antiviral activities.【Conclusion】In general,the efficient methods for purification of the four lipopeptide antibiotics surfactin,bacillomycin L,fengycin and locillomycin from Bacillus subtilis was set up and their biological activities were elucideted,which laid a solid foundation to further elucidate the structure function relationship,biosynthesis pathway and application of these lipopeptide antibiotics.
引文
[1]Patel S,Ahmed S,Eswari J S. Therapeutic cyclic lipopeptides mining from microbes:latest strides and hurdles[J]. World J Microbiol Biotechnol,2015,31(8):1177-1193.
[2]吕应年,杨世忠,牟伯中.脂肽的分离纯化与结构研究[J].微生物学通报,2005,32(1):67-73.
[3]邓建良,刘红彦,刘玉霞,等.解淀粉芽胞杆菌YN-1抑制植物病原真菌活性物质鉴定[J].植物病理学报,2010(2):202-209.
[4]Ongena M,Jacques P. Bacillus lipopeptides:versatile weapons for plant disease biocontrol[J]. Trends Microbiol,2008,16:115-125.
[5]覃尚忠.达托霉素研究进展[J].中国药事,2006(6):375-377.
[6]Miao V,Co3ffet-Legal M F,Brian P,et al. Daptomycin biosynthesis in Streptomyces roseosporus:cloning and analysis of the gene cluster and revision of peptide stereochemistry[J]. Microbiology,2005,151:1507-1523.
[7]Inaoka T,Wang G,Ochi K. ScoC regulates bacilysin production at the transcription level in Bacillus subtilis[J]. J Bacteriol,2009,191(23):7367-7371.
[8]Bais H P,Fall R,Vivanco J M. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J]. Plant Physiol,2004,134:307-319.
[9]Schneider T,Müller A,Miess H,et al. Cyclic lipopeptides as antibacterial agents-Potent antibiotic activity mediated by intriguing mode of actions[J]. Int J Med Microbiol,2014,304(1):37-43.
[10]Luo C,Liu X,Zhou X,et al. Unusual Biosynthesis and Structure of Locillomycins from Bacillus subtilis 916[J]. Appl Environ Microbiol,2015,81(19):6601-6609.
[11]金鑫.稻瘟病生防菌Af1、Af4的初步研究[D].武汉:华中农业大学,2010.
[12]王帅,高圣风,高学文.枯草芽胞杆菌脂肽类抗生素发酵和提取条件[J].中国生物防治,2007,23(4):342-347.
[13]陈华,袁成凌,蔡克周,等.枯草芽胞杆菌JA产生脂肽类抗生素-iturinA的纯化及电喷雾质谱鉴定[J].微生物学报,2008,48(1):116-120.
[14]高学文,姚仕义,Pham H,等.枯草芽胞杆菌B2菌株产生的表面活性素变异体的纯化和鉴定[J].微生物学报,2003,43(5):647-652.
[15]Romero D,Pérez-García A,Rivera M E,et al. Isolation and evaluation of antagonistic bacteria towards the cucurbit powdery mildew fungus Podosphaera fusca[J]. Appl Microbiol Biotechnol,2004,64:263-269.
[16]Romero D,De Vicente A,Rakotoaly R H,et al. The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca[J]. Mol Plant Microbe Interact,2007,20(4):430-440.
[17]Zeriouh H,Romero D,Garcia-Gutierrez L,et al. The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits[J]. Mol Plant Microbe Interact,2011,24:1540-1552.
[18]罗楚平,刘邮洲,吴荷芳,等.脂肽类化合物bacillomycin L抗真菌活性及其对水稻病害的防治[J].中国生物防治学报,2011,27(1):76-81.
[19]Khem Raj Meena,Shamsher S,Kanwar. Lipopeptides as the Antifungal and Antibacterial Agents:Applications in Food Safety and Therapeutics[J]. BioMed Research International,2015:1-9.
[20]Gordillo M C,Maldonado. Purification of peptides from Bacillus strains with biological activity[J]. Chromatography and Its Applications,2012,11:201-225.
[21]周华飞,罗楚平,王晓宇,等.枯草芽胞杆菌Bs916突变体库的构建和抑制水稻细菌性条斑病菌相关基因的克隆[J].中国农业科学,2013(11):2232-2239.
[22]罗楚平,王晓宇,陈志谊,等.枯草芽胞杆菌Bs916中脂肽抗生素Bacillomycin L的操纵子结构及生物活性[J].中国农业科学,2010(22):4624-4634.
[23]向亚萍,陈志谊,罗楚平,等.芽胞杆菌的抑菌活性与其产脂肽类抗生素的相关性[J].中国农业科学学,2015(20):4064-4076.
[24]罗楚平,王晓宇,周华飞,等.生防菌Bs916合成脂肽抗生素泛革素的操纵子结构功能及生物活性[J].中国农业科学,2013,46(24):5142-5149.
[25]Koumoutsi A,Chen X H,Vater J,et al. Deg U and Ycz E positively regulate the synthesis of bacillomycin D by Bacillus amyloliquefaciens strain FZB42[J]. Appl Environ Microbiol,2007,73(21):6953-6964.
[26]Chen Y,Cao S,Chai Y,et al. A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants[J]. Mol Microbiol,2012,85:418-430.
[27]Luo C,Liu X,Zhou H,et al. Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions[J]. Appl Environ Microbiol,2015,81(1):422-431.
[28]Luo C,Wang X,Chen Z,et al. The operon,structure and biological activities of the lipopeptide Bacillomycin L produced by Bacillus subtilis 916[J]. Sci Agric Sin,2010,43(22):4624-4634.
[29]Luo C,Zhou H,Zou J,et al. Bacillomycin L and surfactin contribute synergistically to the phenotypic features of Bacillus subtilis 916and the biocontrol of rice sheath blight induced by Rhizoctonia solani[J]. Appl Microbiol Biotechnol,2014,99(4):1897-1910.
[30]Houry a,Briandet R,Aymerich S,et al. Involvement of motility and flagella in Bacillus cereus biofilm formation[J]. Microbiology,2010,156:1009-10018.
[31]罗楚平,陈志谊,刘永锋,等.生防菌Bs-916合成脂肽类化合物Bac操纵子突变株构建及功能[J].微生物学报,2009(4):445-452.
[32]Rodrigues L,Banat I M,Teixeira J,et al. Biosurfactants:potential applications in medicine[J]. J Antimicrob Chemother,2006,57:609-618.
[33]陈蓉明,林跃鑫,黄谚谚.枯草芽胞杆菌ATCC2233产生表面活性素的研究[J].福建轻纺,2000(12):1-4.
[34]Stein T. Bacillus subtilis antibiotics:structures,syntheses and specific functions[J]. Mol Microbiol,2005,56:845-857.
[35]杨红梅,王浩,刘艳琴,等.高效液相色谱-质谱联用测定乳制品中6种四环素类抗生素[J].中国乳品工业,2007(11):50-52.
[36]Schagger H. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100k Da[J]. Analytical Biochemistry,1998,166(2):368-379.
[37]Volpon L,Tsan P,Majer,et al. NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of LAsn1 in the natural iturinic antibiotics[J]. Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy,2006,67(5):1374-1381.
[2]吕应年,杨世忠,牟伯中.脂肽的分离纯化与结构研究[J].微生物学通报,2005,32(1):67-73.
[3]邓建良,刘红彦,刘玉霞,等.解淀粉芽胞杆菌YN-1抑制植物病原真菌活性物质鉴定[J].植物病理学报,2010(2):202-209.
[4]Ongena M,Jacques P. Bacillus lipopeptides:versatile weapons for plant disease biocontrol[J]. Trends Microbiol,2008,16:115-125.
[5]覃尚忠.达托霉素研究进展[J].中国药事,2006(6):375-377.
[6]Miao V,Co3ffet-Legal M F,Brian P,et al. Daptomycin biosynthesis in Streptomyces roseosporus:cloning and analysis of the gene cluster and revision of peptide stereochemistry[J]. Microbiology,2005,151:1507-1523.
[7]Inaoka T,Wang G,Ochi K. ScoC regulates bacilysin production at the transcription level in Bacillus subtilis[J]. J Bacteriol,2009,191(23):7367-7371.
[8]Bais H P,Fall R,Vivanco J M. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J]. Plant Physiol,2004,134:307-319.
[9]Schneider T,Müller A,Miess H,et al. Cyclic lipopeptides as antibacterial agents-Potent antibiotic activity mediated by intriguing mode of actions[J]. Int J Med Microbiol,2014,304(1):37-43.
[10]Luo C,Liu X,Zhou X,et al. Unusual Biosynthesis and Structure of Locillomycins from Bacillus subtilis 916[J]. Appl Environ Microbiol,2015,81(19):6601-6609.
[11]金鑫.稻瘟病生防菌Af1、Af4的初步研究[D].武汉:华中农业大学,2010.
[12]王帅,高圣风,高学文.枯草芽胞杆菌脂肽类抗生素发酵和提取条件[J].中国生物防治,2007,23(4):342-347.
[13]陈华,袁成凌,蔡克周,等.枯草芽胞杆菌JA产生脂肽类抗生素-iturinA的纯化及电喷雾质谱鉴定[J].微生物学报,2008,48(1):116-120.
[14]高学文,姚仕义,Pham H,等.枯草芽胞杆菌B2菌株产生的表面活性素变异体的纯化和鉴定[J].微生物学报,2003,43(5):647-652.
[15]Romero D,Pérez-García A,Rivera M E,et al. Isolation and evaluation of antagonistic bacteria towards the cucurbit powdery mildew fungus Podosphaera fusca[J]. Appl Microbiol Biotechnol,2004,64:263-269.
[16]Romero D,De Vicente A,Rakotoaly R H,et al. The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca[J]. Mol Plant Microbe Interact,2007,20(4):430-440.
[17]Zeriouh H,Romero D,Garcia-Gutierrez L,et al. The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits[J]. Mol Plant Microbe Interact,2011,24:1540-1552.
[18]罗楚平,刘邮洲,吴荷芳,等.脂肽类化合物bacillomycin L抗真菌活性及其对水稻病害的防治[J].中国生物防治学报,2011,27(1):76-81.
[19]Khem Raj Meena,Shamsher S,Kanwar. Lipopeptides as the Antifungal and Antibacterial Agents:Applications in Food Safety and Therapeutics[J]. BioMed Research International,2015:1-9.
[20]Gordillo M C,Maldonado. Purification of peptides from Bacillus strains with biological activity[J]. Chromatography and Its Applications,2012,11:201-225.
[21]周华飞,罗楚平,王晓宇,等.枯草芽胞杆菌Bs916突变体库的构建和抑制水稻细菌性条斑病菌相关基因的克隆[J].中国农业科学,2013(11):2232-2239.
[22]罗楚平,王晓宇,陈志谊,等.枯草芽胞杆菌Bs916中脂肽抗生素Bacillomycin L的操纵子结构及生物活性[J].中国农业科学,2010(22):4624-4634.
[23]向亚萍,陈志谊,罗楚平,等.芽胞杆菌的抑菌活性与其产脂肽类抗生素的相关性[J].中国农业科学学,2015(20):4064-4076.
[24]罗楚平,王晓宇,周华飞,等.生防菌Bs916合成脂肽抗生素泛革素的操纵子结构功能及生物活性[J].中国农业科学,2013,46(24):5142-5149.
[25]Koumoutsi A,Chen X H,Vater J,et al. Deg U and Ycz E positively regulate the synthesis of bacillomycin D by Bacillus amyloliquefaciens strain FZB42[J]. Appl Environ Microbiol,2007,73(21):6953-6964.
[26]Chen Y,Cao S,Chai Y,et al. A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants[J]. Mol Microbiol,2012,85:418-430.
[27]Luo C,Liu X,Zhou H,et al. Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions[J]. Appl Environ Microbiol,2015,81(1):422-431.
[28]Luo C,Wang X,Chen Z,et al. The operon,structure and biological activities of the lipopeptide Bacillomycin L produced by Bacillus subtilis 916[J]. Sci Agric Sin,2010,43(22):4624-4634.
[29]Luo C,Zhou H,Zou J,et al. Bacillomycin L and surfactin contribute synergistically to the phenotypic features of Bacillus subtilis 916and the biocontrol of rice sheath blight induced by Rhizoctonia solani[J]. Appl Microbiol Biotechnol,2014,99(4):1897-1910.
[30]Houry a,Briandet R,Aymerich S,et al. Involvement of motility and flagella in Bacillus cereus biofilm formation[J]. Microbiology,2010,156:1009-10018.
[31]罗楚平,陈志谊,刘永锋,等.生防菌Bs-916合成脂肽类化合物Bac操纵子突变株构建及功能[J].微生物学报,2009(4):445-452.
[32]Rodrigues L,Banat I M,Teixeira J,et al. Biosurfactants:potential applications in medicine[J]. J Antimicrob Chemother,2006,57:609-618.
[33]陈蓉明,林跃鑫,黄谚谚.枯草芽胞杆菌ATCC2233产生表面活性素的研究[J].福建轻纺,2000(12):1-4.
[34]Stein T. Bacillus subtilis antibiotics:structures,syntheses and specific functions[J]. Mol Microbiol,2005,56:845-857.
[35]杨红梅,王浩,刘艳琴,等.高效液相色谱-质谱联用测定乳制品中6种四环素类抗生素[J].中国乳品工业,2007(11):50-52.
[36]Schagger H. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100k Da[J]. Analytical Biochemistry,1998,166(2):368-379.
[37]Volpon L,Tsan P,Majer,et al. NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of LAsn1 in the natural iturinic antibiotics[J]. Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy,2006,67(5):1374-1381.