产抗大肠杆菌脂肽化合物枯草芽孢杆菌筛选及脂肽化合物的分离纯化
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摘要
采用致病性大肠杆菌作为主要指示菌,运用杯碟法对不同分离株的枯草芽孢杆菌代谢产物进行抑菌效果筛选。在确定GD株作为抗菌物质研究的基础上,采用GD株发酵液,经大孔吸附树脂XD吸附、洗脱,将获取的抗菌产物样品与表面活性素标准品进行薄层色谱(TLC)层析、分析、比较。运用液质联用(LC-MS)分析技术对代谢产物样品中各组分的含量、分子量及结构进行分析。结果显示:GD株在发酵过程中产生的抗菌物质是以表面活性素A、B和C等同系物为主组成的脂肽混合物。表面活性剂含量达77.39%,分子量主要分布在1 036.6933～994.6449之间。
The Bacillus subtilis strains capable of producing antibacterial lipopeptide were selected by cylinder-plate method, with the pathogenic Escherichia coli as the main indicator. According to the experimental results, on the basis of determining the isolate(GD) as an antimicrobial substance, using the fermentation broth of GD strain. After fermentation broth of GD strain adsorbed and eluted by macroporous resin XD in the study, products obtained above and standards of surfactin was carried out by thin layer chromatography(TLC) analysis and comparison, antibacterial substances in GD strain metabolites was proved as cyclic lipopeptide compounds surfactin; and through analysis of content, molecule and structure of metabolites by means of liquid chromatography and mass spectroscopy(LC-MS).The results showed that the antimicrobial substance produced by GD strain during fermentation was A mixture of lipopeptide mainly composed of homolog A, B and C. Surfactin content reached 77.39%, and the molecular weight was mainly distributed between 1 036.6933 to 994.6449.
The Bacillus subtilis strains capable of producing antibacterial lipopeptide were selected by cylinder-plate method, with the pathogenic Escherichia coli as the main indicator. According to the experimental results, on the basis of determining the isolate(GD) as an antimicrobial substance, using the fermentation broth of GD strain. After fermentation broth of GD strain adsorbed and eluted by macroporous resin XD in the study, products obtained above and standards of surfactin was carried out by thin layer chromatography(TLC) analysis and comparison, antibacterial substances in GD strain metabolites was proved as cyclic lipopeptide compounds surfactin; and through analysis of content, molecule and structure of metabolites by means of liquid chromatography and mass spectroscopy(LC-MS).The results showed that the antimicrobial substance produced by GD strain during fermentation was A mixture of lipopeptide mainly composed of homolog A, B and C. Surfactin content reached 77.39%, and the molecular weight was mainly distributed between 1 036.6933 to 994.6449.
引文
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[24]HATHOUT Y,P HO Y,V RYZHOV,et al.Kunstakins:a new class of lipopeptides isolated from Bacillus thuringiensis[J].J Nat Prod,2002(63):1492-1496.
[25]CHUNG Y R,H KIM C,I HWANG,et al.Paenibacillus koreensis sp.nov.,a new species that produces an iturinlike antifungal compound[J].Int J Syst Evol Micr,2000(50):1495-1500.
[2]STEIN T.Bacillus subtilis antibiotics:Structures,syn-theses and specific functions[J].Mol Microbiol,2005,56(4):845-857.
[3]高学文,姚仕义,PHAM H.枯草芽孢杆菌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):647-653.
[4]PEYPOUX F,GUIMAND M,MICHEL G,et al.Structure of iturin A,a peptidolipid antibiotic from Bacillus subtilis[J].Biochemistry,1978,17(19):3992-3996.
[5]PEYPOUX F,BONMAIN J M,WALLACH J.Recent trends in the biochemistry of surfactin[J].Appl Microbiol Biot,1999(51):553-563.
[6]VANITTNAKOM N,KOCH U,SOOJUNG G,et al.Fengycin-A novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3[J].J Antibiot,1986,39(7):888-901.
[7]AKPA E,JACQUES P,WATHELET B,et al.Influence of culture conditions on lipopeptide production by Bacillus subtilis[J].Appl Biochem Biotechnol,2001(91-93):551-561.
[8]KOSARIC N,CAIMS W L,et al.Biosurfactants and Biotechnology[M].Bosa Roca:Taylor&Francis Inc,1987:21245.
[9]BODANSZKY M,PERLMAN D.Peptide Antibiotics[J].Science,1969,163(3865):352-358.
[10]BULLA L A J R,HOCH J A.Biology of the Bacilli[M]//DEMAIN A L,SOLOMN N A.Biology of industrial microorganisns,1985:57-78.
[11]WU H J,WANG S,QIAO J Q,et al.Expression of HpaGXooc protein in Bacillus subtilis and its biological functions[J].J Microbiol Biotechnol,2009,19(2):194-203.
[12]郝士海.现代细菌学培养基和生化试验手册[M].北京:中国科学技术出版社,1992.
[13]徐积恩,朱明珍.抗生素[M].北京:科学出版社,1982:152.
[14]吕应年,杨世忠,牟伯中,等.一种脂肽类生物表面活性剂产生菌的筛选[J].微生物杂志,2005,25(2):428.
[15]冯大兴.抗菌脂肽分离纯化的研究[J].现代畜牧兽医,2015(11):9-16.
[16]庄国宏,宋涛,陆金荣,等.抗鸡源大肠杆菌的枯草芽孢杆菌鉴定及代谢产物理化特性研究[J].中国家禽,2014,36(12):15-18.
[17]HIRADATE S,YOSHIDA S,SUGIE H,et al.Mulberry anthracnose antagonists(lttirins)produced by Bacillus amyloliquefaciens RC-2[J].Phytichemistry,2002(61):693-698.
[18]YOSHIDA S,HIRADATE S,TSULAMOTO T,et al.Antimierobial activity of culture filtrate of Bacillus amyloliquefaciens RC-2 isolated from mulberry leaves[J].Phytopathology,2001(91):181-187.
[19]YU G Y,B SINCLAIR J,L HARTMAN G,et al.Production of iturinA by Bacillus amyloliquefaciens suppressing Rhizoetonia solani[J].Soil Biol Biochem,2002(34):955-963.
[20]MASAAKI MORIKAWA,ITO MIKITO,IMANAKA TADA-YUKL.Isolation of a new surfactin producer Bacillus pumilus A-l,and cloning and nucleotide sequence of the regulator gene,psf-1[J].J Ferment Bioeng,1992,74(5):255-261.
[21]HOROWITZ S,M GTIFFIN W.Structural analysis of Bacillus licheniformis 86 surfactant[J].J Ind Microbiol Biot,1991(7):45-52.
[22]NISHIKORI T,H NAGANAWA,Y MURAOKA,et al.Plipastatins:New inhibitors of phospholipase A2,produced by Bacillus cereus BMG302-fF67.111.Structural elucidation of plipastatins[J].J Antibiot,1986(39):755-761.
[23]KIM P I,BAI H,BAI D,et al.Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26[J].J Appl Microbiol,2004(97):942-949.
[24]HATHOUT Y,P HO Y,V RYZHOV,et al.Kunstakins:a new class of lipopeptides isolated from Bacillus thuringiensis[J].J Nat Prod,2002(63):1492-1496.
[25]CHUNG Y R,H KIM C,I HWANG,et al.Paenibacillus koreensis sp.nov.,a new species that produces an iturinlike antifungal compound[J].Int J Syst Evol Micr,2000(50):1495-1500.