枯草芽孢杆菌MB8儿茶酚型铁载体的合成与结构分析
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摘要
【目的】对枯草芽孢杆菌MB8合成铁载体的条件及铁载体结构进行了研究。【方法】通过正交实验设计分析了铁载体合成的条件,采用质谱分析等技术对铁载体的结构进行分析。【结果】当甘油浓度为4%(mL/L),精氨酸浓度为1.5 g/L,接种量为7%时,铁载体产量达到最大(SU=92.58%)。培养基中游离Fe~(3+)浓度的增加,铁载体产量逐渐下降;当Al~(3+)浓度为100μmol/L时,能够显著提高铁载体的合成。菌株MB8分泌的铁载体能够促进不溶性针铁矿的溶解,溶解速率比对照提高了6倍。最后,采用质谱分析等技术对菌株MB8分泌的铁载体结构进行了分析,结果显示MB8合成的铁载体与已知铁载体Bacillibactin结构相似。【结论】枯草芽孢杆菌MB8分泌的铁载体具有良好的抑菌作用、清除DPPH自由基的抗氧化能力和加速不溶性针铁矿溶解的能力。枯草芽孢杆菌MB8在多种领域可能具有较好的应用前景。
【Objective】The aim of this study is to optimize fermentation parameters and analyze the structure of the siderophore produced by Bacillus subtilis MB8. 【Method】The synthesis conditions of siderophore were analyzed by orthogonal design and the structure of siderophore was analyzed by mass spectrometry.【Result】The siderophore production was at the highest level(SU = 92.58 %) when the medium contained 40 mL/L glycerol, 1.5 g/L arginine, and 7 % inoculum. Along with the increased concentration of Fe~(3+) in medium, MB8 produced less and less siderophore. When Al~(3+) was supplemented at 100 μmol/L in medium, siderophore production were significantly increased. In biomining process the MB8 siderophore could significantly dissolve the ferrin ions from goethite, 6 times higher than the control experiment. Finally, with mass spectrometry analysis, the siderophore was identified with the structure similar to Bacillibactin. 【Conclusion】The siderophore produced by B. subtilis MB8 has good bacteriostatic effect, antioxidant ability of scavenging DPPH free radicals and the ability to accelerate the dissolution of insoluble goeite.B. subtilis MB8 may have good application prospects in many fields.
【Objective】The aim of this study is to optimize fermentation parameters and analyze the structure of the siderophore produced by Bacillus subtilis MB8. 【Method】The synthesis conditions of siderophore were analyzed by orthogonal design and the structure of siderophore was analyzed by mass spectrometry.【Result】The siderophore production was at the highest level(SU = 92.58 %) when the medium contained 40 mL/L glycerol, 1.5 g/L arginine, and 7 % inoculum. Along with the increased concentration of Fe~(3+) in medium, MB8 produced less and less siderophore. When Al~(3+) was supplemented at 100 μmol/L in medium, siderophore production were significantly increased. In biomining process the MB8 siderophore could significantly dissolve the ferrin ions from goethite, 6 times higher than the control experiment. Finally, with mass spectrometry analysis, the siderophore was identified with the structure similar to Bacillibactin. 【Conclusion】The siderophore produced by B. subtilis MB8 has good bacteriostatic effect, antioxidant ability of scavenging DPPH free radicals and the ability to accelerate the dissolution of insoluble goeite.B. subtilis MB8 may have good application prospects in many fields.
引文
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[19]郝莎莎,卫旭芳,侯智霞,等.文冠果根系瘤状物可培养内生细菌的特性[J].应用与环境生物学报,2018(4):1-12.
[20]赵树民,虞方伯,叶正钱,等.耐镉产铁载体菌株的分离及鉴定[J].环境污染与防治,2017,39(9):999-1002.
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[22]Yu X,C Ai,L.Xin,et al.The siderophore-producing bacterium,Bacillus subtilis CAS15,has a biocontrol effect on Fusarium wilt and promotes the growth of pepper[J].European Journal of Soil Biology,2011,47(2):138-145.
[23]Radhakrishnan M,K.J.Samshath,R.Balagurunathan.Hydroxamate Siderophore from Bacillus Sp SD12 Isolated from Iron Factory Soil[J].Current World Environment,2014(9):990-993.
[24]Heidarzadeh N,S.Baghaee-Ravari.Application of Bacillus pumilus as a potential biocontrol agent of Fusarium wilt of tomato[J].Archives of Phytopathology and Plant Protection,2015,48(13-16):841-849.
[25]Kejela T,V.R.Thakkar,P.Thakor.Bacillus species (BT42) isolated from Coffea arabica L.rhizosphere antagonizes Colletotrichum gloeosporioides and Fusarium oxysporum and also exhibits multiple plant growth promoting activity[J].BMC Microbiology,2016,16(1):277.
[26]Wilson M K,R.J.Abergel,K.N.Raymond,et al.Siderophores of Bacillus anthracis,Bacillus cereus and Bacillus thuringiensis[J].Biochemical and Biophysical Research Communications,2006,348(1):320-325.
[27]Temirov Y V,T.Z.Esikova,I.A.Kashparov,et al.A Catecholic Siderophore Produced by the Thermoresistant Bacilluslicheniformis VK21 Strain[J].Russian Journal of Bioorganic Chemistry,2003,29(6):542-549.
[28]Santos S,I.F.F.Neto,M.D.Machado,et al.Siderophore Production by Bacillus megaterium:Effect of Growth Phase and Cultural Conditions[J].Applied Biochemistry and Biotechnology,2014,172(1):549-560.
[2]Hider R C,Kong X.Chemistry and biology of siderophores[J].Natural Product Reports,2010,27(5):637-657.
[3]Chu B C,Herrero A G,Johanson T H,et al.Siderophore uptake in bacteria and the battle for iron with the host:a bird’s eye view[J].Biometals,2010,23:601-611.
[4]Crowley D E,Wang Y C,Reidz C P,et al.Mechanisms of iron acquisition from siderophores by microorganisms and plants[J].Plant and Soil,1991,130:179-198.
[5]Wire′n N,Khodr H,Hider R C.Hydroxylated phytosiderophore species possess an enhanced chelate stability and affinity for Iron(III)[J].Plant Physiology,2000,124:1149-1157.
[6]Yamanaka K,Oikawa H,Ogawa H O,et al.Desferrioxamine E produced by Streptomyces griseus stimulates growth and development of Streptomyces tanashiensis[J].Microbiology,2005,151(9):2899-2905.
[7]Yuqiang B,Hesterberg D L,Duck O W.Siderophore-promoted dissolution of cobalt from hydroxide minerals[J].Geochimica et Cosmochimica Acta,2010,74(10):2915-2925.
[8]Zhu H,H.Yang.Isolation and Characterization of a Highly Siderophore Producing Bacillus subtilis Strain,Berlin[J].Heidelberg,Springer Berlin Heidelberg,2015,332:83-92.
[9]Lin Y,Du D L,Si C C,et al.Potential biocontrol Bacillus sp.Strains isolated by an improved method from vinegar waste compost exhibit antibiosis against fungal pathogens and promote growth of cucumbers[J].Biological Control,2014,71:7-15.
[10]Pérez M S,Cabirol N,George T R,et al.O-CAS,a fast and universal method for siderophore detection[J].Journal of Microbiology Methods,2007,70(1):127-131.
[11]Arnow L E.Colorimetric determination of the components of 3,4-dihydroxy-phenylalaninetyrosine mixtures[J].Journal Biology Chemistry,1937,118:531-537.
[12]朱永平.生物统计学实验教程[M].北京:云南科技出版社,2006:231-234.
[13]Schwertmann U,Cornell R M.Iron oxides in the laboratory[J].Library of Congress Card,2000,10(4):341-345.
[14]Hersman L,Maurice P,Sposito G.Iron acquisition from hydrous Fe(III)-oxides by an aerobic Pseudomonas sp[J].Chemical Geology,1996,132:25-31.
[15]Schwertmann U,Cornell R M.Iron oxides in the laboratory[J].Library of Congress Card,2000,10(4):341-345.
[16]Camberos E P,Díaz E L,Fernandez J M,et al.Evaluation of the inhibition of carbohydrate hydrolyzing enzymes,the antioxidant activity and the polyphenolic content of citrus limetta peel extract[J].The Scientific World Journal,2014(4):1-4.
[17]Hotta K,Kim C Y,Fox D T,et al.Siderophore-mediated iron acquisition in Bacillus anthracis and related strains[J].Microbiology,2010,156:1918-1925.
[18]Achard M S,Kaiwen W C,Sweet M J,et al.An antioxidant role for catecholate siderophores in Salmonella[J].Biochemistry,2013,454:543-549.
[19]郝莎莎,卫旭芳,侯智霞,等.文冠果根系瘤状物可培养内生细菌的特性[J].应用与环境生物学报,2018(4):1-12.
[20]赵树民,虞方伯,叶正钱,等.耐镉产铁载体菌株的分离及鉴定[J].环境污染与防治,2017,39(9):999-1002.
[21]李艳梅,王琼瑶,涂卫国,等.镍胁迫下产铁载体细菌对花生的促生性[J].微生物学通报,2017,44(8):1882-1890.
[22]Yu X,C Ai,L.Xin,et al.The siderophore-producing bacterium,Bacillus subtilis CAS15,has a biocontrol effect on Fusarium wilt and promotes the growth of pepper[J].European Journal of Soil Biology,2011,47(2):138-145.
[23]Radhakrishnan M,K.J.Samshath,R.Balagurunathan.Hydroxamate Siderophore from Bacillus Sp SD12 Isolated from Iron Factory Soil[J].Current World Environment,2014(9):990-993.
[24]Heidarzadeh N,S.Baghaee-Ravari.Application of Bacillus pumilus as a potential biocontrol agent of Fusarium wilt of tomato[J].Archives of Phytopathology and Plant Protection,2015,48(13-16):841-849.
[25]Kejela T,V.R.Thakkar,P.Thakor.Bacillus species (BT42) isolated from Coffea arabica L.rhizosphere antagonizes Colletotrichum gloeosporioides and Fusarium oxysporum and also exhibits multiple plant growth promoting activity[J].BMC Microbiology,2016,16(1):277.
[26]Wilson M K,R.J.Abergel,K.N.Raymond,et al.Siderophores of Bacillus anthracis,Bacillus cereus and Bacillus thuringiensis[J].Biochemical and Biophysical Research Communications,2006,348(1):320-325.
[27]Temirov Y V,T.Z.Esikova,I.A.Kashparov,et al.A Catecholic Siderophore Produced by the Thermoresistant Bacilluslicheniformis VK21 Strain[J].Russian Journal of Bioorganic Chemistry,2003,29(6):542-549.
[28]Santos S,I.F.F.Neto,M.D.Machado,et al.Siderophore Production by Bacillus megaterium:Effect of Growth Phase and Cultural Conditions[J].Applied Biochemistry and Biotechnology,2014,172(1):549-560.