解淀粉芽孢杆菌TF28抗菌脂肽芬芥素的分离鉴定及抑菌作用 <sup>*</sup>

doi:10.13523/j.cb.20181003

中国生物工程杂志

2018, Vol. 38

Issue (10): 20-29 DOI: 10.13523/j.cb.20181003

研究报告

解淀粉芽孢杆菌TF28抗菌脂肽芬芥素的分离鉴定及抑菌作用 ^*

刘宇帅^1,²,张杰³,钟瑾³,李晶^1,²,孟利强^1,²,张淑梅^1,^2,^**(

)

1 黑龙江省科学院微生物研究所哈尔滨 150010 2 黑龙江省科学院高技术研究院哈尔滨 150020
3 中国科学院微生物研究所北京 100101

Isolation and Identification of Antibacterial Lipopeptides Fengycin Produced by Bacillus amyloliquefaciens TF28 and Its Anti-fungal Mechanism Studies

Yu-shuai LIU^1,²,Jie ZHANG³,Jin ZHONG³,Jing LI^1,²,Li-qiang MENG^1,²,Shu-mei ZHANG^1,^2,^**(

)

1 Institute of Microbiology of Heilongjiang Academy of Sciences, Harbin 150010, China
2 Institute of Advanced Technology, Heilongjiang Academy of Sciences, Harbin 150020, China

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摘要：

对一株解淀粉芽孢杆菌TF28产生的抗菌脂肽进行分离鉴定及抑菌活性研究,采用酸沉淀、乙酸乙酯和甲醇萃取技术制备了抗菌脂肽粗提物,经过2次HPLC分离纯化,在保留时间32~42min内获得8个抗菌脂肽纯品,经MALDI-TOF-MS鉴定为芬芥素(fengycins),对尖孢镰刀菌和禾谷镰刀菌显示出较强的抑菌活性,该研究为提高菌株TF28抗菌脂肽产量的定向遗传改造奠定基础。

关键词： 解淀粉芽孢杆菌; 尖孢镰刀菌; 禾谷镰刀菌; 抗菌脂肽; 芬芥素

Abstract:

The antifungal lipopeptides produced by Bacillus amyloliquefaciens TF28 have been isolated and identified and their antifungal activity have been studied. The extract of antifungal lipopeptides was prepared by acid precipitation, ethyl acetate and methanol extraction. After two rounds of HPLC separation, eight antifungal lipopeptides were obtained within 32~42 min of retention time, and they have been identified as fengycins by MALDI-TOF-MS, which show strong antifungal activity against Fusarium oxysporum and Fusarium graminearum. These results will contribute to the directional genetic manipulation of the strain TF28 to improve the yield of antifungal lipopeptides.

Key words: Bacillus amyloliquefaciens Fusarium oxysporum Fusarium graminearum Antifungal lipopeptides Fengycin

收稿日期: 2018-05-14 出版日期: 2018-11-09

ZTFLH:

Q939

基金资助: * 中国科学院微生物研究所微生物资源前期开发国家重点实验室开放课题(SKLMR-20150604);黑龙江省省院科技合作项目(YS16C15);黑龙江省科学院青年创新基金面上项目(CXMS2017SW01)

通讯作者: 张淑梅 E-mail: 1401135157@qq.com

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引用本文:

刘宇帅,张杰,钟瑾,李晶,孟利强,张淑梅. 解淀粉芽孢杆菌TF28抗菌脂肽芬芥素的分离鉴定及抑菌作用 ^*[J]. 中国生物工程杂志, 2018, 38(10): 20-29.

Yu-shuai LIU,Jie ZHANG,Jin ZHONG,Jing LI,Li-qiang MENG,Shu-mei ZHANG. Isolation and Identification of Antibacterial Lipopeptides Fengycin Produced by Bacillus amyloliquefaciens TF28 and Its Anti-fungal Mechanism Studies. China Biotechnology, 2018, 38(10): 20-29.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181003 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I10/20

图1 菌株TF28脂肽粗提物对病原真菌的抑菌作用

图2 菌株TF28脂肽粗提物的HPLC分析

图3 HPLC第一次分离纯化部分组份对尖孢镰刀菌的抑菌效果

图4 3个活性组份第二次HPLC分离纯化结果

图5 第二次分离纯化各组份的抑菌活性

表1 抗菌脂肽MALDI-TOF-MS的结果与分析

图6 菌株TF28的8个活性组分MALDI-TOF-MS的一级质谱图

图7 菌株TF28的8个活性组分MALDI-TOF-MS的二级质谱图

[1]	车晓曦, 李社增, 李校堃 , 等. 1株解淀粉芽孢杆菌发酵培养基的设计及发酵条件的优化. 安徽农业科学, 2010,38(18):9402-9405. doi: 10.3969/j.issn.0517-6611.2010.18.008
	Che X X, Li S Z, Li X K , et al. Research on the design of the medium for the Amyloliquefaciens bacillus fermentation and optimization of its fermentation condition. Journal of Anhui Agricultural Sciences, 2010,38(18):9402-9405. doi: 10.3969/j.issn.0517-6611.2010.18.008
[2]	郝建安, 曹志辉, 赵凤梅 , 等. 解淀粉芽孢杆菌NK10.BAhjaWT 抑真菌作用的研究. 微生物学通报, 2008,35(6):903-908. doi: 10.3969/j.issn.0253-2654.2008.06.013
	Hao J A, Cao Z H, Zhao F M , et al. Exploring the antifungal activity of Bacillus amyloliquefaciens NK10.BAhjaWT. Microbiology China, 2008,35(6):903-908. doi: 10.3969/j.issn.0253-2654.2008.06.013
[3]	Niazi A, Manzoor S, Bejai S , et al. Complete genome sequence of a plant associated bacterium Bacillus amyloliquefaciens subsp. plantarum UCMB5033. Stand Genomic Sci, 2014,9(3):718-725. doi: 10.4056/sigs.4758653 pmid: 4148973
[4]	吴一晶, 林艺芬, 林河通 , 等. 生防菌解淀粉芽孢杆菌研究进展. 包装与食品机械, 2012,30(6) : 49-52. doi: 10.3969/j.issn.1005-1295.2012.06.013
	Wu Y J, Lin Y F, Lin H T , et al. Advances in the researches of biocontrol bacteria Bacillus amyloliquefaciens. Packaging and Food Machinery, 2012,30(6) : 49-52. doi: 10.3969/j.issn.1005-1295.2012.06.013
[5]	张龙来, 康向辉, 魏孝义 , 等. 1株解淀粉芽孢杆菌 HN011抑菌次级代谢产物的分析. 华南农业大学学报, 2016,37(1) : 63-69.
	Zhang L L, Kang X H, Wei X Y , et al. Research on secondary metabolites from Bacillus amyloliquefaciens strain HN011. Journal of South China Agricultural University, 2016,37(1) : 63-69.
[6]	Stein T . Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol Microbiol, 2005,56(4):845-857. doi: 10.1111/j.1365-2958.2005.04587.x
[7]	Montesinos E . Antimicrobial peptides and plant disease control. FEMS Microbiol Lett, 2007,270(1):1-11. doi: 10.1111/j.1574-6968.2007.00683.x pmid: 17371298
[8]	汪静杰, 赵东洋, 刘永贵 , 等. 解淀粉芽孢杆菌 SWB16 菌株脂肽类代谢产物对球孢白僵菌的拮抗作用. 微生物学报, 2014,54(7) : 778-785. doi: 10.13343/j.cnki.wsxb.2014.07.008
	Wang J J, Zhao D Y, Liu Y G , et al. Antagonism against Beauveria bassiana by lipopeptide metabolites produced by entophyte Bacillus amyloliquefaciens strain SWB16. Acta Microbiologica Sinica, 2014,54(7) : 778-785. doi: 10.13343/j.cnki.wsxb.2014.07.008
[9]	Ongena M, Jacques P . Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol, 2008,16(3):115-125. doi: 10.1016/j.tim.2007.12.009
[10]	Alvarez F, Castro M, Principe A , et al. The plant-associated Bacillus amyloliquefaciens strains MEP2 18 and ARP2 3 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease. J Appl Microbiol, 2012,112(1):159-174. doi: 10.1111/j.1365-2672.2011.05182.x pmid: 22017648
[11]	张淑梅, 沙长青, 王玉霞 , 等. 大豆内生细菌的分离及根腐病拮抗菌的筛选鉴定. 微生物学通报, 2008(10):1593-1599. doi: 10.3969/j.issn.0253-2654.2008.10.016
	Zhang S M, Sha C Q, Wang Y X , et al. Isolation and characterization of antifungal endophytic bacteria from soybean. Microbiology China, 2008(10):1593-1599. doi: 10.3969/j.issn.0253-2654.2008.10.016
[12]	Zhang S, Jiang W, Li J , et al. Whole genome shotgun sequence of Bacillus amyloliquefaciens TF28, a biocontrol entophytic bacterium. Stand Genomic Sci, 2016,11:73. doi: 10.1186/s40793-016-0182-6 pmid: 5031281
[13]	Zhang S, Wang Y, Meng L , et al. Isolation and characterization of antifungal lipopeptides produced by endophytic Bacillus amyloliquefaciens TF28. African Journal of Microbiology Research, 2012,6(8) : 1747-1755.
[14]	刘宇帅, 孟利强, 陈静宇 , 等. 解淀粉芽胞杆菌 TF28 产抗菌脂肽培养基优化. 微生物学杂志, 2017,37(3) : 52-58. doi: 10.3969/j.issn.1005-7021.2017.03.010
	Liu Y S, Meng L Q, Chen J Y , et al. Optimization of medium for the production of antifungal lipopeptides by Bacillus amyloliquefaciens TF28. Journal of Microbiology, 2017,37(3) : 52-58. doi: 10.3969/j.issn.1005-7021.2017.03.010
[15]	Vater J, Kablitz B, Wilde C , et al. Matrix-assisted laser desorption ionization--time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol, 2002,68(12):6210-6219. doi: 10.1534/genetics.106.057034 pmid: 12450846
[16]	de Faria A F, Stefani D, Vaz B G , et al. Purification and structural characterization of fengycin homologues produced by Bacillus subtilis LSFM-05 grown on raw glycerol. J Ind Microbiol Biotechnol, 2011,38(7):863-871. doi: 10.1007/s10295-011-0980-1 pmid: 21607611
[17]	Wang J, Liu J, Wang X , et al. Application of electrospray ionization mass spectrometry in rapid typing of fengycin homologues produced by Bacillus subtilis. Lett Appl Microbiol, 2004,39(1):98-102. doi: 10.1111/j.1472-765X.2004.01547.x pmid: 15189295
[18]	李宝庆, 鹿秀云, 郭庆港 , 等. 枯草芽孢杆菌BAB-1产脂肽类及挥发性物质的分离和鉴定. 中国农业科学, 2010,43(17):3547-3554. doi: 10.3864/j.issn.0578-1752.2010.17.008
	Li B Q, Lu X Y, Guo Q G , et al. Isolation and identification of lipopeptides and volatile compounds produced by Bacillus subtilis strain BAB-1, Scientia Agricultura Sinica, 2010,43(17):3547-3554. doi: 10.3864/j.issn.0578-1752.2010.17.008
[19]	姬婧媛, 杨洁, 高小宁 , 等. 植物内生枯草芽孢杆菌E1R-j脂肽类化合物的分离鉴定及抑菌作用. 农药学学报, 2015,17(02):172-178. doi: 10.3969/j.issn.1008-7303.2015.02.08
	Ji J Y, Yang J, Gao X N , et al. Isolation and identification of lipopeptides produced by endophytic bacteria Bacillus subtilis E1R-j and its anti-fungal mechanism studies, Chinese Journal of Pesticide Science, 2015,17(02):172-178. doi: 10.3969/j.issn.1008-7303.2015.02.08
[20]	Deleu M, Paquot M, Nylander T . Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes. Biophys J, 2008,94(7):2667-2679. doi: 10.1529/biophysj.107.114090 pmid: 18178659
[21]	Arrebola E, Jacobs R, Korsten L . Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens. J Appl Microbiol 2010,108(2):386-395. doi: 10.1111/j.1365-2672.2009.04438.x pmid: 19674188
[22]	Maget-Dana R, Thimon L, Peypoux F , et al. Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A. Biochimie, 1992,74(12):1047-1051. doi: 10.1016/0300-9084(92)90002-V pmid: 1292612
[23]	Tao Y, Bie X M, Lv F X , et al. Antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer. J Microbiol, 2011,49(1):146-150. doi: 10.1007/s12275-011-0171-9 pmid: 21369992
[24]	Ongena M, Duby F, Jourdan E , et al. Bacillus subtilis M4 decreases plant susceptibility towards fungal pathogens by increasing host resistance associated with differential gene expression. Appl Microbiol Biotechnol, 2005,67(5):692-698. doi: 10.1007/s00253-004-1741-0 pmid: 15578181
[25]	Ongena M, Jourdan E, Adam A , et al. Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environ Microbiol, 2007,9(4):1084-1090. doi: 10.1111/j.1462-2920.2006.01202.x pmid: 17359279

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