Stability Analysis, Isolation and Purification of Antimicrobial Protein From Brevibacillus laterosporus BL-21

doi:10.11924/j.issn.1000-6850.casb16110071

Abstract

Abstract: To clarify the type of antimicrobial substance produced by Brevibacillus laterosporus BL-21, the stability of the fermentation liquor was analyzed, and the active ingredients were isolated and purified based on this. The thermostability, pH stability and protease stability of antimicrobial metabolites from B laterosporus BL-21 were detected by cylinder plate method. The antimicrobial substances were isolated and purified by ammonium sulfate precipitation, CM- Sepharose Fast Flow ion exchange chromatography, gel filtration chromatography and SDS- PAGE. The results showed that the antimicrobial substance produced by B laterosporus BL-21 was stable below 50℃, its antifungal activity was the best at pH 7-8, and it was sensitive to protease. Several antimicrobial components were isolated from the fermentation liquor, the molecular weight of one of the components was about 20.1 kDa. Fermentation liquor of B. laterosporus BL-21 could inhibit the growth of Alternaria solani and Bacillus cereus, so it was valuable for the research on its application potentiality in the field of biological control.

CLC Number:

Q939.92

Zhang Miao,Zhou Limei,Xu Deyang and Du Chunmei. Stability Analysis, Isolation and Purification of Antimicrobial Protein From Brevibacillus laterosporus BL-21[J]. Chinese Agricultural Science Bulletin, 2017, 33(15): 43-48.

References

[1] 陈中义,张杰,黄大昉,等.植物病害生防芽孢杆菌抗菌机制与遗传改良研究[J].植物病理学报,2003,33(2):97-103.
[2] Li G, Jie X, Wu L, et al. Full genome sequence of Brevibacillus laterosporus strain B9, a biological control strain isolated from Zhejiang, China[J].Journal of Biotechnology,2015,207:77-78.
[3] 王志新,张丹,李兴峰,等.侧孢短芽孢杆菌S62-9的生物学特性研究[J].现代食品科技,2016,32(5):52-58.
[4] Montaldi F A, Roth I L. Parasporal bodies of Bacillus laterosporus sporangia[J].Journal of Bacteriology,1990,172(4):2168-2171.
[5] Shida O, Takagi H, Kadowaki K, et al. Proposal for two new genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov[J].International Journal of Systematic Bacteriology,1996,46(4):939-46.
[6] Marche M G, Mura M E, Ruiu L. Brevibacillus laterosporus, inside the insect body: Beneficial resident or pathogenic outsider?[J].Journal of Invertebrate Pathology,2016,137:58-61.
[7] Pessanha R R, Carramaschi I N, Mallet J R D S, et al. Evaluation of larvicidal activity and effects on post embrionary development of laboratory reared Lucilia cuprina, (Wiedemann, 1830) (Diptera:Calliphoridae), treated with Brevibacillus laterosporus[J].Journal of Invertebrate Pathology,2015,128:44-46.
[8] 任召珍,孙谧,郑媛,等.海洋侧孢短芽孢杆菌Lh-1株所产多肽R-1性质分析及作用原理探讨[J].海洋科学,2010, 34(11):41-45.
[9] Sharma V, Singh P K, Midha S, et al. Genome sequence of Brevibacillus laterosporus strain GI-9[J].Journal of Bacteriology,2012,194(5):1279.
[10] 张楹.侧孢芽孢杆菌产生的抑真菌蛋白酶[J].中国生物防治,2006,22(2):146-149.
[11] 刘蒲临,程德勇,缪礼鸿.产几丁质酶侧孢短芽孢杆菌的筛选及其酶学性质研究[J].生物技术通报,2016,32(6):174-180.
[12] 陈潺,陈升富,王建宇,等.侧孢短芽孢杆菌的应用研究进展[J].山东农业科学,2015,47(2):149-156.
[13] Azizbekyan R R, Didenko L V, Smirnova T A, et al. Biofilm formation and sporulation in Brevibacillus laterosporus[J].Applied Biochemistry & Microbiology,2015,51(9):866-872.
[14] Todd Barsby, Michael T K. Tupuseleiamides and Basiliskamides, New Acyldipeptides and Antifungal Polyketides Produced in Culture by a Bacillus laterosporus Isolate Obtained from a Tropical Marine Habitat[J].Journal of Neuroscience Research,2002,65(10):1447-1451.
[15] 任召珍,郑媛,孙谧,等.海洋侧孢短芽孢杆菌Lh-1抗菌活性物质的分离及特性研究[J].微生物学报,2007,47(6):997-1001.
[16] Zhao J, Guo L, Zeng H, et al. Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60[J].Peptides,2012,33(2):206-211.
[17] 余惠荣,李悦,李晓菲,等.侧孢短芽孢杆菌B8抑菌物质的理化性质及其分离纯化[J].沈阳农业大学学报,2015,46(4):398-403.
[18] 李悦,余惠荣,李蔚,等.侧孢短芽孢杆菌B8对两种植物病原菌抗菌机制初步研究[J].中国植保导刊,2015,35(3):12-16.
[19] 陈武,彭曙光,周清明,等.侧孢芽孢杆菌2-Q-9菌株外泌抗菌肽的纯化与鉴定[J].湖南农业大学学报:自然科学版,2011,37(1):26-30.
[20] 黎定军,陈武,罗宽.侧孢芽孢杆菌2-Q-9外泌抑菌物质性质[J].湖南农业大学学报:自然科学版,2007,33(4):471-474.
[21] Thongwai N, Kunopakarn J. Growth inhibition of Ralstonia solanacearum PT1J by antagonistic bacteria isolated from soils in the northern part of Thailand[J].Chiang Mai J. Sci,2007,34(3):345-354.
[22] Jiang H, Wang X, Xiao C, et al. Antifungal activity of Brevibacillus laterosporus JX-5 and characterization of its antifungal components[J].World Journal of Microbiology & Biotechnology,2015,31(10):1-14.
[23] 马俊美,宁亚维,王志新,等.侧孢短芽孢杆菌抗菌肽的结构与性质[J].食品与生物技术学报,2016,35(6):629-634.
[24] 王岩,王红英,钱斯日古楞,等.海洋侧孢短芽孢杆菌的分离鉴定及其抗菌活性物质初步研究[J].工业微生物,2014,44(6):28-33.
[25] Saikia R, Gogoi D K, Mazumder S, et al. Brevibacillus laterosporus, strain BPM3, a potential biocontrol agent isolated from a natural hot water spring of Assam, India[J].Microbiological Research,2011,166(3):216-225.
[26] Kakar K U, Nawaz Z, Cui Z, et al. Characterizing the mode of action of Brevibacillus laterosporus B4 for control of bacterial brown strip of rice caused by Aavenae subsp avenae RS-1[J].World Journal of Microbiology & Biotechnology,2013,30(2):469-478.
[27] 杨倩,于宏伟,郭润芳,等.侧孢短芽孢杆菌S62-9产抗菌物质的分离纯化及部分特性的研究[J].河北农业大学学报,2010,33(2):74-78.
[28] 赵秀香,张春波,赵柏霞,等.侧孢短芽孢杆菌B8发酵液中抑菌物质理化性质研究[J].河南农业科学,2011,40(6):90-93.