S tudy on Purification and Properties ofAnti bacterialSubstance from Antago-nistic Bacteria P72 againstWheatScabPEITao, REN Da-ming*, SHI JiaoBiological Science& Technology College, Shenyang Agricultural University, Shenyang 110161Abstract [Objective] The study ai med to explore the antibacterial activity and stability ofantagonisticbacteriaP72 againstwheat scab. [M ethod] TheBacillus subtilis P72 was inoculated into fermentationmediawith the inoculation amount 5%, then cultured on shaking tableat 28 for 48 h and centr-ifuged at 8 000 r/m in for 10 min. The supernatant of fermentation brothwas purified and then the genetic stability, thermal stability and pH stabilityweredetected. [Result] ByDEAE-52 ion exchange chromatography, theprotein eluted by0. 5mol/L NaC l solution possessed the strongest antagonistic abilityagainst wheat scab pathogen. The SDS-PAGE electrophoresis showed that themolecularweight of the purified protein with antibacterial effectwas 40 kD.By stability tes,t the antibacterial substanceproduced by P72 strain showed heritable antagonistic activity, high stability below 60 , stability to acid andunstability to alkal. i [Conclusion] The antagonisticbacteria P72 had strong antagonistic ability towheat scab pathogen, stable antibacterial activity andthermal stability, so itwould possess awide development prospec. tKey words Wheat scab; Antibacterial substance; Isolation and purificationR D , y ,* @ 6Scab is a worldwide wheat disease as well as the maindisease in wheat production ofCh ina. As the commonly usedmethod for controlling wheat scab presently, chem ical pest-icides have already caused serious environmental problems,and furthermore, long- term use of them would induce resis- tant pathogenic strains, making itmore difficult to control thedisease effectively[1] .In view of the absence ofwheat varie-ties with h igh resistance to scab, biological control hasuniversal practical significance. Therefore, the author isola-ted and purified an antibacterial substance from Bacillus sub-tilis P72, and then studied some of its properties.Ma te rials and MethodsM aterialsBacterial strains Antagonistic bacteria P72 strain (Bacillussubtilis P72) against wheat scab and Fusarium graminearumwere provided by theBioengineeringLab ofBiologicalScience& Technology College, ShenyangAgriculturalUniversity.M edium Fermentation medium for experm i ental bacteria:1% cornmea,l 0. 5% yeast extrac, t 0. 075% K 2 HPO 4 , 0. 06%NaC. l PDA medium[ 2] :20. 0 g sucrose, 200 g potato,20. 0 g agar, 1 000 ml water , natural pH. The PDA mediawere sterilized at 121 and 0. 1MPa for 30 m in and usedfor the determination ofantagonism.Reagents and instrum ents DEAE- 52 cellulose was pur -chased from Whatman Corporation, and other conventionalchem ical reagents were domestic analytically pure. Themainequ ipmentswere as follows: electric heating constant temper -ature incubator(ShanghaiYuejinMed ical Instruments Facto-ry), HD- 21-2 UV detector ( Shanghai Qingpu-Huxi Instru-ments Factory), TGL-16B high speed centrifuge ( ShanghaiAnting Science Instruments Factory), lam inar flow cab inet(Shanghai Zhengxin E lectronic Equipment Factory), HD-A(S Q x I Ftory), DHL-A constant flow pump (ShanghaiQingpu-HuxiInstrumentsFactory).M ethodsPreparation of ferm en tation filtrate B. subtilis P72 wasfermented under the following conditions: inoculation amount5%, 100ml fermentationmedia in 250m l flask, 28 , sha-king culture for 48 h. Then centrifugation was carried out at8 000 r/min for 10m in to get the supernatant .An tibacterial activity determ ination of the antibacterialsubstance Two oxford cups were placed on the centre ofPDA plate, each ofwh ich accepted 200 l fermentation fi-ltrate. Then F. graminearum was inocu lated at 2. 5 cm awayfrom the edge of oxford cup. After cultured at 28 for 72h, the distance between the colony edge and the oxford cupedge wasmeasured. Three replications weremade[ 3] .Isolation and purification of the antibacterial substanceBy slowly adding solid ammonium sulfate, the fermentationsupernatantwas adjusted to 70% saturation. After stand ingfor 15m in and centrifugation at 8 000 r/m in for20m in, thesupernatantwas obtained, wh ich was then superadded withammon ium sulfate to attain 80% saturation, followed byovern ight standing at 4 and centrifugation at 8 000 r/m infor 20m in. W ith the supernatant abandoned, the precipitatewas redissolved by proper amount ofdistilled water , and thendialysed in d istilledwater overnight . The liquid in the dialy-sis bag was concentrated to 1/10 of the original volume bypolyethylene glycol (PEG) 20000. Subsequently, 5 ml con-centrated solutionwas taken forDEAE-52 ion exchange chro-matography. At the flow rate of0. 5 ml/m in, chromatographycolumn was eluted by 0. 02, 0. 1, 0. 5 and 1. 0 mol/L NaClsolution in turn. The eluent was detected at 280 nm UVwavelength, and the elution peaks were collected and con-centrated for antibacterial activity determ ination[ 4- 5] .Thenthe eluent showing antibacterial activity was concentrated tof f f y SDSG[ 6]G y ff NP lant P rotectionAgriculturalS ci ence& Technology, 2008, 9(6): 124- 126Copyright 2008, Information Ins titute ofHAAS. All rights reserved.eceived: ecember1 2008 Accepted: Januar 14 2009Corresponding author . E-mai: l rendam ing 12 . comcomputergathering hanghai ingpu-Hu i nstruments ac-1/20 o the original volume or puri ication detection b -PA E .enetic stabilit detection o P72 P72 strainwas subcu-ltured or continuous ten tm i es on A slantwith the interval of4 d. The strains obtained from the 2nd , 4 th ,6th ,8thand10thsubculture were uniformly activated on NA slants, andstrain platesweremade by a puncher ( d= 5mm). With du-al culturemethod, the antagonistic distance ofP72 strain wasmeasured after indicator bacteria inoculation and culture at28 for 72 h.Thermal stab ility detection of the supernatant of ferm en-tation broth The supernatant of fermentation broth was re-spectively treated under 40, 60, 80, 100 and 120 for 20or 40m in, 3m l for each temperature[ 7- 8] . Then the antibac-terial effects were determined according to the method men-tioned above.pH stab ility detection of the supernatant of ferm entationbroth The supernatant ofP72 fermentation broth was ad-justed to pH 2 with 1 mol/L HCl or pH 10 with 1 mol/LNaOH. After standing for 12 h, the supernatant and precip-itate were collected respectively, and the latterwas dissolvedwith the equivalent volume of distilled water. Then pH va- lues of the supernatant and the precipitate dissolved solutionwere adjusted to the same value as the original fermentationsupernatant for the determ ination of antibacterial effects.Results and Ana lysisIsolation and purification of the antibacterial substanceAfter ammonium sulfate salting-out , dialysis and con-centration for the P72 strain fermentation broth, three elutionpeakswere obtained byDEAE-52 ion exchange chromatogra-phy (Fig. 1), among which the 2ndone showed antibacterialeffects (F ig. 2), indicating the protein eluted by 0. 5 mol/LNaCl had antibacterial activity. The eluent collected at theelution peak showing antibacterial effectwas concentrated forSDS-PAGE electrophoresis. The fermentation broth samplescollected after salting-out and dialysis were also detected atthe same tm i e. The results showed that the purified antibac-terial substance presented single band ( Fig. 3) and its mo-lecularweight was about 40 kD by comparing with standardprotein (Fig. 4).F ig. 1 TheDEAE-Cellulose ion exchange chrom atography of theantibacterial substanceG yD f ,(T ),ffTable1 Stability test of P72 antibacterial effect and supernatant offerm entation broth at different temperaturesSubcultureti mesAntibacterialdistancemmTemperatureAntibacterial distance mmHeated for20 minH eated for40m in2 11. 8 20 16. 37 16. 414 11. 6 40 16. 58 16. 296 12. 1 60 15. 78 16. 238 11. 6 80 10. 77 9. 9810 11. 9 100 8. 87 7. 54120 5. 32 6. 09Therm al stabilityT y ff 6 ,f f (T ),y 6125 PEITao et al . Study on Purification and Properties ofAntibacterial Substance from Antagonistic BacteriaP72 againstWheat Scabenetic stabilituring the process o continuous subculture the ant-ibacterial distance changed little able 1 indicating P72strain had stable antibacterial e ects.he activit o the antibacterial protein was al most in-variant rom 20 to 0 and then began todecreasewith theurther increase o temperature able1 suggestingthe an-tibacterial protein had a high stabilit below 0 . pH stab ilityAfter treated with acid and alkal,i the supernatant ofP72 fermentation brothwas found to be stable to acid and un-stable to alkali (Table 2).Table 2 Antagon istic ability of P72 antibacterial substance treatedwith acid and alkaliTreat mentAntibacterial distance mmSupernatant PrecipitateHCl 10. 37 5. 56NaOH 0 15. 78Conclus ionBy ion exchange chromatography, the protein eluted by0. 5 mol/L NaCl solution showed the best antagonistic effectagainstF. graminearum, indicating that its polypeptides car -ried plenty ofnegative charges and had strong binding capac-ities with an ion exchanger . The SDS-PAGE electrophoresisshowed that themolecularweight ofthe protein with antibac-terial effect was about 40 kD. Through the stability test , itcould be found that the antibacterial substance produced byP72 strain had heritable antagonistic activity, h igh stabilitybelow 60 , stability to acid and unstability to alkal,iwhich was corresponding with the study ofWANG Zhang-m ing et al[ 9] .References[1] LIUWC, PANHY, XI JH, et al. Biocontrol effects of some antifungalstrains from Bucillus against pathogens ofwheat head blight[ J]. ActaTritical Crops, 2005, 25(4): 95- 100. ( in Chinese).[2] PELCZAR MJ, REID RD, CHAN ECS. M icrobiology[M]. Beijing: Sc-iencePress, 1987. ( in Chinese).[3] LI U ZH. Modern microbiology[M]. Beijing: Science Press, 2002. ( inChinese).[4] SHEN JY, YINWL, CAO Z, et al . Purification and partial characteriza-tion of antagonistic substance from Bacillus subtilus B115 [ J]. ActaHydrobiologica Sinica, 2005, 29(6): 689- 693. ( in Chinese).[5] BAIGH, PLATTNER R, DESJARDINSA, etal . Resistance toF usariumhead blight and deoxynivalenol accumulation in wheat[ J]. Plant Breed-ing, 2001, l20(1): l- 6.[6] WANG JX, SUIXZ, CHEN YH, et al . Research on themolecularweightdetermination ofsmall polypeptides with SDS-PAGE [J]. Silk Monthly,2001, 12: 44- 45. ( in Chi nese).[7] YE HZ, YU GR, YAN JM, et al. Studies on biological control ofwheathead blightwith Bacillus subtilis . Themechanisms of biocontrol headblight scab with B4 and B6 strain[ J]. Journal ofSichuanAgriculturalU-niversity, 2003, 21(1): 18- 22. ( in Chinese).[8] PAULITZTC, BELANGER RR. Biological control in green-house systems[J]. Ann RevPhytopathology, 2001, 39: 103- 133.[9] WANG ZM, CHENQ, HAN CX, et al. Stability and effect ofpathogenicaction on antagonisticbacteria ofRhizoctonia cerealis[J]. Journal ofAn-huiAgricultural Sciences, 2006, 34(4): 702, 809. ( in Chinese).小麦赤霉病拮抗菌 P72抗菌物质的分离纯化和性质研究裴 韬, 任大明*, 石 皎 (沈阳农业大学生物科学技术学院, 辽宁沈阳 110161)摘要 [目的 ]研究小麦赤霉病拮抗菌 P72的抑菌活性及稳定性。[方法]将Bacillus subtilis P72接种于发酵培养液中, 接种量 5%, 28摇床培养 48 h后, 8 000 r/min, 离心 10 min取菌株发酵离心所得上清液进行分离纯化、遗传稳定性、热稳定性及酸碱稳定性测定。P72菌株发酵上清液经过盐析、透析后进行 DEAE- 52离子交换层析, 用 0. 5 mol/L NaCl溶液洗脱的蛋白对小麦赤霉病菌的拮抗能力最强。SDS- PAGE电泳显示具有抗菌作用的蛋白分子量约为 40 kD。经过稳定性的试验测定, 该抗菌物质的拮抗活性具有遗传稳定性, 在 60以下有较高的稳定性, 无菌液对酸具有稳定性, 在碱性溶液中不具有稳定性。 [结论 ]小麦赤霉病拮抗菌 P72对小麦赤霉病菌的拮抗能力较强, 抗菌活性较稳定, 且热稳定性较好, 有较广阔的开发前景。关键词 小麦赤霉病;抗菌物质;分离纯化作者简介 裴韬 (1982- ), 男, 河北盖县人, 硕士研究生, 研究方向: 细胞生物学研究。* 通讯作者。收稿日期 2008-12-01 修回日期 2009-01-14Instructions for AuthorsCon tent requirem en ts AgriculturalScience＆ Technologymainly publishes the original and unpublished research pa-pers, research notes, speed dispatch, agriculturalnew technology, etc, which are related to the applying ofthed isciplines ofagricultural sciences in application basis, h igh tech and other aspects. 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