短短芽孢杆菌XDH的鉴定及其抗菌物质的分离、纯化与部分性质研究
|
摘要
a’311菌株是本实验室从泰山土壤分离获得的一株拮抗细菌。该菌株对多种动、植物病原细菌和真菌如:大肠杆菌、鸡巴氏杆菌、金黄葡萄球菌、黑胸败血病菌、棉花枯萎病菌、黄瓜枯萎病菌、茶轮斑病菌等有较强拮抗作用,该菌株抑菌谱广,菌株发酵性状优异,发酵液性质稳定,显示了诱人的开发前景。本文通过菌体形态特征观察、生理生化指标测定和16S rDNA序列分析,对该菌株进行了鉴定。选择了适合该菌株的发酵条件,并通过硫酸铵沉淀、Sephadex G-50柱层析、高效液相色谱(HPLC)和质谱(MS)分析,对抗菌物质进行了分离纯化,并研究了其部分性质。主要内容如下:
1通过菌体形态特征观察、生理生化指标测定、16S rDNA序列及其系统进化树分析,鉴定a’311菌株为短短芽孢杆菌(Brevibacillus brevis),命名为短短芽孢杆菌XDH。该菌株的16S rDNA序列已在美国Genbank注册,登录号为DQ279738。
2筛选到一个适合该菌株的发酵条件。培养基配方:葡萄糖16g,可溶性淀粉2.5g,蛋白胨10g,黄豆饼粉26g,玉米浆1.0g,NaCl7.5g,(NH4)2SO4 4.0g,蒸馏水1000mL。30℃,200rpm,装液量50mL/250mL,摇瓶振荡培养48h。
3发酵液经40%饱和度硫酸铵盐析和Sephadex G-50柱层析,可将有效物质与其他组分进行粗分离。以乙腈-0.1%TFA水溶液和甲醇-0.4%乙酸水溶液为流动相,选择合适的HPLC条件,确定最佳HPLC条件为甲醇:0.4%乙酸(28:72,v/v),色谱柱Kromasil C18柱(250×4.6mm,5μm),流速1.0mL/min,柱温30℃,270nm检测。HPLC分析结果表明,得到至少A、B两个有效组分,其保留时间分别是6.225min和10.899min,这使得抗菌物质的分离纯化更进一步。质谱分析表明,A、B两组分的分子量分别是218和392。
4抗菌物质粗品在水、甲醇、乙醇中溶解度较大,丙酮和乙醚中次之,在氯仿和苯中不溶解;茚三酮反应为阴性;耐高温;对酸敏感;对蛋白酶和紫外线照射不敏感;在270nm和220nm附近有最大紫外吸收。
5该菌株抑菌谱广,对多种病原细菌和真菌有拮抗作用。其中,抗菌
a’311, isolated from soil, exhibited potent growth inhibitory activities against several pathogens, such as Fusarium oxysporum f.sp. vasinfectum, Fusarium oxysporum, Phyllosticta thesefolia, Escherichia coli, Pasteurella multocida, Staphylococcus aureus and Bacillus sp. It was identified as Brevibacillus brevis XDH by 16S rDNA analysis, conformation, physiological and biochemical features. The best fermentation condition was chosen, and the antibacterial substance was isolated and extracted. The main results were as following:
1 a’311 was identified as Brevibacillus brevis XDH by 16S rDNA analysis, conformation, physiological and biochemical features. The 16S rDNA sequence has been registered at Genbank under the accession number DQ279738.
2 The best fermentation condition was glucose 16g,starch 2.5g,peptone 10g,bean powder 26g,corn plasm 1.0g,NaCl 7.5g,(NH4)2SO4 4.0g,distilled water1000mL,30℃, 200rpm and 48h.
3 The antibacterial substance can be isolated from other groups by salting out with 40% (NH4)2SO4 and Sephadex G-50 analysis. The best HPLC flow phase was chosen when methanol and acetonitrile as organic phase. The HPLC condition was methanol:0.4% acetic acid(28:72,v/v), Kromasil C18 column(250×4.6mm,5μm), flow rate 1.0mL/min, column temperature 30℃and detection wavelength 270nm.The results showed that at least two groups were isolated with the retention time 6.225min and 10.899min, respectively. The preparation column was used to prepare the antibacterial substance under the condition of Kromasil C18 column (250×10mm,5μm), flow rate 3.5mL/min,column temperature 30℃and detection wavelength 270nm. Two
1通过菌体形态特征观察、生理生化指标测定、16S rDNA序列及其系统进化树分析,鉴定a’311菌株为短短芽孢杆菌(Brevibacillus brevis),命名为短短芽孢杆菌XDH。该菌株的16S rDNA序列已在美国Genbank注册,登录号为DQ279738。
2筛选到一个适合该菌株的发酵条件。培养基配方:葡萄糖16g,可溶性淀粉2.5g,蛋白胨10g,黄豆饼粉26g,玉米浆1.0g,NaCl7.5g,(NH4)2SO4 4.0g,蒸馏水1000mL。30℃,200rpm,装液量50mL/250mL,摇瓶振荡培养48h。
3发酵液经40%饱和度硫酸铵盐析和Sephadex G-50柱层析,可将有效物质与其他组分进行粗分离。以乙腈-0.1%TFA水溶液和甲醇-0.4%乙酸水溶液为流动相,选择合适的HPLC条件,确定最佳HPLC条件为甲醇:0.4%乙酸(28:72,v/v),色谱柱Kromasil C18柱(250×4.6mm,5μm),流速1.0mL/min,柱温30℃,270nm检测。HPLC分析结果表明,得到至少A、B两个有效组分,其保留时间分别是6.225min和10.899min,这使得抗菌物质的分离纯化更进一步。质谱分析表明,A、B两组分的分子量分别是218和392。
4抗菌物质粗品在水、甲醇、乙醇中溶解度较大,丙酮和乙醚中次之,在氯仿和苯中不溶解;茚三酮反应为阴性;耐高温;对酸敏感;对蛋白酶和紫外线照射不敏感;在270nm和220nm附近有最大紫外吸收。
5该菌株抑菌谱广,对多种病原细菌和真菌有拮抗作用。其中,抗菌
a’311, isolated from soil, exhibited potent growth inhibitory activities against several pathogens, such as Fusarium oxysporum f.sp. vasinfectum, Fusarium oxysporum, Phyllosticta thesefolia, Escherichia coli, Pasteurella multocida, Staphylococcus aureus and Bacillus sp. It was identified as Brevibacillus brevis XDH by 16S rDNA analysis, conformation, physiological and biochemical features. The best fermentation condition was chosen, and the antibacterial substance was isolated and extracted. The main results were as following:
1 a’311 was identified as Brevibacillus brevis XDH by 16S rDNA analysis, conformation, physiological and biochemical features. The 16S rDNA sequence has been registered at Genbank under the accession number DQ279738.
2 The best fermentation condition was glucose 16g,starch 2.5g,peptone 10g,bean powder 26g,corn plasm 1.0g,NaCl 7.5g,(NH4)2SO4 4.0g,distilled water1000mL,30℃, 200rpm and 48h.
3 The antibacterial substance can be isolated from other groups by salting out with 40% (NH4)2SO4 and Sephadex G-50 analysis. The best HPLC flow phase was chosen when methanol and acetonitrile as organic phase. The HPLC condition was methanol:0.4% acetic acid(28:72,v/v), Kromasil C18 column(250×4.6mm,5μm), flow rate 1.0mL/min, column temperature 30℃and detection wavelength 270nm.The results showed that at least two groups were isolated with the retention time 6.225min and 10.899min, respectively. The preparation column was used to prepare the antibacterial substance under the condition of Kromasil C18 column (250×10mm,5μm), flow rate 3.5mL/min,column temperature 30℃and detection wavelength 270nm. Two
引文
[1]包建中,古德祥.中国生物防治[M].山西科学技术出版社,1998
[2]陈华癸,李阜棣.土壤微生物[M].上海科技技术出版社,1986
[3]陈天寿.微生物培养基的制造与应用[M].中国农业出版社,1995
[4]陈玮.短芽孢杆菌 α-乙酶乳酸脱羧酶基因在大肠杆菌中的克隆和表达[J].微生物学报,1997,37(4):270-275
[5]陈耀祖.有机质谱原理及应用[M].科学出版社,2001
[6]陈仪杰.微生物药物学[M].华东理工大学出版社,1999
[7]程光胜,朱厚础,周方.分析微生物学专辑[M].北京:科学出版社,1988,11
[8]程元荣,郑卫.微生物生物活性物质的研究进展[J].中国抗生素杂志,2002,27(10):632-639
[9]迪芬巴赫.C.W,德维克斯勒.G.S.PCR 技术实验指南[M].北京:科学技术出版社,2000
[10]丁立孝,何国庆,孔青.一种新的农用抗生素初筛方法[J].农业生物技术学报,2002,10(4):409-410
[11]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001
[12]杜秉海.微生物学实验[M].北京农业大学出版社,1994
[13]方中达.植病研究方法(第三版)[M].北京:农业出版社,1998
[14] 高向阳 , 陈念 , 林壁润 . 短杆菌肽 S 的研究进展 [J]. 生命科学研究,2004,6(8):76-79
[15] 顾觉奋 . 微生物药品化学与分析 [M]. 北京 : 军事医学科学出版社,1996,5,166-185
[16]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
[17]胡剑,林心怡,张九一等.拮抗菌 BS-98 分泌抗菌蛋白的条件及其发酵液的特性[J].微生物学通报,1996,2(6):445-449
[18]黄涛.有机化学实验[M].北京,高等教育出版社,1983
[19]蒋振海,黄友亮,汪万青等.新肽类抗生素 B-3543 的研究[J].中国抗生素杂志,1993,18(2):120-124
[20]焦振泉,刘秀梅.16S rRNA 序列同源性分析与细菌系统分类鉴定[J].国外医学卫生学分册,1998,25(1):12-16
[21]金文藻,孟伟,王以光等.新抗生素波拉霉素 A、B 的分离、性质及鉴别[J].中国抗生素杂志,1997,22(1):10-14
[22]李盛,赵志安,李明等.短芽孢杆菌(Bacillus brevis No.G1)几丁质酶的提纯和分离鉴定[J].生物化学与生物物理学报,2002,34(6):690-696
[23]李长松.拮抗性细菌生物防治植物土传病害的研究进展[J].生物防治通报,1992,8(4):168-172
[24]李发美.医药高效液相色谱技术[M].北京:人民卫生出版社,1999,10:46
[25]林东,徐庆,刘忆舟等.枯草芽抱杆菌 S0113 分泌蛋白的抑菌作用及抗菌蛋白的分离纯化[J].农业生物技术学报,2001,9(1):77-80
[26]林万明.细菌分子遗传学分类鉴定法[M].上海:上海科学技术出版社,1990
[27]林卓坤.色谱法[M].科学出版社,1982
[28] 刘 建 国 . 新 型 抗 菌 多 肽 APS 的 抗 真 菌 研 究 [J]. 中 国 生 物 防治,1999,15(3):108-110
[29]刘静,王军,姚建铭等.枯草芽抱杆菌 JA 抗菌物特性的研究及抗菌肤的分离纯化[J].微生物学报,2004,44(4):511-514
[30]刘训理,崔云龙,张国珍等.农用抗生素 S-921 有效组分的分离纯化及部分理化性质研究[J].山东农业大学学报,1995,26(2):160-162
[31] 刘 志 俊 , 段 渝 峰 . 生 物 农 药 新 概 念 新 发 展 [J]. 农 药 科 学 与 管理,2003,24(7):28-32
[32] 马绪荣 , 苏德模 . 药品微生物学检验手册 [M]. 北京 : 科学出版社2000:198-201
[33]彭清忠,张惟材,朱厚础.具有分泌蛋白能力的短芽孢杆菌的筛选及鉴定[J].微生物学报,2002,12,42(6):693-699
[34]彭清忠,张惟材,朱厚础.短芽孢杆菌-大肠杆菌穿梭分泌表达载体的构建[J].生物工程学报,2002,18(4):438-441
[35]钱朝东.抗生素产生菌19-1的分离鉴定及发酵产物的研究[D].浙江大学硕士学位论文,2001,6
[36] 沈寅初 . 农用抗生素研究开发的新进展 [J]. 国外医药抗生素分册,1998,19(2):155-160
[37]师治贤,王俊德.生物大分子的液相色谱分离和制备[M].北京:科学出版社,1996,7:185-200
[38]石炳兴,李景川.抗生索药物面临的挑战和机遇[J]. 过程工程学报,2002,12(5):470-475
[39]宋晓妍.农用抗生素 S-921 发酵的研究[D]山东农业大学硕士论文,1998
[40]孙毓庆.现代色谱法及其在医药中的应用[M].北京:人民卫生出版社,1998,11
[41]田黎,顾振芳,陈杰等.海洋细菌 B-9987 菌株产生的抑菌物质及对几种植物病原真菌的作用[J].植物病理学报,2003,33(1):77-80
[42]童有仁.枯草芽孢杆菌B034拮抗蛋白的分离纯化及特征分析[J].微生物学报,1999,39(4):339-343
[43]涂玉琴.生物农药的研究和应用进展[J].江西植保,1998,(4):32-35
[44]王岳,方金瑞.抗生素[M].科学出版社,1999,500-501
[45] 王槐春 . 蛋白质与核酸序列分析基础 [M]. 北京 : 人民军医出版社,1994:2-10
[46]王雅平.抗真菌蛋白 BII 的分离纯化及其性质研究[J].生物化学与生物物理学报,1993,25(4),391-397
[47]王占武,李晓芝,刘彦利等.拮抗菌防治草莓枯萎病[J].中国生物防治,1999,15(4):187-189
[48]魏群.分子生物学实验技术[M].北京:高等教育出版社,2001
[49]文汇.中国抗生素[J].上海:中国抗生素杂志社,2001:19-21
[50]吴剑波,张致平.微生物制药[M].北京:化学工业出版社,2002
[51]熊强,梁剑光,熊晓辉.微生物-几种溶拴药物的重要来源[J].微生物学通报,2003,30(5):116-119
[52]许光辉,郑洪远.土壤微生物分析方法手册[M].农业出版社,1986
[53]严希康.生化分离技术[M].上海:华东理工大学出版社,1996.12:19-20
[54]杨瑞馥,陶天申.细菌名称英解汉译词典[M].北京:军事医学科学出版社,2000:71
[55]尹萃耘.农用抗生素和抗生菌的筛选[J].抗生素,1983,8(1):64-65
[56] 于世林 . 高效液相色谱方法及应用 [M]. 北京 : 化学工业出版社2000,1:70-71
[57]宁永成.有机化合物鉴定与有机波谱学[M].北京:科学出版社,1993
[58]俞文和,杨纪根.抗生素工艺学[M].辽宁科技出版社,1988:5-6
[59]张龙翔,张庭芳,李令媛.生化实验方法和技术[M].北京:高等教育出版社,1997
[60]张致平,姚天爵.抗生素与微生物产生的生物活性物质[M].化学工业出版社,2005:1-4
[61]赵遥兴,孙祥玉.有机分子结构光谱鉴定[M].北京:科学出版社,2003
[62]中国科学院南京土壤研究所微生物室.土壤微生物研究法[M].北京:科学出版社,1985
[63]周启,王道本.农用抗生素和微生物杀虫剂[M].中国农业出版社,1990,11
[64]朱宝泉.现代生物技术在抗生素研究中的应用.第九次全国抗素(微生物药物学术会议论文汇编)[M].上海:中国抗生素杂志社,2001:19-21
[65]Cohen, Kupiec R, Chet I.The molecular biology of chitin digestion[J].Curr Opin Biotechnol,1998,9:270-277
[66]Deshpande M V.Enzymatic degradation of chitin and its biological applications[J].Sci Ind Res,1986,45:273-281
[67]GoodayG W.Aggressive and defensive roles for chitinases[J].Exe,1999,87: 157-169
[68]Leah R,Tommerup H,Svendsen I,et al.Biochemical and molecular characterization of three barley seed proteins with antifungal properties [J].Biol Chem,1991,266:1564-1573
[69]Majeti N V, Kumar R.A review of chitin and chitosan applications [J].Reactive Functional Polymers,2000,46:1-27
[70]Nielsen M N,Srensen J.Chitin olytic activity of Pseudomonas fluorescens isolates from barley and sugar Beet rhizosphere[J].Fems Microbiol Ecol,1999,30:217-227
[71]Overdijk B, Van Steijn G J, Odds F C.Chitinase levels in guinea pig blood arein creased after systemic infection with Asper gillus fumigatus[J].Glycobiology,1996,6(6):627-634
[72]Patil R S, Ghormade V, Deshpande M V. Chitin olytic enzymes:An exploration[J].Enzyme MicrobTechnol,2000,26:473-483
[73]Sahai A S, Manocha M S.Chitinases of fungi and plants: The involvement in morphogenesis and hostParasite interaction[J].FEMS Microbiol Rev,1993,11:317-338
[74]Svitil A L,Chadhain S M N,Moore J A,et al.Chitin degradation proteins produced by the marine Bacterium Vibrio harveyi growing on different forms of chitin[J].Appl Environ,1998,11(2):45-49
[75]Turner C M, Adler P N.The invitro morphogenesis of Droso phila pupal wings[J].MechDev,1995,52(223):247-256
[76]Wang S L,Chang W T.Purification and characterization of two bifunctional chitinases/lysozymes Extracellularly produced by Pseudomonas aeruginosa K2187 in a shrimp and crab shell powder medium[J].Appl Enviorn Microbiol,1997,63:380-386
[77]Ahimou F. Jacques, Deleu.Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity[J].Microbiol Technol,2000, 27:749-754
[78]Baker CJ, Starvely JR, Thomas CA,et al.Inhibitory effect of Bacillus on Uromyces phaseoli and on development of rust pustules on bean lesves[J].Phytopathology,1983,73:1148-1152
[79]Baneyxf.Recombinant Protein Expression in Escherichia Coli[J].Curr Opin Biotechnol,1999,10:411-421
[80]Broad bent P, Baker K F, Franks N, et al. Effect of Bacillus spp. on increased growth of seeding in steamed and otreated soil[J].Phytopathol, 1997,67:1027-1034
[81]CAUSE G F,Brazhmikova M G..Gramicidin S and its use in the treatment of infected wounds[J].Nature,1944,154:703-709
[82]Chatterjee A,Valasubramanian R, Ma WL,et al.Isolation of Pseudomonas strain pf7-14 altered in antibiotic production, cloning of ant DNA and evaluation of the role of antibioticproduction in cintrol of blast and sheathblight of rice[J].Biological Control,1996,7:185-195
[83]Cinzia Arcelloni,Roberto Vaiani,Rita Paroni. Is the direct quantitation of antibiotics in agar by high-performance liquid chromatography useful [J].Journal of Chromatography A,1998, 812(12):111-116
[84]Cook RJ,Thomashoe LS,Weller DM,et al.Molecular Mechanisma for biological control of plant pathogens [J].Phytopatholgy,1995,31:53-80
[85]Cordula Hopmann,Michael Kurz,Mark Bronstrup,et al.Isolation and structure elucidation of vancoresmycin-a new antibiotic from Amycolatopsis sp. ST 101170[J].Tetrahedron letters,2002,43:435-438
[86]Dai Deshun, Wang Yiming,Luo Guoan. Recent development in high-speed counter-current chromatography[J].Chinese Journal of Analyses Chemistry, 2001,29(5):586-591
[87]David H.Wright, Varen K. Herman, Konstantinides, et al.Determination of quinolone antibiotics in growth media choline,phosphatidyle thanolamine and phosphatidylglycerol lipid bilayer membranes[J].Biochemicaet Biophysica Acta,2001,15,10:83-92
[92]Elmar J,Prenner,Ruthven N A H,et al.The interaction of the antimicrobial peptide gramicidin S with the lipid bilayer model and biological membrane[J].Biochim et Biophysi Acta,1999,1462:201-221
[93]Escott G M,Adams D J.Chitinase activity in human serum and leukocytes[J].Infect Immun,1995,63(12):4770-4773
[94]Fang Hong.New development of capillary electrophoresis[J].Foreign Medical Science:Molecular Biological Section,2002,22(4):124-126
[95]Gao leyi,Fang Yuzhi.On the trends of capillary electrophoresis in the new century[J].Chemistry Analyses:part B,2002,38(1):1-6
[96]GAUSE G F, BRAZHNILOVA M G.Gramicidin S and its use in the treatment of infected wounds[J].Nature,1994,154:703-709
[97]Gould.SJ, He.W, Cone.MC.New cyanocyclines from a cyanide-treated broth of Streptomyces lusitanus[J].Journal of National Production, 1993,56(8):1239-1245
[98]Gu Z R, Ma C Z, Han C A.Screening and indentification of chitinase producing Bacillus spp. and determination of the irchitinase activity reversed-phase high-performance liquid chromatography[J].Journal of Chromatography B,1998,709(1):97-104
[88]De Clercq E.Antivirals for the treatment of herpesvirus infections [J].Antimicrob Chemother,1993,32:121-132
[89]Dubos R J, Hotchkiss R D.Production of bactericidal substance by aerobic sporulating bacilli [J].Exp Med 17:405-422
[90]Efstathiou S, Kemp S, Darby G,et al.The role of herpes simplex virustype 1 thymidine kinase in pathogenesis[J].Gen Virol 1989,70:869-879
[91]Elmar J P,Ruthven N A H,Lewis.Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidyle[J].Acta Agriculture Shanghai,2001,17(3): 92-99
[99]H.KChepkwony,I.Vanderriest, J. M Nguyo,et al.Hoogmartens Separation of erythromycin and related substances on base-deactivated reversed phase silica gel columns[J].Journal of chromatography A,2000,870: 227-235
[100]Harwood C R.Bacillus subtilis and its relatives:Molecular biological and industrial workhorses[J].Trends Biotechnol,1992,10:247-256
[101]HirooY,Koji O,KazuhikO T,et al.Mode of antibacterial action by Gramicidin S[J].Biochem,1986,100:1253-1259
[102]Hook E W,Cannon R O,Nahmias A J,et al.Herpes simplex virus infection as arisk factor for human immunode ciencyvirus infection in heterosexuals[J].Infect Dis,165:251-255
[103]Howell. C R,Stipanovic R D,Lumsden R D.Antibiotic produced bystrains of Gliocladium virens and its relation to the biocontrolof cotton seeding disease[J].Biocongtrol Sci.Technol,1993,3:435-442
[104]Leah R, Tommerup H, Svendsen I, et al. Biochemical and molecular characterization of Three barley seed proteins with antifungal properties [J]. Biol Chem,1991,266:1564-1574
[105]Majeti N V,KumarR.A review of chitin and chitosan applications[J]. Reactive FunctionalPolymers,2000,46:1-8
[106]Jinkui Zhou,Geoi C.Gerhardt,Andrej Baranski,et al.Capillary electrophoresis of some tetrecycline antibiotics coupled with fast cyclic voltammetric detection[J].Journal of Chromatograp,1999,839(12): 193-201
[107]Karel L, Roach E S. A dictionary of antibiosis [M].Columbia University Press,NewYork,1951,135-136
[108]Ken-ichi Harada, Masanao Suzuki, Azusa Kato et al. Separation of WAP-8294A components,a novel antimethicillin resistant staphylococcus aureus antibiotic, using high-speed counter current chromatography [J].Journal of chromatography,2001,932:75-81
[109]Kittelbergerl R,Pavela-Vrancic M,Von D H.Active site titration of gramicidin S synthetase 2:evidence for misaction and editing in non-ribosomal peptide biosynthesis[J].FEBS Letters,1999,461:145-148
[110]Kondejewski L H,Farmes S W,Wishart, et al. Modulation of structure and Antibacterial and Hemolytic activity by ring size in cyclic Gramicidin S analogs[J].Biol Chem,996,271:25261-25268
[111]Kost RG, Hill EL, Tigges M,et al.Brief report: recurrent acyclovir Resistant genital herpes in an immuno competent patient[J].Engl Med,1993,329:1777-1782
[112]L.R.斯奈德,J.J.柯克兰.现代液相色谱法导论[M].NewYork,1991
[113]Leslie H. Kondejewski ,Susan W. Farmer,et al. Modulation of Structure and Antibacterial and Hemolytic Activity by Ring Size in Cyclic Gramicidin S Analogs[J].American Society for Biochemistry and Molecular Biology,1996,27:125261-125268
[114]M.Gupte, P.Kulkarni,Ganguli.Antifungal antibiotics[J].Appl Microbiol Biotechnol,2002,58:46-57
[115]M. Hernandez, F. Borrull, M. Calull. Analysis of antibiotics in biological samples by capillary electrophoresis[J].Trend in Analyses Chernistry,2003,22(78):416-27
[116]Maddox I.S,RichertSH. Use of response surface methodology for the rapid optimization of microbiological media[J].Appl.Bacteriol,1977, 43:197-204
[117]Mao Yu,Xu Jianming.Development of capillary electrophpresis technique and application[J].Chemistry research and application,2001,13(1):4-9
[118]Mc Grath BJ,Newman CL.Genital herpes simplex infections in patients with theacquired immunode ciency syndrome[J].Pharmacotherapy 1994, 14: 529-542
[119]Mekeen C D,Reiliy C C, Pusey P L. Production and partial characterization of antifungal substances antagonistic to Monlinia fructicola from Bacillus subtilics[J].Phytopatholoy,1986,76:136-39
[120]Oka Hisao,Harada Ken-ichi, Ito Yuko,et al.Separation of antibiotics by counter current chromatography[J].Journal of Chromatography A,1998, 812(12):35-52
[121]Oka Hisao,Ikai Yoshitomo,Hayakawa Junko,et al.Separation of ivermectin components by high-speed counter current chromatography [J].Joumal of Chromatography A,1996,23(1):61-68
[122]Roberts WK,Selitrennikoff C P.Plantand bacterial chitinases different in antifungal activity[J].GenMicrobiol,1988,134:169-176
[123]Ueda M,Arai M.Purification and some properties of chitinases from Aeromonassp.No.10S224[J].Biosci Biotech Biochem,1992,56:460-464
[124]Walid Fakhouri,Frank Walker,Bernhard Vogler,et al.Isolation and identification of N-rnercapto-4-formylcarbostyril,an antibiotic produced by Pseudomonas uorescens[J].Phytochemistry,2001,58:1297-1303
[125]WiwatC,Siwayaprahm P,Bhumiratana A.Purification and characterization of chitinase from Bacillus circulans No.4.1[J].Curr Microbiol,1999,39: 134-140
[2]陈华癸,李阜棣.土壤微生物[M].上海科技技术出版社,1986
[3]陈天寿.微生物培养基的制造与应用[M].中国农业出版社,1995
[4]陈玮.短芽孢杆菌 α-乙酶乳酸脱羧酶基因在大肠杆菌中的克隆和表达[J].微生物学报,1997,37(4):270-275
[5]陈耀祖.有机质谱原理及应用[M].科学出版社,2001
[6]陈仪杰.微生物药物学[M].华东理工大学出版社,1999
[7]程光胜,朱厚础,周方.分析微生物学专辑[M].北京:科学出版社,1988,11
[8]程元荣,郑卫.微生物生物活性物质的研究进展[J].中国抗生素杂志,2002,27(10):632-639
[9]迪芬巴赫.C.W,德维克斯勒.G.S.PCR 技术实验指南[M].北京:科学技术出版社,2000
[10]丁立孝,何国庆,孔青.一种新的农用抗生素初筛方法[J].农业生物技术学报,2002,10(4):409-410
[11]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001
[12]杜秉海.微生物学实验[M].北京农业大学出版社,1994
[13]方中达.植病研究方法(第三版)[M].北京:农业出版社,1998
[14] 高向阳 , 陈念 , 林壁润 . 短杆菌肽 S 的研究进展 [J]. 生命科学研究,2004,6(8):76-79
[15] 顾觉奋 . 微生物药品化学与分析 [M]. 北京 : 军事医学科学出版社,1996,5,166-185
[16]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
[17]胡剑,林心怡,张九一等.拮抗菌 BS-98 分泌抗菌蛋白的条件及其发酵液的特性[J].微生物学通报,1996,2(6):445-449
[18]黄涛.有机化学实验[M].北京,高等教育出版社,1983
[19]蒋振海,黄友亮,汪万青等.新肽类抗生素 B-3543 的研究[J].中国抗生素杂志,1993,18(2):120-124
[20]焦振泉,刘秀梅.16S rRNA 序列同源性分析与细菌系统分类鉴定[J].国外医学卫生学分册,1998,25(1):12-16
[21]金文藻,孟伟,王以光等.新抗生素波拉霉素 A、B 的分离、性质及鉴别[J].中国抗生素杂志,1997,22(1):10-14
[22]李盛,赵志安,李明等.短芽孢杆菌(Bacillus brevis No.G1)几丁质酶的提纯和分离鉴定[J].生物化学与生物物理学报,2002,34(6):690-696
[23]李长松.拮抗性细菌生物防治植物土传病害的研究进展[J].生物防治通报,1992,8(4):168-172
[24]李发美.医药高效液相色谱技术[M].北京:人民卫生出版社,1999,10:46
[25]林东,徐庆,刘忆舟等.枯草芽抱杆菌 S0113 分泌蛋白的抑菌作用及抗菌蛋白的分离纯化[J].农业生物技术学报,2001,9(1):77-80
[26]林万明.细菌分子遗传学分类鉴定法[M].上海:上海科学技术出版社,1990
[27]林卓坤.色谱法[M].科学出版社,1982
[28] 刘 建 国 . 新 型 抗 菌 多 肽 APS 的 抗 真 菌 研 究 [J]. 中 国 生 物 防治,1999,15(3):108-110
[29]刘静,王军,姚建铭等.枯草芽抱杆菌 JA 抗菌物特性的研究及抗菌肤的分离纯化[J].微生物学报,2004,44(4):511-514
[30]刘训理,崔云龙,张国珍等.农用抗生素 S-921 有效组分的分离纯化及部分理化性质研究[J].山东农业大学学报,1995,26(2):160-162
[31] 刘 志 俊 , 段 渝 峰 . 生 物 农 药 新 概 念 新 发 展 [J]. 农 药 科 学 与 管理,2003,24(7):28-32
[32] 马绪荣 , 苏德模 . 药品微生物学检验手册 [M]. 北京 : 科学出版社2000:198-201
[33]彭清忠,张惟材,朱厚础.具有分泌蛋白能力的短芽孢杆菌的筛选及鉴定[J].微生物学报,2002,12,42(6):693-699
[34]彭清忠,张惟材,朱厚础.短芽孢杆菌-大肠杆菌穿梭分泌表达载体的构建[J].生物工程学报,2002,18(4):438-441
[35]钱朝东.抗生素产生菌19-1的分离鉴定及发酵产物的研究[D].浙江大学硕士学位论文,2001,6
[36] 沈寅初 . 农用抗生素研究开发的新进展 [J]. 国外医药抗生素分册,1998,19(2):155-160
[37]师治贤,王俊德.生物大分子的液相色谱分离和制备[M].北京:科学出版社,1996,7:185-200
[38]石炳兴,李景川.抗生索药物面临的挑战和机遇[J]. 过程工程学报,2002,12(5):470-475
[39]宋晓妍.农用抗生素 S-921 发酵的研究[D]山东农业大学硕士论文,1998
[40]孙毓庆.现代色谱法及其在医药中的应用[M].北京:人民卫生出版社,1998,11
[41]田黎,顾振芳,陈杰等.海洋细菌 B-9987 菌株产生的抑菌物质及对几种植物病原真菌的作用[J].植物病理学报,2003,33(1):77-80
[42]童有仁.枯草芽孢杆菌B034拮抗蛋白的分离纯化及特征分析[J].微生物学报,1999,39(4):339-343
[43]涂玉琴.生物农药的研究和应用进展[J].江西植保,1998,(4):32-35
[44]王岳,方金瑞.抗生素[M].科学出版社,1999,500-501
[45] 王槐春 . 蛋白质与核酸序列分析基础 [M]. 北京 : 人民军医出版社,1994:2-10
[46]王雅平.抗真菌蛋白 BII 的分离纯化及其性质研究[J].生物化学与生物物理学报,1993,25(4),391-397
[47]王占武,李晓芝,刘彦利等.拮抗菌防治草莓枯萎病[J].中国生物防治,1999,15(4):187-189
[48]魏群.分子生物学实验技术[M].北京:高等教育出版社,2001
[49]文汇.中国抗生素[J].上海:中国抗生素杂志社,2001:19-21
[50]吴剑波,张致平.微生物制药[M].北京:化学工业出版社,2002
[51]熊强,梁剑光,熊晓辉.微生物-几种溶拴药物的重要来源[J].微生物学通报,2003,30(5):116-119
[52]许光辉,郑洪远.土壤微生物分析方法手册[M].农业出版社,1986
[53]严希康.生化分离技术[M].上海:华东理工大学出版社,1996.12:19-20
[54]杨瑞馥,陶天申.细菌名称英解汉译词典[M].北京:军事医学科学出版社,2000:71
[55]尹萃耘.农用抗生素和抗生菌的筛选[J].抗生素,1983,8(1):64-65
[56] 于世林 . 高效液相色谱方法及应用 [M]. 北京 : 化学工业出版社2000,1:70-71
[57]宁永成.有机化合物鉴定与有机波谱学[M].北京:科学出版社,1993
[58]俞文和,杨纪根.抗生素工艺学[M].辽宁科技出版社,1988:5-6
[59]张龙翔,张庭芳,李令媛.生化实验方法和技术[M].北京:高等教育出版社,1997
[60]张致平,姚天爵.抗生素与微生物产生的生物活性物质[M].化学工业出版社,2005:1-4
[61]赵遥兴,孙祥玉.有机分子结构光谱鉴定[M].北京:科学出版社,2003
[62]中国科学院南京土壤研究所微生物室.土壤微生物研究法[M].北京:科学出版社,1985
[63]周启,王道本.农用抗生素和微生物杀虫剂[M].中国农业出版社,1990,11
[64]朱宝泉.现代生物技术在抗生素研究中的应用.第九次全国抗素(微生物药物学术会议论文汇编)[M].上海:中国抗生素杂志社,2001:19-21
[65]Cohen, Kupiec R, Chet I.The molecular biology of chitin digestion[J].Curr Opin Biotechnol,1998,9:270-277
[66]Deshpande M V.Enzymatic degradation of chitin and its biological applications[J].Sci Ind Res,1986,45:273-281
[67]GoodayG W.Aggressive and defensive roles for chitinases[J].Exe,1999,87: 157-169
[68]Leah R,Tommerup H,Svendsen I,et al.Biochemical and molecular characterization of three barley seed proteins with antifungal properties [J].Biol Chem,1991,266:1564-1573
[69]Majeti N V, Kumar R.A review of chitin and chitosan applications [J].Reactive Functional Polymers,2000,46:1-27
[70]Nielsen M N,Srensen J.Chitin olytic activity of Pseudomonas fluorescens isolates from barley and sugar Beet rhizosphere[J].Fems Microbiol Ecol,1999,30:217-227
[71]Overdijk B, Van Steijn G J, Odds F C.Chitinase levels in guinea pig blood arein creased after systemic infection with Asper gillus fumigatus[J].Glycobiology,1996,6(6):627-634
[72]Patil R S, Ghormade V, Deshpande M V. Chitin olytic enzymes:An exploration[J].Enzyme MicrobTechnol,2000,26:473-483
[73]Sahai A S, Manocha M S.Chitinases of fungi and plants: The involvement in morphogenesis and hostParasite interaction[J].FEMS Microbiol Rev,1993,11:317-338
[74]Svitil A L,Chadhain S M N,Moore J A,et al.Chitin degradation proteins produced by the marine Bacterium Vibrio harveyi growing on different forms of chitin[J].Appl Environ,1998,11(2):45-49
[75]Turner C M, Adler P N.The invitro morphogenesis of Droso phila pupal wings[J].MechDev,1995,52(223):247-256
[76]Wang S L,Chang W T.Purification and characterization of two bifunctional chitinases/lysozymes Extracellularly produced by Pseudomonas aeruginosa K2187 in a shrimp and crab shell powder medium[J].Appl Enviorn Microbiol,1997,63:380-386
[77]Ahimou F. Jacques, Deleu.Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity[J].Microbiol Technol,2000, 27:749-754
[78]Baker CJ, Starvely JR, Thomas CA,et al.Inhibitory effect of Bacillus on Uromyces phaseoli and on development of rust pustules on bean lesves[J].Phytopathology,1983,73:1148-1152
[79]Baneyxf.Recombinant Protein Expression in Escherichia Coli[J].Curr Opin Biotechnol,1999,10:411-421
[80]Broad bent P, Baker K F, Franks N, et al. Effect of Bacillus spp. on increased growth of seeding in steamed and otreated soil[J].Phytopathol, 1997,67:1027-1034
[81]CAUSE G F,Brazhmikova M G..Gramicidin S and its use in the treatment of infected wounds[J].Nature,1944,154:703-709
[82]Chatterjee A,Valasubramanian R, Ma WL,et al.Isolation of Pseudomonas strain pf7-14 altered in antibiotic production, cloning of ant DNA and evaluation of the role of antibioticproduction in cintrol of blast and sheathblight of rice[J].Biological Control,1996,7:185-195
[83]Cinzia Arcelloni,Roberto Vaiani,Rita Paroni. Is the direct quantitation of antibiotics in agar by high-performance liquid chromatography useful [J].Journal of Chromatography A,1998, 812(12):111-116
[84]Cook RJ,Thomashoe LS,Weller DM,et al.Molecular Mechanisma for biological control of plant pathogens [J].Phytopatholgy,1995,31:53-80
[85]Cordula Hopmann,Michael Kurz,Mark Bronstrup,et al.Isolation and structure elucidation of vancoresmycin-a new antibiotic from Amycolatopsis sp. ST 101170[J].Tetrahedron letters,2002,43:435-438
[86]Dai Deshun, Wang Yiming,Luo Guoan. Recent development in high-speed counter-current chromatography[J].Chinese Journal of Analyses Chemistry, 2001,29(5):586-591
[87]David H.Wright, Varen K. Herman, Konstantinides, et al.Determination of quinolone antibiotics in growth media choline,phosphatidyle thanolamine and phosphatidylglycerol lipid bilayer membranes[J].Biochemicaet Biophysica Acta,2001,15,10:83-92
[92]Elmar J,Prenner,Ruthven N A H,et al.The interaction of the antimicrobial peptide gramicidin S with the lipid bilayer model and biological membrane[J].Biochim et Biophysi Acta,1999,1462:201-221
[93]Escott G M,Adams D J.Chitinase activity in human serum and leukocytes[J].Infect Immun,1995,63(12):4770-4773
[94]Fang Hong.New development of capillary electrophoresis[J].Foreign Medical Science:Molecular Biological Section,2002,22(4):124-126
[95]Gao leyi,Fang Yuzhi.On the trends of capillary electrophoresis in the new century[J].Chemistry Analyses:part B,2002,38(1):1-6
[96]GAUSE G F, BRAZHNILOVA M G.Gramicidin S and its use in the treatment of infected wounds[J].Nature,1994,154:703-709
[97]Gould.SJ, He.W, Cone.MC.New cyanocyclines from a cyanide-treated broth of Streptomyces lusitanus[J].Journal of National Production, 1993,56(8):1239-1245
[98]Gu Z R, Ma C Z, Han C A.Screening and indentification of chitinase producing Bacillus spp. and determination of the irchitinase activity reversed-phase high-performance liquid chromatography[J].Journal of Chromatography B,1998,709(1):97-104
[88]De Clercq E.Antivirals for the treatment of herpesvirus infections [J].Antimicrob Chemother,1993,32:121-132
[89]Dubos R J, Hotchkiss R D.Production of bactericidal substance by aerobic sporulating bacilli [J].Exp Med 17:405-422
[90]Efstathiou S, Kemp S, Darby G,et al.The role of herpes simplex virustype 1 thymidine kinase in pathogenesis[J].Gen Virol 1989,70:869-879
[91]Elmar J P,Ruthven N A H,Lewis.Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidyle[J].Acta Agriculture Shanghai,2001,17(3): 92-99
[99]H.KChepkwony,I.Vanderriest, J. M Nguyo,et al.Hoogmartens Separation of erythromycin and related substances on base-deactivated reversed phase silica gel columns[J].Journal of chromatography A,2000,870: 227-235
[100]Harwood C R.Bacillus subtilis and its relatives:Molecular biological and industrial workhorses[J].Trends Biotechnol,1992,10:247-256
[101]HirooY,Koji O,KazuhikO T,et al.Mode of antibacterial action by Gramicidin S[J].Biochem,1986,100:1253-1259
[102]Hook E W,Cannon R O,Nahmias A J,et al.Herpes simplex virus infection as arisk factor for human immunode ciencyvirus infection in heterosexuals[J].Infect Dis,165:251-255
[103]Howell. C R,Stipanovic R D,Lumsden R D.Antibiotic produced bystrains of Gliocladium virens and its relation to the biocontrolof cotton seeding disease[J].Biocongtrol Sci.Technol,1993,3:435-442
[104]Leah R, Tommerup H, Svendsen I, et al. Biochemical and molecular characterization of Three barley seed proteins with antifungal properties [J]. Biol Chem,1991,266:1564-1574
[105]Majeti N V,KumarR.A review of chitin and chitosan applications[J]. Reactive FunctionalPolymers,2000,46:1-8
[106]Jinkui Zhou,Geoi C.Gerhardt,Andrej Baranski,et al.Capillary electrophoresis of some tetrecycline antibiotics coupled with fast cyclic voltammetric detection[J].Journal of Chromatograp,1999,839(12): 193-201
[107]Karel L, Roach E S. A dictionary of antibiosis [M].Columbia University Press,NewYork,1951,135-136
[108]Ken-ichi Harada, Masanao Suzuki, Azusa Kato et al. Separation of WAP-8294A components,a novel antimethicillin resistant staphylococcus aureus antibiotic, using high-speed counter current chromatography [J].Journal of chromatography,2001,932:75-81
[109]Kittelbergerl R,Pavela-Vrancic M,Von D H.Active site titration of gramicidin S synthetase 2:evidence for misaction and editing in non-ribosomal peptide biosynthesis[J].FEBS Letters,1999,461:145-148
[110]Kondejewski L H,Farmes S W,Wishart, et al. Modulation of structure and Antibacterial and Hemolytic activity by ring size in cyclic Gramicidin S analogs[J].Biol Chem,996,271:25261-25268
[111]Kost RG, Hill EL, Tigges M,et al.Brief report: recurrent acyclovir Resistant genital herpes in an immuno competent patient[J].Engl Med,1993,329:1777-1782
[112]L.R.斯奈德,J.J.柯克兰.现代液相色谱法导论[M].NewYork,1991
[113]Leslie H. Kondejewski ,Susan W. Farmer,et al. Modulation of Structure and Antibacterial and Hemolytic Activity by Ring Size in Cyclic Gramicidin S Analogs[J].American Society for Biochemistry and Molecular Biology,1996,27:125261-125268
[114]M.Gupte, P.Kulkarni,Ganguli.Antifungal antibiotics[J].Appl Microbiol Biotechnol,2002,58:46-57
[115]M. Hernandez, F. Borrull, M. Calull. Analysis of antibiotics in biological samples by capillary electrophoresis[J].Trend in Analyses Chernistry,2003,22(78):416-27
[116]Maddox I.S,RichertSH. Use of response surface methodology for the rapid optimization of microbiological media[J].Appl.Bacteriol,1977, 43:197-204
[117]Mao Yu,Xu Jianming.Development of capillary electrophpresis technique and application[J].Chemistry research and application,2001,13(1):4-9
[118]Mc Grath BJ,Newman CL.Genital herpes simplex infections in patients with theacquired immunode ciency syndrome[J].Pharmacotherapy 1994, 14: 529-542
[119]Mekeen C D,Reiliy C C, Pusey P L. Production and partial characterization of antifungal substances antagonistic to Monlinia fructicola from Bacillus subtilics[J].Phytopatholoy,1986,76:136-39
[120]Oka Hisao,Harada Ken-ichi, Ito Yuko,et al.Separation of antibiotics by counter current chromatography[J].Journal of Chromatography A,1998, 812(12):35-52
[121]Oka Hisao,Ikai Yoshitomo,Hayakawa Junko,et al.Separation of ivermectin components by high-speed counter current chromatography [J].Joumal of Chromatography A,1996,23(1):61-68
[122]Roberts WK,Selitrennikoff C P.Plantand bacterial chitinases different in antifungal activity[J].GenMicrobiol,1988,134:169-176
[123]Ueda M,Arai M.Purification and some properties of chitinases from Aeromonassp.No.10S224[J].Biosci Biotech Biochem,1992,56:460-464
[124]Walid Fakhouri,Frank Walker,Bernhard Vogler,et al.Isolation and identification of N-rnercapto-4-formylcarbostyril,an antibiotic produced by Pseudomonas uorescens[J].Phytochemistry,2001,58:1297-1303
[125]WiwatC,Siwayaprahm P,Bhumiratana A.Purification and characterization of chitinase from Bacillus circulans No.4.1[J].Curr Microbiol,1999,39: 134-140