大豆过氧化物酶-H_2O_2-KI体系的抗菌作用及其机理研究
|
摘要
过氧化物酶与过氧化氢、卤化物组成的体系具有杀菌／抑菌作用，已广泛被研究。国内外对过氧化物酶体系抗菌作用的研究只限于哺乳动物过氧化物酶(乳过氧化物酶、髓过氧化物酶等)，因此类酶不易获取，且价格昂贵，所以能用价格便宜且在结构和催化活性上都非常相似的一种过氧化物酶类来替代，对理论研究和实践应用都具有潜在的重要意义。
大豆过氧化物酶(soybean peroxidase，SBP)是从大豆加工副产物大豆皮中提取的，以血红素为辅基的Ⅲ类过氧化物酶(EC1．11．1．7)。与其它过氧化物酶在结构和功能上都有许多相似之处，但因其原料价廉易得，底物作用范围广、耐热性能高、酸碱稳定性好、pH适用范围宽等优点，大豆过氧化物酶的研究越来越成为过氧化物酶研究的热点。
实验发现SBP-H_2O_2-KI组成的体系具有杀菌／抑菌作用，抗菌强度受有机物、pH值等的影响。SBP-H_2O_2-KI体系对E.coli、S.aureu的杀菌动力学表明，该体系具有快速的杀菌效果。SBP-H_2O_2-KI体系也具有较好的稳定性，37℃下放置4 h后仍具有杀菌作用。
本文初步研究了SBP-H_2O_3-KI体系的抗菌机理。利用电子顺磁共振波谱法没有检测到SBP-H_2O_2-KI体系中的自由基吸收峰。但在相同的H_2O_2浓度下，Fe~(2+)-H_2O_2体系(产生羟自由基的典型体系)却有明显的自由基吸收峰。比较Fe~(2+)-H_2O_2体系与SBP-H_2O_2-KI体系对E.coli31343与S.aureu的抗菌作用。结果显示，Fe~(2+)-H_2O_2体系几乎没有杀菌效果，而SBP-H_2O_2-KI体系却能完全致死菌体细胞。这也进一步说明了，SBP-H_2O_2-KI体系的抗菌作用不是由于自由基的产生。我们推测在SBP-H_2O_2-KI体系中，I~-离子起了介导作用，抗菌效果是由于碘的活性氧化态。我们在Fe~(2+)-H_2O_2-KI体系中检测到了明显的自由基吸收峰，并发现该体系也具有部分的抗菌作用。但比较Fe~(2+)和SBP对H_2O_2-KI反应的催化活性时，发现Fe~(2+)-H_2O_2-KI体系的抗菌强度远低于SBP-H_2O_2-KI体系。说明SBP对H_2O_2-KI反应的催化活性高于Fe~(2+)的催化活性。Fe~(2+)不能代替SBP在H_2O_2-KI体系中发挥的作用。推测碘的活性氧化态的致死效应可能比自由基更甚。
本文还进一步研究了在SBP-H_2O_2-KI体系的亚致死剂量下连续传代，菌体细胞对SBP-H_2O_2-KI体系敏感性的变化。E.coli31343从第一代传至第十五代，敏感性均未变化，但从第十六代起E.coli31343的敏感性有轻微的降低。而S.aureu从第一代开始敏感性就降低了，但从第二代传至第十五代敏感性上下波动，一定范围内保持恒定。亚致死剂量下菌体敏感性的轻微变化，是由于生理适应还是遗传改变，需深入研究。
A system, made up of peroxidease.hydrogen peroxide and halide, was studied widely for its bactericidal and bacteriostatic action. Nowadays, most of the studies are focus on mammalian peroxidase(Lactoperoxidase,myeloperoxidase). Mammalian peroxidase is hard to be obtained and very expensive. So a new peroxidase which have the similar structure and catalytic activity was expected to take the place of mammalian peroxidase. The peroxidase would have potential significance in academic research and application.
Soybean peroxidase(SBP) obtained from the soybean coats belongs to class III of peroxidase syperfamily(EC1.11.1.7) with hemachrome as its prosthetic group. It is similar to other peroxidase in structure and function. Its raw material was rather cheap and easily obtained. SBP was highly reactive towards both organic and inorganic substrates.SBP was also very stable at high temperature, wide pH and so on. Due to these advantages,more and more attentions are paid to the studies of SBP.
Our studies showed that soybean peroxidase-hydrogen peroxide-potassium iodide system was effective on bactericidal and bacteriostatic action. The system's antimicrobial action can be influenced by organic materials and pH etc. Killing kinetics of the system for E.coli and S.aureu indicated its rapid antimicrobial action. The stability of the system was also very high, as it was still active after being placed for 4 hours at 37℃.
The antibacterial mechanism of SBP-H_2O_2-KI system was studied. The results of ESR experiments demonstrated that there was no free radical formation in SBP-H_2O_2-KI system. In the same H_2O_2 concentration, however, free radical was detected in the ferrous ion-hydrogen peroxide system (a typical system generating hydroxy radical). Campared to the Fe~(2+)-H_2O_2 system, SBP-H_2O_2-KI system had absolute lethal effect on E.coli3\343 and S.aureu. The antibacterial effect of SBP-H_2O_2-KI system was not due to the production of the free radical which generally reported. We speculated that iodide ion performed a mediated role in SBP-H_2O_2-KI system. The antibacterial effect of SBP-H_2O_2-KI system was due to the activating oxidation state of iodine. We detected obvious absorption peak of free radical in the Fe~(2+)-H_2O_2 -KI system which had efficiency on antibacterial action. However, when making a comparison between the catalytic activity of Fe~(2+) and SBP, we found that the antibacterial action of Fe~(2+)-H_2O_2 -K.I system was lower than SBP-H_2O_2-KI system. It explained that the catalytic activity of SBP in the H_2O_2-KI system was higher than Fe~(2+), so the SBP in H_2O_2-KI system cannot be substituted by Fe~(2+) in the aspect of catalytic activity. We speculated the lethal effect of activating oxidation state of iodine was more effective than free radical.
We also made further experiment to study the variety of sensitivity of cells for the SBP-H_2O_2-KI system in the sublethal level. The sensitivity of E.coli3\343 did not vary from the first generation to the fifteenth generation, but reduced a little from the beginning of the sixteenth generation. The sensitivity of S.aureu brought down from the beginning of the first generation, but fluctuated in a certain range and kept steadily from the second generation to the fifteenth generation. The variety of sensitivity of cells in sublethal level may be caused by physiological adaptation or heritable mutation, and the detailed reason can be confirmed in the further study.
大豆过氧化物酶(soybean peroxidase，SBP)是从大豆加工副产物大豆皮中提取的，以血红素为辅基的Ⅲ类过氧化物酶(EC1．11．1．7)。与其它过氧化物酶在结构和功能上都有许多相似之处，但因其原料价廉易得，底物作用范围广、耐热性能高、酸碱稳定性好、pH适用范围宽等优点，大豆过氧化物酶的研究越来越成为过氧化物酶研究的热点。
实验发现SBP-H_2O_2-KI组成的体系具有杀菌／抑菌作用，抗菌强度受有机物、pH值等的影响。SBP-H_2O_2-KI体系对E.coli、S.aureu的杀菌动力学表明，该体系具有快速的杀菌效果。SBP-H_2O_2-KI体系也具有较好的稳定性，37℃下放置4 h后仍具有杀菌作用。
本文初步研究了SBP-H_2O_3-KI体系的抗菌机理。利用电子顺磁共振波谱法没有检测到SBP-H_2O_2-KI体系中的自由基吸收峰。但在相同的H_2O_2浓度下，Fe~(2+)-H_2O_2体系(产生羟自由基的典型体系)却有明显的自由基吸收峰。比较Fe~(2+)-H_2O_2体系与SBP-H_2O_2-KI体系对E.coli31343与S.aureu的抗菌作用。结果显示，Fe~(2+)-H_2O_2体系几乎没有杀菌效果，而SBP-H_2O_2-KI体系却能完全致死菌体细胞。这也进一步说明了，SBP-H_2O_2-KI体系的抗菌作用不是由于自由基的产生。我们推测在SBP-H_2O_2-KI体系中，I~-离子起了介导作用，抗菌效果是由于碘的活性氧化态。我们在Fe~(2+)-H_2O_2-KI体系中检测到了明显的自由基吸收峰，并发现该体系也具有部分的抗菌作用。但比较Fe~(2+)和SBP对H_2O_2-KI反应的催化活性时，发现Fe~(2+)-H_2O_2-KI体系的抗菌强度远低于SBP-H_2O_2-KI体系。说明SBP对H_2O_2-KI反应的催化活性高于Fe~(2+)的催化活性。Fe~(2+)不能代替SBP在H_2O_2-KI体系中发挥的作用。推测碘的活性氧化态的致死效应可能比自由基更甚。
本文还进一步研究了在SBP-H_2O_2-KI体系的亚致死剂量下连续传代，菌体细胞对SBP-H_2O_2-KI体系敏感性的变化。E.coli31343从第一代传至第十五代，敏感性均未变化，但从第十六代起E.coli31343的敏感性有轻微的降低。而S.aureu从第一代开始敏感性就降低了，但从第二代传至第十五代敏感性上下波动，一定范围内保持恒定。亚致死剂量下菌体敏感性的轻微变化，是由于生理适应还是遗传改变，需深入研究。
A system, made up of peroxidease.hydrogen peroxide and halide, was studied widely for its bactericidal and bacteriostatic action. Nowadays, most of the studies are focus on mammalian peroxidase(Lactoperoxidase,myeloperoxidase). Mammalian peroxidase is hard to be obtained and very expensive. So a new peroxidase which have the similar structure and catalytic activity was expected to take the place of mammalian peroxidase. The peroxidase would have potential significance in academic research and application.
Soybean peroxidase(SBP) obtained from the soybean coats belongs to class III of peroxidase syperfamily(EC1.11.1.7) with hemachrome as its prosthetic group. It is similar to other peroxidase in structure and function. Its raw material was rather cheap and easily obtained. SBP was highly reactive towards both organic and inorganic substrates.SBP was also very stable at high temperature, wide pH and so on. Due to these advantages,more and more attentions are paid to the studies of SBP.
Our studies showed that soybean peroxidase-hydrogen peroxide-potassium iodide system was effective on bactericidal and bacteriostatic action. The system's antimicrobial action can be influenced by organic materials and pH etc. Killing kinetics of the system for E.coli and S.aureu indicated its rapid antimicrobial action. The stability of the system was also very high, as it was still active after being placed for 4 hours at 37℃.
The antibacterial mechanism of SBP-H_2O_2-KI system was studied. The results of ESR experiments demonstrated that there was no free radical formation in SBP-H_2O_2-KI system. In the same H_2O_2 concentration, however, free radical was detected in the ferrous ion-hydrogen peroxide system (a typical system generating hydroxy radical). Campared to the Fe~(2+)-H_2O_2 system, SBP-H_2O_2-KI system had absolute lethal effect on E.coli3\343 and S.aureu. The antibacterial effect of SBP-H_2O_2-KI system was not due to the production of the free radical which generally reported. We speculated that iodide ion performed a mediated role in SBP-H_2O_2-KI system. The antibacterial effect of SBP-H_2O_2-KI system was due to the activating oxidation state of iodine. We detected obvious absorption peak of free radical in the Fe~(2+)-H_2O_2 -KI system which had efficiency on antibacterial action. However, when making a comparison between the catalytic activity of Fe~(2+) and SBP, we found that the antibacterial action of Fe~(2+)-H_2O_2 -K.I system was lower than SBP-H_2O_2-KI system. It explained that the catalytic activity of SBP in the H_2O_2-KI system was higher than Fe~(2+), so the SBP in H_2O_2-KI system cannot be substituted by Fe~(2+) in the aspect of catalytic activity. We speculated the lethal effect of activating oxidation state of iodine was more effective than free radical.
We also made further experiment to study the variety of sensitivity of cells for the SBP-H_2O_2-KI system in the sublethal level. The sensitivity of E.coli3\343 did not vary from the first generation to the fifteenth generation, but reduced a little from the beginning of the sixteenth generation. The sensitivity of S.aureu brought down from the beginning of the first generation, but fluctuated in a certain range and kept steadily from the second generation to the fifteenth generation. The variety of sensitivity of cells in sublethal level may be caused by physiological adaptation or heritable mutation, and the detailed reason can be confirmed in the further study.
引文
1 Paul BB,Strauss RR, Selvaraj RJ, Sbarra AJ. Peroxidase mediated antimicrobial activities of alveolar macrophage granules. Science, 1973,181:849-850.
2 Edwin L, Thomas M. Cofactor role of iodide in peroxidase antimicrobial action against Escherichia coll. Antimicrobial agents and chemotherapy, 1978, 13:1000-1005.
3 Adak S, Mazurada A,Banerjee R. Low catalytic turnover of horseradish peroxidase in thiocyanate oxidation. Journal of Biochemistry, 1997, 272(17):11049-11056.
4 曹劲松编著．初乳功能性食品[M]．北京：中国轻工业出版社，2000．
5 Ndewit J. Structure, function and application of lactoperoperoxidase in natural antimicrobial system. [J] Netherlands Milk and Dairy Journal, 1996, 50:227-244.
6 徐天宁．一种天然抗菌活性——乳过氧化物酶系统．中国乳品工业，1994， 22：235-239．
7 李萌．乳过氧化物酶体系的性质及利用．新疆畜牧业，2000，3：20-22．
8 李青．利用牛乳中天然的乳过氧化物酶体系来保存被冷藏的和未经冷却的牛 乳．乳品工业，1982，3：71-76．
9 宋焕禄，马力远．乳过氧化物酶体系的抗菌活性．中国乳品工业，1997，25： 26-27．
10 Hogg B, Jago G. The antibacterial action of lactoperoxidase. Journal of Biochemistry, 1970, 117:779-790.
11 Thomas E, Milligan T,Joyner R. Antibacterial activity of hydrogen peroxide and the lactoperoxidase-hydrogen peroxide-thiocyanate system against oral streptococci.Infection and Immunity, 1994, 62:529-535.
12 Thomas. A, Aune T. Lactoperoxidase, peroxide, thiocyanate antimicrobial system:correlation of sulfhydryl oxidation with antimicrobial??action.Infection and Immunity,1977, 20:456-463.
13 Steele W, Morrison M. Antistreptococcal activity of lactoperoxidase. Journal of Bacteriol,1969,97:635-639.
14 Thomas L, Pera A, Smith W, Chwang K. Inhibition of Streptococcus mutans by the lactoperoxidase antimicrobial system. Infection and Immunity, 1983, 39:767-778.
15 Edwin L,Aune M. Susceptibility of Escherichia coli to bactericidal action of lactoperoxidase, peroxide,and iodide or thiocyanate. Antimicrobial agents and chemotherapy,1978, 13,261-265.
16 张继慈，译．保存牛乳的新方法[J]．国外畜牧科技，1983，3：3-7．
17 方允中，李文杰主编．自由基与酶——基础理论及其在生物学和医学中的应 用[M]．科学出版社，1989．
18 Belding E, Klebanoff S, Ray C. Peroxidase-mediated virucidal systems. Science,1970,167:195-196.
19 Thomas L. Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system:nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli. Infection and Immunity, 1979, 23, 522-531.
20 Rosen H, Orman J, Rakita M, Michel R, Banderanter R. Loss of DNA-membrane interactions and cessation synthesis in myeloperoxidase-treated Escherichia coli. Proc. Natl. Acad. Sci. USA, 1990, 87:10048-10052.
21 Dunford HB. Peroxidase-catalyzed halide ion oxidation[J].Redox Rep,2005, 5:169-171.
22 Thomas L, Aune M. Oxidation of Escherichia coli sulfhydryl components by the peroxidase-hydrogen peroxide-iodide antimicrobialsystem. Antimicrobial agents and chemotherapy, 1978, 13:1006-1010.
23 Borelli B, Vita F,Shankar S,Soranzo M,Banfi E. Human eosinophil peroxidase induces surface alteration,killing,and lysis of Mycobacterium tuberculosis. Infection and immunity,2003,71:605-613.
24 Hansen E, Albertsen L. Curvularia haloperoxidase:antimicrobial activity and potential application as a surface disinfectant. Applied and environmental microbiology, 2003, 69:4611-4617.
25 Hansen E, Schembri M. Elucidation of the antibacterial mechanism of the Curvularia haloperoxidase system by DNA microarray profiling. Applied And environmental microbiology, 2004, 70:1794-1757.
26 Ihalin R, Nuutila J. Susceptibility of fusobacterium nucleatum to Killing by peroxidase-iodide-hydrogen peroxide combination in buffer Solution and in human whole saliva. Anaerobe, 2003, 9:23-30.
27 Sessa D J, Anerson R L. Soybean peroxidase:purification and some properties. J. Agric. Food Chem, 1981, 29(5):960-965.
28 Kamal J K A, Behere D V. Activity, stability and conformational flexibility of seed coat soybean peroxidase. Journal of Inorganic Biochemistry, 2003, 94:236-242.
29 薛广波主编．实用消毒学[M]．人民军医出版社，1986．
30 孙俊主编．消毒技术与应用[M]．化学工业出版社，2003．
31 方允中，郑荣梁主编．自由基生物学的理论与应用[M]．科学出版社，2002．
32 杨保收，陈越，金久善，董梅．氧自由基检测方法在生物学和医学中的应 用．China academic journal electronic publishing house．1999，3：17-18．
33 Drlica K. Antibiotic resistance:can we beat the bugs[J].Drug Discov Today, 2001,6:714-716.
34 Levy SB. Multidrug resistance:a sign of the times[J].N Engl J Med,1998,38:1376-1378.
35 Zhao X. Restricting the selection of antibiotic-resistant mutants:a general strategy derived from fluoroquinolones studies[J]. Clin Infect Dis, 2001,33:147-156.
36 Drilica K. A strategy for fighting antibiotic resistance[J]. ASM News, 2001,67:27-33.
37 闵航主编．微生物学[M]．浙江大学出版社，2005，2：153-154．
38 Kobayashi A, Koguchi Y, Kanzaki H, Kajiyama S, Kawazu K.A new type of antimicrobial phenolics produced by plant peroxidase and its possible role in the chemical defense systems against plant pathogens.Z Naturforch[C], 1994, 49:411-414.
39 Weis VM, Small AL, Mcfall-Ngai MJ. A peroxidase related to the mammalian antimicrobial protein myeloperoxidase in the euprymna-vibrio mutualism. Proc Natl Acad Sci USA,1996, 93:13683-13688.
40 王铨茂，许红．玉米对玉米小斑病菌的抗病性与过氧化物酶活力关系的研究 初报．植物病理学报，1983，18：60-62．
41 Oram D, Reiter B. The inhibition of streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. Biochem. J, 1966, 100: 373-381.
42 Klebanoff J. Myeloperoxidae-halide-hydrogen peroxide antibacterial system. Journal of bacteriology, 1968, 6:2131-2138.
43 Wright D, Nelson D. Candidacidal activity of myeloperoxidase: therapeutic influence of the enzyme in vivo. Infection and immunity, 1985, 2: 363-365.
44 徐之勇．大豆过氧化物酶的酶学特性与应用研究．硕士论文．浙江大学．
45 Yue K.T, Taylor K. L, Powers L.S. X-ray absorption and resonance raman spectroscopy of human myeloperoxidase at neutral and acid pH. Biochem. Biopys. Acta, 1997, 1338: 282-294.
46 Furtmuller P. G, Burner U, Obinger C. Reaction of myeloperoxidase compound I with chloride, bromide, iodide and thiocyanate. Biochemistry, 1998, 37: 17923-17930.
47 Van Dalen C. J, Whitehouse M. W, Winterbourn C. C, Kettle A. J. Thiocyanate and chloride as competing substrates for myeloperoxidase.Biochem. J, 1997, 327: 487-492.
48 Michael A, Purdy, Jorma T. Effect of growth phase and cell envelope structure on susceptibility of salmonella typhimurium to the lactoperoxidase-thiocyanate-hydrogen peroxide system. Infection and??immunity, 1983, 39:1187-1195.
49 Kenneth M, Pruitt, Michael A, Roland A. Lactoperoxidase binding to streptococci. Infection and immunity, 1979, 25:304-309.
50 Clifford D, Wright, James U. Candidacidal activity of myeloperoxidse: Mechanisms of inhibitory influence of soluble cell wall mannan. Infection and immunity, 1983, 42:76-80.
51 Henry R, Seymour J. Oxidation of microbial iron-sulfur centers by the myeloperoxidase-H_2O_2-halide antimicrobial system. Infection and Immunity, 1985,47:613-618.
52 Henry R, Bryce R, Michel. Differential effects of myeloperoxidase-Derived oxidants on escherichia coli DNA replication. Infection and Immunity, 1998, 66:2655-2659.
53 穆光照，甘礼雅，陈敏为译．自由基化学[M]．上海科学技术出版社，1982．
54 方允中．自由基牛物学的理论与应用[M]．科学出版社，2005．
55 胡明．黄孢原毛平革菌胞外低分子物质在木素降解过程中作用的研究．博士论 文．山东大学，2006．
56 庞战军，周玫，陈瑗．自由基医学研究方法[M]．人民卫生出版社，2000．
57 汪守建，刘德启，江飞，王寿武．氧化-萃取光度法检测单线态氧．贵州工业大 学学报，2004，33：12-14．
58 刘玉庆．表征抗菌药物杀菌动力学的新方法以及细菌抗药性与耐药性区分的 研究．博士论文．山东大学，2005．
59 Britigan B, Ratcliffe H, Buettner G, Rosen G. Binding of myeloperoxidase to bacteria:effect on hydroxyl radical formation and susceptibility to oxidant-mediated killing. Biochimica et Biophysica Acta , 1996, 1290:231-240.
60 Kiryu C, Makiuchi M, Miyazaki J, Fujinaga T, Kakinuma K. Physiological production of singlet molecular oxygen in the myeloperoxidase-H2O2-Chloride system. Federation of European Biochemical Societies, 1999, 43 :154-158.
61 Alva KS, Kumar J, Marx AK, et al. Enzymatic synthesis and characterization of a novel water- soluble polyaniline:poly(2, 5-diaminobenzenesulfonate). Macromolecules, 1997, 30:4024-4029.
62 Jin Z, Su Y, Duan Y. A novel method for polyaniline synthesis with the immobilized horseradish peroxidase enzyme. Synthetic Metals, 2001, 122:237-242.
63 Lim CH, Yoo YJ. Synthesis of ortho-directed polyaniline using horseradish peroxidase. Process Biochemistry, 2000, 36:233-241.
2 Edwin L, Thomas M. Cofactor role of iodide in peroxidase antimicrobial action against Escherichia coll. Antimicrobial agents and chemotherapy, 1978, 13:1000-1005.
3 Adak S, Mazurada A,Banerjee R. Low catalytic turnover of horseradish peroxidase in thiocyanate oxidation. Journal of Biochemistry, 1997, 272(17):11049-11056.
4 曹劲松编著．初乳功能性食品[M]．北京：中国轻工业出版社，2000．
5 Ndewit J. Structure, function and application of lactoperoperoxidase in natural antimicrobial system. [J] Netherlands Milk and Dairy Journal, 1996, 50:227-244.
6 徐天宁．一种天然抗菌活性——乳过氧化物酶系统．中国乳品工业，1994， 22：235-239．
7 李萌．乳过氧化物酶体系的性质及利用．新疆畜牧业，2000，3：20-22．
8 李青．利用牛乳中天然的乳过氧化物酶体系来保存被冷藏的和未经冷却的牛 乳．乳品工业，1982，3：71-76．
9 宋焕禄，马力远．乳过氧化物酶体系的抗菌活性．中国乳品工业，1997，25： 26-27．
10 Hogg B, Jago G. The antibacterial action of lactoperoxidase. Journal of Biochemistry, 1970, 117:779-790.
11 Thomas E, Milligan T,Joyner R. Antibacterial activity of hydrogen peroxide and the lactoperoxidase-hydrogen peroxide-thiocyanate system against oral streptococci.Infection and Immunity, 1994, 62:529-535.
12 Thomas. A, Aune T. Lactoperoxidase, peroxide, thiocyanate antimicrobial system:correlation of sulfhydryl oxidation with antimicrobial??action.Infection and Immunity,1977, 20:456-463.
13 Steele W, Morrison M. Antistreptococcal activity of lactoperoxidase. Journal of Bacteriol,1969,97:635-639.
14 Thomas L, Pera A, Smith W, Chwang K. Inhibition of Streptococcus mutans by the lactoperoxidase antimicrobial system. Infection and Immunity, 1983, 39:767-778.
15 Edwin L,Aune M. Susceptibility of Escherichia coli to bactericidal action of lactoperoxidase, peroxide,and iodide or thiocyanate. Antimicrobial agents and chemotherapy,1978, 13,261-265.
16 张继慈，译．保存牛乳的新方法[J]．国外畜牧科技，1983，3：3-7．
17 方允中，李文杰主编．自由基与酶——基础理论及其在生物学和医学中的应 用[M]．科学出版社，1989．
18 Belding E, Klebanoff S, Ray C. Peroxidase-mediated virucidal systems. Science,1970,167:195-196.
19 Thomas L. Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system:nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli. Infection and Immunity, 1979, 23, 522-531.
20 Rosen H, Orman J, Rakita M, Michel R, Banderanter R. Loss of DNA-membrane interactions and cessation synthesis in myeloperoxidase-treated Escherichia coli. Proc. Natl. Acad. Sci. USA, 1990, 87:10048-10052.
21 Dunford HB. Peroxidase-catalyzed halide ion oxidation[J].Redox Rep,2005, 5:169-171.
22 Thomas L, Aune M. Oxidation of Escherichia coli sulfhydryl components by the peroxidase-hydrogen peroxide-iodide antimicrobialsystem. Antimicrobial agents and chemotherapy, 1978, 13:1006-1010.
23 Borelli B, Vita F,Shankar S,Soranzo M,Banfi E. Human eosinophil peroxidase induces surface alteration,killing,and lysis of Mycobacterium tuberculosis. Infection and immunity,2003,71:605-613.
24 Hansen E, Albertsen L. Curvularia haloperoxidase:antimicrobial activity and potential application as a surface disinfectant. Applied and environmental microbiology, 2003, 69:4611-4617.
25 Hansen E, Schembri M. Elucidation of the antibacterial mechanism of the Curvularia haloperoxidase system by DNA microarray profiling. Applied And environmental microbiology, 2004, 70:1794-1757.
26 Ihalin R, Nuutila J. Susceptibility of fusobacterium nucleatum to Killing by peroxidase-iodide-hydrogen peroxide combination in buffer Solution and in human whole saliva. Anaerobe, 2003, 9:23-30.
27 Sessa D J, Anerson R L. Soybean peroxidase:purification and some properties. J. Agric. Food Chem, 1981, 29(5):960-965.
28 Kamal J K A, Behere D V. Activity, stability and conformational flexibility of seed coat soybean peroxidase. Journal of Inorganic Biochemistry, 2003, 94:236-242.
29 薛广波主编．实用消毒学[M]．人民军医出版社，1986．
30 孙俊主编．消毒技术与应用[M]．化学工业出版社，2003．
31 方允中，郑荣梁主编．自由基生物学的理论与应用[M]．科学出版社，2002．
32 杨保收，陈越，金久善，董梅．氧自由基检测方法在生物学和医学中的应 用．China academic journal electronic publishing house．1999，3：17-18．
33 Drlica K. Antibiotic resistance:can we beat the bugs[J].Drug Discov Today, 2001,6:714-716.
34 Levy SB. Multidrug resistance:a sign of the times[J].N Engl J Med,1998,38:1376-1378.
35 Zhao X. Restricting the selection of antibiotic-resistant mutants:a general strategy derived from fluoroquinolones studies[J]. Clin Infect Dis, 2001,33:147-156.
36 Drilica K. A strategy for fighting antibiotic resistance[J]. ASM News, 2001,67:27-33.
37 闵航主编．微生物学[M]．浙江大学出版社，2005，2：153-154．
38 Kobayashi A, Koguchi Y, Kanzaki H, Kajiyama S, Kawazu K.A new type of antimicrobial phenolics produced by plant peroxidase and its possible role in the chemical defense systems against plant pathogens.Z Naturforch[C], 1994, 49:411-414.
39 Weis VM, Small AL, Mcfall-Ngai MJ. A peroxidase related to the mammalian antimicrobial protein myeloperoxidase in the euprymna-vibrio mutualism. Proc Natl Acad Sci USA,1996, 93:13683-13688.
40 王铨茂，许红．玉米对玉米小斑病菌的抗病性与过氧化物酶活力关系的研究 初报．植物病理学报，1983，18：60-62．
41 Oram D, Reiter B. The inhibition of streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. Biochem. J, 1966, 100: 373-381.
42 Klebanoff J. Myeloperoxidae-halide-hydrogen peroxide antibacterial system. Journal of bacteriology, 1968, 6:2131-2138.
43 Wright D, Nelson D. Candidacidal activity of myeloperoxidase: therapeutic influence of the enzyme in vivo. Infection and immunity, 1985, 2: 363-365.
44 徐之勇．大豆过氧化物酶的酶学特性与应用研究．硕士论文．浙江大学．
45 Yue K.T, Taylor K. L, Powers L.S. X-ray absorption and resonance raman spectroscopy of human myeloperoxidase at neutral and acid pH. Biochem. Biopys. Acta, 1997, 1338: 282-294.
46 Furtmuller P. G, Burner U, Obinger C. Reaction of myeloperoxidase compound I with chloride, bromide, iodide and thiocyanate. Biochemistry, 1998, 37: 17923-17930.
47 Van Dalen C. J, Whitehouse M. W, Winterbourn C. C, Kettle A. J. Thiocyanate and chloride as competing substrates for myeloperoxidase.Biochem. J, 1997, 327: 487-492.
48 Michael A, Purdy, Jorma T. Effect of growth phase and cell envelope structure on susceptibility of salmonella typhimurium to the lactoperoxidase-thiocyanate-hydrogen peroxide system. Infection and??immunity, 1983, 39:1187-1195.
49 Kenneth M, Pruitt, Michael A, Roland A. Lactoperoxidase binding to streptococci. Infection and immunity, 1979, 25:304-309.
50 Clifford D, Wright, James U. Candidacidal activity of myeloperoxidse: Mechanisms of inhibitory influence of soluble cell wall mannan. Infection and immunity, 1983, 42:76-80.
51 Henry R, Seymour J. Oxidation of microbial iron-sulfur centers by the myeloperoxidase-H_2O_2-halide antimicrobial system. Infection and Immunity, 1985,47:613-618.
52 Henry R, Bryce R, Michel. Differential effects of myeloperoxidase-Derived oxidants on escherichia coli DNA replication. Infection and Immunity, 1998, 66:2655-2659.
53 穆光照，甘礼雅，陈敏为译．自由基化学[M]．上海科学技术出版社，1982．
54 方允中．自由基牛物学的理论与应用[M]．科学出版社，2005．
55 胡明．黄孢原毛平革菌胞外低分子物质在木素降解过程中作用的研究．博士论 文．山东大学，2006．
56 庞战军，周玫，陈瑗．自由基医学研究方法[M]．人民卫生出版社，2000．
57 汪守建，刘德启，江飞，王寿武．氧化-萃取光度法检测单线态氧．贵州工业大 学学报，2004，33：12-14．
58 刘玉庆．表征抗菌药物杀菌动力学的新方法以及细菌抗药性与耐药性区分的 研究．博士论文．山东大学，2005．
59 Britigan B, Ratcliffe H, Buettner G, Rosen G. Binding of myeloperoxidase to bacteria:effect on hydroxyl radical formation and susceptibility to oxidant-mediated killing. Biochimica et Biophysica Acta , 1996, 1290:231-240.
60 Kiryu C, Makiuchi M, Miyazaki J, Fujinaga T, Kakinuma K. Physiological production of singlet molecular oxygen in the myeloperoxidase-H2O2-Chloride system. Federation of European Biochemical Societies, 1999, 43 :154-158.
61 Alva KS, Kumar J, Marx AK, et al. Enzymatic synthesis and characterization of a novel water- soluble polyaniline:poly(2, 5-diaminobenzenesulfonate). Macromolecules, 1997, 30:4024-4029.
62 Jin Z, Su Y, Duan Y. A novel method for polyaniline synthesis with the immobilized horseradish peroxidase enzyme. Synthetic Metals, 2001, 122:237-242.
63 Lim CH, Yoo YJ. Synthesis of ortho-directed polyaniline using horseradish peroxidase. Process Biochemistry, 2000, 36:233-241.