【作者】 李瑜；

【导师】 赵建； 张为灿；

【作者基本信息】 山东大学 ， 微生物学， 2008， 硕士

【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-H₂O₂-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-H₂O₂-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-H₂O₂-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-H₂O₃-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-H₂O₂-KI体系中的自由基吸收峰。但在相同的H₂O₂浓度下，Fe²⁺-H₂O₂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Fe²⁺-H₂O₂体系与SBP-H₂O₂-KI体系对E.coli31343与S.aureu的抗菌作用。结果显示，Fe²⁺-H₂O₂体系几乎没有杀菌效果，而SBP-H₂O₂-KI体系却能完全致死菌体细胞。这也进一步说明了，SBP-H₂O₂-KI体系的抗菌作用不是由于自由基的产生。我们推测在SBP-H₂O₂-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Fe²⁺-H₂O₂-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Fe²⁺和SBP对H₂O₂-KI反应的催化活性时，发现Fe²⁺-H₂O₂-KI体系的抗菌强度远低于SBP-H₂O₂-KI体系。说明SBP对H₂O₂-KI反应的催化活性高于Fe²⁺的催化活性。Fe²⁺不能代替SBP在H₂O₂-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-H₂O₂-KI体系的亚致死剂量下连续传代，菌体细胞对SBP-H₂O₂-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。更多还原

【Abstract】 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-H₂O₂-KI system was studied. The results of ESR experiments demonstrated that there was no free radical formation in SBP-H₂O₂-KI system. In the same H₂O₂ concentration, however, free radical was detected in the ferrous ion-hydrogen peroxide system （a typical system generating hydroxy radical）. Campared to the Fe²⁺-H₂O₂ system, SBP-H₂O₂-KI system had absolute lethal effect on E.coli3\343 and S.aureu. The antibacterial effect of SBP-H₂O₂-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-H₂O₂-KI system. The antibacterial effect of SBP-H₂O₂-KI system was due to the activating oxidation state of iodine. We detected obvious absorption peak of free radical in the Fe²⁺-H₂O₂ -KI system which had efficiency on antibacterial action. However, when making a comparison between the catalytic activity of Fe²⁺ and SBP, we found that the antibacterial action of Fe²⁺-H₂O₂ -K.I system was lower than SBP-H₂O₂-KI system. It explained that the catalytic activity of SBP in the H₂O₂-KI system was higher than Fe²⁺, so the SBP in H₂O₂-KI system cannot be substituted by Fe²⁺ 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-H₂O₂-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.更多还原