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Mechanism of killing bacteria, expression of gene encoded enzyme and its genetics

杀菌机制、基因编码酶的表达及其遗传学

基本信息

批准号：
14360163
负责人：
SAKAI Senkiti
金额：
$ 10.88万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

L-Amino acid oxidase (LAO) and lactoperoxidase (LPO) of mouse milk are separated by gel-filtration chromatography.LAO generates H_2O_2 in the presence of L-amino acids and subsequently, LPO generates O through the degradation of H_2O_2. Milk is capable of producing the bacterial toxic H_2O_2 and O The quantity of H_2O_2 was around 300 μmoles/ml milk. In order to examine whether or not LAO and LPO is responsible for killing bacteria in the mammary gland, bacteria (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus agalactiae) are isolated from bovine mastitis milk and cultured with LAO in the presence and absence of LPO. In the presence of L-amino acids, LAO inhibits the growth of Klebsiella pneumoniae and Escherichia coli, and kills Staphylococcus aureus. Streptococcus agalactiae grows normally in the presence of LAO and does not grow in the presence of LAO and LPO. Thus, we divide them into two types : one being H_2O_2-sensitive and the other being O^-sensitive. It is concluded that H_2O_2 and O^-generated in milk is able to prevent the growth of bacteria in the mouse mammary gland.Cow milk of Holstein and Japanese Black breeds are collected and separated by gel-filtration chromatography. The H_2O_2 generating enzyme is appeared at the peak fraction of 75, being a molecular weight of about 10 k. The eluting profiles of the two breeds are identical. The quantity.of this enzyme in milk is small at early lactation and at late lactation, but is relatively high at mid-lactation. By the incubation for 2 hr at 37℃, the enzyme generates H_2O_2 about 5-fold as compared to that of the 0 hr-incubation.

采用凝胶过滤色谱法分离小鼠乳中的l-氨基酸氧化酶（LAO）和乳过氧化物酶（LPO）。在l -氨基酸存在下，LAO生成H_2O_2，随后LPO通过H_2O_2的降解生成O。牛奶能产生细菌有毒的H_2O_2和O， H_2O_2的含量约为300 μmol /ml牛奶。为了检验LAO和LPO是否负责杀死乳腺中的细菌，从牛乳腺炎乳中分离出细菌（大肠杆菌、肺炎克雷伯菌、金黄色葡萄球菌和无乳链球菌），在存在和不存在LPO的情况下用LAO进行培养。在l -氨基酸存在的情况下，LAO抑制肺炎克雷伯菌和大肠杆菌的生长，并杀死金黄色葡萄球菌。无乳链球菌在有LAO存在时生长正常，在有LAO和LPO存在时不生长。因此，我们将它们分为两种类型：一种是h_2o_2敏感的，另一种是O^敏感的。由此可见，乳中产生的H_2O_2和O^-对小鼠乳腺细菌的生长具有一定的抑制作用。采用凝胶过滤色谱法对荷斯坦奶牛和日本黑奶牛的牛奶进行分离。H_2O_2生成酶出现在峰分数75处，分子量约为10 k，两个品种的洗脱谱相同。的数量。乳汁中这种酶的含量在泌乳早期和后期较少，但在泌乳中期相对较高。经37℃孵育2h后，酶的H_2O_2产生量是0 h时的5倍左右。