权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Selection of mastitis-resistant cows using the production of L-amino acid oxidase

利用L-氨基酸氧化酶的生产选择抗乳腺炎奶牛

基本信息

批准号：
13356006
负责人：
SAKAI Senkiti
金额：
$ 31.87万
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 (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

项目摘要

Characterization of L-amino acid oxidase (LAO) in mouse milkLAO was purified from mouse milk. LAO reacted with L-amino acids in an apparent order of Phe> Met, Tyr> Cys, Leu> His>> other 11 amino acids and produced hydrogen peroxide (H_2O_2) in a dose-and time-dependent manner. An apparent Km was 1.0μM for Phe, 8.1μM for Leu and 20.2μM for His. LAO in milk had a molecular weight of 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. By ion-exchange chromatography, the LAO activity was detected in three different fractions. LAO consisted of two subunits but is electrically heterogeneous. The amino acid sequences of N- and C-terminals, and lysylendopeptidase-digested fragments were the same to those of mouse milk LAO deduced from cDNA. This is the first demonstration showing that LAO is present in milk.Mechanism of Bacterial Killing by Mouse MilkAfter gel-filtration, we detected lactoperoxidase (LPO) activity. Using LAO, LPO and amino acids, H_2O_2 was maintained between 190 and 23 … More 0 nmol/ml from 1 to 4 h. We cultured Escherichia (E) coli, Klebsiella (K) pneumoniae, Staphylococcus (S) aureus, and Streptococcus (S) agalactiae strains. Bacteria increased with a doubling time of 30-40 min. However, S.agalactiae and S.aureus did not grow ; the number of viable bacteria remained the same at 4 h as at 0 h. Escherichia coli and K.pneumoniae grew with doubling times of 98 and 84 min, respectively, and growth was suppressed. Bacterial growth was suppressed in the LAO/LPO/amino acid system.Hydrogen peroxide generating substance in cow milkCow milk contained a H_2O_2 generating substance. After gel-filtration, the fraction with a molecular weight of 20 kDa had H_2O_2 generating activity, and generated it in a linear manner against the time. A H_2O_2 generating activity increased from early to mid-lactation and decreases at the end of lactation. The activity in mastitis milk was lower than that in normal milk. These results suggest that the H_2O_2 generating substance plays an important role for the activation of the LPO-system which is a potent antibacterial system formed by LPO, H_2O_2, and thiocyanate. Less

小鼠乳中l -氨基酸氧化酶（LAO）的表征。LAO与l -氨基酸的反应顺序为：Phe b> Met, Tyr b> Cys, Leu> His>>其他11种氨基酸，并以剂量和时间依赖的方式产生过氧化氢（H_2O_2）。Phe、Leu和His的表观Km分别为1.0μM、8.1μM和20.2μM。牛奶中的LAO分子量为113 kDa，通过SDS-PAGE转化为60 kDa的蛋白质。采用离子交换色谱法测定了三种不同组分的LAO活性。LAO由两个亚基组成，但具有电异质性。N端、c端和赖氨酸肽酶酶切片段的氨基酸序列与从cDNA中推断的小鼠乳LAO相同。这是第一次证明牛奶中存在LAO。通过凝胶过滤，我们检测了乳酸过氧化物酶（LPO）的活性。利用LAO、LPO和氨基酸，H_2O_2在1 ~ 4 h内维持在190 ~ 23…More 0 nmol/ml。培养大肠杆菌、肺炎克雷伯菌、金黄色葡萄球菌和无乳链球菌。细菌数量增加的时间为30-40 min。然而，无乳链球菌和金黄色葡萄球菌没有生长；4 h和0 h的活菌数量保持不变，大肠埃希菌和肺炎克雷伯菌分别在98 min和84 min增加了一倍，并且生长受到抑制。在LAO/LPO/氨基酸体系中，细菌生长受到抑制。牛奶中的过氧化氢生成物质牛奶中含有一种H_2O_2生成物质。经凝胶过滤后，分子量为20 kDa的馏分具有生成H_2O_2的活性，且生成H_2O_2随时间呈线性关系。A生成H_2O_2活性在泌乳早期至中期呈上升趋势，在泌乳末期呈下降趋势。乳腺炎乳中的活性低于正常乳中的活性。这些结果表明，H_2O_2生成物质对LPO-系统的激活起着重要作用，LPO-系统是由LPO、H_2O_2和硫氰酸盐组成的一个有效的抗菌系统。少