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

小鼠乳中L-氨基酸氧化酶（LAO）的性质LAO是从小鼠乳中分离纯化的。LAO与L-氨基酸的反应顺序为Phe> Met、Tyr> Cys、Leu> His>>其它11种氨基酸，并产生过氧化氢（H_2O_2），且呈剂量和时间依赖性。对苯丙氨酸、亮氨酸和组氨酸的表观Km分别为1.0μM、8.1μM和20.2μM。LAO在牛奶中的分子量为113 kDa，并转化为60 kDa的蛋白质，通过SDS-PAGE。通过离子交换层析，在三个不同的馏分中检测LAO活性。LAO由两个亚基组成，但电异质性。氨基酸序列的N-和C-末端，和赖氨酰内肽酶消化片段是相同的小鼠乳LAO推导的cDNA。这是第一次证明乳中存在LAO。小鼠乳杀菌机理的研究采用凝胶过滤法检测乳过氧化物酶（lactoperoxidase，LPO）活性。使用LAO、LPO和氨基酸，使H_2O_2维持在190 ~ 23 ...更多信息 0 nmol/ml。我们培养了大肠埃希菌、肺炎克雷伯菌、金黄色葡萄球菌和无乳链球菌菌株。细菌在30-40 min的倍增时间内增加。但是，无乳链球菌和金黄色葡萄球菌没有生长; 4 h时的活菌数量与0 h时相同。大肠埃希菌和肺炎克雷伯菌分别以98和84分钟的倍增时间生长，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H_2O_2生成物质。凝胶过滤后，分子量为20 kDa的组分具有H_2O_2生成活性，且生成量与时间呈线性关系。H_2O_2产生活性从哺乳早期到中期增加，到哺乳结束时减少。乳腺炎乳中的酶活性低于正常乳。这些结果表明，H_2O_2产生物质对LPO-体系的活化起着重要作用，而LPO-体系是由LPO、H_2O_2和硫氰酸盐形成的一个有效的抗菌体系。少

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