Linder R, Salton M R
Eur J Biochem. 1975 Jun 16;55(1):291-7. doi: 10.1111/j.1432-1033.1975.tb02162.x.
Conversion of whole cells of Micrococcus lysodeikticus to protoplasts allowed the release of a soluble form of a D-alanine carboxypeptidase into the protoplasting medium. The enzyme cleaves the terminal D-alanine from the radioactively labelled UDP-N-acetylmuramyl-pentapeptide containing L-lysine as the diamino acid. However, the enzyme is only minimally active in this fraction so that it had to be enriched and partially purified before its properties could be studied. Chromatography on carboxymethyl-Sephadex removed the lysozyme used in the protoplasting of the cells. The material which was unadsorbed to the column was applied to an affinity chromatography column of Ampicillin-Sepharose. Most of the contaminating protein was washed from the column while the D-alanine carboxypeptidase adhered to the resin and could be eluted with 0.5 M Tris-HCl buffer pH 8.6. Some of the properties of the enzymic activity were studied using this preparation. The enzyme was activated by Mg2+ ions with a broad optimum from 15--35 mM. It was maximally active when NaCl at a concentrations of 0.06--0.08 M was added to the assay, and the pH curve was biphasic with an alkaline optimum. The Km for substrate was found to be 0.118 mM. Enzymic activity was completely inhibited by low concentrations of Ampicillin and penicillin G.
将溶壁微球菌的全细胞转化为原生质体，使得一种可溶性形式的D - 丙氨酸羧肽酶释放到原生质体形成培养基中。该酶能从含有L - 赖氨酸作为二氨基酸的放射性标记的UDP - N - 乙酰胞壁酰 - 五肽上切割末端的D - 丙氨酸。然而，该酶在这一部分中的活性极低，因此在研究其性质之前必须进行富集和部分纯化。在羧甲基 - 葡聚糖凝胶上进行色谱分离可去除细胞原生质体形成过程中使用的溶菌酶。未吸附到柱上的物质被应用于氨苄青霉素 - 琼脂糖亲和色谱柱。大部分污染蛋白从柱上被洗脱，而D - 丙氨酸羧肽酶则附着在树脂上，可用0.5 M Tris - HCl缓冲液（pH 8.6）洗脱。使用该制剂研究了酶活性的一些性质。该酶被Mg2 +离子激活，在15 - 35 mM范围内有较宽的最佳浓度。当向测定中加入浓度为0.06 - 0.08 M的NaCl时，酶活性最大，pH曲线呈双相，碱性条件下活性最佳。发现底物的Km为0.118 mM。低浓度的氨苄青霉素和青霉素G可完全抑制酶活性。
