Thibault G, Guitard M, Daigneault R
Biochim Biophys Acta. 1980 Aug 7;614(2):339-42. doi: 10.1016/0005-2744(80)90223-5.
We report the purification of chloramphenicol acetyltransferase (acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) by a two-step procecdure involving chromatography on a Sepharose 4B-reduced chloramphenicol matrix and DEAE-Sephadex A-50. This procedure resulted in a 120-fold purification with 50% recovery of the enzyme. Only one band of enzyme activity was present after electrophoresis on polyacrylamide gel. The enzyme is active over a broad pH range, maximal activity being observed near pH 7.6. Both chloramphenicol 1-acetate of chloramphenicol 3-acetate were found to be very stable in Tris-maleate buffer at pH 6.09 with negligible interconversion. The incubation at pH 6.0 of chloramphenicol 1-acetate with the purified chloramphenicol acetyltransferase yielded chloramphenicol 1,3-diacetate. These data indicate that the enzyme acetylates specifically at the 3-hydroxy position and the diacetylation is possible only because of non-enzymatic interconversion of chloramphenicol 3-acetate to chloramphenicol 1-acetate at higher pH values.
我们报道了通过两步法纯化氯霉素乙酰转移酶（乙酰辅酶A：氯霉素3 - O - 乙酰转移酶，EC 2.3.1.28）的过程，该方法包括在琼脂糖4B - 还原氯霉素基质和DEAE - 葡聚糖A - 50上进行色谱分离。此方法使酶纯化了120倍，回收率为50%。在聚丙烯酰胺凝胶上电泳后仅出现一条酶活性带。该酶在较宽的pH范围内具有活性，在pH 7.6附近观察到最大活性。发现在pH 6.09的Tris - 马来酸缓冲液中，氯霉素1 - 乙酸酯和氯霉素3 - 乙酸酯都非常稳定，相互转化可忽略不计。纯化的氯霉素乙酰转移酶与氯霉素1 - 乙酸酯在pH 6.0下孵育产生了氯霉素1,3 - 二乙酸酯。这些数据表明该酶特异性地在3 - 羟基位置进行乙酰化，并且二乙酰化仅由于在较高pH值下氯霉素3 - 乙酸酯非酶促转化为氯霉素1 - 乙酸酯才有可能。
