Mannens G, Slegers G, Lambrecht R, Claeys A
Department of Pharmaceutical Sciences, State Univerisity of Ghent, Belgium.
Biochim Biophys Acta. 1988 Apr 15;959(3):214-9. doi: 10.1016/0005-2760(88)90193-2.
The enzyme acetylcoenzyme A synthetase (acetate-CoA ligase (AMP forming), EC 6.2.1.1) from Saccharomyces cerevisiae (baker's yeast) is used for the synthesis of 1 mumol [11C]acetylcoenzyme A. (CoA-[11C]Ac). A screening of the immobilization of the enzyme on differently derivatized controlled pore glass beads (50 nm pore size and 125-180 micron particle size) was performed. Several silanes, spacer arms and terminal reactive groups were tested. The immobilized enzyme was subjected to storage stability tests. From these experiments, the method of choice was selected: immobilization on CNBr-activated controlled pore glass. The immobilized parameters were optimized further to improve the activity of the enzyme-loaded glass beads. The latter were packed in a glass column. The kinetic properties of the column were investigated and optimized to obtain an almost complete conversion of 1 mumol acetate into acetylcoenzyme A (CoA-Ac) within a few minutes. This is realized with an enzyme reactor (13.0 x 0.5 cm) containing 6.12 U active acetylcoenzyme A synthetase immobilized onto 1 g controlled pore glass.
来自酿酒酵母（面包酵母）的乙酰辅酶A合成酶（乙酸辅酶A连接酶（生成AMP），EC 6.2.1.1）用于合成1微摩尔[11C]乙酰辅酶A（辅酶A-[11C]乙酸）。对该酶固定在不同衍生化的可控孔径玻璃珠（孔径50纳米，粒径125 - 180微米）上的情况进行了筛选。测试了几种硅烷、间隔臂和末端反应基团。对固定化酶进行了储存稳定性测试。通过这些实验，选择了首选方法：固定在溴化氰活化的可控孔径玻璃上。进一步优化固定化参数以提高载酶玻璃珠的活性。将后者装填到玻璃柱中。对该柱的动力学性质进行了研究和优化，以在几分钟内实现将1微摩尔乙酸几乎完全转化为乙酰辅酶A（辅酶A - 乙酸）。这通过一个酶反应器（13.0×0.5厘米）实现，该反应器含有固定在1克可控孔径玻璃上的6.12单位活性乙酰辅酶A合成酶。
