Immobilization of acetylcoenzyme A synthetase and the preparation of an enzyme reactor for the synthesis of [11C]acetylcoenzyme A.
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.
D000106:Acetate-CoA Ligase / Q000378:metabolism*; D000105:Acetyl Coenzyme A / Q000096:biosynthesis*; D002021:Buffers; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008722:Methods; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D002021:Buffers; D004800:Enzymes, Immobilized; D000105:Acetyl Coenzyme A; D003066:Coenzyme A Ligases; D000106:Acetate-CoA Ligase