Purification and immobilization of NAD + synthetase from Escherichia coli

Abstract

The enzyme NAD + synthetase [deamido-NAD +: ammonia ligase (AMP-forming), EC 6.3.1.5] is used for the preparation of 2 μmol isotopically labelled [ 13 N]NAD +, a radiopharmaceutical designed for positron emission tomography. To obtain a rapid and high yield synthesis of [ 13 N]NAD +, the NAD + synthetase is immobilized on porous glass beads and packed in a column. The NAD + synthetase was obtained from Escherichia coli. Different strains were tested; the cell culture technique was optimized. A new, high yield purification was applied. A screening of different immobilization techniques was done. The selected immobilization method was further optimized to increase the enzymatic activity of the enzyme-loaded glass beads. The latter were packed into a glass column. The kinetic properties of this column were investigated and optimized.