Hyperproduction and Purification of Nicotinamide Deamidase, a Microconstitutive Enzyme of Escherichia coli

Abstract

Nicotinamide deamidase is synthesized constitutively in very small amounts by Escherichia coli. Mutants characterized by up to 50-fold higher deamidase activity have been isolated by mutagenesis and selection on minimal plates containing nicotinamide as the sole nitrogen source. These mutants still synthesize the enzyme constitutively. The enzyme was purified 200-fold. The final specific activity from one mutant is 10,000 times greater than the specific activity in the wild type. The molecular weight of the enzyme is about 30,000. Kinetic constants and heat stabilities of purified enzymes from two mutant strains and the wild type are identical. Gel electrophoresis has further suggested that the mutations result in over production of unaltered nicotinamide deamidase. Various hyperconstitutive mutations and the deamidase structural gene are cotransduced at high frequency, and thus lie close together.