Crystalline l-Asparaginase from Escherichia coli B: I. PURIFICATION AND CHEMICAL CHARACTERIZATION

Abstract

Abstract Purification of l-asparaginase from Escherichia coli B is described. The enzyme is crystallized from a partially purified preparation in the presence of cations and aqueous alcohol. Effective crystallization requires a minimum of 4 g atoms of cation to each mole of protein. The crystalline l-asparaginase is homogeneous by disc gel electrophoresis and ultracentrifugation and has a sedimentation coefficient (s020,w) of 7.6 S and a molecular weight (by sedimentation equilibrium) of 133,000 ± 5,000. On gel electrophoresis, the crystalline enzyme migrates similarly to the enzyme present in cell-free extracts. The affinity constants for l-asparagine and l-glutamine are 1.15 x 10-5 and 6.25 x 10-3 m, respectively. The amino acid composition indicates a minimum molecular weight of 33,300. The crystalline enzyme has an E1%278 of 7.1 and a specific activity of 300 ± 15 I.U. per mg, corrected for moisture. The isoelectric point, determined by cellulose acetate electrophoresis, is 5.2. No carbohydrate or phospholipid was detected in the enzyme. The purification procedure provides an over-all yield of about 25% from cell cake to crystals, and is adaptable to large scale isolation of the enzyme.