Novel approaches to the purification of penicillin acylase.
Penicillin acylase of E. coli NCIM 2400 has been purified to homogeneity using a combination of hydrophobic interaction chromatography and DEAE-cellulose treatment. A variety of substituted matrices were synthesized using D- or DL-phenylglycine, norleucine, ampicillin, or amoxycillin as ligands, all of which retained penicillin acylase at high concentrations of ammonium sulfate or sodium sulfate. The enzyme could be eluted nonbiospecifically by buffer of lower ionic strength with over 95% recovery of the activity. Ammonium chloride, ammonium nitrate, sodium chloride, sodium nitrate, and potassium chloride were ineffective in either adsorption or elution of the enzyme on these columns. Further purification of this partially pure enzyme with DEAE-cellulose at pH 7.0-7.2 yielded an enzyme preparation of very high purity according to electrophoretic and ultracentrifugal analyses, its specific activity being as high as 37 U/mg protein. The purified enzyme has a molecular weight of 67,000 a sedimentation coefficient of 4.0S, and resolves into two forms upon isoelectric focusing. Overall recoveries ranged between 75 and 85%. Ease of operation, high recoveries, high purity of the enzyme and prolonged reuse of the conjugates make the process economically feasible and possibly of great commercial importance.
D000581:Amidohydrolases / Q000302:isolation & purification*; D000658:Amoxicillin; D000667:Ampicillin; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods*; D002848:Chromatography, DEAE-Cellulose; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000031:analogs & derivatives; D007525:Isoelectric Focusing; D009646:Norleucine; D010399:Penicillin Amidase / Q000302:isolation & purification*; D012685:Sepharose
D001426:Bacterial Proteins; C008852:2-phenylglycine; D000667:Ampicillin; D000658:Amoxicillin; D009646:Norleucine; D012685:Sepharose; D000581:Amidohydrolases; D010399:Penicillin Amidase; D005998:Glycine