Purification of isopenicillin N synthetase from Streptomyces clavuligerus.
Isopenicillin N synthetase was purified from Streptomyces clavuligerus by sequential salt precipitation, ion-exchange and gel-filtration chromatography using both conventional open column and high-performance liquid chromatographic techniques. Material from the final purification step had a specific activity of 204.1 X 10(-3) units/mg of protein which represented a 130-fold purification over the cell-free extract. The purified isopenicillin N synthetase was determined to have a molecular weight of 33,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have a Km of 0.32 mM with respect to its substrate delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine. The enzyme showed a sensitivity to thiol-specific inhibitors with N-ethylmaleimide giving the strongest inhibitory effect.
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000032:analysis / Q000302:isolation & purification*; D010088:Oxidoreductases; D013302:Streptomyces / Q000201:enzymology*
D004798:Enzymes; D010088:Oxidoreductases; C038370:isopenicillin N synthetase