Purification and properties of ampicillin acylase from Pseudomonas melanogenum.
Ampicillin acylase, which is known to have a novel substrate spectrum, was purified to homogeneity from Pseudomonas melanogenum by the crude extract preparation and chromatography with S-Sepharose, hydroxyapatite, CM-cellulose C-52, and CM-Sepharose. The molecular weight of the native enzyme was calculated to be 146,000 by Protein PAK-300 sw HPLC chromatography. SDS-polyacrylamide gel electrophoresis revealed that the enzyme consisted of two identical subunits with a molecular weight of 72,000. The enzyme was a glycoprotein containing 13% of total carbohydrate, and its isoelectric point was 7.2. The enzyme catalyzed both synthesis and hydrolysis of ampicillin and hydrolysis of the ester bond of phenylglycinemethylester hydrochloride substrate. The substrate specificity showed that the enzyme required a free amino group on the alpha-carbon of the acyl group. Chemical modification by diethylpyrocarbonate or N-bromosuccinimide resulted in time-dependent inactivation of the enzyme, and other results suggest the participation of essential histidine residue(s) in the catalytic activity of ampicillin acylase. Substrates of the enzyme, 6-aminopenicillanic acid and ampicillin, exhibited protective effects against N-bromosuccinimide inactivation, suggesting that the modification occurred near or at the active site.
Kim
D J
DJ
Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, Seoul.
Byun
S M
SM
D000581:Amidohydrolases / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D010399:Penicillin Amidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D006882:Hydroxyapatites; D017886:Durapatite; D000581:Amidohydrolases; C049057:ampicillin acylase; D010399:Penicillin Amidase