Large-scale purification of the chromosomal beta-lactamase from Enterobacter cloacae P99.
Homogeneous beta-lactamase (beta-lactam hydrolase, E.C. 3.5.2.6) from Enterobacter cloacae P99, an enzyme that has an important function in antibiotic resistance, was prepared using a single cation-exchange chromatographic step with CM-Sepharose fast-flow. A 6-g amount of the enzyme was isolated from 5 kg of cell paste, with 84% of the enzyme activity in the cell homogenate being recovered by the single cation-exchange step. The specific activity of the beta-lactamase was 587 U/mg protein. The relative molecular mass of the enzyme was determined to be 45 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and the isoelectric point was 8.95.
Goward
C R
CR
Division of Biotechnology, PHLS, Centre for Applied Microbiology and Research, Salisbury, Wiltshire, U.K.
Stevens
G B
GB
Hammond
P M
PM
Scawen
M D
MD
D000900:Anti-Bacterial Agents / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D002876:Chromosomes, Bacterial / Q000201:enzymology*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004754:Enterobacter / Q000201:enzymology* / Q000254:growth & development / Q000648:ultrastructure; D004755:Enterobacteriaceae / Q000201:enzymology*; D007525:Isoelectric Focusing; D001618:beta-Lactamases / Q000302:isolation & purification* / Q000378:metabolism; D047090:beta-Lactams
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D047090:beta-Lactams; D001618:beta-Lactamases