Purification and characterization of cephalosporin 7 alpha-hydroxylase from Streptomyces clavuligerus.
Cephalosporin 7 alpha-hydroxylase, which catalyses the conversion of cephalosporins into their 7 alpha-hydroxy derivatives, was purified nearly 390-fold from Streptomyces clavuligerus through ion-exchange chromatography, (NH4)2SO4 fractionation, gel filtration and dye chromatography, with the use of h.p.l.c. to monitor enzyme activity. The nearly pure enzyme migrates as a single major band, with an Mr of 32,000 in SDS/PAGE. Its optimum pH is in the range 7.3-7.7. Under our conditions the reaction was fastest at temperatures in the range 20-30 degrees C. The Km for cephalosporin C is 0.72 mM, and the Vmax. is 15.4 mumol of cephalosporin C hydroxylated/min per mg. Cephalosporin 7 alpha-hydroxylase did not show any deacetoxycephalosporin C synthase or deacetoxycephalosporin C hydroxylase activity.
Xiao
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Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Wolfe
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Demain
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D002511:Cephalosporins / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D019751:Intramolecular Transferases; D007535:Isomerases / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature
D002511:Cephalosporins; D046915:Penicillin-Binding Proteins; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C071965:cephalosporin 7-alpha-hydroxylase; C045288:deacetoxycephalosporin C hydroxylase; D007535:Isomerases; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase