Non-radioactive assay and HPLC analysis of cephalosporin C 7?-methoxylation by cell-free extracts of Streptomyces clavuligerus

1991 ◽  
Vol 35 (6) ◽  
Author(s):  
Xinfa Xiao ◽  
RaymondJ. Bowers ◽  
Hee-Sook Shin ◽  
Saul Wolfe ◽  
ArnoldL. Demain
Keyword(s):  
Hplc Analysis ◽  
Streptomyces Clavuligerus ◽  
Cephalosporin C ◽  
Radioactive Assay

scholarly journals The conversion of cephalosporins to 7 α-methoxycephalosporins by cell-free extracts of Streptomyces clavuligerus

1980 ◽  
Vol 186 (2) ◽  
pp. 613-616 ◽  
Author(s):  
J O'Sullivan ◽  
E P Abraham
Keyword(s):  
Methoxy Group ◽  
Biosynthetic Pathway ◽  
Streptomyces Clavuligerus ◽  
Ring System ◽  
Reducing Agent ◽  
Cephalosporin C ◽  
Methoxy Derivative

In the presence of S-adenosylmethionine, 2-oxoglutarate, Fe2+ and a reducing agent, cell-free extracts of Streptomyces clavuligerus convert cephalosporin C and O-carbamoyldeacetylcephalosporin C into 7 alpha-methoxy derivatives. No synthesis of a 7 alpha-methoxy derivative of deacetylcephalosporin C was detected in the system used, and the 7 alpha-methoxy derivative of deacetoxycephalosporin C was produced only in relatively small amounts. It appears that the 7 alpha-methoxy group is introduced after the cephalosporin ring system has been formed and that its introduction may represent the final step in a biosynthetic pathway.

scholarly journals Purification and characterization of cephalosporin 7α-hydroxylase from Streptomyces clavuligerus

1991 ◽  
Vol 280 (2) ◽  
pp. 471-474 ◽  
Author(s):  
X Xiao ◽  
S Wolfe ◽  
A L Demain
Keyword(s):  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
Exchange Chromatography ◽  
Cephalosporin C ◽  
Hydroxylase Activity ◽  
Purification And Characterization ◽  
Major Band ◽  
Sds Page ◽  
Optimum Ph

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.

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