scholarly journals Purification of Protease II from Escherichia coli by Affinity Chromatography and Separation of Two Enzyme Species from Cells Harvested at Late Log Phase

1976 ◽  
Vol 64 (1) ◽  
pp. 199-204 ◽  
Author(s):  
Michele. PACAUD
1978 ◽  
Vol 253 (16) ◽  
pp. 5847-5851 ◽  
Author(s):  
E. Lanka ◽  
C. Edelbluth ◽  
M. Schlicht ◽  
H. Schuster

1984 ◽  
Vol 39 (9-10) ◽  
pp. 908-915 ◽  
Author(s):  
Anna M. Mata ◽  
M. Carmen ◽  
Juan López-Barea

Abstract The glutathione reductase from Escherichia coli strain S33 was purified to homogeneity by a simple and fast procedure consisting of two affinity chromatography steps. After 40-80% ammonium sulfate fractionation, the enzyme was adsorbed to an N6-2′.5′-ADP-Sepharose affinity column from which it was specifically eluted by a 0 - 10 mᴍ NADP+ linear gradient. The enzyme was finally purified to homogeneity after a second affinity chromatography step in a C8-ATPR-Sepharose column, from which it was eluted by means of the same NADP+ gradient. Starting from 182 g of E. coli cells. 6.9 mg of pure enzyme was obtained after a 2632-fold purifi­cation, with a total yield of 63%. The pure enzyme showed a specific activity of 361 U/mg, and its absorption spectrum was characteristic of a flavoprotein. with an A272A450 of 7.84. The enzyme was a dimer with a molecular weight 109 000 and 40 Å hydrodynamic radius. The optimum pH were 7.5 and 4.5 with NADPH and NADH. respectively, as reductants. Apparent K′m values of 16, 377, and 66 μᴍ were determined at pH 7.5 for NADPH, NADH, and GSSG, respectively. Upon storage the enzyme was stable at pH values ranging from 7.5 to 9.5, being additionally stabilized by FAD. NADP+, dithiothreitol, or glycerol. The pure enzyme was quite heat stable, denaturing signifi­cantly only after 10 min at 70 0C. A marked activity loss was observed however, even at 0 °C, in the presence of 20 μᴍ NADPH. The enzyme was inactivated by low concentrations of para- hydroximercuribenzoate: the sensitivity towards such mercurial was greatly enhanced after reduction of the enzyme by NADPH.


1974 ◽  
Vol 52 (12) ◽  
pp. 1087-1090 ◽  
Author(s):  
Michel Guitard ◽  
Réjean Daigneault

Chloramphenicol acetyltransferase (CATase) was purified by affinity chromatography from Escherichia coli W677/HJR66, an R-factor-bearing mutant. The chloramphenicol aryl nitro group had to be reduced to an amino group prior to its coupling to the Sepharose 4B matrix. The single-step isolation procedure resulted in a 237-fold purification of CATase with over 65% recovery of the enzyme.


1999 ◽  
Vol 181 (19) ◽  
pp. 6184-6187 ◽  
Author(s):  
Axel Raisig ◽  
Gerhard Sandmann

ABSTRACT Staphylococcus aureus synthesizes C30carotenoids. Their formation involves the introduction of three double bonds, which is catalyzed by a single enzyme. This enzyme, 4,4′-diapophytoene desaturase from S. aureus, was overexpressed in Escherichia coli and purified in one step by affinity chromatography, and then the protein was characterized with respect to substrate specificity, cofactor requirement, and oligomerization.


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