1.5.1.30: flavin reductase (NADPH)

This is an abbreviated version!
For detailed information about flavin reductase (NADPH), go to the full flat file.

Word Map on EC 1.5.1.30

Reaction

reduced riboflavin
+
NADP+
=
riboflavin
+
NADPH
+
H+

Synonyms

alkanesulfonate FMN reductase, ArsH, azoreductase, AZRü, Biliverdin-IX beta-reductase, CysJ, flavin oxidoreductase, flavin reductase, flavin reductase 1, flavin reductase Nr1, flavin reductase P, FLR, FR1, Frb, Frd188, frd2, fre, Fre oxidoreductase, Fre-1, FRG/FRaseI, FRGvf, FRP, FRPvh, GHBP, Green heme binding protein, HpaC, ipa-43d, More, NAD(P)H-dependent H2O2-forming flavin reductase, NADPH-dependant FMN reductase, NADPH-dependent diaphorase, NADPH-flavin oxidoreductase, NADPH-flavin reductase, NADPH-FMN oxidoreductase, NADPH-FMN reductase, NADPH-nitrofurazone reductase, NADPH-specific flavin reductase, NADPH:FAD oxidoreductase, NADPH:flavin oxidoreductase, NADPH:FMN oxidoreductase, NfrA1, nitro/flavin reductase, non-luminescent-bacterial NfsA/Frp-type enzyme, SsuE, TVAG_517010

ECTree

     1 Oxidoreductases
         1.5 Acting on the CH-NH group of donors
             1.5.1 With NAD+ or NADP+ as acceptor
                1.5.1.30 flavin reductase (NADPH)

Purification

Purification on EC 1.5.1.30 - flavin reductase (NADPH)

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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
bifunctional enzyme flavin reductase (NADPH)/biliverdin-IXbeta reductase
-
cells harvested by centrifugation, washing twice and suspended in 50 mM Tris-HCl buffer, pH 8.0, with 1 mM dithiothreitol and 10% glycerol, cells disrupted with ultraoscillator (20 kHz), debris removed by centrifugation, enzyme purified from cell-free extracts with ÄKTA explorer, applied to Toyopearl DEAE-650 M column, equilibrated with 50 mM Tris-HCl buffer, pH 8.0, washed, proteins eluted with linear gradient of 0-0.5 M KCl, active fractions (at 0.15-0.2 M KCl) combined and concentrated by ultrafiltration (membrane filter YM-30), enzyme purified with HiLoad 16/60 Superdex 200, equilibrated with 50 mM Tris-HCl buffer, pH 8.0 with 0.15 M NaCl, elution with same buffer
-
crude lysate assay with briefly centrifuged cells, resuspended in B-PER bacterial protein extraction reagent, recombinant pZCFRE1 expression product is clarified by centrifugation and applied to a custom nickel affinity column, dialyzed into buffer containing 100 mM Na+/K+ phosphate and 100 mM NaCl, pH 7.0
-
FMN-agarose chromatography as a key step
-
HiTrap DEAE column chromatography, Mono Q column chromatography, and Superdex 75 gel filtration
-
preparation of apoenzyme
-
recombinant enzyme 23fold from Escherichia coli to homogeneity by dialysis, anion exchange chromatography, dialysis, two steps of Blue Sepharose affinity chromatography, and again dialysis
-
recombinant enzyme from Escherichia coli strain BL21(DE3) to over 90% purity, recombinant fusion protein from Escherichia coli strain JM109 to over 80% purity
-
recombinant enzyme from Escherichia coli to homogeneity
-
recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
recombinant wild-type and mutant enzyme from Escherichia coli to homogeneity
-
the flavin reductase-luciferase fusion protein is purified using DEAE-cellulose DE-52 column chromatography, DEAE-Sepharose column chromatography, and Superdex 200 gel filtration
-
ultracentrifugation, G-25 gel filtration, DEAE-Sepharose column chromatography, and Sephadex G25 gel filtration
-