1.14.13.22: cyclohexanone monooxygenase

This is an abbreviated version!
For detailed information about cyclohexanone monooxygenase, go to the full flat file.

Word Map on EC 1.14.13.22

Reaction

cyclohexanone
+
NADPH
+
H+
+
O2
=
hexano-6-lactone
+
NADP+
+
H2O

Synonyms

Bpro_556, CAMO, CHMO, ChnB, chnB protein, chnB1 protein, CHXON, CMO, cycloalkaone monooxygenase, cyclohexanone 1, 2-mono-oxygenase, cyclohexanone mono-oxygenase, cyclohexanone monooxygenase, cyclohexanone oxygenase, cyclohexanone:NADPH:oxygen oxidoreductase (lactone-forming), oxygenase, cyclohexanone mono-

ECTree

     1 Oxidoreductases
         1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen
             1.14.13 With NADH or NADPH as one donor, and incorporation of one atom of oxygen into the other donor
                1.14.13.22 cyclohexanone monooxygenase

Purification

Purification on EC 1.14.13.22 - cyclohexanone monooxygenase

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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
alcohol dehydrogenase(Mesotoga infera)/cyclohexanone monooxygenase(Thermocrispum municipale) fusion construct expressed in Escherichia coli cells
cells centrifuged, washed with reaction buffer (0.1 M glycine/NaOH buffer, pH 9.0), disruption by ultrasonic processor, centrifugation, supernatants used as crude enzyme extracts
-
crude extract of Rhodococcus cells is partially purified using ammonium sulfate fractionation (50-70%), chromatography on Butyl-Toyopearl 650S with a linear gradient of 30%-0% (NH4)2SO4, and gel filtration on a Sephadex G-150 column. Purification of the recombinant enzyme: cells centrifuged and lysed with a French press, crude extract centrifuged, supernatant loaded on a DEAE-Sepharose FF column equilibrated with 50 mM sodium phosphate buffer (pH 7.0), elution with 0-0.2 M NaCl gradient, ammonium sulfate added to active fractions (30%), loaded onto a Butyl-S-Sepharose 6 FF column equilibrated with 30% ammonium sulfate in 50 mM sodium phosphate buffer (pH 7.0), elution with 30-0% ammonium sulfate gradient, active fractions pooled, concentrated, applied to two combined Superose 6 HR (10/300)/Superose 12 HR (10/300) gel filtration columns equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl, elution with same buffer, pooled, concentrated with YM 10 membrane
industrial scale production
-
native enzyme and recombinant His-tagged enzyme from Escherichia coli and Saccharomyces cerevisiae
partial purification of recombinant enzyme
-
partially, recombinant from Escherichia coli
-
recombinant enzyme 21fold from Escherichia coli and native enzyme to homogeneity by ion exchange and affinity chromatography
-
recombinant enzyme using His-tag
recombinant from bacterial expression system
recombinant from Escherichia coli
-
recombinant from Escherichia coli and Saccharomyces cerevisiae
-
recombinant His6-tagged enzyme 8.3fold from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant protein
-
recombinant protein from Escherichia coli
-