Literature summary for 1.3.3.5 extracted from

Gounel, S.; Rouhana, J.; Stines-Chaumeil, C.; Cadet, M.; Mano, N.
Increasing the catalytic activity of bilirubin oxidase from Bacillus pumilus importance of host strain and chaperones proteins (2016), J. Biotechnol., 230, 19-25 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene BPUM_0532, recombinant expression of His-tagged enzyme in Escherichia coli strain Origami B (DE3), evaluation and optimization of a complementary method to optimize the copper oxidase, bilirubin oxidase, enzyme expression in Escherichia coli. Usage of Escherichia coli strain Origami B (DE3), known to promote the formation of disulfide bridges in the bacterial cytoplasm, because the enzyme has a disulfide bridge. In a second step, the effect of coexpression of chaperone proteins (DnaK, DnaJ and GrpE usingthe plasmid pKJE7) on the protein production and specific activity is investigated resulting in an increase of the final amount of enzyme by 858% and its catalytic rate constant by 83%, overview. Comparison of expression efficiency in Escherichia coli strain BL21 (DE3) and Origami B (DE3). When all protein chaperones are expressed together with the r-BOD, the amount of enzyme is largely promoted without impairing the specific activity pointing out the synergy between the two systems, as GroES/GroEL has no effect on the expression alone. Synergy between DnaK, DnaJ, and GrpE, which prevent aggregation of misfolded enzymes, and GroEL and GroES, which promote their proper folding	Bacillus pumilus

Cloned (Comment)

Organism

gene BPUM_0532, recombinant expression of His-tagged enzyme in Escherichia coli strain Origami B (DE3), evaluation and optimization of a complementary method to optimize the copper oxidase, bilirubin oxidase, enzyme expression in Escherichia coli. Usage of Escherichia coli strain Origami B (DE3), known to promote the formation of disulfide bridges in the bacterial cytoplasm, because the enzyme has a disulfide bridge. In a second step, the effect of coexpression of chaperone proteins (DnaK, DnaJ and GrpE usingthe plasmid pKJE7) on the protein production and specific activity is investigated resulting in an increase of the final amount of enzyme by 858% and its catalytic rate constant by 83%, overview. Comparison of expression efficiency in Escherichia coli strain BL21 (DE3) and Origami B (DE3). When all protein chaperones are expressed together with the r-BOD, the amount of enzyme is largely promoted without impairing the specific activity pointing out the synergy between the two systems, as GroES/GroEL has no effect on the expression alone. Synergy between DnaK, DnaJ, and GrpE, which prevent aggregation of misfolded enzymes, and GroEL and GroES, which promote their proper folding

Bacillus pumilus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics	Bacillus pumilus
0.032	-	2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)	recombinant enzyme expressed in Escherichia coli strain BL21(DE3), pH 3.2, 37°C	Bacillus pumilus
0.087	-	2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)	recombinant enzyme expressed in Escherichia coli strain Origami B(DE3), pH 4.0, 37°C	Bacillus pumilus
0.124	-	2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)	recombinant enzyme expressed in Escherichia coli strain BL21(DE3), pH 4.0, 37°C	Bacillus pumilus
0.216	-	2,6-dimethoxyphenol	recombinant enzyme expressed in Escherichia coli strain BL21(DE3), pH 7.0, 37°C	Bacillus pumilus
0.774	-	2,6-dimethoxyphenol	recombinant enzyme expressed in Escherichia coli strain Origami B(DE3), pH 7.0, 37°C	Bacillus pumilus
0.822	-	2,6-dimethoxyphenol	recombinant enzyme expressed in Escherichia coli strain BL21(DE3), pH 6.8, 37°C	Bacillus pumilus

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Cu2+	the enzyme is a multicopper oxidase	Bacillus pumilus

Organism

Organism	UniProt	Comment	Textmining
Bacillus pumilus	A8FAG9	-	-
Bacillus pumilus SAFR-032	A8FAG9	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain Origami B (DE3) by nickel affinity chromatography and dialysis	Bacillus pumilus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + O2	-	Bacillus pumilus	? + H2O	-	?
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + O2	-	Bacillus pumilus SAFR-032	? + H2O	-	?
2,6-dimethoxyphenol + O2	-	Bacillus pumilus	? + H2O	-	?
2,6-dimethoxyphenol + O2	-	Bacillus pumilus SAFR-032	? + H2O	-	?
additional information	usage of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol as substrates, optimization of the assay method	Bacillus pumilus	?	-	?
additional information	usage of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol as substrates, optimization of the assay method	Bacillus pumilus SAFR-032	?	-	?

Subunits

Subunits	Comment	Organism
?	x * 30000, folded recombinant enzyme, SDS-PAGE, x * 60000, unfolded recombinant enzyme, SDS-PAGE	Bacillus pumilus

Synonyms

Synonyms	Comment	Organism
BOD	-	Bacillus pumilus
BPUM_0542	-	Bacillus pumilus
copper oxidase	-	Bacillus pumilus
MCO	-	Bacillus pumilus
multicopper oxidase	-	Bacillus pumilus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Bacillus pumilus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
29	-	2,6-dimethoxyphenol	recombinant enzyme expressed in Escherichia coli strain BL21(DE3), pH 7.0, 37°C	Bacillus pumilus
57	-	2,6-dimethoxyphenol	recombinant enzyme expressed in Escherichia coli strain BL21(DE3), pH 6.8, 37°C	Bacillus pumilus
100	-	2,6-dimethoxyphenol	recombinant enzyme expressed in Escherichia coli strain Origami B(DE3), pH 7.0, 37°C	Bacillus pumilus
322	-	2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)	recombinant enzyme expressed in Escherichia coli strain BL21(DE3), pH 4.0, 37°C	Bacillus pumilus
391	-	2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)	recombinant enzyme expressed in Escherichia coli strain BL21(DE3), pH 3.2, 37°C	Bacillus pumilus
716	-	2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)	recombinant enzyme expressed in Escherichia coli strain Origami B(DE3), pH 4.0, 37°C	Bacillus pumilus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
4.2	7.2	assay at	Bacillus pumilus

pH Range

pH Minimum	pH Maximum	Comment	Organism
additional information	-	inactive at pH 9.2	Bacillus pumilus

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the family of multicopper oxidases (MCOs)	Bacillus pumilus