Engineering of choline oxidase from Arthrobacter nicotianae for potential use as biological bleach in detergents.
In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an alpha-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.
Ribitsch
Doris
D
Austrian Centre of Industrial Biotechnology, c/o Research Centre Applied Biocatalysis, Petersgasse 14, 8010 Graz, Austria. doris.ribitsch@a-b.at
Winkler
Sonja
S
Gruber
Karl
K
Karl
Wolfgang
W
Wehrschütz-Sigl
Eva
E
Eiteljörg
Inge
I
Schratl
Petra
P
Remler
Peter
P
Stehr
Regina
R
Bessler
Cornelius
C
Mussmann
Nina
N
Sauter
Kerstin
K
Maurer
Karl Heinz
KH
Schwab
Helmut
H
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D004396:Coloring Agents / Q000378:metabolism*; D019020:Directed Molecular Evolution; D008744:Methylamines / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013463:Sulfuric Acid Esters / Q000378:metabolism
D001426:Bacterial Proteins; D004396:Coloring Agents; D008744:Methylamines; D050505:Mutant Proteins; D013463:Sulfuric Acid Esters; C027451:methylamine; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; C033522:methyl sulfate