Engineering of choline oxidase from Arthrobacter nicotianae for potential use as biological bleach in detergents

• Published in: Applied microbiology and biotechnology 2010-08, Vol.87 (5), p.1743-1752
• Main Authors: Ribitsch, Doris, Winkler, Sonja, Gruber, Karl, Karl, Wolfgang, Wehrschütz-Sigl, Eva, Eiteljörg, Inge, Schratl, Petra, Remler, Peter, Stehr, Regina, Bessler, Cornelius, Mußmann, Nina, Sauter, Kerstin, Maurer, Karl Heinz, Schwab, Helmut
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Amino Acid Substitution; Arthrobacter - enzymology; Arthrobacter nicotianae; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Biodegradable materials; Biological and medical sciences; Biological bleaching; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Choline oxidase; Cloning; Coloring Agents - metabolism; Detergents; Directed evolution; Directed Molecular Evolution; DNA polymerase; E coli; Engineering; Enzymes; Evolution; Fundamental and applied biological sciences. Psychology; Hydrogen peroxide; Life Sciences; Methods. Procedures. Technologies; Methylamines - metabolism; Microbial Genetics and Genomics; Microbiology; Models, Molecular; Mutagenesis; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation; Mutation, Missense; Oxidation; Plasmids; Protein Engineering; Protein Structure, Tertiary; Studies; Substrates; Sulfuric Acid Esters - metabolism
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-010-2637-9

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 α-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.