BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Engineering of choline oxidase from Arthrobacter nicotianae for potential use as biological bleach in detergents Doris Ribitsch & Sonja Winkler & Karl Gruber & Wolfgang Karl & Eva Wehrschütz-Sigl & Inge Eiteljörg & Petra Schratl & Peter Remler & Regina Stehr & Cornelius Bessler & Nina Mußmann & Kerstin Sauter & Karl Heinz Maurer & Helmut Schwab Received: 17 March 2010 / Revised: 15 April 2010 / Accepted: 21 April 2010 / Published online: 14 May 2010 # Springer-Verlag 2010 Abstract 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. Keywords Choline oxidase . Arthrobacter nicotianae . Directed evolution . Biological bleaching . Detergents . Hydrogen peroxide Introduction In Europe, hydrogen peroxide-based bleaching systems are widely used in detergent formulations to remove stains like tea, coffee, red wine or fruits efficiently from various fabric types. The peroxide is formed by alkaline hydrolysis of perborates and percarbonates but requires temperatures of about 60°C to act sufficiently on stains (Dannacher 2006; Reinhardt 2006). To overcome the need of such high temperatures, so-called activators like N,N,N′,N′-tetraace- tylethylene diamine were introduced into detergents (Cuypers et al. 2005). On the basis of perhydrolysis, these activators generate peracids facilitating bleaching of stains at temperatures of about 40°C with satisfying performance thus representing the current state of the art technology. Due to the new European Detergent Regulation (see References) which came into force in 2005, modern detergents are met with new challenges. The Regulation restricts the use of detergent ingredients which are not highly Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2637-9) contains supplementary material, which is available to authorized users. D. Ribitsch (*) : S. Winkler : K. Gruber : W. Karl : E. Wehrschütz-Sigl : I. Eiteljörg : P. Schratl : P. Remler : H. Schwab Austrian Centre of Industrial Biotechnology (ACIB), c/o Research Centre Applied Biocatalysis, Petersgasse 14, 8010 Graz, Austria e-mail: doris.ribitsch@a-b.at H. Schwab Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria K. Gruber Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/3, 8010 Graz, Austria R. Stehr : C. Bessler : N. Mußmann : K. Sauter : K. H. Maurer Henkel AG & Co. KGaA, Henkelstrasse 67, 40191 Duesseldorf, Germany Appl Microbiol Biotechnol (2010) 87:1743–1752 DOI 10.1007/s00253-010-2637-9