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

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

Convenient microtiter plate‐based, oxygen‐independent activity assays for flavin‐dependent oxidoreductases based on different redox dyes

“…When screening for flavin-dependent oxidases the second reaction product, hydrogen peroxide, is frequently detected in a second, enzyme-coupled reaction. Here, chromogenic substrates such as 4-aminoantipyrine together with a suitable phenolic compound [21], o -dianisidine [22], or 2,2'-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) [23, 24] are used together with horseradish peroxidase or another suitable peroxidase. Obviously, these approaches cannot be used for flavin-dependent dehydrogenases since these only show negligible activity with oxygen.…”

“…When screening for flavin-dependent oxidases the second reaction product, hydrogen peroxide, is frequently detected in a second, enzyme-coupled reaction. Here, chromogenic substrates such as 4-aminoantipyrine together with a suitable phenolic compound [21], o-dianisidine [22],…”

Characterization of a purified decolorizing detergent-stable peroxidase from Streptomyces griseosporeus SN9

“…The stability of the purified enzyme toward SDS and oxidizing agents is an important characteristic for its eventual use in detergent formulations. Bleach stability was also attained through protein engineering [40,41]. The enzyme was highly stable against perfume and anti-redeposition agents, particularly 50 mM Na 2 CO 3 , and of cationic (CTAB) and other detergent compound (Table 4), retaining 95, 104, 107, and 125% of its initial activity after treatment with 1% perfume, 50 mM CTAB, 1% STTP, and 50 mM Na 2 CO 3 , respectively.…”

Biocatalyst development by directed evolution

“…Several traits of this class of enzymes have been improved through directed evolution to be more compatible with current industrial processes (Gupta & Farinas, 2010; Liu et al, 2011b; Ribitsch et al, 2010). For example, García-Ruiz and coworkers generated a mutant library of both a laccase from basidiomycete PM1 and a peroxidase from Pleurotus eryngii via Mutagenic StEP (Staggered Extension Process) (Zhao et al, 1998) followed by in vivo DNA shuffling and IvAM (In vivo Assembly of Mutant) libraries with different mutational spectra.…”