Hydrolysis of polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilis.

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p-nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET-hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2-hydroxyethyl benzoate (HEB), and mono-(2-hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET-hydrolase was isolated from non-denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC-MS/MS analysis. BsEstB was expressed in Escherichia coli with C-terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p-nitrophenyl acetate and 108 U/mg on p-nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half-life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2-hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The kcat values decreased with increasing complexity of the substrate from 6 and 8 (s-1) for p-nitrophenyl-acetate (4NPA) and p-nitrophenyl-butyrate (4NPB), respectively, to 0.14 (s-1) for bis(2-hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water-contact angle (WCA) from 68.2°±1.7° to 62.6°±1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2-(bromomethyl)naphthalene.

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.