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- Title
Hydrolysis of polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilis.
- Authors
Ribitsch, Doris; Heumann, Sonja; Trotscha, Eva; Herrero Acero, Enrique; Greimel, Katrin; Leber, Regina; Birner-Gruenberger, Ruth; Deller, Sigrid; Eiteljoerg, Inge; Remler, Peter; Weber, Thomas; Siegert, Petra; Maurer, Karl-Heinz; Donelli, Ilaria; Freddi, Giuliano; Schwab, Helmut; Guebitz, Georg M.
- Abstract
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. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
- Subjects
AGAR; BACILLUS subtilis; ESTERASES; POLYETHYLENE terephthalate; HYDROLYSIS; POLYACRYLAMIDE; ESCHERICHIA coli; ACETATES
- Publication
Biotechnology Progress, 2011, Vol 27, Issue 4, p951
- ISSN
8756-7938
- Publication type
Article
- DOI
10.1002/btpr.610