title: Hydrolysis of polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilis.
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.
authors.affiliation: Austrian Centre of Industrial Biotechnology ACIB, Petersgasse 14, Graz 8010, Austria.
authors.forename: Doris
authors.initials: D
authors.lastname: Ribitsch
authors.forename: Sonja
authors.initials: S
authors.lastname: Heumann
authors.forename: Eva
authors.initials: E
authors.lastname: Trotscha
authors.forename: Enrique
authors.initials: E
authors.lastname: Herrero Acero
authors.forename: Katrin
authors.initials: K
authors.lastname: Greimel
authors.forename: Regina
authors.initials: R
authors.lastname: Leber
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Gruenberger
authors.forename: Sigrid
authors.initials: S
authors.lastname: Deller
authors.forename: Inge
authors.initials: I
authors.lastname: Eiteljoerg
authors.forename: Peter
authors.initials: P
authors.lastname: Remler
authors.forename: Thomas
authors.initials: T
authors.lastname: Weber
authors.forename: Petra
authors.initials: P
authors.lastname: Siegert
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Maurer
authors.forename: Ilaria
authors.initials: I
authors.lastname: Donelli
authors.forename: Giuliano
authors.initials: G
authors.lastname: Freddi
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
authors.forename: Georg M
authors.initials: GM
authors.lastname: Guebitz
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D011093:Polyethylene Terephthalates / Q000378:metabolism*
chemical_list: D011093:Polyethylene Terephthalates; C091617:4-nitrobenzyl esterase; D002265:Carboxylic Ester Hydrolases