Premium
Hydrolysis of polyethyleneterephthalate by p ‐nitrobenzylesterase from Bacillus subtilis
Author(s) -
Ribitsch Doris,
Heumann Sonja,
Trotscha Eva,
Herrero Acero Enrique,
Greimel Katrin,
Leber Regina,
BirnerGruenberger Ruth,
Deller Sigrid,
Eiteljoerg Inge,
Remler Peter,
Weber Thomas,
Siegert Petra,
Maurer KarlHeinz,
Donelli Ilaria,
Freddi Giuliano,
Schwab Helmut,
Guebitz Georg M.
Publication year - 2011
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.610
Subject(s) - hydrolysis , terephthalic acid , chemistry , bacillus subtilis , hydrolase , substrate (aquarium) , butyrate , polyethylene terephthalate , nuclear chemistry , polymer chemistry , enzyme , organic chemistry , polyester , materials science , bacteria , oceanography , composite material , genetics , geology , biology , fermentation
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 k cat 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