title: Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins.
abstract: Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased kcat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria.
authors: Doris
authors: D
authors: Ribitsch
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Vienna, Tulln, Austria.
authors: Enrique
authors: E
authors: Herrero Acero
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria Microbiology Group, Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
authors: Agnieszka
authors: A
authors: Przylucka
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria.
authors: Sabine
authors: S
authors: Zitzenbacher
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria.
authors: Annemarie
authors: A
authors: Marold
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria.
authors: Caroline
authors: C
authors: Gamerith
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria.
authors: Rupert
authors: R
authors: Tscheließnig
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria Institute of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Vienna, Austria.
authors: Alois
authors: A
authors: Jungbauer
authors: Institute of Physics and Material Sciences, University of Natural Resources and Life Sciences, Vienna, Vienna, Austria.
authors: Harald
authors: H
authors: Rennhofer
authors: Institute of Physics and Material Sciences, University of Natural Resources and Life Sciences, Vienna, Vienna, Austria.
authors: Helga
authors: H
authors: Lichtenegger
authors: Institute for Inorganic Chemistry, University of Technology Graz, Graz, Austria.
authors: Heinz
authors: H
authors: Amenitsch
authors: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.
authors: Klaus
authors: K
authors: Bonazza
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria Microbiology Group, Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.
authors: Christian P
authors: CP
authors: Kubicek
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria Microbiology Group, Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria irina.druzhinina@tuwien.ac.at.
authors: Irina S
authors: IS
authors: Druzhinina
authors: Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Vienna, Tulln, Austria.
authors: Georg M
authors: GM
authors: Guebitz
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D010795:Phthalic Acids / Q000378:metabolism; D011093:Polyethylene Terephthalates / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D010795:Phthalic Acids; D011093:Polyethylene Terephthalates; D011993:Recombinant Fusion Proteins; C011363:terephthalic acid; D002265:Carboxylic Ester Hydrolases; C019952:cutinase