Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins

Doris Ribitsch, Enrique Herrero Acero, Agnieszka Przylucka, Sabine Zitzenbacher, Annemarie Marold, Caroline Gamerith, Rupert Tscheließnig, Alois Jungbauer, Harald Rennhofer, Helga Lichtenegger, Heinz Amenitsch, Klaus Bonazza, Christian P. Kubicek, Irina S. Druzhinina, Georg M. Guebitz

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Pages (from-to)3586-3592
Number of pages7
JournalApplied and Environmental Microbiology
Volume81
Issue number11
DOIs
Publication statusPublished - 2015

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cutinase
Polyethylene Terephthalates
hydrolysis
Hydrolysis
enzyme
Enzymes
protein
enzymes
Hydrophobic and Hydrophilic Interactions
scattering
fold
Thermobifida
Proteins
Trichoderma
Fungal Proteins
Surface Properties
hydrophobicity
physicochemical property
hydrophobins
polyethylene terephthalates

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Food Science
  • Biotechnology
  • Ecology

Cite this

Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins. / Ribitsch, Doris; Acero, Enrique Herrero; Przylucka, Agnieszka; Zitzenbacher, Sabine; Marold, Annemarie; Gamerith, Caroline; Tscheließnig, Rupert; Jungbauer, Alois; Rennhofer, Harald; Lichtenegger, Helga; Amenitsch, Heinz; Bonazza, Klaus; Kubicek, Christian P.; Druzhinina, Irina S.; Guebitz, Georg M.

In: Applied and Environmental Microbiology, Vol. 81, No. 11, 2015, p. 3586-3592.

Research output: Contribution to journalArticleResearchpeer-review

Ribitsch, D, Acero, EH, Przylucka, A, Zitzenbacher, S, Marold, A, Gamerith, C, Tscheließnig, R, Jungbauer, A, Rennhofer, H, Lichtenegger, H, Amenitsch, H, Bonazza, K, Kubicek, CP, Druzhinina, IS & Guebitz, GM 2015, 'Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins' Applied and Environmental Microbiology, vol. 81, no. 11, pp. 3586-3592. https://doi.org/10.1128/AEM.04111-14
Ribitsch, Doris ; Acero, Enrique Herrero ; Przylucka, Agnieszka ; Zitzenbacher, Sabine ; Marold, Annemarie ; Gamerith, Caroline ; Tscheließnig, Rupert ; Jungbauer, Alois ; Rennhofer, Harald ; Lichtenegger, Helga ; Amenitsch, Heinz ; Bonazza, Klaus ; Kubicek, Christian P. ; Druzhinina, Irina S. ; Guebitz, Georg M. / Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins. In: Applied and Environmental Microbiology. 2015 ; Vol. 81, No. 11. pp. 3586-3592.
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AU - Zitzenbacher, Sabine

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AU - Tscheließnig, Rupert

AU - Jungbauer, Alois

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AU - Lichtenegger, Helga

AU - Amenitsch, Heinz

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