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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.

SUBMITTER: Ribitsch D

PROVIDER: S-EPMC4421044 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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

Ribitsch Doris D Herrero Acero Enrique E Przylucka Agnieszka A Zitzenbacher Sabine S Marold Annemarie A Gamerith Caroline C Tscheließnig Rupert R Jungbauer Alois A Rennhofer Harald H Lichtenegger Helga H Amenitsch Heinz H Bonazza Klaus K Kubicek Christian P CP Druzhinina Irina S IS Guebitz Georg M GM

Applied and environmental microbiology 20150320 11

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 Trichoderm ...[more]

PMID: 25795674

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Project description:Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis-diol, is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDOKF1). TPDOKF1 exhibited substrate specificity for TPA (kcat/Km = 57 ± 9 mM-1 s-1). The TPDOKF1 structure harbors characteristic RO features as well as a unique catalytic domain that rationalizes the enzyme's function. The docking and mutagenesis studies reveal that its substrate specificity for TPA is mediated by the Arg309 and Arg390 residues, positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, as its mutation to alanine decreases the activity (kcat) by 80%. This study delineates the structural features that dictate the substrate recognition and specificity of TPDO. IMPORTANCE Global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential for tackling this. Microbial utilization of this released product, TPA, is an emerging and promising strategy for waste-to-value creation. Research in the last decade has identified terephthalate dioxygenase (TPDO) as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

Dataset Information

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

Publications

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

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