Among the enzymes described to hydrolyze synthetic polyesters, cutinases have been shown to be most active on PET. Recently we have identified two cutinases from Thermobifida cellulosilytica (Thc_Cut1 & Thc_Cut2) having only few amino acid differences located outside the active site but yet showing very different kinetic parameters regarding hydrolysis of PET. Therefore, based on exchanges of these critical aminoacids we investigated their influence in hydrolysis of soluble and insoluble substrates and developed mutants more active towards PET.We also fused two binding domains from a PHA-depolymerase from Alcaligenes faecalis (Thc_Cut1+PBM) and from cellobiohydrolase I from Trichoderma reesei (Thc_Cut1+CBM) to Thc_Cut1 in order to improve the sorption process on the polymer surface. We successfully cloned, expressed and characterized these enzymes in terms of hydrolysis of soluble substrates and PET. It was possible to correlate an increase in adsorption measured with Quarz Crystal Microbalance with Dissipation (QCM-D) with a higher activity measured as soluble released products (HPLC-UV). Figure 1. <img src='https://acs-archive-assets2.s3.amazonaws.com/cerm2011/uploads/94392_cerm_2011_structures_for_poster_abstract.jpg' alt='' />

Re-design of cutinases to increase activity on polyesters

245th National Meeting (2013)

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