Fusion of binding-domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis

Doris Ribitsch, Orcal Yebra Antonio, Sabine Zitzenbacher, Jing Wu, Susanne Nowitsch, Georg Steinkellner, Katrin Julia Greimel, Doliška Aleš, Gustav Oberdorfer, Christian Gruber, Karl Gruber, Helmut Schwab, Stana-Kleinschek Karin, Enrique Herrero Acero, Georg Gübitz

Research output: Contribution to journalArticlepeer-review

Abstract

A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli. Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s–1 on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s–1/ μM (native enzyme) to 0.21 and 0.33 s–1/μM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.
Original languageEnglish
Pages (from-to)1769–1776
JournalBiomacromolecules
Volume14
Issue number6
DOIs
Publication statusPublished - 2013

Fields of Expertise

  • Human- & Biotechnology

Treatment code (Nähere Zuordnung)

  • Experimental

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