title: Fusion of binding domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis.
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.
authors.affiliation: Enzymes and Polymers, Austrian Centre of Industrial Biotechnology ACIB , Petergasse 14, 8010, Graz, Austria.
authors.forename: Doris
authors.initials: D
authors.lastname: Ribitsch
authors.forename: Antonio Orcal
authors.initials: AO
authors.lastname: Yebra
authors.forename: Sabine
authors.initials: S
authors.lastname: Zitzenbacher
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.forename: Susanne
authors.initials: S
authors.lastname: Nowitsch
authors.forename: Georg
authors.initials: G
authors.lastname: Steinkellner
authors.forename: Katrin
authors.initials: K
authors.lastname: Greimel
authors.forename: Ales
authors.initials: A
authors.lastname: Doliska
authors.forename: Gustav
authors.initials: G
authors.lastname: Oberdorfer
authors.forename: Christian C
authors.initials: CC
authors.lastname: Gruber
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
authors.forename: Karin
authors.initials: K
authors.lastname: Stana-Kleinschek
authors.forename: Enrique Herrero
authors.initials: EH
authors.lastname: Acero
authors.forename: Georg M
authors.initials: GM
authors.lastname: Guebitz
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D000327:Adsorption; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008958:Models, Molecular; D011093:Polyethylene Terephthalates / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011093:Polyethylene Terephthalates; D011993:Recombinant Fusion Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase