title: Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis.
abstract: Modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 revealed that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site could be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate). To investigate this hypothesis in more detail, selected amino acids of surface regions outside the active site of Thc_Cut2, which hydrolyzes PET much less efficiently than Thc_Cut1 were exchanged by site-directed mutagenesis. The mutants were expressed in E. coli BL21-Gold(DE3), purified and characterized regarding their specific activities and kinetic parameters on soluble substrates and their ability to hydrolyze PET and the PET model substrate bis(benzoyloxyethyl) terephthalate (3PET). Compared to Thc_Cut2, mutants carrying Arg29Asn and/or Ala30Val exchanges showed considerable higher specific activity and higher kcat /KM values on soluble substrates. Exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for 3PET and PET. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on PET films. These findings clearly demonstrate that surface properties (i.e., amino acids located outside the active site on the protein surface) play an important role in PET hydrolysis.
authors.affiliation: ACIB GmbH, Petersgasse 14, Graz, 8010, Austria.
authors.forename: Enrique
authors.initials: E
authors.lastname: Herrero Acero
authors.forename: Doris
authors.initials: D
authors.lastname: Ribitsch
authors.forename: Anita
authors.initials: A
authors.lastname: Dellacher
authors.forename: Sabine
authors.initials: S
authors.lastname: Zitzenbacher
authors.forename: Annemarie
authors.initials: A
authors.lastname: Marold
authors.forename: Georg
authors.initials: G
authors.lastname: Steinkellner
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
authors.forename: Georg M
authors.initials: GM
authors.lastname: Guebitz
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D011091:Polyesters / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013499:Surface Properties
chemical_list: D001426:Bacterial Proteins; D011091:Polyesters; D002265:Carboxylic Ester Hydrolases; C019952:cutinase
keywords: PET degradation; polyesterase; surface engineering