title: Engineered Thermobifida fusca cutinase with increased activity on polyester substrates.
abstract: A bacterial cutinase from Thermobifida fusca, named Tfu_0883, was genetically modified by site-directed mutagenesis to enhance its activity on poly(ethylene terephthalate) (PET). The new mutations tailored the catalytic site for PET, increasing the affinity of cutinase to this hydrophobic substrate and the ability to hydrolyze it. The mutation I218A was designed to create space and the double mutation Q132A/T101A was designed both to create space and to increase hydrophobicity. The activity of the double mutant on the soluble substrate p-nitrophenyl butyrate increased two-fold compared to wild-type cutinase, while on PET both single and double mutants exhibited considerably higher hydrolysis efficiency. The replacement of specific amino acids at the active site was an effective approach for the improvement of the Tfu_0883 cutinase capacity to hydrolyze polyester surfaces. Thus, this study provides valuable insight on how the function and stability of enzymes can be improved by molecular engineering for their application in synthetic fiber biotransformation.
authors.affiliation: University of Minho, Textile Engineering Department, Guimarães, Portugal.
authors.forename: Carla
authors.initials: C
authors.lastname: Silva
authors.forename: Shi
authors.initials: S
authors.lastname: Da
authors.forename: Nádia
authors.initials: N
authors.lastname: Silva
authors.forename: Teresa
authors.initials: T
authors.lastname: Matamá
authors.forename: Rita
authors.initials: R
authors.lastname: Araújo
authors.forename: Madalena
authors.initials: M
authors.lastname: Martins
authors.forename: Sheng
authors.initials: S
authors.lastname: Chen
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.forename: Margarida
authors.initials: M
authors.lastname: Casal
authors.forename: Artur
authors.initials: A
authors.lastname: Cavaco-Paulo
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics*; D004950:Esterases / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011092:Polyethylene Glycols / Q000378:metabolism*; D011093:Polyethylene Terephthalates; D013379:Substrate Specificity; D013784:Textiles; D017155:Wettability
chemical_list: D001426:Bacterial Proteins; D011093:Polyethylene Terephthalates; D011092:Polyethylene Glycols; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D008049:Lipase