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Engineered Thermobifida fusca cutinase with increased activity on polyester substrates
Author(s) -
Silva Carla,
Da Shi,
Silva Nádia,
Matamá Teresa,
Araújo Rita,
Martins Madalena,
Chen Sheng,
Chen Jian,
Wu Jing,
Casal Margarida,
CavacoPaulo Artur
Publication year - 2011
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201000391
Subject(s) - cutinase , protein engineering , hydrolysis , chemistry , biochemistry , directed evolution , polyester , enzyme , mutant , organic chemistry , gene
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.