title: Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach.
abstract: The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C(4) substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(ε-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/μkat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET.
authors.affiliation: Department of Material and Life Science, Osaka University, Suita, Osaka, Japan.
authors.forename: Sintawee
authors.initials: S
authors.lastname: Sulaiman
authors.forename: Saya
authors.initials: S
authors.lastname: Yamato
authors.forename: Eiko
authors.initials: E
authors.lastname: Kanaya
authors.forename: Joong-Jae
authors.initials: JJ
authors.lastname: Kim
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Koga
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Takano
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D054892:Metagenome; D008969:Molecular Sequence Data; D011093:Polyethylene Terephthalates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012987:Soil; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005227:Fatty Acids; D011093:Polyethylene Terephthalates; D011994:Recombinant Proteins; D012987:Soil; D002265:Carboxylic Ester Hydrolases; C019952:cutinase