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.

Fig 1

Substrate specificity of LC-cutinase*. The enzymatic activity was determined at 30°C in 25 mM Tris-HCl (pH 8.0) containing 10% acetonitrile using pNP-acetate (C₂), pNP-butyrate (C₄), pNP-hexanoate (C₆), pNP-caprylate (C₈), pNP-laurate (C₁₂), pNP-myristate (C₁₄), pNP-palmitate (C₁₆), or pNP-stearate (C₁₈) as a substrate. The specific activities relative to that determined for hydrolysis of pNP-butyrate are shown. The experiment was carried out at least twice, and the average values are shown together with error bars.

Fig 2

CD spectra. The far-UV spectra of LC-cutinase* (solid line) and S165A-cutinase* (dotted line) measured in 10 mM Tris-HCl (pH 7.0) containing 1 mM DTT are shown. These spectra were measured at 25°C as described in Materials and Methods.

Fig 3

Degradation of PCL film by LC-cutinase*. A PCL film (20–30 mg) was incubated at 50°C for 6 h in 1 ml of 500 mM Tris-HCl (pH 8.0) containing 5 μg of LC-cutinase* (LCC), or 50 μg of B. cepacia lipase (Bc-Lip) or C. rugosa lipase (Cr-Lip), and its weight loss after incubation was determined. The experiment was carried out at least twice and the average values are shown, together with error bars.

Fig 4

Degradation of PET film by LC-cutinase*. A PET film (20 to 25 mg) was incubated at 50°C for 24 h in 1 ml of 500 mM Tris-HCl (pH 8.0) containing 5 μg of LC-cutinase* (LCC), or 50 μg of Bc-Lip or Cr-Lip, and its weight loss after incubation was determined. The experiment was carried out at least twice, and the average values are shown together with error bars.

Fig 5

Degradation of cutin by LC-cutinase*. LC-cutinase* (50 μg) was incubated with 1% (wt/vol) tomato cutin in 1 ml of 20 mM Tris-HCl (pH 8.0) at 50°C. The released fatty acids were quantified by titration with 50 mM NaOH. The experiment was carried out at least twice, and the average values are shown together with error bars.