Functional characterization and structural modeling of synthetic polyester-degrading hydrolases from Thermomonospora curvata.

Ren Wei,Johannes Then,Markus Barth,Thorsten Oeser,Juliane Schmidt,Wolfgang Zimmermann,Nancy Kühn

doi:10.1186/s13568-014-0044-9

Abstract

Thermomonospora curvata is a thermophilic actinomycete phylogenetically related to Thermobifida fusca that produces extracellular hydrolases capable of degrading synthetic polyesters. Analysis of the genome of T. curvata DSM43183 revealed two genes coding for putative polyester hydrolases Tcur1278 and Tcur0390 sharing 61% sequence identity with the T. fusca enzymes. Mature proteins of Tcur1278 and Tcur0390 were cloned and expressed in Escherichia coli TOP10. Tcur1278 and Tcur0390 exhibited an optimal reaction temperature against p-nitrophenyl butyrate at 60°C and 55°C, respectively. The optimal pH for both enzymes was determined at pH 8.5. Tcur1278 retained more than 80% and Tcur0390 less than 10% of their initial activity following incubation for 60 min at 55°C. Tcur0390 showed a higher hydrolytic activity against poly(ε-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50°C. At 55°C and 60°C, hydrolytic activity against PET nanoparticles was only detected with Tcur1278. In silico modeling of the polyester hydrolases and docking with a model substrate composed of two repeating units of PET revealed the typical fold of α/β serine hydrolases with an exposed catalytic triad. Molecular dynamics simulations confirmed the superior thermal stability of Tcur1278 considered as the main reason for its higher hydrolytic activity on PET.

Highlights

The widespread use of synthetic polyesters such as polyethylene terephthalate (PET) in industry and daily life has resulted in serious environmental pollution over the last decades
We report the identification of two genes coding for the polyester hydrolases Tcur1278 and Tcur0390 by genome mining of T. curvata DSM43183 (Chertkov et al 2011), the characterization of their catalytic properties and thermal stability, as well as the modeling and analysis of their three-dimensional structures
Expression and purification of Tcur1278 and Tcur0390 Synthetic genes encoding Tcur1278 and Tcur0390 were amplified in the pBAD-TOPO expression vector (Invitrogen) for recombinant expression in One Shot E. coli TOP10 (Invitrogen)

Summary

Introduction

The widespread use of synthetic polyesters such as polyethylene terephthalate (PET) in industry and daily life has resulted in serious environmental pollution over the last decades. Enzymes with high PET-hydrolyzing activity are mostly extracellular proteins secreted by thermophilic microorganisms such as Thermomyces insolens (Ronkvist et al 2009) and several Thermobifida species (Müller et al 2005; Eberl et al 2009; Herrero Acero et al 2011; Ribitsch et al 2012a; Ribitsch et al 2012b; Kitadokoro et al 2012; Chen et al 2010; Oeser et al 2010). The biodegradability of PET by these enzymes has been shown to strongly depend on the flexibility of polymer chains that is directly influenced by the hydrolysis reaction temperatures (Ronkvist et al 2009; Wei et al 2013)

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Results

Conclusion