Then J

References (7)

Title : A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate - Then_2016_FEBS.Open.Bio_6_425
Author(s) : Then J , Wei R , Oeser T , Gerdts A , Schmidt J , Barth M , Zimmermann W
Ref : FEBS Open Bio , 6 :425 , 2016
Abstract : Elevated reaction temperatures are crucial for the efficient enzymatic degradation of polyethylene terephthalate (PET). A disulfide bridge was introduced to the polyester hydrolase TfCut2 to substitute its calcium binding site. The melting point of the resulting variant increased to 94.7 degreesC (wild-type TfCut2: 69.8 degreesC) and its half-inactivation temperature to 84.6 degreesC (TfCut2: 67.3 degreesC). The variant D204C-E253C-D174R obtained by introducing further mutations at vicinal residues showed a temperature optimum between 75 and 80 degreesC compared to 65 and 70 degreesC of the wild-type enzyme. The variant caused a weight loss of PET films of 25.0 +/- 0.8% (TfCut2: 0.3 +/- 0.1%) at 70 degreesC after a reaction time of 48 h. The results demonstrate that a highly efficient and calcium-independent thermostable polyester hydrolase can be obtained by replacing its calcium binding site with a disulfide bridge.
ESTHER : Then_2016_FEBS.Open.Bio_6_425
PubMedSearch : Then_2016_FEBS.Open.Bio_6_425
PubMedID: 27419048
Gene_locus related to this paper: thefu-q6a0i4

Title : Effect of Tris, MOPS, and phosphate buffers on the hydrolysis of polyethylene terephthalate films by polyester hydrolases - Schmidt_2016_FEBS.Open.Bio_6_919
Author(s) : Schmidt J , Wei R , Oeser T , Belisario-Ferrari MR , Barth M , Then J , Zimmermann W
Ref : FEBS Open Bio , 6 :919 , 2016
Abstract : The enzymatic degradation of polyethylene terephthalate (PET) occurs at mild reaction conditions and may find applications in environmentally friendly plastic waste recycling processes. The hydrolytic activity of the homologous polyester hydrolases LC cutinase (LCC) from a compost metagenome and TfCut2 from Thermobifida fusca KW3 against PET films was strongly influenced by the reaction medium buffers tris(hydroxymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid (MOPS), and sodium phosphate. LCC showed the highest initial hydrolysis rate of PET films in 0.2 m Tris, while the rate of TfCut2 was 2.1-fold lower at this buffer concentration. At a Tris concentration of 1 m, the hydrolysis rate of LCC decreased by more than 90% and of TfCut2 by about 80%. In 0.2 m MOPS or sodium phosphate buffer, no significant differences in the maximum initial hydrolysis rates of PET films by both enzymes were detected. When the concentration of MOPS was increased to 1 m, the hydrolysis rate of LCC decreased by about 90%. The activity of TfCut2 remained low compared to the increasing hydrolysis rates observed at higher concentrations of sodium phosphate buffer. In contrast, the activity of LCC did not change at different concentrations of this buffer. An inhibition study suggested a competitive inhibition of TfCut2 and LCC by Tris and MOPS. Molecular docking showed that Tris and MOPS interfered with the binding of the polymeric substrate in a groove located at the protein surface. A comparison of the K i values and the average binding energies indicated MOPS as the stronger inhibitor of the both enzymes.
ESTHER : Schmidt_2016_FEBS.Open.Bio_6_919
PubMedSearch : Schmidt_2016_FEBS.Open.Bio_6_919
PubMedID: 27642555

Title : Engineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibition - Wei_2016_Biotechnol.Bioeng_113_1658
Author(s) : Wei R , Oeser T , Schmidt J , Meier R , Barth M , Then J , Zimmermann W
Ref : Biotechnol Bioeng , 113 :1658 , 2016
Abstract : Recent studies on the enzymatic degradation of synthetic polyesters have shown the potential of polyester hydrolases from thermophilic actinomycetes for modifying or degrading polyethylene terephthalate (PET). TfCut2 from Thermobifida fusca KW3 and LC-cutinase (LCC) isolated from a compost metagenome are remarkably active polyester hydrolases with high sequence and structural similarity. Both enzymes exhibit an exposed active site in a substrate binding groove located at the protein surface. By exchanging selected amino acid residues of TfCut2 involved in substrate binding with those present in LCC, enzyme variants with increased PET hydrolytic activity at 65 degrees C were obtained. The highest activity in hydrolyzing PET films and fibers were detected with the single variant G62A and the double variant G62A/I213S. Both variants caused a weight loss of PET films of more than 42% after 50 h of hydrolysis, corresponding to a 2.7-fold increase compared to the wild type enzyme. Kinetic analysis based on the released PET hydrolysis products confirmed the superior hydrolytic activity of G62A with a fourfold higher hydrolysis rate constant and a 1.5-fold lower substrate binding constant than those of the wild type enzyme. Mono-(2-hydroxyethyl) terephthalate is a strong inhibitor of TfCut2. A determination of the Rosetta binding energy suggested a reduced interaction of G62A with 2PET, a dimer of the PET monomer ethylene terephthalate. Indeed, G62A revealed a 5.5-fold lower binding constant to the inhibitor than the wild type enzyme indicating that its increased PET hydrolysis activity is the result of a relieved product inhibition by mono-(2-hydroxyethyl) terephthalate. Biotechnol. Bioeng. 2016;113: 1658-1665. (c) 2016 Wiley Periodicals, Inc.
ESTHER : Wei_2016_Biotechnol.Bioeng_113_1658
PubMedSearch : Wei_2016_Biotechnol.Bioeng_113_1658
PubMedID: 26804057
Gene_locus related to this paper: 9bact-g9by57 , thefu-q6a0i4

Title : A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films - Barth_2016_Biotechnol.J_11_1082
Author(s) : Barth M , Honak A , Oeser T , Wei R , Belisario-Ferrari MR , Then J , Schmidt J , Zimmermann W
Ref : Biotechnol J , 11 :1082 , 2016
Abstract : TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60 degrees C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.
ESTHER : Barth_2016_Biotechnol.J_11_1082
PubMedSearch : Barth_2016_Biotechnol.J_11_1082
PubMedID: 27214855
Gene_locus related to this paper: 9bact-g9by57 , thefu-1831

Title : Ca2+ and Mg2+ binding site engineering increases the degradation of polyethylene terephthalate films by polyester hydrolases from Thermobifida fusca - Then_2015_Biotechnol.J_10_592
Author(s) : Then J , Wei R , Oeser T , Barth M , Belisario-Ferrari MR , Schmidt J , Zimmermann W
Ref : Biotechnol J , 10 :592 , 2015
Abstract : Several bacterial polyester hydrolases are able to hydrolyze the synthetic polyester polyethylene terephthalate (PET). For an efficient enzymatic degradation of PET, reaction temperatures close to the glass transition temperature of the polymer need to be applied. The esterases TfH, BTA2, Tfu_0882, TfCut1, and TfCut2 produced by the thermophilic actinomycete Thermobifida fusca exhibit PET-hydrolyzing activity. However, these enzymes are not sufficiently stable in this temperature range for an efficient degradation of post-consumer PET materials. The addition of Ca2+ or Mg2+ cations to the enzymes resulted in an increase of their melting points between 10.8 and 14.1 degreesC determined by circular dichroism spectroscopy. The thermostability of the polyester hydrolases was sufficient to degrade semi-crystalline PET films at 65 degreesC in the presence of 10 mM Ca2+ and 10 mM Mg2+ resulting in weight losses of up to 12.9% after a reaction time of 48 h. The residues Asp174, Asp204, and Glu253 were identified by molecular dynamics simulations as potential binding residues for the two cations in TfCut2. This was confirmed by their substitution with arginine, resulting in a higher thermal stability of the corresponding enzyme variants. The generated variants of TfCut2 represent stabilized catalysts suitable for PET hydrolysis reactions performed in the absence of Ca2+ or Mg2+.
ESTHER : Then_2015_Biotechnol.J_10_592
PubMedSearch : Then_2015_Biotechnol.J_10_592
PubMedID: 25545638

Title : Functional characterization and structural modeling of synthetic polyester-degrading hydrolases from Thermomonospora curvata - Wei_2014_AMB.Express_4_44
Author(s) : Wei R , Oeser T , Then J , Kuhn N , Barth M , Schmidt J , Zimmermann W
Ref : AMB Express , 4 :44 , 2014
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 degrees C and 55 degrees 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 degrees C. Tcur0390 showed a higher hydrolytic activity against poly(epsilon-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50 degrees C. At 55 degrees C and 60 degrees 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 alpha/beta 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.
ESTHER : Wei_2014_AMB.Express_4_44
PubMedSearch : Wei_2014_AMB.Express_4_44
PubMedID: 25405080
Gene_locus related to this paper: thecd-d1a9g5 , thecd-d1a2h1

Title : Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca - Roth_2014_Appl.Microbiol.Biotechnol_98_7815
Author(s) : Roth C , Wei R , Oeser T , Then J , Follner C , Zimmermann W , Strater N
Ref : Applied Microbiology & Biotechnology , 98 :7815 , 2014
Abstract : Bacterial cutinases are promising catalysts for the modification and degradation of the widely used plastic polyethylene terephthalate (PET). The improvement of the enzyme for industrial purposes is limited due to the lack of structural information for cutinases of bacterial origin. We have crystallized and structurally characterized a cutinase from Thermobifida fusca KW3 (TfCut2) in free as well as in inhibitor-bound form. Together with our analysis of the thermal stability and modelling studies, we suggest possible reasons for the outstanding thermostability in comparison to the less thermostable homolog from Thermobifida alba AHK119 and propose a model for the binding of the enzyme towards its polymeric substrate. The TfCut2 structure is the basis for the rational design of catalytically more efficient enzyme variants for the hydrolysis of PET and other synthetic polyesters.
ESTHER : Roth_2014_Appl.Microbiol.Biotechnol_98_7815
PubMedSearch : Roth_2014_Appl.Microbiol.Biotechnol_98_7815
PubMedID: 24728714
Gene_locus related to this paper: thefu-q6a0i4