Gene_locus Report for: thefu-1831

TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structurefunction relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.

Plastics are highly durable and widely used materials. Current methodologies of plastic degradation, elimination, and recycling are flawed. In recent years, biodegradation (the usage of microorganisms for material recycling) has grown as a valid alternative to previously used methods. The evolution of bioengineering techniques and the discovery of novel microorganisms and enzymes with degradation ability have been key. One of the most produced plastics is PET, a long chain polymer of terephthalic acid (TPA) and ethylene glycol (EG) repeating monomers. Many enzymes with PET degradation activity have been discovered, characterized, and engineered in the last few years. However, classification and integrated knowledge of these enzymes are not trivial. Therefore, in this work we present a summary of currently known PET degrading enzymes, focusing on their structural and activity characteristics, and summarizing engineering efforts to improve activity. Although several high potential enzymes have been discovered, further efforts to improve activity and thermal stability are necessary.

The gram-positive thermophilic actinomycete Thermobifida fusca KW3 secretes a highly hydrophobic carboxylesterase (TfCa) that is able to hydrolyze poly(ethylene terephthalate). TfCa was produced in the Escherichia coli strain BL21(DE3) as a fusion protein consisting of a pelB leader sequence to ensure periplasmic localization of the protein and a His(6) tag for use in its purification. To enhance the recombinant enzyme yield, the tfca gene from T. fusca KW3 was successfully optimized for codon usage in E. coli. In addition, the gene expression induction conditions were optimized and the temperature for cell cultivation was lowered to reduce inclusion body formation. The optimized codons and expression conditions yielded 4500-fold higher TfCa activity than the wild-type strain. Using a pH-controlled bioreactor for cultivation, a TfCa protein concentration of 41.6mg/L was achieved.

TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structurefunction relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.

Plastics are highly durable and widely used materials. Current methodologies of plastic degradation, elimination, and recycling are flawed. In recent years, biodegradation (the usage of microorganisms for material recycling) has grown as a valid alternative to previously used methods. The evolution of bioengineering techniques and the discovery of novel microorganisms and enzymes with degradation ability have been key. One of the most produced plastics is PET, a long chain polymer of terephthalic acid (TPA) and ethylene glycol (EG) repeating monomers. Many enzymes with PET degradation activity have been discovered, characterized, and engineered in the last few years. However, classification and integrated knowledge of these enzymes are not trivial. Therefore, in this work we present a summary of currently known PET degrading enzymes, focusing on their structural and activity characteristics, and summarizing engineering efforts to improve activity. Although several high potential enzymes have been discovered, further efforts to improve activity and thermal stability are necessary.

Synthetic plastics such as polyethylene terephthalate (PET) can be cooperatively degraded by microbial polyester hydrolases and carboxylesterases, with the latter hydrolyzing the low-molecular-weight degradation intermediates. For the identification of PET-degrading enzymes, efficient and rapid screening assays are required. Here we report a novel turbidimetric method in a microplate format for the fast screening of enzyme activities against the PET model substrates with two ester bonds bis-(2-Hydroxyethyl) terephthalate (BHET) and ethylene glycol bis-(p-methylbenzoate) (2PET). The carboxylesterase TfCa from Thermobifida fusca KW3 was used for validating the method. High correlation and regression coefficients between the experimental and fitted data confirmed the accuracy and reproducibility of the method and its feasibility for analyzing the kinetics of the enzymatic hydrolysis of the PET model substrates. A comparison of the hydrolysis of BHET and 2PET by TfCa using a kinetic model for heterogeneous catalysis indicated that the enzyme preferentially hydrolyzed the less bulky molecule BHET. The high-throughput assay will facilitate the detection of novel enzymes for the biocatalytic modification or degradation of PET.

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.

Here, we present the complete genome sequence of Thermobifida fusca strain TM51, which was isolated from the hot upper layer of a compost pile in Hungary. T. fusca TM51 is a thermotolerant, aerobic actinomycete with outstanding lignocellulose-decomposing activity.

We have identified a carboxylesterase produced in liquid cultures of the thermophilic actinomycete Thermobifida fusca KW3 that were supplemented with poly(ethylene terephthalate) fibers. The enzyme hydrolyzed highly hydrophobic, synthetic cyclic poly(ethylene terephthalate) trimers with an optimal activity at 60 degrees C and a pH of 6. V (max) and K (m) values for the hydrolysis were 9.3 micromol(-1) min(-1) mg(-1) and 0.5 mM, respectively. The esterase showed high specificity towards short and middle chain-length fatty acyl esters of p-nitrophenol. The enzyme retained 37% of its activity after 96 h of incubation at 50 degrees C and a pH of 8. Enzyme inhibition studies and analysis of substitution mutants of the carboxylesterase revealed the typical catalytic mechanism of a serine hydrolase with a catalytic triad composed of serine, glutamic acid, and histidine.

The gram-positive thermophilic actinomycete Thermobifida fusca KW3 secretes a highly hydrophobic carboxylesterase (TfCa) that is able to hydrolyze poly(ethylene terephthalate). TfCa was produced in the Escherichia coli strain BL21(DE3) as a fusion protein consisting of a pelB leader sequence to ensure periplasmic localization of the protein and a His(6) tag for use in its purification. To enhance the recombinant enzyme yield, the tfca gene from T. fusca KW3 was successfully optimized for codon usage in E. coli. In addition, the gene expression induction conditions were optimized and the temperature for cell cultivation was lowered to reduce inclusion body formation. The optimized codons and expression conditions yielded 4500-fold higher TfCa activity than the wild-type strain. Using a pH-controlled bioreactor for cultivation, a TfCa protein concentration of 41.6mg/L was achieved.

It is demonstrated that PET, which is usually regarded as non-biodegradable, can effectively be depolymerized by a hydrolase from the actinomycete Thermobifida fusca. Erosion rates of 8 to 17 micro m per week were obtained upon incubation at 55 degC. Lipases from Pseudomonas sp. and Candida antarctica did not degrade PET under comparable conditions. The influences of crystallinity, melting point, and glass transition temperature on the enzymatic attack on PET, PBT, and PHB are discussed.