Gene_locus Report for: thefu-q6a0i3

With the increasing production of polyethylene terephthalate (PET) plastic products, the problem of PET waste has become a serious threat to ecosystem. PET enzymatic biodegradation, due to its environmental friendliness and sustainability, has gradually attracted attention. As a multifunctional hydrolase, cutinase (EC 3.1.1.74) can not only degrade fatty acid esters, soluble synthetic esters, and emulsified triglycerides, but also exhibit potential for PET degradation. In order to enhance the PET degradation activity of cutinase, we functionally screened several PET binding domains, e.g. carbohydrate binding module, anchor peptide, and hydrophobin, that promote the absorption of enzyme to PET substrate, selected Dermaseptin SI (DSI) and fused it onto the N-terminus of Thermobifida fusca cutinase mutant D204C/E253C (Tfuc2), and finally achieved the PET degradation rate up to 57.9% at 70 degreesC for 96 h, which was 22.7-fold of that of Tfuc2 itself. These results indicate that the fusion of PET binding domain is a promising strategy to enhance PET enzymatic degradation.

In the past 20 years, scientific research focused on the identification of valid alternatives to materials of fossil origin, in particular, related to biobased polymers. Recently, the efforts led to the synthesis of thiophene-based polymers (TBPs), a new class of polyesters based on 2,5-thiophenedicarboxylic acid (TPCA) that can be industrially produced using biomass-derived molecules. In this study, TBPs were synthesized using diols with different chain length (from C4 to C6) leading to poly(butylene 2,5-thiophenedicarboxylate) (PBTF), poly(pentamethylene 2,5-thiophenedicarboxylate) (PPeTF), and poly(hexamethylene 2,5-thiophenedicarboxylate) (PHTF), respectively, that were processed to thin films. To investigate enzymatic hydrolysis of these polymer films, cutinase 1 (Thc_cut1) and cutinase 2 (Thc_cut2) from Thermobifida cellulosilytica were recombinantly expressed in the host E. coli and purified. After 72 h of incubation at 65 degreesC with 5 microM Thc_cut1, weight loss and HPLC analysis indicated 9, 100, and 80% degradation of PBTF, PPeTF, and PHTG with a concomitant release of 0.12, 2.70, and 0.67 mM of TPCA. The SEM analysis showed that tiny holes were formed on the surface of the films and after 72 h PPeTF was completely degraded. The LC-TOF/MS analysis indicated that Thc_cut2 in particular released various oligomers from the polymer during the reaction. In addition, the FTIR analysis showed the formation of novel acid and hydroxyl groups on the polymer surfaces. The results showed that the two used thermostable cutinases are promising biocatalysts for the environmentally friendly degradation of TPCA-based polyesters, in view of a possible sustainable recycling of plastic waste through resynthesis processes.

We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;114: 2481-2488. (c) 2017 Wiley Periodicals, Inc.

Poly (ethylene terephthalate) (PET) is a widely used type of general plastic that produces a significant amount of waste due to its non-degradable properties. We propose a novel directional-path modification (DPM) strategy, involving positive charge amino acid introduction and binding groove remodeling, and apply it to Thermobifida fusca cutinase to enhance PET degradation. The highest value of PET degradation (90%) was achieved in variant 4Mz (H184S/Q92G/F209I/I213K), exhibiting values almost 30-fold that of the wild-type. We employed molecular docking, molecular dynamics simulations, and QM/MM MD for the degradation process of PET, accompanied by acylation and deacylation. We found that the distance of nucleophilic attack was reduced from about 4.6 A in the wild type to 3.8 A in 4Mz, and the free energy barrier of 4Mz dropped from 14.3 kcal/mol to 7.1 kcal/mol at the acylation which was the rate-limiting step. Subsequently, the high efficiency and universality of the DPM strategy were successfully demonstrated in LCC, Est119, and BhrPETase enhancing the degradation activity of PET. Finally, the highest degradation rate of the pretreated commercial plastic bottles had reached to 73%. The present study provides insight into the molecular binding mechanism of PET into the PET hydrolases structure and proposes a novel DPM strategy that will be useful for the engineering of more efficient enzymes for PET degradation.

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 degreesC. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

Accelerated hydrolysis of polyethylene terephthalate (PET) by enzymatic surface modification of various hydrolases, which would not degrade the building blocks of PET in order to retain the quality of recycled PET, is a promising research area. Many studies have been reported to identify mutations of different hydrolases that can improve PET degradation. Recently, the mutation of glycine and phenyl alanine with alanine in cutinase was found to improve the activity of PET degradation 6-fold. Yet, a deep insight into the overall structural basis as well as the explicit role played by the amino acid residues for PET degradation is still elusive, which is nevertheless important for comparative analyses, structure-function relations and rational optimization of the degradation process. Our molecular dynamics simulations coupled with quantum mechanical study demonstrate that mutations of anchor residue phenyl alanine to alanine at the PET binding cleft of cutinase unveiled a distal yet novel binding subsite, which alters the nature of dispersive interaction for PET recognition and binding. The phenyl alanine engages in Pi-Pi interaction with the phenyl ring of PET (-8.5 kcal mol(-1)), which on one side helps in PET recognition, but on the other side restricts PET to attain fully extended conformations over the entire binding cleft. The loss of Pi-Pi interaction due to mutation of phenyl alanine to alanine is not only compensated by the favourable cation-Pi and hydrophobic interactions from the arginine residues (-17.1 kcal mol(-1)) found in the newly discovered subsite, but also favours the fully extended PET conformation. This subsequently impacts the overall increased catalytic activity of mutated cutinase.

Polyethylene terephthalate (PET) has caused serious environmental concerns but could be degraded at high temperature. Previous studies show that cutinase from Thermobifida fusca KW3 (TfCut2) is capable of degrading and upcycling PET but is limited by its thermal stability. Nowadays, Popular protein stability modification methods rely mostly on the crystal structures, but ignore the fact that the actual conformation of protein is complex and constantly changing. To solve these problems, we developed a computational approach to design variants with enhanced protein thermal stability by mining Molecular Dynamics simulation trajectories using Machine Learning methods (MDL). The optimal classification accuracy and the optimal Pearson correlation coefficient of MDL model were 0.780 and 0.716, respectively. And we successfully designed variants with high deltaT (m) values using MDL method. The optimal variant S121P/D174S/D204P had the highest deltaT (m) value of 9.3 degreesC, and the PET degradation ratio increased by 46.42-fold at 70 degC, compared with that of wild type TfCut2. These results deepen our understanding on the complex conformations of proteins and may enhance the plastic recycling and sustainability at glass transition temperature.

With the increasing production of polyethylene terephthalate (PET) plastic products, the problem of PET waste has become a serious threat to ecosystem. PET enzymatic biodegradation, due to its environmental friendliness and sustainability, has gradually attracted attention. As a multifunctional hydrolase, cutinase (EC 3.1.1.74) can not only degrade fatty acid esters, soluble synthetic esters, and emulsified triglycerides, but also exhibit potential for PET degradation. In order to enhance the PET degradation activity of cutinase, we functionally screened several PET binding domains, e.g. carbohydrate binding module, anchor peptide, and hydrophobin, that promote the absorption of enzyme to PET substrate, selected Dermaseptin SI (DSI) and fused it onto the N-terminus of Thermobifida fusca cutinase mutant D204C/E253C (Tfuc2), and finally achieved the PET degradation rate up to 57.9% at 70 degreesC for 96 h, which was 22.7-fold of that of Tfuc2 itself. These results indicate that the fusion of PET binding domain is a promising strategy to enhance PET enzymatic degradation.

Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure-function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze PET, PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in-silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically. This article is protected by copyright. All rights reserved.

In the past 20 years, scientific research focused on the identification of valid alternatives to materials of fossil origin, in particular, related to biobased polymers. Recently, the efforts led to the synthesis of thiophene-based polymers (TBPs), a new class of polyesters based on 2,5-thiophenedicarboxylic acid (TPCA) that can be industrially produced using biomass-derived molecules. In this study, TBPs were synthesized using diols with different chain length (from C4 to C6) leading to poly(butylene 2,5-thiophenedicarboxylate) (PBTF), poly(pentamethylene 2,5-thiophenedicarboxylate) (PPeTF), and poly(hexamethylene 2,5-thiophenedicarboxylate) (PHTF), respectively, that were processed to thin films. To investigate enzymatic hydrolysis of these polymer films, cutinase 1 (Thc_cut1) and cutinase 2 (Thc_cut2) from Thermobifida cellulosilytica were recombinantly expressed in the host E. coli and purified. After 72 h of incubation at 65 degreesC with 5 microM Thc_cut1, weight loss and HPLC analysis indicated 9, 100, and 80% degradation of PBTF, PPeTF, and PHTG with a concomitant release of 0.12, 2.70, and 0.67 mM of TPCA. The SEM analysis showed that tiny holes were formed on the surface of the films and after 72 h PPeTF was completely degraded. The LC-TOF/MS analysis indicated that Thc_cut2 in particular released various oligomers from the polymer during the reaction. In addition, the FTIR analysis showed the formation of novel acid and hydroxyl groups on the polymer surfaces. The results showed that the two used thermostable cutinases are promising biocatalysts for the environmentally friendly degradation of TPCA-based polyesters, in view of a possible sustainable recycling of plastic waste through resynthesis processes.

Cutinases are promising agents for poly(ethylene terephthalate) (PET) bio-recycling because of their ability to produce the PET monomer terephthalic acid with high efficiency under mild reaction conditions. In this study, we found that the low-crystallinity PET (lcPET) hydrolysis activity of thermostable cutinase from Thermobifida fusca (TfCut2), was increased by the addition of cationic surfactant that attracts enzymes near the lcPET film surface via electrostatic interactions. This approach was applicable to the mutant TfCut2 G62A/F209A, which was designed based on a sequence comparison with PETase from Ideonella sakaiensis. As a result, the degradation rate of the mutant in the presence of cationic surfactant increased to 31 +/- 0.1 nmol min(-1) cm(-2), 12.7 times higher than that of wild-type TfCut2 in the absence of surfactant. The long-duration reaction showed that lcPET film (200 mum) was 97 +/- 1.8% within 30 h, the fastest biodegradation rate of lcPET film thus far. We therefore believe that our approach would expand the possibility of enzyme utilization in industrial PET biodegradation.

We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;114: 2481-2488. (c) 2017 Wiley Periodicals, Inc.

Polyurethanes (PU) are widely used synthetic polymers. The growing amount of PU used industrially has resulted in a worldwide increase of plastic wastes. The related environmental pollution as well as the limited availability of the raw materials based on petrochemicals requires novel solutions for their efficient degradation and recycling. The degradation of the polyester PU Impranil DLN by the polyester hydrolases LC cutinase (LCC), TfCut2, Tcur1278 and Tcur0390 was analyzed using a turbidimetric assay. The highest hydrolysis rates were obtained with TfCut2 and Tcur0390. TfCut2 also showed a significantly higher substrate affinity for Impranil DLN than the other three enzymes, indicated by a higher adsorption constant K. Significant weight losses of the solid thermoplastic polyester PU (TPU) Elastollan B85A-10 and C85A-10 were detected as a result of the enzymatic degradation by all four polyester hydrolases. Within a reaction time of 200 h at 70 degreesC, LCC caused weight losses of up to 4.9% and 4.1% of Elastollan B85A-10 and C85A-10, respectively. Gel permeation chromatography confirmed a preferential degradation of the larger polymer chains. Scanning electron microscopy revealed cracks at the surface of the TPU cubes as a result of enzymatic surface erosion. Analysis by Fourier transform infrared spectroscopy indicated that the observed weight losses were a result of the cleavage of ester bonds of the polyester TPU.

An extracellular cutinase from Pseudomonas cepacia NRRL B 2320 was purified to apparent homogeneity. Upon biochemical characterization, the purified cutinase was found to be tolerant to organic solvents and surfactants under assay conditions. The molecular mass of cutinase was found to be 26.25 kDa by MALDI-TOF-MS analysis. The enzyme was able to show activity towards synthetic esters of chain length C4C16. The activity of cutinase was enhanced by mono cations and various effectors, whereas it was moderately inhibited by various divalent cations and serine blocking reagent, phenyl methyl sulphonyl fluoride (PMSF). The optimal pH and temperature for highest activity were found to be 7.9 and 36.5 degrees C, respectively. An overall 1.42-fold increase in activity was observed after optimization of both assay and process conditions. The exposure of hydrophobic amino acid to an aqueous environment and change in secondary structure of cutinase was observed from thermodynamic parameters (DeltaH(*), DeltaS(*)), fluorescence and circular dichorism spectra during the deactivation process. Two cutinase encoding genes were identified in P. cepacia, cloned and expressed in E. coli BL21 (DE3).

In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their kcat and Km values on pNP-acetate were in the ranges 2.4-211.9 s-1 and 127-200 micoM while on pNP-butyrate they showed kcat and Km values between 5.3 and 195.1 s-1 and between 1483 and 2133 microM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A1340/A1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.

Thermobifida fusca produces two cutinases which share 93% identity in amino acid sequence. In the present study, we investigated the detailed biochemical properties of T. fusca cutinases for the first time. For a better comparison between bacterial and fungal cutinases, recombinant Fusarium solani pisi cutinase was subjected to the similar analysis. The results showed that both bacterial and fungal cutinases are monomeric proteins in solution. The bacterial cutinases exhibited a broad substrate specificity against plant cutin, synthetic polyesters, insoluble triglycerides, and soluble esters. In addition, the two isoenzymes of T. fusca and the F.solani pisi cutinase are similar in substrate kinetics, the lack of interfacial activation, and metal ion requirements. However, the T.fusca cutinases showed higher stability in the presence of surfactants and organic solvents. Considering the versatile hydrolytic activity, good tolerance to surfactants, superior stability in organic solvents, and thermostability demonstrated by T. fusca cutinases, they may have promising applications in related industries.

A recombinant polyester-degrading hydrolase from Thermobifida sp. BCC23166 targeting on aliphatic-aromatic copolyester (rTfH) was produced in Streptomyces rimosus R7. rTfH was expressed by induction with thiostrepton as a C-terminal His(6) fusion from the native gene sequence under the control of tipA promoter and purified from the culture supernatant to high homogeneity by a single step affinity purification on Ni-Sepharose matrix. The enzyme worked optimally at 50-55 degrees C and showed esterase activity on C3-C16 p-nitrophenyl alkanoates with a specific activity of 76.5 U/mg on p-nitrophenyl palmitate. Study of rTfH catalysis on surface degradation of polyester films using surface plasmon resonance analysis revealed that the degradation rates were in the order of poly-epsilon-caprolactone > Ecoflex > polyhydroxybutyrate. Efficient hydrolysis of Ecoflex by rTfH was observed in mild alkaline conditions, with the highest activity at pH 8.0 and ionic strength at 250 mM sodium chloride, with the maximal specific activity of 0.79 mg(-1)min(-1)mg(-1) protein. Under the optimal conditions, rTfH showed a remarkable 110-time higher specific activity on Ecoflex in comparison to a lipase from Thermomyces lanuginosus, while less difference in degradation efficiency of the two enzymes was observed on the aliphatic polyesters, suggesting greater specificities of rTfH to the aliphatic-aromatic copolyester. This study demonstrated the use of streptomycetes as an alternative expression system for production of the multi-polyester-degrading enzyme of actinomycete origin and provided insights on its catalytic properties on surface degradation contributing to further biotechnological application of this enzyme.

Cutinase, which exists in both fungi and bacteria, catalyzes the cleavage of the ester bonds of cutin. Fungal cutinases have been extensively studied, however, reports on bacterial cutinases have been limited due to the lack of knowledge concerning the identity of their open reading frames. In the present study, the cutinase from Thermobifida fusca was induced by cutin and purified to homogeneity by following p-nitrophenyl butyrate hydrolyzing activity. Peptide mass fingerprinting analysis of the wild-type enzyme matched two proteins, Tfu_0883 and Tfu_0882, which are 93% identical in sequence. Both proteins were cloned and overexpressed in their mature form. Recombinant Tfu_0883 and Tfu_0882 display very similar enzymatic properties and were confirmed to be cutinases by their capability to hydrolyze the ester bonds of cutin. Comparative characterization of Fusarium solani pisi and T. fusca cutinases indicated that they have similar substrate specificity and catalytic properties except that the T. fusca enzymes are thermally more stable. Homology modeling revealed that T. fusca cutinases adopt an alpha/beta-hydrolase fold that exhibits both similarities and variations from the fungal cutinase structure. A serine hydrolase catalytic mechanism involving a Ser(170)-His(248)-Asp(216) (Tfu_0883 numbering) catalytic triad was supported by active site-directed inhibition studies and mutational analyses. This is the first report of cutinase encoding genes from bacterial sources.

Thermobifida fusca is a moderately thermophilic soil bacterium that belongs to Actinobacteria. It is a major degrader of plant cell walls and has been used as a model organism for the study of secreted, thermostable cellulases. The complete genome sequence showed that T. fusca has a single circular chromosome of 3,642,249 bp predicted to encode 3,117 proteins and 65 RNA species with a coding density of 85%. Genome analysis revealed the existence of 29 putative glycoside hydrolases in addition to the previously identified cellulases and xylanases. The glycosyl hydrolases include enzymes predicted to exhibit mainly dextran/starch- and xylan-degrading functions. T. fusca possesses two protein secretion systems: the sec general secretion system and the twin-arginine translocation system. Several of the secreted cellulases have sequence signatures indicating their secretion may be mediated by the twin-arginine translocation system. T. fusca has extensive transport systems for import of carbohydrates coupled to transcriptional regulators controlling the expression of the transporters and glycosylhydrolases. In addition to providing an overview of the physiology of a soil actinomycete, this study presents insights on the transcriptional regulation and secretion of cellulases which may facilitate the industrial exploitation of these systems.

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His(6) tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L(-1) could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.