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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. © 2017 Wiley Periodicals, Inc.

Summary Due to the rising global environment protection awareness, recycling strategies that comply with the circular economy principles are needed. Polyesters are among the most used materials in the textile industry; therefore, achieving a complete poly(ethylene terephthalate) (PET) hydrolysis in an environmentally friendly way is a current challenge. In this work, a chemo-enzymatic treatment was developed to recover the PET building blocks, namely terephthalic acid (TA) and ethylene glycol. To monitor the monomer and oligomer content in solid samples, a Fourier-transformed Raman method was successfully developed. A shift of the free carboxylic groups (1632 cm−1) of TA into the deprotonated state (1604 and 1398 cm−1) was observed and bands at 1728 and 1398 cm−1 were used to assess purity of TA after the chemo-enzymatic PET hydrolysis. The chemical treatment, performed under neutral conditions (T = 250 °C, P = 40 bar), led to conversion of PET into 85% TA and small oligomers. The latter were hydrolysed in a second step using the Humicola insolens cutinase (HiC) yielding 97% pure TA, therefore comparable with the commercial synthesis-grade TA (98%).

Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty- and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

Rayon filaments composed of regenerated cellulose are used as reinforcement materials in tires and to a lower extent in the clothing industry as personal protective equipment e.g. flame retardant cellulosic based materials. After use, these materials are currently transferred to landfills while chemical degradation does not allow the recovery of the cellulose (as glucose) nor the separation of the high valuable flame-retardant pigment. In this study, rayon fibers were enzymatically hydrolyzed to allow recovery of glucose and valuable additives. The glucose was successfully used as carbon source for the production of high value compounds such as itaconic acid, lactic acid and chitosan. 14.2 g/L of itaconic acid, 36.5 g/L of lactic acid and 39.2 g/L of chitosan containing biomass were produced from Escherichia coli, Lactobacillus paracasei and Aspergillus niger, respectively, comparable to yields obtained when using commercial glucose as carbon source.

Poly(lactic acid) as a biodegradable thermoplastic polyester has received increasing attention. This renewable polyester has found applications in a wide range of products such as food packaging, textiles and biomedical devices. Its major drawbacks are poor toughness, slow degradation rate and lack of reactive side-chain groups. An enzymatic process for the grafting of carboxylic acids onto the surface of poly(L-lactic acid) (PLLA) films was developed using Candida antarctica lipase B as a catalyst. Enzymatic hydrolysis of the PLLA film using Humicola insolens cutinase in order to increase the number of hydroxyl and carboxylic groups on the outer polymer chains for grafting was also assessed and showed a change of water contact angle from 74.6 to 33.1° while the roughness and waviness were an order of magnitude higher in comparison to the blank. Surface functionalization was demonstrated using two different techniques, (14) C-radiochemical analysis and X-ray photoelectron spectroscopy (XPS) using (14) C-butyric acid sodium salt and 4,4,4-trifluorobutyric acid as model molecules, respectively. XPS analysis showed that 4,4,4-trifluorobutyric acid was enzymatically coupled based on an increase of the fluor content from 0.19 to 0.40%. The presented (14) C-radiochemical analyses are consistent with the XPS data indicating the potential of enzymatic functionalization in different reaction conditions.

Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased k cat values on soluble substrates ( p -nitrophenyl acetate and p -nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.

The potential of limited enzymatic poly(ethylene terephthalate) (PET) surface hydrolysis for the modification of track-etched (TE) membranes was investigated. Cutinases 1 and 2 from Thermobifida cellulosilytica as well as a fusion protein of cutinase 1 with the polymer binding module from the polyhydroxyalkanoate depolymerase of Alcaligenes faecalis (Thc_Cut1_PBM) were shown to hydrolyse highly crystalline PET TE membranes with a pore diameter of ∼120nm at very narrow size distribution. Furthermore the effects of surface chemistry were investigated by comparison of enzymatic hydrolysis by Thc_Cut1_PBM of "as received" PET TE membranes with two surface functionalized versions towards a "hydrophilic" and a more "hydrophobic" surface. The effects of adsorbed protein and the efficacy of cleaning steps after enzymatic treatment were elucidated by complementary methods for surface analysis and membrane characterization. With the optimized cleaning protocol, all adsorbed protein could be removed from the enzyme-treated membranes and effects of chemical surface functionalization of the PET TE membranes were demonstrated. The highest efficiency of enzymatic surface hydrolysis was observed for the original PET TE membranes, leading to an 0.36% weight loss corresponding to a removal of ∼3nm PET from the entire surface of the porous membrane. This correlates very well with the measured increase of barrier pore diameter by 4nm (a radius reduction? of 2nm), leading to about a two-fold increased water permeability.

Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma . HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S -transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens , with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET.

Modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 revealed that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site could be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate). To investigate this hypothesis in more detail, selected amino acids of surface regions outside the active site of Thc_Cut2, which hydrolyzes PET much less efficiently than Thc_Cut1 were exchanged by site-directed mutagenesis. The mutants were expressed in E. coli BL21-Gold(DE3), purified and characterized regarding their specific activities and kinetic parameters on soluble substrates and their ability to hydrolyze PET and the PET model substrate bis(benzoyloxyethyl) terephthalate (3PET). Compared to Thc_Cut2, mutants carrying Arg29Asn and/or Ala30Val exchanges showed considerable higher specific activity and higher kcat /KM values on soluble substrates. Exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for 3PET and PET. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on PET films. These findings clearly demonstrate that surface properties (i.e., amino acids located outside the active site on the protein surface) play an important role in PET hydrolysis.

Phenol red is a cytocompatible pH sensing dye that is commonly added to cell culture media, but removed from some media formulations due to its structural mimicry of estrogen. Phenol red free media is also used during live cell imaging, to avoid absorbance and fluorescence quenching of fluorophores. To overcome these complications, we developed cytocompatible and degradable phenol red-silk tyrosine cross-linked hydrogels using horseradish peroxidase (HRP) enzyme and hydrogen peroxide (H2O2). Phenol red added to silk during tyrosine crosslinking accelerated di-tyrosine formation in a concentration-dependent reaction. Phenol red diffusion studies and UV–Vis spectra of phenol red-silk tyrosine hydrogels at different pHs showed altered absorption bands, confirming entrapment of dye within the hydrogel network. LC–MS of HRP-reacted phenol red and N-acetyl- l -tyrosine reaction products confirmed covalent bonds between the phenolic hydroxyl group of phenol red and tyrosine on the silk. At lower phenol red concentrations, leak-proof hydrogels which did not release phenol red were fabricated and found to be cytocompatible based on live-dead staining and alamar blue assessments of encapsulated fibroblasts. Due to the spectral overlap between phenol red absorbance at 415 nm and di-tyrosine fluorescence at 417 nm, phenol red-silk hydrogels provide both absorbance and fluorescence-based pH sensing. With an average pKa of 6.8 and good cytocompatibiltiy, phenol red-silk hydrogels are useful for pH sensing in phenol red free systems, cellular microenvironments and bioreactors. Statement of Significance Phenol red entrapped within hydrogels facilitates pH sensing in phenol red free environments. Leak-proof phenol red based pH sensors require covalent binding techniques, but are complicated due to the lack of amino or carboxyl groups on phenol red. Currently, there is no simple, reliable technique to covalently link phenol red to hydrogel matrices, for real-time pH sensing in cell culture environments. Herein, we take advantage of phenolic groups for covalent linkage of phenol red to silk tyrosine in the presence of HRP and H2O2. The novelty of the current system stems from its simplicity and the use of silk protein to create a cytocompatible, degradable sensor capable of real-time pH sensing in cell culture microenvironments.

The polymer industry is under pressure to mitigate the environmental cost of petrol-based plastics. Biotechnologies contribute to the gradual replacement of petrol-based chemistry and the development of new renewable products, leading to the closure of carbon circle. An array of bio-based building blocks is already available on an industrial scale and is boosting the development of new generations of sustainable and functionally competitive polymers, such as polylactic acid (PLA). Biocatalysts add higher value to bio-based polymers by catalyzing not only their selective modification, but also their synthesis under mild and controlled conditions. The ultimate aim is the introduction of chemical functionalities on the surface of the polymer while retaining its bulk properties, thus enlarging the spectrum of advanced applications.

A new cutinase from Thermobifida alba (Tha_Cut1) was cloned and characterized for polyethylene terephthalate (PET) hydrolysis. Tha_Cut1 showed a high degree of identity to a T. cellulolysitica cutinase with only four amino acid differences outside the active site area, according to modeling data. Yet, Tha_Cut1 was more active in terms of PET surface hydrolysis leading to considerable improvement in hydrophilicity quantified based on a decrease of the water contact angle from 87.7° to 45.0°. The introduction of new carboxyl groups was confirmed and measured after esterification with the fluorescent reagent alkyl bromide, 2-(bromomethyl) naphthalene (BrNP), resulting in a fluorescence emission intensity increase from 980 to 1420 a.u. On the soluble model substrates p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB), the cutinase showed Km values of 213 and 1933 μM and kcat values of 2.72 and 6.03 s−1 respectively. The substrate specificity was investigated with bis(benzoyloxyethyl)terephthalate ...

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p-nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET-hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2-hydroxyethyl benzoate (HEB), and mono-(2-hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET-hydrolase was isolated from non-denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC-MS/MS analysis. BsEstB was expressed in Escherichia coli with C-terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p-nitrophenyl acetate and 108 U/mg on p-nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half-life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2-hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The kcat values decreased with increasing complexity of the substrate from 6 and 8 (s-1) for p-nitrophenyl-acetate (4NPA) and p-nitrophenyl-butyrate (4NPB), respectively, to 0.14 (s-1) for bis(2-hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water-contact angle (WCA) from 68.2°±1.7° to 62.6°±1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2-(bromomethyl)naphthalene.

For many years, lipase B from Candida antarctica (CaLB) was the primary biocatalyst used for enzymatic esterification and polycondensation reactions. More recently, the need for novel biocatalysts with different selectivity has arisen in the biotechnology and biocatalysis fields. The present work describes how the catalytic potential of Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) was exploited for polyester synthesis. in a first step, Thc_Cut1 was immobilized on three different carriers, namely Opal, Coral and Amber, using a novel non-toxic His-tag method based on chelated Fe(iii) ions (>99% protein bounded). in a second step, the biocatalyzed synthesis of an array of aliphatic polyesters was conducted. A selectivity chain study in a solvent-free reaction environment showed how, in contrast to CaLB, Thc_Cut1 presents a certain preference for C6-C4 ester-diol combinations reaching monomer conversions up to 78% and Mw of 878 g mol-1 when the Amber immobilized Thc_Cut1 was used. The synthetic potential of this cutinase was also tested in organic solvents, showing a marked activity decrease in polar media like that observed for CaLB. Finally, recyclability studies were performed, which showed an excellent stability of the immobilized Thc_Cut1 (retained activity >94%) over 24 hour reaction cycles when a solvent-free workup was used. Concerning a practical application of the biocatalyst's preparation, the production of oligomers with Mn values below 10 kDa is usually desired for the production of nanoparticles and for the synthesis of functional pre-polymers for coating applications that can be crosslinked in a second reaction step.

Modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 revealed that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site could be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate). To investigate this hypothesis in more detail, selected amino acids of surface regions outside the active site of Thc_Cut2, which hydrolyzes PET much less efficiently than Thc_Cut1 were exchanged by site-directed mutagenesis. The mutants were expressed in E. coli BL21-Gold(DE3), purified and characterized regarding their specific activities and kinetic parameters on soluble substrates and their ability to hydrolyze PET and the PET model substrate bis(benzoyloxyethyl) terephthalate (3PET). Compared to Thc_Cut2, mutants carrying Arg29Asn and/or Ala30Val exchanges showed considerable higher specific activity and higher kcat /KM values on soluble substrates. Exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for 3PET and PET. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on PET films. These findings clearly demonstrate that surface properties (i.e., amino acids located outside the active site on the protein surface) play an important role in PET hydrolysis.