Less is more: Hydrolysis of polyesters is enhanced by a truncated esterase

Polymers frequently used for one-way applications like packaging are preferably biodegradable, albeit non-biodegradable polyesters are mostly used. Biodegradability of the aliphatic/aromatic copolyester poly(butylene adipate-co-terephthalate) (PBAT) has been investigated, showing biological decomposability under composting conditions. However, little is known about its anaerobic hydrolysis while large amounts of food packaging ends up in biogas plants. The enzyme EstA from Clostridium botulinum (Cbotu_EstA) actively hydrolyzed PBAT while it failed to act on polyethylene terephthalate (PET). Yet, enzymes would allow mild decomposition of widely used PET enabling recycling of the monomeric building blocks. The enhancement of the hydrolase activity with regard to polyester hydrolysis can be achieved by fusion of hydrophobic domains, improving the biocatalyst adsorption on the hydrophobic polymer surface, or by substitution of specific residues, enlarging the active site of the enzyme. The deletion of the Cbotu_EstA N-terminal domain can satisfactory combine both approaches. Surface engineering successfully produced a highly active Cbotu_EstA variant which was able to hydrolyze PET. Truncation of the N-terminal domain of Cbotu_EstA improved the adsorption of the enzyme on hydrophobic polyester surfaces and enhanced their hydrolysis eight times more compared to the wild-type enzyme, based on released monomers quantification. Please click Additional Files below to see the full abstract

The Closure of the Cycle: Enzymatic Synthesis and Functionalization of Bio-Based Polyesters

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

Synthetic enzymes for synthetic substrates

In recent years, hydrolases like cutinases, esterases and lipases have been recognized as powerful tools for hydrolysis of synthetic polymers such as polyethylene terephthalate (PET) as an environmentally friendly alternative for environmentally harmful chemical recycling methods1. PET is currently the most common type of aromatic polyester, with widespread application as packaging material, beverage bottles, and synthetic textile fibers. So far, cutinases have been the most active enzyme class regarding PET degradation. In nature, cutinases catalyze the hydrolysis of the aliphatic biopolyester cutin, the structural component of plant cuticle. Although cutinases are able to act on natural insoluble polyesters, their activities on non-natural substrates are quit low. For this reason, different engineering strategies were established to optimize “polyesterases” for synthetic polymers (Fig.1). Thereby, development of rationale enzyme-engineering strategies led to remarkable enhancement of hydrolytic activities on polyesters and clearly showed that the affinity between the enzyme and the substrate plays a key role in the enzymatic hydrolysis of synthetic polyester. Please click Additional Files below to see the full abstract

Effect of Binding Modules Fused to Cutinase on the Enzymatic Synthesis of Polyesters

open9In relation to the development of environmentally-friendly processing technologies for the continuously growing market of plastics, enzymes play an important role as green and sustainable biocatalysts. The present study reports the use of heterogeneous immobilized biocatalysts in solvent-free systems for the synthesis of aliphatic oligoesters with Mws and monomer conversions up to 1500 Da and 74%, respectively. To improve the accessibility of hydrophilic and hydrophobic substrates to the surface of the biocatalyst and improve the reaction kinetic and the chain elongation, two different binding modules were fused on the surface of cutinase 1 from Thermobifida cellulosilytica. The fusion enzymes were successfully immobilized (>99% of bound protein) via covalent bonding onto epoxy-activated beads. To the best of our knowledge, this is the first example where fused enzymes are used to catalyze transesterification reactions for polymer synthesis purposes.openFerrario, Valerio; Todea, Anamaria; Wolansky, Lisa; Piovesan, Nicola; Guarneri, Alice; Ribitsch, Doris; Guebitz, Georg M.; Gardossi, Lucia; Pellis, AlessandroFerrario, Valerio; Todea, Anamaria; Wolansky, Lisa; Piovesan, Nicola; Guarneri, Alice; Ribitsch, Doris; Guebitz, Georg M.; Gardossi, Lucia; Pellis, Alessandr

Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica

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 poly(ethylene terephthalate) (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\u27s 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

A fungal ascorbate oxidase with unexpected laccase activity

Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. Af_AO1 had a 10-fold higher affinity to ascorbic acid (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activityPeer ReviewedPostprint (published version

Characterization of a new alpha/beta hydrolase from Pelosinus fermentans for polyesters hydrolysis

A new alpha/beta hydrolase gene from Pelosinus fermentans (Pfe_Lip) was optimized against Escherichia coli codon usage and cloned into the pET26b(+) vector, containing a 6xHis-Tag downstream the polylinker, and transformed into the bacterial cells. The Pfe_Lip is a member of the serine hydrolases containing a modification in the common –GxSxG– motif in the first glycine to have the –AxSxG– motif. This enzyme can be grouped in the I.5 subfamily (Nardini M, 1999). To improve the expression of this enzyme in the soluble fraction, a lactose-containing medium was used for the expression of the enzyme. Western blotting was carried out for revelation of the enzyme with an antibody against the His-Tag and further purification was carried out by means of Immobilized Ion Metal Affinity Chromatography (IMAC) with Nickel-coated columns for the binding of the His-Tagged enzyme. The Pfe_Lip purified was tested in hydrolase activity assay with p-nitrophenylbutyrate (p-NPB) (Herrero Acero E, 2013). Pfe_Lip had the pH optimal at 8 (0.1 M sodium phosphate buffer). Analysis of homology shows high identity with enzymes containing a zinc ion in the structure and a lid covering the active site, containing serine, aspartic acid and histidine. The enzyme was able to hydrolyse polyesters like Ecoflex based on HPLC quantification of the solubilized hydrolysis products

Truncation of an esterase enhances the hydrolysis on polyesters: Less is more

Polymers, and especially polyesters, frequently used for one-way applications like packaging are preferably biodegradable, albeit non-biodegradable polyesters are mostly used, namely polyethylene terephthalate (PET). Biodegradability of the aliphatic/aromatic copolyester poly(butylene adipate-co- terephthalate) (PBAT) has been investigated, showing biological decomposability under composting conditions. However, little is known about its anaerobic hydrolysis, while large amounts of food packaging end up in biogas plants. Two different enzymes belonging to the carboxylesterase superfamily from Clostridium botulinum (Cbotu_EstA) and from Pelosinus fermentans, were reported to actively hydrolyze PBAT, while they failed to act on PET. Yet, enzymes would allow mild decomposition of widely used PET enabling recycling of the monomeric building blocks. The enhancement of the hydrolase activity with regard to polyester hydrolysis can be achieved by fusion of hydrophobic domains, improving the biocatalyst adsorption on the hydrophobic polymer surface, or by substitution of specific residues, enlarging the active site of the enzyme. Interestingly, analysis of the 3D structure of Cbotu_EstA revealed the presence of an extra domain at the N-terminus of the enzyme which covered the lid structure and a hydrophobic patch. The deletion of the Cbotu_EstA N-terminal domain can satisfactory combine the enlarging of the active site and the presence of hydrophobic domain on the surface of the enzyme for improved sorption properties. Surface engineering successfully produced a highly active Cbotu_EstA variant (del71Cbotu_EstA) which was able to hydrolyze PET. Truncation of the N-terminal domain of Cbotu_EstA improved the adsorption of the enzyme on hydrophobic polyester surfaces and enhanced their hydrolysis eight times more compared to the wild-type enzyme, based on released monomers quantification