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

Exploring mild enzymatic sustainable routes for the synthesis of bio-degradable aromatic-aliphatic oligoesters

The application of Candida antarctica lipase B in enzyme-catalyzed synthesis of aromatic-aliphatic oligoesters is here reported. The aim of the present study is to systematically investigate the most favorable conditions for the enzyme catalyzed synthesis of aromatic-aliphatic oligomers using commercially available monomers. Reaction conditions and enzyme selectivity for polymerization of various commercially available monomers were considered using different inactivated/activated aromatic monomers combined with linear polyols ranging from C2 to C12. The effect of various reaction solvents in enzymatic polymerization was assessed and toluene allowed to achieve the highest conversions for the reaction of dimethyl isophthalate with 1,4-butanediol and with 1,10-decanediol (88 and 87% monomer conversion respectively). Mw as high as 1512 Da was obtained from the reaction of dimethyl isophthalate with ,10-decanediol. The obtained oligomers have potential applications as raw materials in personal and home care formulations, for the production of aliphatic-aromatic block co-polymers or can be further functionalized with various moieties for a subsequent photo- or radical polymerization

Synergistic chemo-enzymatic hydrolysis of poly(ethylene terephthalate) from textile waste

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 in order 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 (1,632 cm-1) of TA into the deprotonated state (1,604 and 1,398 cm-1) was observed and bands at 1,728 and 1,398 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 by using the Humicola insolens cutinase (HiC) yielding 97% pure TA, therefore comparable with the commercial synthesis grade TA (98%)

Synergistic chemo-enzymatic hydrolysis of poly(ethylene terephthalate) from textile waste

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 in order 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 (1,632 cm-1) of TA into the deprotonated state (1,604 and 1,398 cm-1) was observed and bands at 1,728 and 1,398 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 by using the Humicola insolens cutinase (HiC) yielding 97% pure TA, therefore comparable with the commercial synthesis grade TA (98%)

Fully renewable polyesters via polycondensation catalyzed by Thermobifida cellulosilytica cutinase 1: an integrated approach

The present study addresses comprehensively the problem of producing polyesters through sustainable processes while using fully renewable raw materials and biocatalysts. Polycondensation of bio-based dimethyl adipate with different diols was catalyzed by cutinase 1 from Thermobifida cellulosilytica (Thc_cut1) under solvent free and thin-film conditions. The biocatalyst was immobilized efficiently on a fully renewable cheap carrier based on milled rice husk. A multivariate factorial design demonstrated that Thc_cut1 is less sensitive to the presence of water in the system and it works efficiently under milder conditions (50 \ub0C; 535 mbar) when compared to lipase B from Candida antarctica (CaLB), thus enabling energy savings. Experimental and computational investigations of cutinase 1 from Thermobifida cellulosilytica (Thc_cut1) disclosed structural and functional features that make this serine-hydrolase efficient in polycondensation reactions. Bioinformatic analysis performed with the BioGPS tool pointed out functional similarities with CaLB and provided guidelines for future engineering studies aiming, for instance, at introducing different promiscuous activities in the Thc_cut1 scaffold. The results set robust premises for a full exploitation of enzymes in environmentally and economically sustainable enzymatic polycondensation reactions

Enzymatic surface hydrolysis of PET : effect of structural diversity on kinetic properties of cutinases from thermobifida

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 μM while on pNP–butyrate they showed kcat and Km values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. 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.This study was performed within the Austrian Centre of Industrial Biotechnology ACIB, the MacroFun project and COST Action 868. This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. Financial support was also given from Sachsisches Staatsministerium fur Umwelt und Landwirtschaft, Germany. PET was kindly provided by Dr. Vincent Nierstrasz from Ghent University

Enzymatic Functionalization of HMLS-Polyethylene Terephthalate Fabrics Improves the Adhesion to Rubber

Among synthetic thermoplastic fiber materials for reinforcement, high modulus and low shrinkage poly(ethylene terephthalate) (HMLS-PET) became the major carcass material for the low- to medium-end tire segment. Usually cords are coated with a resorcinol-formaldehyde-latex (RFL) dip to achieve acceptable power transmission. However, the low concentration of polar groups on the PET's surface requires an additional activation with costly and potentially toxic chemicals to create additional nucleophilic groups prior to RFL dipping. Here, a green enzyme based alternative to chemical HMLS-PET activation was investigated. Four different cutinase variants from Thermobifida cellulosilytica were shown to hydrolyze HMLS-PET cords, creating new carboxylic and hydroxyl groups with distinct exoendo-wise selectivity. The highest degree of enzymatic functionalization reached a concentration of 0.51 nmol mm-2of COOH with a release of 1.35 mM of soluble products after 72 h. The chemical treatment with 1 M NaOH released more soluble products leading up to a 10% decrease of the tensile strength while the functionalization degree achieved was only 0.21 nmol mm-2. This clearly indicates a more endowise mode of hydrolysis for the enzymatic treatment when compared to chemical hydrolysis. Scanning electron microscopy of the fibers confirmed the aggressiveness of the chemical treatment, whereas the enzymatic approach only led to 0.7% solubilization of the polymer with no loss of mechanical properties and crystallinity changes. The newly created groups were chemically accessible and reactive in the dipping step and led after the vulcanization to a significant improvement of the adhesion between the polymer and a representative carcass rubber compound according to the peel tests