Alessandro Pellis

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.

Biocatalyzed approach for the surface functionalization of poly(L-lactic acid) films using hydrolytic enzymes.

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, 14C‐radiochemical analysis and X‐ray photoelectron spectroscopy (XPS) using 14C‐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 14C‐radiochemical analyses are consistent with the XPS data indicating the potential of enzymatic functionalization in different reaction conditions.

Enzymatic hydrolysis of poly(ethylene furanoate)

The urgency of producing new environmentally-friendly polyesters strongly enhanced the development of bio-based poly(ethylene furanoate) (PEF) as an alternative to plastics like poly(ethylene terephthalate) (PET) for applications that include food packaging, personal and home care containers and thermoforming equipment. In this study, PEF powders of various molecular weights (6, 10 and 40kDa) were synthetized and their susceptibility to enzymatic hydrolysis was investigated for the first time. According to LC/TOF-MS analysis, cutinase 1 from Thermobifida cellulosilytica liberated both 2,5-furandicarboxylic acid and oligomers of up to DP4. The enzyme preferentially hydrolyzed PEF with higher molecular weights but was active on all tested substrates. Mild enzymatic hydrolysis of PEF has a potential both for surface functionalization and monomers recycling.

Ultrasound-enhanced enzymatic hydrolysis of poly(ethylene terephthalate)

The application of ultrasound was found to enhance enzymatic hydrolysis of poly(ethylene terephthalate) (PET). After a short activation phase up to 6.6times increase in the amount of released products was found. PET powder with lower crystallinity of 8% was hydrolyzed faster when compared to PET with 28% crystallinity. Ultrasound activation was found to be around three times more effective on powders vs. films most likely due to a larger surface area accessible to the enzyme.

Enlarging the tools for efficient enzymatic polycondensation: structural and catalytic features of cutinase 1 from Thermobifida cellulosilytica

Cutinase 1 from Thermobifida cellulosilytica is reported for the first time as an efficient biocatalyst in polycondensation reactions. Under thin film conditions the covalently immobilized enzyme catalyzes the synthesis of oligoesters of dimetil adipate with different polyols leading to higher Mw (~1900) and Mn (~1000) if compared to lipase B from Candida antarctica or cutinase from Humicola insolens. Computational analysis discloses the structural features that make this enzyme readily accessible to substrates and optimally suited for covalent immobilization. As lipases and other cutinase enzymes, it presents hydrophobic superficial regions around the active site. However, molecular dynamics simulations indicate the absence of interfacial activation, similarly to what already documented for lipase B from Candida antarctica. Notably, cutinase from Humicola insolens displays a “breathing like” conformational movement, which modifies the accessibility of the active site. These observations stimulate wider experimental and bioinformatics studies aiming at a systematic comparison of functional differences between cutinases and lipases.

Evolving biocatalysis to meet bioeconomy challenges and opportunities

The unique selectivity of enzymes, along with their remarkable catalytic activity, constitute powerful tools for transforming renewable feedstock and also for adding value to an array of building blocks and monomers produced by the emerging bio-based chemistry sector. Although some relevant biotransformations run at the ton scale demonstrate the success of biocatalysis in industry, there is still a huge untapped potential of catalytic activities available for targeted valorization of new raw materials, such as waste streams and CO2. For decades, the needs of the pharmaceutical and fine chemistry sectors have driven scientific research in the field of biocatalysis. Nowadays, such consolidated advances have the potential to translate into effective innovation for the benefit of bio-based chemistry. However, the new scenario of bioeconomy requires a stringent integration between scientific advances and economics, and environmental as well as technological constraints. Computational methods and tools for effective big-data analysis are expected to boost the use of enzymes for the transformation of a new array of renewable feedstock and, ultimately, to enlarge the scope of biocatalysis.

Superhydrophobic functionalization of cutinase activated poly(lactic acid) surfaces

Superhydrophobic materials have focused the interest of many researchers due to their potential in a wide spectrum of applications like microfluidics or biosensors in the biomedical field. Typically, the increased surface roughness at the micro or nano scale needed for superhydrophobic surfaces is achieved by coating of different substances, which in combination with a lower surface energy lead to Water Contact Angle (WCA) values greater than 150°. Here, limited enzymatic surface hydrolyis poly(lactic acid) (PLA) was combined with spin coating of a steraic alkene ketene dimer (AKD) layer. The selective enzymatic hydrolysis creates, in a gentle and controlled way, new hydroxylic and carboxylic groups on the polymer surface without damaging the material bulk properties like alkaline treatment does. The creation of new hydrophilic surface groups lead to a significant increase in the hydrophilicity, decreasing the WCA to less than 30° while raising the roughness from an Rrms of 50.5 to 90.8 nm concomittantly increasing the exposed surface vs. the projected one by 13.2%. Coupling of PLA hydroxy groups with AKD was demonstrated by using a PLA model substrate and subsequent identification of the reaction product via LC-TOF/MS. On the PLA film, FTIR based detection of the characteristic β-ketoester bond peak between the AKD and enzymatically generated hydroxy groups on the surface confirmed successful coupling. Scanning Electron Microscopy (SEM) & Atomic Force Microscopy (AFM) imaging confirmed the presence of fractal structures after curation of the enzymatically activated PLA film. The suitable size, 4.15 μm on the lateral dimension and 0.7 μm on height of the structures, together with the high density of these fractal structures lead to a superhydrophobic surface (WCA >150°). This process represents an alternative to produce chemically inert superhydrophobic bio-based polyesters surfaces, by combining mild biocatalytic activation of a polyester film with non-toxic chemicals in an environmentally friendly manner.

Polymerization of Various Lignins via Immobilized Myceliophthora thermophila Laccase (MtL)

Enzymatic polymerization of lignin is an environmentally-friendly and sustainable method that is investigated for its potential in opening-up new applications of one of the most abundant biopolymers on our planet. In this work, the laccase from Myceliophthora thermophila was successfully immobilized onto Accurel MP1000 beads (67% of protein bound to the polymeric carrier) and the biocatalyzed oxidation of Kraft lignin (KL) and lignosulfonate (LS) were carried out. Fluorescence intensity determination, phenol content analysis and size exclusion chromatography were performed in order to elucidate the extent of the polymerization reaction. The collected results show an 8.5-fold decrease of the LS samples’ fluorescence intensity after laccase-mediated oxidation and a 12-fold increase of the weight average molecular weight was obtained.

Enzymatic surface hydrolysis of poly(ethylene furanoate) thin films of various crystallinities

This work reports on the successful production of poly(ethylene furanoate) (PEF) thin films and a comparison of the enzymatic hydrolysis of PEF and poly(ethylene terephthalate) (PET) films with three different crystallinities (0, 10 and 20%). The data suggest that the PEF films are enzymatically hydrolyzed 1.7 times faster than the commonly investigated PET films. QCM-D and SEM/AFM analyses fully confirm the observed reaction trend. The results also show a negative dependence of the hydrolysis rates with the increasing of the film crystallinity.

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 °C; 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.