Chemo-Enzymatic Derivatization of Glycerol-Based Oligomers: Structural Elucidation and Potential Applications.

Switching from oil-based to bio-based feedstocks to ensure the green transition to a sustainable and circular future is one of the most pressing challenges faced by many industries worldwide. For the cosmetics and personal and house care industries there is a strong drive to accelerate this transition from the customers that starts favoring the purchase of naturally derived and bio-degradable products over the traditionally available products. In this work we developed a series of fully biobased macromolecules constituted of a glycerol-based oligoester backbone. Based on the subsequent derivatization with fatty acids or peptides, the resulting products may find application as emulsifiers, wetting agents, and potential vectors for the delivery of bioactive peptides. All steps of the resulting macromolecules were conducted following the green chemistry principles with no toxic or environmentally damaging compounds that were used in the overall production process.

Biocatalyzed Synthesis of Flavor Esters and Polyesters: A Design of Experiments (DoE) Approach.

In the present work, different hydrolases were adsorbed onto polypropylene beads to investigate their activity both in short-esters and polyesters synthesis. The software MODDE® Pro 13 (Sartorius) was used to develop a full-factorial design of experiments (DoE) to analyse the thermostability and selectivity of the immobilized enzyme towards alcohols and acids with different chain lengths in short-esters synthesis reactions. The temperature optima of Candida antarctica lipase B (CaLB), Humicola insolens cutinase (HiC), and Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) were 85 °C, 70 °C, and 50 °C. CaLB and HiC preferred long-chain alcohols and acids as substrate in contrast to Thc_Cut1, which was more active on short-chain monomers. Polymerization of different esters as building blocks was carried out to confirm the applicability of the obtained model on larger macromolecules. The selectivity of both CaLB and HiC was investigated and best results were obtained for dimethyl sebacate (DMSe), leading to polyesters with a Mw of 18 kDa and 6 kDa. For the polymerization of dimethyl adipate (DMA) with BDO and ODO, higher molecular masses were obtained when using CaLB onto polypropylene beads (CaLB_PP) as compared with CaLB immobilized on macroporous acrylic resin beads (i.e., Novozym 435). Namely, for BDO the Mn were 7500 and 4300 Da and for ODO 8100 and 5000 Da for CaLB_PP and for the commercial enzymes, respectively. Thc_Cut1 led to polymers with lower molecular masses, with Mn < 1 kDa. This enzyme showed a temperature optimum of 50 °C with 63% of DMA and BDO when compared to 54% and 27%, at 70 °C and at 85 °C, respectively.

Sustainable Galactarate-Based Polymers: Multi-Enzymatic Production of Pectin-Derived Polyesters.

Large amounts of agricultural wastes are rich in pectins that, in many cases, disrupt the processing of food residues due to gelation. Despite pectins being a promising sustainable feedstock for bio-based chemical production, the current pathways to produce platform molecules from this polysaccharide are hazardous and entail the use of strong acids. The present work describes a sequence of biocatalyzed reactions that involves 1) the extraction of pectin from sugar beet pulp and enzymatic recovery of galacturonic acid (GalA), followed by 2) the enzymatic oxidation of the GalA aldehyde and the recovery of galactaric acid (GA), and 3) the biocatalyzed polycondensation of GA to obtain fully bio-based polyesters carrying lateral hydroxy functionalities. The acid-free pectin extraction is optimized using enzymes and microwave technology. The conditions for enzymatic oxidation of GalA allow the separation of the GA produced by a simple centrifugation step that leads to the enzyme-catalyzed polycondensation reactions.

Laminaria digitata and Palmaria palmata Seaweeds as Natural Source of Catalysts for the Cycloaddition of CO2 to Epoxides.

Seaweed powder has been found to act as an effective catalyst for the fixation of CO2 into epoxides to generate cyclic carbonates under solvent free conditions. Model background reactions were performed using metal halides and amino acids typically found in common seaweeds which showed potassium iodide (KI) to be the most active. The efficacy of the seaweed catalysts kelp (Laminaria digitata) and dulse (Palmaria palmata) was probed based on particle size, showing that kelp possessed greater catalytic ability, achieving a maximum conversion and selectivity of 63.7% to styrene carbonate using a kelp loading of 80% by weight with respect to epoxide, 40 bar of CO2, 120 °C for 3 h. Maximizing selectivity was difficult due to the generation of diol side product from residual H2O found in kelp, along with a chlorinated by-product thought to form due to a high quantity of chloride salts in the seaweeds. Data showed there was loss of organic matter upon use of the kelp catalyst, likely due to the breakdown of organic compounds and their subsequent removal during product extraction. This was highlighted as the likely cause of loss of catalytic activity upon reuse of the Kelp catalyst.

Small cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilytica.

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.

On the Effect of Microwave Energy on Lipase-Catalyzed Polycondensation Reactions.

Microwave energy (MWe) is, nowadays, widely used as a clean synthesis tool to improve several chemical reactions, such as drug molecule synthesis, carbohydrate conversion and biomass pyrolysis. On the other hand, its exploitation in enzymatic reactions has only been fleetingly investigated and, hence, further study of MWe is required to reach a precise understanding of its potential in this field. Starting from the authors' experience in clean synthesis and biocatalyzed reactions, this study sheds light on the possibility of using MWe for enhancing enzyme-catalyzed polycondensation reactions and pre-polymer formation. Several systems and set ups were investigated involving bulk and organic media (solution phase) reactions, different enzymatic preparations and various starting bio-based monomers. Results show that MWe enables the biocatalyzed synthesis of polyesters and pre-polymers in a similar way to that reported using conventional heating with an oil bath, but in a few cases, notably bulk phase polycondensations under intense microwave irradiation, MWe leads to a rapid enzyme deactivation.

Controlled Enzymatic Synthesis of Polyesters Based on a Cellulose-Derived Triol Monomer: A Design of Experiment Approach.

Regioselective enzymatic polycondensation of the bio-based cellulose derived polyol, Triol-citro, and dimethyl adipate using Candida antarctica Lipase B (CaLB) was investigated. A Design of Experiment approach with MODDE® Pro 13 was used to determine important factors in the branching behavior of this polymer, and reactant ratio, temperature, reaction time and enzyme wt % were the studied factors. Multifunctional polyesters with pendant hydroxy groups were synthesized and fully characterized using 2D NMR techniques to determine degree of branching. Branching was minimal, with a maximum of 16 % observed, and monomer ratio, temperature and reaction time were all determined to be significant factors. In this work, Mn of up to 13 kDa were achieved, while maintaining degree of branching below 15 %, resulting in a linear polyester with the potential to be further functionalized.

Biodegradable and gas barrier polylactic acid/star-shaped polycaprolactone blend films functionalized with a bio-sourced polyelectrolyte coating.

This work aims at improving and disclosing new properties of films based on polylactic acid (PLA) and a star-shaped polycaprolactone (PCL). Indeed, previous works demonstrated that the presence of ad-hoc synthesized PCL, characterized by low molecular weight and carboxyl end groups (coded as PCL-COOH), improves the elongation at break of the films compared to that of neat PLA and increases their functionality. To further improve the properties of the system, alternating layers of chitosan (CH) and DNA were deposited on the surface applying a Layer-by-Layer (LbL) technique. This method was chosen because it allows the properties of the system to be modified without affecting the specific features of the bulk. In addition, the LbL technique is easily scalable and environmentally friendly because it is based on the use of an aqueous solution of two biomaterials, namely DNA and CH, which are not only derived from renewable sources but are also biocompatible and biodegradable. IR measurements on model silicon substrates subjected to the same treatment as the films, pointed out a linear growth of the proposed LbL assembly. Indeed, FE-SEM measurements highlighted the deposition of a uniform coating. The presence of the CH/DNA assembly reduced the oxygen permeability under both dry and humid (50% R.H.) conditions when compared to the uncoated film. In addition, the coating had no relevant effect on the hydrolytic and enzymatic degradation of the system, so that the biodegradability of the film was maintained.

Efficient Depolymerization of Poly(ethylene 2,5-furanoate) Using Polyester Hydrolases.

Poly(ethylene 2,5-furanoate) (PEF) is considered to be the next-generation green polyester and is hailed as a rising star among novel plastics. It is biobased, is nontoxic, and has comparable or improved properties compared to polyethylene terephthalate (PET). Biobased PEF offers lower life-cycle greenhouse gas emissions than PET. However, with its industrial production starting soon, relatively little is known about its actual recyclability. This work reports on the near complete depolymerization of PEF using two efficient PET hydrolases, FastPETase and leaf compost-cutinase (LCC), at loadings 4.5-17 times lower than previously reported. FastPETase and LCC exhibited maximum depolymerization of PEF, measured by weight loss and 2,5-furandicarboxylic acid (FDCA) production, using potassium phosphate-NaOH buffer at 50 and 65 °C, respectively. The 98% depolymerization of 13 g L-1 PEF film was achieved by three additions of the LCC in 72 h, while 78% weight loss was obtained using FastPETase in controlled conditions. Nonetheless, 92% weight loss was obtained with FastPETase when using only 6 g L-1 PEF. The main reaction products were identified as FDCA, ethylene glycol, and mono(2-hydroxyethyl)-furanoate. LCC performed better than FastPETase, in terms of both FDCA release and weight loss. The effect of crystallinity was evident on the enzymes' performance, as only 4% to 7% weight loss of crystalline PEF (32%) was recorded. Microscopy studies of the treated PEF films provided information on the surface erosion processes and revealed higher resistance of the crystalline phase, explaining the low level of depolymerization. The study presents important insights into the enzymatic hydrolysis of biobased PEF material and paves the path toward more viable applications within biopolymer waste recycling.

Controlled Polymer Synthesis Toward Green Chemistry: Deep Insights into Atom Transfer Radical Polymerization in Biobased Substitutes for Polar Aprotic Solvents.

Cyrene (dihydrolevoglucosenone) and its derivative Cygnet 0.0, recognized as eco-friendly alternatives to polar aprotic solvents, were utilized in atom transfer radical polymerization (ATRP) of a wide range of both hydrophobic and hydrophilic (meth)acrylates. The detailed kinetics study and electrochemical experiments of the catalytic complex in these solvents reveal the opportunities and limitations of their use in controlled radical polymerization. Both solvents produce precisely controlled polymers using supplemental activator and reducing agent (SARA) ATRP. They offer an efficient reaction medium for crafting well-defined branched architectures from naturally derived cores such as riboflavin, ß-cyclodextrin, and troxerutin, thereby significantly expanding the application scope of these solvents. Notably, Cygnet 0.0 significantly reduces side reactions between the solvent and the catalyst compared to Cyrene, allowing the catalyst complex to be used at a reduced concentration down to 75 ppm. The effective mass yield values achieved in Cyrene and Cygnet 0.0 underscore a substantial advantage of these solvents over DMF in generating processes that adhere to the principles of green chemistry. Furthermore, the copper residue in the final polymers was several hundred times lower than the permissible daily exposure to orally administered copper in pharmaceuticals. As a result, the resulting polymeric materials hold immense potential for various applications, including the pharmaceutical industry.

Bio-upcycling of multilayer materials and blends: closing the plastics loop.

The urge to discover and develop new technologies for closing the plastic carbon cycle is motivating industries, governments, and academia to work closely together to find suitable solutions in a timely manner. In this review article, a combination of uprising breakthrough technologies is presented highlighting their potential and complementarity to be integrated one with the other, therefore providing a potential solution to efficiently solve the plastics problem. First, modern approaches for bio-exploration and engineering of polymer-active enzymes are presented to degrade polymers into valuable building blocks. Special focus is placed on the recovery of components from multilayered materials since these complex materials can only be recycled insufficiently or not at all by existing technologies. Then, the potential of microbes and enzymes for resynthesis of polymers and reuse of building blocks is summarized and discussed. Finally, examples for improvement of the bio-based content and enzymatic degradability and future perspectives are given.

Enzymatic route for selective glycerol oxidation using covalently immobilized laccases.

Glycerol is an important starting material for the synthesis of many chemical compounds and its selective oxidation represents an efficient way to produce value-added compounds. Glyceric acid, one of these selective oxidation products, is an important intermediate in the food, medicine, cosmetics, and light industries. In this work, four commercially available native laccases were screened for glycerol oxidation using different initiators, and the two most efficient biocatalysts were covalently immobilized on functionalized magnetic and polymethacrylate (Lifetech™) solid supports. Apart from the mostly employed Fe3O4 magnetic particles, in this work Ni-Zn or Ni-Zn-Co spinel ferrite (MFe2O4) microparticles were used. Particularly, the utilization (for the first time for laccase immobilization) of Ni-Zn ferrite support Ni0.7Zn0.3Fe2O4 functionalized with 3-aminopropyl-trimethoxysilane, via crosslinking by glutaraldehyde and reduction with NaBH4 led to excellent biocatalytic efficiency and stability. These results confirm the feasibility of Trametes versicolor laccase for covalent bonding, as presumed by computational modelling. The resulted enzymatic preparations were characterized in detail in terms of stability and reusability, demonstrating enhanced storage, pH and thermal stability compared to the native enzymes. The most active biocatalysts (790.93 [U/g]) were successfully used for glycerol oxidation and the specific conversion in glyceric acid exceeded 50%.

On the Selective Enzymatic Recycling of Poly(pentamethylene 2,5-furanoate)/Poly(lactic acid) Blends and Multiblock Copolymers.

Among novel renewable furanoate-based polyesters, poly(pentamethylene 2,5-furandicarboxylate) (PPeF) shows outstanding gas barrier properties and high flexibility. PPeF blending/copolymerization with another well-known bio-based polymer, poly(lactic acid) (PLA), leads to considerably better mechanical and gas barrier properties of the latter, making it suitable for flexible food packaging applications. In this work, enzymatic depolymerization of PLA/PPeF blends with different compositions (1, 3, 5, 20, 30, and 50 wt % PPeF) and a PLA-PPeF block copolymer (50 wt % PPeF) by cutinase 1 from Thermobifida cellulositilytica (Thc_Cut1) was investigated as a possible recycling strategy. Based on quantification of weight loss and high-performance liquid chromatography (HPLC) analysis of released molecules, faster hydrolysis was seen for PLA/PPeF blends with increasing PPeF content when compared to neat PLA, while the block copolymer (P(LA50PeF50)) was significantly less susceptible to hydrolysis. Surface morphology analysis (via scanning electron microscopy), Fourier transform infrared spectroscopy, and NMR analysis confirmed preferential hydrolysis of the PPeF component. Through crystallization, 2,5-furandicarboxylic acid was selectively recovered from the depolymerized films and used for the resynthesis of the PPeF homopolymer, demonstrating the potential of enzymes for novel recycling concepts. The possibility of selective recovery of 2,5-furandicarboxylic acid from the completely depolymerized films with a 75% yield could bring further evidence of the high value of these materials, both in the form of blends and copolymers, for a sustainable whole packaging life cycle, where PPeF is potentially enzymatically recycled and PLA is mechanically recycled.

Engineering a Carboxyl Methyltransferase for the Formation of a Furan-Based Bioplastic Precursor.

FtpM from Aspergillus fumigatus was the first carboxyl methyltransferase reported to catalyse the dimethylation of dicarboxylic acids. Here the creation of mutant R166M that can catalyse the quantitative conversion of bio-derived 2,5-furandicarboxylic acid (FDCA) to its dimethyl ester (FDME), a bioplastics precursor, was reported. Wild type FtpM gave low conversion due to its reduced catalytic efficiency for the second methylation step. An AlphaFold 2 model revealed a highly electropositive active site, due to the presence of 4 arginine residues, postulated to favour the binding of the dicarboxylic acid over the intermediate monoester. The R166M mutation improved both binding and turnover of the monoester to permit near quantitative conversion to the target dimethyl ester product. The mutant also had improved activity for other diacids and a range of monoacids. R166M was incorporated into 2 multienzyme cascades for the synthesis of the bioplastics precursor FDME from bioderived 5-hydroxymethylfurfural (HMF) as well as from poly(ethylene furanoate) (PEF) plastic, demonstrating the potential to recycle waste plastic.

Chitosan: Sources, Processing and Modification Techniques.

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is derived from chitin. Chitin is found in cell walls of crustaceans, fungi, insects and in some algae, microorganisms, and some invertebrate animals. Chitosan is emerging as a very important raw material for the synthesis of a wide range of products used for food, medical, pharmaceutical, health care, agriculture, industry, and environmental pollution protection. This review, in line with the focus of this special issue, provides the reader with (1) an overview on different sources of chitin, (2) advances in techniques used to extract chitin and converting it into chitosan, (3) the importance of the inherent characteristics of the chitosan from different sources that makes them suitable for specific applications and, finally, (4) briefly summarizes ways of tailoring chitosan for specific applications. The review also presents the influence of the degree of acetylation (DA) and degree of deacetylation (DDA), molecular weight (Mw) on the physicochemical and biological properties of chitosan, acid-base behavior, biodegradability, solubility, reactivity, among many other properties that determine processability and suitability for specific applications. This is intended to help guide researchers select the right chitosan raw material for their specific applications.

Mycobacterium smegmatis acyltransferase: The big new player in biocatalysis.

After several decades during which proteases and after lipases took the biotransformation world scene as the predominant biocatalysts, a new, promising enzyme was discovered and characterized. The acyltransferase from Mycobacterium smegmatis (MsAcT) has in fact an extraordinary activity for a wide array of reactions, such as trans-esterification, amidation, trans-amidation and perhydrolysis, both in water and solvent media, giving rise to a series of interesting compounds including APIs (i.e., active pharmaceutical ingredients), natural flavors and fragrances, monomers for polymer synthesis, and peracids employed as disinfectants or antimicrobials. Although the most used acylating agent has been ethyl acetate (EtOAc), depending on the reaction type also acetamide, dimethyl carbonate and a variety of other esters, have been reported. The best yields were reached using very reactive donors such as vinyl or isopropenyl esters (almost complete conversion in rapid reaction times and water media for condensation reactions). In this review article the most innovative scientific advances on MsAcT, its mechanism and engineering are summarized, putting a particular focus on the different kind of processes (batch and flow) that it is possible to carry out using this enzyme as free or immobilized form. In conclusion, the author personal view on the unexplored reaction possibilities using MsAcT is reported as a window on the future of the topic.

Cutinase-Catalyzed Polyester-Polyurethane Degradation: Elucidation of the Hydrolysis Mechanism.

Polyurethanes (PU) are one of the most-used classes of synthetic polymers in Europe, having a considerable impact on the plastic waste management in the European Union. Therefore, they represent a major challenge for the recycling industry, which requires environmentally friendly strategies to be able to re-utilize their monomers without applying hazardous and polluting substances in the process. In this work, enzymatic hydrolysis of a polyurethane-polyester (PU-PE) copolymer using Humicola insolens cutinase (HiC) has been investigated in order to achieve decomposition at milder conditions and avoiding harsh chemicals. PU-PE films have been incubated with the enzyme at 50 °C for 168 h, and hydrolysis has been followed throughout the incubation. HiC effectively hydrolysed the polymer, reducing the number average molecular weight (Mn) and the weight average molecular weight (Mw) by 84% and 42%, respectively, as shown by gel permeation chromatography (GPC), while scanning electron microscopy showed cracks at the surface of the PU-PE films as a result of enzymatic surface erosion. Furthermore, Fourier Transform Infrared (FTIR) analysis showed a reduction in the peaks at 1725 cm-1, 1164 cm-1 and 1139 cm-1, indicating that the enzyme preferentially hydrolysed ester bonds, as also supported by the nuclear magnetic resonance spectroscopy (NMR) results. Liquid chromatography time-of-flight/mass spectrometry (LC-MS-Tof) analysis revealed the presence in the incubation supernatant of all of the monomeric constituents of the polymer, thus suggesting that the enzyme was able to hydrolyse both the ester and the urethane bonds of the polymer.

Role of Surface Enhancement in the Enzymatic Cross-Linking of Lignosulfonate Using Alternative Downstream Techniques.

Lignosulfonate (LS), one of the byproducts of the paper and pulp industry, was mainly used as an energy source in the last decade until the valorization of lignin through different functionalization methods grew in importance. Polymerization using multicopper oxidase laccase (from the Myceliophthora thermophila fungus) is one of such methods, which not only enhances properties such as hydrophobicity, flame retardancy, and bonding properties but can also be used for food and possesses pharmaceutical-like antimicrobial properties and aesthetic features of materials. Appropriate downstream processing methods are needed to produce solids that allow the preservation of particle morphology, a vital factor for the valorization process. In this work, an optimization of the enzymatic polymerization via spray-drying of LS was investigated. The response surface methodology was used to optimize the drying process, reduce the polymerization time, and maximize the dried mass yield. Particles formed showed a concave morphology and enhanced solubility while the temperature sensitivity of spray-drying protected the phenol functionalities beneficial for polymerization. Using the optimized parameters, a yield of 65% in a polymerization time of only 13 min was obtained. The experimental values were found to be in agreement with the predicted values of the factors (R 2: 95.2% and p-value: 0.0001), indicating the suitability of the model in predicting polymerization time and yield of the spray-drying process.

Renewable polymers and plastics: Performance beyond the green.

Renewable bio-based polymers are one of the effective answers that the bioeconomy offers to solve the environmental emergency connected to plastics and more specifically fossil-based plastics. Previous studies have shown that more than 70 % of the natural capital cost associated with plastic derives from the extraction and processing of fossil raw materials and that the price of fossil plastic would be on average 44 % higher if such impact was fully paid by businesses. The disclosure of the hidden costs of plastics will contribute to dispelling the myth of the expensiveness of renewable polymers. Nevertheless, the adoption of bio-based plastics in the market must be motivated by their functional properties and not merely by their green credentials. This article highlights some successful examples of synergies between chemistry and biotechnology in achieving a new generation of bio-based monomers and polymers. Their success is justified by the combination of scientific advances with positive environmental and social fallouts.

Biotechnological production and high potential of furan-based renewable monomers and polymers.

Of the 25 million tons of plastic waste produced every year in Europe, 40% of these are not reused or recycled, thus contributing to environmental pollution, one of the major challenges of the 21st century. Most of these plastics are made of petrochemical-derived polymers which are very difficult to degrade and as a result, a lot of research efforts have been made on more environmentally friendly alternatives. Bio-based monomers, derived from renewable raw materials, constitute a possible solution for the replacement of oil-derived monomers, with furan derivatives that emerged as platform molecules having a great potential for the synthesis of biobased polyesters, polyamides and their copolymers. This review article summarizes the latest developments in biotechnological production of furan compounds that can be used in polymer chemistry as well as in their conversion into polymers. Moreover, the biodegradability of the resulting materials is discussed.