Unveiling the Enzymatic Degradation Process of Biobased Thiophene Polyesters.

In the past 20 years, scientific research focused on the identification of valid alternatives to materials of fossil origin, in particular, related to biobased polymers. Recently, the efforts led to the synthesis of thiophene-based polymers (TBPs), a new class of polyesters based on 2,5-thiophenedicarboxylic acid (TPCA) that can be industrially produced using biomass-derived molecules. In this study, TBPs were synthesized using diols with different chain length (from C4 to C6) leading to poly(butylene 2,5-thiophenedicarboxylate) (PBTF), poly(pentamethylene 2,5-thiophenedicarboxylate) (PPeTF), and poly(hexamethylene 2,5-thiophenedicarboxylate) (PHTF), respectively, that were processed to thin films. To investigate enzymatic hydrolysis of these polymer films, cutinase 1 (Thc_cut1) and cutinase 2 (Thc_cut2) from Thermobifida cellulosilytica were recombinantly expressed in the host E. coli and purified. After 72 h of incubation at 65°C with 5 µM Thc_cut1, weight loss and HPLC analysis indicated 9, 100, and 80% degradation of PBTF, PPeTF, and PHTG with a concomitant release of 0.12, 2.70, and 0.67 mM of TPCA. The SEM analysis showed that tiny holes were formed on the surface of the films and after 72 h PPeTF was completely degraded. The LC-TOF/MS analysis indicated that Thc_cut2 in particular released various oligomers from the polymer during the reaction. In addition, the FTIR analysis showed the formation of novel acid and hydroxyl groups on the polymer surfaces. The results showed that the two used thermostable cutinases are promising biocatalysts for the environmentally friendly degradation of TPCA-based polyesters, in view of a possible sustainable recycling of plastic waste through resynthesis processes.

In situ based surface-enhanced Raman spectroscopy (SERS) for the fast and reproducible identification of PHB producers in cyanobacterial cultures.

The production of polyhydroxybutyrate (PHB) by autotrophic fermentation of cyanobacteria has received increasing interest in the light of carbon emission reducing process strategies. Biotechnological approaches are in development to optimize the yield of PHB, including adapted cultivation media, characterized by a limitation of key nutrients: cyanobacteria accumulate PHB as energy storage molecules under limited growth conditions. Since there is an increasing demand for fast, simple and reliable analytics, we report the establishment of surface enhanced Raman spectroscopy (SERS) as a suitable monitoring tool for up scaled PHB production processes. Both, pure Ag-colloids mixed with bacterial culture, and in situ prepared colloids (Ag-Synechocystis), generated on the cell surface directly, were successfully applied and evaluated for this purpose. SERS measurements with in situ prepared Ag-colloids improved the reproducibility of Raman signals from 54.8% to 93.9%. The measurement time could be reduced significantly, completing our secondary goal. The quality of classically and in situ prepared Ag-colloids was monitored by zeta potential measurements and scanning electron microscopy (SEM) respectively. For data interpretation and statistical model-building an in house written code in the open source software RStudio was implemented. It was applied for the differentiation of PHB producers at the cellular level, revealing heterogeneities within sample groups regarding the PHB amount accumulated. The results obtained using the statistical model were validated as well and were complementary to the reference HPLC analysis. Therefore, a fast and reliable identification in situ SERS tool for the selection of the most promising cyanobacterial PHB production was established.

High Throughput Screening for New Fungal Polyester Hydrolyzing Enzymes.

There is a strong need for novel and more efficient polyester hydrolyzing enzymes in order to enable the development of more environmentally friendly plastics recycling processes allowing the closure of the carbon cycle. In this work, a high throughput system on microplate scale was used to screen a high number of fungi for their ability to produce polyester-hydrolyzing enzymes. For induction of responsible enzymes, the fungi were cultivated in presence of aliphatic and aromatic polyesters [poly(1,4-butylene adipate co terephthalate) (PBAT), poly(lactic acid) (PLA) and poly(1,4-butylene succinate) (PBS)], and the esterase activity in the culture supernatants was compared to the culture supernatants of fungi grown without polymers. The results indicate that the esterase activity of the culture supernatants was induced in about 10% of the tested fungi when grown with polyesters in the medium, as indicated by increased activity (to >50 mU/mL) toward the small model substrate para-nitrophenylbutyrate (pNPB). Incubation of these 50 active culture supernatants with different polyesters (PBAT, PLA, PBS) led to hydrolysis of at least one of the polymers according to liquid chromatography-based quantification of the hydrolysis products terephthalic acid, lactic acid and succinic acid, respectively. Interestingly, the specificities for the investigated polyesters varied among the supernatants of the different fungi.

Thermal Upgrade of Enzymatically Synthesized Aliphatic and Aromatic Oligoesters.

The enzymatic synthesis of polyesters in solventless systems is an environmentally friendly and sustainable method for synthetizing bio-derived materials. Despite the greenness of the technique, in most cases only short oligoesters are obtained, with limited practical applications or requiring further chemical processing for their elongation. In this work, we present a catalyst-free thermal upgrade of enzymatically synthesized oligoesters. Different aliphatic and aromatic oligoesters were synthesized using immobilized Candida antarctica lipase B (iCaLB) as the catalyst (70 °C, 24 h) yielding poly(1,4-butylene adipate) (PBA, Mw = 2200), poly(1,4-butylene isophthalate) (PBI, Mw = 1000), poly(1,4-butylene 2,5-furandicarboxylate) (PBF, Mw = 600), and poly(1,4-butylene 2,4-pyridinedicarboxylate) (PBP, Mw = 1000). These polyesters were successfully thermally treated to obtain an increase in Mw of 8.5, 2.6, 3.3, and 2.7 folds, respectively. This investigation focused on the most successful upgrade, poly(1,4-butylene adipate), then discussed the possible effect of di-ester monomers as compared to di-acids in the thermally driven polycondensation. The herein-described two-step synthesis method represents a practical and cost-effective way to synthesize higher-molecular-weight polymers without the use of toxic metal catalysts such as titanium(IV) tert-butoxide, tin(II) 2-ethylhexanoate, and in particular, antimony(IV) oxide. At the same time, the method allows for the extension of the number of reuses of the biocatalyst by preventing its exposure to extreme denaturating conditions.

Smart textiles in wound care: functionalization of cotton/PET blends with antimicrobial nanocapsules.

Management of infected wounds is one of the most costly procedures in the health care sector. Burn wounds are of significant importance due to the high infection risk that can possibly lead to severe consequences such as sepsis. Because antibiotic wound treatments have caused increasing antibiotic resistance in bacteria, there is currently a strong need for alternative strategies. Therefore, we developed new antimicrobial wound dressings consisting of pH-responsive human serum albumin/silk fibroin nanocapsules immobilized onto cotton/polyethylene terephthalate (PET) blends loaded with eugenol, which is an antimicrobial phenylpropanoid. Ultrasound-assisted production of eugenol-loaded nanocapsules resulted in particle sizes (hydrodynamic radii) between 319.73 ± 17.50 and 574.00 ± 92.76 nm and zeta potentials ranging from -10.39 ± 1.99 mV to -12.11 ± 0.59 mV. Because recent discoveries have indicated that the sweat glands contribute to wound reepithelialisation, release studies of eugenol were conducted in different artificial sweat formulas that varied in pH. Formulations containing 10% silk fibroin with lower degradation degree exhibited the highest release of 41% at pH 6.0. After immobilization, the functionalized cotton/PET blends were able to inhibit 81% of Staphylococcus aureus and 33% of Escherichia coli growth. Particle uniformity, silk fibroin concentration, and high surface-area-to-volume ratio of the produced nanocapsules were identified as the contributing factors leading to high antimicrobial activities against both strains. Therefore, the production of antimicrobial textiles using nanocapsules loaded with an active natural compound that will not contribute to antibiotic resistance is seen as a potential future alternative to commercially available antiseptic wound dressings.

Enzymatic synthesis of lignin derivable pyridine based polyesters for the substitution of petroleum derived plastics.

Following concerns over increasing global plastic pollution, interest in the production and characterization of bio-based and biodegradable alternatives is rising. In the present work, the synthesis of a series of fully bio-based alternatives based on 2,4-, 2,5-, and 2,6-pyridinedicarboxylic acid-derived polymers produced via enzymatic catalysis are reported. A similar series of aromatic-aliphatic polyesters based on diethyl-2,5-furandicarboxylate and of the petroleum-based diethyl terephthalate and diethyl isophthalate were also synthesized. Here we show that the enzymatic synthesis starting from 2,4-diethyl pyridinedicarboxylate leads to the best polymers in terms of molecular weights (Mn = 14.3 and Mw of 32.1 kDa when combined with 1,8-octanediol) when polymerized in diphenyl ether. Polymerization in solventless conditions were also successful leading to the synthesis of bio-based oligoesters that can be further functionalized. DSC analysis show a clear similarity in the thermal behavior between 2,4-diethyl pyridinedicarboxylate and diethyl isophthalate (amorphous polymers) and between 2,5-diethyl pyridinedicarboxylate and diethyl terephthalate (crystalline polymers).

Lysozyme-Responsive Spray-Dried Chitosan Particles for Early Detection of Wound Infection.

Infections are a severe health issue, and the need for an early point-of-care diagnostic approach for wound infections is continuously growing. Lysozyme has shown a great potential as a biomarker for rapid detection of wound infection. In this study, spray-drying of labeled and derivatized chitosans was investigated for the production of small particles responsive to lysozyme. Therefore, various chitosans, differing in their origin (snow crab, Chionoecetes sp., with medium and low molecular weight or shrimp) were N-acetylated, labeled with reactive black 5, and tested for solubility and spray-drying suitability. Reactive black-5-stained N-acetylated chitosan (low molecular weight, origin crab) was successfully spray-dried, and the obtained particles were characterized regarding size, ζ potential, and morphology. The particles showed an average hydrodynamic radius of 612.5 ± 132.8 nm. ζ potential was measured in the context of a later application as an infection detection system for wound infections in artificial wound fluid (-6.14 ± 0.16 mV) and infected wound fluid (-7.93 ± 1.35 mV). Furthermore, the aggregation behavior and surface structure were analyzed by using scanning electron microscopy and confocal laser scanning microscopy revealing spherical-shaped particles with explicit surface topologies. Spray-dried N-acetylated chitosan particles showed a 5-fold increase in lysozyme-responsive release of dyed chitosan fragments due to the enhanced surface area to volume ratio when compared to non-spray-dried N-acetylated chitosan flakes. On the basis of these results, the study showed the improved properties of N-acetylated spray-dried chitosan particles for future applications for early and rapid infection detection.

Highly Selective Enzymatic Recovery of Building Blocks from Wool-Cotton-Polyester Textile Waste Blends.

In Europe, most of the discarded and un-wearable textiles are incinerated or landfilled. In this study, we present an enzyme-based strategy for the recovery of valuable building blocks from mixed textile waste and blends as a circular economy concept. Therefore, model and real textile waste were sequentially incubated with (1) protease for the extraction of amino acids from wool components (95% efficiency) and (2) cellulases for the recovery of glucose from cotton and rayon constituents (85% efficiency). The purity of the remaining poly(ethylene terephthalate) (PET) unaltered by the enzymatic treatments was assessed via Fourier-transformed infrared spectroscopy. Amino acids recovered from wool were characterized via elementary and molecular size analysis, while the glucose resulting from the cotton hydrolysis was successfully converted into ethanol by fermentation with Saccharomyces cerevisiae. This work demonstrated that the step-wise application of enzymes can be used for the recovery of pure building blocks (glucose) and their further reuse in fermentative processes.