Stability Increase of Phenolic Acid Decarboxylase by a Combination of Protein and Solvent Engineering Unlocks Applications at Elevated Temperatures.

Enzymatic decarboxylation of biobased hydroxycinnamic acids gives access to phenolic styrenes for adhesive production. Phenolic acid decarboxylases are proficient enzymes that have been applied in aqueous systems, organic solvents, biphasic systems, and deep eutectic solvents, which makes stability a key feature. Stabilization of the enzyme would increase the total turnover number and thus reduce the energy consumption and waste accumulation associated with biocatalyst production. In this study, we used ancestral sequence reconstruction to generate thermostable decarboxylases. Investigation of a set of 16 ancestors resulted in the identification of a variant with an unfolding temperature of 78.1 °C and a half-life time of 45 h at 60 °C. Crystal structures were determined for three selected ancestors. Structural attributes were calculated to fit different regression models for predicting the thermal stability of variants that have not yet been experimentally explored. The models rely on hydrophobic clusters, salt bridges, hydrogen bonds, and surface properties and can identify more stable proteins out of a pool of candidates. Further stabilization was achieved by the application of mixtures of natural deep eutectic solvents and buffers. Our approach is a straightforward option for enhancing the industrial application of the decarboxylation process.

Spectrophotometric and Fluorimetric High-Throughput Assays for Phenolic Acid Decarboxylase.

Biocatalytic decarboxylation of hydroxycinnamic acids yields phenolic styrenes, which are important precursors for antioxidants, epoxy coatings, adhesives and other polymeric materials. Bacillus subtilis decarboxylase (BsPAD) is a cofactor-independent enzyme that catalyzes the cleavage of carbon dioxide from p-coumaric-, caffeic-, and ferulic acid with high catalytic efficiency. Real-time spectroscopic assays for decarboxylase reactions remove the necessity of extensive sample workup, which is required for HPLC, mass spectrometry, gas chromatography, or NMR methods. This work presents two robust and sensitive assays based on photometry and fluorimetry that allow decarboxylation reactions to be followed with high sensitivity while avoiding product extraction and long analysis times. Optimized assay procedures were used to measure BsPAD activity in cell lysates and to determine the kinetic constants (KM and Vmax ) of the purified enzyme for p-coumaric-, caffeic- and ferulic acid. Substrate inhibition was shown for caffeic acid.

How to capture the bioeconomy's industrial and regional potential through professional cluster management.

The bioeconomy transforms the fossil-based economy by forming new value chains and linking until now distinct industrial sectors. It provides an opportunity for rural as well as industrialized regions. The transformation process can be accelerated by building bioeconomy clusters comprising industries, academia and investors. Using the model of the German cluster CLIB2021 the role of cluster organisations and professional cluster management in moderating the transformation process and gaining a competetive advantage is discussed. In addition examples of how cluster management supports the formation of an industrial consortium and the analysis of regional options are presented.

A Nanocapsule-Based Approach Toward Physical Thermolatent Catalysis.

Performance under pressure is utilized as a key concept for colloidal control of reaction kinetics. Barrier nanocapsules containing a catalyst and a low-boiling-point release agent enable a highly desired, adjustable, and distinct non-Arrhenius behavior for thermoset curing.

Enzyme-Triggered Antifouling Coatings: Switching Bioconjugate Adsorption via Proteolytically Cleavable Interfering Domains.

Protease activable antifouling coatings based on peptide-poly(ethylene glycol) conjugates are shown. The material-specific adsorption of a bioconjugate is temporarily suppressed by extending a titanium binding sequence with a proteolytically cleavable epitope and a suitable interfering domain. The adsorption of the PEG-peptide conjugates onto titanium substrates can be regained by cleaving the interfering domain with Tobacco Etch Virus protease. This activates peptide-mediated PEGylation of titanium surfaces and results in coatings that are stable against dilution and suppress nonreversible adsorption of blood protein models. Effects of branched and linear peptidic binding domains on coating stability and antifouling properties are elucidated.

Phosphonic Acid-Functionalized Polyurethane Dispersions with Improved Adhesion Properties.

A facile route to phosphorus-functionalized polyurethane dispersions (P-PUDs) with improved adhesion properties is presented. (Bis)phosphonic acid moieties serve as adhesion promoting sites that are covalently attached via an end-capping reaction to isocyanate-reactive polyurethane particles under aqueous conditions. The synthetic approach circumvents solubility issues, offers great flexibility in terms of polyurethane composition, and allows for the synthesis of semicrystalline systems with thermomechanical response due to reversible physical cross-linking. Differential scanning calorimetry (DSC) is used to investigate the effect of functionalization on the semicrystallinity. The end-capping conversion was determined via inductively-coupled plasma optical emission spectroscopy (ICP-OES) and was surprisingly found to be almost independent of the stoichiometry of reaction, suggesting an adsorption-dominated process. Particle charge detection (PCD) experiments reveal that a dense surface coverage of phosphonic acid groups can be attained and that, at high functionalization degrees, the phosphonic adhesion moieties are partially dragged inside the colloidal P-PUD particle. Quartz crystal microbalance with dissipation (QCMD) investigations conducted with hydroxyapatite (HAP) and stainless steel sensors as model surfaces show a greatly enhanced affinity of the aqueous P-PUDs and furthermore indicate polymer chain rearrangements and autonomous film formation under wet conditions. Due to their facile synthesis, significantly improved adhesion, and variable film properties, P-PUD systems such as the one described here are believed to be of great interest for multiple applications, e.g., adhesives, paints, anticorrosion, or dentistry.

Polyurethane Dispersions with Peptide Corona: Facile Synthesis of Stimuli-Responsive Dispersions and Films.

Peptide-polymer hybrid particles of submicron size yielding stimuli-responsive macroscopic films are presented. A thermoplastic polyurethane (PU) carrying polysiloxane and polyester soft segments serves as core material to obtain flexible, yet semicrystalline films with temperature-sensitivity. The synthesis is based on the high-sheer emulsification of isocyanate-terminated PU prepolymers, which in our model system purposefully lack any ability of colloidal self-stabilization. While emulsification in water leads to immediate coagulation, stable dispersions of polyurethane nanoparticles were formed in aqueous solutions of a hydrolyzed protein from wool. A comparison of dispersion and film properties to nonreactive, otherwise identical dispersions suggests covalent attachment of the peptide to the PU backbone. We show that the colloidal stability of the hybrid particles is completely governed by the peptide corona, and hence pH-triggered coagulation can be employed to induce particle deposition and film formation. Differential scanning calorimetry confirms partial crystallinity in the film and reveals strongly modified crystallization behavior due to the peptide.

Unique Curing Properties through Living Polymerization in Crosslinking Materials: Polyurethane Photopolymers from Vinyl Ether Building Blocks.

Photopolymers with unique curing capabilities were produced by combining living cationic polymerization with network formation and restricted polymer motion. A vinyl ether diol was synthesized as a functional building block and reacted with isophorone diisocyanate to form a highly functionalized vinyl ether polyurethane as a model system with high crosslinking ability. When using a cationic photoinitiator, fast polymerization is observed upon short UV irradiation. Curing proceeds in the absence of light and under ambient conditions without oxygen inhibition. Cationic active sites become trapped dormant species upon network-induced vitrification and surprisingly remain living for several days. The polymerization can be reactivated by additional UV irradiation and/or raised temperature. The curing behavior was studied in detail by using UV and FT-NIR coupled rheology and photo-DSC to simultaneously study spectroscopic and mechanical information, as well as thermal effects.

Controlled formation of polymer nanocapsules with high diffusion-barrier properties and prediction of encapsulation efficiency.

Polymer nanocapsules with high diffusion-barrier performance were designed following simple thermodynamic considerations. Hindered diffusion of the enclosed material leads to high encapsulation efficiencies (EEs), which was demonstrated based on the encapsulation of highly volatile compounds of different chemical natures. Low interactions between core and shell materials are key factors to achieve phase separation and a high diffusion barrier of the resulting polymeric shell. These interactions can be characterized and quantified using the Hansen solubility parameters. A systematic study of our copolymer system revealed a linear relationship between the Hansen parameter for hydrogen bonding (δh ) and encapsulation efficiencies which enables the prediction of encapsulated amounts for any material. Furthermore EEs of poorly encapsulated materials can be increased by mixing them with a mediator compound to give lower overall δh values.

Water-based adhesives with tailored hydrophobic association: dilution resistance and improved setting behavior.

Hydrophobic association and stimuli-responsiveness is a powerful tool towards water-based adhesives with strongly improved properties, which is demonstrated based on the example of hydrophobically modified alkali-soluble latexes (HASE) with modulated association. Their rheological properties are highly tunable due to the hydrophobic domains that act as physical crosslinking sites of adjustable interaction strength. Ethanol, propanol, and butanol are used as water-soluble model additives with different hydrophobicity in order to specifically target the association sites and impact the viscoelastic properties and stimuli-responsiveness. The rheological and mechanical property response upon dilution with water can be tailored, and dilution-resistant or even dilution-thickening systems are obtained. The investigations are of high importance for water-based adhesives, as our findings provide insight into general structure-property relationships to improve their setting behavior, especially upon contact with wet substrates.

Alternative Pathway for the Stabilization of Reactive Emulsions via Cross-Linkable Surfactants.

Highly reactive emulsions were stabilized by employing a surfmer analogous concept. An interfacial reaction between an emulsion droplet and a cross-linkable reactive surfactant was used to provide colloidal stability and simultaneously maintain the majority of the reactive groups. Polyaddition-type reaction between epoxy and amine was chosen as a model system to spontaneously and covalently bond the surfactant to the emulsion droplets. The interfacial reaction was monitored via isothermal titration calorimetry analysis. With this method, the increased colloidal stability could be attributed to a reaction rather than a pure physical adsorption. The maintained reactivity of the emulsion droplets enables consecutive conversions with coreactive components, e.g., for cross-linking reactions, corrosion protection, or functional coatings.

Crystallization of dyes by directed aggregation of colloidal intermediates: a model case.

The crystallization of two oil-soluble dyes, oil red and oil blue, from the 125-nm-sized nanodroplets of miniemulsions, results in larger well-crystallized species of high quality, which must have been grown by aggregation of colloidal intermediates. This model case not only allows the existence of this nonclassical crystallization process to be proven but also gives evidence for the presence of "super-van der Waals" forces operating between the single nanocrystalline moieties.