An enzymatic continuous-flow reactor based on a pore-size matching nano- and isoporous block copolymer membrane.

Continuous-flow biocatalysis utilizing immobilized enzymes emerged as a sustainable route for chemical synthesis. However, inadequate biocatalytic efficiency from current flow reactors, caused by non-productive enzyme immobilization or enzyme-carrier mismatches in size, hampers its widespread application. Here, we demonstrate a general-applicable and robust approach for the fabrication of a high-performance enzymatic continuous-flow reactor via integrating well-designed scalable isoporous block copolymer (BCP) membranes as carriers with an oriented and productive immobilization employing material binding peptides (MBP). Densely packed uniform enzyme-matched nanochannels of well-designed BCP membranes endow the desired nanoconfined environments towards a productive immobilized phytase. Tuning nanochannel properties can further regulate the complex reaction process and fortify the catalytic performance. The synergistic design of enzyme-matched carriers and efficient enzyme immobilization empowers an excellent catalytic performance with >1 month operational stability, superior productivity, and a high space-time yield (1.05 × 105 g L-1 d-1) via a single-pass continuous-flow process. The obtained performance makes the designed nano- and isoporous block copolymer membrane reactor highly attractive for industrial applications.

Adhesion Peptide-Functionalized Biobased Microgels for Controlled Delivery of Pesticides.

Widespread use of plant protection agents in agriculture is a major cause of pollution. Apart from active ingredients, the environmental impact of auxiliary synthetic polymers should be minimized if they are highly persistent. An alternative to synthetic polymers is the use of natural polysaccharides, which are abundant and biodegradable. In this study, we explore pectin microgels functionalized with anchor peptides (P-MAPs) to be used as an alternative biobased pesticide delivery system. Using copper as the active ingredient, P-MAPs effectively prevented infection of grapevine plants with downy mildew under semi-field conditions on par with commercial copper pesticides. By using anchor peptides, the microgels tightly bind to the leaf surface, exhibiting excellent rain fastness and prolonged fungicidal activity. Finally, P-MAPs are shown to be easily degradable by enzymes found in nature, demonstrating their negligible long-term impact on the environment.

Bifunctional peptides as alternatives to copper-based formulations to control citrus canker.

Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial "killer" peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 µg mL-1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 µg mL-1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations. KEY POINTS: • The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri. • Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane. • Mel-CgDEF protected citrus leaves against citrus canker disease.

Brush-Like Coatings Provide a Cloak of Invisibility to Titanium Implants.

Orthopedic implants such as knee and hip implants are one of the most important types of medical devices. Currently, the surface of the most advanced implants consists of titanium or titanium-alloys with high porosity at the bone-contacting surface leading to superior mechanical properties, excellent biocompatibility, and the capability of inducing osseointegration. However, the increased surface area of porous titanium provides a nidus for bacteria colonization leading to implant-related infections, one of the main reasons for implant failure. Here, two readily applicable titanium-coatings based on hydrophilic carboxybetaine polymers that turn the surface stealth thereby preventing bacterial adhesion and colonization are developed. These coatings are biocompatible, do not affect cell functionality, exhibit great antifouling properties, and do not cause additional inflammation during the healing process. In this way, the coatings can prevent implant-related infections, while at the same time being completely innocuous to its biological environment. Thus, these coating strategies are a promising route to enhance the biocompatibility of orthopedic implants and have a high potential for clinical use, while being easy to implement in the implant manufacturing process.

BioAdhere: tailor-made bioadhesives for epiretinal visual prostheses.

Introduction: Visual prostheses, i.e. epiretinal stimulating arrays, are a promising therapy in treating retinal dystrophies and degenerations. In the wake of a new generation of devices, an innovative method for epiretinal fixation of stimulator arrays is required. We present the development of tailor-made bioadhesive peptides (peptesives) for fixating epiretinal stimulating arrays omitting the use of traumatic retinal tacks. Materials and methods: Binding motifs on the stimulating array (poly[chloro-p-xylylene] (Parylene C)) and in the extracellular matrix of the retinal surface (collagens I and IV, laminin, fibronectin) were identified. The anchor peptides cecropin A (CecA), KH1, KH2 (author's initials) and osteopontin (OPN) were genetically fused to reporter proteins to assess their binding behavior to coated microtiter plates via fluorescence-based assays. Domain Z (DZ) of staphylococcal protein A was used as a separator to generate a bioadhesive peptide. Following ISO 10993 "biological evaluation of medical materials", direct and non-direct cytotoxicity testing (L-929 and R28 retinal progenitor cells) was performed. Lastly, the fixating capabilities of the peptesives were tested in proof-of-principle experiments. Results: The generation of the bioadhesive peptide required evaluation of the N- and C-anchoring of investigated APs. The YmPh-CecA construct showed the highest activity on Parylene C in comparison with the wildtype phytase without the anchor peptide. eGFP-OPN was binding to all four investigated ECM proteins (collagen I, laminin > collagen IV, fibronectin). The strongest binding to collagen I was observed for eGFP-KH1, while the strongest binding to fibronectin was observed for eGFP-KH2. The selectivity of binding was checked by incubating eGFP-CecA and eGFP-OPN on ECM proteins and on Parylene C, respectively. Direct and non-direct cytotoxicity testing of the peptide cecropin-A-DZ-OPN using L-929 and R28 cells showed good biocompatibility properties. Proof-of-concept experiments in post-mortem rabbit eyes suggested an increased adhesion of CecA-DZ-OPN-coated stimulating arrays. Conclusion: This is the first study to prove the applicability and biocompatibility of peptesives for the fixation of macroscopic objects.

Rational Design Yields Molecular Insights on Leaf-Binding of Anchor Peptides.

In times of a constantly growing world population and increasing demand for food, sustainable agriculture is crucial. The rainfastness of plant protection agents is of pivotal importance to reduce the amount of applied nutrients, herbicides, and fungicides. As a result of protective agent wash-off, plant protection is lost, and soils and groundwater are severely polluted. To date, rainfastness of plant protection products has been achieved by adding polymeric adjuvants to the agrochemicals. However, polymeric adjuvants will be regarded as microplastics in the future, and environmentally friendly alternatives are needed. Anchor peptides (APs) are promising biobased and biodegradable adhesion promoters. Although the adhesion of anchor peptides to artificial surfaces, such as polymers, has already been investigated in theory and experimentally, exploiting the adhesion to biological surfaces remains challenging. The complex nature and composition of biological surfaces such as plant leaves and fruit surfaces complicate the generation of accurate models. Here, we present the first detailed three-layered atomistic model of the surface of apple leaves and use it to compute free energy profiles of the adhesion and desorption of APs to and from that surface. Our model is validated by a novel fluorescence-based microtiter plate (MTP) assay that mimics these complex processes and allows for quantifying them. For the AP Macaque Histatin, we demonstrate that aromatic and positively charged amino acids are essential for binding to the waxy apple leaf surface. The established protocols should generally be applicable for tailoring the binding properties of APs to biological interfaces.

Endogenous Nitric Oxide-Releasing Microgel Coating Prevents Clot Formation on Oxygenator Fibers Exposed to In Vitro Blood Flow.

BACKGROUND: Clot formation on foreign surfaces of extracorporeal membrane oxygenation systems is a frequent event. Herein, we show an approach that mimics the enzymatic process of endogenous nitric oxide (NO) release on the oxygenator membrane via a biomimetic, non-fouling microgel coating to spatiotemporally inhibit the platelet (PLT) activation and improve antithrombotic properties. This study aims to evaluate the potential of this biomimetic coating towards NO-mediated PLT inhibition and thereby the reduction of clot formation under flow conditions. METHODS: Microgel-coated (NOrel) or bare (Control) poly(4-methyl pentene) (PMP) fibers were inserted into a test channel and exposed to a short-term continuous flow of human blood. The analysis included high-resolution PLT count, pooled PLT activation via ß-Thromboglobulin (ß-TG) and the visualization of remnants and clots on the fibers using scanning electron microscopy (SEM). RESULTS: In the Control group, PLT count was significantly decreased, and ß-TG concentration was significantly elevated in comparison to the NOrel group. Macroscopic and microscopic visualization showed dense layers of stable clots on the bare PMP fibers, in contrast to minimal deposition of fibrin networks on the coated fibers. CONCLUSION: Endogenously NO-releasing microgel coating inhibits the PLT activation and reduces the clot formation on PMP fibers under dynamic flow.

A plea for the integration of Green Toxicology in sustainable bioeconomy strategies - Biosurfactants and microgel-based pesticide release systems as examples.

A key aspect of the transformation of the economic sector towards a sustainable bioeconomy is the development of environmentally friendly alternatives for hitherto used chemicals, which have negative impacts on environmental health. However, the implementation of an ecotoxicological hazard assessment at early steps of product development to elaborate the most promising candidates of lowest harm is scarce in industry practice. The present article introduces the interdisciplinary proof-of-concept project GreenToxiConomy, which shows the successful application of a Green Toxicology strategy for biosurfactants and a novel microgel-based pesticide release system. Both groups are promising candidates for industrial and agricultural applications and the ecotoxicological characterization is yet missing important information. An iterative substance- and application-oriented bioassay battery for acute and mechanism-specific toxicity within aquatic and terrestrial model species is introduced for both potentially hazardous materials getting into contact with humans and ending up in the environment. By applying in silico QSAR-based models on genotoxicity, endocrine disruption, skin sensitization and acute toxicity to algae, daphnids and fish, individual biosurfactants resulted in deviating toxicity, suggesting a pre-ranking of the compounds. Experimental toxicity assessment will further complement the predicted toxicity to elaborate the most promising candidates in an efficient pre-screening of new substances.

Unraveling the Mechanism and Kinetics of Binding of an LCI-eGFP-Polymer for Antifouling Coatings.

The ability of proteins to adsorb irreversibly onto surfaces opens new possibilities to functionalize biological interfaces. Herein, the mechanism and kinetics of adsorption of protein-polymer macromolecules with the ability to equip surfaces with antifouling properties are investigated. These macromolecules consist of the liquid chromatography peak I peptide from which antifouling polymer brushes are grafted using single electron transfer-living radical polymerization. Surface plasmon resonance spectroscopy reveals an adsorption mechanism that follows a Langmuir-type of binding with a strong binding affinity to gold. X-ray reflectivity supports this by proving that the binding occurs exclusively by the peptide. However, the lateral organization at the surface is directed by the cylindrical eGFP. The antifouling functionality of the unimolecular coatings is confirmed by contact with blood plasma. All coatings reduce the fouling from blood plasma by 8894% with only minor effect of the degree of polymerization for the studied range (DP between 101 and 932). The excellent antifouling properties, combined with the ease of polymerization and the straightforward coating procedure make this a very promising antifouling concept for a multiplicity of applications.

Enzyme mimetic microgel coating for endogenous nitric oxide mediated inhibition of platelet activation.

Nitric oxide (NO) continuously generated by healthy endothelium prevents platelet activation and maintains vascular homeostasis. However, when artificial surfaces, like of extracorporeal membrane oxygenator comes in contact with blood, protein adsorption and thereby platelet activation takes place, which eventually leads to thrombus formation. To overcome this, we present an antifouling microgel coating mimicking the function of enzyme glutathione peroxidase to endogenously generate NO in the blood plasma from endogenous NO-donors and maintain a physiological NO flux. Microgels are synthesized by copolymerization of highly hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) and glycidyl methacrylate (GMA) with diselenide crosslinks. For immobilization of the microgels on hydrophobic poly(4-methylpentene) (TPX) membranes bioengineered amphiphilic anchor peptides with free thiols are used. The anchor peptide attaches to the TPX membranes by hydrophobic interactions while the free thiols are presented for crosslinking with the microgels. The hydrophilic nature of the microgel coating prevents protein adsorption while the reversible diselenide bridges make the microgels responsive to the reducing environment and lead to the formation of reactive selenols/selenolates. The generated selenols/selenolates provide an efficient and sustained NO-release from endogenous S-nitrosothiols (RSNOs) mimicking the enzymatic function of glutathione peroxidase. On exposure to the whole blood, the microgel coating inhibited platelet activation and prolonged the blood clotting time.

A peptide-based coating toolbox to enable click chemistry on polymers, metals, and silicon through sortagging.

A versatile peptide-based toolbox for surface functionalization was established by a combination of a universal material binding peptide (LCI-anchor peptide) and sortase-mediated bioconjugation (sortagging). This toolbox facilitates surface functionalization either as a one- or a two-step strategy. In the case of the one-step strategy, the desired functionality was directly introduced to LCI. For the two-step strategy, LCI was modified with a reactive group, which can be further functionalized (e.g., employing "click" chemistry). Sortagging of LCI, employing sortase A from Staphylococcus aureus, was achieved with six different amine compounds: dibenzocyclooctyne amine, biotin-polyethylene glycol amine, Cyanine-3 amine, kanamycin, methoxypolyethylene glycol amine (Mn = 5000 Da), and 2,2,3,3,4,4,4-Heptafluorobutylamine. The purification of LCI-amine sortagging products was performed by a negative purification using Strep-tag II affinity chromatography, resulting in LCI-amine conjugates with purities >90%. For the two-step strategy, the LCI-dibenzocyclooctyne sortagging product was purified and enabled, through copper-free azide-alkyne "click" chemistry, universal surface functionalization of material surfaces such as polypropylene, polyethylene terephthalate, stainless steel, gold, and silicon. The click reaction was performed before or after surface binding of LCI-dibenzocyclooctyne. Finally, in the case of the one-step strategy, polypropylene was directly functionalized with Cyanine-3 and biotin-polyethylene glycol amine.

MicroGelzymes: pH-Independent Immobilization of Cytochrome P450 BM3 in Microgels.

Microgels are an emerging class of "ideal" enzyme carriers because of their chemical and process stability, biocompatibility, and high enzyme loading capability. In this work, we synthesized a new type of permanently positively charged poly(N-vinylcaprolactam) (PVCL) microgel with 1-vinyl-3-methylimidazolium (quaternization of nitrogen by methylation of N-vinylimidazole moieties) as a comonomer (PVCL/VimQ) through precipitation polymerization. The PVCL/VimQ microgels were characterized with respect to their size, charge, swelling degree, and temperature responsiveness in aqueous solutions. P450 monooxygenases are usually challenging to immobilize, and often, high activity losses occur after the immobilization (in the case of P450 BM3 from Bacillus megaterium up to 100% loss of activity). The electrostatic immobilization of P450 BM3 in permanently positively charged PVCL/VimQ microgels was achieved without the loss of catalytic activity at the pH optimum of P450 BM3 (pH 8; ∼9.4 nmol 7-hydroxy-3-carboxy coumarin ethyl ester/min for free and immobilized P450 BM3); the resulting P450-microgel systems were termed P450 MicroGelzymes (P450 µ-Gelzymes). In addition, P450 µ-Gelzymes offer the possibility of reversible ionic strength-triggered release and re-entrapment of the biocatalyst in processes (e.g., for catalyst reuse). Finally, a characterization of the potential of P450 µ-Gelzymes to provide resistance against cosolvents (acetonitrile, dimethyl sulfoxide, and 2-propanol) was performed to evaluate the biocatalytic application potential.

Designed Streptococcus pyogenes Sortase A Accepts Branched Amines as Nucleophiles in Sortagging.

Sortase-mediated ligation (sortagging) is commonly performed using the Staphylococcus aureus sortase A (SaSrtA) that strictly recognizes the N-terminal glycine residue. In this work, a rational design of Streptococcus pyogenes sortase A (SpSrtA) for improved transpeptidase activity toward different N-terminal amino acid residues was conducted. The generated variant SpSrtA M3 (E189H/V206I/E215A) showed up to 6.6-fold (vs SpSrtA wild-type) enhanced catalytic efficiency. Additionally, M3 retains the specificity toward N-terminal alanine, glycine, serine residues, as well as branched (at α-carbon) primary amines as wild-type parent. Furthermore, M3 was applied for head-to-tail backbone cyclization of proteins.

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization.

Invited for the cover of this issue is Ulrich Schwaneberg and co-workers at RWTH Aachen University and DWI Leibniz-Institut für Interaktive Materialien. The image depicts a loop engineered, and backbone cyclized Staphylococcus aureus sortase A which shows enhanced robustness in site-specific protein and peptide modifications. Read the full text of the article at 10.1002/chem.202002740.

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization.

Staphylococcus aureus sortase A (SaSrtA) is widely used for site-specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presence of denaturing agents. Loop engineering and subsequent head-to-tail backbone cyclization of SaSrtA yielded the cyclized variant CyM6 that has a 7.5 °C increased melting temperature and up to 4.6-fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6-fold (vs. parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions.

Gas-Phase Transformation of Fluorinated Benzoporphyrins to Porphyrin-Embedded Conical Nanocarbons.

Geodesic nitrogen-containing graphene fragments are interesting candidates for various material applications, but the available synthetic protocols, which need to overcome intrinsic strain energy during the formation of the bowl-shaped skeletons, are often incompatible with heteroatom-embedded structures. Through this mass spectrometry-based gas-phase study, we show by means of collision-induced dissociation experiments and supported by density functional theory calculations, the first evidence for the formation of a porphyrin-embedded conical nanocarbon. The influences of metalation and functionalization of the used tetrabenzoporphyrins have been investigated, which revealed different cyclization efficiencies, different ionization possibilities, and a variation of the dissociation pathway. Our results suggest a stepwise process for HF elimination from the fjord region, which supports a selective pathway towards bent nitrogen-containing graphene fragments.

Matter-tag: A universal immobilization platform for enzymes on polymers, metals, and silicon-based materials.

Enzyme immobilization is extensively studied to improve enzyme properties in catalysis and analytical applications. Here, we introduce a simple and versatile enzyme immobilization platform based on adhesion-promoting peptides, namely Matter-tags. Matter-tags immobilize enzymes in an oriented way as a dense monolayer. The immobilization platform was established with three adhesion-promoting peptides; Cecropin A (CecA), liquid chromatography peak I (LCI), and Tachystatin A2 (TA2), that were genetically fused to enhanced green fluorescent protein and to two industrially important enzymes: a phytase (from Yersinia mollaretii) and a cellulase (CelA2 from a metagenomic library). Here, we report a universal and simple Matter-tag-based immobilization platform for enzymes on various materials including polymers (polystyrene, polypropylene, and polyethylene terephthalate), metals (stainless steel and gold), and silicon-based materials (silicon wafer). The Matter-tag-based enzyme immobilization is performed at ambient temperature within minutes (<10 min) in an aqueous solution harboring the phytase or cellulase by immersing the targeted material. The peptide LCI was identified as universal adhesion promoter; LCI immobilized both enzymes on all investigated materials. The attachment of phytase-LCI onto gold was characterized with surface plasmon resonance spectroscopy obtaining a dissociation constant value (KD ) of 2.9·10-8 M and a maximal surface coverage of 504 ng/cm².

Rapid and Robust Coating Method to Render Polydimethylsiloxane Surfaces Cell-Adhesive.

Polydimethylsiloxane (PDMS) is a synthetic material with excellent properties for biomedical applications because of its easy fabrication method, high flexibility, permeability to oxygen, transparency, and potential to produce high-resolution structures in the case of lithography. However, PDMS needs to be modified to support homogeneous cell attachments and spreading. Even though many physical and chemical methods, like plasma treatment or extracellular matrix coatings, have been developed over the last decades to increase cell-surface interactions, these methods are still very time-consuming, often not efficient enough, complex, and can require several treatment steps. To overcome these issues, we present a novel, robust, and fast one-step PDMS coating method using engineered anchor peptides fused to the cell-adhesive peptide sequence (glycine-arginine-glycine-aspartate-serine, GRGDS). The anchor peptide attaches to the PDMS surface predominantly by hydrophobic interactions by simply dipping PDMS in a solution containing the anchor peptide, presenting the GRGDS sequence on the surface available for cell adhesion. The binding performance and kinetics of the anchor peptide to PDMS are characterized, and the coatings are optimized for efficient cell attachment of fibroblasts and endothelial cells. Additionally, the applicability is proven using PDMS-based directional nanotopographic gradients, showing a lower threshold of 5 µm wrinkles for fibroblast alignment.

Selective Functionalization of Microgels with Enzymes by Sortagging.

Enzyme immobilization has been widely used to improve the stability and recyclability of enzymes in industrial processes. In this work, a sortase-mediated and therefore selective covalent immobilization strategy (sortagging) for enzymes on microgels (GelZyms) was investigated. Aqueous microgels were synthesized from poly(N-vinylcaprolactam)/glycidyl methacrylate (PVCL/GMA) and tagged with the sortase A recognition peptide sequence (LPETG) or its nucleophilic counterpart-tag (GGG). General applicability and selective immobilization were confirmed by subsequent sortagging of five different enzymes (Bacillus subtilis lipase A (BSLA), Yersinia mollaretii phytase (Ym-phytase), Escherichia coli copper efflux oxidase (CueO laccase), cellulase A2, and Bacillus megaterium monooxygenase P450 BM3). The latter was performed directly from the cell lysate to ensure cost-effective immobilization. All five immobilized enzymes were catalytically active and could be recycled (e.g., laccase CueO and monooxygenase P450 BM3 F87A; >55% residual activity after six cycles). Application potential was demonstrated by using CueO decorated microgels for bleaching of the synthetic dye indigo carmine.

Biadhesive Peptides for Assembling Stainless Steel and Compound Loaded Micro-Containers.

Biadhesive peptides (peptesives) are an attractive tool for assembling two chemically different materials-for example, stainless steel and polycaprolactone (PCL). Stainless steel is used in medical stents and PCL is used as a biodegradable polymer for fabrication of tissue growth scaffolds and drug delivering micro-containers. Biadhesive peptides are composed of two domains (e.g., dermaseptin S1 and LCI) with different material-binding properties that are separated through a stiff peptide-spacer. The peptesive dermaseptin S1-domain Z-LCI immobilizes antibiotic-loaded PCL micro-containers on stainless steel surfaces. Immobilization is visualized by microscopy and field emission scanning electron microscopy analysis and released antibiotic from the micro-containers is confirmed through growth inhibition of Escherichia coli cells.