KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding

The functionalization of polymer surfaces by polymer-binding peptides offers tremendous opportunities for directed immobilization of enzymes, bioactive peptides, and antigens. The application of polymer-binding peptides as adhesion promoters requires reliable and stable binding under process conditions. Molecular modes of interactions between material surfaces, peptides, and solvent are often not understood to an extent that enables (semi-) rational design of polymer-binding peptides, hindering the full exploitation of their potential. Knowledge-gaining directed evolution (KnowVolution) is an efficient protein engineering strategy that facilitates tailoring protein properties to application demands through a combination of directed evolution and computational guided protein design. A single round of KnowVolution was performed to gain molecular insights into liquid chromatography peak I peptide, 47 aa (LCI)-binding to polypropylene (PP) in the presence of the competing surfactant Triton X-100. KnowVolution yielded a total of 8 key positions (D19, S27, Y29, D31, G35, I40, E42, and D45), which govern PP-binding in the presence of Triton X-100. The recombination of two of the identified amino acid substitutions (Y29R and G35R; variant KR-2) yielded a 5.4 ± 0.5-fold stronger PP-binding peptide compared to LCI WT in the presence of Triton X-100 (1 mM). The LCI variant KR-2 shows a maximum binding capacity of 8.8 ± 0.1 pmol/cm2 on PP in the presence of Triton X-100 (up to 1 mM). The KnowVolution approach enables the development of polymer-binding peptides, which efficiently coat and functionalize PP surfaces and withstand surfactant concentrations that are commonly used, such as in household detergents

Adhesion promoting peptides for polymer surface functionalization

Synthetic polymers are used in numerous applications (e.g. packaging, automobiles or furniture) and therefore play an essential role in our daily life. Surface functionalization is essential to combine required surface properties e.g. wettability, dyability, biocompatibility, or anti-biofouling, with polymer bulk properties. Standard surface modifications are based on photo-, plasma- or chemo-treatments. Surface modification based on peptide or protein adsorption on the other hand can reduce energy and water consumption of the process considerably and lead to fewer toxic waste. Material binding anchor peptides are ideal candidates for biological surface functionalization due to their propensity to selectively bind to surfaces. The knowledge about molecular modes of interactions between peptides, surfaces and solvents is until now limited. The directed evolution of binding peptides is challenging due to the limits in diversity generation in short peptides and in the screening of binding peptides. Knowledge gaining directed evolution approaches can provide first insights into peptides surface interactions while simultaneously tailoring the binding strength for application demands. Therefore, the aim of this work was to identify and characterize a polymer binding peptide and to provide first insight into its surface interactions in application conditions through the KnowVolution approach. A novel toolbox for identification of polymer binding anchor peptides was developed. Fusion proteins composed of anchor peptides (adenoregulin, catelicidin-BF, cecropin A, reutericin, and LCI) and enhanced green fluorescent protein (EGFP) were designed and their binding potential towards polypropylene surfaces was analyzed by fluorescence microscopy. LCI was identified as a high potential polypropylene binding peptide. Protein coatings of EGFP-LCI were characterized by fluorescence and scanning force microscopy. EGFP-LCI formed a densely packed monolayer of 4.1 ± 0.2 nm thickness and with a coating density of 0.8 pmol/cm2. The applicability of the anchor peptide toolbox for polypropylene functionalization was verified by equipping polypropylene with the fluorescent dye ThioGlo-1 via the anchor peptide LCI. Application of anchor peptides as adhesion promoters requires stable binding under process conditions (e.g. presence of surfactants). A robust directed evolution protocol (PePevo) was developed to tailor polymer binding peptides to specific application conditions. Keys for success were the development of an epPCR protocol with an extremely high mutation frequency (60 mutations/kb) to ensure sufficient mutations in polymer binding peptides and the optimization of screening assay (e.g. protein concentration and surfactant concentration) to achieve a standard deviation of ±14.4% (selection pressure 1 mM Triton X 100). Interactions between material surfaces, peptides, and solvent are often not sufficiently understood to enable a rational polymer binding peptide design. By employing the KnowVolution (knowledge gaining directed evolution) protein properties can be adapted to application demands. One round of KnowVolution was performed to gain insights into interactions between LCI and polypropylene in presence of the surfactant Triton X-100. KnowVolution yielded 8 LCI positions (D19, S27, Y29, D31, G35, I40, E42, and D45) which influence the binding to PP in presence of Triton X-100. Amino acid substitutions Y29R and G35R were recombined in variant LCI KR-2 with a 5.4 ± 0.5-fold stronger PP-binding in presence of Triton X-100 (1 mM). The variant LCI KR-2 shows a maximal binding capacity of 8.8 ± 0.1 pmol/cm2 on PP with a Triton X-100 up to 1 mM. The variant LCI KR-2 furthermore yields a 5.1-fold increased maximal binding capacity and a 1.9-fold increased dissociation constant. Interactions of anchor peptide LCI, PP, and Triton X-100 were shown to be affected by LCI’s negatively charged amino acid content. Variants with substituted negatively charged amino acids (D19, D31, E42, and D45) or variants with introduced positively charged amino acids bound stronger to PP than wild type LCI. Furthermore, it was noted that Triton X-100 influences the binding of LCI KR-2 very likely in its monomer form since an increase in concentration above critical micelle concentration (CMC: 0.2-0.9 mM) up to 10 mM did not influence the binding further. EGFP-LCI’s high coating density, the number and diversity of provided functional groups, and the adaptability to process conditions offer a feasible alternative to conventional modification strategies

KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding

The functionalization of polymer surfaces by polymer-binding peptides offers tremendous opportunities for directed immobilization of enzymes, bioactive peptides, and antigens. The application of polymer-binding peptides as adhesion promoters requires reliable and stable binding under process conditions. Molecular modes of interactions between material surfaces, peptides, and solvent are often not understood to an extent that enables (semi-) rational design of polymer-binding peptides, hindering the full exploitation of their potential. Knowledge-gaining directed evolution (KnowVolution) is an efficient protein engineering strategy that facilitates tailoring protein properties to application demands through a combination of directed evolution and computational guided protein design. A single round of KnowVolution was performed to gain molecular insights into liquid chromatography peak I peptide, 47 aa (LCI)-binding to polypropylene (PP) in the presence of the competing surfactant Triton X-100. KnowVolution yielded a total of 8 key positions (D19, S27, Y29, D31, G35, I40, E42, and D45), which govern PP-binding in the presence of Triton X-100. The recombination of two of the identified amino acid substitutions (Y29R and G35R; variant KR-2) yielded a 5.4 ± 0.5-fold stronger PP-binding peptide compared to LCI WT in the presence of Triton X-100 (1 mM). The LCI variant KR-2 shows a maximum binding capacity of 8.8 ± 0.1 pmol/cm2 on PP in the presence of Triton X-100 (up to 1 mM). The KnowVolution approach enables the development of polymer-binding peptides, which efficiently coat and functionalize PP surfaces and withstand surfactant concentrations that are commonly used, such as in household detergents

Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels

This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study, we address the following question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents, and organic solvents. The nanogels were constructed via direct chemical cross-linking of water-soluble reactive copolymers poly­(<i>N</i>-vinylpyrrolidone-<i>co</i>-<i>N</i>-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive succinimide groups and narrow dispersity were synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization. Poly­(ethylene glycol) bis­(3-aminopropyl) and branched polyethylenimine were utilized as model cross-linkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying cross-linking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their cross-linking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly cross-linked nanogels were strongly altered compared with loosely cross-linked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower cross-linking degree enable higher substrate transport rate, providing easier access to the active site of the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields

KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding

The functionalization of polymer surfaces by polymer-binding peptides offers tremendous opportunities for directed immobilization of enzymes, bioactive peptides, and antigens. The application of polymer-binding peptides as adhesion promoters requires reliable and stable binding under process conditions. Molecular modes of interactions between material surfaces, peptides, and solvent are often not understood to an extent that enables (semi-) rational design of polymer-binding peptides, hindering the full exploitation of their potential. Knowledge-gaining directed evolution (KnowVolution) is an efficient protein engineering strategy that facilitates tailoring protein properties to application demands through a combination of directed evolution and computational guided protein design. A single round of KnowVolution was performed to gain molecular insights into liquid chromatography peak I peptide, 47 aa (LCI)-binding to polypropylene (PP) in the presence of the competing surfactant Triton X-100. KnowVolution yielded a total of 8 key positions (D19, S27, Y29, D31, G35, I40, E42, and D45), which govern PP-binding in the presence of Triton X-100. The recombination of two of the identified amino acid substitutions (Y29R and G35R; variant KR-2) yielded a 5.4 ± 0.5-fold stronger PP-binding peptide compared to LCI WT in the presence of Triton X-100 (1 mM). The LCI variant KR-2 shows a maximum binding capacity of 8.8 ± 0.1 pmol/cm2 on PP in the presence of Triton X-100 (up to 1 mM). The KnowVolution approach enables the development of polymer-binding peptides, which efficiently coat and functionalize PP surfaces and withstand surfactant concentrations that are commonly used, such as in household detergents

Anchor peptides: A green and versatile method for polypropylene functionalization

Polypropylene is one of the widest spread commodity polymers in plastic industry with an estimated global consumption of 62.4 million tons in 2020. Surface modification of polypropylene is required for its application as textile fibers, packaging material or filtration membranes. Modification of polypropylene is challenging due to absent functional surface groups. An anchor-peptide-based toolbox for green and versatile polypropylene functionalization was developed. Fusion proteins composed of enhanced green fluorescent protein (EGFP) and anchor peptides (e.g. cecropin A or LCI) were designed and applied to polypropylene surfaces. Resulting protein coatings of EGFP-LCI were characterized by fluorescence and scanning force microscopy. The fusion protein EGFP-LCI formed densely packed monolayers of 4.1 +/- 0.2 nm thickness. A microtiter plate-based fluorescence assay was developed to analyze the coating in presence of surfactants. Washing of EGFP-LCI coated polypropylene with 10 mM non-ionic surfactant (Triton X-100) did not detach the protein film, whereas EGFP was removed completely. Anchor peptides promote binding to polypropylene by simple dip-coating at room temperature in water. The high coating density (0.8 pmol/cm(2)) as well as the number and diversity of provided functional groups offer a viable alternative to conventional modification strategies of functionalizing polypropylene. LCI's role as broadly applicable adhesion promoter was demonstrated by equipping polypropylene with the fluorescent dye ThioGlo-1 via the anchor peptide LCI

Water-Soluble Reactive Copolymers Based on Cyclic N -Vinylamides with Succinimide Side Groups for Bioconjugation with Proteins

Reversible addition–fragmentation chain transfer (RAFT) copolymerizations of methacrylic acid <i>N</i>-hydroxysuccinimide ester and cyclic <i>N</i>-vinylamide derivatives (<i>N</i>-vinylpyrrolidone, <i>N</i>-vinylpiperidone, and <i>N</i>-vinylcaprolactam) were successfully performed with methyl 2-(ethoxycarbonothioylthio)­propanoate as chain transfer agent (CTA). Effects of different reaction parameters, such as solvent type, temperature, and CTA-to-initiator (C/I) ratio, were studied to optimize the polymerization conditions in order to obtain copolymers with variable chemical composition, controlled molecular weight, and narrow polydispersity index (PDI). The solvent type has a high impact on the polymerization reaction, and a high C/I ratio decreases polydispersity as well as conversion. Increased steric hindrance through an enlarged lactam ring offsets the monomer reactivity. The controlled character of RAFT polymerization was evidenced by the low PDI of the copolymers and a linear relationship between conversion and molecular weight. Biohybrid nanogels were synthesized by direct coupling between reactive copolymers and enhanced green fluorescent protein (EGFP) or cellulase (CelA2_M2) at room temperature in a water-in-oil emulsion. The EGFP-conjugated nanogels were fluorescent, while the CelA2_M2 encapsulated in nanogels retained its catalytic activity, as demonstrated by the hydrolysis of 4-methylumbelliferyl-β-d-cellobioside