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Accumulation of plastics in the environment became a geological indicator of the Anthropocene era. An effective reduction of long-lasting plastics requires a treatment with micro-organisms that release polymer-degrading enzymes. Polymer binding peptides function as adhesion promoters and enable a targeted binding of whole cells to polymer surfaces. An esterase A-based Escherichia coli cell surface display screening system was developed, that enabled directed evolution of polymer binding peptides for improved binding strength to polymers. The E. coli cell surface screening system facilitates an enrichment of improved binding peptides from a culture broth through immobilization of whole cells on polymer beads. The polypropylene (PP)-binding peptide liquid chromatography peak I (LCI) was simultaneously saturated at five positions (Y29, D31, G35, E42, and D45; 3.2 million variants) and screened for improved PP-binding in the presence of the anionic surfactant sodium dodecylbenzenesulfonate (LAS; 0.25 mM). The cell surface system enabled efficient screening of the generated LCI diversity (in total ~10 million clones were screened). Characterization of identified LCI binders revealed an up to 12-fold improvement (eGFP-LCI-CSD-3: E42V/D45H) in PP-binding strength in the presence of the surfactant LAS (0.125 mM). The latter represents a first whole cell display screening system to improve adhesion peptides which can be used to direct and to immobilize organisms specifically to polymer surfaces (e.g., PP) and novel applications (e.g., in targeted plastic degradation)., (© 2019 Wiley Periodicals, Inc.)

Adhesion promoting peptides have been reported to enable efficient enzyme immobilization on various material surfaces. Here we report the first immobilization of a synthetic Grubbs-Hoveyda (GH) type catalyst on two different materials (silica and polypropylene). To this end, the GH catalyst was coupled to an engineered (F16C) variant of the adhesion promoting peptide LCI through thiol-maleimide "click" reaction. Immobilization was performed in an oriented manner through the adhesion promoting peptide by simple incubation with the materials in water and subsequent washing with water and tetrahydrofuran. The immobilized GH catalyst was probed in ring-opening metathesis polymerization of a norbornene derivative to alter the surface properties in a layer-by-layer fashion.

Herein we report the synthesis of new reactive stimuli-responsive polymers by RAFT copolymerization of glycidyl methacrylate and three cyclic N-vinyllactam derivatives. The copolymerization process was thoroughly investigated and the influence of the steric hindrance originating from the monomer structure of cyclic N-vinyllactams on the polymerization process and the properties of obtained copolymers were studied. A series of water-soluble copolymers with variable chemical composition, controlled molecular weight and narrow dispersity ( Đ) were synthesized and their properties are systematically investigated. Experimentally determined cloud points for different copolymers in aqueous solutions indicate shift of lower critical solution temperature (LCST) to lower values with the increase of GMA content in copolymers and increase of the lactam ring size. The obtained reactive stimuli-responsive copolymers can be efficiently used for encapsulation of cellulase in water-in-oil emulsions forming biohybrid nanogels. The enzymes entrapped in nanogels demonstrated significantly improved resistance against harsh store conditions, chaotropic agents, and organic solvents.

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., Competing Interests: The authors declare no conflict of interest.

Sortase-catalyzed ligations have emerged as powerful tools for the site-specific ligation of peptides and proteins in material science and biocatalysis. In this work, a directed sortase evolution strategy (SortEvolve) has been developed as a general high-throughput screening (HTS) platform to improve activity of sortase A (application 1) and to perform directed laccase evolution through a semipurification process in 96-well microtiter plate (MTP) (application 2). A semipurification process in polypropylene MTP (PP-MTP) is achieved through the anchor peptide LCI, which acts as adhesion promoter. To validate the SortEvolve screening platform for both applications, three site-saturation mutagenesis (SSM) libraries of sortase A (Sa-SrtA) from Staphylococcus aureus (application 1) and two SSM libraries of the copper efflux oxidase (CueO laccase) from Escherichia coli (application 2) were generated at literature reported positions. After screening and rescreening, an array of Sa-SrtA variants (including the previously reported P94S, D160N, and D165A) and CueO variants (including the previously reported D439A and P444A) were identified. Further recombinant Sa-SrtA variant P94T/D160L/D165Q and CueO variant D439V/P444V were characterized with 22-fold and 103-fold improvements in catalytic efficiency compared with corresponding wild-types, respectively. An important advantage of the SortEvolve screening platform in comparison to many MTP-based screening systems is that the background noise was minimized (decreased 20-fold; application 2) due to the employed semipurification process. In essence, SortEvolve provides a universal surface-functionalized screening platform for sortases and enzymes in which especially background activity can be minimized to enable successful directed evolution campaigns.

Surface functionalization of biological inert polymers (e.g., polypropylene PP; polystyrene PS) with material binding peptides facilitates an efficient immobilization of enzymes, bioactive peptides or antigens at ambient temperature in water. The developed robust directed evolution protocol enables to tailor polymer binding anchor peptides (PBPs) for efficient binding under application conditions. Key for a successful directed evolution campaign was to develop an epPCR protocol with a very high mutation frequency (60 mutations/kb) to ensure sufficient diversity in PBPs (47 aas LCI: "liquid chromatography peak I"; 44 aas TA2: "Tachystatin A2"). LCI and TA2 were genetically fused to the reporter egfp to quantify peptide binding on PP and PS by fluorescence analysis. The Peptide-Polymer evolution protocol (PePevo protocol) was validated in two directed evolution campaigns for two PBPs and polymers (LCI: PP; TA2: PS). Surfactants were used as selection pressure for improved PBP binders (non-ionic surfactant Triton X-100; 1 mM for LCI-PP // anionic surfactant LAS; 0.5 mM for TA2-PS). PePevo yielded an up to three fold improved PP-binder (LCI-M1-PP: I24T, Y29H, E42 K and LCI-M2-PP: D31V, E42G) and an up to six fold stronger PS-binder (TA2-M1-PS: R3S, L6P, V12 K, S15P, C29R, R30L, F33S, Y44H and TA2-M2-PS: F9C, C24S, G26D, S31G, C41S, Y44Q)., (© 2017 Wiley Periodicals, Inc.)

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(N-vinylpyrrolidone-co-N-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.

Novel redox-responsive polymeric nanogels that allow highly efficient enzyme encapsulation and reversible modulation of enzyme activity are developed. The nanogel synthesis and encapsulation of enzyme are performed simultaneously via in situ crosslinking of pyridyldisulfide-functionalized water-soluble reactive copolymers, which are synthesized via reversible addition-fragmentation chain transfer copolymerization. Obtained nanogels with loaded cellulase demonstrate very good colloidal stability in aqueous solutions. The enzymatic activity of cellulase is greatly reduced when encapsulated in the nanogels and rapidly recovered in 10 × 10 -3 m dithiothreitol solution. Fluorescence resonance energy transfer (FRET)-based experiments indicate that the recovered enzymatic activity is mainly ascribed to the release of the enzyme due to the degradation of the disulfide crosslinking network after addition of dithiothreitol (DTT), instead of the enhanced substrate transport rate. The developed enzyme immobilization method opens new possibilities for reversible activation/deactivation of enzymes and opens up new directions for targeted protein therapy and biotechnology applications., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The causal association between hepatitis virus infections and lichen planus (LP) remains a matter of controversy. Reliable figures for German patients are still lacking., Patients and Methods: We analyzed the prevalence of serum antibodies against hepatitis B and C viruses (HBV, HCV) in 265 LP patients and compared the results to 257 patients with chronic urticaria (URT) and 222 patients with malignant melanoma (MM). Additionally, we analyzed age- and sex-specific differences., Results: The prevalence of HBV (13.2 % or 14.7 %) antibodies was significantly higher in patients with LP and URT patients than in the MM control group (HBV: 5.4 %, p < 0.001). The prevalence of HCV antibodies among LP and URT patients (2.6 % or 0.8 %) was not significantly greater (p > 0.05) than in MM patients (0.4 %). The prevalences of HBV and HCV in the MM control group were comparable to those in the general German population (HBV: 5-8 %, HCV: 0.4-0.7 %). An analysis by sub-groups showed that these differences resulted from sex- and age-dependent prevalences. HBV antibodies were significantly more common only in male LP patients (16.1 %) and in male (20.0 %) and female URT patients (15.6 %) aged 31-60 years. The greater prevalence of HCV in female LP patients older than 60 years of age was not significant., Conclusions: LP is not specifically associated with hepatitis B or C antibodies in the Caucasian population of Germany. The higher prevalences of HBV antibodies found in LP and URT patients may be a reflection of age- and sex-related factors rather than disease-specific exposure to HBV infections. The increased prevalences seen in patients with LP or URT compared to MM patients may suggest that HBV infection serves as an unspecific trigger for a specific immune reaction of another origin., (© The Authors • Journal compilation © Blackwell Verlag GmbH, Berlin.)

Patients with the depigmentation disorder vitiligo have low catalase expression/activities and constantly accumulate 10(-3) M hydrogen peroxide (H(2)O(2)) in their skin. Such high concentrations of H(2)O(2) oxidize L-methionine residues in proteins and peptides to (R and S)-methionine sulfoxide diasteriomers. In vivo FT-Raman Spectroscopy revealed the presence of methionine sulfoxide in the depigmented skin of patients with active vitiligo. In normal healthy human skin, methionine sulfoxide reductases A and B specifically reduce methionine sulfoxides (S) and (R), respectively, back to L-methionine consequently repairing oxidatively damaged proteins and peptides. In this report, we show that the expression/activities of MSRA and MSRB are significantly decreased in the epidermis of patients with vitiligo compared to healthy controls. Also, we used recombinant human MSRA and MSRB1 to show that both enzymes are deactivated by 10(-3) M H(2)O(2) by 85 and 40%, respectively. Structural modelling based on the crystal structure of human MSRA revealed that the active site of this enzyme is significantly altered after H(2)O(2)-mediated oxidation of L-methionine, L-tryptophan, and L-cysteine residues in its active site. Taken together, our results confirm that very important anti-oxidant enzymes are seriously affected in acute vitiligo.

Oxidation of methionine residues by reactive oxygen (ROS) in protein structures leads to the formation of methionine sulfoxide which can consequently lead to a plethora of impaired functionality. The generation of methionine sulfoxide yields ultimately a diastereomeric mixture of the S and R sulfoxides. So far two distinct enzyme families have been identified. MSRA reduces methionine S-sulfoxide, while MSRB reduces the R-diastereomer. It has been shown that these enzymes are involved in regulation of protein function and in elimination of ROS via reversible methionine formation besides protein repair. Importantly, both enzymes require coupling to the NADPH/thioredoxin reductase/thioredoxin electron donor system. In this report, we show for the first time the expression and function of both sulfoxide reductases together with thioredoxin reductase in the cytosol as well as in the nucleus of epidermal melanocytes which are especially sensitive to ROS. Since this cell resides in the basal layer of the epidermis and its numbers and functions are reduced upon ageing and for instance also in depigmentation processes, we believe that this discovery adds an intricate repair mechanism to melanocyte homeostasis and survival.