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Turning a Killing Mechanism into an Adhesion and Antifouling Advantage
Mild and universal methods to introduce functionality in polymeric surfaces remain a challenge. Herein, a bacterial killing mechanism based on amphiphilic antimicrobial peptides is turned into an adhesion advantage. Surface activity (surfactant) of the antimicrobial liquid chromatography peak I (LCI) peptide is exploited to achieve irreversible binding of a protein–polymer hybrid to surfaces via physical interactions. The protein–polymer hybrid consists of two blocks, a surface-affine block (LCI) and a functional block to prevent protein fouling on surfaces by grafting antifouling polymers via single electron transfer-living radical polymerization (SET-LRP). The mild conditions of SET-LRP of N-2-hydroxy propyl methacrylamide (HPMA) and carboxybetaine methacrylamide (CBMAA) preserve the secondary structure of the fusion protein. Adsorption kinetics and grafting densities are assessed using surface plasmon resonance and ellipsometry on model gold surfaces, while the functionalization of a range of artificial and natural surfaces, including teeth, is directly observed by confocal microscopy. Notably, the fusion protein modified with poly(HPMA) completely prevents the fouling from human blood plasma and thereby exhibits a resistance to protein fouling that is comparable to the best grafted-from polymer brushes. This, combined with their simple application on a large variety of materials, highlights the universal and scalable character of the antifouling concept. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2-Methyl-2,4-pentanediol (MPD) boosts as detergent-substitute the performance of ß-barrel hybrid catalyst for phenylacetylene polymerization
Covering hydrophobic regions with stabilization agents to solubilize purified transmembrane proteins is crucial for their application in aqueous media. The small molecule 2-methyl-2,4-pentanediol (MPD) was used to stabilize the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA) utilized as host for the construction of a rhodium-based biohybrid catalyst. Unlike commonly used detergents such as sodium dodecyl sulfate or polyethylene polyethyleneglycol, MPD does not form micelles in solution. Molecular dynamics simulations revealed the effect and position of stabilizing MPD molecules. The advantage of the amphiphilic MPD over micelle-forming detergents is demonstrated in the polymerization of phenylacetylene, showing a ten-fold increase in yield and increased molecular weights.
Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability
Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15–21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15–22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.
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.
Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?
Despite the significant advances in the field of protein engineering, general design principles to improve organic cosolvent resistance of enzymes still remain undiscovered. Previous studies drew conclusions to engineer enzymes for their use in water-miscible organic solvents based on few amino acid substitutions. In this study, we conduct a comparison of a Bacillus subtilis lipase A (BSLA) library—covering the full natural diversity of single amino acid substitutions at all 181 positions of BSLA—with three state of the art random mutagenesis methods: error-prone PCR (epPCR) with low and high mutagenesis frequency (epPCR-low and high) as well as a transversion-enriched Sequence Saturation Mutagenesis (SeSaM-Tv P/P) method. Libraries were searched for amino acid substitutions that increase the enzyme’s resistance to the water-miscible organic cosolvents 1,4-dioxane (DOX), 2,2,2-trifluoroethanol (TFE), and dimethyl sulfoxide (DMSO). Our analysis revealed that 5%–11% of all possible single substitutions (BSLA site-saturation mutagenesis (SSM) library) contribute to improved cosolvent resistance. However, only a fraction of these substitutions (7%–12%) could be detected in the three random mutagenesis libraries. To our knowledge, this is the first study that quantifies the capability of these diversity generation methods generally employed in directed evolution campaigns and compares them to the entire natural diversity with a single substitution. Additionally, the investigation of the BSLA SSM library revealed only few common beneficial substitutions for all three cosolvents as well as the importance of introducing surface charges for organic cosolvent resistance—most likely due to a stronger attraction of water molecules. © 2017 by the authors.
One‐Pot Two‐Step Chemoenzymatic Cascade for the Synthesis of a Bis‐benzofuran Derivative
Chemoenzymatic cascades enable reactions with the high productivity of chemocatalysts and high selectivity of enzymes. Nevertheless, the combination of these different fields of catalysis is prone to mutual deactivation of metal- and biocatalysts. In this study, a one-pot sequential two-step catalytic cascade reaction was successfully implemented for the synthesis of a methylene-bridged bis(2-substituted benzofuran). In the first step, a palladium-free Sonogashira reaction is used for the synthesis of a benzofuran derivative. In the subsequent step, the formed 2-substituted benzofuran is hydroxylated by the monooxygenase P450 BM3 variant (A74S-F87V-L188Q) and undergoes further elimination reactions. The study proofs that combination of Cu scorpionate catalyzed Sonogashira cross-coupling and P450 mediated oxidation is possible and results in up to 84 % yield of the final product. The oxidation reaction is boosted by capturing inhibiting reaction components.
A bifunctional dermaseptin–thanatin dipeptide functionalizes the crop surface for sustainable pest management
To reduce pesticide use while preserving crop productivity, alternative pest and disease control measures are needed. We thought of an alternative way of functionalizing leaves of soybean to fight its most severe disease, Asian soybean rust (Phakopsora pachyrhizi). To do so, we produced bifunctional peptides that adhere to the soybean leaf surface and prevent the germination of P. pachyrhizi spores. In detail, amphiphilic peptides liquid chromatography peak I (LCI), thanatin (THA), tachystatin A2 (TA2), and lactoferricin B (LFB) were all fused to enhanced green fluorescent protein (eGFP). Of these fusion peptides, eGFP–LCI and eGFP–THA bound strongly and in a rainfast manner to the surface of soybean, barley, and corn leaves. eGFP–THA binding to soybean also withstood high temperature, sunlight and biotic degradation for at least 17 days. The dipeptides seem to bind mainly to the surface wax layer of leaves because eGFP–THA and eGFP–LCI did not stick to the wax-depleted cer-j59 mutant of barley or to corn leaves with their surface wax removed. A fusion of the antimicrobial peptide dermaseptin 01 and THA (DS01–THA) inhibits the germination of P. pachyrhizi spores in vitro and reduces Asian soybean rust disease in a rainfast manner. Therefore, this study reveals that bifunctional peptides can be used to functionalize the crop surface for sustainable disease management.
Correction: A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels
Correction for ‘A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels’ by Nina Lülsdorf et al., Chem. Commun., 2015, 51, 8679–8682.
Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles
The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation.
CaLB Catalyzed Conversion of ε-Caprolactone in Aqueous Medium. Part 1: Immobilization of CaLB to Microgels
The enzymatic ring-opening polymerization of lactones is a method of increasing interest for the synthesis of biodegradable and biocompatible polymers. In the past it was shown that immobilization of Candida antarctica lipase B (CaLB) and the reaction medium play an important role in the polymerization ability especially of medium ring size lactones like ε-caprolactone (ε-CL). We investigated a route for the preparation of compartmentalized microgels based on poly(glycidol) in which CaLB was immobilized to increase its esterification ability. To find the ideal environment for CaLB, we investigated the acceptable water concentration and the accessibility for the monomer in model polymerizations in toluene and analyzed the obtained oligomers/polymers by NMR and SEC. We observed a sufficient accessibility for ε-CL to a toluene like hydrophobic phase imitating a hydrophobic microgel. Comparing free CaLB and Novozym® 435 we found that not the monomer concentration but rather the solubility of the enzyme, as well as the water concentration, strongly influences the equilibrium of esterification and hydrolysis. On the basis of these investigations, microgels of different polarity were prepared and successfully loaded with CaLB by physical entrapment. By comparison of immobilized and free CaLB, we demonstrated an effect of the hydrophobicity of the microenvironment of CaLB on its enzymatic activity.