Hanna Gracz

Photobactericidal Porphyrin-Cellulose Nanocrystals: Synthesis, Characterization, and Antimicrobial Properties

Adherence and survival of pathogenic bacteria on surfaces leading to concomitant transmission to new hosts significantly contributes to the proliferation of pathogens, which in turn considerably increases the threat to human health, particularly by antibiotic-resistant bacteria. Consequently, more research into effective surface disinfection and alternative materials (fabrics, plastics, or coatings) with antimicrobial and other bioactive characteristics is desirable. This report describes the synthesis and characterization of cellulose nanocrystals that were surface-modified with a cationic porphyrin. The porphyrin was appended onto the cellulose surface via the Cu(I)-catalyzed Huisgen-Meldal-Sharpless 1,3-dipolar cycloaddition having occurred between azide groups on the cellulosic surface and porphyrinic alkynes. The resulting, generally insoluble, crystalline material, CNC-Por (5), was characterized by infrared and diffusion (1)H NMR spectroscopies, gel permeation chromatography, and thermogravimetric analysis. Although only suspended, and not dissolved, in an aqueous system, CNC-Por (5) showed excellent efficacy toward the photodynamic inactivation of Mycobacterium smegmatis and Staphylococcus aureus , albeit only slight activity against Escherichia coli . The synthesis, properties, and activity of CNC-Por (5) described herein serve as a benchmark toward our overall objectives of developing novel, potent, bioactive, photobactericidal materials that are effective against a range of bacteria, with potential utilization in the health care and food preparation industries.

Lignin extraction from biomass with protic ionic liquids

A highly effective method has been developed for the simple extraction of lignin from lignocellulosic biomass using a potentially inexpensive protic ionic liquid (PIL). After the lignin-extraction step, the PIL is easily recovered using distillation leaving the separated lignin and cellulose-rich residues available for further processing. Biopolymer solubility tests indicate that increasing the xylan (i.e., hemicellulose) solubility in the PIL results in greater fiber disruption/penetration, which significantly enhances the effectiveness of the lignin extraction.

Structural Analysis of Residual and Technical Lignins by 1H-13C Correlation 2D NMR-Spectroscopy

Summary Structural analysis was conducted on residual lignin from pine Kraft AQ pulp, Eucalyptus Kraft lignin from Eucalyptus globulus and Repap Organosolv lignin by 2D 13C-1H correlation NMR spectroscopic techniques such as HMQC sequence. These lignins contain a rather wide variety of saturated aliphatic groups. The HMQC NMR spectra of the lignins do not verify the presence of diarylmethane moieties in any lignin investigated. The type and amount of other condensed structures depend on the nature of lignin preparation. All the lignins investigated still contained β-O-4′, pino- and syringayresinol (β-β′) and phenylcoumarane (β-5′) structures. Stilbene structures were also identified. Vinyl ether structures were present only in Eucalyptus Kraft lignin. All the lignins contain α-carbonyl groups conjugated to aromatic moieties as terminal side chains rather than involving β-O-4′ structures. No coniferyl alcohol and coniferyl aldehyde type structures are detected in the lignins after pulping. The spectra of kraft lignins show some new signals, the origin of which is discussed.

Different Modes of Binding of Mono-, Di-, and Trihalogenated Phenols to the Hemoglobin Dehaloperoxidase from Amphitrite ornata †

The hemoglobin dehaloperoxidase (DHP), found in the coelom of the terebellid polychaete Amphitrite ornata, is a dual-function protein that has the characteristics of both hemoglobins and peroxidases. In addition to oxygen transport function, DHP readily oxidizes halogenated phenols in the presence of hydrogen peroxide. The peroxidase activity of DHP is high relative to that of wild-type myoglobin or hemoglobin, but the most definitive difference in DHP is a well-defined substrate-binding site in the distal pocket, which was reported for 4-iodophenol in the X-ray crystal structure of DHP. The binding of 2,4,6-trihalogenated phenols is relevant since 2,4,6-tribromophenol is considered to be the native substrate and 2,4,6-trichlorophenol also gives high turnover rates in enzymatic studies. The most soluble trihalogenated phenol, 2,4,6-trifluorophenol, acts as a highly soluble structural analogue to the native substrate 2,4,6-tribromophenol. To improve our understanding of substrate binding, we compared the most soluble substrate analogues, 4-bromophenol, 2,4-dichlorophenol, and 2,4,6-trifluorophenol, using (1)H and (19)F NMR to probe substrate binding interactions in the active site of the low-spin metcyano adduct of DHP. Both mono- and dihalogenated phenols induced changes in resonances of the heme prosthetic group and an internal heme edge side chain, while (1)H NMR, (19)F NMR, and relaxation data for a 2,4,6-trihalogenated substrate indicate a mode of binding on the exterior of DHP. The differences in binding are correlated with differences in enzymatic activity for the substrates studied.

Biobleaching of pulp with dioxygen in the laccase-mediator system — reaction mechanisms for degradation of residual lignin

Abstract Pine Kraft-AQ pulp was biobleached with pressurized dioxygen at 40°C in laccase-mediator system (LMS), i.e. in acetate buffer (pH 4.5) containing Coriolus-laccase and 1-hydroxy-benzotriazole (HOBT), the latter being as a mediator. The LMS-treatment was followed by alkaline extraction (E) under standard conditions. The structures of the residual lignins before and after the biobleaching did not differ appreciably. This indicates that only a part of the residual lignin in the pulp undergoes oxidative degradation in the LMS treatment. In contrast, the treatment resulted in strong changes in the structure of the lignin isolated from E-effluents. The 2D HMQC (1H13C correlation) spectra showed the disappearance of β-O-4′, β-β′ and β-5′ bonds in the structure of the alkaline soluble lignin (ASL) from E-effluents, which are present in the 2D spectrum of the original residual lignin (RKL). In addition, the spectra exhibited new signals that are assigned to ArCOOH in biphenyl (5-5′) moieties. This implies that oxidative cleavage of side chains plays an important role in the delignification of pulp. The NMR studies also indicated that intensive degradation of aromatic ring has occurred in the biobleaching. However, premethylation of neither benzyl alcohol nor phenolic hydroxyl groups of the residual lignin in pulp before the biobleaching affected the rate of delignification. The latter indicates that phenolic moieties participate not only in oxidative degradation but also dehydrogenative polymerization reactions in the biobleaching. This is consistent with an appreciable increase in the proportion of fractions with higher molecular mass in lignin isolated from E-effluents.

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine 86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D [Protein Data Bank (PDB) entry 3MYN ] and M86E (PDB entry 3MYM ) mutants reveal the structural origins of a stable catalytic triad in DHP A. A decrease in the rate of H(2)O(2) activation as well as a lowered reduction potential versus that of the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine in binding to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is five-coordinate, and this structure resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a six-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a six-coordinate low-spin adduct, which inhibits the ligation and activation of H(2)O(2) as required for peroxidase activity. The results suggest that the proximal charge relay in peroxidases regulate the redox potential of the heme Fe but that the trans effect is a carefully balanced property that can both activate H(2)O(2) and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, we studied three M86 mutants, M86A, M86D, and M86E, by spectroelectrochemistry and nuclear magnetic resonance spectroscopy of (13)C- and (15)N-labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the N(δ) hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.

Cyclodextrin facilitated electrospun chitosan nanofibers

The use of chitosan, a cationic, biodegradable polysaccharide derived from sea-shells, in nanofibrous form offers a powerful platform to exploit its inherent benefits. However, chitosan nanofiber formation is difficult, requiring corrosive solvents or a carrier polymer blend to successfully electrospin. Our approach entails blending chitosan with a functional small molecule, cyclodextrin, to facilitate nanofiber formation of chitosan in acetic acid and trifluoroacetic acid. In this case the cyclodextrin, with its complexation properties, could serve to improve chitosan fiber formation, thus serving as a multi-functional blend. In this study, we examine the role of each component and the possibility of synergistic effects in nanofiber formation. Significant improvements in chitosan fiber formation were observed in concert with cyclodextrin at solvent concentrations not possible with just the individual components. Multiple fiber morphologies including three-dimensional fiber mats were also achieved. We examine the improved nanofiber formation in relation to solution viscosity, polymer entanglement, and chitosan–cyclodextrin associations. Rheological studies provide evidence of interactions between cyclodextrin and chitosan. NMR and FTIR studies further validate complexation between these two components.

Evaluation of physical and chemical properties and their interactions in fat, oil, and grease (FOG) deposits

Fat, oil and grease (FOG) blockages in sewer systems are a substantial problem in the United States. It has been estimated that over 50% of sewer overflows are a result of FOG blockages. In this work, a thorough laboratory study was undertaken to examine key variables that contribute to FOG deposit formation under controlled conditions. Physical and chemical properties and their interactions were evaluated and conditions that generated deposits that mimicked field FOG deposits were identified. It was found that 96 of the of 128 reaction conditions tested in the laboratory formed FOG deposits with similar physical and chemical characteristics as field FOG deposits. It was also found that FOG deposits can be created through fatty acid crystallization and not just saponification. Furthermore FOG deposits were found to be more complex than previously documented and contain free fatty acids, fatty acid metal salts, triacylglycerols, diacylglycerols and, monoacylglycerols. Lastly it was found that FOG deposits that only contained saturated fatty acids were on average 2.1 times higher yield strength than deposits that contained unsaturated fatty acids.

Dynamics of dehaloperoxidase-hemoglobin A derived from NMR relaxation spectroscopy and molecular dynamics simulation

Dehaloperoxidase-hemoglobin is the first hemoglobin identified with biologically-relevant oxidative functions, which include peroxidase, peroxygenase and oxidase activities. Herein we report a study of the protein backbone dynamics of DHP using heteronuclear NMR relaxation methods and molecular dynamics (MD) simulations to address the role of protein dynamics in switching from one function to another. The results show that DHPs backbone helical regions and turns have average order parameters of S2 = 0.87 ± 0.03 and S2 = 0.76 ± 0.08, respectively. Furthermore, DHP is primarily a monomer in solution based on the overall tumbling correlation time τm is 9.49 ± 1.65 ns calculated using the prolate diffusion tensor model in the program relax. A number of amino acid residues have significant Rex using the Lipari-Szabo model-free formalism. These include Lys3, Ile6, Leu13, Gln18, Arg32, Ser48, Met49, Thr56, Phe60, Arg69, Thr71 Cys73, Ala77, Asn81, Gly95, Arg109, Phe115, Leu127 and Met136, which may experience slow conformational motions on the microseconds-milliseconds time scale according to the model. Caution should be used when the model contains >4 fitting parameters. The program caver3.0 was used to identify tunnels inside DHP obtained from MD simulation snapshots that are consistent with the importance of the Xe binding site, which is located at the central intersection of the tunnels. These tunnels provide diffusion pathways for small ligands such as O2, H2O and H2O2 to enter the distal pocket independently of the trajectory of substrates and inhibitors, both of which are aromatic molecules.