Single-quantum Coherence Nuclear Magnetic Resonance Research Articles

Acid-Catalyzed Solvolysis of Softwood in Caprylyl Glycol to Produce Lignin Derivatives.

Glycol lignin (GL) is produced via the acid-catalyzed solvolysis of softwood meal using glycols such as polyethylene glycol. The physicochemical and thermal properties of GL are expected to be controllable by varying the glycol type. In this study, caprylyl glycol (CG), which is a biobased glycol derived from the caprylic acid component of coconut and palm oils, was used to investigate the effects on the acid-catalyzed solvolysis of softwood. The reactions were performed at 140, 150, and 160 °C for 30-120 min to obtain lignin derivatives (CG-lignin: CGL). The chemical and physical properties of CGLs were investigated. Two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance analysis suggested that CGL possesses an α-CG-β-O-4 structure as CG is grafted onto the lignin structure. CGLs prepared at higher reaction temperatures exhibited lower molecular weights. The thermomechanical analysis of CGL revealed a glass transition temperature of 89-96 °C and a viscous thermal flow temperature of 134-155 °C.

The Limitation of Unproductive Binding of Cellulases to Lignin by Ozone Pretreatment

The limitation of enzymatic saccharification of lignocellulose is attributed to the nonproductive adsorption between lignin and cellulase. This study aims to investigate the effects of ozone pretreatment on the physical structure and chemical properties of milled wood lignin (MWL). The objective is to reduce the non-productive adsorption of cellulase on lignin. The structure–activity relationship between the physical structure of MWL and the occurrence of nonproductive adsorption was analysed using two-dimensional heteronuclear single quantum coherence–nuclear magnetic resonance (HSQC-NMR) and phosphorus-31 nuclear magnetic resonance spectrum (31P-NMR), etc. The results indicate that ozone pretreatment resulted in a decrease in the phenolic hydroxyl content and S/G ratio, an increase in the carboxyl content, and a negative zeta potential of MWL. The maximum adsorption capacity decreased from 25.77 mg/g to 10.09 mg/g, the Langmuir constant decreased from 13.86 mL/mg to 10.11 mL/mg, and the binding strength decreased from 357.14 mL/g to 102.04 mL/g, as determined by Langmuir isothermal adsorption. This suggests that ozone pretreatment resulted in a reduction in the hydrophobicity of lignin and a weakening of the electrostatic attraction between lignin and cellulase, thereby effectively reducing the non-productive adsorption of cellulase on lignin. This study provides an environmentally friendly pretreatment technique and comprehensively analyses the structural changes of ozone-treated MWL. These findings contribute to a deeper understanding of the interaction between lignin and cellulase.

1H-13C HSQC-NMR Analysis of Cranberry (Vaccinium Macrocarpon) Juice Defines the Chemical Composition of Juice Precipitate.

Shelf-stable cranberry juice precipitate has not been well characterized. Here, we describe using 1H-13C heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR) spectroscopy for cranberry juice analysis, focusing on proanthocyanidins and the precipitate. HSQC-NMR cross-peaks from juices were categorized as aliphatic, olefinic, aromatic, carbohydrate backbone, or anomeric signals. An average cranberry juice precipitate had significantly more aromatic and significantly less carbohydrate backbone signals than an average supernatant. The precipitate was a collection of biomolecules held together by a mix of weak and strong intermolecular forces. Proanthocyanidin signals from precipitates of juices showed 22 ± 2 to 29.9 ± 0.7% A-type interflavan linkages and 34 ± 2 to 48 ± 3% of flavan-3-ol units with trans stereochemistry between the C2 and C3 positions. Based on this work, 1H-13C HSQC-NMR is useful to analyze cranberry juice and reveals the complex chemical nature of components in the soluble and insoluble phases.

Deciphering the contribution of microbial biomass to the properties of dissolved and particulate organic matter in anaerobic digestates

The recalcitrant structures either from substrate or microbial biomass contained in digestates after anaerobic digestion (AD) highly influence digestate valorization. To properly assess the microbial biomass contribution to the digested organic matter (OM), a combination of characterization methods and the use of various substrate types in anaerobic continuous reactors was required. The use of totally biodegradable substrates allowed detecting soluble microbial products via fluorescence spectroscopy at emission wavelengths of 420 and 460 nm while the protein-like signature was enhanced by the whey protein. During reactors' operation, a transfer of complex compounds to the dissolved OM from the particulate OM was observed through fluorescence applied on biochemical fractionation. Consequently, the fluorescence complexity index of the dissolved OM increased from 0.59–0.60 to 1.06–1.07, whereas it decreased inversely for the extractable soluble from the particulate OM from 1.16–1.19 to 0.42–0.54. Accordingly, fluorescence regional integration showed differences among reactors based on visual inspection and orthogonal partial latent structures (OPLS) analysis. Similarly, the impact of the substrate type and operation time on the particulate OM was revealed by 13C nuclear magnetic resonance using OPLS, providing a good model (R2X = 0.93 and Q2 = 0.8) with a clear time-trend. A high signal resonated at ∼30 ppm attributed to CH2-groups in the aliphatic chain of lipid-like structure besides carbohydrates intensities at 60–110 ppm distinguished the reactor fed with whey protein from the other, which was mostly biomass related. Indeed, this latter displayed a higher presence of peptidoglycan (δH/C: 1.6–2.0/20–25 ppm) derived from microbial biomass by 1H-13C heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance. Interestingly, the sample distribution obtained by non-metric multidimensional scaling of bacterial communities resembled the attained using 13C NMR properties, opening new research perspectives. Overall, this study discloses the microbial biomass contribution to digestates composition to improve the OM transformation mechanism knowledge.

Hydrogen Transfer Hydrogenolysis of Organosolv Chinese Fir Lignin to Monophenols over NiZnAlOx Catalyst

Lignin is the only naturally renewable aromatized polymer consisting of several phenyl propane structures linked by C–O and C–C bonds, so lignin can be depolymerized into value-added chemicals or liquid fuels. In this study, M5Zn5AlOx (M = Co, Ni and Cu) catalysts were obtained by the co-precipitation method and then were used in organosolv lignin depolymerization. Among these catalysts, the Ni5Zn5AlOx catalyst possessed the largest surface area and abundant surface oxygen vacancies as well as strong acidic sites on the surface, giving the highest yield of monophenols (about 14.49 wt %). The effect of Ni/Zn ratios on the lignin depolymerization was also investigated, and it was found that the surface area and the proportion of surface oxygen vacancies and strong acidic sites of the NiZnAlOx catalysts increased and then decreased with the Ni/Zn ratios increasing. Similarly, the yield of monomeric compounds increased and then decreased with the Ni/Zn ratios increasing. The highest yield of monophenols was 17.18 wt % obtained over the Ni3Zn7AlOx catalyst, a remarkable monomer yield from organosolv lignin. The two-dimensional 1H-13C heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of bio-oil revealed that the linkage bonds in lignin could be effectively broken over the Ni3Zn7AlOx catalyst. This study provided an effective route to obtain high-value chemicals from organosolv lignin under nickel-based catalysts.

Production of Exopolysaccharides by Cultivation of Halotolerant Bacillus atrophaeus BU4 in Glucose- and Xylose-Based Synthetic Media and in Hydrolysates of Quinoa Stalks

A halotolerant, exopolysaccharide-producing bacterium isolated from the Salar de Uyuni salt flat in Bolivia was identified as Bacillus atrophaeus using next-generation sequencing. Comparisons indicate that the genome most likely (p-value: 0.0024) belongs to a subspecies previously not represented in the database. The growth of the bacterial strain and its ability to produce exopolysaccharides (EPS) in synthetic media with glucose or xylose as carbon sources, and in hydrolysates of quinoa stalks, was investigated. The strain grew well in all synthetic media, but the growth in glucose was better than that in xylose. Sugar consumption was better when initial concentrations were low. The growth was good in enzymatically produced cellulosic hydrolysates but was inhibited in hemicellulosic hydrolysates produced using hydrothermal pretreatment. The EPS yields were up to 0.064 g/g on initial glucose and 0.047 g/g on initial xylose, and was higher in media with relatively low sugar concentrations. The EPS was isolated and purified by a sequential procedure including centrifugation, cold ethanol precipitation, trichloroacetic acid treatment, dialysis, and freeze-drying. Glucose and mannose were the main sugars identified in hydrolyzed EPS. The EPS was characterized by size-exclusion chromatography, Fourier-transform infrared (FTIR) spectroscopy, heteronuclear single-quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopy, scanning electron microscopy, X-ray diffraction, and thermogravimetric analysis. No major differences were elucidated between EPS resulting from cultivations in glucose- or-xylose-based synthetic media, while some divergences with regard to molecular-weight averages and FTIR and HSQC NMR spectra were detected for EPS from hydrolysate-based media.

Chemical Structures of Adhesive and Interphase Parts in Sucrose/Citric Acid Type Adhesive Wood-Based Molding Derived from Japanese Cedar (Cryptomeria japonica).

In sucrose/citric acid based wood adhesive, the detailed bonding mechanism has still been unknown. Here, we investigated the detailed chemical structures of this adhesive wood (Japanese cedar)-based molding by using heteronuclear single quantum coherence–nuclear magnetic resonance (HSQC-NMR). NMR peaks associated with the furan-type structure appeared, suggesting that the furan compound was formed from sucrose and converted to a furan polymer during the adhesive process and that some of the furan structures in the polymers were ester-bonded with citric acid. The secondary forces between the furan polymers and wood components were thought to contribute to the adhesive effect. In our analysis of the interphase structure, primary hydroxyl groups of both polysaccharides and of lignin substructures were found to be esterified with citric acid. Additionally, some of the glycosidic bonds in polysaccharides were cleaved during the acidic condition produced by citric acid. The above results provided evidence of the polymerization of sucrose-derived 5-HMF, the esterification of wood components, and the degradation of polysaccharides during the molding process. Citric acid functioned as a clamp between the obtained furan polymer and the wood components. The sucrose/citric acid based wood adhesive can be defined as a hybrid-type wood adhesive, involving both secondary forces and chemical bonding interactions.

Repurposing Acitretin as an Antipseudomonal Agent Targeting the Pseudomonas aeruginosa Iron-Regulated Heme Oxygenase.

Iron is an essential micronutrient for the survival and virulence of the bacterial pathogen Pseudomonas aeruginosa. To overcome iron withholding and successfully colonize a host, P. aeruginosa uses a variety of mechanisms to acquire iron, including the secretion of high-affinity iron chelators (siderophores) or the uptake and utilization of heme. P. aeruginosa heme oxygenase (HemO) plays pivotal roles in heme sensing, uptake, and utilization and has emerged as a therapeutic target for the development of antipseudomonal agents. Using a high-throughput fluorescence quenching assay combined with minimum inhibitory concentration measurements, we screened the Selleck Bioactive collection of 2100 compounds and identified acitretin, a Food and Drug Administration-approved oral retinoid, as a potent and selective inhibitor of HemO. Acitretin binds to HemO with a KD value of 0.10 ± 0.02 μM and inhibits the growth of P. aeruginosa PAO1 with an IC50 of 70 ± 18 μg/mL. In addition, acitretin showed good selectivity for HemO, which uniquely generates BVIXβ/δ, over human heme oxygenase (hHO1) and other BVIXα-producing homologues such as the heme oxygenases from Neisseria meningitidis (nmHO) and Acinetobacter baumannii (abHO). The binding of acitretin within the HemO active site was confirmed by 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance, and molecular modeling provided further insight into potential interactions of acitretin with residues specific for orienting heme in the β/δ selective HemO. Moreover, at 20 μM, acitretin inhibited the enzymatic activity of HemO in P. aeruginosa cells by >60% and effectively blocked the ability of P. aeruginosa to sense and acquire heme as demonstrated in the β-galactosidase transcriptional reporter assay.

Chemical Composition, Antioxidant and Antibacterial Activities of Essential Oil From Cymbopogon densiflorus (Steud.) Stapf Flowers

This study reports, for the first time, the chemical composition and biological activity of essential oil from Brazilian Cymbopogon densiflorus flowers. Thirty chemical compounds were identified by gas chromatography coupled with mass spectrometry (GC/MS), corresponding to 95 % of total compounds from an essential oil chromatogram obtained by gas chromatography-flame ionization detection (GC-FID ). The essential oil was mainly composed of cis-p-mentha-1(7),8-dien-2-ol (26 %), trans-p-mentha-1(7),8-dien-2-ol (18 %), trans-p-mentha-2,8-dien-1-ol (13 %) limonene (9 %), cis-p-mentha-2,8-dien-1-ol (7 %), cis-carveol (7 %) and carvone (5 %). The identity confirmation of the major compounds was performed by 1H/13C heteronuclear single-quantum coherence nuclear magnetic resonance (HSQC NMR). The essential oil exhibited a weak antioxidant activity by the DPPH method and a wide spectrum of antimicrobial activity against the pathogenic bacterial strains tested, showing some efficacy against 7 of the 10 strains with the greatest inhibition observed with the Gram-negative bacterium Shigella sonnei.

Bond Structures between Wood Components and Citric Acid in Wood-Based Molding.

Citric acid-based wood adhesive is considered a chemical-bonding wood adhesive. However, the detailed structures of the bonds between wood components and citric acid remain unknown. Here, we examine the chemical bonding structures between citric acid and wood by heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR) analysis of wood-based molding using Japanese cedar (Cryptomeria japonica) and citric acid. In the HSQC-NMR spectrum of the wood molding, some esterified C/H correlation peaks appeared. The primary hydroxyl groups of polysaccharides, such as cellulose and galactoglucomannan, and the primary hydroxyl groups of the β-O-4 and β-5 substructures in lignin were found to be esterified with citric acid. In contrast, the secondary hydroxyl groups, except for xylan, barely reacted because of the steric hindrance. Additionally, the C/H correlation peak volumes of the reducing ends of mannan and xylan in the anomeric region increased after molding. It was clarified that the glycosidic bonds in the hemicelluloses were cleaved under the acidic molding condition with citric acid. The HSQC-NMR analysis revealed that the esterification of hemicellulose and lignin, and degradation of hemicellulose, proceeded under the molding condition. These results will promote understanding of the adhesive mechanism of citric acid-based wood adhesive and of the properties of the molding.

Characterization of Immature Bamboo (Phyllostachys nigra) Component Changes with Its Growth via Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance Spectroscopy.

A 6.2 m high immature bamboo (Phyllostachys nigra) was divided into seven fractions. The bamboo cell walls and lignin samples from young to old were characterized by 1H-13C correlation heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy both qualitatively and semiquantitatively. Mature bamboo and bamboo shoot samples were used as comparison references. HSQC-NMR analysis proved that cellulose and arabinoxylan have already deposited in bamboo shoot, and cellulose amount increased during growth. Lignin side chain linkage formation started from β-ether (β-O-4), then phenylcoumaran (β-5), and finally resinol (β-β). Ferulic acid and p-coumaric acid (pCA) were formed at the earlier stages in the immature bamboo, and the pCA proportion decreased throughout the lignification process. We propose that the bamboo lignification process is distinct from both woody and other herbaceous plants, where syringyl units deposited at the early stage and polymerized with the β-O-4 linkage. Then guaiacyl units formed gradually, and finally, p-hydroxyphenyl units formed.

Synergistic effect of hydrogen peroxide and ammonia on lignin

To reveal the synergistic effect of hydrogen peroxide and ammonia on lignin, four kinds of ball-milled wood lignins, named as UN-L, HP-L, AFEX-L and H-AFEX-L respectively, were isolated from corn stover of untreated, hydrogen peroxide pretreatment alone, ammonia fiber expansion pretreatment alone and combined hydrogen peroxide and ammonia fiber expansion pretreatment. Modern analytical techniques including elemental analysis, gel permeation chromatography (GPC), Fourier transform infrared (FT-IR) spectroscopy and two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (2D-HSQC NMR) spectroscopy were applied to analyze the characteristics of the obtained lignins. The results showed that oxidation, ammonolysis were main chemical reaction during H-AFEX pretreatment. The action site of oxidation was S unit, while that of ammonolysis were ferulate and p-coumarate ester bonds. The synergistic effect of hydrogen peroxide and ammonia strengthened ammonolysis effect whereas weakened oxidation effect. Ammonia selectively degraded the ester bond, while hydrogen peroxide selectively degraded the resinol structure. The synergistic effect of hydrogen peroxide and ammonia could not only effectively degrade lignin, but also greatly reduce its PI to obtain homogeneous lignin fragments.

Study on the chemical modification of alkali lignin towards for cellulase adsorbent application

The research of cost-efficient lignin-based adsorbents is a practical strategy for the recovery of cellulase. In this study, alkali lignin was modified to increase the phenolic hydroxyl (Ph-OH) content for cellulase adsorption applications. After phenolation, compared with the lignin reference, the maximum adsorption cellulase capacity of lignoresorcinol (LigR) and lignopyrogallol (LigP) was improved from 76.5 mg/g to 842.1 mg/g and 911.4 mg/g, respectively. The enzyme activity of the adsorbed cellulase on LigR was higher than that on LigP, which could migrate to the fresh substrates during enzymatic hydrolysis. The adsorbed cellulase could be easily recovered from two lignin-based adsorbents by adjusting pH. The distinct cellulase adsorption behavior of two lignin-based adsorbents was closely related to the high Ph-OH contents and low S/G ratio in phenolated lignin samples characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Heteronuclear Single Quantum Coherence-Nuclear Magnetic Resonance (HSQC-NMR).

Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A.

Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to require acidic lipids in the cytoplasmic membrane, whereas its close homologue colicin N shows neither behavior. Compared to that of ColN-P, the ColA-P primary structure reveals the replacement of several uncharged residues with aspartyl residues, which upon replacement with alanine induce an unfolded state at neutral pH. Here we investigate ColA-P’s structural requirement for these critical aspartyl residues that are largely situated at the N-termini of α helices. As previously shown in model peptides, the charged carboxylate side chain can act as a stabilizing helix N-Cap group by interacting with free amide hydrogen bond donors. Because this could explain ColA-P destabilization when the aspartyl residues are protonated or replaced with alanyl residues, we test the hypothesis by inserting asparagine, glutamine, and glutamate residues at these sites. We combine urea (fluorescence and circular dichroism) and thermal (circular dichroism and differential scanning calorimetry) denaturation experiments with 1H–15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of ColA-P at different pH values to provide a comprehensive description of the unfolding process and confirm the N-Cap hypothesis. Furthermore, we reveal that, in urea, the single domain ColA-P unfolds in two steps; low pH destabilizes the first step and stabilizes the second.

Molecular characterization of particulate organic matter in full scale anaerobic digesters: An NMR spectroscopy study

This study assesses the molecular characteristics of particulate organic matter (POM) in agricultural and food waste digesters and elucidates the molecular properties of the recalcitrant POM fraction, which remains in the digestate after AD process. Molecular properties of POM in influent (substrate) and effluent (digestate) of seven full-scale AD plants (three agricultural waste and four food waste digesters) were characterized and compared using solid-state 13C cross-polarization magic angle spinning (CP-MAS) and solution-state 1H,13C heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy. Comparison of the POM structural compositions of substrate and digestate from each AD plant revealed an enrichment of protein structures relative to the carbohydrates in most cases, implying a preferential degradation of the carbohydrates over proteins and/or increase of microbial biomass upon AD of agricultural and food wastes. Distinctive molecular structures of labile and recalcitrant fractions of POM, subjected to AD, were identified by comparing the NMR spectra of all substrate and digestate POM. Accordingly, the labile POM fraction in food and agricultural solid wastes is characterized by structural entities of lipids and starch-like carbohydrates, whereas recalcitrant POM structures resemble alkyl and aromatic subunits of amino acids, lignin, and polysaccharides with β-glycosidic linkages. This information serves as a basis to further explore optimization approaches for improving AD of the underutilized POM and the fate of organic matter in digestate-amended arable lands.

Recombinant Butelase-Mediated Cyclization of the p53-Binding Domain of the Oncoprotein MdmX-Stabilized Protein Conformation as a Promising Model for Structural Investigation

Cyclization of the polypeptide backbone has proven to be a powerful strategy for enhancing protein stability for fundamental research and pharmaceutical application. The use of such an approach is restricted by how well a targeted polypeptide can be efficiently ligated. Recently, an Asx-specific peptide ligase identified from a tropical cyclotide-producing plant and named butelase 1 exhibited excellent cyclization kinetics that cannot be matched by other known ligases, including intein, PATG, PCY1, and sortase A. In this work, we aimed to examine whether butelase 1 facilitated protein conformational stability for structural investigation. First, we successfully expressed recombinant butelase 1 (rBTase) in the yeast Pichia pastoris. Next, rBTase was shown to be highly efficient in the cyclization of the p53-binding domain (N-terminal domain) of murine double minute X (N-MdmX), an important target for designing anticancer drugs. The cyclized N-MdmX (cMdmX) exhibited increased conformational stability and improved interaction with the ligand compared with those of noncyclized N-MdmX. Importantly, the thermal melting process was completely reversible, contrary to noncyclized N-MdmX, and the melting temperature ( Tm) of cMdmX was increased to 47 from 43 °C. This stable conformation of cMdmX was further confirmed by 15N-1H heteronuclear single-quantum coherence nuclear magnetic resonance (NMR) spectroscopy. The complex of cMdmX and the ligand was tested for protein crystallization, and several promising findings were revealed. Therefore, our work not only provides a recombinant version of butelase 1 but also suggests a conventional approach for preparing stable protein samples for both protein crystallization and NMR structural investigation.

Comprehensive understanding the chemical structure evolution and crucial intermediate radical in situ observation in enzymatic hydrolysis/mild acidolysis lignin pyrolysis

The in-depth understanding of free radical involved in lignin pyrolysis, is the most important technical barrier for pyrolysis mechanism research. Herein, we report an in situ project by using EPR high temperature cavity for the observation of generated radical in lignin pyrolysis, along with the other state-of-the-art techniques such as two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (2D HSQC-NMR) and Py-GCMS combined with high temperature oven. The result showed that the originate lignin structure had significant influence on the pyrolytic products and the radical process. Compared with hardwood and non-wood lignin, softwood lignin tented to produce more amount of radical, due to the higher content of guaiacyl type subunits and phenylcoumaran structure. Corresponding with the two mainly pyrolytic reaction stage according the activation energy, the radical reaction of lignin pyrolysis was divided into three stages: radical inducing stage, the main reacting stage and the quenching stage.

Modified Hydrotropic Pretreatment of Eucalyptus under Alkali and Acid Conditions for Lignin Removal and Enhancing Enzymatic Hydrolysis

Hydrotropic pretreatment is an environmentally friendly technology that can be applied for lignin isolation from lignocellulosic biomass to enhance enzymatic hydrolysis. In this study, conventional hydrotropic pretreatment was modified with additional alkali and acid to investigate the lignin removal mechanism. Lignin recovered from the hydrotropic solution was analyzed by Heteronuclear Single Quantum Coherence-Nuclear Magnetic Resonance (HSQC-NMR). It was found that the amounts of β-O-4 linkages in the different types of hydrotropic lignin were more than 50%. Alkali-hydrotropic lignin particularly contained 74.9% of β-O-4 linkages. Also, the cleavage of α-O-4 formed more phenolic groups in alkali-hydrotropic lignin. The addition of acid in the hydrotropic treatment could reinforce the broken β-O-4 bond. Modified hydrotropic pretreatments more easily displaced the lignin from the fiber surface and improved the enzymatic accessibility of the pretreated substrates. Considering other impact factors including the fiber structure and total lignin content, enzymatic hydrolysis was heavily influenced and facilitated by the acid-hydrotropic method.

Influence of drying process on reactivity of cellulose and xylan in acetylation of willow (Salix schwerinii E. L. Wolf) kraft pulp monitored by HSQC-NMR spectroscopy

The acetylation behavior of xylan and cellulose in kraft pulp (KP) was investigated. Heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR) spectroscopy was used to identify the positions at which the hydroxyl groups of polysaccharides were acetylated. X-ray diffraction analysis was used to determine the acetylation of cellulose in the crystal region. It was found that only xylan was obviously acetylated in the initial stages of the reaction. In the next stage of the acetylation, however, both the xylan and surface of the cellulose crystals reacted. Then, acetylation of the inner crystal region of the cellulose started before the completion of the xylan modification. To improve the reactivity of xylan in KP, the effect of drying or the dehydration of never-dried KP prior to acetylation was also investigated. The use of supercritical CO2 (scCO2) drying produced a KP with a higher specific surface area. This caused the xylan to exhibit a higher reactivity than that resulting from freeze drying and vacuum drying after solvent exchange. Furthermore, never-dry dehydration pretreatment produced xylan with the highest reactivity. This suggests that the cellulose–cellulose and/or cellulose–hemicellulose adhesion in the cell wall that arises as a result of drying reduces the reactivity of the xylan.

Identification of major matrix metalloproteinase-20 proteolytic processing products of murine amelogenin and tyrosine-rich amelogenin peptide using a nuclear magnetic resonance spectroscopy based method

ObjectiveThe aim of this study was to identify major matrix metalloproteinase-20 (MMP20) proteolytic processing products of amelogenin over time and determine if the tyrosine-rich amelogenin peptide (TRAP) was a substrate of MMP20. DesignRecombinant15N-labeled murine amelogenin and 13C,15N-labeled TRAP were incubated with MMP20 under conditions where amelogenin self-assembles into nanospheres. Digestion products were fractionated by reverse-phase high-performance liquid chromatography at various time points. Product identification took advantage of the intrinsic disorder property of amelogenin that results in little change to its fingerprint 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectrum in 2% acetic acid upon removing parts of the protein, allowing cleavage site identification by observing which amide cross peaks disappear. ResultsThe primary product in five out of the six major reverse-phase high-performance liquid chromatography bands generated after a 24 h incubation of murine amelogenin with MMP20 were: S55-L163, P2-L147, P2-E162, P2-A167, and P2-R176. After 72 h these products were replaced with five major reverse-phase high-performance liquid chromatography bands containing: L46-A170, P2-S152, P2-F151, P2-W45, and short N-terminal peptides. TRAP was completely digested by MMP20 into multiple small peptides with the initial primary site of cleavage between S16 and Y17. ConclusionsIdentification of the major MMP20 proteolytic products of amelogenin confirm a dynamic process, with sites towards the C-terminus more rapidly attacked than sites near the N-terminus. This observation is consistent with nanosphere models where the C-terminus is exposed and the N-terminus more protected. One previously reported end-product of the MMP20 proteolytic processing of amelogenin, TRAP, is shown to be an in vitro substrate for MMP20.