In vitro degradation of insoluble lignin in aqueous media by lignin peroxidase and manganese peroxidase

The abilities of lignin peroxidase (LIP) and manganese peroxidase (MNP) fromPhanerochaete chrysosporium to degrade an insoluble hardwood lignin in vitro in aqueous media were tested. Neither LIP nor MNP appreciably changed the mass or lignin content, although both produced small amounts of unique solubilized lignin fragments. Treatment with both LIP and MNP, however, decreased the mass by 11%, decreased the lignin content by 5.1% (4.2% as total weight), and solubilized unique lignin-derived molecules. These results suggest that LIP and MNP synergistically degrade high molecular weight insoluble lignin, but singly, neither enzyme is sufficient to effect lignin degradation.     

Effect of strong detergents and chaotropes on the detection of proteins in two-dimensional gels.

The solubilization of a particular protein is mandatory for its subsequent resolution and detection in two-dimensional gels. However, the extraction solutions, that are compatible with the first-dimensional separation step, such as urea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), do not solubilize all proteins in a sample. We studied the effect of various common, strong detergents and chaotropes, widely used as solubilizing agents, such as sodium dodecyl sulfate, lithium dodecyl sulfate and guanidine hydrochloride, on the solubilization of the total and membrane proteins of the bacterium Haemophilus influenzae. The proteins solubilized with each system were analyzed by two-dimensional electrophoresis and these of interest were identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Use of sodium dodecyl sulfate, lithium dodecyl sulfate or guanidine hydrochloride for the solubilization of total proteins of the microorganism resulted in the detection of several additional spots, representing mainly outer membrane proteins, in comparison with those detected in the soluble protein fraction. Solubilization of the proteins of the cell envelope fraction with sodium dodecyl sulfate did not result in a more efficient protein detection when compared to the extraction with the urea/CHAPS system. When the dry immobilized pH gradient strips were rehydrated in a solution containing the proteins of the membrane fraction solubilized with sodium dodecyl sulfate or lithium dodecyl sulfate, a larger number of protein spots were detected in comparison with strips that were rehydrated in the urea/CHAPS solution. However, no improvement was observed in comparison with protein application in sample cups. The additional proteins detected with the use of strong detergents and chaotropes are in the majority difficult to solubilize and less hydrophobic proteins.     

Depolymerization of water soluble coal polymer from subbituminous coal and lignite by lignin peroxidase

Coal polymers, water soluble at pH 3.5, were prepared from North Dakota lignite and German subbituminous coal in 35–61% yield. Gel permeation chromatography showed a major component of relatively narrow molecular weight range >75,000. The material did not dialyze through a 12,000-14,000 MW cutoff membrane under several conditions. Minor amounts of smaller fragments were present, but monomeric components were not detected. Incubation of soluble polymer with lignin peroxidase ofPhanerochaete chrysosporium caused substantial disappearance of the high molecular weight polymer and formation of smaller amounts of both higher and lower molecular weight components, but not of monomeric compounds. Addition of veratryl alcohol enhanced depolymerization. Coal polymer competitively inhibited veratryl alcohol oxidation by lignin peroxidase.

     

Catalyzed sulphite and semichemical pulping

Very high yield sulphite pulps were produced by cooking black spruce wafers in pulping liquors at pH 7 or 10, containing 0.1% (on O.D. wood) of soluble anthraquinone (SAQ). These pulps had better strength properties relative to controls prepared without SAQ, breaking length and burst index being greater, on average, by 20%. Other improvements included: increased pulping rate, lower lignin contents at comparable pulp yields, and higher carbohydrate content at the same level of residual lignin in pulp (this resulted in an increase of total pulp yield by 2%). Results of cooks in liquors ranging in pH from 4 to 10, and under variable conditions of time (20–60 min) and temperature (120–160°C) suggested that: firstly, AQ does not act as a pulping catalyst at pH 4, and secondly, the sulphonate contents of AQ-catalyzed pulps are lower than those of the uncatalyzed controls. In the light of the lower sulphonate content, the higher strength is unexpected.     

Structure of Alkali Lignins Fractionated from Ricinus communis and Bagasse. 3. IR Spectra

Abstract

The lignin fractions isolated by one- and multistage soda and sulfate cookings showed almost identical IR spectra, indicating the similarity of the lignin skeletal structure throughout the plant. However, the absorbances reveal some differences. Similarity of the spectra includes: 1) chelation and bonding of the hydroxyl groups. 2) Stretching vibration of C-H bonds in methyl, methoxyl, and methylene groups. 3) Stretching vibration of C≡N. 4) Carbonyl unconjugated β-ketone, conjugated acids, or esters at 1725 cm^?1. There is no change in the intensity of absorption at this band from that at 1515 cm^?1 with the cooking stage. 5) Aromatic skeletal vibration at 1610 and 1515 cm^?1, affected by ring substituents at 1425 cm^?1. 6) The band at 1465 cm^?1 showed a higher intensity for soda and soluble kraft lignins than for insoluble kraft ones. 7) The band at 1370 cm^?1, assigned to phenolic OH bending, is affected by the methoxyl group. 8) The absence of condensed guaiacyl and the presence of syringyl and uncondensed guaiacyl. Assignments for hardwood lignin are shown for soda and soluble kraft lignins of bagasse, while those for softwood lignin are shown for soda, soluble, and insoluble kraft lignins of Ricinus communis and for insoluble kraft lignin of bagasse. A relation exists between the carbohydrate's lignin and the band at 920 cm^?1. Lignins from Ricinus communis are of higher guaiacyl to syringyl ratios than those from bagasse. The presence of C—S vibration and the absence of thiol groups for kraft lignins are indicated.     

Fractionation of aluminum in natural waters by fluorometry based on the competitive complexation

A novel strategy for the speciation of aluminum has been developed by fluorometry using selective analytical reagents under different pH conditions, which is based on the measurement of the complexation ability of Al for analytical reagents and native organic matters. With Eriochrome Blue Black R, labile monomeric Al and total monomeric Al fractions are determined at pH 4.5 and pH 8.0, respectively. With morin only the labile monomeric Al is determined at pH 4.0, while with 8-hydroxyquinoline the total monomeric Al is determined at pH 5.5. So, acid soluble Al fraction is obtained indirectly. This method is successfully applied to the fractionation of Al in natural waters. The results well agree with those obtained by Driscoll’s 8-hydroxyquinoline/cation-exchange protocol. This fluorometric method possesses some advantages such as high sensitivity, easy manipulation, no separation step and less time-consuming.     

Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly.

Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg2+, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES--1 m M EGTA--1 mM MgCl2--0.02% NaN3, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg2+ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl2 prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl2 is raised to 10 mM, reassembly occurs. The results suggest that Mg2+ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg2+ (10 microM--10 mM) increases reassembly whereas chelation of Mg2+ by (EGTA) inhibits reassembly. Coats reassembled in low-Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl2 do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low-ionic-strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice.     

次烟煤与木质素共热溶物的液化及其Ni-Mo-S/Al2O3催化剂循环利用性能

对神府次烟煤与木质素共热溶得到的四种具有不同热溶产率的热溶物进行元素分析、红外光谱、同步荧光光谱等表征,对四种热溶物和神府原煤的加氢液化性能进行比较,并进行了催化剂在热溶物液化过程的循环利用性能研究。结果表明,神府煤热溶物较同一温度得到的神府煤与木质素共热溶物具有更多的芳香组分和四环及其以上的多环芳烃。热溶物较神府原煤在液化时具有更高的转化率和油收率。在催化剂Ni-Mo-S/Al₂O₃作用下,热溶物在液化过程中几乎全部转化,并具有很高的油收率,且神府煤与木质素共热溶物较神府煤热溶物具有更高的油收率。在神府煤与木质素共热溶物的液化过程中,催化剂Ni-Mo-S/Al₂O₃表现出优异的可循环利用性能,经过四次循环利用后没有观察到催化剂表面的炭沉积现象。     

Separation of tubulin subunits by reversed-phase high-performance liquid chromatography

When properly solubilized with trifluoroacetic acid (TFA), alpha- and beta-tubulin subunits from a variety of sources may be resolved at high yield by reversed-phase high-performance liquid chromatography (HPLC), using a Waters muBondapak C18 column and simple linear aqueous acetonitrile gradients containing TFA. The tubulin subunits are typically the most non-polar proteins present, with the beta-tubulin subunit eluting before the alpha. Column temperature above ambient improve both the resolution and the yield; less polar solvent systems do not. Tubulins not freely soluble in aqueous TFA may be solubilized in 6 M guanidine-hydrochloric acid with no change in retention time. Other columns with shorter carbon chain lengths and larger pore size produce a single, unresolved tubulin peak. Reversed-phase HPLC analysis provides an independent comparative evaluation of organelle-specific tubulins, with characteristic retention time differences observed between homologous ciliary and flagellar outer doublet tubulin subunits and also between them and their cytoplasmic counterparts.     

Structure of Alkali Lignins Fractionated from Ricinus communis and Bagasse. 1. Chemical Constituents

Fractionation of alkali lignins of the soda and sulfate pulping processes of Ricinus communis and bagasse was carried out by using successive equal concentrations of the alkaline reagent. Soda lignins were soluble in organic solvents, while the sulfate ones were sparingly soluble. Thus, two fractions of the sulfate lignins, soluble and insoluble, could be obtained from acetone. The different alkali lignin fractions were subjected to elemental and functional group analyses. For both Ricinus communis and bagasse, the carbon content of the fractions of the various types of lignin is in the order: soluble sulfate > soda > insoluble sulfate, while the methoxy is in the order: soda > soluble sulfate > insoluble sulfate. The phenolic OH content, as well as OH/C₉ of soda lignins of bagasse, are lower than those of soluble sulfate lignins. For Ricinus communis, the phenolic OH content and OH/C₉ is higher for some of the fractions (first three stages of cooking) of soda lignin than the corresponding fractions of kraft (sulfate) lignin, while the reverse takes place for the other fractions. For the same type of lignin, the fractions showed changes in their carbon, methoxyl, and phenolic OH contents. The change may be regular, i.e., increase or decrease with the order of stage of cooking, or irregular. Molecular weights of the different alkali lignins which are soluble in organic solvents ranged between 750 and 840.     

Derivatizing Reagents Based on Ferrocene for HPLC-Ecd Determination of Peptides and Proteins

Abstract

Several ferrocenic compounds for derivatization of peptides and proteins were synthesized and then tested by reaction with bovine serum albumin (BSA). The reactivity of the reagents and the electroactivity of the derivatized BSA were estimated by the height of the ECD-signal after an HPLC analysis run. The most suitable reagent was 3-ferrocenylpropionic anhydride which reacts with BSA within 15 minutes at room temperature. The anhydride presented itself as a stable compound which can be synthesized in high yields. Up to pH 9–10 it is only slowly hydrolyzed, and its derivatization products are highly electroactive. Another reagent is especially suited to derivatize those proteins whose isoelectric points are higher than 10: Ferrocenylmethyl-succinimidyl-glycine-hydrochloride. This compound develops its derivatization activity only with pH values higher than 9, but it is rather difficult to prepare.     

Characterization of hemicelluloses isolated with tetraacetylethylenediamine activated peroxide from ultrasound irradiated and alkali pre-treated wheat straw

Post-treatment with 2% H₂O₂-0.2% tetraacetylethylenediamine (TAED) at pH 11.8 for 12 h at 48 °C solubilized 27.1-28.1% of the original hemicelluloses and 43.3-46.2% of the original lignin from the ultrasound irradiated and alkali pre-treated wheat straw. It was found that the bleaching activator TAED forms peracetic acid with hydroperoxide anion in aqueous alkali and improves the brightness of the solubilized hemicelluloses. Xylose was a predominant sugar in all the hemicellulosic preparations, comprising 72.0-73.1% of the total sugars. Arabinose (13.4-13.9%) and glucose (6.7-7.9%) were detected as the second and third major neutral sugar constituents. In addition, the hemicelluloses also contained a noticeable amount of 4-O-methyl-d-glucuronic acid (6.0-7.0%) together with a small amount of galactose (4.3-4.9%) and minor quantities of rhamnose (1.5-1.8%) and mannose (0.5-0.8%). Furthermore, the hemicellulosic preparations obtained by TAED activated peroxide extraction showed a relatively lower content of associated lignin (3.8-4.4%), but a higher molecular weight (34,210-40,310 g mol⁻¹). No significant differences in the structural features such as oxidation of the hemicelluloses by TAED were observed as compared to those obtained by alkaline peroxide without bleaching activator.     

UV-INDUCED DAMAGE OF PHOTORECEPTOR PROTEINS IN THE PARAFLAGELLAR BODY OF Euglena gracilis

Abstract— The paraflagellar body (PFB), the putative photoreceptor for phototaxis in the flagellate Euglena gracilis , was isolated still attached to the flagellum. After solubilization the proteins were separated and analyzed by two-dimensional gel electrophoresis. To discriminate the PFB-specific proteins from the flagella proteins, flagella without PFB were analyzed, isolated from the flagellate Astasia longa (a chlorophyll-free relative of E. gracilis ), which has no PFB and lacks phototaxis. The absorption spectra of solubilized PFB samples showed a maximum at 415 nm, two shoulders around 380 and 410 nm and two additional small peaks at 515 and 540 nm not present in the control sample without PFB. Two-dimensional gel electrophoresis showed eight PFB-specific proteins with molecular masses in the range of 25000–45000 and isoelectric points in the range of pH 3.5–7. Ultraviolet radiation strongly affects some of these PFB-specific proteins, but also flagella proteins are damaged by UV treatment. There is also a drastic decrease in the PFB-specific absorption maxima after UV irradiation.     

Size-exclusion chromatography of the soluble lignin products from hydrogen peroxide brightening of mechanical pulps

The molar mass distributions and yields of soluble lignin degradation products formed during the alkaline hydrogen peroxide brightening of spruce mechanical pulp have been determined. These distributions depend on pH, the extent to which the pulp was subjected to solvent extractions, H₂O₂ concentration and reaction time. At and below pH 11, there is an excellent correlation between the brightness and b values on pulp and the amounts of residual peroxide and lignin derived products in solution. Brightening reactions lead primarily to products with intermediate molecular mass values between 1500 and 6000. During multistage brightening, the products formed are initially those with high ionizable phenolic hydroxyl and low carbohydrate contents, and carbonyl-containing moieties. NMR and UV spectroscopic analysis show that products formed include lignin and carbohydrate-containing species. The lignin-originating products from the later stages contain a lower relative concentration of ionizable phenolic groups and more visible-absorbing and carbohydrate-containing species. Brightness and b gains during multistage brightening were exponential with product yield.     

DNA fingerprinting of Vibrio cholerae and Aeromonas species by pulsed-field minigel electrophoresis

DNA molecules of Vibrio cholerae and Aeromonas species were prepared by incubating immobilized cells for 4 and 2 h, respectively, with a nonenzymatic solution that contains chemical reagents only (NDSUPlus). This method gave results as reproducible as the enzymatic one that uses proteinase K, and rendered DNA molecules suitable for fingerprinting by mini-CHEF electrophoresis. As rapid DNA separations at high electric field are achieved in mini-CHEF chamber with low heat evolution, DNA restriction fragments were separated in 5 h at 10 V/cm in a single resolution window. Then, fragment separations in three resolution windows were done in 15 h. This time is shorter than the one needed by the large CHEF chamber for resolving fragments in a single resolution window. Three windows permitted to include larger numbers of restriction fragments in the calculation of isolate similarities. Both sample preparation and mini-CHEF electrophoresis may represent an alternative for performing massive epidemiological studies of V. cholerae and Aeromonas species.     

Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

Introduction CardiactroponinI(cTnI),aspecificproteinof cardiacmusclecells,showsa40%dissimilarity withskeletaltroponinI(sTnI)inaminoacidse- quence.Moreover,humancardiacTnIhas31addi- tionalresiduesonitsN-terminalend,whichare notpresentinskeletalforms,thusprovidingahigh potentialforobtainingcardiac-specificantibod- ies[1,2].Themolecularweightofthisproteinis29 kDaandtherefore,itwillbereleasedreasonably rapidlyafteracutemyocardialinfarction(AMI). CTnIoftenappearsinbloodwithinafewhoursaf- ter…     

Strategy for selective chemical cross-linking of tyrosine and lysine residues

Chemical cross-linking of proteins combined with mass spectral analysis is a powerful technique that can be utilized to yield protein structural information, such as the spatial arrangement of multi-protein complexes or the folding of monomeric proteins. The succinimidyl ester cross-linking reagents are commonly used to cross-link primary amine-containing amino acids (N-terminus and lysine). However, in this study they were used to react with tyrosines as well, which allowed for the formation of cross-links between two primary amines, one primary amine and one tyrosine, or two tyrosines. This result is extremely important to the chemical cross-linking community for two reasons: (1) all possible cross-linked residues must be considered when analyzing data from these experiments to generate correct distance constraints and structural information, and (2) utilizing the versatility of these cross-linking reagents allows more information content to be generated from a single cross-linking reagent, which may increase the number of cross-links obtained in the experiment. Herein, we study the reactivity of the succinimidyl ester labeling and cross-linking reagents with angiotensin I and oxidized insulin beta-chain. Using the succinimidyl acetate labeling reagent, the reactivity of the N-terminus was found to be greater than either lysine or tyrosine. However, a selectivity of the cross-linking reagent was observed for either tyrosine or lysine depending on the pH of the reaction solution. In acidic pH, it was observed that tyrosine was more reactive, while in alkaline pH lysine was more reactive. Exploiting this selectivity predominantly N-terminus-tyrosine or tyrosine-tyrosine cross-links were favored at acidic pH, while N-terminus-tyrosine or tyrosine-lysine cross-links were favored at alkaline pH.     

Separation of proteins and peptides by capillary electrophoresis in acid buffers containing high concentrations of surfactants.

Separations of proteins at acid pH in the presence of a high concentration of surfactant [sodium laurylsulfate (SDS), 50 mmol/l] was investigated. The purpose of using high concentrations of SDS as background electrolyte modifier was threefold: First, the surfactant exerts a washing effect upon the capillary wall thus preventing binding of analytes and possible clogging of the capillary. Second, it was revealed that even under very acid conditions (below pH 3) the surfactant is capable of forming associates with protein analytes which still bear considerable negative charge and can be separated on this basis. Third, the system can be applied not only for protein mixtures sufficiently soluble in neutral to alkaline media (leukocyte lysates, standard proteins), but it can be used also with proteins, that are under such conditions virtually insoluble and their solubilization is possible in acid buffers only (eggshell proteins or collagen CNBr fragments). The result was that adsorption to the capillary wall was minimized and the analytes were separated as negatively charged associates with high efficiency. With collagen fragments partition was possible on the affinity differences of the peptides to the surfactant micelles and inner wall of the capillary. Theoretical plate counts approaching 100,000 were easily achieved even with proteins which under the more conventional operation conditions exhibit considerable sticking to the capillary wall. The other feature of this system is that the associates move very rapidly to the anode. Owing to the low pH, endoosmotic flow is negligible, and therefore the system has to be operated at reversed polarity.     

Fabrication of Environmentally Biodegradable Lignin Nanoparticles

We developed a method for the fabrication of novel biodegradable nanoparticles (NPs) from lignin which are apparently non‐toxic for microalgae and yeast. We compare two alternative methods for the synthesis of lignin NPs which result in particles of very different stability upon change of pH. The first method is based on precipitation of low‐sulfonated lignin from an ethylene glycol solution by using diluted acidic aqueous solutions, which yields lignin NPs that are stable over a wide range of pH. The second approach is based on the acidic precipitation of lignin from a high‐pH aqueous solution which produces NPs stable only at low pH. Our study reveals that lignin NPs from the ethylene glycol‐based precipitation contain densely packed lignin domains which explain the stability of the NPs even at high pH. We characterised the properties of the produced lignin NPs and determined their loading capacities with hydrophilic actives. The results suggest that these NPs are highly porous and consist of smaller lignin domains. Tests with microalgae like Chlamydomonas reinhardtii and yeast incubated in lignin NP dispersions indicated that these NPs lack measurable effect on the viability of these microorganisms. Such biodegradable and environmentally compatible NPs can find applications as drug delivery vehicles, stabilisers of cosmetic and pharmaceutical formulations, or in other areas where they may replace more expensive and potentially toxic nanomaterials.     

Synthesis and evaluation of fluorogenic reagents for simultaneous detection of peptides and proteins by HPLC in two different samples

To find the pairs of fluorogenic reagents having similar retention times in HPLC but with different fluorescent characteristics, six fluorogenic reagents bearing benzoxadiazole or benzoselenadiazole skeletons were synthesized. The resultant derivatives obtained from the reaction of peptides and proteins with reagents which have a benzoselenadiazole skeleton showed different fluorescence characteristics from those with a benzoxadiazole skeleton. Since each corresponding derivatives of trypsin inhibitor and BSA with DAABD-Cl and 7-fluoro-N-[2-(diethylamino)ethyl]-2,1,3-benzoselenadiazole-4-sulfonamide (DEAEABSeD-F) have similar retention times, the pair of reagents was adopted for the sensitive simultaneous detection of proteins in two different samples. When the soluble fraction of mouse hippocampus was divided into the two samples (A and B), each was reacted with DEAEABSeD-F for A and DAABD-Cl for B, respectively. The two reaction solutions were combined and subjected to HPLC analysis with two fluorescent detectors in series (excitation and emission at different wavelengths for A and B, respectively). The resultant two chromatograms had quite similar patterns for each other. The new pair of fluorogenic reagents (DAABD-Cl and DEAEABSeD-F) would be applicable to proteomics studies using the previously reported FD-LC-MS/MS method.