Biodegradation of lignin-carbohydrate complexes

Covalent lignin-carbohydrate (LC) linkages exist in lignocellulose from wood and groups herbaceous plants. In wood, they consist of ester and ether linkages through sugar hydroxyl to the -carbanol of phenylpropane subunits in lignin. In grasses, ferulic and p-coumaric acids are esterified to hemicelluloses and lignin, respectively. Hemicelluloses also contain substitutents and side groups that restrict enzymatic attack. Watersoluble lignin-carbohydrate complexes (LCCs) often precipitate during digestion with polysaccharidases, and the residual sugars are more diverse than the bulk hemicellulose. A number of microbial esterases and hemicellulose polysaccharidases including acetyl xylan esterase, ferulic acid esterase, and p-coumaric esterase attack hemicellulose side chains. Accessory hemicellulases include -l-arabinofuranosidase and -methyl-glucuranosidase. Both of these side chains are involved in LC bonds. -Glucosidase will attach sugar residues to lignin degradation products and when carbohydrate is attached to lignin, lignin peroxidase will depolymerize the lignin more readily.Abbreviations APPL acid precipitable polymeric lignin - CBQase cellobioquinone oxidoreductase - LC lignincarbohydrate - LCC(s) lignin-carbohydrate complex - DHP Dehydrogenative polymerisate - DMSO dimethylsulfoxide - DP degree of polymerisation - MWEL milled wood enzyme lignin - MWL milled wood lignin (not digested with carbohydrases)     

Fractionation of lignin-carbohydrate complexes by hydrophobic-interaction chromatography

Hydrophobic properties of lignin-carbohydrate complexes (LCC) isolated from Pinus densiflora Sieb. et Zucc. have been analysed by hydrophobic-interaction chromatography on Phenyl- and Octyl-Sepharose CL-4B gels. The ability of LCC to be adsorbed by these hydrophobic gels was exclusively dependent on their lignin content. Materials adsorbed on Octyl-Sepharose were desorbed with a lower concentration of 2-ethoxyethanol than those adsorbed on Phenyl-Sepharose. In the adsorption of LCC by Phenyl-Sepharose, ππ interactions between the aromatic ligands and the benzene skeletons of lignin play an important role, whereas hydrophobic interaction is the exclusive driving-force for adsorption in the case of Octyl-Sepharose.     

Investigation of lignin-carbohydrate complexes in kraft pulps by selective enzymatic treatments

The occurrence of covalent bonds between residual lignin and polysaccharides in birch and pine kraft pulps was investigated by specific enzymatic treatments. Pure enzymes degrading cellulose, xylan and mannan were used both separately and in combination. Comparison of the molar masses of polysaccharides and lignin in the orginal pulps and in the residual pulps after enzymatic treatments showed that residual lignin in birch kraft pulp is linked at least to xylan. A minor portion may also be linked to cellulose. In pine kraft pulp some of the residual lignin appears to be linked to cellulose, glucomannan and xylan. The linkages between lignin and cellulose and hemicelluloses may be either native or formed during pulp processing. The results also provided new information on the synergistic action of cellulose- and hemicellulose-degrading enzymes on pulp fibres. The synergism appears to be mainly due to the structure of the pulp fibres, with different layers of cellulose sheets, hemicelluloses and lignin. On the other hand the results also provided information about fibre structure. The degradation of xylan clearly enhanced the action of enzymes on cellulose, suggesting that xylan partially covers the cellulose. A similar phenomenon was not observed in the simultaneous hydrolysis of glucomannan and cellulose. However, the results suggest that glucomannan does interact with cellulose, possibly by non-covalent linkages. Received: 8 July 1998 / Received revision: 7 October 1998 / Accepted: 11 October 1998     

Presence of soluble lignin-carbohydrate complexes in the bovine rumen.

The cell-free rumen liquor of a steer on a diet of spear grass has been shown to contain macromolecular substances in which carbohydrates and lignin-derived compounds are covalently bound to each other. The lignin-carbohydrate complexes are soluble at pH 7 or higher, but precipitate at pH 3. At the latter pH, small amounts of a polymer, assumed to be glycoprotein, remain in solution. Some of the lignin-carbohydrate linkages are broken by treatment with alkali. Treatment with 50mM sulphuric acid for a few minutes at room temperature converts part of the complex into an acetone-soluble product, which still contains both carbohydrate and lignin-derived compounds. The formation of soluble lignin-carbohydrate complexes by the action of rumen micro-organisms on the grass may account for the dissolution (and hence the apparent digestion) of about half of the total lignin-intake.     

Magnetic detection of injury-induced ionic currents in bean plants

A superconducting quantum interference device (SQUID) multichannel magnetometer was used to measure the temporal and spatial evolution of the magnetic field accompanying stimulation by burning and/or cutting of Vicia faba plants. These magnetic fields are caused by ionic currents that appear after injury in different parts of the plant. All measured V. faba plants responded to the burning stimulation with detectable quasi-d.c. magnetic signals. In order to measure these signals, a suitable modulation had to be used. The covariance method was applied to analyse the measured data. The results demonstrate a dipolar-like magnetic signal, exponentially decreasing in time, above the cutting type of injury. After the burning stimulation, the magnetically detected activity was concentrated predominantly above the leaves/petioles and less above the stem. Possible mechanisms for this behaviour are suggested. A comparison with previously known electrical measurements of plant injury is given.     

Fluorescence detection of cardenolides in reversed-phase high-performance liquid chromatography after post-column derivatization

Methods for the fluorescence derivatization of cardiac glycosides with concentrated acids from TLC are adopted to HPLC for post-column derivatization. The column effluent is blended with concentrated acids in a knitted tube reactor, which enables derivatization with negligible increase in chromatographic peak width. The selectivity of the reaction is temperature-dependent and influenced by the respective acid. Reactivity increases from H₃PO₄→CH₃SO₃H ≡ H₂SO₄. The conversion of digoxigenin, digitoxigenin and their digitoxosides is accelerated by Cu(II) acetate or Co(II) nitrate in H₂SO₄. Combined with a new two-mode, single-column solid-phase sample preparation, cardiac glycoside levels of less than 100 pg/glycoside in 1 ml plasma are detectable.     

Femtomole peptide mapping by derivatization, high-performance liquid chromatography, and fluorescence detection.

A highly sensitive peptide mapping method using derivatization and fluorescence detection is described. Bovine cytochrome c was digested using a buffer compatible with the derivatization that followed. The derivatization was performed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The peptide mapping of the tagged digest was conducted with both HPLC and capillary LC (CLC) systems. A capillary LC-electrospray ionization mass spectrometer (MS) was set up for measuring the molecular weights of the tagged peptides. Optimization was made of the conditions used for digestion, derivatization, and mapping. MS measurements of the tagged peptides suggested that there was only one derivatization product produced from all peptides (except one) and that all the identified peptides were fully tagged. Peptide mapping of the tagged digest reviews a larger number of peptides, covering almost the entire sequence. Peptide mapping of a 20 fmol amount of tagged digest was readily performed with the CLC system. By using derivatization and fluorescence detection, the sensitivity of peptide mapping could be improved 2000 times compared to that observed with uv detection of untagged peptides.     

Trace analysis of tobramycin in human plasma by derivatization and high-performance liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method is established for the trace determination of tobramycin in human plasma by derivatization. The method is based on the chemical derivatization of aminoglycoside antibiotic, tobramycin in human plasma, with 1-naphthyl isothiocyanate (NITC) in pyridine at 70 degrees C. After derivatization reaction, a methylamine/acetonitrile solution was added to the reaction mixture to eliminate the excess derivatizing agent and shorten the analysis time. The resulting derivative was separated using a Purospher STAR RP-18e column and a water-acetonitrile (50:50, v/v) mobile phase (detection at 230 nm). Optimization conditions for the derivatization of tobramycin were investigated by HPLC. The linear range for the quantitation of tobramycin in spiked plasma was over 0.93-9.34 mg/l; the detection limit (signal-to-noise ratio=3; injection volume, 10 microl) was about 0.23 mg/l. The relative standard deviation was less than 2.1% for intra-day assay (n=6) and 5.2% for inter-day assay (n=6) and relative recoveries were found greater than 99%.     

Quantitative determination of glufosinate in biological samples by liquid chromatography with ultraviolet detection after p-nitrobenzoyl derivatization

We have established a new HPLC method for derivatizing and quantifying glufosinate (GLUF) in human serum and urine using p-nitrobenzoyl chloride (PNBC). The p-nitrobenzoyl derivative of GLUF (PNB-GLUF) was produced quantitatively over 10 min at room temperature. PNB-GLUF possesses the property of ultraviolet (UV) light absorption with a lambda(max) of 272.8 nm, and was isolated from biological specimens by reversed-phase chromatography using Inertsil Ph-3. In experiments at a UV wavelength of 273 nm, GLUF has a quantitative detection limit of 0.005 microg/ml, and when it was added to both serum and urine to yield concentrations of 0.1-1000 microg/ml, its recovery rate was quite satisfactory: at least 93.8% in all cases. Further, the measured amounts of GLUF in 23 serum samples from patients intoxicated by ingestion of GLUF compared favorably with those obtained by fluorescence derivatization-HPLC using 9-fluorenylmethyl chloroformate (R=0.998). This technique of analysis is, in addition, applicable for Glyphosat, which possesses a chemical structure resembling that of GLUF, and it will be of great use in the determination of these two compounds.     

Determination of amphetamine in human urine by dansyl derivatization and high-performance liquid chromatography with fluorescence detection

A simple procedure for the determination of amphetamine in urine with minimal sample preparation is described. This method involves direct addition of human urine to an acetone-dansyl chloride solution for simultaneous deproteinization and fluorescence derivatization. The derivatized amphetamine is then measured by HPLC with fluorescence detection. It eliminates the extraction procedures often required by other HPLC or GC methods. The effects of pH, temperature and reaction time on the derivatization reaction were investigated. The stability of amphetamine-dansyl chloride in different storage conditions was examined. The detection limit and linearity associated with this assay are discussed.     

Isolation and analysis of lignin-carbohydrate complexes from Lolium multiflorum

Lignin-carbohydrate complexes were extracted from grass cell walls by a variety of solvents. The yield of complexes was greatly enhanced if the sample was finely milled in a ball mill; dimethyl sulphoxide and N alkali extractions gave the highest yields. Hydrolysis showed that the carbohydrate fraction of the alkali-extracted complex contained mainly xylose (ca. 70%) and arabinose (ca. 20 %) whereas the dimethyl sulphoxide extracted complex contained glucose (ca. 50 %), xylose (ca. 30%), arabinose (ca. 12 %) and galactose (ca. 5 %). The UV spectrum of the dimethyl sulphoxide extracted complex showed lignin absorbance at 280 nm, but, in addition, ester bonding was also observed by the presence of a secondary absorbing region near 325 nm. This secondary absorbing region was absent from the spectrum of the alkali-extracted complexes. Fractionation of the complexes by ethanol precipitation gave a major component which appeared homogeneous by molecular sieve chromatography and had a MW of ? 150,000.     

Molecular requirements of lignin-carbohydrate complexes for expression of unique biological activities

Lignins are major cell wall components formed by the dehydrogenative polymerization of three monolignols, p-coumaryl, coniferyl and sinapyl alcohols. We prepared lignin-carbohydrate complexes (Fr. VI and Fr. VII) from pine cones by acid and ethanol precipitation, and investigated which part of these molecules is essential for expression of biological activities. They showed potent antiviral activity upon direct interaction with the virus. The antiviral activity of Frs. VI and VII required the higher-order structure of polyphenols without polysaccharides. Pretreatment of mice with Fr. VI or VII induced higher antiparasite activity than those of natural and chemically modified antitumor polysaccharides. Fr. VI or VII at higher concentrations enhanced the radical intensity and cytotoxic activity of vitamin C, whereas tannins counteracted the effect of vitamin C. Fr. VI at lower concentrations enhanced the O2(-)-scavenging activity of vitamin C. Frs. VI and VII stimulated mouse macrophage-like cells Raw 264.7 to produce nitric oxide (NO), citrulline (CIT) and asparagine (ASN), via the enhanced expression of iNOS and ASN synthetase, whereas phenylpropenoid monomers and polymers inhibited NO/CIT/ASN production. These data suggest that the polymerized structure of phenylpropenoids in lignin-carbohydrate complexes is required for the induction of antiviral activity, and that the higher-order structure of phenylpropenoid polymers and polysaccharides is required for immunopotentiation, including macrophage activation.     

Determination of methylguanidine in plasma and urine by high-performance liquid chromatography with fluorescence detection following postcolumn derivatization

A high-performance liquid chromatographic method was developed for the determination of methylguanidine in biological fluids. Methylguanidine and the internal standard were isolated from plasma by cation-exchange solid-phase extraction prior to chromatographic analysis. Urine samples were diluted and injected directly onto the analytical column. Chromatographic separation was carried out on an Ultrasil cation-exchange column using a mixture of methanol and monochloroacetate (15/85, v/v) as the mobile phase. Postcolumn derivatization of methylguanidine was carried out using alkaline ninhydrin reagent and the resulting fluorescent product was detected on-line. The method was specific, sensitive, reproducible, and linear over a wide a range of concentrations. The lower limit of detection for methylguanidine in plasma and urine was 1 and 100 ng/ml, respectively. The method was successfully employed for quantification of the levels of methylguanidine in normal and uremic human subjects, normal dogs, and dogs with ischemic-induced acute or spontaneous chronic renal failure.     

Structural differences between the lignin-carbohydrate complexes present in wood and in chemical pulps

Lignin-carbohydrate complexes (LCCs) were prepared in quantitative yield from spruce wood and from the corresponding kraft and oxygen-delignified pulps and were separated into different fractions on the basis of their carbohydrate composition. To obtain an understanding of the differences in lignin structure and reactivity within the various LCC fractions, thioacidolysis in combination with gas chromatography was used to quantify the content of beta-O-4 structures in the lignin. Periodate oxidation followed by determination of methanol was used to quantify the phenolic hydroxyl groups. Furthermore, size exclusion chromatography (SEC) of the thioacidolysis fractions was used to monitor any differences between the original molecular size distribution and that after the delignification processes. Characteristic differences between the various LCC fractions were observed, clearly indicating that two different forms of lignin are present in the wood fiber wall. These forms are linked to glucomannan and xylan, respectively. On pulping, the different LCCs have different reactivities. The xylan-linked lignin is to a large extent degraded, whereas the glucomannan-linked lignin undergoes a partial condensation to form more high molecular mass material. The latter seems to be rather unchanged during a subsequent oxygen-delignification stage. On the basis of these findings, a modified arrangement of the fiber wall polymers is suggested.     

Soil depth detection by seeds and diurnally fluctuating temperatures: different dynamics in 10 annual plants

Background and Aims

Seeds buried in the soil detect burial depth through light and diurnally fluctuating temperatures (DFT) and in this way limit losses due to germination too deep in the soil. DFTs and germination also increase in vegetation gaps. However, dry open environments with high DFTs can also increase seedling mortality, creating conflicting selection pressures for reaction to DFTs. Since this questions the general function of DFT detection, we therefore tested if interspecific differences in DFT detection are related to mortality in different soil depths.

Methods

We buried seeds of ten annual plants including species pairs of increasing and decreasing germination in response to DFTs. Seeds were buried in 5, 10 and 25cm soil depth and exhumed after two different burial times. Seed viability was tested using germination in growth chambers and tetrazolium. We also measured DFTs at these depths using temperature data loggers.

Results

DFT detection was not related to differences in mortality at three burial depths. Three species showed a clear pattern of depth dependent mortality, however inconsistently related to DFT detection.

Conclusions

Depth detection mechanisms are more species-specific than expected. Hence, interspecific differences in seed mortalities are difficult to predict by DFT detection alone and alternative soil depth sensing mechanisms should be explored in future.     

Determination of cycloserine in human plasma by high-performance liquid chromatography with fluorescence detection,using derivatization with p-benzoquinone

A new method for determining cycloserine in plasma samples is described. This method is based on the derivatization of cycloserine with p-benzoquinone, a reaction that takes place at the same time as the process of plasma deproteinization due to the presence of ethanol as solvent in the solution of the derivatization reagent. Four derivatives are obtained from this reaction. The main derivative is well correlated with the cycloserine concentration. The ratio between the volumes of the plasma sample and the reagent solution is 1:2 for a p-benzoquinone concentration of 1000 μg/mL. Elution from a C₁₈ column was isocratic, using a mobile phase containing (v/v) 85% aqueous 0.1% formic acid solution, and 15% (v/v) of a mixture of methanol and acetonitrile (1:1), with a flow-rate of 1 mL/min, at 25°C. Determinations by fluorescence detection were achieved with excitation at 381 nm and emission at 450 nm, with a detection limit of 10 ng/mL for an injection volume of 5 μL. This method was validated and applied to the determination of cycloserine in blood plasma samples of several healthy volunteers.     

Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C₂ cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C₁₈ reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.     

Separation of keratan-sulfate-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection

In this paper, we report a rapid, sensitive, and quantitative procedure to conduct disaccharide compositional analyses of keratan sulfates (KS) by means of high-performance liquid chromatography (HPLC) separation and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase II or Escherichia freundii endo-beta-galactosidase. Following E. freundii endo-beta-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6sbeta(1-->3)gal, accounting for approximately equals 95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-beta-N-acetylglucosaminidase (keratanase II) from Bacillus sp. generates two major products, the monosulfated disaccharide galbeta(1-->4)glcNAc6s ( approximately equals 50% nmol product) and the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s ( approximately equals 40% nmol product) for over 90% nmol products. These disaccharides are separated and readily determined within 30 min by using a linear-gradient strong anion-exchange separation. A linear relationship was found for the two purified disaccharides over a wide range of concentrations, from approximately equals 108 pmol, 50 ng, to 2,160 pmol, 1,000 ng, for the disaccharide galbeta(1-->4)glcNAc6s, and from 92 pmol, 50 ng, to 1,840 pmol, 1,000 ng, for the disaccharide gal6sbeta(1-->4)glcNAc6s. HPLC analysis was applied to the quantitative and qualitative determination of KS produced by 3T3-J2 murine fibroblasts in the cell medium. The amount of KS was found to be 2.80+/-0.34 microg/ml/10(6) cells and composed of approximately equals 71% nmol of disaccharide galbeta(1-->4)glcNAc6s and 18% nmol of the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s having approximately equals 1.20 sulfate groups/disaccharide. Our data illustrate that the HPLC procedure reported represents an improved approach for the quantitative and compositional microanalyses of KS, especially applicable to experimentation involving small amounts ( approximately 50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.     

High-performance liquid chromatographic determination of tazobactam by precolumn derivatization

A new reversed-phase high-performance liquid chromatography (RP-HPLC) method for the detection and quantification of tazobactam in serum and haemofiltration fluid is described. The assay for these biological fluids involves an extraction with diethyl ether followed by derivatization using 1,2,4-triazole. The mobile phase consisted of phosphate buffer-methanol and the detection wavelength was 325 nm. The limit of detection was 0.05 μg/ml in the two fluids and the calibration curves were linear over the range 0.1–50 μg/ml. For a tazobactam concentration equal to 1, 5 or 20 μg ml⁻¹, the coefficients of variation were less than 5%. The assay was successfully applied to the analysis of samples from drug monitoring in a patient with renal insufficiency undergoing continuous venovenous haemofiltration (CVVH).