Gary Gamble

Extracellular laccases and peroxidases from sycamore maple (Acer pseudoplatanus) cell-suspension cultures

We have investigated the abilities of extracellular enzymes from dark-grown cell-suspension cultures of sycamore maple (Acer pseudoplatanus L.) to oxidize monolignols, the precursors for lignin biosynthesis in plants, as well as a variety of other lignin-related compounds. Laccase and peroxidase both exist as a multiplicity of isoenzymes in filtrates of spent culture medium, but their abilities to produce water-insoluble, dehydrogenation polymers (DHPs) from the monolignols (in the presence of hydrogen peroxide for the peroxidase reaction) appear identical whether or not the enzymes are purified from the concentrated filtrates or left in a crude mixture. The patterns of bonds formed in these DHPs are identical to those found in DHPs synthesized using horseradish peroxidase or fungal laccase, and many of these bonds are found in the natural lignins extracted from different plant sources. On the other hand, sycamore maple laccase is very much less active on phenolic substrates containing multiple aromatic rings than is sycamore maple peroxidase. We suggst that whereas laccase may function during the early stages of lignification to polymerize monolignols into oligo-lignols, cell-wall peroxidases may function when H2O2 is produced during the later stages of xylem cell development or in response to environmental stresses.

Cotton Fiber Chemistry and Technology

Cotton fiber chemistry and technology , Cotton fiber chemistry and technology , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Chemical and structural analysis of fibre and core tissues from flax

Samples of flax (Linum usitatissimum) stems from the cultivars ‘Natasja’ and ‘Ariane’ were separated into fibre and core fractions and analysed by gas–liquid chromatographic methods, 13C CPMAS NMR spectrometry, histochemistry, electron microscopy and UV absorption microspectrophotometry to assist in determining the structure and composition of these cell walls in relation to quality and utilisation. Analyses from chromatography and NMR gave similar results for carbohydrate and phenolic constituents in various samples and in the lower, more mature regions of the stem. Amounts of uronic acids and xylose were lower while amounts of mannose, galactose and glucose were higher in fibre vs core fractions. Quantities of phenolic constituents were significantly higher in the core than the fibre, with groups representative of both guaiacyl and syringyl lignins; amounts of phenolic acids were low. NMR showed a low intensity signal for aromatics in fibre, and it is possible that such signals arise from compounds in the cuticle rather than the fibre. Microscopic studies indicated that aromatic constituents were present in core cell walls, cuticle of the epidermis, and cell corners and middle lamellae of some regions within the fibre tissues. The lignin in fibre appeared to be of the guaiacyl type and may be too low in concentration to be unambiguously detected by NMR. Aromatic compounds were not observed in the epidermis or parenchyma cell walls. Similar analyses of dew-retted (unscutched) samples indicated that core tissues were mostly unchanged from unretted samples. Retted fibre tissues still contained lignified cell corners and middle lamellae in some regions. The cuticle, which was associated with retted fibres, was not degraded by dew-retting fungi. Fungi removed interfibre materials in some places and at times degraded the secondary wall near the cell lumen of fibre cells. Results indicate that microspectrophotometry and histochemistry are useful to identify the location and type of aromatics in fibre cell walls.

Effect of Retting Enzymes on the Structure and Composition of Flax Cell Walls

Commercial enzyme mixtures are tested for their possibly selective degradation of flax (Linum usitatissimum L.) stem components in relation to the retting process in producing linen. Structural and chemical compositional results from treatments are obtained using scanning electron microscopy, histochemistry, gas-liquid chromatography, 13C cp mas nmr spectrometry, and mid-infrared spectroscopy. Flaxzyme and Ultrazym and an enriched pectinase mixture (epm), which was not developed for flax retting but is included for comparison, are tested for their activity toward cell wall components and used in various concentrations for “enzyme-retting” of flax. Ariane flax stem sections are incubated with enzymes in a rotary incubator and the fibers are manually separated from the residual core. All of the commercial enzyme mixtures have cellulase, pectinase, and hemicellulase activities, but individual enzyme activities vary. Activities against the soluble test substrates do not predict the activity against natural fibers. At about equal protein concentrations, Flaxzyme treatment appears to facilitate bast fiber removal better than the other enzymes, with Ultrazym nearly as effective and epm the least effective. The ranking of effectiveness is generally supported by the amounts of uronic acid, arabinose, and xylose removed from the stems analyzed chemically. Increased enzyme levels generally facilitate removal of matrix carbohydrates from the flax. All enzymes separate bast fibers from the lignified core and partially from the cuticle near the cut surface of the stem sections, but the enzymes do not work far from the exposed ends. Retting quality is defined more by the degree of cell wall degradation and fiber separation than by any differences in kinds of cell walls degraded by the various enzymes. The cuticle remains attached to the fiber at times, apparently reducing access of the enzymes to the matrix polysacchrides and suggesting some recalcitrance of epidermal cells (and therefore loss of cuticle) to biodegradation. Lignin remains in the middle lamellae after enzyme retting and would likely prevent separation of the fiber bundles. Some solubilzation of the inner secondary wall of the flax fiber appears to occur with Flaxzyme. The structural and chemical analyses characterize alterations in flax bast after enzyme retting and would be useful in ranking the specificity and effectiveness of cell wall degradation.

Biological delignification of plant components by the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus

Lignocelluloses from diverse plant types were treated with the white rot fungi Ceriporiopsis subvermispora (strains CZ-3-8497 and FP-90031-sp) and Cyathus stercoreus. Sources of lignocellulose included: the warm-season grasses sorghum (leaf blades, sheaths, and stems), pearl millet, napiergrass, and maize (stems); the cool-season grass wheat (leaf blades, sheaths, and stems); the legumes alfalfa (stems) and lespedeza (leaflets and stems). Fungus-treated residues were compared with untreated, control samples and with plants treated with a non-delignifying isolate of Trichoderma. Residues were evaluated for improved biodegradability by ruminal microorganisms and modifications in cell wall chemistry by nuclear magnetic resonance, gas chromatography, and ultraviolet absorption microspectrophotometry. Specific plant—fungus interactions were identified that resulted in selective removal of lignin and improved biodegradability by white rot fungi but not the Trichoderma sp. All white rot fungi removed ester-linked p-coumaric and ferulic acids from grass stems, and this phenomenon appeared to account for the significant reduction in aromatic components and improved biodegradability of fungus-treated grass lignocellulose. Cell walls in alfalfa stems were more resistant to biological delignification than those in grasses, with only C. stercoreus removing significant amounts of aromatics and improving biodegradability. All white rot fungi improved the biodegradability of tannin-rich lespedeza samples.

Limitation of the butanol–hydrochloric acid–iron assay for bound condensed tannins

Abstract The butanol–HCl–iron method is widely used for measurement of extractable condensed tannins ( syn . proanthocyanidins) in foods and feeds. As the method is based on acid catalysed oxidative depolymerization of condensed tannins into anthocyanidins, this method has also been used for determination of bound condensed tannins. The recovery of bound condensed tannins by the butanol–HCl–iron assay was monitored by subjecting the residues left after the assay to solid-state 13 C NMR spectroscopy. The signal at δ 155.0, indicative of condensed tannins, remained relatively high in the residues following the butanol–HCl–iron assay, suggesting an incomplete recovery of bound condensed tannins by the assay. The results obtained using the butanol–HCl–iron assay should therefore be interpreted with caution.

Development of Fourier transform infrared spectroscopy in direct, non-destructive, and rapid determination of cotton fiber maturity

Fourier transform infrared (FTIR) spectra of seed and lint cottons were collected to explore the potential for the discrimination of immature cottons from mature ones and also for the determination of actual cotton maturity. Spectral features of immature and mature cottons revealed large differences in the 900—1200 cm1 region, and such spectral distinctions formed the basis on which to develop a simple three-band ratio algorithm for classification analysis. Next, an additional formula was created to assess the degree of cotton fiber maturity by converting the three-band ratios into an appropriate FTIR maturity (MIR) index. Furthermore, the MIR index was compared with parameters derived from traditional image analysis (IA) and advanced fiber information system (AFIS) measurements. Results indicated strong correlations (R2 > 0.89) between MIR and M AFIS and between MIR and MIA among either International Cotton Calibration standards or selected cotton maturity references. On the other hand, low correlations between the pairs were observed among regular cotton fibers, which likely resulted from the heterogeneous distribution of structural, physical, and chemical characteristics in cotton fibers and subsequent different sampling specimens for individual and independent measurement.

Phenolic constituents in flax bast tissue and inhibition of cellulase and pectinase.

Flax bast tissue was sequentially extracted using hexane, propanol, methanol and water as solvents and extracts were analyzed using reverse phase HPLC and 13C NMR. Results indicated a large variety of aromatic constituents including flavonoids and hydroxy-methoxy cinnamic acids linked to oligosaccharides and hydroxy acids through glycosidic linkages. The extracts inhibited cellulase and pectinase activities and can thus influence retting.

Hyperspectral imaging using RGB color for foodborne pathogen detection

Abstract. This paper reports the development of a spectral reconstruction technique for predicting hyperspectral images from RGB color images and classifying food-borne pathogens in agar plates using reconstructed hyperspectral images. The six representative non-O157 Shiga-toxin producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) grown on Rainbow agar plates were used for the study. A line-scan pushbroom hyperspectral imaging spectrometer was used to scan full reflectance spectra of pure non-O157 STEC cultures in the visible and near-infrared spectral range from 400 to 1000 nm. RGB color images were generated by simulation from hyperspectral images. Polynomial multivariate least-squares regression analysis was used to reconstruct hyperspectral images from RGB color images. The mean R-squared value for hyperspectral image reconstruction was ∼0.98 in the spectral range between 400 and 700 nm for linear, quadratic, and cubic polynomial regression models. The accuracy of the hyperspectral image classification algorithm based on k-nearest neighbors algorithm of principal component scores was validated to be 92% with the test set (99% with the original hyperspectral images). The results of the study suggested that color-based hyperspectral imaging would be feasible without much loss of prediction accuracy compared to true hyperspectral imaging.

Thermochemical Degradation of Melezitose and Trehalulose as Related to Cotton Stickiness

The melezitose and trehalulose contents of a cotton sample heavily contaminated by whitefly honeydew are analyzed by high performance anion exchange chromatography (HPAEC) as a function of heating time at 200°C. Results indicate that the concentrations of melezitose and trehalulose on the surface of the cotton decrease exponentially as a result of thermochemical reactions. The mechanism and rate of each reaction is elucidated by similar heat treatment of pure melezitose and trehalulose with subsequent analysis of reaction products. The rate of thermochemical degradation in the case of trehalulose is of sufficient magnitude that its concentration on the cotton surface may be substantially decreased under controlled conditions of heat and time. The degradation rate of melezitose is nearly an order of magnitude smaller, and its concentration on the cotton surface is not substantially affected under similar conditions.