Danny E. Akin

Feruloyl and p-coumaroyl esterase from anaerobic fungi in relation to plant cell wall degradation

SummaryTrans-feruloyl and trans-p-coumaroyl esterases were found in the culture filtrates of two monocentric (Piromyces MC-1, Neocallimastix MC-2) and three polycentric (Orpinomyces PC-2, Orpinomyces PC-3, and PC-1, an unnamed genus with uniflagellated zoospores) isolates of anaerobic rumen fungi. Treatment of cell walls of Coastal bermudagrass shoots with the filtrates released the trans isomers of ferulic and p-coumaric acids; results of microscopic observations indicated that fungal isolates degraded primarily unlignified cell walls in leaf blades and stems. A greater proportion of ferulic than p-coumaric acid was released by this treatment when compared with the amounts of the acids released by saponification of the walls with 1 M NaOH. The filtrates also showed esterase activities against the trans isomers of methyl ferulate and methyl p-coumarate, with ferulic acid being released at a faster rate than p-coumaric acid. Assays for other cell-wall-degrading enzymes (xylanase, β-xylosidase, α-l-arabinosidase, cellulase, β-glucosidase) indicated that only β-xylosidase correlated with ferulate and p-coumarate esterase activities. The monocentric isolate MC-2 had the highest esterase activity against both the plant cell wall and methyl ester substrates and the highest specific activities of acetyl esterase, β-xylosidase, α-l-arabinosidase, cellulase and β-glucosidase. Isolate MC-2 produced substantially greater amounts of feruloyl and p-coumaroyl esterase when the growth substrate contained higher levels of saponifiable ferulic and p-coumaric acids.

Cell wall alterations in loblolly pine wood decayed by the white-rot fungus, Ceriporiopsis subvermispora

Ultrastructural, immunocytochemical and UV absorption spectroscopy techniques were used to elucidate the progressive changes that occur within woody cell walls during decay by Ceriporiopsis subvermispora. After only 2 weeks of incubation, uranyl acetate staining revealed a diffuse electron dense zone in the secondary wall near hyphae and around the outer circumference of the wall. The extent of cell wall staining increased with longer fungal incubation. No staining occurred in sound unaltered cell walls. Proteins of varying molecular weights (insulin, 5730 Da; myoglobin, 17 600 Da; ovalbumin, 44 287 Da) were infiltrated into sound and decayed wood followed by immunogold labelling and transmission electron microscopy. Insulin readily penetrated into the outer most regions of secondary walls of wood cells after 2 weeks of decay. Myoglobin was first observed to penetrate cell walls at 4 weeks of degradation and ovalbumin was found after 8 weeks in wood with advanced stages of decay where extensive cell wall disruption was evident. None of the proteins used were localized within cell walls of untreated, control wood samples. UV microspectrophotometry demonstrated a progressive loss of absorbance at 240 and 280 nm within the secondary walls and middle lamellae at various sampling times throughout the duration of the decay study.

Structural and chemical properties of grass lignocelluloses related to conversion for biofuels

Grass lignocelluloses, such as those in corn and switchgrass, are a major resource in the emerging cellulose-to-ethanol strategy for biofuels. The potential bioconversion of carbohydrates in this potential resource, however, is limited by the associated aromatic constituents within the grass fiber. These aromatics include both lignins, which are phenylpropanoid units of various types, and low-molecular weight phenolic acids. Structural and chemical studies over the years have identified the location and limitation to fiber degradation imposed by a variety of these aromatic barriers. For example, coniferyl lignin appears to be the most effective limitation to biodegradation, existing in xylem cells of vascular tissues. On the other hand, cell walls with syringyl lignin, e.g., leaf sclerenchyma, are often less recalcitrant. Ferulic and p-coumaric acids that are esterified to hemicellulosic sugars constitute a major limitation to biodegradation in non-lignified cell walls in grass fibers, especially warm season species. Non-chemical methods to improve bioconversion of the lignocelluloses through modification of aromatics include: (1) use of lignin-degrading white rot fungi, (2) pretreatment with phenolic acid esterases, and (3) plant breeding to modify cell wall aromatics. In addition to increased availability of carbohydrates for fermentation, separation and collection of aromatics could provide value-added co-products to improve the economics of bioconversion.

Enzyme-retting of flax and characterization of processed fibers

Enzyme-retting formulations consisting of Viscozyme L, a pectinase-rich commercial enzyme product, and ethylenediaminetetraacetic acid (EDTA) were tested on Ariane fiber flax and North Dakota seed flax straw residue. Flax stems that were crimped to disrupt the outer layers were soaked with various proportions of Viscozyme-EDTA solutions, retted, and then cleaned and cottonized with commercial processing equipment. Fiber properties were determined and crude test yarns were made of raw and Shirley cleaned flax fibers and cotton in various blend levels. Cleaned fibers were obtained from both seed and fiber flax types, but with variations due to treatment. Retting formulations produced fibers having different properties, with enzyme levels of 0.3% (v/v as supplied) giving finer but weaker fibers than 0.05% regardless of EDTA level. Experimental yarns of blended flax and cotton fibers varied in mass coefficient of variation, single end strength, and nep imperfections due to sample and formulation. With cost and fiber and yarn quality as criteria, results established a range in the amounts of components comprising retting formulations as a basis for further studies to optimize enzyme-retting formulations for flax. Under conditions examined herein, Viscozyme L at 0.3% (v/v) plus 25 mM EDTA produced the best test yarns and, therefore, established a base for future studies to develop commercial-grade, short staple flax fibers for use in textiles.

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.

Production of highly efficient enzymes for flax retting by Rhizomucor pusillus

The fungus Rhizomucor pusillus grew well on flax (Linum usitatissimum L.) stems and on pectin as the sole source of carbon. Of several fungal isolates from dew-retted flax, R. pusillus produced enzyme filtrates that were the most effective in retting flax as evaluated by the Fried test. Addition of the chelator oxalic acid enhanced the retting efficiency of culture filtrates from the fungal isolates, with those of R. pusillus giving the highest degree of retting. Approximately one tenth of the protein concentration of R. pusillus as that of the commercial product Flaxzyme produced the same degree of flax retting. The culture filtrate of R. pusillus contained high levels of pectinase activity, low levels of pectin methyl esterase activity, low cellulase and mannase activities, and no pectin lyase or xylanase activities. In our assay, pectinase activity was highest at pH 6.0 and 40°C. The enzyme mixture in the filtrates of cultures grown on citrus-pectin appeared to contain relatively few proteins according to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. R. pusillus produces a simple and efficient enzyme mixture that could provide an opportunity to determine the contribution of specific enzymes to flax retting. Results further suggest that hemicellulase and cellulase may not be required to ret flax. These studies provide information on enzymes towards the goal of developing a commercial process for enzymatic retting of flax.

Influence of Chelating Agents and Mechanical Pretreatment on Enzymatic Retting of Flax

Adding chelating agents, i.e., oxalic acid and ethylenediamine-tetra-acetic acid (edta), substantially increases the retting effect on flax by the commercial enzyme products Ultrazym and Flaxzyme (Novo Nordisk), as shown by scanning electron microscopy, release of reducing sugars, and the Fried test. Degradation of pectin-rich citrus peel by these enzymes also increases with the addition of oxalic acid and edta, while citric acid has a low or insignificant effect. Oxalic acid at 50 mmol concentration reduces the amount of Flaxzyme required to effectively ret flax stems, according to the Fried test, by a factor of about 50. Retting with Flaxzyme and 50 mmol oxalic acid is completed in approximately half the time at 45°C, compared with that at 22°C. A mechanical pretreatment that crushes flax stems by pulling them over a surface at a 90° angle opens the flax structure and further increases the efficiency of enzymatic retting. These procedures appear to modify both the chemical and structural features of flax, and they reduce the time as well as the amount of enzyme required to ret flax, therefore improving technical efficiency and economic attractiveness at the commercial level.

Chemical and physical characterization of water- and dew-retted flax fibers

Abstract The composition of dew-retted and water-retted flax fibers were evaluated by chemical and mass spectral analyses to determine their chemical differences. Phenolics, waxes, cutin, and carbohydrates were determined by gas liquid chromatography. Water-retted fibers contained more residual wax and lower arabinose content than the dew-retted and were finer and stronger. Pyrolysis mass spectrometric analysis differentiated water- and dew-retted fibers. Principal component analysis of the chemical data including both strength and fineness measurements produced a grouping of the water-retted samples distinct from the dew-retted fibers. Principal component analysis of the mass spectral data produced the same grouping based mass markers characteristic of the chemical components that were associated with the initial grouping with fineness and strength measurements.

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.

Flax-retting by polygalacturonase-containing enzyme mixtures and effects on fiber properties

Enzyme-retting of flax was accomplished via individual treatment with four polygalacturonase (PGase) containing solutions of various fungal sources and the resulting fibers were characterized. The retting solutions were equilibrated to contain 2.19 U of PGase activity as determined via a dinitrosalicylic acid (DNS) reducing sugar assay. As compared with the buffer control, treatment with the various enzyme solutions increased the yield of fine fibers. Treatment with Aspergillus niger PGase resulted in a 62% increase in fine fiber yield as compared with the buffer control and fiber strength did not statistically differ (P</=0.05) between these treatments. Retting via PGases of Rhizopus origin produced the weakest fibers. These results illustrate that the crude PGases differ in their ability to ret flax and that under the defined experimental conditions the A. niger PGase is a better retting agent. Light microscopy demonstrated the ability of all enzymes to separate fiber from shive and epidermal tissues. Enzyme profiles of the solutions were determined via viscometric assays. Pectinolytic activity was the predominant activity of all enzymes tested. Activity against carboxymethyl cellulose (CMC) was a minor component of all solutions except A. niger PGase for which no activity was detected.