Alex H. Gordon

Xylan-degrading enzyme system produced by the fungus Aspergillus awamori : Isolation and characterization of a feruloyl esterase and a p-coumaroyl esterase

Abstract Feruloyl esterase and p -coumaroyl esterase activities were separated by ion-exchange chromatography on a column of DEAE-Sepharose CL 6B and then purified by hydrophobic interaction chromatography and anion-exchange chromatography, respectively. Feruloyl esterase had an apparent molecular mass of 112 kDa (SDS-PAGE) and an isoelectric pH of 3.7; the p -coumaroyl esterase was much smaller (75 kDa) and had a pI of 4.2. The feruloyl esterase was highly specific for the methyl ester of ferulic acid and for feruloyl esters associated with a water-soluble xylan polysaccharide from wheat straw; p -coumaroyl esterase was also highly specific for the corresponding p -coumaroyl ester substrates. The wheat straw xylan was de-esterified by the esterases without prior degradation of the polysaccharide; however, esterified short-chain xylooligosaccharides which were generated by a purified fungal xylanase were better substrates. The apparent K m values for the feruloyl and p -coumaroyl residues were 0.93 ± 0.05 and 0.031 ± 0.02 mM, respectively, using the methyl ester substrates; the respective V max values were 2.82 ± 0.05 and 0.57 ± 0.03 μ mol min −1 mg −1 protein. The esterases had only a small capacity to release ferulic and p -coumaric acids from cell walls of various plants, including mesophyll cell walls from rye grass, but they were more effective when acting in concert with the commercially available plant cell wall-degrading enzyme Celluclast.

Methylation analysis of mesophyll, epidermis, and fibre cell-walls isolated from the leaves of perennial and Italian ryegrass

Abstract Intact, finely milled mesophyll, epidermis, and fibre cell-walls prepared from the leaves of perennial and Italian ryegrass have been subjected to methylation analysis. Methylation of the cell-walls led to a consistently higher recovery of glucose residues than that obtained by analysis of monosaccharide residues as their alditol acetates. Values for other sugars were in close agreement. The partially methylated sugars formed were consistent with the presence, in order of decreasing concentration, of cellulose, (glucurono)arabinoxylan, xyloglucan, rhamnogalacturonan, (1→3),(1→4)-linked glucan, (1→4)-linked galactan, and (1→3),(1→6)-linked galactan. The relative proportions of these polysaccharides differed between the various types of cell. Arabinoxylan comprised 21.6%, 26.7%, and 36.5% of the total sugars recovered from mesophyll, epidermis, and fibre cell-walls, respectively. Mixed-linked glucan and rhamnogalacturonan were found in epidermis walls in amounts 2- to 3-fold higher than in other cell-walls. The xylan backbone of arabinoxylan was more heavily substituted in primary than in secondary-thickened (fibre) cell-walls. Arabinose, found largely as terminal residues in the cell-walls, carried various amounts of alkali-labile substituents, particularly at position 5. The extent of 5-substitution reflected the phenolic content and was substantially higher in fibre cell-walls. The methylation data, coupled with the analytical data for uronic acids and non-carbohydrate components, accounted for ∼98% of the cell-wall dry matter.

Methylation of unfractionated, primary and secondary cell-walls of plants, and the location of alkali-labile substituents

Abstract A procedure has been developed for the methylation of intact (mature) plant cell-walls in good yield. The method depended on the reduction of particle size by dry milling in liquid nitrogen to a level that allowed plant fragments to swell and disperse in the methylation solvent. Quantification of the results was achieved by using methyl β- d -allopyranoside as internal standard. Perennial ryegrass cell-walls, whole barley straw, and beechwood, with degrees of lignification of 1.93, 12.66, and 19.33%, respectively, were used to determine the efficiency of methylation after milling. The recoveries of parent sugars, calculated by summing values for the appropriate partially methylated sugars, were 116, 102, and 109%, respectively, of those determined on the basis of alditol acetates. Acetalation of the free hydroxyl groups of milled, whole cell-walls, using methyl vinyl ether, enabled selective methylation to be performed at sites of alkali-labile substitution. The resulting recovery of pentoses was nearly quantitative, but that of (cellulosic) glucose was very low. These procedures allow linkage analysis of the structural polysaccharides and determination of the location of sites of alkali-labile substitution in the matrix polysaccharides of the intact cell-wall.

Polysaccharides and associated components of mesophyll cell-walls prepared from grasses

Abstract Mesophyll cells have been prepared from rye-grass leaves by a procedure similar to that used by Edwards and Black 1 for crab-grass. Successive filtrations through nylon cloth of 300-, 80-, 45-, and 15-μm mesh yielded suspension consisting chiefly of single cells, some broken. Passage through a French pressure cell brought the total breakage to near 100%. Microscopic examination of the walls showed that very little of the cell contents remained. The walls were thin (0.1–0.2 μm) and had numerous pit fields; torn surfaces revealed a randomly oriented, fibrillar structure. Determinations of the polysaccharide content, protein, phenolic esters, lignin, and ash accounted for ∼85% of the dry matter. The proportion of glucose in the polysaccharides of these walls was similar to that in the leaf cell-wall fraction as a whole, but the proportions of arabinose, galactose, and uronic acid were higher, and that of xylose lower. The acetyl content was 1.0–1.2%. The walls were readily digested by fungal culture filtrates, by snail digestive juice, and by immersion in the sheep rumen in nylon bags. These findings are discussed in relation to the digestion of forage cell-walls in ruminants.

d-Xylan-degrading enzyme system from the fungus Phanerochaete chrysosporium. isolation and partial characterisation of an α-(4-O-methyl)-d-glucuronidase

A number of fungi were screened for their capacities to produce extracellular alpha-(4-O-methyl)-D-glucuronidase. Of those tested, Phanerochaete chrysosporium ATCC 24725 produced the enzyme in greatest yield. The single alpha-(4-O-methyl)-D-glucuronidase produced by this fungus was purified by a series of chromatographic methods involving anion exchange, hydrophobic interaction and chromatofocusing. Isolated in this way, the enzyme had an apparent molecular mass of 112 kDa in sodium dodecyl sulphate polyacrylamide gels, and a pI of 4.6 when determined by isoelectric focusing in polyacrylamide gels. The enzyme was optimally active at pH 3.5, but showed significant activity over the pH range 3-5. In the absence of substrate the enzyme was inactivated at pH 3.5 in 2 h at 50 degree C: at pH 5.0 it retained 42% of its activity for 24 h at this temperature. The enzyme showed little activity on glucuronoxylan polysaccharides, but some short-chain xylo-oligosaccharides which were substituted with alpha-linked 4-O-methyl-D-glucopyranosyl uronic acid attached to the 2-position of the non-reducing D-xylopyranosyl residue were readily hydrolysed. There were marked synergistic effects apparent in the release of 4-O-methyl-D-glucopyranosyl uronic acid from various glucuronoxylans when the alpha-(4-O-methyl)-D-glucuronidase was acting in concert with endo-(1-->4)-beta-D-xylanase, and with beta-D-xylosidase and/or an alpha-L-arabinofuranosidase.

A multiple-column approach to the methylation analysis of plant cell-walls

Abstract A procedure has been developed for the improved analysis of the neutral-sugar glycosidic linkages in plant cell-walls, utilising capillary g.l.c. and columns of three different phases for the separation of the products of methylation analysis. Retention coefficients are reported for a wide range of partially methylated alditol acetates on columns of CP-Sil88 and a bonded phase (BP-1) equivalent to OV-1. Using these phases and SP-1000, all cases of co-chromatography can be resolved. Computers were used to process the large amounts of data produced, to identify peaks and to assist in merging the results obtained using the three phases.

Deacetylation and enhancement of digestibility

Abstract The nylon bag technique has been used to study the effects of various reagents on the digestion of grass cell walls and straws in the sheep rumen. A large proportion of the acetyl groups were removed by sodium alkoxides in alcoholic solution with appreciable improvement in digestibility. Aqueous sodium hydroxide removed virtually all the acetyl, producing a correspondingly greater improvement. When straw was treated with increasing amounts of sodium hydroxide, up to 10 g/ 100 g straw, the enhancement of digestibility was linear but not the release of acetyl; 2 g/100 g removed half the acetyl groups. After extraction with neutral detergent solution, followed by chlorite delignification, about 70% of the acetyl groups were left; sodium hydroxide treatment then had no effect upon the rate or extent of digestion of this residue. Physical methods, based on X-ray diffraction and infrared absorption, showed that the degree of order of the cellulose component, which sets an upper limit to its rate of digestion, was not affected by concentrations of sodium hydroxide up to 20 g/100 g straw, more than sufficient to produce the maximum enhancement of digestibility. Both electron micrography and chemical analysis indicated that the major components of the cell wall were removed simultaneously. It is concluded that the effects of basic reagents must be sought in the lignin-hemicellulose fraction of the cell wall, and that both covalent and hydrogen bonds may be responsible for its resistance to digestion.

Pectic polysaccharides of mesophyll cell walls of perennial ryegrass leaves

Abstract Extraction of mesophyll cell walls from leaves of perennial ryegrass with CDTA, a chelating agent, removed 25 mg uronic acid g − , largely in the form of a polymer which spontaneously precipitated on removal of the CDTA or during subsequent purification. Methylation analysis, before and after reduction, showed that the precipitated polymer was a 1,4-linked homogalacturonan essentially free from neutral sugar residues, with a low degree of acetylation (3.6%) and methyl esterification (3.3%). Hot water (HW) extracted further acidic material (5 mg uronide g −1 cell wall) which could be resolved by ion-exchange chromatography into neutral mixed-linked glucan and bound rhamnogalacturonan fractions. The latter co-chromatographed with sugar residues typical of 3-, 4- and 6-linked galactan and arabinoxylan. Pectin esterase promoted the release from cell walls of HW-soluble rhamnogalacturonan by polygalacturonase, but had no effect on the release of CDTA-soluble uronide. The presence of both homogalacturonan and rhamnogalcturonan, typical of dicotyledons, suggested that the pectic polysaccharides of the Gramineae differed from those of other plants in amount only, rather than nature.