Donald J. Nevins

Isolation and identification of O-(5-O-feruloyl-α-l-arabinofuranosyl)-1(→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose as a component of Zea shoot cell-walls

Abstract Zea shoot cell-walls were hydrolyzed with 30m m oxalic acid followed by treatment with “Driselase” (a Basidiomycetes enzyme preparation) to obtain carbohydrate fragments containing ferulic acid. The structure of the major feruloyl compound was identified as O -(5- O -feruloyl-α- l -arabinofuranosyl)-(1→3)- O -β- d -xylopyranosyl-(1→4)- d -xylopyranose on the basis of 13 C-n.m.r., methylation analysis, and partial acid-hydrolysis, alkali hydrolysis, or esterase hydrolysis followed by analyses of the hydrolyzate.

Partial purification of endo- and exo-β-D-glucanase enzymes from Zea mays L. seedlings and their involvement in cell-wall autohydrolysis.

The proteins dissociated from isolated Zea seedling cell wall using high-ionic-strength salt solutions have been found to include a number of enzymes which appear to participate in autolytic reactions of the cell wall. These enzymes caused extensive degradation of enzymatically inactive cell wall, liberating as much as 100 μg/mg dry weight over a 48-h period. Lithium chloride (3M) was shown to be most effective in yielding protein and wall-degrading activities.Molecular-sieve chromatography of the cell-wall protein resolved endo-β-D-glucanase and exo-β-1,3-glucanase (EC 3.2.1.58) activities when Avena glucan and laminarin, respectively, were employed as substrates. The exoenzyme (molecular weight around 60,000) was strongly inhibited by inorganic mercury at a concentration which suppressed the release of monosaccharide from autolytically active cell wall. The endo-β-D-glucanase (MW around 26,000), which showed a marked preference for substrates of mixed-linkage, exhibited features indicating that it initiates the autolytic solubilization of wall glucan.Cell-wall β-D-glucan, recovered as a component of an alkali-soluble cell-wall fraction, served as a substrate for the purified glucanases. Their hydrolysis pattern, assessed using gel exclusion chromatography and product analysis, confirmed that they hydrolyze β-D-glucan. The products generated by the endoglucanase were similar in molecular-size distribution to those liberated from autolytically active-wall. Exoglucanase activity was required for extensive hydrolysis of β-D-glucan in vitro.During coleoptile development the autolytic activity of the cell wall increased dramatically. This increased activity, however, did not parallel the growth potential of the tissue, but more closely reflected an increase in cell-wall β-D-glucan, the primary substrate for autolytic reactions.

Cell wall β-d-glucans of five grass species

Abstract Structural features of noncellulosic β- d -glucans of Zea mays, Hordeum vulgare, Triticum vulgare, Secale cereale , and Sorghum bicolor were compared. Treatment of cell walls derived from these species with specific Bacillus subtilis or Rhizopus glucanases yields virtually identical profiles upon Bio-Gel P-2 fractionation of the liberated oligosaccharides. The two predominant reaction products, a trisaccharide and tetrasaccharide, were identified as 3- O -β-cellobiosyl- d -glucose and 3- O -β-cellotriosyl- d -glucose respectively by virtue of the specificity of these enzymes and by paper chromatography and electrophoresis. The similarity of the reaction product profiles indicates a rather regular repeating sequence in all β- d -glucans examined. The ratios of 3- O -β-cellobiosyl- d -glucose to 3- O -β-cellotriosyl- d -glucose indicates that 30.4–30.9% of the β-glucosyl linkages in the intact molecule are 1 → 3. The yields of wall glucan as estimated from the quantity of oligosaccharides released, range from 41 μg/mg wall in Hordeum to 97 μg/mg wall from Sorghum .

Distribution of noncellulosic β-d-glucans in grasses and other monocots

Abstract Cell wall specimens from nonendospermic tissues of six grasses and representatives of nine other monocot families were treated with a specific glucanase in order to liberate wall-bound noncellulosic β- d -glucans. Gel filtration chromatography profiles of the oligosaccharides released from all grass species indicated the presence of a mixed linkage β-(1→3):(1→4)-glucan. The results also indicate that this glucan was not present in the other monocots examined. The evidence from this and previous studies indicates that the mixed linkage glucan may be restricted in the monocots to the Gramineae.

Degradation of a glucan containing β-(1→3) and β-(1→6) linkages by exo-(1→3)-β-D-glucanase

Abstract A β- d -glucan isolated from a Japanese marine alga known as “Arame” ( Eisenia bicyclis ) was shown by methylation analysis to be composed of (1→3) and (1→6) linkages and (1→3), (1→6) branch points. The 13 C-n.m.r. spectrum confirmed these linkages and also verified the absence of β-(1→4) linkages. The endo-(1→3)-β- d -glucanase of Rhizopus liberates from the native glucan oligosaccharides having an odd number of glucosyl residues, such as 3, 5, 7, and 9. The exo-(1→3)-β- d -glucanase of Basidiomyete QM 806 produces β-glucose, gentiobiose, and 6- O -β-laminarabiosyl- d -glucose, and a tetrasaccharide, β- d -Glc-(1→6)-β- d -Glc-(1→3)-β- d -Glc-(1→6)- d -Glc. Thus, the exo-enzyme appears to bypass glucosyl substituents at O-6 of 3-O-linked glucose in this polymer and, because it liberates oligosaccharides, it behaves as if it mediates an endo-type hydrolysis of the glucan.

Purification and some properties of β-transglucosylases of sclerotinia libertiana, having the ability to synthesize higher cello-oligosaccharides from cellotriose or cellotetraose

Abstract Two specific transglucosylases were extracted from the culture of Sclerotinia libertiana . The enzymes were separated from cellulases and cellobiases. Each of the enzymes moved as a single band in poly(acrylamide)-gel electrophoresis. The molecular weights of the enzymes were estimated to be 188,000 by electrophoresis, and 165,000 and 175,000, respectively, by chromatography on Bio Gel P-200. The optimum pH of the reaction mediated by the enzymes was 5.0. The enzymes synthesized a tetrasaccharide from cellotriose and a pentasaccharide from cellotetraose, without production of d-glucose; they also synthesized larger oligosaccharides, in a long-term action, from either substrate.