Enzymatic Maceration of Plant Tissues by Endo-Pectin Lyase and Endo-Polygalacturonase from Aspergillus japonicus

Ishii, Shigetaka

doi:10.1094/phyto-66-281

Cited by 46 publications

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“…These results and other evidence lead to the well known conclusion that pectic polysaccharides are the principal binding agents between higher plant cells (7). This is true in the tissues of dicots and some monocots such as Liliaceae and Araceae (12,14), but not always true in the case of graminaceous tissues. It has been demonstrated in many species that pectic polysaccharides are very minor components in cell walls of Gramineae, suggesting that they do not play as important a role in the cell walls as they do in cell walls of dicots (9).…”

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confidence: 74%

Cell Wall Cementing Materials of Grass Leaves

Ishii¹

1984

Plant Physiol.

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Treatment of grass leaves with either a purified pectin lyase of Aspergillus japonicus or a purified xylanase of Trichoderma viride could lead to the isolation of some single leaf cells. However, a mixture of pectin lyase and xylanase brought about more rapid isolation of single cells than did either of the two enzymes alone, indicating a synergistic effect. Analysis of the components released from oat cell walls by the enzymes indicated that both homogalacturonans with a high degree of esterification and a kind of glucuronoarabinoxylan with ferulic acid ester may play a role in cell wall cementing in grass leaves.The enzymic maceration of plant tissues, in which cells are freed from one another, is the first reaction step of enzymic protoplast isolation. Breakdown of intercellular cementing materials leads to separation of individual cells. Endo-types of PG' (2,3,25), PL (5,16,22), and pectate lyase (8,10,20) Purified Enzymes. Purified PG and PL were obtained from the culture medium of Aspergillus japonicus by the methods described previously (17,18). Xylanase (fraction P4) was purified from a cellulase preparation of Trichoderma viride by the method described in a previous paper (15).Assay of Macerating Activity. The lower epidermis of oat leaves was removed, and the leaves (140 mg) were placed in a 50-ml Erlenmeyer flask containing 0.8 M mannitol, 20 mm Mes buffer (pH 5.5), 10 mM cysteine, enzymes, and water to a final volume of 5.0 ml. Mannitol was added as an osmotic stabilizer. The flasks were shaken on a rotary shaker at 120 rpm and 25°C. After 2 h the mixture was filtered through Miracloth, and the OD ofthe filtrate was measured at 660 nm. A linear relationship exists between the OD at 660 nm and the number of single cells counted in a hemocytometer.Treatment of Oat Cell Walls by Enzyme. The cell walls (100 mg) were placed in 150-ml Erlenmeyer flasks containing 50 ml of 0.1 M ammonium acetate buffer (pH 5.5) and 370 gg of PL or 1.65 mg ofxylanase. The flasks were shaken on a rotary shaker at 100 rpm and 30°C. After 3 h the reaction mixtures were filtered through Toyo No. SC paper and the residues were repeatedly washed with 0.1 M ammonium acetate buffer (pH 5.5). The filtrate and washings were combined and lyophilized.Gel Filtration. Gel filtration was carried out on a Sephadex G-100 column (1.9 x 100 cm) equilibrated with 0.1 M ammonium acetate buffer (pH 5.5). Samples were applied in 2.0 ml to the column, and the elution was carried out with 0.1 M ammonium acetate buffer (pH 5.5) at a flow rate of 20 ml/h. Analysis of Sugars. The uronide content was estimated by the m-hydroxydiphenyl method using galacturonic acid as a standard (4) by TLC using ethyl acetate-pyridine-acetic acid-water (5:5:1:3, by vol) and the organic phase from a mixture of 100 g of phenol, 100 ml of water, and 1 ml of 85% HCOOH (23)

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Cell Wall Cementing Materials of Grass Leaves

Ishii¹

1984

Plant Physiol.

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“…nidulans appear to be identical to the enzymes isolated from A . niger, also with respect to their activities on complex substrates, as is indicatcd by the ability of the PGII isolated from A. nidulans transformants to macerate cucumber tissue [41]. We therefore intend to use the expression of subcloned polygalacturonase genes in A .…”

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confidence: 99%

The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes

Bussink

Buxton

Fraaye

et al. 1992

European Journal of Biochemistry

117

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Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases T and I1 (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lEMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergilh nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A . niduluns transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pguC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intronlexon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A . niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to rcpresent a binding site for a regulatory protein as it shows a high similarity to the yeast CYCl upstream activation site recognized by the HAP2/3/4 activation complex.Polygalacturonases are involved in the degradation orpectin, a complex polysaccharide that is primarily found in the middle lamella and primary cell wall of higher plants [I]. Polygalacturonases as well as other pectolytic enzymes are produced by many organisms (reviewed in [2]), and microbial pectolytic enzymes have been extensively studied in relation to plant pathogenesis. The molecular biology of the bacterial enzymes, especially the pectate lyases of Erwiniu, is well developed compared to that of the pectolytic enzymes of fungal pathogens [3, 41. Recently, genomic or cDNA clones coding for pectinesterase [5], pectin lyases [6, 71, pectate lyase [8] and polygalacturonases [9 -121 have been obtained from Aspei-gillus species, which are considered to be saprophytes. The availability of commercial pectinase preparations derived from Aspergillus niger has contributed to the rapid progress in gene Correspondence to J. Visser, Section

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“…The mixture of enzymes constituting Pectolyase increased ethylene production 15-to 25-fold when introduced into tomato and orange fruits. The enzymes purified from Pectolyase all increased ethylene production in the fruits but the lyases were generally more effective than the hydrolases.Pectolyase is a commercially available enzyme mixture prepared from culture filtrates of Aspergillus japonicus (8,13). These enzymes in combination with cellulases are very effective in degrading cell walls and thereby liberating protoplasts.…”

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“…Pectolyase is a commercially available enzyme mixture prepared from culture filtrates of Aspergillus japonicus (8,13). These enzymes in combination with cellulases are very effective in degrading cell walls and thereby liberating protoplasts.…”

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Pectic Enzymes in Pectolyase

Baldwin¹,

Pressey²

1989

Plant Physiol.

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The pectic enzymes in Pectolyase were separated by ion exchange chromatography on Q-Sepharose. Three pectin lyases, two polygalacturonases, and a pectinmethylesterase were resolved. The enzymes were further purified on Mono 0 and/or Mono S columns to remove traces of cellulase. The enzymes had molecular weights ranging from 25,000 to 36,000 daltons. They were optimally active between pH 4.0 and 6.2 and were not greatly affected by ions. The pectin lyases and polygalacturonases were endo-enzymes. They solubilized uronic acids from washed cell wall fragments, but the lyases were much more effective than the polygalacturonases. The mixture of enzymes constituting Pectolyase increased ethylene production 15-to 25-fold when introduced into tomato and orange fruits. The enzymes purified from Pectolyase all increased ethylene production in the fruits but the lyases were generally more effective than the hydrolases.Pectolyase is a commercially available enzyme mixture prepared from culture filtrates of Aspergillus japonicus (8,13). These enzymes in combination with cellulases are very effective in degrading cell walls and thereby liberating protoplasts. For this reason, Pectolyase is widely used to generate protoplasts in cell culture research. There have been several attempts to identify the cell wall degrading enzymes in Pectolyase to explain the effectiveness of this preparation. Ishii and coworkers (10, 11) separated an endo-PG' (EC 3.2.1.15) and an endo-PL (EC 4.2.2.3) from the culture filtrates. They also obtained evidence that the solutions contained a maceration stimulation factor which enhanced the action of both enzymes on cell walls (9). The combination of enzymes able to degrade pectate and pectin plus the enzyme activator appeared to be the basis for the potency of Pectolyase in liberating protoplasts.Our interest in the pectic enzymes in Pectolyase stems from the observation that purified tomato PG induced ethylene production and accelerated ripening when infiltrated into green tomato fruit (4). We wanted to expand these studies to microbial pectic enzymes and selected Pectolyase as a source. Attempts to separate the PL and PG (10, 11) revealed that the Pectolyase contained several isoenzymes of each. This ' Abbreviations: PG, polygalacturonase; PL, pectin lyase; PME, pectinmethylesterase.paper describes the separation and properties of the enzymes and their effectiveness in inducing ethylene production in tomato and orange fruit. MATERIALS AND METHODS SubstratesCitrus pectin (Sigma Chemical Co.) was purified by precipitation from a 1% aqueous solution by the addition of two volumes of ethanol, followed by washing with ethanol and acetone. Polygalacturonic acid was prepared from pectate (Sigma Chemical Co.) by partial hydrolysis as described earlier (15). Cell walls were prepared from the fruits of apple, nectarine, cucumber, and tomato and the roots of carrot and radish by homogenizing 100 g of tissue with 200 mL of0.5 M sodium phosphate (pH 7.0). The insoluble material was collected by centrifuga...

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Enzymatic Maceration of Plant Tissues by Endo-Pectin Lyase and Endo-Polygalacturonase from Aspergillus japonicus

Cited by 46 publications

References 0 publications

Cell Wall Cementing Materials of Grass Leaves

Cell Wall Cementing Materials of Grass Leaves

The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes

Pectic Enzymes in Pectolyase

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