Studies on Pectic Enzymes of Microorganisms

To pick out potent strains which specifically produce one of several pectic enzymes, endo- and exo-polygalacturonase, pectin esterase, macerating, and apple juice clarifying activities were examined with regard to 344 strains of mold (containing 71 strains of phytopathogenic mold) grown on a bran culture medium and 56 strains of shakingly cultured yeast. As the result of screening, Asper gillus saitoi and Penicillium islandicum were isolated as potent specific producers of endo-polygalacturonase. And the composition of pectic enzymes of mold was found to be rather genus or species specific. So far as examined in crude enzyme systems, there was no parallelism between anyone of pectic enzyme activities and apple juice clarifying or macerating activities.     

Role of Cell-wall-degrading Enzymes in the Development of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

Extracts of limited and spreading lesions caused by Mycosphaerellapinodes on detached pea leaflets contained proteolytic, cellulolytic,and pectolytic enzymes although only in spreading lesions wasthere much degradation of cell walls. The brown tissue fromlimited M. pinodes lesions was resistant to maceration by enzymesfrom spreading lesions. Limited lesions contained water-soluble,95 per cent ethanol insoluble, partially dialysable, inhibitorsof pectin transeliminase which is probably the macerating enzyme. Green, spreading M. pinodes lesions developed only on leafletsfloating on water. Growth of these lesions was accompanied bycontinous loss of phenolic substances to the water while thephenol content in infected tissue remained similar to that inuninoculated controls. In contrast, the phenol content in mature,limited M. pinodes lesions on leaflets suspended just abovethe water level was about four times that in healthy tissue.It is suggested that loss of phenolics from floating leafletsprevents tissue browning and the development of resistance ofthe cell walls to maceration. But this type of resistance doesnot appear to be a major factor in the limitation of lesionson suspended tissue. Extracts of limited Ascochyta pisi lesions on leaflets floatingon water contained pectolytic and hemicellulolytic enzymes.Some cellulase (Cx) activity was detected although there waslittle evidence of cellulose degradation in cell walls in infectedtissue. The nature of the macerating factor remains uncertainbut it was found that extracts from lesions contained inhibitorsof pectic enzymes and that tissue just beyond that colonizedby the fungus was resistant to maceration; this resistance isprobably important in restricting the growth of the pathogenin the leaf.     

Pectic Enzymes and Phenolic Substances in Apples Rotted by Fungi

The activities of pectic enzymes in extracts from sound applesand from apples rotted by different fungi are described. Sclerotiniafructigenaand Botrytis cinerea rots had little or no polygalacturonaseor macerating enzyme activity, but Penicillium expansum rotswere very active in these respects. Extracts from each of therots had very high pectinesterase activity, and contained galacturonicacid. None of the rots had any cellulase activity. Each of thefungi produced polygalacturonase, macerating enzymes, and pectinesterasein liquid media. The effects of adding extracts of apples tothese media are described. Filtrates from cultures of S. fructigenaand P. expansum liberated galacturonic acid from apple fruitfibre which had been thoroughly extracted with cold water. The phenolic jsubstances present in healthy and rotted tisueswere estimated. B. cinerea and S. fructigena rots containedvery little, but P. expansum rots contained as much as healthytissue which had been allowed to brown. An extract of healthyapple tissue reduced the activity of the polygalacturonase ina culture filtrate of S. fructigena. The substances responsiblefor this were tentatively identified as leuco-anthocyanins whichhad been changed to other compunds following the action of polyphenoloxidase.Thej significance of these results is discussed.     

Studies in the Physiology of Parasitism: XXI. The Production and Properties of Pectic Enzymes secreted by Fusarium moniliforme Sheldon

Fusarium moniliforme secreted macerating enzymes in liquid mediaonly when these contained certain natural extracts, pectic substances,or galacturonic acid. Apple extract was unsuitable for enzymesecretion and also inhibited enzyme secretion in synthetic mediaotherwise suitable. Protopectinase activity of solutions was highest in the pH range8·0–9·0, was rapidly lost at temperaturesabove 50–60° C., and was reduced by concentrationsof phosphate higher than 0·02 M. The enzyme was partiallypurified by precipitation in 60 per cent. acetone at pH 6·0. Protopectinase solutions also contained an enzyme which reducedthe viscosity of solutions of various pectic substances. Theproperties of this enzyme were, in general, similar to thoseof protopectinase. When activity of enzyme solutions was measured by the liberationof reducing groups, pectate solutions were more rapidly degradedthan were solutions of a high methoxyl pectin, particularlyin the early stages of the reaction. Paper chromatography ofthe products formed showed that pectate and pectin were degradedin different ways. Although the pathogen readily secreted protopectinase in potatoextract, potato tubers were not readily parasitized. In contrast,Fusarium avenaceum which readily attacked tubers, secreted littleprotopectinase in potato extract.     

Enzymatic Deconstruction of Backbone Structures of the Ramified Regions in Pectins

The pectic enzymes are a diverse group of enzymes that collectively degrade pectin, a mixture of highly heterogeneous and branched polysaccharides rich in d-galacturonic acids forming a major component of the primary cell wall of plants. This review covers key enzymes that function to deconstruct the “ramified region” of pectin. The enzymes include glycoside hydrolases and polysaccharide lyases that degrade complex pectic domains consisting of rhamnogalacturonans, xylogalacturonans, and other heterogeneous polymers. The chemical nature of the pectic substrates for the enzymes is presented. The biochemical properties of the enzymes, the mechanisms of enzyme actions, and related structures and functions, are described. Applications of these enzymes in fruit juice processing and in the production of bioactive compounds, as well as their technological relevance to the deconstruction of cell wall structures for biomass conversion are discussed.     

Application of Pectic Enzymes to Maceration of Plant Tissues for Microscopic Study

A new method of maceration of relatively soft plant parts, such as petioles, fruits, and fruit skins, which depends upon the treatment of the material with pectic enzyme solutions is described. The commerical preparation Pectinol W, (manufactured by Rohm and Haas Co., Washington Square, Philadelphia S, Pa.) as well as pectic enzymes secreted by growing Aspergillus species upon pectin-containing media were effective in macerating a variety of plant materials.     

Green mould of oranges caused by Penicillium digitatutn Sacc.; effect of additives on spore germination and infection

Water extracts of rind, essential oil and juice from oranges, also citrus pectin and citric acid promoted the formation of lesions when spores of Penicillium digitatum were placed in wounds 1·0 mm deep in flavedo of oranges; fructose, glucose and sucrose had little effect. Rind extracts were less effective in wounds 0·5 mm deep but orange juice and pectin still increased infection. None of the substances allowed the parasite to infect fruit through unwounded surfaces. Germination of spores in water increased as spore concentration decreased but was poor even at low concentrations. Almost all spores germinated in aqueous extracts of flavedo, albedo or whole rind, or in wounds on the surface of fruit. Fructose, glucose, sucrose and xylose were less effective but still caused over two-thirds of spores to germinate but only in the presence of phosphate buffer. Without buffer, germination was little different from that in water. Arabinose and galactose stimulated germination to a lesser extent but with the same phosphate effect. Carboxymethylcellulose and pectin did not affect germination. A variety of substances containing nitrogen increased germination but to different degrees, decreasing in the order, casamino acids, yeast extract, ammonium salts, nitrate. Thiamin and to a lesser extent biotin were also effective. Volatile substances from rind infected with P. digitatum stimulated spore germination and growth of germ tubes. The significance of these results is discussed in relation to infection.     

Growth of Lesions Caused by Botrytis fabae on Field Bean Leaves in Relation to Foliar Bacteria, Non-enzymic Phytotoxins, Pectic Enzymes and Osmotica

Extracts from spreading chocolate spot lesions contained a heat-stable,water-soluble, ether- or ethanol-insoluble phytotoxic fraction.Elution from Sephadex G75 indicated that the molecular weightsof the toxic compounds were between 10000 and 30000 daltons.The toxins were adsorbed on DEAE Sephadex and eluted from itwith 0.1–0.2 M NaCl. Toxic activity was enhanced in solutionswith low osmotic potentials similar to those found in lesions.Controlling the growth of contaminating bacteria in lesionswith antibiotics appeared to reduce the levels of heat-stabletoxins extracted and to reduce the rate of increase in lesionsize. There were high levels of polygalacturonase (PG) activity inlesion extracts; large quantitites of PG, but little or no trans-eliminases,were produced by Botrytis fabae in liquid culture. A pectolyticstrain of Bacillus lentus was associated with trans-eliminasesin lesion extracts, and produced transeliminases, but not PG,in liquid culture. Activities of both heat-stable phytotoxins and of pectic enzymesmay depend on fungal isolate and types and populations of contaminatingbacteria. Botrytis fabae L., Bacillus, Vicia faba L., phytotoxins, pectic enzymes, chocolate spot     

Studies in the Physiology of Parasitism: XX. The Pathogenicity of Botrytis cinerea, Sclerotinia fructigena, and Sclerotinia laxa, with special reference to the part played by pectolytic enzymes

1. Though Sclerotinia fructigena, S. laxa, and Botrytis cinereacause rotting of apple tissue and death of the protoplasts,little or no pectolytic activity was detectable in extractsof the rotted tissue. 2. Pectic materials were extracted from normal and parasitizedapple tissue in three fractions and precipitated as calciumpectate. There was a loss of total, total insoluble, and solublepectic substances in the invaded tissues. This was most markedwith B. cinerea and S. laxa and least with S. fructigena. 3. Pectolytic activity was measured by methods involving (a)maceration of plant tissues, (b) viscosity and reducing groupdeterminations in pectic substrates, (c) increase in acidityof pectin. By these methods it was shown that pectolytic enzymeswere produced by all three fungi in synthetic media. With S.fructigena, which was the only fungus studied in detail, replacementof glucose by pectin increased the formation of pectolytic enzymes. 4. When various apple extracts were used as culture media, littleor no pectolytic activity was detectable. With all three fungithe presence of apple juice in a culture medium, which by itselfwas suitable for enzyme formation, resulted in the suppressionof pectolytic activity. 5. Oxidized apple juice had a pronounced effect in deactivatingcertain pectolytic enzymes, an effect which was especially markedwith B. cinerea. This points to an interaction between the pectolyticand oxidizing systems and introduces a new line of approachto the study of the biochemical interaction between host andparasite.     

Studies on Pectolytic Enzymes of Molds

Two hundred and fifty strains of molds including plant pathogenic microorganisms were cultured on solid media, and the production of pectolytic enzymes was followed by the clarification of fruit juice. Forty-four of them were found to have the action of clarifying fruit juice.

Out of the said 44 strains, the following 7 strains, Coniothyrium diplodiella, Agaricus capentris, Botrytis cinerea, Penicillium citrinum, Sclerotinia libertiana, Carpenteles javanicus and Aspergillus niger, were choosen as producers of effective pectolytic enzymes, and C. diplodiella proved the most active of all in clarifying fruit juice and hydrolyzing pectin or pectic acid.     

The Relationship Between Oil Gland and Fruit Development in Washington Navel Orange (Citrus sinensis L. Osbeck)

Changes in structure, size and number of oil glands locatedin the fruit rind were assessed in developing fruit of the WashingtonNavel orange (Citrus sinensis L. Osbeck) from pre-anthesis tofruit maturity. Initiation of oil glands was found to be restrictedto early fruit development. Glands continued to develop throughoutfruit growth, until all reached maturity by a fruit size of30 to 50 mm diameter. Mature glands continued to enlarge withfruit growth. Mature fruit had between 8 000 and 12 000 oilglands. Anatomical studies of the fruit rind were carried outusing light microscopy on samples prepared by different tissueprocessing methods. Glands were found to develop from a clusterof cells adjacent to the fruit epidermis, into a structure consistingof a central cavity surrounded by several layers of epithelialcells. All glands were joined to the fruit epidermis, irrespectiveof their stage of development. Neither lignin nor suberin waspresent in the gland. Gland cavity formation appeared to involveschizogeny. Copyright 2001 Annals of Botany Company Washington Navel orange, Citrus sinensis L. Osbeck, fruit development, secretory cavity, oil gland, image analysis, light microscopy     

外源GA3和ABA对红肉脐橙果实品质的影响

以生长一致的红肉脐橙成年植株为试材,研究了果实转色前外源GA₃和ABA处理对果实品质的影响。结果表明,外源GA₃处理提高了果皮亮度,但降低了果皮红色度、黄色度和单果重;ABA处理提高了果皮红色度,但降低了果皮亮度;GA₃100mg/L处理虽然极显著降低了果皮厚度,极显著提高了果皮亮度、可溶性固形物和VitC含量,但同时极显著降低了果皮红色度、糖酸比,并极显著提高了果实含酸量;外源ABA处理还极显著降低了果实可食率、出汁率和VitC含量。因此,在果实转色前用外源GA₃和ABA处理红肉脐橙果实均不利于其综合品质的提高。     

The Separation of Pectic Polymers from Apple Fruit Tissue 3

A column chromatographic system utilizing diethylaminoethyl(DEAE) cellulose equilibrated with phosphate buffer at pH 6.5has been found satisfactory for the separation of pectic polysaccharidesfrom apple fruit tissue. Increasing phosphate concentrationseluted in order from a sample of Bramley Seedling whole pectin,a neutral arabinan-galactan, a polyuronide and a ‘ poly-aldo-uronide’. Similar components were found in extracts from Cox's OrangePippin tissue. The pectic fraction soluble in neutral bufferat 20 °C was found to contain largely polyuronide. Sodiumhexametaphosphate at 95 °C extracted a much larger proportionof poly-aldo-uronide but this component was partially degradedduring extraction. A proportion of polyuronide remaining afterthis extraction could be liberated only by markedly degradativeprocedures and was therefore not characterised.     

Heterogeneity of Rice Glutelin

Three forms of endopolygalacturonase from Saccharomyces fragilis (Kluyveromyces fragilis) were separated by a procedure including adsorption on Amberlite IRC-50, CM Sephadex C-50 column chromatography and repeated preparative disc electrophoresis. Each endo-PG was almost homogenoeus as judged by polyacrylamide gel electrofocusing and disc electrophoresis. The three enzyme were designated as enzymes I, II and III. Enzymes I and II were similar but enzyme HI different from I and II in isoelectric point. The three enzymes resembled one another in eznyme action on pectic acid and other properties. All the three enzymes showed macerating activity toward the potato and carrot tissues.     

The role of wounds in the infection of oranges by Penicillium digitatum Sacc

Inocula of spores of Penicillium digitatum in water applied to apparently uninjured skin of oranges do not cause lesions to develop. Addition of citric acid, orange juice, or various extracts of rind had little effect on susceptibility to infection. When spores in water are applied to wounds made between oil vesicles, lesions develop only from wounds that penetrate deeply into the albedo. The flavedo of most oranges seems to be resistant to infection even when damaged, but in a few consignments it showed much less resistance. Increasing the number of conidia in the inoculum caused more lesions to develop but some fruits developed lesions from inocula containing very few spores. The method and timing of spore application to wounds had a considerable effect on the incidence of lesions; emanations from infected fruit had no effect. Lesions developed more rapidly and readily when suspensions of spores in water were applied to wounds in the skin that damaged oil vesicles; wounds as shallow as 0–25 mm allowed lesions to develop.     

Changes in Cell Wall Composition and Water-soluble Polysaccharides During Kiwifruit Development

Changes in both cell wall and water-soluble polysaccharide compositionduring the growth of kiwifruits [Actinidia deliciosa (A. chev.) C. F. Liang and A. R. Ferguson var. deliciosa ‘Hayward’]were investigated. Cellulose was the major wall polysaccharide,with galactose and uronics the main non-cellulosic sugars. Muchsolubilization of cell wall pectic polysaccharides was detected.While wall-galactose solubilization started 3 months after anthesis,polyuronide degradation did not start until the fifth month,1 month prior to the harvest date. Parallel to these processes,a linear increase in water-soluble polysaccharides was detected.These mainly comprised galactose-rich polymers in the first3 months and little-branched polyuronides after the fifth month.Two different mechanisms for galactose and uronic acid solubilizationfrom kiwifruit cell walls during fruit development are proposed. Actinidia deliciosa ; cell wall; fruit growth; kiwifruit; water-soluble polysaccharides     

Fractionation and Partial Characterization of Pectic Polysaccharides in Cell Walls from Liverwort (Marchantia polymorph) Cell Cultures

Konno, H., Yamasalu, Y. and Katoh, K. 1987. Fractionation andpartial characterization of pectic polysaccharides in cell wallsfrom liverwort (Marchantia polymorpha) cell cultures.—Jexp. Bot. 38: 711–722. Pectic polysaccharides were extracted from the starch-free cellwall preparation of cell suspension cultures of Marchantia polymorpha.The polysaccharides were fractionated by DEAE-Sephadex A-50ion-exchange chromatography yielding the five fractions, andthe degree of polymerization and glycosyl composition determinedfor each fraction. The neutral rich and acidic pectic polymerswere depolymerized by purified endoglucanase (l,4-ß-D-glucan4-glucanohydrolase, E.C. 3.2.1.4 [EC] .) and endopolygalacturonase(poly-l,4--Dgalacturonide glycanohydrolase, E.C. 3.2.1.15 [EC] ),respectively. The degraded pectic fractions were fractionatedby gel filtration chromatography on Bio-Gel A-5m and Bio-GelP-2, and glycosyl composition determined for each fraction.The results indicate that pectic polysaccharides contain glucose-richpolymer, rhamnogalacturonan and homogalacturonan in a ratioof 1:4:0–6. In addition, pectic polysaccharides were releasedas five pectic fragments from the cell walls by purified endopectatelyase (poly-l,4--D-galacturonide lyase, E.C. 4.2.2.2 [EC] ). Basedon the analysis of glycosyl composition of each fragment, thepectic polysaccharides of Marchantia cell walls are characterized Key words: Cell suspension culture, cell wall, liverwort, Marchantia polymorpha, pectic polysaccharides     

Fungal Associations: III. The Role of Pectic Enzymes on the Synergistic Relation between Rhizoctonia solani Kuhn and Fusarium solani Snyder and Hansen, in the Rotting of Potato Tubers

The greatest activity of protopectinase obtained from the growthof Rhizoctonia solani and Fusarium solani on autoclaved potatoplugs occurred at pH 6.5, and greatest activity of the ‘lossof viscosity’ enzyme was found at 6–5 for Rhizoctonia,and between 6.5 and 8.3 for Fusarium. Protopectinase enzymeobtained from double infections of the Fusarium spp. with Rhizoctonia,or by mixing the enzymes of individual Fusarium spp. with Rhizoctoniaenzyme, were more active than the enzymes from single inoculations.Cylindrocarpon radicicola enzyme was more active when obtainedfrom a pure culture than from double infection. Similarly, mixingthis enzyme with the enzyme of Rhizoctonia reduced its activity.The evidence indicated that the protopectinase of Rhizoctoniawas similar to that of Cylindrocarpon and differed from thatof the Fusarium spp. Using paper partition chromatography, two bands from Rhizoctoniacrude enzyme had a stimulatory effect on Fusarium enzyme, whileonly one band from Fusarium enzyme stimulated Rhizoctonia enzyme. The purified enzyme of Rhizoctonia degraded pectin to galacturonicacid. Fusarium pure enzyme degraded pectin to an intermediatestage. A mixture of the two enzymes degraded pectin to galacturonicacid, without the intermediate stage formed by Fusarium alonebeing detected. The role played by pectic enzymes upon the synergistic relationof Rhizoctonia solani and Fusarium solani on rotting potatotubers is discussed.     

Histological and Physiological Characterization of Rind Breakdown of 'Navelate' Sweet Orange

Rind breakdown of ‘Navelate’ sweet orange is characterizedby sunken colourless areas of the peel which develop into reddish-brown,dry areas partially covering the exposed portion of the maturefruit. Sudden changes in relative humidity at fruit colour breakseem to be responsible for the natural development of this disorder,which begins at the transitional zone of the flavedo-albedoand advances across the flavedo reaching the epidermis. Affectedcells have reduced amounts of cytoplasm located in a centralposition and have twisted and squashed walls, forming areasof collapsed cells amongst the healthy cells of the flavedoand albedo. Comparisons of healthy and damaged areas of affectedfruits showed no significant differences in wax morphology andcuticular thickness or permeability. Our results suggest thatan excessive loss of water from hypodermal and albedo cellsis responsible for the disorder. Copyright 2001 Annals of BotanyCompany Citrus, physiological disorder, cell collapse, rind blemish, fruit quality     

Pectic enzyme production and morphogenesis inHelminthosporium atypicum

The production of pectic enzymes byHelminthosporium atypicum and its morphogenesis on different media were studied. It was observed that the fungus produces pectic enzyme (macerating enzyme) adaptively. Increasing concentrations of glucose had an inhibitory effect on enzyme production. Glucose promotes profuse growth and early sporulation whereas presence of pectin slows down the growth and delays sporulation. Delay in sporulation is the effect of presence of pectin and not of the low pH of the medium. It is also suggested that in the case ofH. atypicum low pH of the medium does not allow the fungus to utilize a carbon source as efficiently as at higher pH.