Production of Pectolytic Enzymes by Rhizopus stolonifer Sporangiospores After “Lethal” Gamma Irradiation

Irradiated sporangiospores of Rhizopus stolonifer excreted pectolytic enzymes, which hydrolyzed pectin and macerated potato tuber discs, into the suspending medium. Pectin glycosidase, but not pectin methylesterase, activity developed regardless of the amount of radiation the spores had received, unless the dose exceeded about 1 megarad. The ability to produce pectolytic enzymes was found to be more radiation-resistant than the potential for colony formation or the ability to germinate. Spores made incapable, through irradiation, of forming colonies continued to produce pectolytic enzymes after a 6-day period following irradiation treatment.     

Pectin methylesterases induce an abrupt increase of acidic pectin during strawberry fruit ripening

The decrease of strawberry (Fragariaxananassa Duch.) fruit firmness observed during ripening is partly attributed to pectolytic enzymes: polygalacturonases, pectate lyases and pectin methylesterases (PMEs). In this study, PME activity and pectin content and esterification degree were measured in cell walls from ripening fruits. Small green, large green, white, turning, red and over-ripe fruits from the Elsanta cultivar were analyzed. Using the 2F4 antibody directed against the calcium-induced egg box conformation of pectin, we show that calcium-bound acidic pectin was nearly absent from green and white fruits, but increased abruptly at the turning stage, while the total pectin content decreased only slightly as maturation proceeded. Isoelectrofocalisation performed on wall protein extracts revealed the expression of at least six different basic PME isoforms. Maximum PME activity was detected in green fruits and steadily decreased to reach a minimum in senescent fruits. The preliminary role of PMEs and subsequent pectin degradation by pectolytic enzymes is discussed.     

Studies on Pectolytic Enzymes of Molds

Experiments have been made on fractionation of the pectolytic enzymes produced by Coniothyrium diplodiella. It has been observed that 30 to 35% of the polygalacturonase (PG) activity of the pectolytic enzymes of the said microorganism is salted out with ammonium sulfate, and this portion contains cndo-PG I, endo-PG II and pectin esterase (PE) (with a trace of exo-PG). The endo-PG I accounts for 60 to 65% of the total PG activity, and the endo-PG II, 25 to 30%. Both types of endo-PG scarcely act on pectin, and hydrolyze pectic acid to the extent of 65 to 70%.     

Production of a toxic metabolite and pectolytic enzyme by Pythium butleri

Pythium butleri which causes root-rot of Belladonna produced a heat stable toxic metabolitein vitro. The culture filtrate induced wilting when applied to cut shoots. The pathogen produced significant amount of endo-polygalacturonase, endo-polymethylgalacturonase, exo-polygalacturonase, polygalacturonate transeliminase and pectin methyl-trans-eliminase in media containing glucose and pectin as carbon source. The fungus did not produce pectin methyl estrase and was very weakly cellulolytic. The extensive distintegration of host tissue during pathogenesis can be considered due to the pectolytic enzymes produced by the pathogen. The physiology of this fungus differs markedly from other species ofPythium and justifies its taxonomic position as a distinct species.     

Production of pectolytic enzymes – a review

Pectolytic enzymes play an important role in food processing industries and alcoholic beverage industries. These enzymes degrade pectin and reduce the viscosity of the solution so that it can be handled easily. These enzymes are mainly synthesized by plants and microorganisms. Aspergillus niger is used for industrial production of pectolytic enzymes. This fungus produces polygalacturonase, polymethylgalacturonase and pectinlyase. This review mainly concerns with the production of pectolytic enzymes using different carbon sources. It also deals with the effect of operating parameters such as temperature, aeration rate, agitation and type of fermentation on the production of these enzymes.     

Pectolytic enzymes in Rhizobium

A sensitive pectin agar plate assay was used to demonstrate low levels of pectolytic enzymes in infective and noninfective strains of Rhizobium. The possible relation of this characteristic to legume infection is discussed.     

Production of pectolytic enzymes fromErwinia grown on different carbon sources

A quantitative analysis of pectolytic enzymes (polygalacturonase (PG), pectin methyl esterase (PME) and six isoenzymes of pectate lyase (PL)) produced byErwinia bacteria in the presence of diverse carbon sources was made by preparative electrophoresis. Synthesis of each of these enzymes was regulated independently; different induction and repression ratios (about 10- to 1000-fold) were observed for diverse PL isoenzymes, PG and PME. The possibility of using specially constructed media for the production of pectinase complexes with a specific spectra of pectolytic enzymes has been demonstrated.     

Pectolytic enzymes in Rhizobium.

A sensitive pectin agar plate assay was used to demonstrate low levels of pectolytic enzymes in infective and noninfective strains of Rhizobium. The possible relation of this characteristic to legume infection is discussed.     

The effect of iron concentration on pectin decomposition by Aspergillus niger and Aspergillus awamori]

The effect of cations Fe2+ and Fe3+ on the decomposition of apple pectin by the enzymic preparations of Asp. niger and Asp. awamori has been examined. Fe ions have delayed the process of enzymic decomposition of the pectin molecule. In the presence of Fe3+ far less amounts of monogalacturonic acid are formed. The presence of Fe ions makes the pectin molecule more stable to the effect of pectolytic enzymes.     

Pectolytic moulds in Nigeria

Eighty moulds isolated from decayed root tubers were screened for pectolytic activities. Of these 82.5% were pectolytic, 38.75% showed both polygalacturonase and pectin lyase activities, 21.5% showed polygalacturonase activity and 22.50% showed pectin lyase activity only. The aspergilli formed the largest group of pectolytic isolates. Other mould isolates with fairly high pectolytic activities include Absidia corymbifera, Cunninghamella elegans, Fusarium pallidoroseum, F. solani, Penicillium aurantiogriseum, P. brevicompactum and Rhizopus oryzae .     

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.     

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.     

Induction and Inducers of the Pectolytic System in Aureobasidium pullulans

The strain of Aureobasidium pullulans NRRL Y-2311 (CCY 27-1-98), known as a hyperproducer of endo-1,4-β-xylanase, exhibited good growth on pectin or pectate. Growth on these carbon sources is associated with an inducible production of significant amounts of pectolytic enzymes, of which exopolygalacturonase (EC 3.2.1.67) and endopolygalacturonase (EC 3.2.1.15) were identified. The two enzymes are not produced on D-glucose or under carbon starvation conditions. The enzymes can be induced in glucose-grown cells by D-galacturonic acid and its oligomers. Thus, D-galacturonic acid, the monomer derived from the polysaccharide, appears to be the natural inducer or a precursor of an inducer of pectolytic enzymes in the studied yeast. Received: 4 November 1995 / Accepted: 11 December 1995     

Purification and partial characterisation of pectin methylesterase produced by Fusarium asiaticum

Fusarium asiaticum is the predominant causal agent of Fusarium head blight (FHB) in China. When grown in liquid cultures containing potato tuber extract as the sole carbon source, F. asiaticum (strain 7071) from wheat (China) produced pectin methylesterase (PME), polygalacturonase (PG), and pectin lyase (PNL). The activity of these pectolytic enzymes was detected by a gel diffusion assay. Three forms of PME were identified in a culture filtrate of F. asiaticum. Two forms of PME with molecular weights of 31 kDa and 42.5 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), were purified using a combination of chromatographic techniques. These PMEs did not bind to Concanavalin A (Con A), which was confirmed by rechromatography using a Con A agarose column. The 31 kDa purified PME was thermostable in a temperature range of 25-55 °C. The optimal pH for the PME of F. asiaticum was 6.5. This research provides the basis for future investigations of pectolytic enzymes from F. asiaticum.     

Microorganisms from composting leaves: Ability to produce extracellular degradative enzymes

Mixed populations of bacteria, fungi, and actinomycetes in a leaf compost pile were examined over a 100-day test period for their ability to produce extracellular proteolytic, lipolytic, amylolytic, cellulolytic, pectolytic, and ureolytic enzymes and ability to utilize alkanes. Urea was added to the leaves to adjust the carbon to nitrogen ratio but was of little value in maintaining the proper ratio since it was degraded within the first few days. The degradative enzymes excreted by microorganisms was dependent on the temperature of the pile. In many cases organisms able to produce specific extracellular enzymes at medium temperatures were able to grow at high temperatures, but either did not excrete the specific enzymes or the enzymes were inactivated by the high temperature.     

A New Pectolytic Enzyme in Erwinia aroideae Formed in the Presence of Nalidixic Acid

Pectolytic enzyme formation by whole cells of Erwinia aroideae was markedly stimulated when nalidixic acid was added to a culture medium. The activity of pectolytic enzyme was markedly stimulated by nalidixic acid when the activity was measured by the decrease of viscosity of pectin, while activities of both polygalacturonic acid trans-eliminase and polygalacturonase which were measured respectively by the increase of optical density at 230 nm and the liberation of aldehyde groups, were not stimulated. The analysis of pectolytic enzyme by carboxymethyl cellulose column chromatography indicated that there was a significant difference in the elution profiles between the pectolytic enzyme induced by nalidixic acid and that synthesized under normal conditions. Therefore, we conclude that two enzymes are distinct protein species.     

Production of polygalacturonase byByssochlamys fulva

Summary Byssochlamys fulva was grown in two fermentation media using shake flasks, stirred fermentor and disc fermentor under conditions to give maximum production of pectolytic enzymes. Only polygalacturonase activity was detected in the culture filtrates during all fermentations. In all production conditions studied, no evidence of pectin methylesterase, pectin lyase, cellulase or proteinase activities were found. The maximum polygalacturonase activity (4.5 units/ml) was achieved when the microorganism was grown on medium II in shake flasks at pH 4.0–4.5 and 30°C after 12 days of fermentation.     

Characteristics of the immobilized pectin lyase activity from a commercial pectolytic enzyme preparation

A commercial pectolytic enzyme preparation has been immobilized onto a nylon-polyethyleneimine copolymer, in order ot study the contribution of the pectin lyase activity inthe overall pectindegrading activitg. Optimal conditions for the immobilization process of the pectin lyase activity, such as chemical modification of the support, coupling pH and protein concentration, were determined. Kinetic parameters and temperature behaviour of both the soluble and immobilzied pectin lyase activitgy of the derivative were also determined. OPerational stability of the pectin lyase and overall viscosity reducring activities resulting in a halflife times of 3.8 and 8.5 days, respectively, for both pectolytic activities, when the immobilized derivatives were tested in a cross-flow reactor, using a highly esterified pedtin as substrate.     

Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus,specific for the deacetylation of hairy (ramified) regions of pectins

Rhamnogalacturonan acetylesterase, able to specifically hydrolyse the acetyl asters present in modified hairy (ramified) regions (MHR) of apple pectin, was identified. The enzyme removed about 70% of the total acetyl groups in MHR. This acetylesterase did not cause the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, including the acetylated smooth regions present in beet pectin. Pretreatment of pectic polysaccharides in order to remove arabinose side chains had no effect on the acetyl release, wor was an effect found on the rate or degree of acetyl release, when the purified acetylesterase was combined with pectolytic enzymes, pectin methylesterase or arabinanases. Correspondence to: A. G. J. Voragen     

Comparison of the structural features of apple and citrus pectic substances

Pectic substances were extracted from Alcohol Insoluble Solids from lemon peel (albedo) and fractionated by ion exchange chromatography and gelfiltration. The pectin molecules contained rhamnose, arabinose, galactose, glucose and galacturonic acid residues; xylose residues were almost absent. Degradation with purified pectolytic enzymes and subsequent gelfiltration of the resulting pectin fragments showed that the neutral sugar side chains were present in ‘hairy regions’ (blocks of neutral sugar side chains). The distribution of the methoxyl groups was studied by HPLC analysis of enzyme-degraded pectins. Some influence of native pectinesterase on the distribution of the methoxyl groups was found. The results are compared with those of similarly extracted and purified apple pectic substances.