endo-PG Production Research Articles

Production and characterization of endo-polygalacturonase from Aspergillus niger in solid-state fermentation in double-surface bioreactor

Endo-polygalacturonase (endo-PG) production by Aspergillus niger T0005/007-2 in solid medium with 170 mm of height was evaluated in a cylindrical double surface bioreactor in 96-h experiments. Cell concentration close to 92 mg.g -1 dm (mg per g of dry medium) in the standard condition (static) was achieved, whereas in tests under forced aeration of 1.4 and 2.8 L.min-1. Kg-1 mm (L of air per minute per Kg of moist medium) and with the central shaft fungal biomass attained approximately 100 mg.g-1 dm. Superior endo-PG activity was obtained with the central-shaft system, 78 U.g-1 dm (units per g of dry medium). Forced aeration and pressure pulse showed no positive effect on the production of endo-PG, 45 U.g-1 dm and 28 U.g-1 dm, respectively. None of the conditions evaluated was efficient for medium temperature control. Endo-PG was stable up to 40oC. The activity decreased in 50% after 120 minutes at 50oC, which is a temperature normally found during this process.

Melting of ‘Big Top’ Nectarine Fruit: Some Physiological, Biochemical, and Molecular Aspects

‘Big Top’ nectarine [Prunus persica (L.) Batsch] has appreciable keeping quality because it resembles, at ripening, the stony hard (SH) peach (P. persica) in firmness and crispness but melts at a slow speed at full ripening. We have characterized the postharvest behavior of ‘Big Top’ fruit, treated or not with ethylene for 5 days after harvest (DAH), and compared it with that of a SH peach (‘Ghiaccio’). Pp-ACS1 expression, ethylene evolution, endo-polygalacturonase (endo-PG) production, and softening were evaluated and compared with those of the physiologically ripe melting flesh (M) cultivar Bolero. Like ‘Bolero’, ‘Big Top’ fruit expressed Pp-ACS1 and evolved ethylene but with a 5-day delay. Pp-endo-PG expression, production of an active endo-PG, and fruit melting showed a parallel behavior; ethylene treatment enhanced all these features. In SH ‘Ghiaccio’ Pp-ACS1 expression, ethylene evolution, endo-PG production, and softening were absent during the first 5 DAH in air. ‘Ghiaccio’ neither expressed Pp-ACS1 nor evolved ethylene even after ethylene treatment but responded by accumulating Pp-endo-PG transcripts and an active endo-PG protein, with flesh melting. A ‘Big Top’ Pp-endo-PG clone showed several single nucleotide (SNP) and insertion-deletion (InDel) polymorphisms in comparison with the M Pp-endo-PG clone of ‘Bolero’ and substantial similarity with the Pp-endo-PG clone of ‘Ghiaccio’. In ‘Big Top’, we identified a peculiar SNP (bp 348) and InDels shared with ‘Ghiaccio’, possibly suitable for discriminating among different genotypes. Overall, the data confirm the pivotal role of ethylene in the regulation of endo-PG production and in the determination of peach flesh texture and support the evidence that ‘Big Top’ could be classified as a melting (slow-melting) phenotype.

The effects of different carbon sources on biosynthesis of pectinolytic enzymes by Aspergillus niger

The aim of this work was to investigate the effects of different carbon sources on the nourishing base on the production of pectinolytic enzymes by Aspergillus niger with the aim of optimizing the medium for maximal enzyme production. Growth and enzymes production by Aspergillus niger were evaluated on glucose, fructose,galactose, xylose, lactose, apple pectin and the dry apple pulp. Results of different carbon sources on base showed maximal endo-pectinolytic activity, endo-PG/328 U L-1 with the pressed apple pulp, compared with endo-PG/140 U L-1 with apple pectin, endo-PG/62 U L-1 with galactose, endo-PG/28 U L-1 with lactose, endo-PG/0.0 U L-1 with glucose and fructose and endo-PG/5.0 U L-1 without carbon source (control). The growth of the microorganism (dry biomass) on different carbon sources showed maximum dry biomass, 4.5 g L-1 with glucose, compared with dry biomass, 4.3 g L-1 with fructose, 4.0 g L-1 with the pressed apple pulp, 3.5 g L-1 with galactose, 3.0 g L-1 with lactose, 2.2 g L-1 with apple pectin and 0.8 g L-1 without carbon source (control). Maximal endo-PG production, 328 U L-1 and dry biomass, 4.0 g L-1 by fungus Aspergillus niger was observed in a medium at pH initial, 4.0. The results presented here will be of commercial importance for using apple pulp as a carbon source for production of pectinolytic enzymes in submerged fermentation.

Influence of immobilization parameters on endopolygalacturonase productivity by hybrid Aspergillus sp. HL entrapped in calcium alginate

Culture cells of the hybrid Aspergillus HL were immobilized in calcium alginate beads. Different spore concentrations and bead sizes produced surfaces with different mechanical resistances. Bead size of 2.2 mm made with 2% alginate and inoculum of 4 × 10 7 spores/ml gel was determined to be adequate to reduce bead fracture by 32%. The production of endopolygalacturonases (endoPG) was strongly dependent on the temperature alone or in interaction ( P < 0.05) with others factors, particularly with the pH of the medium. High temperature (39 °C) increased the production of endoPG 10-fold in comparison with low temperature (27 °C) without modifying the growth of HL (2.2 g/l). Acid pH (2.8) facilitated the production of the enzyme (235 U/(l h −1)), achieving a reduction in growth of 71% in relation to the maximum obtained (3.2 g/l) at pH 4.5. The best endoPG efficiency was found at 39 °C, pH 2.8 using 5 ml of bead volume. Under these conditions, the immobilized cells doubled the production of endoPG in comparison with free cells, and minimum support cell detachment.

Production and partial characterization of polygalacturonases produced by thermophilic Monascus sp N8 and by thermotolerant Aspergillus sp N12 on solid-state fermentation

Polygalacturonases production by newly isolated Monascus sp N8 and Aspergillus sp N12 strains was carried out in solid-state fermentation using mixtures of wheat bran, sugar cane bagasse and orange bagasse as carbon sources. The maximal activity values of exo-polygalacturonases (exo-Pg) from Monascus sp and Aspergillus sp were obtained using wheat bran/sugar cane bagasse/orange bagasse mixture (6.6 U/mL) and wheat bran/orange bagasse mixture (10 U/mL), respectively. Enzyme production by both strains was higher at 45oC after 72 h and 1.6 U/mL at 50oC after 120 h. Endo-polygalacturonase (endo-Pg) production was higher in wheat bran/orange bagasse mixture and was not affected by temperature of incubation for both fungi. Endo-Pg production by Monascus was 1.8 U/mL at 45oC and 50oC, after 72. Similar values were obtained in Aspergillus sp culture, 1.9 U/mL at 45oC and 1.8 U/mL at 50oC. Exo-Pg from both strains showed optimum activity at pH 5.5. Maximal activity was determined at 60oC for enzyme from Monascus sp and 50oC for that produced by Aspergillus sp. Exo-Pg from Monascus sp was stable at pH range 4.5-6.0 whereas that from Aspergillus sp enzyme was stable at pH 4.0. Both enzymes showed stability when incubated at 50oC for 1 h, in absence of substrate.

Production of pectinase by solid-state fermentation with Penicillium viridicatum RFC3

Endo-polygalacturonase (endo-PG), exo-polygalacturonase (exo-PG) and pectin liase (PL) were produced by solid-state fermentation of a mixture of orange bagasse and wheat bran (1:1) with the filamentous fungus Penicillium viridicatum RFC3. This substrate was prepared with two moisture contents, 70% and 80%, and each was fermented in two types of container, Erlenmeyer flask and polypropylene pack. When Erlenmeyer flasks were used, the medium containing 80% of initial moisture afforded higher PL production while neither exo- nor endo-PG production was influenced by substrate moisture. The highest enzyme activities obtained were 0.70 U mL −1 for endo-PG, 8.90 U mL −1 for exo-PG, and 41.30 U mL −1 for PL. However, when the fermentation was done in polypropylene packs, higher production of all three enzymes was obtained at 70% moisture (0.7 and 8.33 U mL −1 for endo- and exo-PG and 100 U mL −1 for PL). An increase in the pH and decrease in the reducing sugar content of the medium was observed. The fungus was able to produce pectin esterase and other depolymerizing enzymes such as xylanase, CMCase, protease and amylase.

Evaluation of Filamentous Fungi and Inducers for the Production of Endo-Polygalacturonase by Solid State Fermentation

Aspergillus oryzae CCT 3940, Aspergillus awamori NRRL 3112 and a Trichoderma sp.) were compared for their capacity to produce endo-polygalacturonase (endo-PG) in solid state fermentation. Maximum pectinolytic activity was reached in 72 h of growth, the best two fungal strains being A. niger T0005007-2 and A. oryzae CCT 3940. Three types of commercial purified pectin and four of unprocessed pectin (tangerine, orange, Tahiti lime and sweet lime rind) were used to assess the effect of pectin on the production of endo-PG by A. niger T0005007-2. Maximum pectinolytic activity was achieved using 6 and 10% (w/w) of purified pectin as inducer. Depending on the origin of the commercial pectin used as inducer, maximum endo-PG levels varied from 223 to 876 units per gram of dry medium (one endo-PG unit (U) was defined as the quantity of enzyme which caused a reduction in viscosity of 50% in a 1% w/v solution of pectin in 30 min), indicating that care should be taken when choosing this component of the medium. When the crude pectins were used as inducers at the same concentration as purified pectin, maximum endo-PG activities were 250-300 units/g. However, by increasing the amount of Tahiti lime rind to 50% (w/w) maximum endo-PG was 919 U/g, thus opening up the possibility of a low cost medium for endo-PG production.

Polygalacturonase production by Aspergillus awamori on wheat in solid-state fermentation.

The production of exo-polygalacturonase (exo-PG) and endo-PG by Aspergillus awamori grown on wheat in solid-state fermentation was studied. Endo- and exo-PG activities were detected after 24 h of inoculation. Glucose released from starch hydrolysis acted as a catabolite repressor for the exo-PG enzyme. In contrast, endo-PG production was not affected by glucose repression. When milled grains were used, the particle-size distribution and the chemical composition of the medium influenced the rate of micro-organism growth and therefore the trend followed by endo- and exo-PG production. However, these two parameters did not affect the maximum production of exo-PG and endo-PG. For one of the milled samples, three different moisture contents were used (50, 55, 60%). Moisture contents of 60% provide a higher yield of pectinases by A. awamori.

Endo-polygalacturonase production from untreated lemon peel byAspergillus sp. CH-Y-1043

Endo-polygalacturonase (endo-PG) production byAspergillus sp. CH-Y-1043 using untreated lemon peel as the sole carbon source was investigated. This strain was observed to produce more activity of endo-PG at 37°C than at 29°C. Untreated lemon peel proved to be a beeter substrate than citrus pectin for endo-PG production. Modification of the culture medium and lowering of the initial pH to 2.8 caused a 10-fold increase in the production of endo-PG activity using lemon peel.

In Vitro Production of Pectolytic Enzymes by Pleospora infectoria (Group) and Nigrospora sphaerica (Sacc.) Causing Pod Spots of Pea (Pisum sativum) and Bean (Dolichos lablab)

Nigrospora sphaerica and Pleospora infectoria produced hydrolytic and non-hydrolytic pectic enzymes in vitro. Culture conditions and length of the incubation period greatly influenced the type and quantity of enzymes. Endo-polygalacturonase (Endo-PG), pectate lyase (PGTE), pectin lyase (PTE) and pectin methyl esterase (PME) were most actively secreted by P. infectoria. Pectin and pectate lyase production was associated with increase in p H of the culture media while endo-PG production was associated with decrease in p H of the medium. No correlation was found between the growth of the pathogen and the pectic enzyme secretion.

Induction and repression of endopolygalacturonase synthesis by Pyrenochaeta terrestris.

Endopolygalacturonase (endo-PG) synthesis was inducible in standing and replacement liquid culture by Pyrenochaeta terrestris. Inducers were galacturonic acid, its polymers (pectin and polypectate), and structural relatives (mucic acid, tartronic acid, and dulcitol). On pectin, fungus growth and endo-PG synthesis were proportional to initial pectin concentration.Synthesis of endo-PG on pectin was stimulated by hexose supplements at 0.005 M and repressed by supplements at 0.05 M and above. Thus, repression of induced endo-PG synthesis was not dominant at low supplement concentrations as was found for induced cellulase synthesis by the same organism. This suggests that the production of endo-PG by the parasite during pathogenesis is less repressed by host sugars than is cellulase synthesis.