Thin-layer isoelectric focusing of multiple forms of tomato pectinesterase

Commercial tomato pectinesterase has been separated into at least eight multiple forms by thin-layer isoelectric focusing. The enzyme components were basic proteins in the range pH 7–9.3, the predominant form having an isoelectric point of 8.6. The enzyme was detected with a staining procedure, employing the reaction of hydroxylamine with the ester groups of pectin. The MW's of the multiple forms of pectinesterase were in the range of 27000 ± 5000.     

Production of pectinesterase and polygalacturonase by Aspergillus niger in submerged and solid state systems

Production of pectinesterase and polygalacturonase by Aspergillus niger was studied in submerged and solid-state fermentation systems. With pectin as a sole carbon source, pectinesterase and polygalacturonase production were four and six times higher respectively in a solid state system than in a submerged fermentation system and required a shorter time for enzyme production. The addition of glucose increased pectinesterase and polygalacturonase production in the solid state system but in submerged fermentation the production was markedly inhibited. A comparison of enzyme productivities showed that those determined for pectinesterase and polygalacturonase with pectin as a carbon source were three and five times higher by using the solid state rather than the submerged fermentation system. The productivities of the two enzymes were affected by glucose in both fermentation systems. The membranes of cells from the solid state fermentation showed increased levels of C18:1, C16:0 and C18:0 fatty acids. Differences in the regulation of enzyme synthesis by Aspergillus niger depended on the fermentation system, favoring the solid state over the submerged fermentation for pectinase production. Received 12 May 1997/ Accepted in revised form 19 September 1997     

Coordinated action of pectinesterase and polygalacturonate lyase complex of Clostridium multifermentans.

The polygalacturonate lyase and pectinesterase activities of Clostridium multifermentans, both produced extracellularly when the organism grows on pectin or polygalacturonate, have been suggested to be associated in a single complex. Both enzymic sites act on their respective substrates by single-chain action patterns, as shown by equivalent release of terminal tritium label and total product throughout the reaction. From these results, the Km and V of the lyase, and the amount of lyase activity present, we calculate the steady-state concentration of lyase substrate expected during action of the two sites on pectin if the sites are independent. No such steady-state concentration of lyase substrate was observed. Therefore, we conclude that the two types of active site act in a coordinated manner; the polysaccharide chain passes from the esterase site to the lyase site without intermediate dissociation and rebinding. This 'molecular disassembly line' constituted by the two sites may represent a system of general significance in synthesis and degradation of biological polymers.     

Purification and characterization of pectinesterase and polygalacturonase from Trichoderma reesei

Abstract Exopolygalacturonase, endopolygalacturonase and pectinesterase were separated from culture filtrates of Trichoderma reesei QM9414 by Sephadex chromatography. Exopolygalacturonase was characterized by specific cleavage of pectic acid to form d -galactopyranuronic acid, and by the hydrolysis of oligomers (highest reaction rate at pentamer). Polygalacturonase exhibited 2 pH-optima peaks (at 4.8 and 5.1) and 10 bands with enzyme activity by isoelectric focusing (IEF) (p I 4.6–8.5). Pectinesterase showed a pH-optimum at 7.6, and 6 enzyme-activity bands on an IEF zymogram which seemed identical with those of higher plants (tomato, alfalfa).     

Agrobacterium adherence involves the pectic portion of the host cell wall and is sensitive to the degree of pectin methylation

Cell walls isolated from dicotyledon tissues compete with natural plant host sites for Agrobacterium tumefaciens (strain B6) when co-inoculated with infectious bacteria, thereby reducing tumor initiation. Removal of the pectic fraction from the cell walls results in loss of inhibition and the soluble pectic fraction is inhibitory. On treatment with pectin methyl transferase plus S-adenosyl-L-methionine these cell walls become less inhibitory and this change is reversible by pectinesterase. Cell walls isolated from monocotyledons, crown gall tumors or embryonic dicotyledons do not compete for Agrobacterium in the infection assay. These cell walls become inhibitory on treatment with pectinesterase and this is partially reversed by pectin methyl transferase. These data indicate that the pectic portion of the host cell wall is involved in the Agrobacterium -host adherence which is essential for tumor initiation and that the degree of methylation of polygalacturonic acid is critical to this adherence.     

β-Galactosidase, polygalacturonase and pectinesterase in differential softening and cell wall modification during papaya fruit ripening

Papaya ( Carica papaya L. cv. Eksotika) fruit softens differentially in relation to position of the tissue. The inner mesocarp tissue is softer, and its firmness decreases more rapidly during ripening than that of the outer mesocarp tissue. As the fruit ripens, pectin solubility and depolymerisation increase. Hemicellulose, too, appears to be depolymerised but, unlike pectins, this apparent degradation of hemicellulose is associated with an increase rather than a decrease in its level. Pectin and hemicellulose depolymerisation began in the inner mesocarp tissue at about the same time as β-galactosidase (EC 3.2,1.23) activity started to increase and tissue firmness began to decrease more rapidly. In contrast, pectin solubilisation in both outer and inner mesocarp tissues occurred steadily throughout ripening at a comparable rate and paralleled closely the increase of polygalacturonase (PG; EC 3.2.1.67) and pectinesterase (EC 3.1.1.11). In general, irrespective of enzyme distribution, tissue softening during ripening was more closely related to changes in β-galactosidase activity than to PG or pectinesterase activity. Papaya, β-galactosidase appears to be an important wall degrading enzyme and may contribute significantly to differential softening, perhaps by complementing the action of polygalacturonase. Polygalacturonase activity increased with increasing depth of the mesocarp tissue, as did softening of the fruit.     

Purification and characterization of pectinesterase produced by a strain ofAspergillus niger

After an 88-fold purification, pectinesterase produced by a strain ofAspergillus niger, isolated from rotten lemons, showed the following main characteristics: maximum activity at 45°C, pH 5; Km, with pectin as substrate, 1.01 mg/L; G^*, 4750 Cal/mol. Polygalacturonic acid and methanol acted as competitive and non-competitive inhibitors, respectively. The activity of the enzyme was impaired by MgCl₂ and stimulated by NaCl.     

An investigation into the pectolytic activity of the yeast Saccharomycopsis fibuliger

Saccharomycopsis fibuliger cells produce an inducible hydrolase, tentatively characterized as a polygalacturonase [poly(1,4-α-d-galacturonide) glycanohydrolase, EC 3.2.1.15], which is associated with the yeast cells and which causes the partial hydrolysis of pectin or poly-d-galacturonic acid. No evidence of pectinesterase (pectin pectyl hydrolyase, EC 3.1.1.11) or pectate lyase [poly(1,4-α-d-galacturonide) lyase, EC 4.1.1.1] activity has been found. Enzyme production took place at an optimum temperature of 28°C, whereas optimum activity was at ～45°C. The optimum pH for pectolytic activity was similar to the optimum pH for cell growth. A reduction in the concentration of dissolved oxygen in the culture medium and an increase in cell age caused an increase in the rate of pectin decomposition within the limits employed. Products of pectin decomposition consisted of a mixture of uronides including d-galacturonic acid.     

Molecular cloning and sequencing of a pectinesterase gene from Pseudomonas solanacearum

Two pectinesterase-positive Escherichia coli clones, differing in expression levels, were isolated from a genomic library of Pseudomonas solanacearum. Both clones contained a common DNA fragment which included the pectinesterase-encoding region. The different expression levels found with the two clones could be ascribed to different positioning of the pectinesterase gene with respect to a vector promoter. Restriction analysis, subcloning, and further exonuclease deletion mapping revealed that the genetic information for pectinesterase was located within a 1.3 kb fragment. A protein of 41 to 42 kDa was expressed from this fragment. Nucleotide sequence analysis of the respective region disclosed an open reading frame of 1188 bp. The deduced polypeptide had a calculated molecular mass of 41,004 Da, which is consistent with the determined size of the pectinesterase protein. The predicted amino acid sequence showed significant homology to pectinesterases from Erwinia chrysanthemi and tomato. In cultures of E. coli clones up to 30% of total pectinesterase activity was transported into the medium. However, no significant pectinesterase activity could be detected in the periplasm.     

Multiple forms of pectinesterase in limes and oranges

Two forms of pectinesterase, apparently similar in MW but differing in charge and response to cation concentration, have been demonstrated in West Indian limes and Washington Navel oranges, and these enzymes have been purified at least 100-fold. In the orange one pectinesterase is located almost exclusively in the peel while the other is located within the segment covers and juice sacs. The location of the two pectinesterases in lime has not been determined.     

Influence of environmental conditions on the production of pectinesterase and polygalacturonase byAspergillus niger

Summary A pectinesterase- and polygalacturonase-producer strain ofAspergillus niger showed a diauxic growth when cultivated in a medium containing pectin as the carbon source. Diauxie was suppressed by adding yeast extract to the medium. The specific production of polygalacturonase was decreased by increasing the initial concentration of pectin, while that of pectinesterase was enhanced with up to 15 g pectin/l. Yeast extract increased the production of biomass but decreased that of the enzymes. The temperature of incubation (25 to 40°C) did not affect the production of biomass or that of polygalacturonase, but production of pectinesterase was markedly affected, 30°C being the optimal temperature.

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Influence de la composition du milieu et les conditions des cultures sur la production de pectinestérase et polygalacturonase par Aspergillus niger

Résumé Aspergillus niger que produit pectinestérase et polygalacturonase, quand est developpé dans un milieu avec pectine comme source de carbone a presenté le phenomene de diauxie. Cela ne succedait pas quand le milieu avait aussi extrait de levure dans sa composition. L'effect de la concentration initiale de pectine sur la production specifique des enzymes des cellules (unité d'enzyme g^–1 de cellules poid sec) a été différent selon la phase du developpement de la culture, sugérant une rélation étroite parmi la synthèse des enzymes et l'état physiologique des cellules. L'augmentation de la concentration de pectine a troublé beaucoup la production spécifique de polygalacturonase. Au contraire, jusqu'à une concentration de 15 g/l a augmenté la synthèse de pectinestérase. La grandeur et structure des pellets ont changé avec la variation de la concentration de pectine, ces modifications peuvent avoir eu influence sur les valeurs des coefficients de transfert de masse et en conséquence sur la cinétique de production des enzymes. L'augmentation de la concentration iniciale d'extrait de levure dans le milieu que contenait pectine comme source de carbone a augmenté la production de biomasse, mais a baisse la synthèse des enzymes surtout celle de pectinestérase. Températures parmi 25°C et 40°C n'ont pas modifié la production de biomasse et non plus polygalacturonase. Au contraire la production de pectinesterase a été bien différente selon la température utilisé ayant un optimum de 30°C.

     

Isolation and properties of pectinases from the fungus Aspergillus japonicus

Using anion-exchange chromatography on different carriers and phenyl-Sepharose hydrophobic chromatography, five pectolytic enzymes were isolated from the culture liquid of a mutant strain of Aspergillus japonicus: two endo-polygalacturonases (I and II, 38 and 65 kD, pI5.6 and 3.3), pectin lyase (50 kD, pI3.8), and two pectinesterases (I and II) with similar molecular weights (46 and 47 kD) and the same pI(3.8). The pectinesterases apparently represent two isoforms of the same enzyme. All purified enzymes were homogenous according to SDS-PAGE and polyacrylamide gel-IEF, except for endo-polygalacturonase II that gave two bands on isoelectric focusing, but one band on electrophoresis. All enzymes had maximal activity in an acid medium (at pH 4.0-5.5). The pectin lyase and pectinesterase were stable at 40-50°C. The thermal stability of both endo-polygalacturonases was much lower (after 3 h of incubation at 30°C, endo-polygalacturonases I and II lost 40 and 10% of the activity, respectively). The activity of endo-polygalacturonases I and II towards polygalacturonic acid strongly depended on NaCl concentration (optimal concentration of the salt was 0.1-0.2 M); the enzymes were also capable of reducing the viscosity of pectin solution, but rather slowly. The pectin lyase had no activity towards polygalacturonic acid. The activity of the pectin lyase increased with increasing degree of methylation of pectins. Both endo-polygalacturonases demonstrated synergism with the pectinesterase during the hydrolysis of highly methylated pectin. On the contrary, in the mixture of pectin lyase and pectinesterase an antagonism between the two enzymes was observed.     

果实软化的胞壁物质和水解酶变化

果实软化通常被认为是由于胞壁水解酶如多聚半乳糖醛酸酶,果胶酯酶,纤维素酶降解胞壁物质导致。本文概述了这三种酶分子与果实软化关系的研究进展。反义基因证明,这三种酶基因的任一种表达被报制,果实能够正常软化,暗示果实的软化有其它因子的参与。其中由细胞内的淀粉酶和蔗糖酶引起的细胞膨压的变化及果胶的溶解可能是引起果肉软化的重要原因。     

Simultaneous co-suppression of polygalacturonase and pectinesterase in tomato fruit: inheritance and effect on isoform profiles

The simultaneous down regulation of two, or more, genes can be brought about by the transformation of a plant with a single chimeric transgene containing homologous sequences to both target genes. This has been achieved for the two cell wall hydrolases — polygalacturonase and pectinesterase — in tomato fruit. This paper reports the stable inheritance of this co-ordinated gene silencing over two generations. It has also been shown that only two of the three isoforms of pectinesterase in the tomato fruit are silenced by this chimeric construct thus providing some indication of the relative homologies between the gene sequences for these isoforms.     

Role of Pectinesterase in pH-Dependent Interactions between Pea Cell Wall Polymers

Extracts of etiolated pea (Pisum sativum L.) shoots converted soluble pectin from the seedlings to a trichloroacetic acid-insoluble form. This activity coincided with pectinesterase peaks separated from the extracts by gel filtration and ion exchange. The conversion of pectin to the trichloroacetic acid-insoluble form and pectinesterase exhibited identical responses to pH, with activity only above pH 6. The formation of trichloroacetic acid-insoluble pectin in pea cell walls and their extracts is due to de-esterification of the pectin by pectinesterase and not to binding between pectin and a protein, as reported in the literature.     

Use of the Hydroxamic Acid Reaction for Determining Pectinesterase Activity

The technique is: React pectinesterase contained on filter paper discs on a pectin-agar plate, flood the plate with a solution of hydroxylamine hydrochloride and then a solution of sodium hydroxide, acidify with hydrochloric acid, and then add a ferric chloride solution. The areas of substrate, desterified by the enzyme, appear as clear zones on a red background. The formation of the insoluble, red-colored ferric-pectin hydroxamic acid is characteristic of the presence of pectin carbomethoxy (methyl ester). Areas which are not colored are indicative of pectinesterase activity and this test can be used for qualitative and semi-quantitative test for approximately 1 × 10^-3 to 1 × 10^-4 pectinesterase units.     

Pectic Enzyme Activities of Bacteria Associated with Rotted Onions (Allium cepa)

The aerobic bacteria associated with soft rot in onions (Allium cepa) were isolated and identified as a Vibrio sp., Micrococcus epidermidis, Pseudomonas cepacia, an Acinetobacter sp., a Xanthomonas sp., Bacillus polymyxa, and Bacillus megaterium. With the cup-plate assay method, no pectin hydrolase could be detected from any of these isolates when they were cultured in pectin medium, but lyase and pectinesterases were detectable. Onion tissue cultures showed pectin hydrolase activity for P. cepacia and B. polymyxa and lyase and pectinesterase activities for all of the isolates, usually at higher levels of activity than those of the pectin medium culture filtrates. In both culture media, Vibrio sp. showed the highest lyase and pectinesterase activities. In the viscometric test, all of the isolates achieved at least a 50% decrease in viscosity for lyase enzyme, with M. epidermidis and Vibrio sp. recording viscosity decreases as high as 83%. The ability to cause soft rot in onion bulbs was demonstrated by P. cepacia and Xanthomonas sp. Benzoic acid at a concentration of 0.8 mg/ml caused total suppression of enzyme production, whereas sodium benzoate at this concentration reduced pectinesterase production by 71% and lyase production by 72%. The possible use of these preservatives in the control of soft rot in onions is noted.     

Purification and biochemical characteristics of pectinesterase from Malatya apricot (Prunus armeniaca L.)

Pectinesterase (PE) in Malatya apricot pulp (Prunus armeniaca L.) was extracted and purified through (NH(4))(2)SO(4) precipitation, dialysis, and DEAE-Sephadex gel filtration chromatography. The samples obtained from the dialysis procedure, named partially purified enzyme, were used for characterization of the apricot pectinesterase. The effect of various factors such as pH, temperature, heat, and storage stability on the partially purified apricot PE enzyme was investigated. Optimum pH value was 9.0 for PE with 1% pectin in 0.1 N NaCl (w/v). The optimum temperature for apricot PE was found to be 60 degrees C on standard analysis conditions. Heat inactivation studies showed a decrease in enzymatic activity at temperatures above 70 degrees C. Km and V(max) values were 0.77 mM and 1.75 micromol min(-1) mg(-1) for apricot PE. Five inhibitors were tested in the study; the most effective inhibitor was found to be sodium carbonate (100% inhibition). The order of inhibitory effectiveness was: Na(2)CO(3), iodine, lauril sulphate, AgNO(3), EDTA. Thermal inactivation data indicated that apparent activation energy with pectin substrate was 2.96 kcal mol(-1) for the enzyme. Ascorbic acid, CaCl(2), and KCl showed activatory effect on the apricot PE enzyme.     

Changes in the properties of cell wall pectin methylesterase along the Vigna radiata hypocotyl

The properties of cell wall pectinesterase (EC 3.1.1.11) from Vigna radiata (L.) Wilczek hypocotyl were investigated with immobilized and solubilized wall enzymes. Along the hypocotyl, the decrease of growth potential coincides with significant changes of the characteristics of the pectinesterase activities. As hypocotyl cells grow older, the proportion of ionically bound enzymes decreases and pH profile, sensitivity to cations and affinity for pectin change. The possible significance of these observations is discussed.     

Synergistic effect of endo-D-polygalacturonase on coimmobilized pectinesterase

Summary Two pectic enzymes, pectinesterase and endo-D-polygalacturonase were covalently coimmobilized into a Glycophase-coated controlled pore-glass. The parameters of the immobilization process were characterized. The efficiency of the coimmobilized derivative with respect to the homologous system in separate particles was proved.