A rapid assay for pectinesterase activity which can be used as a prescreen for pectinesterase inhibitors.

A rapid assay for inhibition of pectinesterase has been developed to facilitate large scale screening for possible inhibitors. The assay gives quantitative results which can be confirmed by other assay techniques. The assay uses an agar plate in which pectin and an indicator have been incorporated. The hydrolysis of pectin to pectinic acid by pectinesterase causes a lowering of the pH of the medium and produces a color change. The area of the color change produced by the hydrolysis of pectin is directly proportional to the activity of the pectinesterase.     

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.     

Disulfide bridges in tomato pectinesterase: variations from pectinesterases of other species; conservation of possible active site segments.

Analysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys-98 with Cys-125, and Cys-166 with Cys-200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18-33% residue identities) from microorganisms. However, one half-Cys (i.e., Cys-166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N-terminal part of the tomato pectinesterase, another in the C-terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to high-light a functional site in a midchain segment.     

Purification and properties of two pectinesterases from tomatoes.

Pectinesterase is present in green tomato fruit and increases several-fold during ripening. Several isoenzymes of pectinesterase are known to exist in tomatoes, but one isoenzyme predominates in the fruit of most cultivars. A few cherry tomato cultivars have been identified that contain low levels of this isoenzyme and much higher levels of another pectinesterase that is unique to those cultivars. The two major pectinesterases were purified to homogeneity and characterized. There were significant differences in the pectinesterases but they cross-reacted with antibodies raised against them and their N-terminal amino acid sequences were similar.     

A new method for immobilization of pectic enzymes

Summary Polygalacturonase and pectinesterase have been successfully immobilized on gamma alumina by activation of the support with glutaraldehyde at pH 3.0. The half life of the enzymes increased by four and two fold compared to the immobilization on gamma alumina without activation.     

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.     

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.     

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.     

Preparation and characterisation of immobilised metal ion hollow-fibre polysulphone membranes. Their application in high-speed pectic enzyme fractionation

Chelating hollow-fibre membranes were prepared from epoxy-activated polysulphone microfiltration fibres by introducing iminodiacetic acid (IDA) groups in the presence of dimethyl sulphoxide. Fibres with 160, 350 and 620 μmol epoxy groups/ml provided ligand densities of 69, 134 and 203 μmol IDA/ml and pure water fluxes of 7.8, 5.8 and 0.42 cm/min, respectively. However, lysozyme capacity was close to 4 μmol/ml for all fibres. Adsorption isotherms for lysozyme and pectinesterase did not fit Langmuir-type curves and the existence of two types of ligand (A and B) with different accessibility to proteins was assumed. For pectinesterase, maximum capacities of 5100 and 2900 U/ml and dissociation constants of 25 and 316 U/ml were found, respectively, for ligands A and B. For lysozyme, maximum capacities were 2.9 and 0.9 μmol/ml and dissociation constants 5.0 and 102 μM, respectively, for said ligands. A cartridge assembled with IDA hollow fibres had a dynamic capacity for pectinesterase of 7509 U/ml. Productivity of this cartridge for pectic enzyme fractionation was 750 pectinesterase U/ml min, far higher than that obtained with a chelating soft gel (81 pectinesterase U/ml min).     

Pectinolytic enzymes of anaerobic fungi

Pectinolytic enzymes of four rumen fungi have been described. Three fungal species were monocentric Neocallimastix spp. H15, JL3 and OC2, and one isolate was a polycentric strain of Orpinomyces joyonii , A4. They differed in degree of pectin degradation and utilization. Only the strain Neocallimastix sp. H15 and partially Orpinomyces joyonii A4 were able to utilize pectin to a higher extent. The most important pectinolytic activity in all these isolates represented pectin lyase (EC 4.2.2.10) and polygalacturonase (EC 3.2.1.15). Their specific activities were in the range of 100–900 and 10–450 μg galacturonic acid h^-1 mg protein^-1 for pectin lyase and polygalacturonase, respectively. Polygalacturonase, located mainly in the endocellular fraction, was inhibited by calcium ions and had the main pH optimum at pH 6.0. All strains produced pectate lyase (EC 4.2.2.2). None of the strains tested produced pectinesterase (EC 3.1.1.11).     

植物果胶甲酯酶及其花粉特异基因的分离

果胶甲酯酶与植物的多种重要生长发育过程有关,是目前植物生物学研究中的一个热点。根据相关文献,对植物果胶甲酯酶的结构模型、作用方式以及花粉特异的果胶甲酯酶基因的分离进行了综述。     

Similarities in the Action Patterns of Exopolygalacturonate Lyase and Pectinesterase from Clostridium multifermentans

Exopolygalacturonate lyase and pectinesterase from Clostridium multifermentans were assayed simultaneously in the same reaction mixture which contained a highly esterified pectin, polymethyl polygalacturonic acid methyl glycoside. Lyase is specific for unesterified galacturonide residues and cannot degrade this substrate in the absence of the esterase. The rate for esterase was twice the rate for lyase throughout the entire course of the combined reaction. Thus, the molar ratio of the two enzyme activities was the same since the product of the lyase is an unsaturated digalacturonic acid containing two free carboxyl groups. Since clostridial exopolygalacturonate lyase is known to degrade polygalacturonate in a linear manner beginning from the reducing ends of polygalacturonate chains, it was apparent that clostridial pectinesterase must hydrolyze methyl groups in highly esterified pectins with an action pattern similar to that of the lyase. Otherwise it would be impossible for the two enzyme rates to have corresponded on the basis of a 2:1 ratio.     

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     

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.     

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.     

Molecular biology of fruit ripening and its manipulation with antisense genes

Considerable progress in tomato molecular biology has been made over the past five years. At least 19 different mRNAs which increase in amount during tomato fruit ripening have been cloned and genes for enzymes involved in cell wall degradation (polygalacturonase and pectinesterase) and ethylene synthesis (ACC synthase) have been identified by conventional procedures. Transgenic plants have been used to identify regions of DNA flanking fruit-specific, ripening-related and ethylene-regulated genes and trans-acting factors which bind to these promoters have also been identified.Antisense genes expressed in transgenic plants have proved to be highly effective for inhibiting the specific expression of ripening-related genes. These experiments have changed our understanding of how softening occurs in tomato fruit. Antisense techniques have also been used to identify genes encoding enzymes for carotenoid biosynthesis (phytoene synthase) and ethylene biosynthesis (the ethylene-forming enzyme). The altered characteristics of fruit transformed with specific antisense genes, such as retarded ripening and resistance to splitting, may prove to be of value to fruit growers, processors and ultimately the consumer.     

Changes in the Pattern of Enzyme Development in Gibberellin-treated Pea Internodes

A study was made on the effect of gibberellic acid on amylase,cellulase, ß-fructofuranosidase, pectinesterase, andstarch phosphorylase activities in elongating dwarf-pea internodes. Hormonal stimulation of amylase and ß-fructofuranosidaseactivities correlated closely with internode growth, the activityof starch phosphorylase less so, and gibberellic acid had noimmediate effect on cellulase and pectinesterase activities. Injection of glucose (or glucose derivatives) into pea internodesmimicked the effect of gibberellic acid on fresh- and dry-weightaccumulation, cell elongation, cell division, and cell-wallsynthesis. It is proposed that the over-all effect of gibberellic acidon enzyme development is to provide more substrate (particularlyglucose) for general cell metabolism and wall synthesis withinelongating internodes.     

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.     

Studies in the Physiology of Parasitism: XXV. Some Propertles of the Pectic Enzymes secreted by Pythium debaryanum

The pectic enzymes of Pythium debaryanum have been comparedwith those from two other soft-rot causing organisms, Erwiniaaroideae and Botrytis cinerea, by their effects (viscosity reduction,acid production, and reducing power) on 1 per cent, solutionsof (a) high methoxyl pectin, (b) sodium polypectate, and (c)sodium pectate (2 types). The Pythium debaryanum preparationdiffered particularly in giving no increase in reducing poweror evidence of galacturonic-acid-like derivatives. It maceratedthe walls of potato-tissue freely but had nopolygalacturonaseor pectinesterase activity.     

Polyuronide solubilization during ripening of normal and mutant tomato fruit

The amount and molecular size of soluble polyuronide extractable from ripening tomatoes is markedly affected by residual enzyme activity. The efficacy of phenol-acetic acid-water treatment to remove this residual activity is demonstrated. Data obtained using treated wall preparations confirms that there is an increase in soluble polyuronide during normal ripening and that this also occurs in the ‘Never-ripe’ mutant, and to a lesser degree in the ‘ripening-inhibitor’ mutant. However, changes in the molecular size of this polyuronide during normal ripening were not as extensive as previously reported and few changes were apparent in either of the mutants.Measurements were also made of polygalacturonase (EC 3.2.1.15) and pectinesterase (EC 3.1.1.11) activity during ripening. The level of polygalacturonase activity does not appear to correlate with the amount of soluble polyuronide released, but may be related to the extent of depolymerisation. No relationship was apparent between the level of pectinesterase and either soluble polyuronide released or depolymerization.