Gelation of high-methoxy pectin by enzymic de-esterification in the presence of calcium ions: a preliminary evaluation

Cohesive gels have been obtained by de-esterification of 1.0 wt % high-methoxy citrus pectin (degree of esterification ≈ 68%) in the presence of Ca²⁺ cations, using a commercial preparation (NovoShape) of fungal methyl esterase cloned from Aspergillus aculeatus. A convenient rate of network formation (gelation within ∼30 min) was achieved at an enzyme concentration of 0.2 PEU/g pectin. At a Ca²⁺-concentration of 40 mM and incubation temperature of 20 °C, severe syneresis (>7% of sample mass) was observed, but release of fluid decreased with decreasing concentration of Ca²⁺ and increasing temperature of incubation, becoming undetectable for 10 mM Ca²⁺ at 30 °C. Under these conditions, progressive development of solid-like character (storage modulus, G′) was observed during 160 min of enzymic de-esterification, and the mechanical spectrum recorded at the end of the incubation period had the form typical of a biopolymer gel. On subsequent heating to 70 °C, dissociation of the gel network (sigmoidal reduction in G′ and G″) was observed. At or above the midpoint temperature of this melting process (∼50 °C), there was no indication of gel formation on enzymic de-esterification (at 50 or 60 °C). At lower temperatures (20, 30 and 40 °C), the rate of gelation (assessed visually) showed no systematic increase as the incubation temperature was increased towards the temperature-optimum of the enzyme (∼50 °C). This unexpected behaviour is attributed to competition between faster de-esterification and slower formation of Ca²⁺-induced ‘egg-box’ junctions.     

Role of pectin methylesterases in cellular calcium distribution and blossom-end rot development in tomato fruit

Blossom-end rot (BER) in tomato fruit (Solanum lycopersicum) is believed to be a calcium (Ca(2+) ) deficiency disorder, but the mechanisms involved in its development are poorly understood. Our hypothesis is that high expression of pectin methylesterases (PMEs) increases Ca(2+) bound to the cell wall, subsequently decreasing Ca(2+) available for other cellular functions and thereby increasing fruit susceptibility to BER. The objectives of this study were to evaluate the effect of PME expression, and amount of esterified pectins and Ca(2+) bound to the cell wall on BER development in tomato fruit. Wild-type and PME-silenced tomato plants were grown in a greenhouse. At full bloom, flowers were pollinated and Ca(2+) was no longer provided to the plants to induce BER. Our results show that suppressing expression of PMEs in tomato fruit reduced the amount of Ca(2+) bound to the cell wall, and also reduced fruit susceptibility to BER. Both the wild-type and PME-silenced fruit had similar total tissue, cytosolic and vacuolar Ca(2+) concentrations, but wild-type fruit had lower water-soluble apoplastic Ca(2+) content and higher membrane leakage, one of the first symptoms of BER. Our results suggest that apoplastic water-soluble Ca(2+) concentration influences fruit susceptibility to Ca(2+) deficiency disorders.     

Immunogold localization of pectin methylesterases in the cortical tissues of flax hypocotyl

Summary Pectin methylesterases (PMEs, EC 3.1.1.11) catalyse the deesterification of pectins. Up to now, most information concerning their location was obtained from biochemical analyses. Taking advantage of specific anti-PME antibodies, we report the precise localization of PMEs at the electron microscopy level within the different cortical tissues of flax hypocotyl. Quantitative data on the densities of immunolabelling have been collected, using anti-PME antibodies as well as JIM5 and JIM7 monoclonal antibodies. Our findings show a co-localization of PMEs and acidic pectins (as revealed by JIM5 antibodies) within specific cell wall microdomains. Moreover, PME epitopes are associated with the cellular membranes, particularly with the plasmalemma.Abbreviations Cdta diamino-1,2 cyclohexane tetra-acetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PME pectin methylesterase - TEM transmission electron microscopy     

A hydrodynamic study of the depolymerisation of a high methoxy pectin at elevated temperatures

The hydrodynamic properties (intrinsic viscosity, [η]; infinite dilution sedimentation coefficient, s_20,w⁰; weight average molecular weight, M_w and translational frictional ratio, f/f₀) of a high methoxy pectin have been evaluated at various temperatures (20–60°C). A reduction in the value of all four hydrodynamic parameters is indicative of depolymerisation and is in agreement with an earlier study using viscometry [Axelos, M.A.V., & Branger, M., (1993). Food Hydrocolloids, 7, 91–102]. The apparent linearity of the Mark – Houwink plot of log[η] vs log M_w suggests that the conformation of the pectin molecule does not change significantly over the temperature range studied. The evaluation of the Mark–Houwink viscosity exponent (a=0.84) indicates a moderately extended structure. This then allows the calculation of the number of Kuhn statistical lengths per chain from the adapted ‘blob’ theory of Dondos [Dondos A. (2001). Polymer, 42, 897–901]. The weight average number of Kuhn statistical lengths per chain is reduced from (170±10) to (125±10) when the temperature is increased from 20–60°C. This may be of significance as many high methoxy pectins are exposed to high temperatures during processing in both the food and pharmaceutical industries.     

Comparative structural and functional analysis of genes encoding pectin methylesterases in Phytophthora spp.

We have scanned the Phytophthora infestans, P. ramorum, and P. sojae genomes for the presence of putative pectin methylesterase genes and conducted a sequence analysis of all gene models found. We also searched for potential regulatory motifs in the promoter region of the proposed P. infestans models, and investigated the gene expression levels throughout the course of P. infestans infection on potato plants, using in planta and detached leaf assays. We found that genes located on contiguous chromosomal regions contain similar motifs in the promoter region, indicating the possibility of a shared regulatory mechanism. Results of our investigations also suggest that, during the pathogenicity process, the expression levels of some of the analyzed genes vary considerably when compared to basal expression observed in in vitro cultures of non-sporulating mycelium. These results were observed both in planta and in detached leaf assays.     

Measurement of pectin methylation in plant cell walls

A procedure was developed to measure the degree of pectin methylation in small samples of isolated cell walls from nonlignified plant tissues or pectin solutions. Galacturonic acid was determined colorimetrically with the 3,5-dimethylphenol reagent. Methylation was measured by base hydrolysis of galacturonic acid methyl esters, followed by gas chromatographic determination of released methanol. Estimates of the precision of analysis of pectin and cell wall samples were made. The coefficient of variation for estimates of the pectin esterification in cell walls isolated from 10-g samples of cucumber tissue ranged from 7.7 to 13.2%.     

Functional characterization of the gels prepared with pectin methylesterase (PME)-treated pectins

High- and low-methoxyl pectins were treated with pectin methylesterase (PME) and the functional properties of the resulting pectin gels were characterized. The degree of esterification of high- and low-methoxyl pectins decreased from 74.5% to 6.3% and 40.0% to 6.5%, respectively while not changing their molecular weight. Also, the addition of glucono-delta-lactone (GDL) dramatically affected the gel strength and the pH reduction by the GDL led to the increased syneresis of the pectin gels, which was also observed in the PME-treated samples. When flavor compounds were incorporated into the pectin gels, the flavor release from the gels increased with decreasing the degree of esterification due to increased hydrophilic properties.     

Effect of phytic acid,tannic acid and pectin on fasting iron bioavailability both in the presence and absence of calcium

ObjectiveTo determine the effect of phytic acid, tannic acid and pectin on fasting non-heme iron bioavailability in both the presence and absence of calcium.Research methodsTwenty-eight apparently healthy adult females participated in two iron absorption studies using radioactive iron isotopes (⁵⁹Fe and ⁵⁵Fe). One group received 5 mg of iron (as FeSO₄) alone (control), together with 10 mg of phytic acid, 100 mg of tannic acid and 250 mg of pectin (study A), on different days. The second group received the same iron doses and compounds as the other group, plus 800 mg of calcium (CaCl₂) (study B). The compounds were administered after an overnight fast, and no food or beverages were consumed for the following 3 h. Iron status and circulating radioactivity were measured in venous blood samples.ResultsThe geometric means of iron bioavailability (range ± 1SD) for iron alone, iron with phytic acid, iron with tannic acid, and iron with citrus pectin were 25.0% (11.9–52.0); 18.9% (9.9–35.8); 16.8% (8.7–32.3); and 21.1% (10.2–43.9), respectively (repeated-measures ANOVA, p < 0.02 (Dunnett's post hoc: control vs tannic acid p < 0.05). When 800 mg of calcium was added (study B), iron bioavailability was 16.7% (10.1–27.5); 13.2% (7.1–24.6); 14.8% (8.8–25.1); and 12.6% (5.5–28.8), respectively (repeated-measures ANOVA, NS).ConclusionsTannic acid decreases the fasting bioavailability of non-heme iron, however this effect did not exist in the presence of calcium. No effect was observed by phytic acid or citrus pectin on fasting non-heme iron bioavailability in both the presence and absence of calcium.     

Conformations and interactions of pectins: II. Models for junction zones in pectinic acid and calcium pectate gels

Pectinic acid and calcium pectate gels condensed into uniaxially oriented fibers have been studied by X-ray diffraction. Although the diffraction patterns correspond to systems of only limited order, they show that both systems conserve the 1.3 nm axial period and 0.43 nm pseudo-period observed in sodium pectate. Pectinic acid further resembles sodium pectate in packing isometrically in a hexagonal net of side 0.84 nm. On the other hand, calcium pectate fibers contain the 1.2 nm lateral spacing observed in pectic acid. Speculative models for pectinic acid and calcium pectate have been developed. The former structure could be stabilized by hydrophobic binding from columns of methyl groups as well as by specific intermolecular hydrogen bonds. In the latter, the main interactions between pairs of chains could be bridges formed by calcium ions, which incorporate into their co-ordination shells two polyanion oxygen atoms from one chain and three from another. These model-building studies provide plausible visualizations of two different kinds of junction zones that may exist in pectic gels.     

Cell-wall pectin and its degree of methylation in the maize root-apex: significance for genotypic differences in aluminium resistance

Cell-wall (CW) pectin content and its degree of methylation in root apices of selected maize cultivars were studied in relation to genotypic Al resistance. Maize cultivars differing in Al resistance were grown in nutrient solution treated with or without Al, and pectin content of the root tips was determined. Control plants did not differ in pectin content in the 5 mm root apex. Al treatment increased the pectin content of the root apex in all cultivars but more prominently in the Al-sensitive cultivars. Pectin and Al contents in 1 mm root sections decreased from the apex to the 3–4 mm zone. Pectin contents of the apical root sections were consistently higher although significantly different only in the 1–2 mm zone in the Al-sensitive cv Lixis. Al contents in most root sections were significantly higher in cv Lixis than in Al-resistant cv ATP-Y. Localization of pectins by immunofluorescence revealed that Al-sensitive cv. Lixis has a higher proportion of low-methylated pectin and thus a higher negativity of the cell wall than Al-resistant cv ATP-Y. This is in agreement with the higher Al content and Al sensitivity of cv Lixis. It is concluded that differences in CW pectin and its degree of methylation contribute to genotypic differences in Al resistance in maize in addition to the release of organic acid anions previously reported.     

Enzymatic decomposition of pectin in the presence of amino acids and proteins]

The influence of amino acids--glycine, alpha-alanine, lysine, phenylalanine, serine, methionine, cysteine, tryptophan, aspartic acid and asparagine--and proteins--chick egg albumin and autolysate of Aspergillus niger biomass--on the disintegration of beet pectine by the Asp. niger enzymic preparation was investigated. This process was shown to depend on the chemical composition and concentration of amino acids and the protein nature. All the tested amino acids, except tryptophan, at doses of 0.0001 to 0.5% stimulated the activity of pectolytic enzymes. The effect of proteins was different: Asp. niger autolysate showed no essential influence and egg albumin inhibited pectine disintegration.     

Determination of the degree of N-acetylation (DA) of chitin and chitosan in the presence of water by first derivative ATR FTIR spectroscopy

A new method for the determination of the degree of N-acetylation (DA) of chitin and chitosan is described using first derivative diamond ATR FTIR spectroscopy. Applying the derivative values of the amide III band at 1327 cm⁻¹ and the CH deformation band of the N-acetyl group at 1383 cm⁻¹ as measure of the N-acetyl content of the sample in relation to the derivative value of the bridge oxygen vibration at 1163 cm⁻¹ as internal standard, a linear correlation to the results of first derivative UV spectroscopy was obtained and confirmed by elemental analysis and Raman spectroscopy. The described method allows the determination of the degree of N-acetylation of chitosan and chitin in the presence of water thus making drying procedures unnecessary.     

Different calcium sensitivity in osteoclasts on glass and on bone and maintenance of cytoskeletal structures on bone in the presence of high extracellular calcium

The sensitivity of rat osteoclasts to increased extracellular calcium concentrations ([Ca²⁺]_e) was investigated by single cell measurements of free cytosolic calcium concentrations ([Ca²⁺]_i), by changes in microfilament organization of resorbing osteoclasts, and by in vitro bone resorption assays. Osteoclasts cultured on glass and on bone showed clear differences in their responses, as in 44% and 52% of osteoclasts on glass but in only 21% and 25% of osteoclasts on bone [Ca²⁺]_i increased when [Ca²⁺]_e was increased from 2 mM to 6 or 10 mM via perfusion, respectively. Bone resorption was inhibited without changes in the osteoclast numbers only by 10 mM [Ca²⁺]_e in 2 day cultures. Furthermore, there were no changes in the organization of microfilament structures in resorbing osteoclasts after increased [Ca²⁺]_e (up to 20 mM [Ca²⁺]_e, 30 min incubation). These results suggest that the sensitivity of osteoclasts to increased [Ca²⁺]_e is dependent on their activation phase (resting/migrating vs. resorbing) and that resorbing osteoclasts are not sensitive to increased [Ca²⁺]_e or that the sensing system cannot be reached in polarized resorbing osteoclasts. In contrast, increasing [Ca²⁺]_i through the use of calcium ionophores dispersed specific microfilament structures at the sealing zone transiently in a few minutes. This shows that [Ca²⁺]_i is used as a signaling mechanism to inactivate osteoclasts, with a similar end result on microfilament structures at the sealing zone as caused by increased concentration of cAMP and activation of protein kinase C. © 1996 Wiley-Liss, Inc.     

Structural studies of arabinogalactan and pectin from Silene vulgaris (M.) G. callus

Arabinogalactan and pectin (named silenan) were isolated from Silene vulgaris (M.) G. callus. Fractionation by ion-exchange chromatography on DEAE-cellulose and digestion with pectinase demonstrated that silenan from S. vulgaris callus (80% of D-galacturonic acid) and silenan from the aerial part of the campion S. vulgaris are similar: both pectins contain a high quantity of homogalacturonan segments. The NMR spectral data and mass spectrometry of the purified polysaccharide and its fragment obtained by Smith degradation confirmed that the core of the arabinogalactan consisted of the different segments of β-1,3-D-galactopyranan. Some of the β-galactopyranose residues of the backbone are branched at O-6. The side chains of the arabinogalactan were shown to contain residues of terminal and 3-O-substituted β-galactopyranose, terminal α-arabinofuranose and α-rhamnopyranose, and 2-O-substituted α-rhamnopyranose. The α-rhamnopyranose residues in the sugar chain appeared to be 2-O-glycosylated by the β-1,4-D-galactopyranosyl uronic acid residues. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 6, pp. 798–807.     

Dissociation and aggregation of calpain in the presence of calcium

Calpain is a heterodimeric Ca(2+)-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca(2+) on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca(2+) and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca(2+) concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca(2+), but this was fully reversible by EDTA. With Ca(2+) in 0.2 m NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca(2+) gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca(2+) was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca(2+)-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I.     

Preparation and properties of oxidized starch with high degree of oxidation

A highly efficient method for preparing oxidized starches with a high degree of oxidation (DO) was developed, using CuSO₄ and H₂O₂ respectively as a catalyst and an oxidant. The effect of different parameters including starch origin, oxidant content, temperature, catalyst content, and reaction time on the DO was investigated systematically. In the present study, only 0.5% of catalyst was added, and the reaction time could be reduced to 1 h, while in the previous study the reaction time of 72 h was necessary to achieve almost the same DO without a catalyst. The structures and properties of oxidized starches were characterized by FT-IR, DSC, TGA, XRD, and transmittance light testing. The oxidation reduced the intrinsic viscosity and thermal stability of the oxidized starches, and could change the crystalline structures into amorphous states when the DO reached 56.3%. When temperature and/or DO increased, the transmittance of suspended solution of oxidized starch increased correspondingly.     

Structural characterization, degree of esterification and some gelling properties of Krueo Ma Noy (Cissampelos pareira) pectin

Pectins extracted from Krueo Ma Noy (Cissampelos pareira) leaves mainly consisted of galacturonic acid with trace amount of neutral sugars. The dominant structure of Krueo Ma Noy pectin was established as a 1,4-linked -D-galacturonan by a combination of carboxyl reduction and methylation analysis, and confirmed by FT-IR spectroscopy. The degree of esterification of Krueo Ma Noy pectins was 41.7 and 33.7% for crude and dialyzed pectins, respectively. Krueo Ma Noy pectin has an average molecular weight of 55 kDa, radius of gyration of 15.2 nm and intrinsic viscosity of 2.3 dl/g. Krueo Ma Noy pectin exhibited gelling properties in aqueous solutions at 0.5% (w/v) at 5 °C. Gels were formed at concentrations of 1.0% (w/v) and above even at room temperature. The gel strength, melting point, and melting enthalpy of Krueo Ma Noy pectin increased with polysaccharide concentration.     

Modeling of quantal neurotransmitter release kinetics in the presence of fixed and mobile calcium buffers

The local calcium concentration in the active zone of secretion determines the number and kinetics of neurotransmitter quanta released after the arrival of a nerve action potential in chemical synapses. The small size of mammalian neuromuscular junctions does not allow direct measurement of the correlation between calcium influx, the state of endogenous calcium buffers determining the local concentration of calcium and the time course of quanta exocytosis. In this work, we used computer modeling of quanta release kinetics with various levels of calcium influx and in the presence of endogenous calcium buffers with varying mobilities. The results of this modeling revealed the desynchronization of quanta release under low calcium influx in the presence of an endogenous fixed calcium buffer, with a diffusion coefficient much smaller than that of free Ca²⁺, and synchronization occurred upon adding a mobile buffer. This corresponds to changes in secretion time course parameters found experimentally (Samigullin et al., Physiol Res 54:129–132, 2005; Bukharaeva et al., J Neurochem 100:939–949, 2007).     

Cobtorin target analysis reveals that pectin functions in the deposition of cellulose microfibrils in parallel with cortical microtubules

Cellulose and pectin are major components of primary cell walls in plants, and it is believed that their mechanical properties are important for cell morphogenesis. It has been hypothesized that cortical microtubules guide the movement of cellulose microfibril synthase in a direction parallel with the microtubules, but the mechanism by which this alignment occurs remains unclear. We have previously identified cobtorin as an inhibitor that perturbs the parallel relationship between cortical microtubules and nascent cellulose microfibrils. In this study, we searched for the protein target of cobtorin, and we found that overexpression of pectin methylesterase and polygalacturonase suppressed the cobtorin-induced cell-swelling phenotype. Furthermore, treatment with polygalacturonase restored the deposition of cellulose microfibrils in the direction parallel with cortical microtubules, and cobtorin perturbed the distribution of methylated pectin. These results suggest that control over the properties of pectin is important for the deposition of cellulose microfibrils and/or the maintenance of their orientation parallel with the cortical microtubules.     

绿原酸对莴苣生长的化感作用及其机理研究

酚酸类化合物是一类重要的化感物质,广泛存在于农作物等植物体内以及耕作土壤中,与植物的化感效应以及连作障碍密切相关。该研究以蔬菜类植物莴苣为受体,采用植物细胞和生理学方法,分析酚酸类化感物质绿原酸抑制莴苣生长的活性效应及其作用机理,以揭示绿原酸介导的化感作用及连作障碍机制。结果显示:(1)绿原酸对莴苣的根长、茎长以及鲜重等生长指标均表现出低浓度(0.1和1.0μmol/L)促进、高浓度(10、100和1 000μmol/L)抑制的活性作用模式,其在10μmol/L及以上浓度时对根长和鲜重具有显著抑制作用,但在各浓度下对莴苣茎长无显著影响。(2)绿原酸处理后,莴苣根尖细胞分裂指数明显下降,随处理浓度的升高,各分裂时期的细胞比例也显著降低,导致细胞分裂过程受阻。(3)较低浓度(0.1、1.0和10μmol/L)绿原酸对莴苣根尖细胞活力无明显影响,较高浓度绿原酸(100和1 000μmol/L)使莴苣根尖死细胞显著增多,根尖端绝大部分细胞失去活力。(4)DHE荧光染色显示,与对照相比,低浓度(0.1和1.0μmol/L)绿原酸对莴苣根部的活性氧积累无明显影响,当绿原酸处理浓度大于10μmol/L时,随处理浓度升高莴苣根部的活性氧积累明显大量增多。研究表明,绿原酸能够诱导莴苣体内活性氧产生并积累,从而导致莴苣细胞分裂受阻、细胞活力降低,最终影响莴苣幼苗的生长发育。