Pectate concentration

At the optimal sodium pectate concentration, the efflux time of the substrate solution in the viscometer should be in the range of 115 to 120 sec. 

The viscosity of sodium pectinate was previously examined by Pals and Hermans [44]. In salt excess (NaCI 0.1 M), the viscosity of a pectate sodium form solution (ri) is related to the polymer concentration (c) and the molecular weight (or [rj] = KM ) by the relation ... 

The rheological behaviour in the range of LM pectin was analyzed and the sol-gel diagram established [59] for different stoichiometric ratios. In their paper, these authors determined the gel times for sodium pectate during calcium-induced gelation and the variation of the gel time with polymer concentration, stoichrometric ratio and temperature. 

Figure 9. The cable model for the structure of concentrated calcium pectate gels. Egg-box dimers link single-chains segments (top left) and are themselves ed together by larger aggregates of either egg-box or 3i helical chains (lower right)...

F ure 10. solid-state NMR spectra of calcium pectate in the solid (A) and gel (B) forms. Inset resolution-enhanced spectra. The gel concentration was 290 g/1. 

Figure 12. Binding of calcium ions by pectate and apple pectin, measured by equilibrium dialysis gainst citrate to buffer the concentration of free calcium ions at low levels.

Some divalent cations such as Cu and Pb form very stable complexes with pectate, but are unlikely to be present at sufiScient concentration in the apoplast of plants to form a major fraction of the counterions associated with the pectic fraction in vivo. The Al ion may deserve closer examination, as it is certainly able to displace Ca from cell walls and reaches substantial concentrations in plant roots under some conditions [60,61]. aluminium is not usually considered to be freely translocated, however. Basic peptides with their negative charges spaced at a similar interval to galacturonans (0.43 nm or a small multiple thereof) can in principle have a very high afiBnity for pectate [62,63], but the extensins that are associated with the most insoluble pectic fractions [M] do not appear to have this type of structure. The possibility that the non-extractable pectic polymers in most cell walls are very strongly complexed with some cation other than Ca " cannot be ruled out, but there is little evidence to support it at present. 

Ultrafiltration of heterogenous colloidal suspensions such as citrus juice is complex and many factors other than molecular weight contribute to fouling and permeation. For example, low MW aroma compounds were unevenly distributed in the permeate and retentate in UF in 500 kd MWCO system (10). The authors observed that the 500 kd MWCO UF removed all suspended solids, including pectin and PE. If PE is complexed to pectate in an inactive complex, then it is conceivable that release of PE from pectin with cations will enhance permeation in UF. At optimum salt concentration, less PE activation was observed at lower pH values than at higher pH (15). In juice systems, it is difficult to separate the effect of juice particulates on PE activity. Model studies with PE extracts allows UF in the absence of large or insoluble particulates and control of composition of the ultrafilter. In... 

The enzyme had a requirement for calcium. The addition of EDTA to the reaction mixtures, resulted in complete loss of activity, whereas the addition of CaCl2 increased the activity (figure 8). Presumably, sufficient contaminating calcium ions were present in the dialyzed enzyme and substrate mixture to permit the limited activity of the controls, but apparently these were removed by chelation with EDTA. The optimum concentration was in the range of 5 to 15 M, and higher concentration resulted in a decrease in activity. Phoma medicaginis var. pinodella synthesizes a pectin lyase that lacked an absolute requirement for calcium ions but maximum enzyme activity required the presence of 1 mM Ca [25]. The lyase from Fusarium solani f sp. phaseoli, that is active on pectin and pectic acid, is calcium-dependent [30]. Most of the pectate lyases characterized are calcium-dependent the pectate lyase from Rhizoctonia solani [34] and the endopectate lyase fi om Fusarium solani f sp. pisi [31]. 

Table 1 shows some biochemical properties of the pectolytic enzymes present in pool 1. The pectin lyase/pectate lyase activities (pool I) and polygalacturonase activity (pool II) were not significantly affected by NH4+, Na+ and K+ (0,25 - 2,5mM), while Al +, p-mercaptoethanol, Hg2+, EDTA, Ba + and Zn+2 (2,5mM) inhibited 30-100% these activities. On the other hand, Ca2+, Mg + and Mn + at 2,5mM concentration activated 20-100% pectin/pectate lyases but Ca " " and Cu " " (2,5mM) inhibited polygalacturonase activity about 42 - 70%. 

Figure 3. Determination of the optimal parameters of PelZ activity. A pectate lyase activity in a reaction medium containing PGA 0.05%, CaCl2 0.2 mM and Tris-HCl 50 mM at various pH. B pectate lyase activity in a reaction medium containing PGA 0.05%, Tris-HCl 50 mM pH 8.5 and increasing concentrations of CaCl2- C pectate lyase activity in a reaction medium containing CaCl2 0.2 mM, Tris-HCl 50 mM pH 8.5 and as substrate either PGA 0.05% or pectins 0.05%, with different degrees of methylation (from Copenhagen Pectin).

Based on the same principle, there are monomeric / -helical proteins that carry at their extremities a cluster of helical or nonrepetitive structures that could act as a capping element covering their exposed ends (Emsley et al., 1996 Lietzke et al, 1994 Petersen et al, 1997 Steinbacher et al, 1994). For example, the last 40 residues of pectate lyase C form a large loop that partially covers the surface of the /Hielix (Yoder et al, 1993). The fibrous (or otherwise elongated) domain of these natural /f-stranded proteins is not stable in isolation, as for example in the case of the P22 tailspike where bacterially expressed isolated /Hielix domain, at high concentrations, forms fibrous aggregates that bind Congo red (Schuler et al, 1999). 

A unit of activity is arbitrarily defined as that amount of enzyme that will reduce the viscosity of the sodium pectate solution by 50% in a 5-min period. The time required to reach A = 50% is typically inversely proportional to the concentration of enzyme (Mill and Tuttobello, 1961 Gusakov et al., 2002). 

If steps are not taken to stabilize cloud, most citrus juices will clarify when allowed to stand. Clarification occurs when native PE lowers the ester content of juice soluble pectin until it becomes susceptible to precipitation as insoluble pectates (23). If pectin levels are high enough, as in concentrates, these pectates may form a gel. 

Calcium and magnesium are very abundant in soils, and soils deficient in Ca are rare, calcium status is maintained when lime is added to correct acidity. Plant cells contain relatively large concentrations of Ca, but most of it is bound in the cell-wall as the pectate (about 60%) or sequestered in different organelles (Clarkson, 1984). Ca affects the permeability of the cytoplasmic membrane and its deficiency leads to malformation of the growing parts of the plant. Hewitt and Smith (1975) have discussed the early experiments on the morphological effects of Ca deficiency. Calcium is often found in combination with organic acids, for example oxalic acid, a soluble, toxic metabolic by-product is converted to insoluble calcium oxalate. 

Endogenous pectin esterases play an important role in citrus processing they have therefore been studied intensively. They cause cloud loss in citrus juices, which is due to the calcium precipitation of enzymatically de-esterified pectin. This is desirable in the production of lemon and lime juices but undesirable in the production of orange juice. In orange juice concentrates strong calcium pectate gels may form which cannot be reconstituted... 

Because of the polyelectroly tic nature, pectin solutions need to be made in excess of salt, usually in 0.05 0.1 M sodium chloride or phosphate, and use the same solvent for dilution (isoionic dilution) (Pals and Hermans, 1952). This is because, unlike neutrol polymers, the viscosity of dilute solution of polyelectrolytes displays unique dependence on concentration. As shown in Figure 9.5, the qsp of sodium pectate exhibits a maximum in pure water and low concentration of salt, a phenomenon caused by the so-called electroviscous effect. When the salt concentration is... 

FIGURE 9.5 Zero shear rate reduced viscosity (r sp0/c) vs. concentration of sodium pectate at various NaCl concentrations 1-7 0, 0.2, 0.4, 1.0, 2.5, 5.0 and 50 mM. From Pals and Hermans (1952), with permission of Elsevier Applied Science. 

In spite of usefulness of the simplification obtained by decreasing the experimental substrate concentration, many studies are aimed at the investigation of kinetic properties of immobilized biocatalysts within broader concentration ranges. In a previous paper [29], cells of Escherichia coli with penicillin acylase activity were immobilized by entrapment in calcium pectate gel and tested on the transformation of penicillin G to 6-amino penicillanic acid. Figure 9 shows experimental data from a microcalorimetric investigation of the penicillin G transformation in steady state. As appreciable particle-mass transfer was expected, the mathematical model that includes particle-mass balance was used. 

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