Pectin endopolygalacturonase

Hydrolysis pectate pectin endopolygalacturonase endopolymethylgalacturonase exopolygalacturonase exopolymethylgalacturonase... 

Immobilization. The fixing property of PEIs has previously been discussed. Another appHcation of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on soHd supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus faciHtating preparative work. This technique has been appHed to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

Other polysaccharides of primary cell walls.-A complex mixture of enzymes including endopolygalacturonase, pectin methylesterase, and/or pectin lyase solubilizes a mixture of polysaccharides from the primary cell walls of fruits [57-64]. Food scientists have referred for some 15 years to this mixture of polysaccharides as the hairy region to describe the highly branched character of the polysaccharides in the fraction and to emphasize the contrast to unbranched homogalacturonan. The recent discovery of rhamnogalacturonan hydrolase [65,66], which selectively cleaves the backbone of RG-I, led to the realization that the hairy... 

The mode of action and substrate specificity of all pectin lyases together with the endopolygalacturonases is currently one of the main topics of our research. 

As shown for the pectin lyases endopolygalacturonases are also present as a gene family in A. niger. The individual endopolygalacturonases are also characterized both at the genetic and biochemical level. 

Figure 3 Gel-permeation chromatography on Sephacryl S-200 of the products obtained after endopolygalacturonase degradation of the (a) water-soluble pectins from extruded citrus fibres (SME = 250 kWh/t) and (b) the acid-extracted pectins from the same raw material.

Pectins were extracted from isolated cell walls of 5-week-old wheat plants using different methods. Enzymic digestions of the cell walls involved pectinases such as a commercial pectolayse or recombinant endopolygalacturonase [Maness Mort, 1989]. Chemical extractions involved the chelating agent imidazole [Mort et al., 1991] or solvolysis with anhydrous HF at 0 °C in a closed teflon line [Mort et al., 1989] followed by imidazole extraction. 

Figure 1. Transverse section of barley leaf epidermal cells taken perpendicular to the long axis of the cells and anticlinal to the leaf surface. The section has been labeled by the EMSIL technique (see Methods) utilizing purified C. sativus endopolygalacturonase and monoclonal antibody EPG-4, which is specific for this enzyme, in order to localize the substrate of the enzyme at the typical site penetrated by the fungal pathogen. Bar = 1 pm. Inset Comparable cell wall region as in Fig. 1 but labeled with monoclonal antibody JIM 5 to localize non-esterified pectin. Bar = 1 pm. Note the identical labeling patterns obtained with either method.

Pectinesterase activity expressed as a unit corresponding to the microequivalent of ester bonds of pectin molecule, which were hydrolyzed during 1 min. at 45 °C and pH 5.0 under the conditions, which were optimum for these enzymes. Endopolygalacturonase and exopolygalacturonase activities were determined using a technique determined by Ufshitz [8]. Activity of pectintranseliminase was determined by procedure (lOJ. 

In a recycling system, the aqueous discharge effluent from both centrifiiges is returned to the extractors for additional oil recovery, the water being reused. During this extraction process the viscosity of the emulsions increases because peel polysaccharides, mainly pectins, are transported with the emulsion. Enzymatic breakdown of the internal links of the pectin, catalysed by endopolygalacturonase activity, produces an important decrease in the viscosity of the emulsion [16]. In addition, enzymatic treatment removes pectins from the emulsion and contributes to it destabilization [17]. 

A commercial pectinase, immobilised on appropriately functionalised y-alumina spheres, was loaded in a packed bed reactor and employed to depolymerise the pectin contained in a model solution and in the apple juice. The activity of the immobilized enzyme was tested in several batch reactions and compared with the one of the free enzyme. A successful apple juice depectinisation was obtained using the pectinase immobilised system. In addition, an endopolygalacturonase from Kluyveromyces marxianus, previously purified in a single-step process with coreshell microspheres specifically prepared, was immobilised on the same active support and the efficiency of the resulting catalyst was tested. 

Mixtures of the polygalacturonases or of the pectinesterases were also ineffectual. Only the combined action of at least one of the polygalacturonase fractions with one of the pectinesterase fractions was effective in clarification. Purified endopolygalacturonase alone could decrease the viscosity of a solution of citrus pectin (64% esterification), but it was completely ineffectual when apple pectin was used [90% esterified (iS6)]. 

Degradation studies. Degradation limits by endopolygalacturonase and by p-elimination were determined as described by Thibault (17) and by endopectate lyase as described by Rombouts a] (4). For pectin lyase the reaction conditions were 0.25 (w/v) pectin, 0.01 M sodium phosphate buffer, pH 5.2 and 0.52 U/ml of pectin lyase, 30°C, 24 h. The pectin lyase reaction was monitored by measuring nm of aliquots, diluted thirtyfold with 0.1 N hydrochloric aciQ and the degradation limit was calculated, using a molar extinction coefficient of 5500 (18). 

Fig. 3 Gel-permeation chromatogram of acid-soluble sugar-beet pectin degraded with endopolygalacturonase after pec-tinesterase demethoxylation.

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