Pectin enzyme action

The conversion of sucrose into a branched polysaccharide of the amylo-pectin-glycogen type by a bacterial-enzyme system was discovered by Hehre and his coworkers. Enzyme action involves the synthesis of polymeric chains of (1— 4)-linked a-D-glucose residues by a transglucosylase named amylosucrase, followed by the action of a branching enzyme. 

Both processes involve the action of enzymes used by retting organisms to dissolve pectin. Enzymes can also be used directly to degum the straw. For flax a pectolytic activity (°PM) of 210 U cm is required. 

Pectins represent complex molecules providing many opportunities for different types of enzyme action. Our understanding of the enz unic hydrolysis of protopectin is so unsatisfactory that a discussion of protopectinase seems unwarranted. The pol3uiionic skeleton of pectinic acids and pectic acids can be d raded through glycosidic hydrolysis by the various poly-Re/erences p. 244... 

The differentiation of cells occurs concomitantly to modifications of wall components. The nature of the pectins of the walls changes under the action of enzymes, among which esterases, secreted between the apical meristematic cells and the more basal differentiated cells. The apposition of new layers of pectins with different compositions at the inner surface of the walls is another mechanism by which the cells adapt their immediate environment. Using the 2F4 antibody, we have observed, in plant suspensions as well as in tissues, a third mechanism involved in wall modification. Numerous invaginations of the... 

The discovery of these enzymes enables a better structural characterisation of the hairy (ramified) regions of pectin, as already demonstrated by Schols et al. (1990b) and also of native plant cell wall pectin (Schols et al., 1995). In this study we show how the two exo-enzymes of the above described series, the RG-rhamnohydrolase and the RG-galacturonohydrolase, can be used as tools in the characterisation of unknown RG fragments. These unknown fragments were the products of RG-hydrolase or RG-lyase action toward linear RG oligomers (RGO s), which were produced by acid hydrolysis of sugar beet pulp. 

HPAEC analyses were carried out to determine the oligomeric products released from various pectic substrates after depolymerization by the PL isoenzymes. Action pattern analyses for the concerted action of PL isoenzymes utilized 68% esterified pectin as substrate. One-ml reaction mixtures in a buffer system as detailed in section 2.2. comprising 0.5% (w/v) substrate and 5 U of enzyme were incubated for 30 s to 18 h, and then thermoinactivated. Samples of 750 pi were applied to a Carbopac PA-1 (Dionex) column before the carbohydrates were eluted over a period of 70 min using a gradient of 0.2 M KOH, 0.05 M K-acetate to 0.2 M KOH, 0.7 M K-acetate. Detection employed a Pulsed Electrochemical Detector (PED, Dionex) in the integrated amperometry mode according to the manufacturer s recommendations. 

Intrigued by the finding that Eca PLs exhibit notable differences in their kinetics, HPAEC analyses were carried out to examine the products from the depolymerization of PGA and 31% esterified pectin. After 18 h of incubation with PGA, PL1 and PL2 had produced mainly di- and trimers. Similariy, main products of PL3 action were trimers, followed by dimers. Moreover, it was the only enzyme found to produce monomers from unesterified substrates with a degree of polymerization >3. Using 31% esterified pectin as a substrate, similar end products were released by the PLs as from PGA. In addition to the products described, traces of tetra- up to octamers were detectable. While PL1 and PL2 released di- and trimers at almost... 

The degradation of pectin is initiated by the action of PME, an enzyme removing methylester groups from highly methoxylated pectins. Production of incompletely deesterified pectin, probably due to end-product inhibition of PME (19, 20), may explain that an affinity of PL for both pectate and pectin is required for full pathogenicity of the bacteria. 

Action pattern analyses of pectin degradation. HPAEC data of oligomers released from 68% esterified pectin by combinations of Eca PLs are graphically represented. Arrows indicate addition of the third enzyme. Products with degrees of polymerization ranging from 2 to 9 were detected. The graphs illustrate the generation of dimers (A), trimers ( ) and pentamers ( ). 

Pectin degradation requires fee combined action of various enzymatic activities. However, evaluation of fee contribution of individual pectinases in Suit juice extraction and clarification is rather complicated. Most commercial pectinolytic enzyme preparations are produced by fermentation wife filamentous fungi, mostly strains belonging to fee genus Aspergillus,. plication studies with mixtures of isolat enzymes obtained by fermentation or by means of fractionation of commercial enzyme preparations can be used to assess the importance of fee various individual enzymes. Subsequently, molecular biology and fermentation technology can be used to enhance specific desirable enzymatic activities. 

The r2 isolate of Fusarium oxysporum f. sp. radicis-lycopersici (FORL) produced several pectic enzymes that differ in substrate preference, reaction mechanism, and action pattern. We have detected three forms that have lyase activity, an absolute requirement for calcium, and pis of 9.20, 9.00 and 8.65. The two most alkaline forms had a weak preference for pectin whereas the other was more active on pectate. The three lyases were produced when the fungus grew on pectin and on restricted galacturonic acid (data presented in the "XV Congreso Nacional de Microbiologia" [21] and sent for publication). 

Mode of action and substrate specificity of the purified enzyme were determined by following the decrease in viscosity and the increase in absorbance at 235 nm in reaction mixtures in the presence of 0.187 % substrate (pectin or pectate) at pH 8.0. 

Substrate specificity and mode of action. Previous information, which we had obtained from FORL crude culture filtrates, showed that the pectin lyase (characterized by an isoelectric point of 9.2) had a predominantly "endo" way of action. This fact has been confirmed with the purified protein it decreased the viscosity of reaction mixtures with pectin, but no increase in absorbance was detected in standard conditions. Moreover, the enzyme showed a great specificity for the substrate, as no activity was detected when the decrease in viscosity of pectate was tried. So, properties of the purified enzyme were studied by using pectin as substrate and following the decrease in viscosity of the reaction mixtures. 

The more abundant lyase produced by FORL has been purified to homogeneity as it is shown by analytical isoelectric focusing (figure 5). The data in Table 1 show that a 32.48-fold increase in specific activity is achieved with a recovery of approximately 2.36%. The enzyme showed an "endo" type of action and a great specificity for pectin. 

A decrease in the pectic molecular weight by pectin lyase resulted in an increase in the activity of PAE (Figure 2). This could be caused by easier access of the enzyme to the substrate, but PAE is not limited in its action by the substrate size as was found for FAE. 

Some properties of Penicillium fellutanum pectinesterase were studied. The optimum of pectinesterase action was detected at pH 5 and 45 °C. The enzyme was stable at pH 4 — 5 and 40 °C (pH 5) for 240 min. and was specific towards lemon pectin. An enzyme preparation composed mainly of pectinesterase was partially purified by gel filtration. Pectinesterase activity was accumulated in one of the obtained fractions. Molecular weights of fraction determined were found to be 46,000 and 1,200. Disk electrophoresis in polyacrilamide gel of the purified preparation revealed two protein bonds with one active component. The partially purified enzyme had the kinetic characteristics =... 

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