Action pattern enzymes

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. 

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 ( ). 

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). 

The courve of pH optima determination indicated a presence of acidic exopolygalacturonase form as it was in Fraction A but with slight shift to pH 3.6 (Fig. 2). It was impossible to commute this enzyme form with the acidic exopolygalacturonase from Fraction A because of its molecular mass about 30000 and action pattern identical with form with pH optimum 5.4. Further characterization of this form was not made because of its low content in lyophilizate. 

In further work on this series, the same methods were used for the examination of the action pattern of highly purified pectinesterase produced by Fusarium oxysporum f. sp. vasinfectum.49 This pectinesterase was found to affect highly esterifled pectin by a mechanism similar to that of tomato pectinesterase, that is, more than half of the enzymic activity occurred at the reducing ends of the molecules, and the rest attacked a different locus or loci of the pectin chains. These conclusions were supported by a comparison of the effect of clostridial lyase on pectin partly de-esterified in an alkaline solution with its effect on pectin partly de-esterified by Fusarium oxysporum pectinesterase. The lyase did not act on the randomly... 

The difference in the effect of various endo-D-galacturonanases on oligomeric substrates [in particular, the difference in the rate of degradation of tri(D-galactosiduronic acid) and in the action pattern toward the tetrasaccharide] indicates, however, that it is not the substrate, but rather, the properties of the enzyme (in particular, the character of its active center) that constitute the determining factor. 

The activity of tomato endo-D-galacturonanase follows action pattern B (see Scheme 1), which is characterized by an alternative cleavage of tetra(D-galactosiduronic acid) and by a relatively rapid degradation of trisaccharide.128 In the tetrasaccharide, bond 1 is split faster than bond 2. Bond 3 is not broken by the enzyme. Both the reduced and oxidized tetra(D-galactosiduronic acid) derivatives are hydrolyzed by this enzyme at bond 2. 

The action pattern and the specificity of enzymes acting on homo-polymeric substrates are determined by the nature of the active center.134-137 Hence, it may be assumed that the active centers of enzymes exhibiting the aforementioned action-patterns are different. 

A similar interpretation of action patterns B and C (Scheme 1), and of the number of productive complexes, indicates that, in the corresponding enzymes, there is a binding site composed of three (B) or five (C) subsites having the catalytic groups situated between the first and second (B) and the second and third (C) subsites. Another indication of a binding site containing three subsites for enzymes of... 

Viscosimetric determination of activity is used only with pectic enzymes displaying the random-action pattern. The activity is mostly expressed as the time required for attaining a 50% decrease of viscosity (in s) or as the amount of enzyme required for attaining a certain decrease of viscosity per unit time.223... 

Action pattern A was observed with extracellular, endopectate lyase of Bacillus polymyxa,4,240 with extra- and intra-cellular lyase of Erwinia carotovora,241 with extracellular enzyme of Xanthomonas campestris,23S and with lyase produced by Bacteroides ruminicola242 isolated from the rumen fluid of sheep.243... 

An enzyme having the same action-pattern was found in Erwinia... 

Amylol3rtic glucosylases are probably the most widely studied of the carbohydrate enz3nnes, due to their biological and industrial importance, as well as their ubiquitous distribution. Many different types of amyloglucosylases exist, with a variety of physical, chemic and catalytic properties. Recent reviews 28-30) describe the classification, characterization, action patterns and biochemistry of different enzymes. 

P-Mannanases are generally prepared by conventional chromatographic procedures, which can lead to high yields of enzyme, but in some cases only a poor state of purity. Small quantities of highly purified p-mannanases have been prepared by substrate affinity chromatography of partially purified enzymes on a column of glucomannan immobilised on aminohexane Sepharose 4B (6). The action patterns of enzymes described in this paper were determined using enzymes purified by this latter procedure. 

The difference in action patterns between these p-mannanases is shown clearly by fractionating and characterising the reaction products (Table I) (5). These products are a consequence of the abihty of the enzyme to cleave in the vicinity of D-mannosyl residues substituted by D-galactose, as well as the length of the 1,4-P-D-manno-oligosaccharide chain required by the enzyme for binding. The favoured conformation of the (l-4)-p-D-linked mannan chain is a flat... 

The lytic enzyme systems, active against yeast cell walls, usually contain l,3-/ -glucanases, proteases, mannanases, chitinases, and 1,6-) -glucanases. The proportion of those enzyme activities, their action pattern, synergism, and dependence on inhibitors constitute the activity profile... 

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