Lubomíra Rexová-Benková

Selective purification of Aspergillus niger endopolygalacturonase by affinity chromatography on cross-linked pectic acid.

Abstract Incubation of a mixture of extracellular pectolytic enzymes of Aspergillus niger with insoluble pectic acid cross-linked by epichlorhydrine under optimum conditions for endopolygalacturonase (poly-α-1,4-galacturonide glycanohydrolase, EC 3.2.1.15) action results in a selective adsorption of endopolygalacturonase by the insoluble support. The quantitative liberation of the enzyme into solution is achieved either in the presence of soluble sodium pectate or at pH values over pH 6. On the basis of these observations a chromatographic method for endopolygalacturonase purification was developed.

PectinaseAspergillus sp. polygalacturonase: Multiplicity, divergence, and structural patterns linking fungal, bacterial, and plant polygalacturonases

Nine forms ofAspergillus sp. polygalacturonase were purified from a commercial preparation of pectinase Rohament P using chromatographies and chromatofocusing. Individual forms differ in isoelectric point, and at least five differ in structure; whereas molecular masses and enzymatic properties are largely identical. Four forms with freea-amino groups have identical start positions but internal amino acid replacements. Therefore, the multiplicity is derived from true heterogeneities and not from N-terminal truncations. Peptide analysis of the major polygalacturonase reveals large variations toward the enzyme from otherAspergillus species (72–75% residue differences, depending on species) but additional similarities with the enzyme from bacterial and plant sources (only 66–71% residue differences toward theErwinia, tomato, and peach enzymes). Combined with previous data, these facts show polygalacturonase to exhibit extensive multiplicity and much variability, but also unexpected similarities between distantly related forms with conserved functional properties

On the character of the interaction of endopolygalacturonase with cross-linked pectic acid

Abstract The combination of cross-linked pectic acid with endopolygalacturonase (poly-α-1,4-galacturonide glycanohydrolase, EC 3.2.1.15) and its effect on the kinetics of action on sodium pectate as well as on the thermal stability of the enzyme was studied. It was shown that endopolygalacturonase is competitively inhibited by cross-linked pectic acid. The value of the inhibitory constant K i obtained (3.24 mg of cross-linked pectic acid per ml) was found to be very close to the value of the association constant of the complex of endopolygalacturonase with cross-linked pectic acid formed in the absence of the substrate (3.47 mg of cross-linked pectic acid per ml). The character of the inhibition as well as the similarity of the constant values indicate that the process of the complex formation is due exclusively to a functional affinity of the binding site of endopolygalacturonase to the adsorbent. The binding of endopolygalacturonase to cross-linked pectic acid considerably increases the thermal stability of the enzyme.

Endopolygalacturonase immobilized on a porous poly(2,6-dimethyl-p-phenyleneoxide)

SummaryEndopolygalacturonase was immobilized by coupling on to porous poly(2,6-dimethyl-p-phenyleneoxide) activated by adsorbed glutaraldehyde. Catalytic properties, stability and action pattern of the immobilized enzyme are described.

Effect of the Pore Size of a Support on Activity and Action Pattern of Immobilized Endopolygalacturonase

Endopolygalacturonase (E.C. 3.2.1.15) was covalently bound to silica supports of different porosity treated with 3-(2′,3′-epoxypropoxy)propyltrimethoxysilane. The activity and action pattern on sodium pectate and tetra(D-galactosiduronic acid) were investigated in batch and continuous flow-reactors. Pore size of the supports affected the loading of the enzyme as well as its action pattern and kinetics. A decrease in randomness of degradation of D-galacturonan, loss of specificity of (3 + 1) splitting of tetra(D-galactosiduronic acid) and decrease in Km value were found with the supports containing predominantly micropores. The extent of the changes decreased with increasing pore size of the support. The catalytic behaviour of endopolygalacturonase bound on the supports with large pores was quite analogous to that of the free enzymes.

Mechanism of action of d-galacturonan digalacturonohydrolase of Selenomonas ruminantium on oligogalactosiduronic acids

The mechanism of action of the specific D-galacturonan digalacturonohydrolase [poly-(1----4)-alpha-D-galactosiduronate digalacturonohydrolase, EC 3.2.1.82] of Selenomonas ruminantium was investigated by using reducing-end [1-3H]-labeled oligogalactosiduronates having degree of polymerization 3-5 as the substrates. The reaction products, incorporation and distribution of radioactivity in products, and the frequency of oligogalactosiduronate bond-cleavage were quantitatively estimated as functions of the substrate concentration. An alternative cleavage of tri(D-galactosiduronate) occurred during the enzyme reaction, indicating the participation of some bimolecular mechanism in addition to unimolecular hydrolysis in the action of the enzyme. Unimolecular hydrolysis takes place at low initial concentration of the substrate. The shifted termolecular enzyme-substrate complex formation and the subsequent galactosyluronic transfer is the predominant mechanism in degradation of tri(D-galactosiduronate) at high concentration. Tetra(D-galactosiduronate) and penta(D-galactosiduronate) are degraded by unimolecular hydrolysis at low, as well as high concentration of the substrate.

Endopolygalacturonase immobilized on epoxide-containing supports

SummaryEndopolygalacturonase was immobilized onto 3-(2′,3′-epoxypropoxypropyl)-silica and oxirane-acrylic beads. Optimum conditions of immobilization and catalytic properties of the immobilized enzyme preparations are described.

THE EFFECTS OF THE POROSITY OF A SUPPORT AND OF ATTACHMENT ON THE MODE OF ACTION OF IMMOBILIZED ENDOPOLYGALACTURONASE

Endopolygalacturonase of Aspergillus sp. was immobilized by three different methods; viz. (a) via amino groups, (b) via carboxyl groups and (c) by means of epoxy groups to a nonporous microparticular silicon dioxide (Cabosil), functionalized by 3-(amino)-propyl groups and 3-(2′,3′-epoxypropoxy)-propyl groups, respectively. The conjugates were compared in their mode of action with corresponding immobilized preparations based on microporous ceramics. The binding via amino groups and via carboxyl groups lead, by itself, to changes in the mode of action of the enzyme, consisting of a decrease in randomness of glycosidic linkage splitting. The changes were greater in microporous support conjugates due to additional size-exclusion effects. The action pattern of endopolygalacturonase bound by means of epoxy groups was modulated exclusively by the porosity of the support, whereas the binding alone did not play any role.