Inversion of configuration during hydrolysis of alpha-1,4-galacturonidic linkage by three Aspergillus polygalacturonases.

Endopolygalacturonases I and II (PGI and PGII) of Aspergillus niger and an exopolygalacturonase (ExoPG) of A. tubingensis were investigated to reveal the stereochemistry of their hydrolytic action. Reduced pentagalacturonic acid (pentaGalU-ol) and reduced trigalacturonic acid (triGalU-ol) were used as non-reducing substrates for the enzymes. The configuration of the reducing ends in the products formed in D2O reaction mixtures was followed by 1H-NMR spectroscopy. It has been unambiguously established that primary cleavage of pentaGalU-ol by both PGI and PGII leads to diGalU-ol and the beta-anomer of triGalUA. The primary products of hydrolysis of triGalUA-ol by ExoPG were diGal-ol and the beta-anomer of GalUA. Thus, all three Aspergillus polygalacturonases belong to the so-called inverting glycanases, i.e. they utilize the single displacement mechanism of hydrolysis of the glycosidic linkage.

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.

Effects of polyethylene terephthalate on yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) was adsorbed onto polyethylene terephthalate, a synthetic polymer. The effects of the polymer on the properties of the enzyme were studied. The specific activity of the bound enzyme on protein basis was only 1.2 per cent of the specific activity of the soluble enzyme. The optimum pH for the catalytic activity was strongly shifted toward acidic direction. The apparent temperature optimum of the bound enzyme was identical with that of the soluble form. The apparent Michaelis constants of the bound enzyme were higher for both ethanol and NAD+. The conformational stability of the enzyme against heat treatment and urea was decreased as a consequence of adsorption.

The pectinolytic enzyme of Selenomonas ruminantium.

A cell-bound pectinolytic enzyme was isolated from cells of Selenomonas ruminantium and purified about 360-fold. The optimum pH and temperature for enzyme activity was 7.0 and 40 degrees C. The enzyme degraded polymeric substrates by hydrolysis of digalacturonic acid units from the non-reducing end; the best substrate was nonagalacturonic acid. Unsaturated trigalacturonate was also degraded, but 30% slower than the saturated analogue. The enzyme was classified as a poly (1,4-alpha-D-galactosiduronate) digalacturono-hydrolase; EC 3.2.1.82. Another enzyme, hydrolysing digalacturonic acid to monomers, was also produced in a very small amount by this organism.

An exo-D-galacturonanase of Butyrivibrio fibrisolvens from the bovine rumen.

An intracellular pectinolytic enzyme was isolated from a cell extract of Butyrivibrio fibrisolvens and purified. The optimum pH for enzyme activity was 5.6. The enzyme preferentially degraded de-esterified substrates by hydrolysis of monosaccharide units from the non-reducing end; the only product of degradation was D-galacturonic acid. Values of Km and Vmax for oligo- and polygalacturonates indicated that the best substrate was digalacturonic acid; oligogalacturonates containing either a saturated or a delta 4,5-unsaturated non-reducing end were both degraded. The enzyme was classified as an exo-D-galacturonanase [poly(1,4-alpha-D-galacturonide) galacturonohydrolase (EC 3.2.1.67)].

An exopectate lyase of Butyrivibrio fibrisolvens from the bovine rumen.

An extracellular pectinolytic enzyme produced by Butyrivibrio fibrisolvens isolated from the bovine rumen was studied. The enzyme had a pH optimum of 8.0 to 8.5 and was stimulated by Ca2+ and inhibited by EDTA. The products of pectinolysis had an absorption peak at 235 nm and reacted with thiobarbituric acid, indicating a lyase type of action. The enzyme cleaved the substrates terminally from the reducing end; action on poly- and oligogalacturonates resulted in the formation of an unsaturated trigalacturonate. The enzyme was classified as an exopectate lyase (EC 4.2.2.9). A pectinesterase was also produced by B. fibrisolvens but polygalacturonase was not detected.

A polygalacturonate lyase produced by Lachnospira multiparus isolated from the bovine rumen.

A poly(1,4-alpha-D-galacturonide) lyase (EC 4.2.2.2) from the culture fluid of Lachnospira multiparus was purified about 20-fold. The optimum pH and temperature for enzyme activity were 8.0 and 40 degrees C. The enzyme required Ca2+ and was inhibited by EDTA; it preferred polygalacturonate as substrate, cleaving 1,4-alpha-glycosidic linkages randomly to form unsaturated galacturonates, mainly the unsaturated digalacturonate. Some properties of the crude and purified enzyme preparations are described. An exopolygalacturonase is also produced by this organism.

Purification and characterization of an extracellular exo-D-galacturonanase of Aspergillus niger.

A D-galacturonanase (EC 3.2.1.67) catalyzing the degradation of D-galacturonans by terminal action pattern was purified from a culture filtrate of Aspergillus niger by a procedure including the salting-out with ammonium sulfate, precipitation by ethanol, chromatography on DEAE-cellulose, and gel chromatography on Sephadex G-100. The obtained preparation was slightly contaminated by an enzymically inactive protein fraction. Maximum activity and stability of the enzyme was observed at pH 5.2. The enzyme degrades digalacturonic acid, p-nitrophenyl-alpha-D-galactopyranuronide, as well as oligogalacturonides containing at the nonreducing end 4-deoxy-L-threo-hexa-4-enopyranosyluronate. It differs from all A. niger enzymes so far described which degrade D-galaturonans by the terminal action pattern, in not clearly preferring low-molecular substrates. It is therefore classified as an exo-D-galacturonanase.