O. S. Pilipenko

Dissociation and catalytic activity of oligomer forms of β-galactosidases

The dissociation of oligomer forms of bacterial Escherichia coli, yeast Kluyveromices fragilis, and bovine liver β-galactosidases was studied. The catalytic constants for the dimers and tetramers of the bacterial enzyme, dimers and monomers of the animal enzyme, and dimers of the yeast enzyme in the reaction of hydrolysis of 2-nitrophenyl-β-D-galactopyranoside were determined. At 25°C, these values were found to be 180 and 400 s−1 for the bacterial enzyme, 0.01 and 0.08 s−1 for the bovine liver enzyme, and 45.4 s−1 for the yeast enzyme, respectively. The other oligomer forms of the β-galactosidases were inactive under conditions of these experiments.

The catalytic properties and stability of β-galactosidases from fungi

The catalytic activity of β-galactosidases from fungi Penicillium canescens and Aspergillus oryzae is maximum in a weakly acidic medium and does not depend on the presence of magnesium cations in the reaction medium. The enzyme from Aspergillus oryzae fungi is more active, and that from Penicillium canescens is stabler. One of stability indications is the presence of an induction period in the kinetic curves of thermal inactivation. This period disappears at 54°C for the enzyme from Aspergillus oryzae and at 59°C for the enzyme from Penicillium canescens. The temperature dependences of the effective rate constants for the inactivation of the tetrameric enzyme from Penicillium canescens show that the main reason for enzyme inactivation is the dissociation of oligomeric forms below 66°C (Eact = 85 kJ/mol) and enzyme denaturation at higher temperatures (Eact = 480 kJ/mol). The dissociation stage is absent for monomeric β-galactosidase from Aspergillus oryzae fungi, and the activation energy of inactivation is 450 kJ/mol over the whole temperature range studied (53–60°C).

Mechanism of the dimerization of enzymes upon adsorption on silicate adsorbents using the example of lysozyme and β-galactosidase

It is shown by the kinetic analysis of lysozyme and β-galactosidase adsorption on silochrome, silica gel, and mesoporous silica that the adsorption follows a two-stage scheme, including reversible pre-adsorption and the irreversible binding of dimmers. The corresponding rate constants of adsorption, desorption, and dimerization were calculated. It was found that β-galoctosidase adsorbed on silica gel and mesoporous silica retained ∼20% of its activity, while β-galoctosidase adsorbed on silochrome retained more than 30% of its activity.

The Special Features of Protein Adsorption Isotherms on Silica Adsorbents

The adsorption isotherms of hemoglobin, peroxidase, and β-galactosidase on silochrome and mesoporous and biporous silicas were comparatively studied. Adsorption developed in two stages, including fast “reversible” protein adsorption (equilibrium was reached in t ≤ 1–2 h) and a “slow stage” of irreversible binding in t ≫ 24 h (multipoint adsorption). The corresponding equilibrium constants were determined. The mechanism of unlimited linear association of peroxidase in the adsorption layer on the surface of silochrome was established.

Inhibition of β-galactosidases with mono- and disaccharides

It was demonstrated that, in reactions of the hydrolysis of model substrate 2-nitrophenyl-β-D-galactopyranoside (2-NPGP) monosaccharides D-fructose and D-xylose with hydroxyl substituents oppositely directed at the neighboring carbon atoms in the furan ring, as in D-glucose, act as noncompetitive inhibitors of β-galactosidase from E. coli; for mushroom, β-galactosidases from P. canescens and A. oryzae D-galactose is a stronger inhibitor. It was also found that the inhibition constant is the highest in the case of the most active enzyme (E. coli) and is the lowest for the least active one (P. canescens).

A comparative study of the structure and properties of β-galactosidases

A comparative study of β-galactosidase amino acid sequences of E. coli and another four out of 11 microorganisms selected at the first stage was performed. It was shown that the functional amino acid residues in the catalytic domain and the ligand environment of the magnesium cation for all five sequences are identical. The mechanism of the catalytic action of E. coli and K. lactis β-galactosidases was investigated by the method of nucleophilic competition. It was shown that the mechanism of the effects of nucleophilic agents is kinetically identical both enzymes: the presence of methanol or butanediols affects the stage of degalactosylation; the presence of magnesium cations promotes the activity of both β-galactosidases; and the mechanisms of the thermal inactivation of E. coli and K. lactis β-galactosidases are different.

Adsorption of β-galactosidase on silica and aluminosilicate adsorbents

It is shown that adsorption of β-galactosidase of Aspergillus oryzae fungi on mesoporous and biporous silica and aluminosilicate adsorbents and the rate of the process grow along with the diameter of the pores of the adsorbent. It is found that the shape of the adsorption isotherms changes as well, depending on the texture of the adsorbent: the Michaelis constant rises from 0.3 mM for the enzyme in solution to 0.4–0.5 mM for the enzyme on a surface in the hydrolysis of o-nitrophenyl-β-D-galactopyranoside. It is concluded that β-galactosidase displays its maximum activity on the surface of biporous adsorbents.

Adsorption characteristics of lysozyme on silochrome at different pH values

It is established that the adsorption of lysozyme on silochrome proceeds by a two-stage scheme that includes equilibrium adsorption and irreversible multipoint binding, complicated by the association of surface protein. It is shown that, a substantial change in the mechanism of adsorption occurs in dependence on pH. The density of the monolayer increases with increasing pH in the range of 6–10. The rate constants of adsorption and sites occupied by a molecule of protein at different pH values are calculated.

Similarity of and differences between the mechanisms of thermal inactivation of β-galactosidases of different origins

A comparative study of the thermal stabilities of five β-galactosidases of different origins in buffer solutions at pH of their highest activity was performed. The thermal inactivation of these enzymes was found to occur via different mechanisms. The thermal inactivation of four β-galactosidases followed the mechanism with intermediate stages not accompanied by catalytic activity loss. The dissociative mechanism of inactivation, including the reversible dissociation of the oligomeric enzyme and the irreversible dissociation of the monomeric enzyme, was observed for bacterial (Escherichia coli) and yeast (Kluyveromices fragilis) β-galactosidases. The kinetic parameters of dissociative thermal inactivation of these enzymes and the stability parameters of β-galactosidases studied were determined. The latter included the critical temperature of changes in the kinetic regime of inactivation, the smallest number of intermediate stages without catalytic activity loss, the temperature of the disappearance of the induction period of thermal inactivation, and induction period duration at the given temperature (40°C).

Effect of magnesium cations on the activity and stability of β-galactosidases

It was shown that the presence of magnesium cations in the reaction mixture increases, approximately twofold, the activity of bacterial Escherichia coli and yeast Kluyveromyces lactis β-galactosidases but does not affect the activity of bovine liver and fungous Penicillium canescens β-galactosidases. The catalytic constants for E. coli and yeast K. lactis β-galactosidases in the presence of 0.01 M and in the absence of Mg2+ cations were determined (490 and 220 s−1 and 59.8 and 37.4 s−1, respectively). It was shown that the Michaelis constants for these two enzymes are higher in the presence of Mg2+ cations, that the thermal stability of E. coli and K. Lactis β-galactosidases is higher in the presence of 0.01 M Mg2+, and that the effective rate constants of thermal inactivation of the enzymes are two-to eightfold lower, depending on conditions, in the presence of Mg2+ cations. The maximum stabilizing effect of magnesium cations was observed at weak alkaline pH values (7.5–8.5).