A study is performed of the adsorption of lysozyme on biporous silica, silicalite-1 (silica with the structure of MFI zeolite), ZSM-5 zeolite, and two halloysite samples (natural aluminosilicate nanotubes). Adsorption isotherms are obtained, and the limiting values of adsorption and the areas on surfaces of the adsorbent per enzyme molecule are found. The maximum adsorption per unit of adsorbent mass (350 mg/g) is obtained for biporous silica along with the one per unit of surface area (0.962 mg/m(2)) on halloysite.

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.

Показано, что величина адсорбции ?-галактозидазы грибов Aspergillus oryzae на мезопористых и бипористых кремнеземных и алюмосиликатных адсорбентах и скорость процесса увеличивается с ростом диаметра пор адсорбента. Установлено, что в зависимости от текстуры адсорбента меняется также и вид изотерм адсорбции; константа Михаэлиса в реакции гидролиза о-нитрофенил-?-D-галактопиранозида возрастает от 0.3 мМ для фермента в растворе до 0.4–0.5 мМ для фермента на поверхности. Сделан вывод, что максимальной активностью обладает ?-галактозидаза на поверхности бипористых адсорбентов.

Установлено, что адсорбция лизоцима на силохроме протекает по двухстадийной схеме, включающей равновесную адсорбцию и необратимое многоточечное связывание, осложненное ассоциацией белка на поверхности. Показано, что в зависимости от рН происходит существенное изменение механизма адсорбции. Плотность монослоя увеличивается с ростом рН в интервале 6–10. Вычислены константы скорости адсорбции и площадки, занимаемые молекулой белка при различных значениях рН.

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.

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.

Проведено сравнительное исследование изотерм адсорбции гемоглобина, пероксидазы и -галактозидазы на силохроме, мезопористом и бипористом кремнеземах. Установлена двухстадийность адсорбции – “быстрая” обратимая адсорбция белка (равновесие достигается при t 1–2 ч) и “медленная стадия” – необратимое связывание при t 24 ч (многоточечная адсорбция). Определены соответствующие константы равновесия. Установлен механизм неограниченной линейной ассоциации пероксидазы в адсорбционном слое на поверхности силохрома.

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.

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 ).

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 ( E act = 85 kJ/mol) and enzyme denaturation at higher temperatures ( E act = 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).

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).

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.

Показано, что присутствие катионов магния в реакционной смеси увеличивает приблизительно вдвое активность -галактозидаз из бактерий Escherichia coli и дрожжей Kluyveromyces lactis и не влияет на активность -галактозидаз из печени быка и грибов Penicillium сanescens. Определены каталитические константы для фермента из E. coli (490 и 220 с-1 в присутствии 0.01 М и в отсутствие Mg2+ соответственно), для -галактозидазы из дрожжей K. lactis (59.8 и 37.4 с-1). Показано, что значения констант Михаэлиса для этих ферментов в присутствии катионов Mg2+ несколько выше, чем в их отсутствие; в присутствии 0.01 М Mg2+ увеличивается термостабильность -галактозидаз из E. coli и K. Lactis; эффективные константы скорости их термоинактивации в присутствии катионов Mg2+ в зависимости от условий уменьшаются в 2–8 раз. Наиболее сильно стабилизирующее действие катионов магния выявлено при слабощелочных значениях рН (7.5–8.5).

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 Mg 2+ 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 Mg 2+ cations, that the thermal stability of E. coli and K. Lactis β-galactosidases is higher in the presence of 0.01 M Mg 2+ , and that the effective rate constants of thermal inactivation of the enzymes are two-to eightfold lower, depending on conditions, in the presence of Mg 2+ cations. The maximum stabilizing effect of magnesium cations was observed at weak alkaline pH values (7.5–8.5).

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.

An acid-base mechanism for glycoside bond hydrolysis by P-galactosidase including the formation of two intermediate enzyme-substrate complexes ES1 and ES2 with two bond redistribution chains is suggested. A kinetic analysis was performed using a three-step kinetic scheme including the formation of glycosylated enzyme (E-Gal). This allowed us to calculate the elementary kinetic constants k(2) k(3), and K-S for the reaction with o-nitrophenyl-beta-D-galactopyranoside as a model substrate.