A comparison of the kinetic properties of free and immobilized Aspergillus oryzae β-galactosidase

The objective of this work was to compare the properties of free and immobilized β-galactosidase (Aspergillus oryzae), entrapped in alginate–gelatin beads and cross-linked with glutaraldehyde. The free and immobilized forms of the enzyme showed no decrease in enzyme activity when incubated in buffer solutions in pH ranges of 4.5–7.0. The kinetics of lactose hydrolysis by the free and immobilized enzymes were studied at maximum substrate concentrations of 90 g/L and 140 g/L, respectively, a temperature of 35 °C and a pH of 4.5. The Michaelis–Menten model with competitive inhibition by galactose fit the experimental results for both forms. The K_m and V_m values of the free enzyme were 52.13 ± 2.8 mM and 2.56 ± 0.3 g_glucose/L min mg_enzyme, respectively, and were 60.30 ± 3.3 mM and 1032.07 ± 51.6 g_lactose/min m³_catalyst, respectively, for the immobilized form. The maximum enzymatic activity of the soluble form of β-galactosidase was obtained at pH 4.5 and 55 °C. Alternatively, the immobilized form was most active at pH 5.0 at 60 °C. The free and immobilized enzymes presented activation energies of 6.90 ± 0.5 kcal/mol and 7.7 ± 0.7 kcal/mol, respectively, which suggested that the immobilized enzyme possessed a lower resistance to substrate transfer.     

Effects of galactose and glucose on the hydrolysis reaction of a thermostable β-galactosidase from Caldicellulosiruptor saccharolyticus

A recombinant β-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg^?1 by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80ºC, and the half-life at 70ºC was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-β-d-galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-β-d-glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of β-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 β-galactosidases. The results indicated that the enzyme was a β-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among β-galactosidases. When 50 g l^?1 galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l^?1 glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l^?1), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l^?1) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min.     

Mycelia-associated β-galactosidase activity in microbial pellets of Aspergillus and Penicillium strains

Pellet formation and production of mycelia-associated -galactosidase were investigated in 15 Aspergillus and Penicillium strains. Mycelia-associated enzyme activity was measured in sonicated homogenates. The properties of the mycelia-associated -galactosidase of A. phoenicis QM 329 was investigated. The pH optimum of the mycelia-associated enzyme was 4.0. The optimum temperature under assay conditions was 70°C and the optimum temperature for repeated lactose hydrolysis was 60°C. Repeated batch hydrolysis of lactose was made with pellets from five Aspergillus strains. A. phoenicis QM 329 showed the least enzyme leakage from the pellets during hydrolysis. From repeated lactose hydrolysis experiments it was estimated that 50% of the mycelia-associated -galactosidase activity remained after 1300 h. Correspondence to: F. Tjerneld     

Synthesis of propyl-β-d-galactoside with free and immobilized β-galactosidase from Aspergillus oryzae

Synthesis of propyl-β-galactoside catalyzed by Aspergillus oryzae β-galactosidase in soluble form was optimized using response surface methodology (RSM). Temperature and 1-propanol concentration were selected as explanatory variables; yield and productivity were chosen as response variables. Optimal reaction conditions were determined by weighing the responses through a desirability function. Then, synthesis of propyl-β-galactoside was evaluated at the optimal condition previously determined, with immobilized β-galactosidase in glyoxyl-agarose and amino-glyoxyl-agarose, and with cross-linked aggregates (CLAGs). Yields of propyl-β-galactoside obtained with CLAGs, amino-glyoxyl-agarose and glyoxyl-agarose enzyme derivatives were 0.75, 0.81 and 0.87 mol/mol and volumetric productivities were 5.2, 5.6 and 5.9 mM/h, respectively, being significantly higher than the corresponding values obtained with the soluble enzyme: 0.47 mol/mol and 4.4 mM/h. As reaction yield was increased twofold with the glyoxyl-agarose derivative, this catalyst was chosen for evaluating the synthesis of propyl-β-galactoside in repeated batch operations. Then, after ten sequential batches, the efficiency of catalyst use was 115% higher than obtained with the free enzyme. Enzyme immobilization also favored product recovery, allowing catalyst reuse, and avoiding browning reactions. Propyl-β-galactoside was recovery by extraction in 90%v/v acetone with a purity higher than 99% and its synthesis was confirmed by mass spectrometry.     

Effect of pH on transfructosylation and hydrolysis by β-fructofuranosidase from Aspergillus oryzae

 β-Fructofuranosidase was purified from commercial alkaline protease (Aspergillus oryzae origin). The optimal pH of its transfructosylating activity was more alkaline (pH 8) than that of its hydrolyzing activity (pH 5). In the case of a 24-h reaction with sucrose, the hydrolysis and transfructosylation reaction were optimal at pH 4–5 and pH 8, respectively. In the reaction at pH 8 1-kestose and nystose were the main fructooligosaccharides produced. The transfer ratio was hardly different between pH 5 and pH 8 early in the reaction, but the transfer products (1-kestose and nystose) were decreased at pH 5 as the reaction proceeded because of their hydrolysis. Received: 18 January 1995/Received last revision: 23 August 1995/Accepted: 13 September 1995     

Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula

We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°_20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K _m=0.75 mm and molecular activity (k _cat)= 63.1 s^–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K _m=0.04 mm k _cat= 55.8 s^–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl₂) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl₂). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose. Correspondence to: T. Nakayama     

The effect of some ions on the activity of β-galactosidase and the inhibitory effect of tris (hydroxymethyl) aminomethane

The stimulating effect of phosphate and the inhibitory effect of tris-HCl on the activity of β-galactosidase inEscherichia coli was studied. The phosphate anion antagonizes the inhibitory effect of chloride. Since a similar effect is displayed by sulphate and arsenate no specific “stimulating” effect of phosphate can take place. The tris cation has also an inhibitory effect which is antagonized by univalent cations (K⁺). The resulting β-galactosidase activity reflects the antagonisms between cations and anions present in the reaction medium.     

Purification and characterization of a recombinant β-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96

A beta-galactosidase isoenzyme, beta-Gall, from Bifidobacterium infantis HL96, was expressed in Escherichia coli and purified to homogeneity. The molecular mass of the beta-Gall subunit was estimated to be 115 kDa by SDS-PAGE. The enzyme appeared to be a tetramer, with a molecular weight of about 470 kDa by native PAGE. The optimum temperature and pH for o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose were 60 degrees C, pH 7.5, and 50 degrees C, pH 7.5, respectively. The enzyme was stable over a pH range of 5.0-8.5, and remained active for more than 80 min at pH 7.0, 50 degrees C. The enzyme activity was significantly increased by reducing agents. Maximum activity required the presence of both Na+ and K+, at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, divalent metal cations, and Cr3+, and to a lesser extent by EDTA and urea. The hydrolytic activity using lactose as a substrate was significantly inhibited by galactose. The Km, and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. beta-Gall possesses strong transgalactosylation activity. The production rate of galactooligosaccharides from 20% lactose at 30 and 60 degrees C was 120 mg/ml, and this rate increased to 190 mg/ml when 30% lactose was used.     

Biocatalytic characterization of Aspergillus oryzae β-galactosidase immobilized on functionalized multi-walled carbon nanotubes

The objectives of this work were to immobilize commercial Aspergillus oryzae β-galactosidase on functionalized multi-walled carbon nanotubes (MWCNTs) using different treatments and to characterize the products. Treatments were performed with glutaraldehyde, ethylenediamine and a mixture of concentrated H₂SO₄:HNO₃. The MWCNTs and their derivatives were characterized by thermogravimetric analysis. The immobilized enzymes were evaluated using inactivation kinetics, operating conditions, that is pH and temperature, kinetic parameters and lactose hydrolysis reusability. Immobilization yield and efficiency were significantly higher for β-galactosidase immobilized on MWCNTs functionalized by the acid mixture (Ac-Gal-MWCNTs). These values were 97% and 82%, respectively, after 3?h of immobilization. The activity of the Ac-Gal-MWCNTs was maintained at ～51% of their initial activity after being stored for 90 days at 4?°C. The Ac-Gal-MWCNTs retained more than 90% of their initial activity up to the fourth recycle. As the acid functionalization was the most efficient method tested for immobilizing A. oryzae β-galactosidase on MWCNTs, this method shows promise for industrial applications.     

Synthesis of galacto-oligosaccharides by β-galactosidase from Aspergillus oryzae using partially dissolved and supersaturated solution of lactose

The effect of enzyme to substrate ratio, initial lactose concentration and temperature has been studied for the kinetically controlled reaction of lactose transgalactosylation with Aspergillus oryzae β-galactosidase, to produce prebiotic galacto-oligosaccharides (GOS). Enzyme to substrate ratio had no significant effect on maximum yield and specific productivity. Galacto-oligosaccharide syntheses at very high lactose concentrations (40, 50 and 60%, w/w, lactose monohydrate) were evaluated at different temperatures (40, 47.5 and 55°C). Within these ranges, lactose could be found as a supersaturated solution or a heterogeneous system with precipitated lactose, resulting in significant effect on GOS synthesis. An increase in initial lactose concentration produced a slight increase in maximum yield as long as lactose remained dissolved. Increase in temperature produced a slight decrease in maximum yield and an increase in specific productivity when supersaturation of lactose occurred during reaction. Highest yield of 29 g GOS/100 g lactose added was obtained at a lactose monohydrate initial concentration of 50% (w/w) and 47.5°C. Highest specific productivity of 0.38 g GOSh(-1) mg enzyme(-1) was obtained at lactose monohydrate initial concentration of 40% (w/w) and 55°C, where a maximum yield of 27 g GOS/100 g lactose added was reached. This reflects the complex interplay between temperature and initial lactose concentration on the reaction of synthesis. When lactose precipitation occurred, values of yields and specific productivities lower than 22 g GOS/100 g lactose added and 0.03 gGOSh(-1) mg enzyme(-1) were obtained, respectively.     

Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

Stabilization effect of polyvinyl alcohol on horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase

Summary The stabilization effect of different molecular weights of polyvinyl alcohol (PVA) upon solutions of commonly used analytical enzymes was studied. It was found that various PVA conditions could stabilize the activity of horseradish peroxidase and glucose oxidase but not -galactosidase or alkaline phosphatase. It is expected that PVA can stabilize conjugate forms of glucose oxidase as well as horseradish peroxidase (as previously shown) but not the conjugate forms of -galactosidase or alkaline phosphatase.     

Structural and conformational changes of β-lactoglobulin B: an infrared spectroscopic study of the effect of pH and temperature

Infrared spectra of 2.5 mM solutions of β-lactoglobulin B were recorded as a function of pH (from pH 2 to pH 13) and as a function of temperature (from −100°C to +90°C). An analysis of the pH- and temperature-induced changes in the secondary structures was performed based on changes in the conformation-sensitive amide I bands of β-lactoglobulin. Whereas the total of β-structure remains constant (56–59%) between pH22 and pH 10, the proportions of the various β-components do change. In particular, the dimerization of the monomeric protein, induced by raising the pH from 20 to 3, leads to an increase in the intensity of the 1636 cm⁻¹ band (associated with antiparallel β-sheet), at the expense of the 1626 cm⁻¹ band (associated with exposed β-strands). Both the thermal and alkaline denaturation of β-lactoglobulin occur in two distinct stages. Although the spectra (i.e., the structures) after complete thermal or alkaline denaturation are clearly different, the spectrum of the protein after the first stage of thermal denaturation (at about 60°C) is the same as that after the first stage of alkaline denaturation (at pH 11), suggesting a common denaturation intermediate, which probably represents a crossover point in a complex potential hypersurface.     

Screening of β-fructofuranosidase-producing microorganisms and effect of pH and temperature on enzymatic rate

Seventeen different strains of filamentous fungi were grown in batch cultures to compare their abilities for the production of β-fructofuranosidase. Three of them, Aspergillus oryzae IPT-301, Aspergillus niger ATCC 20611 and strain IPT-615, showed high production with total fructosyltransferase activity higher than 12,500 units l⁻¹. In addition, the β-fructofuranosidases of those strains have a high fructosyltransferase activity-to-hydrolytic activity ratio. The temperature and pH effects on the sucrose-β-fructofuranosidase reaction rate were studied using a 2² factorial experimental design. The comparative analysis of the tested variable coefficients shows that the variable pH contributes mostly to the changes in the fructosyltransferase and hydrolytic rates and in the V _t/V _h ratio. At 40 and 50°C, there were no significant differences between the fructosyltransferase and hydrolytic velocities of these enzymes.     

The effect of glucose on cellobiose uptake and β-D-glucosidase activity inStreptomyces granaticolor

Glucose inhibits the inducible synthesis of β-D-glucosidase inStreptomyces granaticolor. Neither cAMP nor cGMP influence the inhibitory effect of glucose. Glucose also inhibits the inducible synthesis of the cellobiose uptake system but has no effect on its activity. This may be the mechanism underlying glucose inhibition of induction of β-D-glucosidase inS.granaticolor.     

Enzymatic synthesis of 3-aminopropyl-1-O-β-D-galactopyranoside catalyzed by Aspergillus oryzae β-galactosidase

Glycosidases represent excellent green chemistry alternatives as catalysts for the synthesis of glycosides, and in particular their stereoselectivity allows the production of anomerically pure glycosides, in only one reaction step using mild reaction conditions. Here, we report the enzymatic synthesis and structural characterization of 3-aminopropyl-1-O-β-D-galactopyranoside. Optimal reaction conditions for the transgalactosylation reaction were 100?mM lactose, 500?mM 3-amino-1-propanol and 24 h of incubation at 50?°C with 6 U/mL of β-galactosidase from Aspergillus oryzae. The fact that the synthesis of 1-propyl-2-O-β-D-galactopyranoside using 1-amino-2-propanol as acceptor was not achieved, and that N-glycoside formation was not observed, confirms the selectivity of β-galactosidase for the synthesis of O-glycosides, and particularly for primary alcohols. The synthesized galactosides were evaluated for their ability to interact with bovine spleen galectin-1 (Gal-1) by using the hemagglutination inhibition assay; results demonstrated that 3-aminopropyl-1-O-β-D-galactopyranoside may be considered as a functionalized galactose moiety more than an efficient Gal-1 inhibitor. The proposed approach constitutes a promising tool for the generation of glycopolymers and glyconanoparticles with potential applications in the development of biosensors as well as construction blocks in chemical synthesis.     

Purification of α-galactosidase and invertase by three-phase partitioning from crude extract of Aspergillus oryzae

alpha-Galactosidase and invertase were accumulated in a coherent middle phase in a three-phase partitioning system under different conditions (ammonium sulphate, ratio of tert-butanol to crude extract, temperature and pH). alpha-Galactosidase and invertase were purified 15- and 12-fold with 50 and 54% activity recovery, respectively. The fractions of interfacial precipitate arising from the three-phase partitioning were analyzed by SDS-PAGE. Both purified preparations showed electrophoretic homogeneity on SDS-PAGE.     

Reaction pH and protein affect the oxidation products of β-pentagalloyl glucose

To better understand the biochemical consequences when polymeric polyphenols serve as biological antioxidants, we studied how reaction pH (pH 2.1–7.4) and protein affected the oxidation of pentagalloyl glucose (PGG) by NaIO₄ in aqueous solution. PGG oxidation produced an o-semiquinone radical intermediate, which tended to form polymeric products at pH values below 5, and o-quinones at higher pH. The model protein bovine serum albumin promoted the formation of quinone even at low pH. Two other polyphenols, procyanidin (epicatechin₁₆-(4→8)-catechin) and epigallocatechin gallate, had similar pH-dependent oxidation patterns.     

Factors influencing synthesis and activity of β-galactosidase inLactobacillus acidophilus

Summary In the type-strainLactobacillus acidophilus ATCC 4356 -galactosidase (-gal) was inducible; lactose, galactose, melibiose and probably maltose, but not glucose, fructose, mannose, sucrose and cellobiose, induced -gal synthesis. Glucose partially inhibited -gal-induction by lactose but not by isopropyl--D-thiogalactoside. -gal synthesis during cell growth was maximal at 0.4% lactose, stimulated by Ca²⁺ but inhibited by Mg²⁺ and Mn²⁺. -gal in the cell-free extract had optimum activity at pH 6.5 and at 45°C. The enzyme activity was stimulated by Mg²⁺, inhibited by Ca²⁺, destroyed by oxidizing agents and protected by reducing agents.