Rat small-intestinal beta-galactosidases. Influence of pH on the hydrolysis of different substrates.
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ABSTRACT:
SUBMITTER: Dahlqvist A
PROVIDER: S-EPMC1270372 | biostudies-other | 1967 Apr
REPOSITORIES: biostudies-other
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Project description:1. Three fractions of beta-galactosidase activity from the rat small-intestinal mucosa were separated chromatographically. Two of these fractions had an acid pH optimum at 3-4, and the third one had a more neutral pH optimum at 5.7. 2. The two ;acid' beta-galactosidase fractions had considerably lower K(m) values for hetero beta-galactosides than for lactose. The V(max.) values were similar for all the substrates used (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside). No difference could be detected between the two ;acid' fractions with respect to their enzymic properties (pH optimum, K(m) for the different substrates, K(i) for lactose as an inhibitor of the hydrolysis of hetero beta-galactosides, K(i) for phenyl beta-galactoside as an inhibitor of the hydrolysis of lactose, and relative V(max.) for the hydrolysis of different substrates). These two fractions probably represent different forms of the same enzyme. 3. The ;neutral' fraction had similar K(m) values for all the substrates hydrolysed, but with lactose as substrate the V(max.) was much higher than with the hetero beta-galactosides. This fraction did not split phenyl beta-galactoside or 6-bromo-2-naphthyl beta-galactoside at a measurable rate. 4. Lactose was a competitive inhibitor of the hetero beta-galactosidase activities of all the three fractions, and K(i) for lactose as an inhibitor in each case was the same as K(m) for the lactase activity. Phenyl beta-galactoside was a competitive inhibitor of the lactase activity of all the three fractions. These facts strongly indicate that in all the three fractions lactose is hydrolysed by the same active sites as the hetero beta-galactosides. 5. Human serum albumin stabilized the separated enzymes against inactivation by freezing and thawing.
| S-EPMC1187119 | biostudies-other
Project description:1. The chromatography of rat small-intestinal beta-galactosidase activities on gel-filtration and ion-exchange columns has been studied. Five different substrates were used to measure beta-galactosidase activity (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside) and the activity was measured at one acid and one more neutral pH value. 2. By gel filtration one acid beta-galactosidase, hydrolysing lactose and the hetero-beta-galactosides at about the same rate, and one more neutral beta-galactosidase, hydrolysing lactose much more rapidly than the hetero-beta-galactosides, were separated. 3. By ion-exchange chromatography the acid enzyme was fractionated into two components. These may be individual enzymes or different forms of the same enzyme.
| S-EPMC1198588 | biostudies-other
Project description:1. Two beta-galactosidases from human small-intestinal mucosa were separated by gel-filtration chromatography and the properties of the two enzymes were studied. Lactose and four hetero beta-galactosides were used as substrates. 2. One of the enzymes was particle-bound and could be partially solubilized with papain. Of the substrates hydrolysed by this enzyme, lactose was hydrolysed most rapidly. This enzyme is thus essentially a disaccharidase and is named lactase. It is presumably identical with the ;lactase 1' described earlier. 3. The other enzyme was mainly soluble and hydrolysed all artificial substrates used, whereas no lactase activity could be detected. This enzyme has therefore been designated hetero beta-galactosidase. 4. p-Chloromercuribenzoate (0.1mm) inhibited the hetero beta-galactosidase completely but did not influence the activity of the lactase. Tris was a competitive inhibitor of both enzymes. 5. The residual lactase activity in the mucosa of lactose-intolerant patients may be exerted by a small amount of remaining lactase as such, or possibly by a third enzyme with a more acid pH optimum.
| S-EPMC1184862 | biostudies-other
Project description:1. Different forms of the rat small-intestinal ;acid' beta-galactosidase were separated by using the isoelectric-focusing technique. The isoelectric points of the different forms were at pH4.2, 4.6, 5.4, 6.1 and approx. 8. 2. The two forms of ;acid' beta-galactosidase isoelectric at pH4.2 and 4.6 were completely excluded from the Sephadex G-200 gel, whereas the form isoelectric at pH8 had K(av.) 0.4. The concentration and pH of the elution buffer influenced the distribution of enzyme activity between different forms. Thus, under certain conditions of ionic strength and pH, the enzyme seems to form high-molecular-weight aggregates with low isoelectric points. These may be homopolymeric aggregates or the result of binding of enzyme to, for example, membrane fragments. The forms isoelectric at pH5.4 and 6.1 are probably aggregates of intermediate size. 3. During ion-exchange chromatography at pH6.0 one fraction of ;acid' beta-galactosidase was not retained on the column and was isoelectric at pH8 and another fraction was eluted when the buffer concentration in the eluate had increased to about 50mm. The main part of enzyme eluted in this second fraction was also isoelectric at pH8, indicating that the elution of this fraction is not a simple ion-exchange procedure but probably also involves a splitting of high-molecular-weight aggregates, originally retained because of their low isoelectric points. The enzyme subunits have a higher isoelectric point, and are therefore no longer bound to the ion-exchange resin.
| S-EPMC1178870 | biostudies-other
Project description:We describe here a new enzyme-coupled assay for the quantitation of d-xylose using readily available enzymes that allows kinetic evaluation of hemicellulolytic enzymes using natural xylooligosaccharide substrates. Hydrogen peroxide is generated as an intermediary analyte, which allows flexibility in the choice of the chromophore or fluorophore used as the final reporter. Thus, we present d-xylose quantitation results for solution-phase assays performed with both the fluorescent reporter resorufin, generated from N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), whose corresponding radical cation has an absorbance maximum at approximately 400 nm. We also describe a useful solid-phase variation of the assay performed with the peroxidase substrate 3,3'-diaminobenzidine tetrahydrochloride, which produces an insoluble brown precipitate. In addition, kinetic parameters for hydrolysis of the natural substrates xylobiose and xylotriose were obtained using this assay for a glycosyl hydrolase family 39 beta-xylosidase from Thermoanaerobacterium sp. strain JW/SL YS485 (Swiss-Prot accession no. O30360). At higher xylobiose substrate concentrations the enzyme showed an increase in the rate indicative of transglycosylation, while for xylotriose marked substrate inhibition was observed. At lower xylobiose concentrations k(cat) was 2.7 +/- 0.4 s(-1), K(m) was 3.3 +/- 0.7 mM, and k(cat)/K(m) was 0.82 +/- 0.21 mM(-1) . s(-1). Nonlinear curve fitting to a substrate inhibition model showed that for xylotriose K(i) was 1.7 +/- 0.1 mM, k(cat) was 2.0 +/- 0.1 s(-1), K(m) was 0.144 +/- 0.011 mM, and k(cat)/K(m) was 14 +/- 1.3 mM(-1) . s(-1).
| S-EPMC1214693 | biostudies-literature
Project description:1. An acid beta-galactosidase, optimum pH4.0-4.5, in the human small-intestinal mucosa was separated and characterized. 2. Autolysis of mucosal homogenates at acid pH inactivated the lactase and hetero beta-galactosidase; the total activity of the acid beta-galactosidase was only slightly depleted, but a greater proportion of the enzyme was solubilized by this treatment. 3. Separation on a Sephadex G-200 column revealed that the acid beta-galactosidase could occur in at least three different forms, probably representing monomer, dimer and octamer or polymer of the enzyme. 4. The properties of the different forms of the acid beta-galactosidase were studied with regard to pH optimum, K(m), rate of hydrolysis of different substrates, and sensitivity to p-chloromercuribenzoate and tris as inhibitors. All these properties were the same for the different forms of the enzyme. 5. The acid beta-galactosidase hydrolyses lactose as well as hetero beta-galactosides and contributes to the lactase activity of intestinal biopsies also when measured at pH 6. This enzyme may therefore be responsible for a considerable part of the residual lactase activity found in lactose-intolerant patients.
| S-EPMC1176570 | biostudies-other
Project description:The ability to predict the transglycosylation activity of glycosidases by in silico analysis was investigated. The transglycosylation abilities of 7 different β-d-galactosidases from GH family 2 were tested experimentally using 7 different acceptors and p-nitrophenyl-β-d-galactopyranoside as a donor of galactosyl moiety. Similar transglycosylation abilities were confirmed for all enzymes originating from bacteria belonging to Enterobacteriaceae, which were able to use all tested acceptor molecules. Higher acceptor selectivity was observed for all others used bacterial strains. Structure models of all enzymes were constructed using homology modeling. Ligand-docking method was used for enzymes-transglycosylation products models construction and evaluation. Results obtained by in silico analysis were compared with results arisen out of experimental testing. The experiments confirmed that significant differences in transglycosylation abilities are caused by small differences in active sites composition of analyzed enzymes. According to obtained result, it is possible to conclude that homology modeling may serve as a quick starting point for detection or exclusion of enzymes with defined transglycosylation abilities, which can be used for subsequent synthesis of e.g., pharmaceutically interesting glycosides.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-021-02715-w.
| S-EPMC7960840 | biostudies-literature
Project description:Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized β-galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger. All enzymes were active toward p-nitrophenyl-β-D-galactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose β-1,4 linked to either N-acetylglucosamine or fucose, such as N-glycans derived from bovine fibrin and Caenorhabditis elegans. In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae, all enzymes also displayed various degrees of activity toward complex oligosaccharides containing β-1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications.
| S-EPMC3973953 | biostudies-literature
Project description:The techniques of metagenomics have allowed researchers to access the genomic potential of uncultivated microbes, but there remain significant barriers to determination of gene function based on DNA sequence alone. Functional metagenomics, in which DNA is cloned and expressed in surrogate hosts, can overcome these barriers, and make important contributions to the discovery of novel enzymes. In this study, a soil metagenomic library carried in an IncP cosmid was used for functional complementation for β-galactosidase activity in both Sinorhizobium meliloti (α-Proteobacteria) and Escherichia coli (γ-Proteobacteria) backgrounds. One β-galactosidase, encoded by six overlapping clones that were selected in both hosts, was identified as a member of glycoside hydrolase family 2. We could not identify ORFs obviously encoding possible β-galactosidases in 19 other sequenced clones that were only able to complement S. meliloti. Based on low sequence identity to other known glycoside hydrolases, yet not β-galactosidases, three of these ORFs were examined further. Biochemical analysis confirmed that all three encoded β-galactosidase activity. Lac36W_ORF11 and Lac161_ORF7 had conserved domains, but lacked similarities to known glycoside hydrolases. Lac161_ORF10 had neither conserved domains nor similarity to known glycoside hydrolases. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain. By discovering founding members of three novel β-galactosidase families, we have reinforced the value of functional metagenomics for isolating novel genes that could not have been predicted from DNA sequence analysis alone.
| S-EPMC5342196 | biostudies-literature
Project description:Soluble inorganic aluminium compounds like aluminium sulfate or aluminium chloride have been challenged by the European Chemical Agency to induce germ cell mutagenicity. Before conducting mutagenicity tests, the hydrolysis products in water and in physiological solutions should be determined as a function of the concentration and pH. We used different 27Al NMR spectroscopic techniques (heteronuclear Overhauser effect spectroscopy (HOESY), exchange spectroscopy (EXSY), diffusion ordered (DOSY)) in this work to gain the information to study the aluminium species in solutions with Al?(SO?)? concentrations of 50.0, 5.0, and 0.5 g/L and their pH and time dependent transformation. At low pH, three different species were present in all physiological solutions and water: [Al(OH)n(H?O)6 - n](3 - n)+ (n = 0-2), [Al(H?O)?SO?]?, and [Al?(OH)?(H?O)?]4+. Increasing pH reduced the amounts of the two monomer species, with a complete loss at pH 5 for solutions with a concentration of 50.0 g/L and at pH 4 for solutions with a concentration of 5.0 g/L. The dimer species [Al?(OH)?(H?O)?]4+ is present in a pH range between 3 and 6. Less symmetric oligomeric and probably asymmetric aluminium species are formed at pH of 5 and 6. The pH value is the driving force for the formation of aluminium species in all media, whereas the specific medium had only minor effect. No conclusive information could be obtained at pH 7 due to signal loss related to fast quadrupole relaxation of asymmetric aluminium species. A slight reduction of the content of the symmetric aluminium species due to the formation of oligomeric species was observed over a period of 6 weeks. Reference 27Al NMR experiments conducted on saturated water solutions of AlCl? and those with a concentration of 50 g/L show that the type of salt/counter ion at the same concentration and pH influences the hydrolysis products formed.
| S-EPMC6017892 | biostudies-literature