Inhibition of glycosidases by aldonolactones of corresponding configuration. The C-4- and C-6-specificity of beta-glucosidase and beta-galactosidase.
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ABSTRACT:
SUBMITTER: Conchie J
PROVIDER: S-EPMC1270457 | biostudies-other | 1967 Jun
REPOSITORIES: biostudies-other
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Project description:1. The previous study (Conchie, Gelman & Levvy, 1967b) of the specificity of beta-glucosidase, beta-galactosidase and beta-d-fucosidase in barley, limpet, almond emulsin and rat epididymis was extended to alpha-l-arabinosidase. 2. The inhibitory action of l-arabinono-(1-->5)-lactone was tested against all four types of enzyme, and alpha-l-arabinosidase was examined for inhibition by glucono-, galactono- and d-fucono-lactone. 3. In emulsin, the enzyme that hydrolyses beta-glucosides, beta-galactosides and beta-d-fucosides also hydrolyses alpha-l-arabinosides. Rat epididymis resembles emulsin except that, as already noted, it lacks beta-glucosidase activity. 4. In the limpet, alpha-l-arabinosidase activity is associated with the enzyme that hydrolyses beta-glucosides and beta-d-fucosides, and not with the separate beta-galactosidase. 5. The effects of the different lactones on the barley preparation suggest that alpha-l-arabinosidase activity is associated with the beta-galactosidase rather than with the enzyme that hydrolyses beta-glucosides and beta-d-fucosides. Fractionation and heat-inactivation experiments indicate that there is also a separate alpha-l-arabinosidase in the preparation.
| S-EPMC1198478 | biostudies-other
Project description:Stevia rebaudiana Bertoni is a plant cultivated worldwide due to its use as a sweetener. The sweet taste of stevia is attributed to its numerous steviol glycosides, however, their use is still limited, due to their bitter aftertaste. The transglycosylation of steviol glycosides, aiming at the improvement of their taste, has been reported for many enzymes, however, glycosyl hydrolases are not extensively studied in this respect. In the present study, a β-glucosidase, MtBgl3a, and a β-galactosidase, TtbGal1, have been applied in the transglycosylation of two steviol glycosides, stevioside and rebaudioside A. The maximum conversion yields were 34.6 and 33.1% for stevioside, while 25.6 and 37.6% were obtained for rebaudioside A conversion by MtBgl3a and TtbGal1, respectively. Low-cost industrial byproducts were employed as sugar donors, such as cellulose hydrolyzate and acid whey for TtbGal1- and MtBgl3a- mediated bioconversion, respectively. LC-HRMS analysis identified the formation of mono- and di- glycosylated products from stevioside and rebaudioside A. Overall, the results of the present work indicate that both biocatalysts can be exploited for the design of a cost-effective process for the modification of steviol glycosides.
| S-EPMC8220073 | biostudies-literature
Project description:1. A method was devised for the preparation of (1-->5)-lactones from pyranose sugars and uronic acids by platinum-catalysed oxidation with gaseous oxygen in aqueous solution at acid pH. It was applied to mannose, N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, galacturonic acid, galactose, l-arabinose and d-fucose. 2. Only the first three yielded products that could be obtained in the solid state without decomposition. In every case, however, the oxidation product in aqueous solution behaved as the aldono-(1-->5)-lactone, and was more inhibitory towards the appropriate glycosidases than any aldonolactone preparation hitherto examined. 3. The stabilities of the oxidation products were studied, and their interconversion with the (1-->4)-lactones was demonstrated. Ring-opening does not appear to be mandatory for this isomeric change, which in some instances is very rapid. 4. To explain all the inhibitory effects observed with aldonolactones on glycosidases of corresponding configuration, it is tentatively postulated that inhibition may be due entirely to the (1-->5)-lactone, and that any inhibitory effect seen with the (1-->4)-lactone is a measure of the extent and speed of its conversion into the (1-->5)-lactone in aqueous solution.
| S-EPMC1270347 | biostudies-other
Project description:An alpha-D-galactosidase (EC 3.2.1.22) and a beta-D-mannanase (EC 3.2.1.78), which were secreted into the growth medium when Aspergillus tamarii was cultivated in the presence of galactomannan, were purified by a procedure including chromatography on hydroxyapatite and DEAE-cellulose columns. Each of these enzymes showed a single protein band, corresponding to their respective activities, on polyacrylamide-gel electrophoresis. Both enzymes were shown to be glycoproteins containing N-acetylglucosamine, mannose and galactose, with molar proportions of 1:6:1.5 for alpha-D-galactosidase and 1:13:8 for beta-D-mannanase. Mr values as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and by the electrophoretic method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164] were 56000 and 53000 respectively. The alpha-D-galactosidase differed markedly from the mycelial forms I and II studied in the preceding paper [Civas, Eberhard, Le Dizet & Petek (1984) Biochem. J. 219, 849-855] with regard to both its kinetic and structural properties.
| S-EPMC1153554 | biostudies-other
Project description:Alpha-galactosidases catalyze the hydrolysis of terminal alpha-1,6-galactosyl units from galacto-oligosaccharides and polymeric galactomannans. The crystal structures of tetrameric Saccharomyces cerevisiae alpha-galactosidase and its complexes with the substrates melibiose and raffinose have been determined to 1.95, 2.40, and 2.70 A resolution. The monomer folds into a catalytic (alpha/beta)(8) barrel and a C-terminal beta-sandwich domain with unassigned function. This pattern is conserved with other family 27 glycosidases, but this enzyme presents a unique 45-residue insertion in the beta-sandwich domain that folds over the barrel protecting it from the solvent and likely explaining its high stability. The structure of the complexes and the mutational analysis show that oligomerization is a key factor in substrate binding, as the substrates are located in a deep cavity making direct interactions with the adjacent subunit. Furthermore, docking analysis suggests that the supplementary domain could be involved in binding sugar units distal from the scissile bond, therefore ascribing a role in fine-tuning substrate specificity to this domain. It may also have a role in promoting association with the polymeric substrate because of the ordered arrangement that the four domains present in one face of the tetramer. Our analysis extends to other family 27 glycosidases, where some traits regarding specificity and oligomerization can be formulated on the basis of their sequence and the structures available. These results improve our knowledge on the activity of this important family of enzymes and give a deeper insight into the structural features that rule modularity and protein-carbohydrate interactions.
| S-EPMC2934667 | biostudies-literature
Project description:BackgroundCold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds.ResultsA bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and β-linked oligosaccharides, and had remarkably high β-galactosidase, β-glucosidase and β-fucosidase activities. Because of the lack of information about the usefulness of β-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for β-glucosidase or β-galactosidase. The BglMKg had maximal β-galactosidase and β-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for β-galactosidase activity was 6.5, whereas the optimum pH for β-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed β-glucosidase substrates than β-galactosidase ones.ConclusionsBglMKg is a small, monomeric, cold-active β-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.
| S-EPMC3605331 | biostudies-literature
Project description:Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. Based upon the X-ray structure of Zea mays beta-glucosidase, we generated a three-dimensional model of hCBG by homology modelling. The enzyme exhibited the (beta/alpha)(8)-barrel fold characteristic of family 1 beta-glucosidases, with structural differences being confined mainly to loop regions. Based on the substrate specificity of the human enzymes, sequence alignment of family 1 enzymes and analysis of the hCBG structural model, we selected and mutated putative substrate (aglycone) binding site residues. Four single mutants (Val(168)-->Tyr, Phe(225)-->Ser, Tyr(308)-->Ala and Tyr(308)-->Phe) were expressed in Pichia pastoris, purified and characterized. All mutant proteins showed a decrease in activity towards a broad range of substrates. The Val(168)-->Tyr mutation did not affect K (m) on p -nitrophenyl ( p NP)-glycosides, but increased K (m) 5-fold on flavonoid glucosides, providing the first biochemical evidence supporting a role for this residue in aglycone-binding of the substrate, a finding consistent with our three-dimensional model. The Phe(225)-->Ser and Tyr(308)-->Ala mutations, and, to a lesser degree, the Tyr(308)-->Phe mutation, resulted in a drastic decrease in specific activities towards all substrates tested, indicating an important role of those residues in catalysis. Taken together with the three-dimensional model, these mutation studies identified the amino-acid residues in the aglycone-binding subsite of hCBG that are essential for flavonoid glucoside binding and catalysis.
| S-EPMC1223474 | biostudies-other
Project description:beta-D-Galactosidase and beta-D-glucosidase activities were determined in homogenates of marmoset kidney by using the appropriate 4-methylumbelliferyl glycoside, beta-D-Galactosidase activity was separated into two main components by ion-exchange chromatography on DEAE-cellulose, starch-gel electrophoresis, isoelectric focusing and gel filtration on Sephadex G-200. One form designated A had a pI of 5.1, was loosely bound to DEAE-cellulose at pH7.0, remained near the origin on starch-gel electrophoresis at pH 7.0 and had an apparent molecular weight of 160000. The second beta-D-galactosidase component, designated B, was associated with the total beta-D-glucosidase activity, had a pI of 4.3, was firmly bound to DEAE-cellulose, migrated rapidly towards the anode on starch-gel electrophoresis and had an apparent molecular weight of 50000. The optimum pH values of beta-D-galactosidase A and B were 4.5 and 6.0 respectively. beta-D-Galactosidase A was activated by 0.1 M-NaC1 but the activity of the B form was inhibited by 1 M-NaC1 at pH 4.5. beta-D-galactosidase had a bimodal distribution, the A form being recovered in the lysosomal fraction whereas the B form was present in the soluble fraction, as was the major portion of the beta-D-glucosidase activity. The lysosomal and soluble forms were further characterized by DEAE-cellulose chromatography.
| S-EPMC1183725 | biostudies-other
Project description:1. The activities of beta-galactosidase, beta-glucosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7.0 indicated slow- and fast-moving components of rat-kidney beta-galactosidase. 3. The fast-moving component is also associated with the total beta-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the beta-glucosidase component is a small acidic molecule, of molecular weight approx. 40000-50000, with optimum pH5.5-6.0 for beta-galactosidase and beta-glucosidase activities. 5. The major beta-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3.7.
| S-EPMC1270276 | biostudies-other
Project description:In an effort to examine similarities in the active sites of glycosidases within the GH35 family, we performed a structure-activity-relationship study using our recently described library of galactonoamidines. The kinetic evaluation based on UV/Vis spectroscopy disclosed inhibition of β-galactosidase (bovine liver) in the picomolar concentration range indicating significantly higher inhibitor affinity than previously determined for β-galactosidase (A. oryzae). Possible alterations in the secondary protein structure or folding were excluded after further examination of the inhibitor binding using CD spectroscopy. Molecular dynamics studies suggested loop closing interactions as a rationale for the disparity of the active sites in the β-galactosidases under investigation.
| S-EPMC5632213 | biostudies-literature