Glycosidases induced in Aspergillus tamarii. Mycelial alpha-D-galactosidases.
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ABSTRACT:
SUBMITTER: Civas A
PROVIDER: S-EPMC1153553 | biostudies-other | 1984 May
REPOSITORIES: biostudies-other
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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:A Thermus thermophilus selector strain for production of thermostable and thermoactive alpha-galactosidase was constructed. For this purpose, the native alpha-galactosidase gene (agaT) of T. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis of agaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (alpha-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal(+) phenotype was assumed to be essential for growth on melibiose. In a Gal(-) background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant alpha-galactosidase. Moreover, the AgaT(-) strain had to be Km(s) for establishment of the plasmids containing alpha-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT(-) Gal(+) Km(s)) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous alpha-galactosidase in T. thermophilus, the isogenes agaA and agaB of Bacillus stearothermophilus KVE36 were cloned into an Escherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.
| S-EPMC93147 | 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:Six putative α-galactosidase genes (α-Gals), three acid forms (CsGAL1, CsGAL2, CsGAL3) and three alkaline forms (CsAGA1, CsAGA2, CsAGAL3), were found in the cucumber genome. It is interesting to know the expression pattern and possible function of these α-Gals in the cucumber plant since it is a stachyose-translocating species. In this study, full-length cDNAs of six α-Gals were cloned and heterologously expressed. The result showed that all recombinant proteins revealed acid or alkaline α-Gal activities with different substrate specificities and pH or temperature responding curves, indicating their distinct roles in cucumber plants. Phylogenetic analysis of collected α-Gal amino acid sequences from different plants indicated that the ancestor of both acid and alkaline α-Gals existed before monocots and dicots separated. Generally, six α-Gal genes are universally expressed in different cucumber organs. CsGAL2 highly expressed in fasting-growing leaves, fruits and germinating seeds; CsGAL3 mainly distributes in vacuoles and significantly expressed in cucumber fruits, senescent leaves and seeds during late stage germination; The expression of CsAGA1 increased from leaf 1 to leaf 3 (sink leaves) and then declined from leaf 4 to leaf 7 (source leaves), and this isoform also highly expressed in male flowers and germinating seeds at early stage; CsAGA2 significantly expressed in cucumber leaves and female flowers; CsAGA3 is localized in plastids and also actively expressed in senescent leaves and germinating seeds; The role of CsGAL1 in cucumber plants is now unclear since its expression was relatively low in all organs. According to their expression patterns, subcellular localizations and previously reported functions of these isoforms in other plants, combining the data of soluble sugars contents in different tissues, the possible functions of these α-Gals were discussed.
| S-EPMC7802950 | biostudies-literature
Project description:In search of alpha-galactosidases with improved kinetic properties for removal of the immunodominant alpha1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of alpha-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454-464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galalpha1-3(Fucalpha1-2)Gal, whereas linear oligosaccharides terminated by alpha1,3-linked galactose such as the immunodominant xenotransplantation epitope Galalpha1-3Galbeta1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific alpha1,3-galactosidases that act equally well on both branched blood group B and linear alpha1,3Gal structures. We determined by one-dimensional (1)H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known alpha-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant alpha3Gal xenotransplantation epitope.
| S-EPMC2417185 | biostudies-literature
Project description:alpha-Galactosidases from thermophilic organisms have gained interest owing to their applications in the sugar industry. The alpha-galactosidases AgaA, AgaB and AgaA A355E mutant from Geobacillus stearothermophilus have been overexpressed in Escherichia coli. Crystals of AgaB and AgaA A355E have been obtained by the vapour-diffusion method and synchrotron data have been collected to 2.0 and 2.8 A resolution, respectively. Crystals of AgaB belong to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 113.3, c = 161.6 A. Crystals of AgaA A355E belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 150.1, c = 233.2 A.
| S-EPMC2150962 | biostudies-literature
Project description:The gut microbiota offers an extensive resource of enzymes, but many remain uncharacterized. To distinguish the activities of similar annotated proteins and mine the potentially applicable ones in the microbiome, we applied an effective Activity-Based Metaproteomics (ABMP) strategy using a specific activity-based probe (ABP) to screen the entire gut microbiome for directly discovering active enzymes and their potential applications, not for exploring host-microbiome interactions. By using an activity-based cyclophellitol aziridine probe specific to α-galactosidases (AGAL), we successfully identified and characterized several gut microbiota enzymes possessing AGAL activities. Cryo-electron microscopy analysis of a newly characterized enzyme (AGLA5) revealed the covalent binding conformations between the AGAL5 active site and the cyclophellitol aziridine ABP, which could provide insights into the enzyme's catalytic mechanism. The four newly characterized AGALs have diverse potential activities, including raffinose family oligosaccharides (RFOs) hydrolysis and enzymatic blood group transformation. Collectively, we present a ABMP platform that facilitates gut microbiota AGALs discovery, biochemical activity annotations and potential industrial or biopharmaceutical applications.
| S-EPMC11329505 | biostudies-literature
Project description:The α-galactosidase AgaA from the thermophilic microorganism Geobacillus stearothermophilus has great industrial potential because it is fully active at 338 K against raffinose and can increase the yield of manufactured sucrose. AgaB has lower affinity for its natural substrates but is a powerful tool for the enzymatic synthesis of disaccharides by transglycosylation. These two enzymes have 97% identity and belong to the glycoside hydrolase (GH) family GH36 for which few structures are available. To understand the structural basis underlying the differences between these two enzymes, we determined the crystal structures of AgaA and AgaB by molecular replacement at 3.2- and 1.8 Å-resolution, respectively. We also solved a 2.8-Å structure of the AgaA(A355E) mutant, which has enzymatic properties similar to those of AgaB. We observe that residue 355 is located 20 Å away from the active site and that the A355E substitution causes structural rearrangements resulting in a significant displacement of the invariant Trp(336) at catalytic subsite -1. Hence, the active cleft of AgaA is narrowed in comparison with AgaB, and AgaA is more efficient than AgaB against its natural substrates. The structure of AgaA(A355E) complexed with 1-deoxygalactonojirimycin reveals an induced fit movement; there is a rupture of the electrostatic interaction between Glu(355) and Asn(335) and a return of Trp(336) to an optimal position for ligand stacking. The structures of two catalytic mutants of AgaA(A355E) complexed with raffinose and stachyose show that the binding interactions are stronger at subsite -1 to enable the binding of various α-galactosides.
| S-EPMC3501081 | biostudies-literature
Project description:Two forms of alpha-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2.8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. alpha-Galactosidases I and II showed different pH optima and K(m) and V(max.) values with p-nitrophenyl alpha-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.
| S-EPMC1184603 | biostudies-other
Project description:The effect of monanchomycalin B, monanhocicidin A, and normonanhocidin A isolated from the Northwest Pacific sample of the sponge Monanchora pulchra was investigated on the activity of α-galactosidase from the marine γ-proteobacterium Pseudoalteromonas sp. KMM 701 (α-PsGal), and α-N-acetylgalactosaminidase from the marine bacterium Arenibacter latericius KMM 426T (α-NaGa). All compounds are slow-binding irreversible inhibitors of α-PsGal, but have no effect on α-NaGa. A competitive inhibitor d-galactose protects α-PsGal against the inactivation. The inactivation rate (kinact) and equilibrium inhibition (Ki) constants of monanchomycalin B, monanchocidin A, and normonanchocidin A were 0.166 ± 0.029 min-1 and 7.70 ± 0.62 μM, 0.08 ± 0.003 min-1 and 15.08 ± 1.60 μM, 0.026 ± 0.000 min-1, and 4.15 ± 0.01 μM, respectively. The 2D-diagrams of α-PsGal complexes with the guanidine alkaloids were constructed with "vessel" and "anchor" parts of the compounds. Two alkaloid binding sites on the molecule of α-PsGal are shown. Carboxyl groups of the catalytic residues Asp451 and Asp516 of the α-PsGal active site interact with amino groups of "anchor" parts of the guanidine alkaloid molecules.
| S-EPMC6356649 | biostudies-literature