Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “GALACTOSIDASE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 523 records · Page 29Linked to original sources
PubMed2006

The effect of oral alpha-galactosidase on intestinal gas production and gas-related symptoms.

Bloating, abdominal distention, and flatulence represent very frequent complaints in functional disorders but their pathophysiology and treatment are largely unknown. Patients frequently associate these symptoms with excessive intestinal gas and the reduction of gas production may represent an effective strategy. The aim was to evaluate the effect of alpha-galactosidase administration, in a randomized double-blind placebo-controlled protocol, on intestinal gas production and gas-related symptoms after a challenge test meal in healthy volunteers. Eight healthy volunteers ingested 300 or 1200 GalU of alpha-galactosidase or placebo during a test meal containing 420 g of cooked beans. Breath hydrogen excretion and occurrence of bloating, abdominal pain, discomfort, flatulence, and diarrhea were measured for 8 hr. The administration of 1200 GalU of alpha-galactosidase induced a significant reduction of both breath hydrogen excretion and severity of flatulence. A reduction in severity was apparent for all considered symptoms, but both 300 and 1200 GalU induced a significant reduction in the total symptom score. Alpha-galactosidase reduced gas production following a meal rich in fermentable carbohydrates and may be helpful in patients with gas-related symptoms.

Adult↗
PubMed1990

Multiple replacements establish the importance of tyrosine-503 in beta-galactosidase (Escherichia coli).

Tyr-503 of beta-galactosidase was specifically replaced with Phe, His, Cys, and Lys using site-directed mutagenesis. The normal enzyme and the substituted enzymes were purified. The activities of each of the substituted enzymes with o-nitrophenyl-beta-D-galactopyranoside (ONPG) and p-nitrophenyl-beta-D-galactopyronoside (PNPG) were very low and Y503K-beta-galactosidase was essentially inactive, showing that Tyr-503 is important for activity. The stability (including tetrameric stability) of the enzymes at 4 and 25 degrees C was essentially the same as that of the wild-type enzyme and the cleavage patterns on sodium dodecyl sulfate gels after protease action were unchanged. These studies thus indicate that Tyr-503 has no noticeable influence on stability under normal conditions. The substitutions for Tyr-503 had some small effects on the binding of both substrate and inhibitor. However, both kappa 2 (glycosidic bond cleavage rate) and kappa 3 (hydrolysis rate constant) were dramatically reduced. Each substitution except that of Lys (which can be explained by electrostatic effects) gave decreases in kappa 2 and kappa 3 of roughly the same magnitude regardless of whether the substitutions were conservative or not. This strongly implies that the changes in rate were not due to conformational changes as it is very unlikely that there would be such similar decreases in the values of kappa 2 and kappa 3 for amino acids with such different structures and chemical properties if the changes in rate were due to conformational differences. The data suggest that one possible role of Tyr-503 is as a general acid/base catalyst. Profiles of the kinetic data of the enzymes as functions of pH supported the suggestion that Tyr-503 normally acts as a general acid and base catalyst. When Tyr-503 was substituted by His, a small amount of base catalytic activity seemed to be restored. The strongest evidence that Tyr-503 acts as an acid catalyst came from studies with isoquinolinium-beta-D-galactopyranoside as the substrate. The kappa cat(s) of Y503F-beta-galactosidase and of Y503C-beta-galactosidase decreased by about an order of magnitude while the rate decreases were about 3 orders of magnitude with ONPG and PNPG. The breakdown of isoquinolinium-beta-D-galactopyranoside cannot be catalyzed by acids.

Base Sequence↗
PubMed1991

The conversion of group B red blood cells into group O by an alpha-D-galactosidase from taro (Colocasia esculenta).

An alpha-D-galactosidase (EC 3.2.1.21), capable of converting group B into group O red cells, was isolated from the stem portion of taro. It was purified about 3000 fold by gel filtration and ion-exchange chromatography. The blood group-converting activity was demonstrated by hemolysis and hemagglutination studies. This activity is comparable to that of alpha-D-galactosidase isolated from coffee beans. Taro alpha-D-galactosidase also hydrolyzes (1----4)- and (1----6)-linked alpha-D-galactopyranosyl groups from D-galactose-containing glycoconjugates. Taro alpha-D-galactosidase has a low Km value (0.28mM), a low molecular weight (40,000), and a neutral optimal pH (6.0). At a final enzyme concentration of 30 units/mL in the incubation mixture, the conversion of group B into group O activity was completed within two hours, without apparent changes in the shape of the red cells.

ABO Blood-Group System↗
PubMed1996

Only sphingolipid activator protein B (SAP-B or saposin B) stimulates the degradation of globotriaosylceramide by recombinant human lysosomal alpha-galactosidase in a detergent-free liposomal system.

The degradation of globotriaosylceramide (GbO-se3Cer) by insect-cell derived recombinant human alpha-galactosidase (EC 3.2.1.22) was carried out in a detergent-free liposomal system in order to mimic intralysosomal conditions. GbOse3Cer incorporated into unilamellar liposomes was used as the substrate, and naturally occurring sphingolipid activator proteins, rather than detergents, were used to stimulate the enzyme reaction. The degradation of GbOse3Cer was dependent on the presence of both alpha-galactosidase and sphingolipid activator protein B (SAP-B or saposin B). It proceeded optimally at pH 4.6, and was enhanced by increasing amounts of both alpha-galactosidase (0.24-24 mU/50 microliters assay) and SAP-B (0-5 micrograms/50 microliters assay). The enzyme reaction was not affected by SAP-A, SAP-C, or SAP-D. Therefore, our results indicate that only SAP-B is essential for the degradation of GbOse3Cer by alpha-galactosidase.

Animals↗
PubMed1994

Conjugation of recombinant reverse transcriptase of HIV-1 to beta-D-galactosidase from Escherichia coli for ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti-HIV-1 IgG.

Recombinant reverse transcriptase (RT) of HIV-1 was conjugated to beta-D-galactosidase from Escherichia coli in three different ways. Maleimide groups were introduced into beta-D-galactosidase molecules using N,N'-o-phenylenedimaleimide in the absence (method I) or presence (method II) of N-ethylmaleimide or into beta-D-galactosidase molecules, which had been treated with excess of 4,4'-dithiodipyridine to block thiol groups, using N-succinimidyl-6-maleimidohexanoate (method III). Subsequently, the maleimide groups were reacted with thiol groups introduced into recombinant RT molecules using N-succinimidyl-S-acetylmercaptoacetate. The conjugates were tested by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay). The immune complex consisting of 2,4-dinitrophenyl-bovine serum albumin-recombinant RT conjugate, anti-HIV-1 IgG and recombinant RT-beta-D-galactosidase conjugate was captured by polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG, eluted with N epsilon-2,4-dinitrophenyl-L-lysine and transferred to polystyrene beads with (anti-human IgG gamma chain) IgG. The conjugate prepared by method III, which showed the least polymerization, the least loss of the specific enzyme activity and the lowest nonspecific binding, improved the sensitivity of the enzyme immunoassay for anti-HIV-1 IgG approximately 30-fold compared with RT-horseradish peroxidase conjugate.

Animals↗
PubMed1992

Effect of pH on subunit association and heat protection of soybean alpha-galactosidase.

Soybeans contain the enzyme alpha-galactosidase, which hydrolyzes alpha-1, 6 linkages in stachyose and raffinose to give sucrose and galactose. We have found that galactose, a competitive product inhibitor of alpha-galactosidase, strongly promotes the heat stability of the tetrameric form of the enzyme at pH 4.0 and at temperatures of up to 70 degrees C for 60 min. Stachyose and raffinose also protect alpha-galactosidase from denaturation at pH 4.0 although to a lesser extent. Glucose and mannose have little effect. At pH 7.0 the enzyme is a monomer, and galactose has no effect on the heat stability of the enzyme. In the absence of heat protection of the enzyme by added sugars, a series deactivation mechanism was found to describe the deactivation data. In comparison, a unimolecular, non-first order deactivation model applies at pH 4.0, where heat protection effects were observed. At a temperature above 60 degrees C, simple deactivation is a suitable model. The results suggest that alpha-galactosidase conformation and heat stability are directly related.

Enzyme Stability↗
PubMed1993

Borate ion-assisted stabilization of beta-galactosidase from Aspergillus oryzae by polyhydroxy compounds in water-miscible organic solvents.

The stability of beta-galactosidase from Aspergillus oryzae in water-miscible organic solvents in different buffers at various pH values ranging from 4.6 to 8.0 was studied. The stability of the enzyme in all six organic solvents studied was dependent on pH and on the type of buffer ions present. At a given pH, destabilization by organic solvents was highest in sodium borate buffer. The destabilization of beta-galactosidase by these solvents could be reversed by addition of sugars or polyhydroxy compounds exclusively in sodium borate, suggesting a role of borate ions in stabilization. A similar effect of addition of mannitol was observed on deactivation of beta-galactosidase by N, N-dimethylformamide (DMF). Exclusively in sodium borate, at pH 8.0, the addition of mannitol (0.02 M) not only prevented the deactivation by DMF (8%, v/v) but increased the enzyme activity to the level at its optimum pH. Since beta-galactosidase from Aspergillus oryzae is a glycoprotein, complexation of the borate ions to the carbohydrate part may result in change in protein conformation, which, without leading to denaturation or inactivation of the enzyme, may facilitate interaction of the organic solvents with the enzyme leading to its denaturation. Such a denaturation is probably prevented by addition of polyhydroxy compounds, which appear to compete favorably with the carbohydrate moiety of the protein in complexing with borate ions. This should result in the enzyme regaining its native conformation.

Aspergillus oryzae↗
PubMed1996

Influence of gelatin matrices cross-linked with transglutaminase on the properties of an enclosed bioactive material using beta-galactosidase as model system.

Transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) from Streptoverticillium mobaraense has been used to stabilize immobilisates produced with beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus oryzae and acid-processed gelatins of different qualities as support. The isopeptide level of N epsilon-(gamma-L-glutamyl)-L-lysine bonds formed by transglutaminase was determined to estimate their influence on the kinetic properties of the enclosed beta-galactosidase. An HPLC procedure using precolumn derivatization of the gelatin hydrolysates with FMOC-chloride was chosen which permits the analysis of cross-linked lysine with satisfactory precision. Depending on the gelatin quality, the degree of cross-links necessary for the transformation of gelatin into an insoluble protein was in the range 0.3-32.3% of the available lysine residues. beta-Galactosidase was entrapped in the gelatin matrices with a yield of 8-46% of the initial activity. Long reaction times for cross-linking were due to low yields rather than to the number of isopeptide bonds. Repeated use of the immobilisates did not lead to an appreciable loss of activity. The Vmax of beta-galactosidase were diminished by immobilization caused by a tighter package of the protein chains rather than by the extent of cross-links, while the obtained Km values of the free enzyme and the immobilisates were quite similar. Also, the pH and temperature of optima of the free enzyme and the gelatin immobilisates differ only slightly. The data suggest that the immobilization procedure only moderately affects the activity of enzymes catalysing the reaction of a small compound if gelatin with high jelly strength is cross-linked in a 10% solution with transglutaminase.

Aspergillus oryzae↗
PubMed1990

A glycoprotein gX-beta-galactosidase fusion gene as insertional marker for rapid identification of pseudorabies virus mutants.

We describe the isolation and characterization of infectious pseudorabies virus (PrV) mutants expressing functional beta-galactosidase. To obtain high level expression of the enzyme, sequences of the bacterial beta-galactosidase gene starting with codon 8 were inserted in frame behind the promoter and the first seven codons of the nonessential PrV glycoprotein gX-gene. Cotransfection of this construct with viral DNA yielded PrV mutants that could be easily identified after plaque staining with a chromogenic substrate. These mutants carry the gX-beta galactosidase fusion gene inserted into the authentic gX-gene leading to loss of gX-expression. The gX-beta galactosidase fusion gene could be excised as an expression cassette and placed into other non-essential PrV genomic regions, such as the thymidine kinase gene and the glycoprotein gI-gene, resulting in inactivation of the target genes. The fusion gene remains stably integrated in the viral genome at all three locations tested. It therefore appears ideal as an insertional and easily identifiable marker and greatly facilitates isolation and purification of PrV mutants.

Animals↗
PubMedDate not supplied

Influence of the helper virus on expression of beta-galactosidase from a defective HSV-1 vector, pHSVlac.

Defective herpes simplex virus type 1 (HSV-1) vectors can deliver genes into both mitotic and postmitotic cells, including neurons and these vectors, therefore, have great potential use. pHSVlac, the prototype HSV-1 vector, expresses the E. coli Lac Z gene from the HSV-1 immediate early 4/5 promoter. pHSVlac can stably express beta-galactosidase in a range of mammalian cell lines and in neurons from throughout the nervous system, both in culture and in the adult rat brain. Thus, HSV-1 vectors may be useful for studying HSV-1 latency, neuronal physiology, and performing gene therapy for neurological conditions. A virus stock of pHSVlac consists of identical HSV-1 particles containing either pHSVlac DNA or the HSV-1 helper virus DNA. Thus, a cell can be infected with the pHSVlac virus, the helper virus, or both; consequently, it is important to determine if the helper virus influences the behavior of pHSVlac. The effect of the helper virus on expression of beta-galactosidase from pHSVlac was investigated: it was demonstrated first, that pHSVlac can be efficiently packaged into HSV-1 virus particles using each of five HSV-1 temperature sensitive (ts) mutants as helper virus. Second, pHSVlac grown with each of the five HSV-1 ts mutants expressed high levels of beta-galactosidase. Third, pHSVlac grown with HSV-1 strain 17 ts K as helper virus expresses the same amount of beta-galactosidase in the absence or presence of ts K. Thus, pHSVlac can efficiently express a gene independent of the HSV-1 helper virus.

Adrenal Gland Neoplasms↗
PubMed1994

Characterization of dairy-related Bifidobacterium spp. based on their beta-galactosidase electrophoretic patterns.

Numerical analysis of phenotypic characteristics based on enzymatic activity and carbohydrate fermentation allowed the discrimination of most strains of bifidobacteria of animal origin from those of human origin. Strains of bifidobacteria studied were separated into nine groups based on numerical analysis. Three groups contained most strains of animal origin, three groups comprised both strains of animal and human origin, and three groups were strictly composed of strains of human origin. The results indicate that one group of animal origin (group II) contained all reference strains of Bifidobacterium animals and 10 strains isolated from fermented milks or commercial preparations. Although numerical analysis of enzymatic activities and carbohydrate fermentation patterns allowed the differentiation of 'wild' strains of B. animalis and B. longum isolated from commercial preparations, this method failed to confirm the species. In the present study, the determination of electrophoretic patterns of beta-galactosidases resulted in the development of a new technique for the differentiation of Bifidobacterium species. Several isoenzymes of beta-galactosidase were detected among strains of bifidobacteria. Each species had a specific electrophoretic pattern. The detection of beta-galactosidase by electrophoresis is a new tool for distinguishing between dairy- and non-dairy-related bifidobacteria. Dairy-related bifidobacteria (B. bifidum, B. breve, B. infantis and B. longum) as well as B. animalis could be better differentiated from other bifidobacteria by comparison of their beta-galactosidase electrophoretic patterns, rather than by numerical analysis of their phenotypic characteristics.

Animals↗
PubMed1992

Continuous beta-galactosidase production with a recombinant baculovirus insect-cell system in bioreactors.

Insect cells were exploited to produce bacterial beta-galactosidase by infecting them with a recombinant nuclear polyhedrosis virus (baculovirus) of Autographa californica. The insect cells were cultured in a continuous stirred tank reactor (CSTR) and led to a second CSTR where they were infected with a recombinant virus in which the lacZ gene from Escherichia coli was inserted. In the effluent of the production reactor, maximum activities of 15 units beta-galactosidase per 10(6) cells were measured. For about 25 d beta-galactosidase production remained constant, but then rapidly declined. This drop was due to a decrease in production of active beta-galactosidase rather than to inactivation of this enzyme. It was concluded that the reduced production was due to reduced polyhedrin promoter-driven synthesis.

Animals↗
PubMed1994

Normal serum beta-galactosidase in juvenile GM1 gangliosidosis.

GM1 gangliosidosis is a genetic disease with lysosomal beta-galactosidase deficiency caused by mutations of the gene coding for this enzyme. However, apparently normal enzyme activity was found in plasma or serum from juvenile GM1 gangliosidosis patients homozygous for a mutation, R201C (201Arg-->Cys), after clotting for 30 min. This extracellular fluid finding is unusual in patients with primary and genetic deficiency of beta-galactosidase. The serum enzyme activity became relatively low only after 3 1/2-hour clotting because its increase in normal controls was not observed in these patients. beta-Galactosidase assay is not always reliable, particularly with serum or plasma as an enzyme source, for the diagnosis of hereditary beta-galactosidase deficiency, unless it is conducted under well-controlled conditions.

Adolescent↗
PubMed2005

Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts.

Senescence-associated beta-galactosidase activity is a widely used biomarker for assessing replicative senescence in mammalian cells. This enzymatic activity has generally been measured by staining cells with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) at pH 6.0, a reaction condition that suppresses lysosomal beta-galactosidase activity sufficiently to ensure that most nonsenescent cells will appear unstained. This article describes a quantitative method for measuring this activity and characterizes the method using extracts from senescent, quiescent, and presenescent human fibroblasts. The assay is capable of detecting relatively subtle changes in activity and confirms previous indications based on staining that confluency and contact inhibition of growth can cause a small increase in the expression of this biomarker. Investigation of the pH dependence of the activity in the cell extracts suggests that the senescent phenotype is correlated with an increase in total beta-galactosidase rather than with a shift in the pH optimum of the enzyme. This assay for measuring senescence-associated changes in beta-galactosidase is suitable for mechanistic studies of senescence regulation in which graduated changes in biomarker expression may be anticipated.

Aging↗
PubMed2006

A novel thermostable alpha-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: purification and characterization.

High levels of an extracellular alpha-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH(4))(2)SO(4) fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric alpha-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5-5.5 and 65 degrees C, respectively. The purified enzyme retains more than 90% of its activity at 45 degrees C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-alpha-galactopyranoside, raffinose and stachyose and very similar K(m) values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn(++) ions activates enzyme activity, whereas inhibitory effects can be observed with Ca(++), Zn(++) and Hg(++). Five min incubation at 65 degrees with 10 mM Ag(+) results in complete inactivation of the purified alpha-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the alpha-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.

Amino Acid Sequence↗
PubMed2006

Alpha-galactosidase production by Aspergillus oryzae in solid-state fermentation.

Comparisons were made for alpha-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for alpha-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced alpha-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 x 10(6) spores) of inoculum volume and four days incubation were optimum for alpha-galactosidase production. Increase in substrate concentration (RGPW+WB) did not decrease enzyme yield in trays.

Aspergillus oryzae↗
PubMed2004

Enzymatic synthesis of a beta-D-galactopyranosyl cyclic tetrasaccharide by beta-galactosidases.

The galactosyl transfer reaction to cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS) was examined using lactose as a donor and beta-galactosidases from Aspergillus oryzae and Bacillus circulans. The A. oryzae beta-galactosidase produced three galactosyl derivatives of CTS. The main galactosyl derivative produced by the A. oryzae enzyme was identified as 6-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-[beta-D-Galp-(1-->6)]-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. The B. circulans beta-galactosidase also synthesized three galactosyl-transfer products to CTS. The structure of main transgalactosylation product was 3-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-D-Galp-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These results showed that beta-galactosidase transferred galactose directly to the ring glucose residue of CTS.

Aspergillus oryzae↗
PubMed2005

High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions.

We describe a completely in vitro high-throughput screening system for directed evolution of enzymes based on in vitro compartmentalization (IVC). Single genes are transcribed and translated inside the aqueous droplets of a water-in-oil emulsion. Enzyme activity generates a fluorescent product and, after conversion into a water-in-oil-in-water double emulsion, fluorescent droplets are sorted using a fluorescence-activated cell sorter (FACS). Earlier in vivo studies have demonstrated that Ebg, a protein of unknown function, can evolve to allow Escherichia coli lacking the lacZ beta-galactosidase gene to grow on lactose. Here we demonstrate that we can evolve Ebg into an enzyme with significant beta-galactosidase activity in vitro. Only two specific mutations were ever seen to provide this improvement in Ebg beta-galactosidase activity in vivo. In contrast, nearly all the improved beta-galactosidases selected in vitro resulted from different mutations.

Directed Molecular Evolution↗