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PubMed2005

Lactobacillus reuteri beta-galactosidase activity and low milk acidification ability.

Beta-galactosidase activity was studied as a possible cause of the low milk acidification ability observed in Lactobacillus reuteri NRRL 14171. Enzymatic activity was determined in MRS broth supplemented with either glucose or lactose and milk at the middle and final stage of the exponential phase, as well as at the stationary phase. Results were compared with beta-galactosidase activity in Lactobacillus casei NRRL-B1922, a strain that shows the milk acidification ability. The effects of the types of carbon and nitrogen sources were established by comparison of growth parameters (higher maximum cell concentration and specific growth rate) in broth culture and skim milk supplemented with 2% glucose or 1% casein peptone. In milk, L. reuteri showed higher beta-galactosidase activity in all growth phases compared with L. casei. Greater cell concentration maxima, specific growth rates, and acidification abilities were observed in L. reuteri when it was cultured in milk supplemented with 1% casein peptone compared with non-supplemented milk cultures. Results suggest that the poor milk acidification ability observed in L. reuteri may be more related to a weak proteolytic system than to deficient beta-galactosidase activity.

Animals↗

PubMed2005

Identification, cloning, and characterization of a Sporobolomyces singularis beta-galactosidase-like enzyme involved in galacto-oligosaccharide production.

Sporobolomyces singularis can be used as a biocatalyst in galacto-oligosaccharide production. We isolated 2-deoxy-D-glucose-resistant mutants of S. singularis ATCC 24193 and recovered a mutant that showed 10-fold higher beta-galactosidase-like activity than the parent strain and which was insensitive to catabolite repression. Thereafter, the beta-galactosidase-like enzyme was purified from the mutant and revealed to be a glycoprotein with both beta-glucosidase- and beta-galactosidase-like activity, the Michaelis-Menten constants of which for o-nitrophenyl-beta-D-galactopyranoside and p-nitrophenyl-beta-D-glucopyranoside were 5.40 and 1.96 mM, respectively, and the maximum velocities were 3.07 and 2.30 micromol/min per mg of protein, respectively. Its molecular mass was estimated to be 73.9 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 146 kDa by gel filtration, suggesting that it has a homodimeric structure. We sequenced the N-terminus and internal peptides of this protein and isolated both a cDNA and a gene with degenerate primers. The gene, named bg l A, has 18 introns and 19 exons and encodes a polypeptide of 594 amino acids. The Bg l A protein was approximately 35% identical and 50% similar to plant beta-glucosidases belonging to family 1 glycosyl hydrolases, but with a unique 110-amino-acid sequence at the N-terminus. The beta-galactosidase-like enzyme (i.e., Bg l A protein) in S. singularis is a beta-glucosidase with high transgalactosidase activity.

Amino Acid Sequence↗

PubMed2003

Beta-D-galactopyranosyl azide: its one-step quantitative synthesis using E461G-beta-galactosidase (Escherichia coli) and a demonstration of its potential as a reagent for molecular biology.

A simple one-step synthesis of beta-D-galactopyranosyl azide from o-nitrophenyl-beta-D-galactopyranoside and azide catalyzed by E461G-beta-galactosidase is described. The synthesis is quantitative in the presence of excess azide and only the beta anomer is produced. The product was purified (71% yield) from the other reaction components by extraction with ethyl acetate, silica gel chromatography, and crystallization. The purity was verified by GLC, TLC, and NMR. Thus, E461G-beta-galactosidase is able to specifically and quantitatively form beta-D-galactopyranosyl-azide. The purified beta-D-galactopyranosyl azide inhibited the growth of Escherichia coli that express beta-galactosidase but not of E. coli that do not. Growth is stopped because beta-galactosidase catalyzes the hydrolysis of the beta-galactopyranosyl-azide, and the azide that is produced inhibits cell growth. This selective inhibition of growth has potential application in molecular biology screening.

Azides↗

PubMed1982

Delivery of fungal beta-galactosidase to rat brain by means of liposomes.

A significant increase in beta-galactosidase activity was observed in the brain of rats 1 hr after an intravenous injection of liposomes containing beta-galactosidase purified from Aspergillus oryzae. The increased activity was proved to have features of the fungal enzyme by differentiating it from rat's native beta-galactosidase in both heat stability and immunochemical studies. Blood content of rat brain tissue under the experimental conditions employed was estimated as 0.83% (v/w) from an infusion experiment of 131I-labeled human serum albumin. The net uptake of fungal beta-galactosidase by rat brain was calculated as equal to 10 micrograms protein of the fungal enzyme or 0.31% of the injected dose/g tissue, which gave rise to 4.4-fold net increase in enzyme activity above control levels. The experiments clearly demonstrated that liposome-entrapped fungal enzyme was allowed to penetrate the blood-brain barrier and to gain access to rat brain, suggesting liposomes as an effective carrier for exogenous enzyme delivering to the central nervous system of patient with inherited lysosomal storage diseases.

Animals↗

PubMed1994

Identification of beta-galactosidase activity in purified bovine retinal rod outer segments.

We have identified beta-galactosidase activity in purified bovine rod outer segments (ROS), using rho-nitrophenyl-beta-D-galactopyranoside (PNPG) and chlorophenol red-beta-D-galactopyranoside (CPRG) as substrates. This glycosylhydrolase activity did not appear to represent contamination from other retinal subcellular fractions, based upon the relative specific activities of beta-galactosidase vs. other hydrolases (N-acetyl-beta-glucosaminidase, alpha- and beta-mannosidase, alpha-fucosidase, and acid phosphatase) in bovine retina and ROS homogenates. Using PNPG as a substrate, two pH optima were observed (at 3.5 and 5.5), while the hydrolysis of CPRG exhibited a single, broad pH optimum centered at 5.5. In contrast, hydrolysis of PNPG and CPRG by retinal homogenates exhibited single pH optima, at 3.5 and 5.5., respectively. ROS beta-galactosidase activity increased linearly with time, temperature, and protein concentration, and obeyed Michaelis-Menten kinetics with both substrates. For PNPG, Vmax approximately 88 nmol/h/mg protein and the apparent Km approximately 147 microM. For CPRG, Vmax approximately 33 nmol/h/mg protein and the apparent Km approximately 50 microM. ROS beta-galactosidase activity was affected by carbohydrates and their derivatives: glucose, fucose, sucrose, maltose and N-acetyl-galactosamine were found to stimulate the activity, while D-galactono-gamma-lactone and, to a lesser extent, D-galactose were inhibitory. The enzyme activity also was slightly stimulated by [Cl-] and markedly by dithiothreitol (DTT), while rho-chloro-mercuribenzoic acid (PCMB) and rho-hydroxymercuribenzoic acid (PHMB) inactivated the enzyme. In addition, the enzymatic activity was also found to be differentially sensitive to various anionic and nonionic detergents. However, n-octyl-beta-D-glucoside was slightly stimulatory.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

Use of an alpha-galactosidase gene as a food-grade selection marker for Streptococcus thermophilus.

The alpha-galactosidase gene (aga) of Lactococcus raffinolactis ATCC 43920 was previously shown to be an efficient food-grade selection marker in Lactococcus lactis and Pediococcus acidilactici but not in Streptococcus thermophilus. In this study, we demonstrated that the alpha-galactosidase of L. raffinolactis is thermolabile and inoperative at 42 degrees C, the optimal growth temperature of S. thermophilus. An in vitro assay indicated that the activity of this alpha-galactosidase at 42 degrees C was only 3% of that at 30 degrees C, whereas the enzyme retained 23% of its activity at 37 degrees C. Transformation of Strep. thermophilus RD733 with the shuttle-vector pNZ123 bearing the aga gene of L. raffinolactis (pRAF301) generated transformants that were stable and able to grow on melibiose and raffinose at 37 degrees C or below. The transformed cells possessed 6-fold more alpha-galactosidase activity after growth on melibiose than cells grown on lactose. Slot-blot analyses of aga mRNA indicated that repression by lactose occurred at the transcriptional level. The presence of pRAF301 did not interfere with the lactic acid production when the transformed cells of Strep. thermophilus were grown at the optimal temperature in milk. Using the recombinant plasmid pRAF301, which carries a chloramphenicol resistance gene in addition to aga, we showed that both markers were equally efficient at differentiating transformed from nontransformed cells. The aga gene of L. raffinolactis can be used as a highly efficient selection marker in Strep. thermophilus.

Dairy Products↗

PubMedDate not supplied

[Mutation of the alpha-galactosidase A gene in two unusual variations of Fabray's disease].

The mutation analysis of alpha-galactosidase A gene was carried out in two families with Fabry disease described by us earlier. In the family P. a new point mutation E341K (a G to A transition at position 10,999 of the gene) was identified. The mutation causes a Glu341Lys substitution in alpha-galactosidase A molecule. Another point mutation was identified in a patient from family N. who had unusual unusually high residual activity of alpha-galactosidase A. The mutation was identified as R112C (a C to T transition at position 5233 of alpha-galactosidase A gene) and it caused the Arg112Cys substitution in the enzyme molecule. This mutation was earlier described in Japanese patient with showed a complete loss of enzyme activity. However, in this case the mutation was combined with another mutation Glu66Gln. The relationship between genetic heterogeneity and clinical manifestation of Fabry disease is discussed.

Amino Acid Substitution↗

PubMed1975

Purification and properties of a soluble factor required for the deoxyribonucleic acid-directed in vitro synthesis of beta-galactosidase.

The DNA-directed in vitro synthesis of beta-galactosidase has been investigated in a system dependent on Escherichia coli ribosomes, a salt wash of the ribosomes, and a supernatant fraction. Fractionation of the supernatant has made it possible to obtain dependencies on RNA polymerase and another protein factor for beta-galactosidase synthesis. The other factor (called L factor) cannot be replaced by a variety of proteins known to be required for transcription and translation. It has been purified to homogeneity and has a molecular weight of approximately 65,000. Although it is required for the in vitro synthesis of beta-galactosidase, it has no effect on total DNA-dependent amino acid incorporation under the conditions of the incubation. However, total RNA synthesis is depressed by the addition of L factor in a manner similar to what is observed with rho factor could not replace L factor in beta-galactosidase synthesis.

Amino Acyl-tRNA Synthetases↗

PubMed2000

[Detection of secretion of beta-galactosidase by plant cells].

A simple, sensitive and reproducible method of detection of extracellular beta-galactosidase was worked out. beta-Galactosidase secreted by the callus culture, or the roots of sprouting plants, hydrolyzes the substrate (6-bromo-2-naphthyl-beta-D-galactopyranoside) to produce beta-D-galactose and 6-bromo-2-naphthol, which after simultaneous azocoupling with hexazotized p-rosaniline, or basic fuchsine, produce a reddish brown compound, nearly insoluble in water (a diazonium salt). The colouring under the callus culture and around it, as well as the colouring of the roots of the sprouting plants, is a manifestation of the activity of extracellular beta-galactosidase by the objects under study. The intensity of the colouring is a measure of their enzymatic activity. The share of intracellularly localized enzyme represents the majority and the share of extracellular beta-galactosidase the minority.

Plant Roots↗

PubMed1975

Activity of human hepatic beta-galactosidase toward natural glycosphingolipid substrates.

1. Human hepatic "acid" beta-galactosidase preparations, which had been purified approximately 250-fold, were examined for activities toward 4-methylumbelliferyl beta-galactoside, galactosylceramide, lactosylceramide, galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosyl-glucosylceramide (GM1-Ganglioside) and galactosyl-Cacetylgalactosaminyl-galactosyl-glucosylceramide (asialo GM1-ganglioside). 2. The enzyme was active toward the synthetic substrate, GM1-ganglioside and asialo GM1-ganglioside but was inactive toward galactosylceramide. Under our assay conditions, optimized for lactosylceramidase II, the preparations were as active toward lactosylceramide as toward GM1-ganglioside or its asialo derivative. Teh apparent Km values for the three natural substrates were similar. When determined by the assay system of Wenger, D.A., Sattler, M., Clark, C. and McKelvey, H. (1974) Clin. Chim. Acta 56, 199-206, lactosylceramidecleaving activity was 0.2% of that determined by our assay system. This confirmed our previous suggestion that the Wenger assay system determines exclusively the activity of lactosylceramidase I, which is probably identical with galactosylceramide beta-galactosidase. 3. Crude sodium taurocholate was far more effective than pure taurocholate in stimualting hydrolysis of the three glycosphingolipids by the beta-galactosidase. However, crude tauroxycholate, suggesting that the unique activating capacity of the crude taurocholate might be due to taurodeoxycholate present as the major impurity. 4. Cl- was generally stimulatory for hydrolysis of the natural glycosphingolipids by our enzyme preparation. Effects of additional oleic acid and Triton X-100 Were generally minor in either direction. 5. When the enzyme preparation was diluted with water, activity toward the synthetic substrate declined rapidly while those toward the natural substrates were essentially stable. Activity toward the synthetic substrate remained much more stable when the enzyme was diluted with 0.1 M sodium citrate/phosphate buffer, pH 5.0. 6. These observations provide insight into the complex relationship among the human hepatic beta-galactosidases.

Chlorides↗

PubMed2002

Beta-galactosidase activity in saliva is associated with oral malodor.

Deglycosylation of oral mucins may be a critical initial step leading to their subsequent proteolysis and putrefaction. The present study was undertaken to determine whether activity in saliva of a major glycosidic enzyme (beta-galactosidase) is associated with oral malodor in a group of 64 subjects. Enzyme activity was detected by the use of a chromogenic substrate (X-Gal) impregnated on paper discs. Malodor-related measurements included two odor judges' assessments of whole-mouth and tongue malodor, and volatile sulfide levels measured by a portable sulfide monitor (Interscan Corp.). Beta-galactosidase assay scores were significantly associated with both odor judges' scores for whole-mouth (p < or = 0.002; Spearman) and tongue malodor (p < or = 0.001; Spearman). Beta-galactosidase activity and sulfide monitor measurements both factored significantly into multiple regression equations for odor judge scores, yielding multiple r-values ranging from 0.47 (p = 0.0007) to 0.60 (p < 0.0001). Analysis of the data presented indicates that beta-galactosidase activity in saliva is correlated with oral malodor.

Adult↗

PubMed2002

A simple method for the identification and assay of extracellular plant beta-galactosidase.

A simple, rapid and reproducible procedure for the identification of extracellular Californian poppy (Eschscholzia californica Cham.) beta-galactosidase is described using callus cultures of seedlings from the tested plant, roots of 4-days-old seedlings of Californian poppy germinating on agar plates and cell suspension cultures cultivated from callus cultures. 6-Bromo-2-naphthyl-beta-D-galactopyranoside and p-nitrophenyl-beta-D-galactopyranoside were used as substrates for the determination of the intracellular and extracellular activities of beta-galactosidase. The extracellular beta-galactosidase activity was identified by evaluating the dye-zones in an agar medium. The enzyme from Californian poppy callus cultures or from seedling roots cultivated on agar plates supplemented with 6-bromo-2-naphthyl-galactopyranoside hydrolyzed this substrate releasing 6-bromo-2-naphthol. By simultaneous coupling with hexazonium p-rosaniline the corresponding (reddish-brown) azo-dye was formed. The agar plate method described permits rapid, simple and specific detection of plant producers of extracellular beta-galactosidase.

Cells, Cultured↗

PubMedDate not supplied

[Induction of endo-1,4-beta-xylanase and beta-galactosidase in the original and recombinant strains of the fungus Penicillium canescens].

The induction of the synthesis of secreted enzymes endo-1,4-beta-xylanase (EC 3.2.1.8) and beta-galactosidase (EC 3.2.1.23) in the original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for the induction: the synthesis of beta-galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4-beta-xylanase was observed at 5 to 10 mM. An increase in the number of endo-1,4-beta-xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of beta-galactosidase; the synthesis of endo-1,4-beta-xylanase in the high-copy-number recombinant producing beta-galactosidase was affected to a lesser extent. The amount of the enzymes synthesized did not depend on the saccharide used as a sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.

Arabinose↗

PubMed1991

Substrate specificity of alpha-galactosidase from Aspergillus niger 5-16.

This paper describes the specificity of Aspergillus niger 5-16 alpha-galactosidase toward various oligosaccharides having terminal galactose or stub galactose or both on the oligosaccharide. The galactosidase rapidly hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside, but hardly liberated galactose from melibiose, manninotriose, 6(3)-alpha-D-galactosylmannotriose, etc. On the other hand, the enzyme tore off the stub galactoses attached to the inner mannoses of the main-chain of galactomannooligosaccharides, but not the terminal galactoses attached to the non-reducing-end mannoses of the main-chain. Thus, the substrate specificity of A. niger 5-16 alpha-galactosidase is quite different from that of Mortierella vinacea alpha-galactosidase.

Aspergillus niger↗

PubMed2004

A novel alpha-galactosidase a mutant (M42L) identified in a renal variant of Fabry disease.

A 65-year-old man presented to our institution for workup of proteinuria. His serum creatinine level was 1.7 mg/dL (130 micromol/L), and he had proteinuria with protein of almost 5 g/24 h. Fabry disease was diagnosed by means of kidney biopsy and low serum and leukocyte levels of alpha-galactosidase A. Review of his history, family history, physical examinations, and diagnostic studies did not show other findings typical of this disease. His renal function continued to decline, and he eventually underwent a living unrelated renal transplantation 5 years later. Three years after transplantation, his creatinine level is 1.7 mg/dL (130 micromol/L), and corrected iothalamate clearance is 53 mL/min/1.73 m2 . Genetic studies showed that he has a novel missense mutation (M42L) in exon 1. Methionine at codon 42 is highly conserved in eukaryotic alpha-galactosidase A orthologues. This genotype predicts a minor misfolding of alpha-galactosidase A because of a small difference in hydrophobicity between methionine and leucine. His mutation resulted in a very low, but detectable, serum level of alpha-galactosidase A (0.002 U/L; normal range, 0.016 to 0.2 U/L). Cases of Fabry disease that present with predominantly renal manifestations are rare and require a high index of suspicion for diagnosis. Because treatment for Fabry disease recently has become available, it is important for clinicians to be aware of this disease and pursue the diagnosis in cases of otherwise unexplained renal dysfunction.

Aged↗

PubMedDate not supplied

[Patterns of growth and beta-galactosidase production by bifidobacteria].

We investigated the patterns of growth and beta-galactosidase formation in the strains Bifidobacterium adolescentis GO-13, MS-42, 91-BIM, and 94-BIM, and B. bifidum No. 1, LVA-3, 791 on media with various carbon sources. The synthesis of beta-galactosidase was shown to be associated with exponential growth of the cultures involved. The maximum specific rate of beta-galactosidase synthesis of 0.20 U mg(-1) h(-1) was observed in B. bifidum LVA-3 after 3-6 h of cultivation. This value for B. adolescentis 91-BIM and 94-BIM was lower and amounted to 0.03-0.08 U mg(-1) h(-1). On the medium with lactose, the highest specific growth rates for B. bifidum LVA-3 and B. bifidum No.1 were 0.38 and 0.60 h(-1), respectively, after 3-6 h of cultivation. For B. adolescentis 91-BIM and 94-BIM, this parameter peaked at 12-15 h of cultivation at 0.13 and 0.22 h(-1), respectively. The hydrolytic activity of beta-galactosidase in the growth medium decreased during the stationary growth phase of the tested cultures.

Bifidobacterium↗

PubMed1975

Isolation and characterization of jack bean beta-galactosidase.

A simple procedure has been devised to isolate beta-galactosidase from jack bean meal. The final preparation gives one major protein banc in disc gel electrophoresis. The substrate specificity of this enzyme toward some natural oligosaccharides, glycoproteins, and sphingoglycolipids has been examined in detail. Among three isomers of N-acetyllactosamine, Galbeta1leads to4GlcNAc; while Galbeta1leads to3GlcNAc was hydrolyzed very slowly. This property can be used to distinguish the galactose linkage in asialo-GM1 (Galbeta1leads to3GalNAcbeta1leads to4Galbeta1leads to4Glcleads toCer) and that in lacto-N-neotetraosylceramide (Galbeta1leads to4GlcNAcbeta1leads to 3Galbeta1leads to4Glcleads toCer). For hydrolyzing glycolipids, the effect of sodium taurodeoxycholate and sodium taurochenodeoxycholate on the rate of hydrolysis was carefully examined. This enzyme hydrolyzes lactosylceramide and asialo-GM1 faster than GM1. These results suggest that in addition to the type and linkage of the penultimate sugar unit, the sugar unit at the distal position of the saccharide chain also affects the hydrolysis rate. It also readily liberates 80% D-galactosyl units from asialo alpha1-acid glycoprotein. Escherichia coli beta-galactosidase on the other hand cannot hydrolyze asialo-alpha1-acid glycoprotein, lactosylceramide, GM1, asialo-GM1, and lacto-N-neotetraosylceramide. The molecular weight of this enzyme is about 75,000 and the isoelectric point is pH 8.0. With p-nitrophenyl beta-D-galactopyranoside as substrate, optimal activity occurs at pH 2.8 with glycine-HCl buffer and at pH 3.5 with citrate-phosphate buffer. With lactose as substrate, the pH optimum in these two buffers are 2.8 and 4.0, respectively. Km values for p-nitrophenyl beta-D-galactopyranoside, o-nitrophenyl beta-D-galactopyranoside and lactose are 0.51 mM, 0.63 mM, and 12.23 mM, respectively. Many inhibitors for this enzyme including inorganic ions, monosaccharides, and glycosides are investigated. In contrast to E. coli beta-galactosidase, jack bean beta-galactosidase is not inhibited by p-aminophenyl thio-beta-D-galactopyranoside.

Carbohydrates↗

PubMed1980

Dog GM1 gangliosidosis: characterization of the residual liver acid beta-galactosidase.

The residual liver acid beta-galactosidase activity from the first documented case of GM1 gangliosidosis in dogs was partially purified and characterized with respect to kinetic properties, thermostability, isoelectric point, molecular weight, and antigenicity. The GM1 dog liver beta-galactosidase appears to be identical with the normal dog liver enzyme in all properties examined. The canine disease is strikingly different from the human disease in the amount of enzyme that is present in the tissue. Unlike the human disease, in which normal amounts of catalytically defective beta-galactosidase are present, in dog GM1 gangliosidosis, only 1% of normal beta-galactosidase protein is detectable.

Animals↗