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At least 1,189 records · Page 66Linked to original sources
PubMed2000

Effect of beta-galactosidase hydration on alcoholysis reaction in organic one-phase liquid systems.

Alcoholysis reactions were performed in organic one-phase liquid systems with E. coli beta-galactosidase to produce heptyl-beta-galactoside from lactose and 1-heptanol. The reaction rate was highly dependent on the amount of water solubilized in the alcohol. A larger amount of water led to a system of two liquid phases in which the alcoholysis rate was 73% faster than in the one-phase system. No hydrolysis reaction of either lactose or product was observed in one-phase liquid systems up to 20 h, independent of the water content. Solubility of lactose in the organic phase increased with the water content in the system and the reaction followed the Michaelis-Menten model. Water activity was calculated for heptanol containing different amounts of water and the obtained values were used to estimate the hydration of beta-galactosidase from known models. Enzyme activity correlated with sorbed water, similar to the behavior reported for lysozyme in low water environments. It is concluded that water contribution to enzyme hydration dominates the rate of reaction compared to its effect on lactose solubilization.

Alcohols↗
PubMed1990

Combined deficiency of beta-galactosidase and neuraminidase: natural history of the disease in the first 18 years of an American patient with late infantile onset form.

We describe the clinical findings over the first 18 years of a patient with a novel phenotype for galactosialidosis, the storage disease produced by the combined deficiency of beta-galactosidase and neuraminidase. Clinical findings in the first few months included somewhat unusual appearance and hepatosplenomegaly. Dysostosis multiplex was evident by age 2 1/2 years. Mitral and aortic valvular disease appeared over the next few years and cardiac disease has become the most important clinical problem. Foam cells were present in the bone marrow, and vacuolated lymphocytes were present in the peripheral blood smear. The patient had no neurological symptoms, cherry red spots, or intellectual deterioration during the first 18 years. Evidence presented elsewhere indicates that the basic defect in this late infantile form of galactosialidosis (as is thought to be true for the other forms of galactosialidosis) is a reduced amount of the 32 kDa phosphoglycoprotein which associates with beta-galactosidase and alpha-neuraminidase in lysosomes.

Aortic Valve Insufficiency↗
PubMed1983

beta-Galactosidase-neuraminidase deficiency (galactosialidosis): clinical, pathological, and enzymatic studies in a postmortem case.

Three male siblings in a Japanese family were affected with beta-galactosidase-neuraminidase deficiency (galactosialidosis). One patient died at 45 years of age, and postmortem liver and brain tissues were studied enzymatically. The residual activity of neuraminidase was relatively high in these tissues. Neuraminidase activity did not change in the tissues after repeated freezing and thawing of the homogenates, whereas this enzyme in control tissues lost 30 to 60% of its activity. There was a profound deficiency of beta-galactosidase in the tissues of the patient. Michaelis constant, pH profiles, and cryostability were identical for the patient and control patients. Cathepsin B was moderately increased in activity in the patient's tissues.

Adult↗
PubMed2006

Production of galacto-oligosaccharides by immobilized recombinant beta-galactosidase from Aspergillus candidus.

The preparation of galacto-oligosaccharides (GOSs) was studied using the immobilized recombinant beta-galactosidase from Aspergillus candidus CGMCC3.2919. The optimal pH and temperature for the immobilized enzyme were observed at pH 6.5 and 40 degrees C, respectively. Increasing the initial lactose concentration increased the yield of GOSs. The dilution rate was found to be a key factor during the continuous production of GOSs. The maximum productivity, 87 g/L.h was reached when 400 g/L lactose was fed at dilution rate of 0.8/h. The maximum GOS yield reached 37% at dilution rate of 0.5/h. Continuous operation was maintained for 20 days in a packed-bed reactor without apparent decrease in GOS production. The average yield of GOSs was 32%, corresponding to the average productivity of 64 g/L.h, which implied that the immobilized recombinant beta-galactosidase has potential application for GOS production.

Aspergillus↗
PubMed2005

Folding of a misfolding-prone beta-galactosidase in absence of DnaK.

In absence of chaperone DnaK, bacterially produced misfolding-prone proteins aggregate into large inclusion bodies, but still a significant part of these polypeptides remains in the soluble cell fraction. The functional analysis of the model beta-galactosidase fusion protein VP1LAC produced in DnaK(-) cells has revealed that the soluble version exhibits important folding defects and that it is less stable and less active than when produced in wild-type DnaK(+) cells. In addition, we have observed that the induction of gene expression at the very late exponential phase enhances twofold the stability of VP1LAC, a fact that in DnaK(-) background results in a dramatic increase of its specific activity up to phenotypically detectable levels. These results indicate that the chaperone DnaK is critical for the folding of misfolding-prone proteins and also that the soluble form reached in its absence by a fraction of polypeptides is not necessarily supportive of biological activity. In the case of E. coli beta-galactosidase, the catalytic activity requires assembling into tetramers and the fine organization of the activating interfaces holding the active sites, what might not be properly reached in absence of DnaK.

Enzyme Activation↗
PubMed1997

Effect of bacterial galactosidase treatment on the nutritional status of soybean seeds and its milk derivative.

Four field strains of Lactobacillus plantarum (LS 4, 19, 21, 133) obtained from fufu (a semi-solid product obtained by boiling fermented cassava--Manihot esculenta Crantz) and a type strain DSM 2017 were grown on different carbon sources to induce galactosidase production. LS 21 produced the highest concentration of alpha- and beta-galactosidase with 0.28 mumol/l and 0.28 mumol/l respectively on lactose and galactose. Milk obtained from soybean seeds treated with the enzyme mixture for 24 h showed a 99, 98 and 96% reduction respectively in the raffinose, stachyose and sucrose content when compared with the dry soybean seed. Glucose and galactose which were not detected in the dry seeds became readily available after soaking in both enzyme mixture and distilled water. Although there was reduction in the nutritional composition of both milk samples, reduction of phytic acid and trypsin inhibitor is beneficial to the consumers. The result of the sensory evaluation showed that the milk prepared from enzyme-treated soybean seeds was rated better in terms of flavour, texture, appearance and palatability.

Dietary Carbohydrates↗
PubMed2003

Fabry disease: characterization of alpha-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele.

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the gene encoding the lysosomal exoglycohydrolase, alpha-galactosidase A (alpha-Gal A; GLA). In two unrelated classically affected males, two alpha-Gal A missense mutations were identified: R112C + D313Y (c.334C>T + c.937G>T) and C172G + D313Y (c.514T>G + c.937G>T). The D313Y lesion was previously identified in classically affected males as the single mutation [Eng et al., 1993] or in cis with another missense mutation, D313Y + G411D (c.937G>T + c.1232G>A) [Guffon et al., 1998]. To determine whether the D313Y mutation was a deleterious mutation or a coding region sequence variant, the frequency of D313Y in normal X-chromosomes, as well as its enzymatic activity and subcellular localization in COS-7 cells was determined. D313Y occurred in 0.45% of 883 normal X-chromosomes, while the R112C, C172G, and G411D missense mutations were not detected in over 500 normal X-chromosomes. Expression of D313Y in COS-7 cells resulted in approximately 60% of wild-type enzymatic activity and showed lysosomal localization, while R112C, C172G, G411D, and the double-mutated constructs had markedly reduced or no detectable activity and were all retained in the endoplasmic reticulum. The expressed D313Y enzyme was stable at lysosomal pH (pH 4.6), while at neutral pH (pH 7.4), it had decreased activity. A molecular homology model of human alpha-Gal A, based on the X-ray crystal structure of chicken alpha-galactosidase B (alpha-Gal B; alpha-N-acetylgalactosaminidase) was generated [Garman et al., 2002], which provided evidence that D313Y did not markedly disrupt the alpha-Gal A enzyme structure. Thus, D313Y is a rare exonic variant with about 60% of wild-type activity in vitro and reduced activity at neutral pH, resulting in low plasma alpha-Gal A activity.

Alleles↗
PubMed1983

72 residues of gal repressor fused to beta-galactosidase repress the gal operon of E. coli.

An active gene has been constructed which produces a chimera consisting of the N-terminal domain of the gal repressor and all but the first five residues of beta-galactosidase. Seventy two residues of gal repressor fused to beta-galactosidase as tetrameric core are sufficient to repress the gal operon in vivo and to bind to the gal operator in vitro.

Amino Acid Sequence↗
PubMed1996

Effect of medium components and metabolic inhibitors on beta-galactosidase production and secretion by Penicillium notatum 1.

Factors affecting the beta-galactosidase production by Penicillium notatum 1 were studied using fermentation media of different chemical composition. The medium containing lactose, salts, peptone and yeast extract with initial pH 2.5 was selected as the best for enzyme production. Monobasic ammonium phosphate (0.9%) was found to be the best inorganic nitrogen source for lactase production. Various extraction media and metabolic inhibitors were examined for effective releasing of beta-galactosidase from the fungal cells. Using a simple method of mycelium extraction with 0.1 Triton X-100, it was possible to obtain about 4-fold higher amounts of enzyme in the cell free extracts, than those excreted into the post-culture liquid.

Culture Media↗
PubMed2002

'Senescence-associated' beta-galactosidase activity in the upper gastrointestinal tract.

Beta-galactosidase activity at pH 6 is associated in vitro with senescence and cellular death, but in vivo data are sparse. This study undertook firstly to map 'senescence-associated' beta-galactosidase activity (SAbetaG) at pH 6 in normal epithelia and mucosae of the upper gastrointestinal tract. As escape from senescence confers a proliferative advantage, a reduction in SAbetaG activity might be predicted in neoplasia and their precursors in vivo. This prediction was tested in metaplastic, dysplastic, and neoplastic epithelium of the upper gastrointestinal tract. Histochemical staining for SAbetaG was performed at pH 6 on cryostat sections of 350 endoscopic biopsies from sites including oesophagus, stomach, and duodenum of 46 patients: 28 with Barrett's oesophagus (two with adenocarcinoma), 15 with gastric adenocarcinoma, and three with oesophageal squamous cancer. A staining score (range 0-6) was assigned to epithelial cells in all mucosae and scores were calculated for surface (luminal), intermediate, and deep (basal) layers. The strongest SAbetaG activity was in surface luminal cells of normal duodenal mucosa (mean score 3.6+/-0.5; n=19), 'specialized' Barrett's mucosa (mean 2.2+/-0.12; n=105), and intestinal metaplasia in the stomach (mean 2.4+/-0.40; n=16). Squamous epithelium was consistently negative for SAbetaG activity. Low- and high-grade Barrett's dysplasia showed no decrease in SAbetaG activity, but reduced activity was seen in gastric and oesophageal adenocarcinomas (mean 1.24+/-0.29; n=17; p=0.012). In six gastric adenocarcinomas, there was no detectable activity. Whether SAbetaG is truly a marker of cellular senescence in vivo remains to be determined. Activity is low in mucosal proliferation compartments and increases with cellular differentiation, especially in native or metaplastic intestinal mucosae. SAbetaG activity persists in dysplastic mucosae but may show some reduction or loss in adenocarcinomas (p=0.0012). Loss of SAbetaG activity is not, therefore, an early event in glandular dysplasia-neoplasia of the upper gastrointestinal tract.

Adenocarcinoma↗
PubMed2000

The carboxyl terminus of coffee bean alpha-galactosidase is critical for enzyme activity.

The role of the carboxyl (C)-terminal region of coffee bean alpha-galactosidase (alpha-GAL) has been studied by expressing C-terminal deletion mutants in the methylotrophic yeast strain Pichia pastoris. A previous study of human alpha-galactosidase determined that enzyme activity increased when up to 10 amino acid residues were deleted. Deleting 11 residues reduced activity, and deleting 12 residues abolished activity. In our studies, alpha-GAL activity is reduced when one or two amino acids are deleted, as is enzyme secretion directed by P. pastoris signal sequences. The pH profile is similar to that of the wild-type enzyme. Deleting 3 or more residues from the C-terminal end results in a complete loss of both enzyme secretion and activity. The C-terminus of alpha-GAL seems to play an important role in overall enzyme conformation and may directly affect the proper conformation of the active site.

Amino Acid Sequence↗
PubMed1994

Differential suppression of background mammalian lysosomal beta-galactosidase increases the detection sensitivity of LacZ-marked leukemic cells.

A method is described for the detection of Escherichia coli beta-galactosidase-expressing leukemic cells in ex vivo bone marrow samples. 4-Methylumbelliferyl-beta-D-galactopyranoside is used as a substrate in a kinetic assay. D-Galactose is used to suppress endogenous lysosomal beta-galactosidase activity, yielding a sixfold increase in sensitivity. With this assay, the detection limit is one leukemic cell per 10(4) normal bone marrow cells.

Animals↗
PubMed1995

Fluorescence-based enzymatic assay by capillary electrophoresis laser-induced fluorescence detection for the determination of a few beta-galactosidase molecules.

beta-Galactosidase can be assayed by monitoring the generation of the fluorescent products, fluorescein-mono-beta-D-galactopyranoside and fluorescein, when the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside is used. We have used capillary electrophoresis with ultrasensitive laser-induced fluorescence detection to monitor the formation of the fluorescent products of off-column enzymatic reactions. By analyzing as little as 40 pl of the enzymatic mixture we obtain limits of detection of 6.5 x 10(-14) M beta-galactosidase or 1.6 molecules based on the detection of fluorescein-mono-beta-D-galactopyranoside.

Electrophoresis↗
PubMed1993

Characterization of a mutant alpha-galactosidase gene product for the late-onset cardiac form of Fabry disease.

The expression product of a mutant alpha-galactosidase gene, Q279E (279Gln-->Glu), found in an atypical variant (cardiac form) of Fabry disease, was purified and characterized. It had kinetic properties similar to those of normal alpha-galactosidase, but was markedly thermolabile at neutral pH. Galactose and melibiose at high concentrations stabilized the mutant enzyme in vitro. Its catalytic activity was 15% of that for the normal enzyme, when it was expressed in COS-1 cells at 37 degrees C. The activity increased at 30 degrees C or in the presence of galactose at 37 degrees C. An increase was also observed in lymphoblasts from a patient with this mutation in the presence of galactose or melibiose. We conclude that this mutant protein is posttranslationally inactivated under the neutral conditions in the cells. The possibility of a new therapeutic approach is suggested.

Age of Onset↗
PubMed1995

Galactose stabilizes various missense mutants of alpha-galactosidase in Fabry disease.

The effect of galactose on alpha-galactosidase missense mutants causing Fabry disease was investigated in the COS-1 cell expression system and lymphoblasts. Three mutant enzymes, A156V, L166V and Q279E, showed increases in activity and amount in COS-1 cells cultured with galactose. Another mutant without catalytic activity, C142Y, did not show any changes. In lymphoblasts cultured with galactose, the enzyme activity increased significantly in four classical Fabry patients with the respective mutations, A156V, L166V, G260A and G373S, and in three atypical Fabry patients with the respective mutations, Q279E, R301Q and M296I. Such an increase was not observed in the other four classical Fabry patients, with C142Y, E66Q/R112C, G328R and N320K, respectively. This suggests that many missense mutations in the alpha-galactosidase gene causing Fabry disease allow the expression of catalytically active mutant enzymes regardless of the clinical phenotype, which are rapidly degraded under physiological conditions and stabilized by galactose.

Amino Acid Sequence↗
PubMed1996

Aggregation of the inactive form of human alpha-galactosidase in the endoplasmic reticulum.

35S-Labeled mutant alpha-galactosidases (Q279E and R301Q) expressed in COS1 cells were detected as two forms (46-kDa protein and gel-top aggregate) on SDS-polyacrylamide gel electrophoresis under nonreducing conditions. The 46-kDa protein disappeared rapidly, but the aggregate decreased slowly. The accumulation of aggregate was observed in COS1 cells expressing either Q279E or R301Q on Western blot analysis. The aggregate was mainly recovered in the fraction extracted with 1% Triton X-100 and had no catalytic activity. The COS1 cells expressing Q279E were treated with 10 microgram/ml brefeldin A or 5 microM monensin. Treatment with brefeldin A caused a decrease in the alpha-galactosidase activity and an increase in the amount of aggregate, but the amount of aggregate did not change on monensin treatment.

Animals↗
PubMed1997

Recombinant Mycobacterium aurum expressing Escherichia coli beta-galactosidase in high throughput screening of antituberculosis drugs.

Mycobacterium aurum is considered a surrogate of M. tuberculosis and recently has been proposed as test organism in high throughput screening of antituberculosis drugs (3). In this investigation, we suggest use of a recombinant M. aurum expressing E. coli lacZ gene, in which beta-galactosidase production is the reporter system as recently reported by us (6). The assay is based on production of beta-galactosidase in presence of drugs during growth. Enzyme production was inhibited within 4 h by frontline antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, ofloxacin, and sparfloxacin at their MICs. The assay could be performed conveniently in 96-well microtiter plate format.

Antitubercular Agents↗
PubMed1998

2-Methoxyestradiol, an endogenous metabolite of estrogen, enhances apoptosis and beta-galactosidase expression in vascular endothelial cells.

2-Methoxyestradiol (2ME) is an endogenous metabolite of estradiol (E2) and is known to inhibit tumor angiogenesis. In the present study, the direct effects of 2ME on the vascular endothelial cells were examined. 2ME enhanced apoptosis and beta-galactosidase expression in bovine vascular endothelial cells. A nitric oxide (NO) donor S-nitroso-N-acetyl penicillamin (SNAP) also enhanced beta-galactosidase expression, suggesting a possible role of NO in mediating the action of 2ME. 2ME increased the cellular content of nitric oxide synthase (NOS) and the production of NO. In addition, 2ME altered the membrane localization pattern of NOS. These suggest that the effects of 2ME on apoptosis and senescence of vascular endothelial cells were mediated, at least partly, by NOS and NO.

2-Methoxyestradiol↗