SEARCH · PubMed Health

Results for “GALACTOSIDASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 559 records · Page 31Linked to original sources

PubMed2005

Ground-state, transition-state, and metal-cation effects of the 2-hydroxyl group on beta-D-galactopyranosyl transfer catalyzed by beta-galactosidase (Escherichia coli, lac Z).

Substitution of the C2-OH group by C2-H at 4-nitrophenyl-beta-d-galactopyranoside to give 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside causes (1) a change in the rate-determining step for beta-galactosidase-catalyzed sugar hydrolysis from formation to breakdown of a covalent intermediate; (2) a 14 000-fold decrease in the second-order rate constant k(3)/K(d) for enzyme-catalyzed transfer of the beta-d-galactopyranosyl group from the substrate to form a covalent adduct to the enzyme; and (3) a larger 320 000-fold decrease in the first-order rate constant k(s) for hydrolysis of this covalent adduct. Only a small fraction (ca. 7%) of the 2-OH substituent effect is expressed in the ground-state Michaelis complex, so that the (apparent) strong interactions between the enzyme and 2-OH group that stabilize the transition state for beta-d-galactopyranosyl transfer only develop upon moving from the Michaelis complex to the transition state. Mg(2+) activates beta-galactosidase for cleavage of both 4-nitrophenyl-beta-d-galactopyranoside and 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside. This suggests that Mg(2+) activation does not involve interactions with the 2-OH group. The removal of Mg(2+) from beta-galactosidase causes a change in the rate-determining step for enzyme-catalyzed hydrolysis of 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside from breakdown to formation of the covalent intermediate. The observed 2-OH effect would require a very large (10-11 kcal/mol) stabilization of the transition state for beta-d-galactopyranosyl group transfer to water by interactions between beta-galactosidase and the neutral 2-OH group. We suggest that the apparent effect of the neutral substituent is more simply rationalized by ionization of the 2-OH to form a 2-O(-) anion, which provides effective electrostatic stabilization of the cationic transition state for glycoside cleavage at an active site of relatively low dielectric constant.

Deoxy Sugars↗

PubMedDate not supplied

Extractive cultivation of Escherichia coli using poly(ethylene glycol)/phosphate aqueous two-phase systems to produce intracellular beta-galactosidase.

Escherichia coli cells were found to grow in poly(ethylene glycol) (PEG)/phosphate aqueous two-phase systems by selecting the phase-forming components and their concentrations, the tie-line length, and the phase volume ratio properly. The cells cultivated up to an optical density at 660 nm of 1.0 were disrupted by ultrasonic irradiation to release and recover overproduced beta-galactosidase. The surviving cells were found to grow immediately after ultrasonic irradiation. The PEG/phosphate (KH2-PO4-K2HPO4) system with added Na2SO4 was the one optimized for extractive cultivation of E. coli cells, where beta-galactosidase was selectively partitioned to the top phase while total soluble proteins and cells partitioned to the bottom phase. This integrated process was extended to a semicontinuous operating mode, where the top phase containing beta-galactosidase was removed following intermittent ultrasonic irradiation and the bottom phase containing cells was recycled together with the new top phase solution to repeat production and recovery of beta-galactosidase.

Cell Division↗

PubMedDate not supplied

Protein purification by ultrafiltration using a beta-galactosidase fusion tag.

The use of beta-galactosidase (465 kDa) as a fusion tag for ultrafiltration-based protein purification has been investigated. The target protein studied was thermophilic glucose dehydrogenase (157 kDa, GDH) from Thermoplasma acidophilum. An expression vector was constructed comprising the lacZ gene fused to a factor Xa cleavage sequence that was attached to the 5' end of the GDH gene. This gene fusion was expressed in Escherichia coli JM109 to yield a soluble protein that exhibited activities for both enzymes. Cleavage of this fusion protein (622 kDa) by factor Xa gave two smaller proteins that showed individual beta-galactosidase and GDH activity. A two-stage diafiltration process for protein purification was used in an ultrafiltration stirred cell. In the first stage, a 500 kDa membrane was used to retain the fusion protein and transmit smaller E. coli host proteins. Approximately 80% of the GDH activity was retained in this step. Following cleavage, the second stage utilized a 300 kDa membrane to fractionate the beta-galactosidase and GDH. No beta-galactosidase was detected in the permeate solutions, and 97% of the GDH activity was recovered in the permeate.

Chemical Fractionation↗

PubMed2000

Isolation and characterization of the normal canine beta-galactosidase gene and its mutation in a dog model of GM1-gangliosidosis.

The acid beta-galactosidase cDNA of Portuguese Water dogs was isolated and sequenced. The entire coding region of the gene consists of 2004 nucleotides encoding a protein of 668 amino acids. Its encoding sequence indicates approximately 86.5% identity at the nucleotide level and about 81% identity at the amino acid level with the encoding region of the human acid beta-galactosidase gene. The deduced amino acid sequence contains a 24-amino-acid putative signal sequence, six possible glycosylation sites, and seven cysteine residues. A homozygous recessive mutation, causing canine GM1-gangliosidosis, was identified at nucleotide G200-->A in exon 2 resulting in an Arg60-->His (mutation R60H) amino acid substitution. The mutation creates a new restriction enzyme site for Pml1. Genotyping 115 dog samples for this acid beta-galactosidase gene alteration readily distinguished affected homozygous recessives (n=5), heterozygous carriers (n=50) and normal homozygotes (n=60). DNA mutation analysis provided a method more specific than enzyme assay of beta-galactosidase for determination of carriers.

Amino Acid Sequence↗

PubMed1998

One step partial purification of beta-D-galactosidase from Kluyveromyces marxianus CDB 002 using STREAMLINE-DEAE.

The intracellular enzyme beta-D-galactosidase provides interesting applications in the dairy industry, which are able to solve problems related to product processing, or can alleviate lactose intolerance in some populations. In order to obtain a technical enzyme, yeast cells of Kluyveromyces marxianus CDB 002 were disrupted by high pressure homogenization and an innovative chromatographic technique was tested for the recovery of beta-D-galactosidase. A STREAMLINE 25 column, containing 65 ml STREAMLINE-DEAE was equilibrated with 50 mM potassium phosphate buffer pH 7.5 at an upward flow of 250 cmh-1. 100-200 ml cell homogenate were applied onto the expanded gel. After unbound proteins and cellular debris were washed out, the bed was allowed to sediment and beta-D-galactosidase was eluted with a downward flow of 0.2 M NaCl in the same buffer. A 6-fold purification factor was achieved with 63% activity recovery, while removing cell debris at a single step, thus avoiding a centrifugation step. Concentration and volume of the applied sample affected purification and gel performance. The results presented show STREAMLINE-DEAE chromatography to be an interesting method for the production of beta-D-galactosidase as a technical enzyme, since it can also be applied on a large scale without much modification.

Biotechnology↗

PubMed1993

The effect of salts on the stability of beta-galactosidase in aqueous solution, as related to the water mobility.

The effect of salts (KI, KBr, NaCl, KCl, KF, phosphate, and Na2SO4) on the stability of beta-galactosidase in aqueous solution was studied from the aspect of changes in water mobility. At salt concentrations up to 200 mM, the inactivation rate of beta-galactosidase in all the salt solutions studied increased with increasing salt concentration. At higher concentrations, those salts which had little effect on the spin-lattice relaxation time, T1, of water (KI, KBr, and KCl) continued to increase the inactivation rate of beta-galactosidase with increasing concentration, while those salts which decreased the T1 of water (KF, phosphate, and Na2SO4) decreased the inactivation rate. It appeared that the decrease in water mobility caused by KF, phosphate, and Na2SO4 resulted in stabilization of beta-galactosidase. The results indicate that water mobility is an important factor in the denaturation rate of proteins.

Bromides↗

PubMed1993

Stability of beta-galactosidase, a model protein drug, is related to water mobility as measured by 17O nuclear magnetic resonance (NMR).

The inactivation of freeze-dried beta-galactosidase during storage was studied, focusing on the effect of water mobility as measured by the spin-lattice relaxation time, T1, of water using 17O NMR. Inactivation of beta-galactosidase lyophilized from phosphate buffer solution was studied as a function of water content, which in turn affected the T1 of water. An increase in the water content of freeze-dried beta-galactosidase brought about an increase in the T1 of water, as well as a rise in pH. For the freeze-dried enzyme with sufficient water content to be dissolved, the inactivation rate was related to the T1 of water rather than to the pH change. It is suggested that as the water content increases, the mobility of water around the enzyme increases, resulting in enhanced enzyme inactivation. The freeze-dried samples with limited moisture showed inactivation rates faster than those expected from the pH and water mobility, suggesting that the inactivation mechanism is different from that for the freeze-dried enzyme with a larger amount of water. Inactivation of beta-galactosidase in solutions was also studied as a function of phosphate buffer and sodium chloride concentrations, which in turn affected the T1 of water. Because the inactivation rate increased with increasing salt concentrations and the rate extrapolated to zero concentration was negligible, inactivation of the freeze-dried enzyme was apparently induced by the salts used as additives for lyophilization. The enhancing effect of phosphate buffer components, however, was reduced at higher concentrations, an effect related to the decrease in the T1 of water. This result may be ascribed to the decrease in water mobility caused by phosphate buffer components and is consistent with the observation that the inactivation rate of the freeze-dried enzyme with a relatively large amount of water decreased with decreasing T1 of water.

Aspergillus oryzae↗

PubMed1993

Aggregates formed during storage of beta-galactosidase in solution and in the freeze-dried state.

Aggregates formed during storage of freeze-dried beta-galactosidase were compared with those formed in solutions. Freeze-dried beta-galactosidase aggregated during storage in the presence of moisture, producing a protein precipitate which was soluble in guanidine hydrochloride solution but not in buffer solution. The protein precipitate dissolved in guanidine solution exhibited a large molecular size by high-performance size exclusion chromatography and converted to proteins of original size in the presence of dithiothreitol. It is suggested that the aggregation involves chemical interaction via covalent disulfide bonding. In contrast, beta-galactosidase in aqueous solution aggregated without formation of protein precipitates. Soluble aggregates were converted to proteins of original size in guanidine solution without dithiothreitol, suggesting noncovalent bonding. The difference in aggregation behavior may be ascribed to the difference in the water:protein ratio. We propose that inactivation of beta-galactosidase is due to formation of thermally denatured (unfolded) protein, which aggregates dependent on the water:protein ratio, either via noncovalent interactions at a high water:protein ratio in solution or via covalent interaction at a low water:protein ratio in the freeze-dried state.

Buffers↗

PubMed2003

Alpha and beta-galactosidase activities and oligosaccharide content in peanuts.

Thirty-three peanut cultivars were examined for their alpha-1,6 and beta-1,4 galactosidase activities and oligosaccharide contents along with proximate compositions. The average moisture, protein, fat, ash, and carbohydrate contents were: 4.9%, 26.6%, 43.1%, 2.3% and 23.1%, respectively. The corresponding coefficients of variation were: 5.2%, 10.1%, 7.2%, 7.8% and 15.7%, respectively. Raffinose and stachyose contents (%) ranged from 0.05 to 0.12 and 0.31 to 0.61, respectively. The specific activity (micromol product/min/mg protein) of crude preparation of alpha-galactosidase for the 33 cultivars ranged from 1.096 to 2.784 for the non-germinated seeds and from not being detected in some samples up to 2.432 for the germinated seeds; the mean values for non-germinated and germinated seeds were: 1.781 and 1.410, respectively. The specific activity of beta-galactosidase ranged from 0.101 to 1.727 in the non-germinated seeds and from not being detected in some samples up to 0.898 in the germinated seeds. Germination decreased the activity of both galactosidases significantly (p < or = 0.05).

Arachis↗

PubMed1999

Successful expression of beta-galactosidase and factor IX transgenes in fetal and neonatal sheep after ultrasound-guided percutaneous adenovirus vector administration into the umbilical vein.

In utero somatic gene therapy in the later stages of pregnancy may allow targeting of organ systems which are difficult to reach later in life and to prevent the development of tissue damage otherwise caused by the early onset of inherited diseases. We report here on the percutaneous delivery of two adenoviral vectors, containing the beta-galactosidase reporter gene and the human Factor IX gene respectively, to the fetal liver and circulation by ultrasound-guided umbilical vein puncture similar to procedures used in human pregnancy. Vector spread, as detected by PCR analysis for the beta-galactosidase encoding vector, was found in almost all fetal and neonatal organs and in the maternal liver. Expression of the beta-galactosidase transgene was detected in many fetal tissues by RT-PCR. High beta-galactosidase production was shown by immuno-histochemistry predominantly in the liver, where about 30percent of the hepatocytes stained positive, and in the adrenal cortex. Production of factor IX was determined by ELISA in the plasma of treated fetuses and newborn lambs and reached at birth up to 80percent of the normal human plasma concentration. This demonstrates a very hopeful proof of principle for the development of prenatal treatment of many genetic diseases but also requires more detailed investigations with respect to the observed systemic spread of the vector.

Adenoviridae↗

PubMed1967

Separation and properties of beta-galactosidase, beta-glucosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase from rat kidney.

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.

Animals↗

PubMed1967

Pool sizes of metabolic intermediates and their relation to glucose repression of beta-galactosidase synthesis in Escherichia coli.

1. The intermediary metabolism of two strains of Escherichia coli has been examined. One strain (Q22) exhibits acute transient repression of beta-galactosidase synthesis when glucose is supplied to cells growing on glycerol; the other strain (W3110) does not. The two strains do not differ genetically in their lac operons. 2. Strain Q22 uses about twice as much glucose as strain W3110 per unit of cell mass produced. 3. Pentose phosphate-cycle activity in the presence of glucose is much stronger in strain Q22 than in strain W3110. 4. In strain Q22 the pool sizes of glucose 6-phosphate, 6-phosphogluconate, fructose 1,6-diphosphate and NADPH increase when glucose is added to cells growing on glycerol, and beta-galactosidase synthesis is severely inhibited. After about 1hr. the synthesis of beta-galactosidase is partly resumed, and the pool sizes of the four compounds fall. ATP, NADH and several other phosphorylated compounds show no concentration changes. 5. These concentration changes do not occur in strain W3110, in which beta-galactosidase synthesis is only rather weakly repressed by glucose. 6. It is suggested that repression of enzyme synthesis by glucose requires the rapid operation of the pentose phosphate cycle, and is mediated by one of the four substances whose concentration rises and later falls in strain Q22. A definite choice of effector from among these four possibilities cannot at present be made.

Adenosine Triphosphate↗

PubMed1968

Rat small-intestinal beta-galactosidases. Separation by ion-exchange chromatography and gel filtration.

1. The chromatography of rat small-intestinal beta-galactosidase activities on gel-filtration and ion-exchange columns has been studied. Five different substrates were used to measure beta-galactosidase activity (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside) and the activity was measured at one acid and one more neutral pH value. 2. By gel filtration one acid beta-galactosidase, hydrolysing lactose and the hetero-beta-galactosides at about the same rate, and one more neutral beta-galactosidase, hydrolysing lactose much more rapidly than the hetero-beta-galactosides, were separated. 3. By ion-exchange chromatography the acid enzyme was fractionated into two components. These may be individual enzymes or different forms of the same enzyme.

Animals↗

PubMed1970

Enzymes of Trichomonas foetus. Separation and properties of two beta-galactosidases.

The beta-galactosidase activity in extracts of Trichomonas foetus is separable into two fractions by gel filtration on Sephadex G-200. When o-nitrophenyl beta-d-galactoside is used as substrate the first fraction to be eluted, beta-galactosidase 1, has 50 times the activity (units per mg of protein) of the crude preparation. This fraction is activated by Mn(2+) and Co(2+) and inhibited by Hg(2+) and EDTA. In the presence of Mn(2+) the pH optimum for the hydrolysis of o-nitrophenyl beta-d-galactoside or lactose is 5.8-6.0. beta-Galactosidase 1 is an exoglycosidase that releases beta-linked galactose joined to aliphatic and various carbohydrate aglycones. Hydrolysis is prevented, however, by a substituent on either the subterminal sugar or the terminal non-reducing beta-galactosyl residue in an oligosaccharide. The second fraction, beta-galactosidase 2, is not activated by metal ions or inhibited by EDTA and has a broad pH optimum from 4.5 to 6.0.

Chromatography, Gel↗

PubMed2003

Isolation and characterization of psychrophiles producing cold-active beta-galactosidase.

AIMS: The present study was conducted to screen for psychrophilic micro-organisms that are able to hydrolyse lactose at low temperature, and to examine the cold-active beta-galactosidase produced by the isolated psychrophilic micro-organisms. METHODS AND RESULTS: Psychrophilic bacteria, which grow on lactose as a sole carbon source, were isolated from soil from Hokkaido, Japan. The phenotype and sequence of 16S rDNA of the isolated strains indicated a taxonomic affiliation to Arthrobacter psychrolactophilus. The isolated A. psychrolactophilus strains were able to grow on lactose at below 5 degrees C, and showed cold-active beta-galactosidase activity, which was highly specific at even 0 degrees C. CONCLUSIONS: Facts in this study may indicate the possibility that the isolated strains produce novel beta-galactosidases that are able to hydrolyse lactose at low temperature, although some strains have isozymes. SIGNIFICANCE AND IMPACT OF THE STUDY: It may be possible that the cold active beta-galactosidases from the isolated strains can be applied to the food industry, e.g. processing of milk and whey below 5 degrees C.

Arthrobacter↗

PubMed2003

Growth and beta-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure.

AIMS: To investigate the effect of total air pressure raise on cell growth and intracellular beta-galactosidase activity in batch cultures of Kluyveromyces marxianus CBS 7894. METHODS AND RESULTS: A pressurized bioreactor was used for K. marxianus batch cultivation under increased air pressure from 1.2 to 6 bar. Under these conditions no inhibition of cell growth was observed. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization led to an enhancement of the cell growth rate obtained at atmospheric pressure without aeration. The specific beta-galactosidase productivity increased from 5.8 to 17.0 U gCD-1 h-1 using a 6-bar air pressure instead of air at atmospheric pressure. The antioxidant enzyme superoxide dismutase (SOD) was slightly induced by the air pressure raise, which indicates that the defensive mechanisms of the cells can cope with an air pressure up to 6 bar. CONCLUSIONS: These experiments showed that the increase of air pressure up to 6 bar is an alternative to other methods of preventing the oxygen limitation and can be applied in the beta-galactosidase production by K. marxianus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results here reported proved that, in what biological aspects are concerned, it is possible to use the air pressure increase as an optimization parameter of beta-galactosidase production in high-density cell cultures of K. marxianus strains.

Air Pressure↗

PubMed1981

Three cases of beta-galactosidase deficiency.

A clinical description of three cases of beta-galactosidase deficiency is presented. Two cases are classical for infant type GM1-gangliosidosis, the third is characterised by dysostosis multiplex with growth retardation and of normal intelligence. Laboratory data revealed mucopolysaccharides in the urine of all described patients in a high level of normal values and increased excretion of oligosaccharides in the urine. Both patients with a classical picture of GM1-gangliosidosis have a very low activity of beta-galactosidase, activity of beta-galactosidase in their parents cells is diminished by 50% of control. In the third nontypical case enzyme deficiency was less expressed. These cases emphasize the variability of the clinical expression in beta-galactosidase deficiency.

Child↗

PubMed1973

Evolution of a second gene for beta-galactosidase in Escherichia coli.

Mutants of E. coli K12 with deletions of the beta-galactosidase gene (lacZ) can reacquire the ability to hydrolyze beta-galactosides during prolonged intense selection for growth on lactose. Full lactose competence is restored through a sequence of at least five mutations. Cell extracts of these derived strains hydrolyze o-nitrophenyl-beta-D-galactoside, the standard substrate for assay of beta-galactosidase. The enzyme responsible for this activity differs in its immunological, kinetic, and sedimentation characteristics from the lacZ beta-galactosidase of wild-type E. coli. Its genetic determinant, designated ebg-5, maps at 59 min on the E. coli chromosome, whereas the lac operon maps at 10 min. We suggest that a gene not involved in lactose utilization has been progressively changed into a form capable of specifying a beta-galactosidase and that this process is similar to that whereby genes with new functions are evolved by natural selection.

Centrifugation, Density Gradient↗