Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of beta-galactosidase activity was 55 degrees C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04+/-0.26 mM, 28.8+/-0.2 micromol D-glucose released per min per mg protein, and 0.73+/-0.07 mM, 361+/-12 micromol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7+/-3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other beta-galactosidases from lactobacilli. Beta-galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.
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PMID:Characterization and molecular cloning of a heterodimeric beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22. 1722 58

Beta-galactosidase was induced in E. coli wild type strains ATCC 8677 and 35321 in the presence of various protease inhibitors. Single enzyme molecule assays were performed using a capillary electrophoresis based protocol. The presence of the protease inhibitors had a minimal effect on the average and distribution of single molecule activities. Two novel capillary electrophoresis based single enzyme molecule assays for beta-galactosidase were developed using DDAO-beta-D-galactopyranoside and fluorescein-beta-D-digalactopyranoside as substrates. Double incubations were performed on individual enzyme molecules to demonstrate the reproducibility of the assays. Assays performed on beta-galactosidase from strains 8677 and 35321 demonstrated that the relative activities of the enzyme for the different substrates differed between the strains. Sequencing showed that these two strains differ in their primary sequence by a single amino acid substitution in position 280, which is in the region of the active site.
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PMID:Single molecule assays of beta-galactosidase from two wild-type strains of E. coli: effects of protease inhibitors on microheterogeneity and different relative activities with differing substrates. 1725 28

Beta-galactosidase is a widely used reporter enzyme, but although several substrates are available for in vitro detection, its application for in vivo optical imaging remains a challenge. To obtain a probe suitable for in vivo use, we modified our previously developed activatable fluorescence probe, TG-betaGal (J. Am. Chem. Soc. 2005, 127, 4888-4894), on the basis of photochemical and photophysical experiments. The new probe, AM-TG-betaGal, provides a dramatic fluorescence enhancement upon reaction with beta-galactosidase, and further hydrolysis of the ester moiety by ubiquitous intracellular esterases affords a hydrophilic product that is well retained within the cells without loss of fluorescence. We used a mouse tumor model to assess the practical utility of AM-TG-betaGal, after confirming that tumors in the model could be labeled with an avidin-beta-galactosidase conjugate. This conjugate was administered to the mice in vivo, followed by AM-TG-betaGal, and subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 microm. This strategy has potential clinical application, for example, in video-assisted laparoscopic tumor resection.
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PMID:An enzymatically activated fluorescence probe for targeted tumor imaging. 1735 71

Beta-galactosidase, commonly named lactase, is one of the most important enzymes used in dairy processing; it catalyzes the hydrolysis of lactose to its constituent monosaccharides glucose and galactose. Here, a thermostable beta-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. subtilis WB600. The recombinant enzyme was purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques. The purified beta-galactosidase appeared as a single protein band in sodium dodecyl sulfate-PAGE gel with a molecular mass of approximately 70 kDa. Its isoelectric point, determined by polyacryl-amide gel isoelectric focusing, was close to 5.1. The optimum temperature and pH for this beta-galactosidase activity were 70 degrees C and pH 7.0, respectively. Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70 degrees C were 50 and 9 h, respectively, and the K(m) and V(max) values were 2.96 mM and 6.62 micromol/min per mg. Metal cations and EDTA could not activate this thermostable enzyme, and some divalent metal ions, namely, Fe(2+), Zn(2+), Cu(2+), Pb(2+), and Sn(2+), inhibited its activity. Thiol reagents had no effect on the enzyme activity, and sulfhydryl group blocking reagents inactivated the enzyme. This enzyme possessed a high level of transgalactosylation activity in hydrolysis of lactose in milk. The results suggest that this recombinant thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galactooligosaccharides in milk processing.
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PMID:Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus. 1842 Jun 5

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3-8 codon minigenes containing AGAAGA codons before the stop codon. Beta-galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, beta-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 microg/microl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of beta-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNA(Arg4). These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA.
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PMID:Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon. 1858 64

The PentC promoter of the entCEBA operon encoding enzymes for enterobactin biosynthesis in Escherichia coli is tightly regulated by the availability of iron in the culture medium. In iron-rich conditions, the PentC promoter activity is strongly repressed by the global transcription regulator Fur (ferric uptake regulator), which complexes with ferrous ions and binds to the Fur box 19-bp inverted repeat. In this study, we have constructed the expression vector pOS2 containing the PentC promoter and characterized its repression, induction, and modulation by quantifying the expression of the lacZ reporter gene encoding beta- galactosidase. Beta-galactosidase activities of E. coli transformants harboring pOS2-lacZ were highly induced in the presence of divalent metal ion chelators such as 2,2'-dipyridyl and EDTA, and were strongly repressed in the presence of excess iron. It was also shown that the basal level beta-galactosidase expression by the PentC promoter was drastically decreased by incorporating the fur gene into the expression vector. Since the newly developed iron chelator-inducible expression system is efficient and cost-effective, it has wide applications in recombinant protein production.
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PMID:Iron chelator-inducible expression system for Escherichia coli. 1875 94

A rapid and convenient assay system was developed to detect viable Escherichia coli in water. The target bacteria were recovered from solution by immunomagnetic separation and incubated in tryptic soy broth with isopropyl-beta-D-thiogalactopyranoside, which induces formation of beta-galactosidase in viable bacteria. Lysozyme was used to lyse E. coli cells and release the beta-galactosidase. Beta-galactosidase converted 4-methylumbelliferyl-beta-D-galactoside to 4-methylumbelliferone (4-MU), which was measured by fluorescence spectrophotometry using excitation and emission wavelengths of 355 and 460 nm, respectively. Calibration graphs of 4-MU fluorescence intensity versus E. coli concentration showed a detection range between 8 x 10(4) and 1.6 x 10(7) cfu mL(-1), with a total analysis time of less than 3 h. The advantage of this method is that it detects viable cells because it is based on the activity of the enzyme intrinsic to live E. coli.
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PMID:Determination of viable Escherichia coli using antibody-coated paramagnetic beads with fluorescence detection. 1909 33

TDP-43 is a DNA/RNA-binding protein implicated in multiple steps of transcriptional and post-transcriptional regulation of gene expression. Alteration of this multifunctional protein is associated with a number of neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin positive inclusions. Whereas a pathological link to neurodegenerative disorders has been established, the cellular and physiological functions of TDP-43 remain unknown. In this study, we show that TDP-43 is a nuclear protein with persistent high-level expression during embryonic development and with progressively decreased protein levels during postnatal development. In mice where the TDP-43 gene (Tardbp) was disrupted using a gene trap that carries a beta-galactosidase marker gene, heterozygous (Tardbp(+/-)) mice are fertile and healthy, but intercrosses of Tardbp(+/-) mice yielded no viable homozygotic null (Tardbp(-/-)) mice. Indeed, Tardbp(-/-) embryos die between 3.5 and 8.5 days of development. Tardbp(-/-) blastocysts grown in cell culture display abnormal expansion of their inner cell mass. The pattern of beta-galactosidase staining at E9.5 Tardbp(+/-) embryos is predominantly restricted to the neuroepithelium and remains prominent in neural progenitors at E10.5-12.5. TDP-43 is detected in spinal cord progenitors and in differentiated motor neurons as well as in the dorsal root ganglia at E12.5. Beta-galactosidase staining of tissues from adult Tardbp(+/-) mice shows widespread expression of TDP-43, including prominent levels in various regions of the central nervous system afflicted in neurodegenerative disorders. These results indicate that TDP-43 is developmentally regulated and indispensible for early embryonic development.
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PMID:TDP-43 is a developmentally regulated protein essential for early embryonic development. 2004 Jun 2

Beta-galactosidase of Aspergillus sp. AF crude homogenate was immobilized in Ca-alginate gel beads and used for the production of galactooligosaccharides (GOS) from lactose. Optimum pH and temperature, thermal and storage stability of the enzyme activity were investigated and compared with those of the free enzyme. The study on the improvement of mechanical strength of the alginate beads was carried out through various methods, which demonstrates that the hardening process, where the alginate beads were treated with 0.225 M CaCl(2) solution after three batches to compensate the lost of calcium in the beads, provided a high mechanical stability for repeated use in large-scale production. The experiment results show that GOS yield increased with the increase of lactose concentration, and also increased with excessive addition of lactose (exceeding its solubility) at the beginning of the reaction. The immobilization of beta-galactosidase of Aspergillus sp. AF crude homogenate is cheap in processing cost and easy to carry out, and the immobilized enzyme possesses high performance for industrial application.
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PMID:Stabilities of immobilized beta-galactosidase of Aspergillus sp. AF for the optimal production of galactooligosaccharides from lactose. 2008

Cerebral amyloid angiopathy (CAA) caused by amyloid beta (Abeta) deposition around brain microvessels results in vascular degenerative changes. Antiangiogenic Abeta properties are known to contribute to the compromised cerebrovascular architecture. Here we hypothesize that Abeta peptides impair angiogenesis by causing endothelial cells to enter senescence at an early stage of vascular development. Wild-type (WT) Abeta and its mutated variant E22Q peptide, endowed with marked vascular tropism, were used in this study. In vivo, in zebrafish embryos, the WT or E22Q peptides reduced embryo survival with an IC(50) of 6.1 and 4.7 microM, respectively. The 2.5 microM concentration, showing minimal toxicity, was chosen. Alkaline phosphatase staining revealed disorganized vessel patterning, narrowing, and reduced branching of vessels. Beta-galactosidase staining and the cyclin-dependent kinase inhibitor p21 expression, indicative of senescence, were increased. In vitro, WT and E22Q reduced endothelial cell survival with an IC(50) of 12.3 and 8.8 microM, respectively. The 5 microM concentration, devoid of acute effects on the endothelium, was applied chronically to long-term cultured human umbilical vein endothelial cells (HUVECs). We observed reduced cumulative population doubling, which coincided with beta-galactosidase accumulation, down-regulation of telomerase reverse-transcriptase mRNA expression, decreased telomerase activity, and p21 activation. Senescent HUVECs showed marked angiogenesis impairment, as Abeta treatment reduced tube sprouting. The endothelial injuries caused by the E22Q peptide were much more aggressive than those induced by the WT peptide. Premature Abeta-induced senescence of the endothelium, producing progressive alterations of microvessel morphology and functions, may represent one of the underlying mechanisms for sporadic or heritable CAA.
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PMID:Abeta peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis. 2020 41


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