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)

A single-stage fed-batch bioprocess for the production of a recombinant protein beta-galactosidase, by E. coli has been developed. The XL1-blue strain of E. coli which harbors a multi-number foreign plasmid PT was cultured in a reformulated medium. Critical medium components were selected and their respective concentrations were optimized with the Orthogonal Table method. An exponential substrate feeding schedule was used to maintain optimum conditions. Inhibition of growth and protein expression caused by excessive concentrations of glucose and acetate was investigated and subsequently minimized with an incremental nutrient feeding schedule which limited the specific growth rate of a culture. The program necessary to facilitate the control of substrate addition is fully described. This program has been used with a 2.5 l bioreactor and a commercially available software package for optimization without on-line or off-line measurement of optical density (OD), CO2, glucose or acetate. The optimized fed-batch process limited the acetate concentration to less than 20 mM; maintained an exponential growth phase for 50 h; and produced a cell density of 51 g l-1 dry cell weight (DCW) or 154 OD600 with a beta-galactosidase activity of 990 U ml-1.
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PMID:Optimization of a cultivation process for recombinant protein production by Escherichia coli. 136 46

Defined minimal media conditions were used to assess and subsequently enhance the production of subtilisin by genetically characterized Bacillus subtilis strains. Subtilisin production was initiated by the exhaustion or limitation of ammonium in batch and fed-batch cultures. Expression of the subtilisin gene (aprE) was monitored with a chromosomal aprE::lacZ gene fusion. The beta-galactosidase production driven by this fusion reflected subtilisin accumulation in the culture medium. Subtilisin gene expression was temporally extended in sporulation-deficient strains (spoIIG), relative to co-genic sporogenous strains, resulting in enhanced subtilisin production. Ammonium exhaustion not only triggered subtilisin production in asporogenous spoIIG mutants but also shifted carbon metabolism from acetate production to acetate uptake and resulted in the formation of multiple septa in a significant fraction of the cell population. Fed-batch culture techniques, employing the spoIIG strain, were investigated as a means to further extend subtilisin production. The constant provision of ammonium resulted in linear growth, with doubling times of 11 and 36 h in each of two independent experiments. At the lower growth rate, the responses elicited (subtilisin production, glucose metabolism, and morphological changes) during the feeding regime closely approximated the ammonium starvation response, while at the higher growth rate a partial starvation response was observed.
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PMID:Physiological and genetic strategies for enhanced subtilisin production by Bacillus subtilis. 136 58

Sugar specific lectins (PNA, RCA I, LPA, SBA, DBA, GSA IB4, GSA II, WGA, LTA, UEA I, Con A, LCA) with and without prior selective glycosidase digestion (sialidase, alpha-fucosidase, alpha-mannosidase, beta-N-acetylglucosaminidase, alpha- and beta-galactosidase, beta-glucosidase) were used in order to investigate the distribution of native accessible carbohydrates and obtain information dealing with the composition of terminal disaccharides within glycoconjugates present in acinar compartments and ductal segments of mammalian (mouse, rat, hare, and rabbit) parotid glands. Glycoconjugates containing variable amounts of mannose, glucose, N-acetylgalactosamine and N-acetylglucosamine were present in the parotid glands of all species. However, these carbohydrate chains exhibited a different composition of terminal sequences within each type of gland. For example, sialylated components having the terminal dimers sialic acid-galactose and sialic acid-N-acetylgalactosamine were found in all acinar cells, whereas fucoglycoconjugates with terminal disaccharide fucose-galactose were localized in the rat striated ducts and hare acinar cells. The terminal sequence alpha-galactose-beta-galactose was demonstrated in the mouse acinar cells. Finally, glycoconjugates characterized by the terminal dimer beta-galactose-N-acetylgalactosamine were demonstrated in the mouse acinar and ductal cells and the rat ductal ones. Thus, present findings outlined and further confirmed the possibility to elucidate the oligosaccharide structure in situ using lectin histochemistry combined with enzymatic degradation.
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PMID:Glycoconjugate composition of mammalian parotid glands elucidated in situ by lectins and glycosidases. 137 7

Lactase-phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl beta-lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO-3' and HO-2' are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical beta-galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4' in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO-6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin-hydrolase activity has also been developed.
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PMID:Substrate specificity of small-intestinal lactase. Assessment of the role of the substrate hydroxyl groups. 139 15

Kinetic parameters for the hydrolysis of a series of deoxy and deoxyfluoro analogues of 2',4'-dinitrophenyl beta-D-galactopyranoside by Escherichia coli (lacZ) beta-galactosidase have been determined and rates found to be two to nine orders of magnitude lower than that for the parent compound. These large rate reductions result primarily from the loss of transition-state binding interactions due to the replacement of sugar hydroxy groups, and such interactions are estimated to contribute at least 16.7 kJ (4 kcal).mol-1 to binding at the 3, 4 and 6 positions and more than 33.5 kJ (8 kcal).mol-1 at the 2 position. The existence of a linear free-energy relationship between log(kcat./Km) for these compounds and the logarithm of the first-order rate constant for their spontaneous hydrolysis demonstrates that electronic effects are also important and provides direct evidence for oxocarbonium ion character in the enzymic transition state. A covalent intermediate which turns over only extremely slowly (t1/2 = 45 h) accumulates during hydrolysis of the 2-deoxyfluorogalactoside, and kinetic parameters for its formation have been determined. This intermediate is nonetheless catalytically competent, since it re-activates much more rapidly in the presence of the transglycosylation acceptors methanol or glucose, thereby providing support for the notion of a covalent intermediate during hydrolysis of the parent substrates.
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PMID:Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) beta-galactosidase. 141 31

Promastigote culture forms of the log growth phase of Leishmania donovani stock LRC L 51 were investigated for expression of cell-surface carbohydrate-binding sites using 15 types of a chemically glycosylated enzyme termed neoglycoenzyme. Carbohydrate conjugation and coupling yield were kept constant to ensure that the type of carbohydrate moiety was the only variable feature of the applied tools. Para-aminophenyl derivatives of the following carbohydrate residues were used for the glycosylation of beta-galactosidase from Escherichia coli: beta-D-lactose, beta-D-thiogalactose, alpha-D-mannose, alpha-L-rhamnose, alpha-D-N-acetylgalactosamine, beta-D-N-acetylgalactosamine, beta-D-N-acetylglucosamine, the alpha- and beta-glucosides maltose and cellobiose, beta-D-xylose, alpha-D-mannose-6-phosphate, the alpha-galactoside melibiose, alpha-L-fucose, and beta-D-glucuronic acid as well as sialic acid. Only melibiose, fucose, and glucuronic acid showed no binding affinity for the cultured flagellates; this served as an internal control reaction to exclude any binding to the linker group. This result demonstrates that many but not all sugar types can be recognized by appropriate receptor structure(s) on the surface of the promastigote Leishmania. Transformation of the binding data for neoglycoenzymes exposing lactose, mannose, rhamnose, and N-acetylated hexose residues, which was carried out to obtain the dissociation constants and to estimate the number of binding sites at saturation, revealed KD values of around 100 mM and around 10(4) binding sites for the polyvalent ligands.
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PMID:Detection and quantitation of cell-surface sugar receptor(s) of Leishmania donovani by application of neoglycoenzymes. 143 41

The beta-galactosidase activities arising from Tn5lac insertions in several genes required for antibiotic TA production were measured under different growth conditions. In all of the non-TA-producing mutants, the beta-galactosidase specific activity was higher when the cells were grown in nutrient-limited 0.5CTS medium (0.5% Casitone plus alanine, serine, and glucose) than in rich 2CT medium (2% Casitone). One of the mutants, 420, had low beta-galactosidase specific activity in both media. The other seven mutants containing inserts in genes essential for TA production had specific activities of 139 to 367 U/mg of protein in 0.5CTS medium and 11 to 48 U/mg of protein in 2CT medium. The beta-galactosidase specific activities of two strains, 1030 and 420, increased during exponential growth in 0.5CTS medium. The beta-galactosidase specific activities of both strains increased greatly when the cells were grown in the presence of magnesium phosphate, which traps ammonium ions. The Tn5lac insertions in 1030 and 420 were used to screen for mutants with increased levels of transcription. An N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in 1030 that mapped 17 kb from the omega 1010 insert increased the specific activity of beta-galactosidase 21 times in 2CT medium. The regulatory mutation appears to release the repression caused by 2CT medium. A UV-induced mutation in 420 increased the beta-galactosidase specific activity 1.4 to 2.4 times. Medium conditions that affect the transcription of TA genes are discussed in terms of enhanced antibiotic TA production.
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PMID:Use of Tn5lac to study expression of genes required for production of the antibiotic TA. 144 12

The production of protective antigen (PA), the common component of the two anthrax toxins, is influenced by the environment. In order to examine factors involved in its regulation, a transcriptional fusion between the promoter region of the PA gene (pag) and the lacZ gene was constructed and introduced into Bacillus anthracis Sterne. Activity of the pag promoter was followed by measuring beta-galactosidase activities under various growth and medium conditions. Expression from the pag promoter was observed throughout exponential-phase and was maximal in early stationary phase. The activity of the pag promoter was stimulated by the addition of glucose in the medium.
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PMID:Regulation of pag gene expression in Bacillus anthracis: use of a pag-lacZ transcriptional fusion. 145 23

Previous work in our laboratory has shown that the 5' nontranscribed promoter region of the gene for ribosomal protein (rp) S16A-1 of Saccharomyces cerevisiae, when fused to a lacZ gene, is necessary and sufficient to cause an increase in expression of the heterologous lacZ gene fusion product after cells have been shifted from a glycerol to glucose carbon source. This increase in expression is characteristic of that observed with the native rp gene. We have sought to define more precisely those areas of the promoter that may be involved in the differential expression/regulation of RPS16A-1 when host cells are subjected to a variety of nutritional environments. It has already been demonstrated by others that the promoter regions of most rp genes contain at least one consensus element, designated UASrpg, which is necessary for the transcriptional activation and maintenance of expression of the gene during steady-state growth in rich media. Our main experimental approach has been to create a series of 5' end deletions in the promoter region of RPS16A-1. The individual truncated promoter fragments were then ligated to a lacZ fusion reporter construct. By assaying the cells for production of beta-galactosidase and determining the abundance of lacZ mRNA, we have been able to determined the extent of fusion product expression. We assayed cells under three physiological conditions: steady-state growth in glucose, steady-state growth in glycerol and during sporulation. We report four main findings of our work.
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PMID:The role of promoter elements of a ribosomal protein gene in Saccharomyces cerevisiae under various physiological conditions. 149 81

A well-defined set of isogenic yeast strains has been constructed whereby each strain contains a different LPD::lacZ gene fusion integrated at the ura3 locus. These LPD::lacZ fusions differ in the amount of the LPD1 gene (encoding lipoamide dehydrogenase) that is fused to the lacZ reporter. Comparison of the beta-galactosidase activities of each strain during growth on glucose or ethanol revealed that some part of the LPD1 coding region between +13 and +700 is involved in activating gene expression in a carbon source-dependent manner. This activation occurs at the mRNA level, and is not mediated by changes in mRNA stability. Therefore, the LPD1 gene appears to contain a transcriptional enhancer that lies 3' to the transcriptional start site, and which responds to carbon source.
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PMID:A 3' transcriptional enhancer within the coding sequence of a yeast gene encoding the common subunit of two multi-enzyme complexes. 154 8


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