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

---

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

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.
...
PMID:Physiological and genetic strategies for enhanced subtilisin production by Bacillus subtilis. 136 58

Subtilisin expression as a function of growth and sporulation was determined using a presubtilisin-beta-galactosidase gene fusion. An approximately 500-base-pair region upstream of the subtilisin gene and including the first eight codons of the presubtilisin protein was fused at the eighth codon of beta-galactosidase in the integrative vector pJF751. This gene fusion does not carry a signal sequence, and therefore its synthesis is uncoupled from maturation of presubtilisin. The fusion protein gene was integrated into a variety of recipient strains to test for the effect of various mutations on the initial rate of presubtilisin-beta-galactosidase synthesis. Among the spo0 mutations tested, the spo0A mutations showed a strong, 10-fold decrease in the rate of beta-galactosidase synthesis. This effect of the spo0A mutations was not evident when the presubtilisin-beta-galactosidase fusion was present on a multicopy plasmid. The sacU mutation, which was known to increase the extracellular level of levansucrase and proteases, was found to increase the synthesis of the presubtilisin-beta-galactosidase gene fusions 7-fold, and the hpr mutations were shown to increase the rate of presubtilisin-beta-galactosidase gene fusions 17-fold, indicating that these mutations influence either transcription or translation of the presubtilisin gene. However, the effect of these mutations was only observed in the stationary phase of growth, indicating they did not render synthesis constitutive. By using multicopy plasmids and an integrated gene fusion, it was shown that there is likely to be a titratable repressor controlling subtilisin synthesis.
...
PMID:Effect of stage 0 sporulation mutations on subtilisin expression. 308 52

The sensitivity of highly purified human fibroblast interferon and partially purified human leukocyte interferon to several proteolytic and glycolytic enzymes was determined with respect to antiviral activity, isoelectric point, molecular weight, and thermal stability. Leucine aminopeptidase altered the distribution of isoelectric points for both interferons but produced little change in molecular weights; this enzyme somewhat reduced the activity of only leukocyte interferon. Treatment of fibroblast interferon with carboxypeptidases A and B did not greatly decrease antiviral activity, but it did slightly reduce the molecular weight of the interferon and substantially altered the distribution of isoelectric point values; similar treatment of leukocyte interferon caused some loss in activity, especially of the 17,000-molecular-weight species. Both interferons were inactivated rapidly by treatment with the endoproteases trypsin, pepsin, bromelain, and subtilisin. Chymotrypsin shifted the isoelectric points of both interferons, but only leukocyte interferon was significantly inactivated. Treatment with neuraminidase and beta-galactosidase changed the isoelectric point distribution but did not affect the activity or thermal stability of either interferon; such a treatment reduced the molecular weight of fibroblast interferon and the size heterogeneity of leukocyte interferon. Treatment with neuraminidase and then leucine aminopeptidase greatly reduced the activity of both interferons, especially leukocyte interferon. The data indicate that biologically active forms of fibroblast and leukocyte interferons can be distinguished by their relative sensitivity to certain proteases.
...
PMID:Enzymatic modifications of human fibroblast and leukocyte interferons. 616 Feb 60

In order to study carbohydrate-induced protein stabilization bovine testis beta-galactosidase and human serum albumin were conjugated with dextran, partially acetylated dextran and partially methylated dextran. The conjugates and the free proteins were compared with respect to thermal stability at 50 degrees C and resistance to proteolytic digestion by subtilopeptidase A. Both beta-galactosidase and serum albumin were stabilized by conjugation with polysaccharide. However, higher stability was achieved by conjugating the proteins with the hydrophilic polysaccharides, dextran and acetylated dextran, than by conjugation with the hydrophobic polysaccharide, methylated dextran. The results are discussed in relation to possible explanations of carbohydrate-induced protein stabilization.
...
PMID:Effect of dextran and dextran modifications on the thermal and proteolytic stability of conjugated bovine testis beta-galactosidase and human serum albumin. 618 68

In order to enhance the stability of beta-galactosidase, we conjugated the enzyme with dextran T-10 (Mr approx. 10 000). The conjugate contained 9-10 mol dextran/mol protein (beta-galactosidase, Mr 68 000), and the specific activity retained after conjugation was 90 +/- 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated beta-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when beta-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated beta-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50 degrees C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.
...
PMID:Enhanced stability of beta-galactosidase in parenchymal and nonparenchymal liver cells by conjugation with dextran. 618 20

High-level synthesis of exportable beta-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent mutations in cis, which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional B. subtilis secretion mutant after nitrosoguanidine mutagenesis. At 42 degrees C, but not at 30 degrees C, this mutant displayed extreme growth retardation when the LacZ fusion protein was produced, and and was also defective in the secretion of subtilisin Carlsberg. The processing kinetics and secretion of a subtilisin Carlsberg-alkaline phosphatase fusion derivative were found to be defective specifically at the non-permissive temperature. The secretion defect was not linked to the secA/div locus.
...
PMID:Identification of a Bacillus subtilis secretion mutant using a beta-galactosidase screening procedure. 755 Oct 41

Filtration membranes carrying strong cation- or anion-exchange groups on their surface were evaluated for their potential as membrane adsorber stationary phases in the high-performance liquid chromatography of proteins. The membranes are commercially available and can be obtained inserted into ready-to-use filter holders. Owing to their thinness (170-190 microns), the pressure drop of the membranes is extremely low. Flow-rates of up to 65 ml min-1 per unit became thus possible. The low pressure drop of a single membrane layer also permitted an effortless scaling up, as a stack of several membranes or filter units could be used, if necessary. Sample distribution, protein binding capacity, elution conditions, separation efficiency and recovery were investigated as a function of the flow-rate. The time required for the separation of certain protein mixtures could be reduced to less than 1 min. Appropriate conditions were defined for the separation of human serum and for the isolation of subtilisin Carlsberg and beta-galactosidase from cell culture supernatants.
...
PMID:Characterization and application of strong ion-exchange membrane adsorbers as stationary phases in high-performance liquid chromatography of proteins. 827 77

S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).
...
PMID:Ribonuclease S-peptide as a carrier in fusion proteins. 845 73

Human lactase-phlorizin hydrolase (LPH, EC 3.2.1.23/62) is synthesized as a single-chain precursor glycoprotein (pro-LPH) with a relative molecular mass of just over 200 kDa. Maturation to the mature enzyme (m-LPH, 160 kDa) occurs after passage of pro-LPH through the Golgi complex and involves the proteolytic removal of a 849 amino acid propeptide. The role of this propeptide as well as its removal is not fully understood and the proteolytic enzyme or enzymes involved are unknown. We studied the potential role of five different members of the family of subtilisin-like proprotein processing proteases in the maturation process of human LPH using a vaccinia virus based coexpression system in pig kidney PK(15) cells. Infected/transfected PK(15) cells expressed full-length pro-LPH but no maturation to m-LPH was observed. Coexpression of human pro-LPH with human furin, human PC1/PC3, human PC2, human PACE4 and mouse PC6A in PK(15) cells did not result in maturation of the enzyme. Cleavage and secretion of von Willebrand factor precursor (pro-vWF) was used as a positive control. None of the five proprotein processing proteases tested were capable of cleaving human pro-LPH, strongly suggesting that they are not involved in the maturation of this enzyme.
...
PMID:Human lactase-phlorizin hydrolase is not processed by furin, PC1/PC3, PC2, PACE4 and PC5/PC6A of the family of subtilisin-like proprotein processing proteases. 866 47

The effect of some culture variables in the production of beta-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), beta-galactosidase production was partially growth-associated, as 40%-60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex medium was used, beta-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific beta-galactosidase activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-1 fermenter scale, a three-fold increase in volumetric beta-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented. Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset of stationary phase, glucose depletion and biomass concentration.
...
PMID:Improvement of culture conditions to overproduce beta-galactosidase from Escherichia coli in Bacillus subtilis. 903 9


1 2 Next >>

---