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PubMed2005

Effect of exogenous beta-galactosidase in patients with lactose malabsorption and intolerance: a crossover double-blind placebo-controlled study.

OBJECTIVE: To evaluate the efficacy of the addition to milk, 5 min and 10 h before its consumption, of a lactase obtained from Kluyveromyces lactis in lactose malabsorbers with intolerance. DESIGN: Double-blind, placebo-controlled, crossover study. SETTING: University Hospital. SUBJECTS: In total, 11 male and 19 female (aged from 18 to 65 y, mean age 43.3 y) lactose malabsorbers with intolerance participated. INTERVENTIONS: Each patient underwent three H(2) breath tests, in a random order. We used 400 ml of cow's semiskimmed milk as substrate and a beta-galactosidase obtained from K. lactis. The test A was carried out adding to the milk the enzyme (3000 UI), 10 h before its consumption; the test B was performed adding the beta-galactosidase (6000 UI) 5 min before milk ingestion and the test C was made using placebo. We evaluated the maximum breath H(2) concentration, the cumulative H(2) excretion and a clinical score based on intolerance symptoms (bloating, abdominal pain, flatulence and diarrhoea). RESULTS: Our study showed a significant reduction of the mean maximum H(2) concentration after both test A (12.07 +/- 7.8 p.p.m.) and test B (13.97 +/- 7.99 p.p.m.) compared with test C (51.46 +/- 16.12 p.p.m.) (ANOVA F = 54.33, P < 0.001). Similarly, there was a significant reduction of the mean cumulative H(2) excretion after both test A (1428 +/- 1156 p.p.m.) and test B (1761 +/- 966 p.p.m.) compared with test C (5795 +/- 2707 p.p.m.) (ANOVA F = 31.46, P < 0.001). We also observed a significant reduction of the mean clinical score after both test A (0.36 +/- 0.55) and test B (0.96 +/- 0.85) compared with test C (3.7 +/- 0.79) (ANOVA F = 106.81, P < 0.001). Moreover, with regard to the mean clinical score, there was a significant reduction after test A with respect to test B (Bonferroni's P = 0.03). CONCLUSIONS: Our study shows that in lactose malabsorbers with intolerance, the lactase obtained from K. lactis can represent a valid therapeutic strategy, with objective and subjective efficacy and without side effects.

Adolescent↗
PubMed1997

Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and beta-galactosidase expression by Saccharomyces cerevisiae.

The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne beta-galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (delta mu) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited delta mu-dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. delta mu decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or delta mu, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased delta mu values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of delta mu may have conferred was counteracted by an increased R value. Increased beta-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity.

Culture Media↗
PubMed1998

Expression immunoassay based on antibodies labeled with a deoxyribonucleic acid fragment encoding the alpha-peptide of beta-galactosidase.

An immunoassay is reported which uses, as a label, an expressible DNA fragment encoding the alpha-peptide of beta-galactosidase. This inactive peptide consists of 97 amino acid residues containing an amino-terminal portion of the enzyme. Antigen (an anti-thyrotropin immunoglobulin) immobilized in microtiter wells is allowed to react with specific antibodies which are then linked to the DNA label via biotin-streptavidin interaction. After completion of the immunoreaction, the solid phase bound DNA is subjected to a cell-free, one-step transcription/translation reaction to produce the alpha-peptide. The alpha-peptide is allowed to react (complementation reaction) with the remaining part of the beta-galactosidase (M15 protein, also inactive) to give fully active enzyme molecules. 4-Methylumbelliferyl galactoside is used as a substrate. The fluorescence is linearly related to the amount of antigen in the well. As little as 3 fmol of antigen can be detected. The RSDs (within-run) obtained for 8 and 20 fmol of antigen were 10.7 and 9.3%, respectively (n = 4). The present work illustrates the utility of expressing a non-detectable peptide capable of triggering a signal generating system.

Antigens↗
PubMed2003

On-chip electrochemical measurement of beta-galactosidase expression using a microbial chip.

[small beta]-Galactosidase expression in a small number of Escherichia coli cells has been measured in real time with an electrochemical sensor chip. E. coli cells were embedded using collagen gel within a micropore which was microfabricated onto a chip. The activity of the expressed [small beta]-galactosidase was determined using p-aminophenyl [small beta]-d-galactopyranoside (PAPG) as a substrate.

Aminophenols↗
PubMed2002

Production and characterization of a thermostable beta-galactosidase from Bacillus coagulans RCS3.

A strain of Bacillus coagulans RCS3 isolated from a hot-water spring produced significant beta-galactosidase activity at 10 days of growth in a flask. While enzyme production was maximum at 50 degrees C, the highest activity was at 65 degrees C, where the half-life was 2 h. A 2 degrees C decrease in temperature increased the half-life to 15 h without significantly changing the activity, suggesting that 63 degrees C is the temperature of preference compared with 65 degrees C for a combination of good activity and stability. The beta-galactosidase was also stable over pH 5-8, with peak activity at pH 6-7. It was strongly and competitively inhibited by the hydrolysis product galactose. Bivalent cations (Cu(2+), Ni(2+) and Hg(2+)) in the concentration range of 0.5-2.0 mM also inhibited enzyme activity. Both lactose solution and whey could be hydrolysed substantially within 36 h at 50 degrees C. The thermostability and pH-stability and good hydrolytic capability make this enzyme potentially useful in the dairy industry.

Animals↗
PubMed1966

Catabolite repression of beta-galactosidase synthesis in Escherichia coli.

1. Repression by glucose of beta-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i(-)) and permease-less (y(-)) cells as well as in the corresponding i(+) and y(+) strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-beta-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of beta-galactosidase synthesis (e.g. isopropyl thio-beta-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-beta-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.

Carbon Isotopes↗
PubMed1969

Purification and properties of alpha-galactosidases from Vicia faba seeds.

Two forms of alpha-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2.8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. alpha-Galactosidases I and II showed different pH optima and K(m) and V(max.) values with p-nitrophenyl alpha-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.

Amino Acids↗
PubMed1970

Interactions between metabolic intermediates and beta-galactosidase from Escherichia coli.

1. 5-Phosphorylribose 1-pyrophosphate, in the presence of beta-mercaptoethanol, protected beta-galactosidase from heat inactivation. Many other substances, including 3':5'-cyclic-AMP, were without effect. 2. The efficiency of complementation in vitro of beta-galactosidase segments was decreased by 5-phosphorylribose 1-pyrophosphate but not by 3':5'-cyclic-AMP. Neither substance affected the activity of the complete enzyme. 3. Some indications as to the possible identity of the catabolite repression effector are presented.

Cyclic AMP↗
PubMed1970

Molecular weight of Escherichia coli beta-galactosidase in concentrated solutions of guanidine hydrochloride.

The molecular weight of Escherichia coli beta-galactosidase was determined in 6m- and 8m-guanidine hydrochloride by meniscus-depletion sedimentation equilibrium, sedimentation velocity and viscosity. Sedimentation equilibrium revealed heterogeneity with the smallest component having a molecular weight of about 50000. At lower speeds, the apparent weight-average molecular weight is about 80000. By use of a calculation based on an empirical correlation for proteins that are random coils in 6m-guanidine hydrochloride, sedimentation velocity gave a molecular weight of 91000, and the intrinsic viscosity indicated a viscosity-average molecular weight of 84000. Heating in 6m-guanidine hydrochloride lowered the viscosity of beta-galactosidase in a variable manner.

Centrifugation↗
PubMed1971

-galactosidase-catalysed hydrolysis of -D-galactopyranosyl azide.

1. beta-d-Galactopyranosyl azide is hydrolysed by the beta-galactosidase of Escherichia coli to galactose and azide ion at a mechanistically significant rate. 2. Methyl 1-thio-beta-d-galactopyranoside is a competitive inhibitor of the hydrolysis of the azide and of o-nitrophenyl beta-d-galactopyranoside with K(i) 1.8mm. 3. beta-Galactosidase can thus hydrolyse a range of substrates of general structure beta-d-galactopyranosyl-X(Y), where the atom X has a lone pair of electrons on which the enzyme may act as a Lewis or Brønsted acid, but in which the length of the bond cleaved varies significantly, which is inconsistent with the orbital steering hypothesis.

Azides↗
PubMed1973

Hydrolysis of ceramide trihexoside by a specific -galactosidase from human liver.

1. Partially purified ceramide trihexoside alpha-galactosidase from human liver was studied by using ceramide trihexoside specifically tritiated in the terminal galactose. 2. The hydrolysis of ceramide trihexoside was absolutely dependent on a mixture of sodium taurocholate and Triton X-100 and was markedly inhibited by human serum albumin and by NaCl. 3. The Lineweaver-Burk plot for ceramide trihexoside hydrolysis was upward curving. Ceramide lactoside inhibited hydrolysis of all concentrations of ceramide trihexoside. Ceramide digalactoside stimulated hydrolysis of low concentrations of ceramide trihexoside, but inhibited hydrolysis of high concentrations of the lipid. 4. alpha-Galactosidase activity assayed with the synthetic substrate 4-methylumbelliferyl alpha-d-galactopyranoside fractionated together with activity assayed with the natural substrate ceramide trihexoside. Both activities had identical heat-inactivation kinetics. 5. Characteristics of the hydrolysis of the synthetic substrate differed considerably from those of the natural substrate, including pH optimum, shape of the Lineweaver-Burk plot, and differential effects of inhibitors and activators. Mutual inhibition of hydrolysis between the synthetic and natural substrates was predominantly non-competitive. 6. These results are discussed in the light of special problems involved in the hydrolysis of lipids in an aqueous milieu.

Cerebrosides↗
PubMed1974

The beta-galactosidase-catalysed hydrolyses of beta-d-galactopyranosyl pyridium salts. Rate-limiting generation of an enzyme-bound galactopyranosyl cation in a process dependent only on aglycone acidity.

1. beta-d-Galactopyranosyl pyridinium salts are well-behaved substrates for the beta-galactosidase of Escherichia coli, catalysis occurring by the interaction of the salt itself with the normal active site of the protein. 2. logk(cat.) values for seven such salts show a linear relationship (correlation coefficient=-0.997) with the pK(a) of the parent pyridine. 3. The beta-d-galactopyranosyl derivatives of pyridine and 4-bromoisoquinoline exhibit alpha-deuterium kinetic isotope effects of 1.136+/-0.040 and 1.187+/-0.046 on their enzymic hydrolysis, indicating formation of a galactopyranosyl cation in the rate-limiting step. 4. This behaviour of the pyridinium salts contrasts with the behaviour of aryl galactosides and this contrast can be accommodated by the beta-galactosidase mechanism of Sinnott & Souchard (1973). 5. The alpha-deuterium kinetic isotope effect for the hydrolysis of beta-d-galactopyranosyl azide is 1.098+/-0.033; comparison of the k(cat.) value of the azide with that of a pyridinium salt of the same aglycone pK(a) enables a maximum factor of 70 to be ascribed to the acceleration of the departure of azide by intracomplex general acid catalysis. 6. The possibility of the rate-limiting process in the glycosidase-catalysed hydrolysis of aryl glycosides being a conformation change is considered for a number of glycosidases where correlations of k(cat.) with aglycone acidity, reported in the literature, have been unsuccessful.

Azides↗
PubMed1975

Synthesis time of beta-galactosidase in Escherichia coli B/r as a function of growth rate.

By analysing the kinetics of beta-galactosidase accumulation after induction, the synthesis time of beta-galactosidase in Escherichia coli B/r was found to be 75s in rapidly growing cells (1.36 and 2.1 doublings/h), and 90s in slowly growing cells (0.63 doubling/h). These values correspond to peptide-chain-elongation rates of 16 and 13 amino acids/s respectively, in agreement with previous findings, indicating that the peptide-chain growth rate is constant (presumably maximal) in fast-growing bacteria, but decreased in slowly growing bacteria [Forchhammer & Lindahl (1971) J. Mol. Biol. 55, 563-568].

Amino Acids↗
PubMed1976

A cyanogen bromide fragment of beta-galactosidase from Escherichia coli with alpha-donor activity in complementation of the enzyme from mutant M15.

Aminoethylated beta-galactosidase from Escherichia coli was cleaved by CNBr. The fragment C4a was purified by gel filtration and ion-exchange chromatography. The molecular weight of the fragment C4a was determined to be 9000 +/- 600. The N-terminal amino acid was found to be isoleucine. Qualitative examination of homogeneity was carried out by disc-gel electrophoresis. The fragment C4a was shown to be active as an alpha donor in complementation of beta-galactosidase activity in vitro with E. coli mutant M15, which has a deletion in the alpha region of the z gene. The molecular weights of complementable fractions from mutant M15 were found to be 123 000 +/- 2500 and 507 000 +/- 11 000, and of the complemented enzyme 522 500 +/- 11 400.

Amino Acids↗
PubMed1994

Substitutions for Glu-537 of beta-galactosidase from Escherichia coli cause large decreases in catalytic activity.

Glu-537 of beta-galactosidase (EC 3.2.1.23) was replaced by Asp, Gln and Val using synthetic oligonucleotides. The kcat values of the purified enzyme mixtures were reduced by about 100-fold for the Asp mutant, 30,000-60,000-fold for the Val mutant and 160,000-300,000-fold for the Gln mutant. The greatest differences in properties from the wild-type enzyme were found for the Asp-substituted enzyme: the Km values increased (from 0.12 to 0.42 mM for o-nitrophenyl beta-D-galactopyranoside), and from 0.04 to 0.37 mM for p-nitrophenyl beta-D-galactopyranoside), the Ki value for isopropyl beta-D-galactopyranoside increased (from 0.11 to 0.30 mM), the stability to heat decreased and methanol did not act as an acceptor. The enzymes with the other two substitutions had properties similar to those of the wild-type. For all three substituted enzymes, the inhibitory effects of the transition-state analogues (2-deoxy-2-amino-D-galactose and L-ribose) and the Mg2+ effects were similar to those of the normal enzyme. As all of the properties (except the kcat values) of the Gln- and Val-substituted enzyme preparations were similar to those of the wild-type enzyme, the activities in those preparations were probably due to the presence of a few wild-type enzyme molecules (formed from misreads) among the substituted enzymes. The enzymes with Gln and Val substitutions appear to be totally inactive. The results obtained support a recent suggestion that Glu-537 is an important catalytic residue of beta-galactosidase.

Amino Acid Sequence↗
PubMed1996

A defect in inducible beta-galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse.

Macrophages were activated by administration of an inflammatory lipid metabolite, lysophosphatidylcholine (lyso-Pc), to wild type mice but not murine (microphthalmic) osteopetrotic (mi/mi) mutant mice. In vitro treatment of wild type mouse peritoneal cells with lyso-Pc efficiently activated macrophages whereas lyso-Pc-treatment of mi mutant mouse peritoneal cells resulted in no activation of macrophages. Generation of macrophage activating factor requires a precursor protein, serum vitamin D binding protein (DBP), and participation of lyso-Pc-inducible beta-galactosidase of B lymphocytes. Lyso-Pc-inducible beta-galactosidase of B lymphocytes was found to be defective in mi mutant mice.

Animals↗
PubMed2000

The gene for a halophilic beta-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum.

Investigations of transcriptional regulation and the characterization of promoters in homologous expression systems are most easily performed using suitable reporter genes. Presumably because of the high internal salt concentration in halophilic Archaea, the successful application of the commonly used reporter genes has not been reported so far. Recently, the gene for an extremely halophilic beta-galactosidase (bgaH) from Haloferax alicantei has become available. After transformation of Halobacterium salinarum with a vector-carrying bgaH, the enzyme activity in cell lysates could be readily determined by a simple colorimetric assay and colonies could be screened for activity on plates containing Xgal substrate. Expression of bgaH under the control of various halobacterial promoters of known strength led to different specific beta-galactosidase activities in the lysates. Using Northern blot hybridization and semiquantitative RT-PCR, it was shown that the bgaH transcript level corresponded to the specific enzyme activity. Therefore, the bgaH gene of Haloferax alicantei appears to be a useful tool for in vivo studies of gene expression in Halobacterium salinarum and possibly other halophilic Archaea.

Gene Expression Regulation, Archaeal↗
PubMed1998

Influence of polyols on the structural properties of Kluyveromyces lactis beta-galactosidase under high hydrostatic pressure.

The conformational changes in dimeric Kluyveromyces lactis beta-galactosidase induced by hydrostatic pressure were investigated by means of its intrinsic tryptophan fluorescence. At high pressure, the fluorescence emission spectrum was shifted to the red, indicating the exposure of buried Trp residues to the aqueous solvent. This spectral change was paralleled by a loss of enzyme activity. The shift of the emission spectrum was quantified by evaluating the centre of spectral mass ((nu(g))), which is an intensity-weighted mean wavenumber. The experimental data could be fitted to a two-state transition (native<-->denatured), corrected for a linear pressure dependence of (nu(g)), and allowed the determination of thermodynamic parameters deltaG0(app), V(app) and P(1/2). The results were consistent with a partial unfolding of the protein and not simply with dissociation of this dimeric enzyme. In the presence of polyols, the native conformation of beta-galactosidase was considerably more resistant to pressure. This protective effect of polyols is probably due to a reduced accessibility of water inside the protein structure, through the direct or indirect action of these additives on the enzyme.

Animals↗