Extracellular beta-galactosidase activity of a Fibrobacter succinogenes S85 mutant able to catabolize lactose

Fibrobacter succinogenes S85 is unable to grow with lactose as the source of carbohydrate, although it does exhibit low beta-galactosidase (EC 3.2.1.23) activity. Spontaneous mutants of strain S85 able to grow on lactose were isolated after spreading cells on a chemically defined agar medium with lactose as the carbohydrate source. A lactose-catabolizing isolate, designated L2, exhibited a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile and an immunoblot profile with polyclonal antibodies to whole cells of S85 which were identical to those observed for S85. Strain L2 exhibited both cell-associated and extracellular beta-galactosidase activity with either p-nitrophenyl-beta-D-galactopyranoside or lactose as the substrate. The cell-associated enzyme exhibited the greatest activity in the periplasmic space. Enzyme production was partially inhibited by glucose. The beta-galactosidase was activated by divalent cations and exhibited a pH optimum of 6.5. Analysis of the extracellular culture fluid revealed that glucose derived from the hydrolysis of lactose was used for growth, but galactose was not metabolized further. Cells were unable to take up the lactose analog, methyl-beta-D-thiogalactopyranoside. These data suggest that beta-galactosidase of F. succinogenes L2 cleaves lactose outside the cells and that the glucose released is catabolized while the galactose accumulates in the extracellular culture fluid.     

Extracellular beta-galactosidase activity of a Fibrobacter succinogenes S85 mutant able to catabolize lactose.

Fibrobacter succinogenes S85 is unable to grow with lactose as the source of carbohydrate, although it does exhibit low beta-galactosidase (EC 3.2.1.23) activity. Spontaneous mutants of strain S85 able to grow on lactose were isolated after spreading cells on a chemically defined agar medium with lactose as the carbohydrate source. A lactose-catabolizing isolate, designated L2, exhibited a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile and an immunoblot profile with polyclonal antibodies to whole cells of S85 which were identical to those observed for S85. Strain L2 exhibited both cell-associated and extracellular beta-galactosidase activity with either p-nitrophenyl-beta-D-galactopyranoside or lactose as the substrate. The cell-associated enzyme exhibited the greatest activity in the periplasmic space. Enzyme production was partially inhibited by glucose. The beta-galactosidase was activated by divalent cations and exhibited a pH optimum of 6.5. Analysis of the extracellular culture fluid revealed that glucose derived from the hydrolysis of lactose was used for growth, but galactose was not metabolized further. Cells were unable to take up the lactose analog, methyl-beta-D-thiogalactopyranoside. These data suggest that beta-galactosidase of F. succinogenes L2 cleaves lactose outside the cells and that the glucose released is catabolized while the galactose accumulates in the extracellular culture fluid.     

Purification and properties of an inducible beta-galactosidase isolated from the yeast Kluyveromyces lactis.

beta-Galactosidase (EC 3.2.1.32) was purified 80-fold from the yeast Kluyveromyces lactis induced for this enzyme by growth on lactose. When the purified enzyme was subjected to electrophoresis on an acrylamide gel in the presence of sodium dodecyl sulfate, one protein with an apparent molecular weight of 135,000 was observed. The enzyme has a sedimentation coefficient of 9.6S. This beta-galactosidase and the one from Escherichia coli are not antigenically related. Maximal enzyme activity requires Na+ and Mn2+ and a reducing agent. beta-Galactosidase has Km values of 12 to 17 and 1.6 mM for lactose and o-nitrophenyl-beta-D-galactoside, respectively. The hydrolase and transgalactosylase activities of the enzyme are similar to those of E. coli beta-galactosidase.     

Human small-intestinal β-galactosidases. Separation and characterization of one lactase and one hetero β-galactosidase

1. Two beta-galactosidases from human small-intestinal mucosa were separated by gel-filtration chromatography and the properties of the two enzymes were studied. Lactose and four hetero beta-galactosides were used as substrates. 2. One of the enzymes was particle-bound and could be partially solubilized with papain. Of the substrates hydrolysed by this enzyme, lactose was hydrolysed most rapidly. This enzyme is thus essentially a disaccharidase and is named lactase. It is presumably identical with the ;lactase 1' described earlier. 3. The other enzyme was mainly soluble and hydrolysed all artificial substrates used, whereas no lactase activity could be detected. This enzyme has therefore been designated hetero beta-galactosidase. 4. p-Chloromercuribenzoate (0.1mm) inhibited the hetero beta-galactosidase completely but did not influence the activity of the lactase. Tris was a competitive inhibitor of both enzymes. 5. The residual lactase activity in the mucosa of lactose-intolerant patients may be exerted by a small amount of remaining lactase as such, or possibly by a third enzyme with a more acid pH optimum.     

Induction of beta-galactosidase in Streptomyces violaceus.

Synthesis of beta-galactosidase by Streptomyces violaceus was induced by D-galactose and L-arabinose, and to a lesser extent by lactose, D-arabinose, and methyl-beta-D-galactopyranoside. The synthesis of the enzyme was linear and started to increase 2--3 h after induction by galactose, reaching a maximum after 5--7 h. The highest level of specific activity was observed in 2% galactose, with an increase of 45 times over the basal level in glycerol. Isopropyl-beta-D-thiogalactopyranoside (IPTG) and methyl-beta-D-thiogalactopyranoside (TMG) inhibited induction by D-galactose, but did not influence enzymatic activity. Cellular extracts hydrolyzed O-nitrophenyl-beta-D-galactopyranoside, but did not significantly hydrolyze lactose, melibiose, p-nitrophenyl-alpha-D-galactopyranoside, p-nitrophenyl-beta-D-fucoside, or p-nitrophenyl-beta-D-glucopyranoside. Rifampicin and chloramphenicol inhibited beta-galactosidase synthesis in non-preinduced and in preinduced cells. The inhibition by chloramphenicol was reversible.     

Sialidase assay by luminescence in the low picomole-range of sialic acid. Its application to the measurement of this activity in influenza virus

A new procedure for a sialidase assay, by bioluminescence, has been developed. The substrate, N- acetylneuraminyllactose (sialyllactose), hydrolysed by the sialidase activity, releases lactose. This lactose is hydrolysed with beta-galactosidase. The released galactose is oxidized with galactose dehydrogenase and NAD. The NADH produced in the last step is measured by a luminescence system, coupling two enzymes, NAD(P)H dehydrogenase (FMN) and luciferase. This microassay, which is specific, rapid, simple and ultra-sensitive, is a measure for amounts as little as (at least) 5 pmol of N-acetylneuraminic acid (corresponding to 0.15 ng of the released sialic acid). It uses commercialized reagents (non-radioisotopic) and avoids interferences common in other procedures. This method has been used for measuring sialidase activity directly on intact virus, avoiding inconvenient modifications produced in the extraction of the enzyme. The specific activity of sialidase of influenza virus X31 (H3N2), determined by this procedure, is 0.65 U/mg of total virus protein.     

Purification and characterization of a thermotolerant beta-galactosidase from Thermomyces lanuginosus.

A new inducible intracellular beta-galactosidase (EC 3.2.1.23) of the thermophilic fungus Thermomyces lanuginosus was purified by fractional salt precipitation, hydrophobic interaction, and anion exchange chromatography. The first 22 amino acid residues were determined by N-terminal sequencing. Electrophoretic investigations revealed a dimeric enzyme with a molecular mass of 75 to 80 kDa per identical subunit and an isoelectric point of 4.4 to 4.5. The native beta-galactosidase was identified as a glycoprotein by the enzyme-linked immunosorbent assay technique. The beta-galactosidase activity was optimal at pH 6.7 to 7.2, and the enzyme displayed stability between pH 6 and 9. It was completely stable at pH 6.8 and 47 degrees C for 2 h. After 191 h at 50 degrees C, the remaining beta-galactosidase activity of an enzyme fraction after salt precipitation was 58%. The beta-galactosidase hydrolyzed p- and o-NO2-phenyl-beta-D-galactopyranoside, lactose, lactulose, MeOH-beta-D-galactopyranoside, phenyl-beta-D-galactopyranoside, and p-NO2-phenyl-alpha-L-arabinopyranoside. The kinetic constants (Km) measured for p- and o-NO2-phenyl-beta-D-galactopyranoside and beta-lactose were 4.8, 11.3, and 18.2 mM, respectively.     

Induction and characterization of -galactosidase in an extreme thermophile

A thermostable beta-galactosidase (EC 3.2.1.23; beta-dgalactoside galactohydrolase) was found to be inducible in an extreme thermophile resembling Thermus aquaticus. Enzyme induction was achieved by the addition of lactose, galactose, or the alpha-galactoside, melibiose, to growing cultures. The addition of glucose to induced cultures had a repressive effect on further enzyme synthesis. The enzyme was purified 78-fold, and the optimum temperature and pH for activity were determined to be 80 C and pH 5.0, respectively. The enzyme was activated by both manganese and ferrous iron. Sulfhydryl activation and thermal stabilization indicate that the thermophilic beta-galactosidase is a sulfhydryl enzyme. Kinetic determinations at 80 C established a K(m) of 2.0 x 10(-3)m for the chromogenic substrate o-nitrophenyl beta-d-galactopyranoside (ONPG) and a K(1) of 7.5 x 10(-3)m for lactose. The Arrhenius energy of activation (for the hydrolysis of ONPG) was calculated to be 13.7 kcal/mole. A molecular weight of 5.7 x 10(5) daltons was estimated by elution of the enzyme from Sephadex 4B.     

Co-induction of beta-galactosidase and the lactose-P-enolpyruvate phosphotransferase system in Streptococcus salivarius and Streptococcus mutans.

The addition of lactose, galactose, or isopropyl-beta-D-thiogalactoside (IPTG) to glucose-grown cells of Streptococcus salivarius 25975 resulted in the co-induction of both the lactose-P-enolpyruvate phosphotransferase system (lactose-PTS) and beta-galactosidase, with the latter the predominant metabolic system. With various strains of Streptococcus mutans and Streptococcus sanguis 10556, on the other hand, the lactose-PTS was the major metabolic pathway with beta-galactosidase induced either to low or negligible levels. In all cases, induction of the lactose-PTS resulted in the concomitant induction of 6-P-beta-galactosidase. The induction by lactose of both the lactose-PTS and beta-galactosidase in all strains was repressed by glucose and other catabolites, notably, fructose. Induction of beta-galactosidase in S. salivarius 25975 by IPTG was, however, relatively resistant to glucose repression. Induction experiments with IPTG and lactose suggested that a cellular metabolite of lactose metabolism was a repressor of enzyme activity. Exogenous cAMP was shown to reverse the transient repression by glucose of beta-galactosidase induction in cells of S. salivarius 25975 receiving lactose, provided the cells were grown with small amounts of toluene to overcome the permeability barrier to this nucleotide, cAMP, was however, unable to overcome the permanent repression of beta-galactosidase activity to a significant extent under these conditions.     

Diet and carbohydrate digestion in the yellow-spotted longicorn beetle Psacothea hilaris

Larval and adult Psacothea hilaris feed on mulberry wood and leaves, respectively. High levels of endogenous activity against the major dietary carbohydrates, cellulose, hemicellulose, starch and soluble sugars were secreted in the gut of larvae and adults. Activity against pectin was also high and multiple polygalacturonase (EC 3.2.1.15) components were secreted in the gut of larvae. One glycanase component, beta-EG1, which was primarily an endo-beta-1,4-glucanase (EC 3.2.1.4) and another, beta-EG2, which was mostly an endo-beta-1,4-xylanase (EC 3.2.1.8), were also secreted, while at least four additional components hydrolysed laminarin, lichenin and crystalline cellulose. The beta-glycosidase component beta-GD1 was associated with most of the beta-mannosidase (EC 3.2.1.25) and beta-xylosidase (EC 3.2.1.37) activity secreted in the gut of larvae, while another, beta-GD2, was a beta-glucosidase (EC 3.2.1.21), the activity of which was directed against cellobiose and other beta-linked disaccharides, and a beta-fucosidase (EC 3.2.1.38). A beta-galactosidase (EC 3.2.1.23), which did not hydrolyse lactose, was also secreted, as were distinct beta-N-acetylhexosaminidase (EC 3.2.1.52), trehalase (EC 3.2.1.28), alpha-L-arabinosidase (EC 3.2.1.55), alpha-galactosidase (EC 3.2.1.22) and a minimum of four alpha-glucosidase (EC 3.2.1.20) components, one of which was also likely to be associated with a peak of alpha-mannosidase (EC 3.2.1.24) activity. The alpha-glucosidase components varied in their specificity for alpha-linked disaccharides, but none was active against sucrose, which was hydrolysed by a beta-fructofuranosidase (EC 3.2.1.26) component. Overall average levels of activity in larvae were twice those of adults, but the secretion of individual carbohydrases in both was not regulated in response to the relative abundance of particular carbohydrate components in their respective diets.     

Carbohydrases of the rumen ciliate Epidinium ecaudatum (Crawley). Hydrolysis of plant hemicellulose fractions and beta-linked glucose polymers

1. Cell-free extracts from Epidinium ecaudatum (Crawley) hydrolysed the three hemicellulose fractions of pasture plants, but at different rates. 2. All of the constituent monosaccharides are released from the hemicellulose fractions, galactose and uronic acids being liberated at much slower rates than pentoses. 3. An arabinofuranosidase, which removes arabinose from highly branched arabinoxylan before the xylan chain can be hydrolysed, was isolated free from other pentosanases. 4. A xylanase hydrolysing xylan (by random cleavage) and xylodextrins of degree of polymerization (D.P.) > 3 to xylotriose and xylobiose was isolated free from other pentosanases. 5. A separate xylodextrinase hydrolysing (by random cleavage) xylodextrins of D.P. > 2 to xylobiose and xylose was also obtained; this enzyme did not hydrolyse xylan or xylobiose and the original extracts themselves possessed very weak xylobiase activity. 6. The epidinial extracts hydrolysed laminaribiose, laminarin, lichenin and cellodextrins of D.P. < 7 rapidly, cellobiose and gentiobiose slowly but cellulose not at all. 7. Polysaccharide glucose associated with plant linear B hemicellulose was liberated with cellobiose and possibly laminaribiose as intermediates. 8. The cellodextrinase hydrolysed cellopentaose initially to cellobiose plus cellotriose and is a distinctly different enzyme from the xylanase and xylodextrinase. 9. Extracts from Entodinium species and Eremoplastron bovis also hydrolysed all three types of plant hemicellose.     

Physiological studies of beta-galactosidase induction in Kluyveromyces lactis

We examined the kinetics of beta-galactosidase (EC 3.2.1.23) induction in the yeast Kluyveromyces lactis. Enzyme activity began to increase 10 to 15 min, about 1/10 of a cell generation, after the addition of inducer and continued to increase linearly for from 7 to 9 cell generations before reaching a maximum, some 125- to 150-fold above the basal level of uninduced cells. Thereafter, as long as logarithmic growth was maintained, enzyme levels remained high, but enzyme levels dropped to a value only 5- to 10-fold above the basal level if cells entered stationary phase. Enzyme induction required the constant presence of inducer, since removal of inducer caused a reduction in enzyme level. Three nongratuitous inducers of beta-galactosidase activity, lactose, galactose, and lactobionic acid, were identified. Several inducers of the lac operon of Escherichia coli, including methyl-, isopropyl- and phenyl-1-thio-beta-d-galactoside, and thioallolactose did not induce beta-galactosidase in K. lactis even though they entered the cell. The maximum rate of enzyme induction was only achieved with lactose concentrations of greater than 1 to 2 mM. The initial differential rate of beta-galactosidase appearance after induction was reduced in medium containing glucose, indicating transient carbon catabolite repression. However, glucose did not exclude lactose from K. lactis, it did not cause permanent carbon catabolite repression of beta-galactosidase synthesis, and it did not prevent lactose utilization. These three results are in direct contrast to those observed for lactose utilization in E. coli. Furthermore, these results, along with our observation that K. lactis grew slightly faster on lactose than on glucose, indicate that this organism has evolved an efficient system for utilizing lactose.     

Purification and characterisation of an intracellular enzyme with beta-glucosidase and beta-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS 236.58

An intracellular beta-glycoside hydrolase with beta-glucosidase and beta-galactosidase activity, designated beta-glucosidase BGL1, was isolated to apparent homogeneity from the thermophilic ascomycete Talaromyces thermophilus CBS 236.58. The monomeric enzyme has a molecular mass of 50 kDa (SDS-PAGE) and an isoelectric point of 4.5-4.6. The enzyme is active with both glucosides such as cellobiose and galactosides including lactose; based on the catalytic efficiencies determined glucosides are the preferred substrates. beta-Galactosidase activity of BGL1 is activated by various mono and divalent cations including Na+, K+ and Mg2+, and it is moderately inhibited by its reaction products glucose and galactose. Its pH optimum for the hydrolysis of galactosides is in the range of 5.5-6.0, and its optimum temperature was found to be 50 degrees C (15 min assay). In addition to its hydrolytic activity, BGL1 shows a significant transferase activity which results in the formation of galacto-oligosaccharides. These have recently attracted interest because of possible applications in food industry. The highest yields of oligosaccharides was approximately 20% when using 38 gl(-1) lactose as the starting material.     

Rat small-intestinal β-galactosidases. Kinetic studies with three separated fractions

1. Three fractions of beta-galactosidase activity from the rat small-intestinal mucosa were separated chromatographically. Two of these fractions had an acid pH optimum at 3-4, and the third one had a more neutral pH optimum at 5.7. 2. The two ;acid' beta-galactosidase fractions had considerably lower K(m) values for hetero beta-galactosides than for lactose. The V(max.) values were similar for all the substrates used (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside). No difference could be detected between the two ;acid' fractions with respect to their enzymic properties (pH optimum, K(m) for the different substrates, K(i) for lactose as an inhibitor of the hydrolysis of hetero beta-galactosides, K(i) for phenyl beta-galactoside as an inhibitor of the hydrolysis of lactose, and relative V(max.) for the hydrolysis of different substrates). These two fractions probably represent different forms of the same enzyme. 3. The ;neutral' fraction had similar K(m) values for all the substrates hydrolysed, but with lactose as substrate the V(max.) was much higher than with the hetero beta-galactosides. This fraction did not split phenyl beta-galactoside or 6-bromo-2-naphthyl beta-galactoside at a measurable rate. 4. Lactose was a competitive inhibitor of the hetero beta-galactosidase activities of all the three fractions, and K(i) for lactose as an inhibitor in each case was the same as K(m) for the lactase activity. Phenyl beta-galactoside was a competitive inhibitor of the lactase activity of all the three fractions. These facts strongly indicate that in all the three fractions lactose is hydrolysed by the same active sites as the hetero beta-galactosides. 5. Human serum albumin stabilized the separated enzymes against inactivation by freezing and thawing.     

Induction of intracellular and extracellular beta-galactosidase activity in Phycomyces blakesleeanus

The inductive effect of different sugars on beta-galactosidase synthesis in Phycomyces blakesleeanus has been studied. The enzyme was inducible by galactose and fructose. When grown on these sugars the enzyme level was 10-20 times greater than when grown on glucose. We have detected both intra- and extracellular beta-galactosidase activity when Phycomyces blakesleeanus was grown on galactose, but only extracellular beta-galactosidase activity when grown on fructose plus lactose.     

Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates

β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor.     

Stimulation of Lactic Streptococci in Milk by β-Galactosidase

Acid production in milk by lactic streptococci was stimulated by added beta-galactosidase. Both glucose and galactose accumulated rapidly in the presence of this enzyme. Glucose accumulation ceased as the culture entered the most rapid period of acid production, whereas galactose accumulation continued. In cultures without added beta-galactosidase, a low concentration of galactose accumulated in the milk, whereas glucose was not detected after 2 hr of incubation. Cultures grew and produced acid faster in broth containing glucose rather than galactose or lactose. These observations suggest that the lactic streptococci do not metabolize the lactose in milk efficiently enough to permit optimum acid production and that a phenomenon such as catabolite repression functions to allow for a preferential use of glucose over either galactose or lactose. In addition to providing the culture with a more readily available energy source, it is possible that the culture produced more acidic metabolites as a result of preferentially utilizing the glucose released by the action of the beta-galactosidase.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.