Genetic Diversity in the Lactose Operons of Lactobacillus helveticus Strains and Its Relationship to the Role of These Strains as Commercial Starter Cultures

Two novel insertion sequence elements, ISLhe1 and ISLhe15, were located upstream of the genes encoding the β-galactosidase enzyme in Lactobacillus helveticus commercial starter strains. Strains with the IS982 family element, ISLhe1, demonstrated reduced β-galactosidase activity compared to the L. helveticus type strain, whereas strains with the ISLhe15 element expressed β-galactosidase in the absence of lactose.     

Heterologous Expression of Lactose- and Galactose-Utilizing Pathways from Lactic Acid Bacteria in Corynebacterium glutamicum for Production of Lysine in Whey

The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and β-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited β-galactosidase activity in excess of 1,000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and β-galactosidase genes exhibited β-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability.     

Galactose Metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Properties of Lactose Plasmid pLY101 in Lactobacillus casei

A starter strain, Lactobacillus casei C257, was found to carry a lactose plasmid, pLY101. Restriction mapping showed that pLY101 DNA was 68.2 kilobases long. Since a non-lactose-utilizing variant of C257, MSK248, lost phospho-β-galactosidase (P-β-gal) activity and pLY101 DNA had a sequence(s) homologous to the streptococcal fragment including a P-β-gal gene, pLY101 is likely to encode a P-β-gal gene required for lactose metabolism in C257. MSK248 grew in galactose medium at a rate identical to that of C257 and retained phosphoenolpyruvate-dependent phosphotransferase system activity for lactose similar to that of C257. Therefore, the C257 chromosome appears to encode a complete set of genes for the lactose-phosphotransferase system and the predominant galactose metabolic pathway in C257. pLY101 DNA had a sequence homologous to a lactobacillus insertion sequence, ISL1, which mapped more than 12 kilobases from the sequence homologous to the streptococcal P-β-gal fragment.     

Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on β-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain β-galactosidase activity during storage in liquid nitrogen at −196°C and during subsequent storage in milk at 5°C was tested. The level of β-galactosidase activity varied among the three strains (0.048 to 0.177 U/10⁷ organisms). Freezing and storage at −196°C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5°C. The strains varied in the extent of the losses of viability and β-galactosidase activity during both types of storage. There was not a significant interaction between storage at −196°C and subsequent storage at 5°C. The strains that exhibited the greatest losses of β-galactosidase activity during storage in milk at 5°C also exhibited the greatest losses in viability at 5°C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed β-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate β-galactosidase activity is provided.     

Catabolite repression of β-galactosidase synthesis in Escherichia coli

1. Repression by glucose of β-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-β-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of β-galactosidase synthesis (e.g. isopropyl thio-β-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-β-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.     

Mutations Affecting Synthesis of β-Galactosidase Activity in the Yeast KLUYVEROMYCES LACTIS

Fifty-one mutants of Kluyveromyces lactis that cannot grow on lactose (Lac^-) were isolated and characterized. All of the mutations are in nuclear genes, are recessive in their wild-type allele and define seven complementation groups, which we designate lac3 through lac9. Strains bearing mutations in lac3, lac5, lac7, lac8 and lac9 are also unable to grow on galactose (Gal^-). Since the Gal^- and Lac^- phenotype co-segregate, they are probably due to a single mutation. Strains bearing mutations in any of the seven complementation groups grow normally on glucose. However, strains bearing mutations in lac3, lac5 and lac6 do not grow on glucose if lactose is also present in the medium. Likewise, strains bearing mutations in lac3 and lac5 do not grow on glucose in the presence of galactose. Complementation groups lac4 and lac5 are loosely linked and map within a cluster of auxotrophic mutations on a chromosome that we designate chromosome 2. The remaining five groups are unlinked. Thus, there is no evidence for clustering of Lac genes into an operon-like regulatory unit.——To further characterize the nature of the Lac^- phenotype, the basal and inducible level of β-galactosidase activity were measured. All mutants had nearly normal basal enzyme levels, except those in lac4, which had barely detectable levels. Inducible enzyme levels varied from barely detectable levels in mutants bearing lac4 mutations up to four-fold inducible levels in strains bearing mutations in other complementation groups. In all cases, however, induction levels were below the 30-fold level obtained in wild-type cells. Three strains bearing lac5 mutations contain increased enzyme activity in the absence of inducer, indicating constitutive synthesis of β-galactosidase. In summary, these data indicate that several genes are necessary for synthesis of β-galactosidase activity.     

Characterization of Thermoanaerobacter Glucose Isomerase in Relation to Saccharidase Synthesis and Development of Single-Step Processes for Sweetener Production

Regulation of glucose isomerase synthesis was studied in Thermoanaerobacter strain B6A, which fermented a wide variety of carbohydrates including glucose, xylose, lactose, starch, and xylan. Glucogenic amylase activities and β-galactosidase were produced constitutively, whereas the synthesis of glucose isomerase was induced by either xylose or xylan. Production of these saccharidase activities was not significantly repressed by the presence of glucose or 2-deoxyglucose in the growth media. Glucose isomerase production was optimized by controlling the culture pH at 5.5 during xylose fermentation. The apparent temperature and pH optima for these cell-bound saccharidase activities were as follows: glucose isomerase, 80°C, pH 7.0 to 7.5; glucogenic amylase, 70°C, pH 5.0 to 5.5; and β-galactosidase, 60°C, pH 6.0 to 6.5 Glucose isomerase, glucogenic amylase, and β-galactosidase were produced in xylose-grown cells that were active and stable at 60 to 70°C and pH 6.0 to 6.5. Under single-step process conditions, these saccharidase activities in whole cells or cell extracts converted starch or lactose directly into fructose mixtures. A total of 96% of initial liquefied starch was converted into a 49:51 mixture of glucose and fructose, whereas 85% of initial lactose was converted into a 40:31:29 mixture of galactose, glucose, and fructose.     

Lactose and galactose uptake by genetically engineered Pediococcus species

The ability to utilize lactose is requisite for lactic acid bacteria used as starters in the dairy industry. Modern genetic recombination techniques have facilitated the introduction of the lactose-positive phenotype into bacteria such as Pediococcus species, which traditionally have not been used as dairy starters. This study investigated lactose and galactose uptake along with phospho-β-galactosidase activity in pediococci that had been transformed with a Latococcus lactis lactose plasmid. Lactose-positive transformants, Pediococcus acidilactici SAL and Pediococcus pentosaceus SPL-2, demonstrated an ability to accumulate [¹⁴C]lactose at a rate greater than the Lactococcus lactis control. Phospho-β-galactosidase activity was also higher in transformants versus Lactococcus lactis. Studies of [³H]galactose uptake suggested that a wild-type galactose transport system and the introduced lactose phosphotransferase system both functioned in galactose uptake by Pediococcus spp. transformants. Significantly lower levels of free galactose were detected in milk fermented with Lactobacillus helveticus LH100 and SAL or SPL-2 than in milk fermented with a LH100 plus Streptococcus thermophilus TA061 control starter blend. Received: 16 September 1997 /  Received revision: 11 November 1997 / Accepted: 21 November 1997     

Postnatal development of β-galactosidase activity in the small intestine of the rat. Effect of adrenalectomy and diet

1. β-Galactosidase activity was studied in homogenates of the proximal and distal thirds of the small intestine from adult and infant rats. o-Nitrophenyl β-d-galactoside served as the substrate. 2. Activity in suckling rats is highest in the distal part of the small intestine. 3. The pH optimum was 3·5 in the distal third of the small intestine in rats aged 5 and 15 days, whereas in the proximal third the maximum was not clearly defined. 4. Activity was higher in both thirds in newborn than in adult rats, expressed per wet wt. or per wt. of protein. In the proximal third activity continually decreases with age, whereas in the distal part there is a rise up to day 15 and then a sudden decrease. Total β-galactosidase activity changes very little in the proximal third during postnatal development; the greatest changes occur in the distal third. 5. Adrenalectomy performed on day 15 postnatally slows down the decrease in β-galactosidase activity, particularly in the distal part. 6. Feeding a lactose diet to infant rats from day 14 postnatally in the presence of the mother rat also slows down the decrease in β-galactosidase activity and this is not found with a diet containing glucose and galactose instead of lactose.     

Towards Enhanced Galactose Utilization by Lactococcus lactis

Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.Lactococcus lactis is a lactic acid bacterium widely used in the dairy industry for the production of fermented milk products. Because of its economic importance, L. lactis has been studied extensively in the last 40 years. A small genome, a large set of genetic tools, a wealth of physiological knowledge, and a relatively simple metabolic potential render L. lactis an attractive model with which to implement metabolic engineering strategies (reviewed in references 21 and 57).In the process of milk fermentation by L. lactis, lactose is taken up and concomitantly phosphorylated at the galactose moiety (C-6) by the lactose-specific phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS^Lac), after which it is hydrolyzed to glucose and galactose 6-phosphate (Gal6P) (64). The glucose moiety enters the glycolytic pathway upon phosphorylation via glucokinase to glucose 6-phosphate (G6P), whereas Gal6P is metabolized to triose phosphates via the d-tagatose 6-phosphate (Tag6P) pathway, encompassing the steps catalyzed by galactose 6-phosphate isomerase (LacAB), Tag6P kinase (LacC), and tagatose 1,6-bisphosphate aldolase (LacD) (Fig. ​(Fig.1).1). Curiously, during the metabolism of lactose by L. lactis, part of the Gal6P is dephosphorylated and excreted into the growth medium, while the glucose moiety is readily used (2, 7, 51, 56, 60).Open in a separate windowFIG. 1.Schematic overview of the alternative routes for galactose uptake and further catabolism in L. lactis. Galactose can be imported by the non-PTS permease GalP and metabolized via the Leloir pathway (galMKTE) to α-G1P, which is converted to the glycolytic intermediate G6P by α-phosphoglucomutase (pgmH). Alternatively, galactose can be imported by PTS^Lac (lacFE) and further metabolized to triose phosphates by the Tag6P pathway (lacABCD). Here, we propose a new uptake route consisting of galactose translocation via the galactose PTS, followed by dephosphorylation of the internalized Gal6P to galactose, which is further metabolized via the Leloir pathway (highlighted in the gray box). galP, galactose permease; galM, galactose mutarotase; galK, galactokinase; galT, galactose 1-phosphate uridylyltransferase; galE, UDP-galactose-4-epimerase; pgmH, α-phosphoglucomutase; lacAB, galactose 6-phosphate isomerase; lacC, Tag6P kinase; lacD, tagatose 1,6-bisphosphate aldolase; lacFE, PTS^Lac; PTS^Gal, unidentified galactose PTS; Phosphatase; unidentified Gal6P-phosphatase; pgi, phosphoglucose isomerase; pfk, 6-phosphofructo-1-kinase; fba, fructose 1,6-bisphosphate aldolase; tpi, triose phosphate isomerase; α-Gal1P, α-galactose 1-phosphate; α-G1P, α-glucose 1-phosphate; UDP-gal, UDP-galactose; UDP-glc, UDP-glucose; G6P, glucose 6-phosphate; Gal6P, galactose 6-phosphate; Tag6P, tagatose 6-phosphate; TBP, tagatose 1,6-bisphosphate; FBP, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate. The dotted arrow represents the conversions of GAP to pyruvate via the glycolytic pathway. Steps essential to improve galactose consumption are shown in black boxes.As a result of incomplete lactose utilization, some fermented dairy products contain significant residual amounts of galactose. The presence of galactose has been associated with shoddier qualities of the fermented product (6, 27, 43). In particular, galactose is a major contributor to the browning that occurs when dairy products (e.g., yogurt and mozzarella, Swiss, and cheddar cheese) are cooked or heated in the manufacture of pizzas, sauce preparation, or processed cheese. In addition, availability of residual galactose may result in production of CO₂ by heterofermentative starters and, consequently, in textural defects such as the development of slits and fractures in cheeses. Therefore, the availability of starter strains with improved galactose utilization capacity is desirable to develop higher-quality dairy products. Additionally, strains with increased galactose metabolism could provide galactose-free foods for individuals and, in particular, children suffering from the rare disease galactosemia (36). To this end, a comprehensive understanding of galactose catabolism is essential.Galactose metabolism in L. lactis was thoroughly studied in the past and has been and still is the subject of some controversy. Indeed, conflicting results regarding the type of PTS involved in galactose uptake have been published. Some authors advocated that galactose is exclusively transported via the plasmid-encoded PTS^Lac, whereas others proposed transport via a galactose-specific PTS (PTS^Gal) to the extreme of questioning the contribution of the PTS^Lac (17, 20, 50, 59). However, a gene encoding PTS^Gal has never been identified in L. lactis. Independently of the nature of the PTS, it is generally accepted that the resulting Gal6P is metabolized via the Tag6P pathway (lac operon) (Fig. ​(Fig.1).1). On the other hand, galactose translocated via the highly specific galactose permease (GalP) is metabolized via the Leloir pathway to α-glucose 1-phosphate (α-G1P) through the sequential action of galactose mutarotase (GalM), galactokinase (GalK), and galactose 1-phosphate uridylyltransferase (GalT)/UDP-galactose-4-epimerase (GalE) (gal operon). Entry in glycolysis is preceded by the α-phosphoglucomutase (α-PGM)-catalyzed isomerization of α-G1P to G6P. The use of the Leloir and/or the Tag6P pathway for galactose utilization is currently viewed as being strain dependent (9, 16, 25, 32, 33, 58), but the relative efficacy in the degradation of the sugar has not been established.The ultimate aim of this study was to engineer L. lactis for improved galactose-fermenting capacity as a means to minimize the galactose content in dairy products. To gain insight into galactose catabolism via the Leloir (gal genes) and the Tag6P (lac genes) pathways, a series of L. lactis subsp. cremoris NZ9000 isogenic gal and lac mutants were constructed. Carbon 13 labeling experiments coupled with nuclear magnetic resonance (NMR) spectroscopy were used to investigate galactose metabolism in the gal and lac strains. The data obtained revealed a novel route for galactose dissimilation and provided clues to further enhance galactose utilization.     

Klebsiella aerogenes Strain Carrying Drug-Resistance Determinants and a lac Plasmid

In addition to carrying determinants conferring resistance to at least two antibiotics, chloramphenicol and streptomycin, a Klebsiella aerogenes strain contains a plasmid responsible for increased β-galactosidase activity. The plasmid can be transferred to Escherichia coli and Salmonella typhimurium strains. K. aerogenes segregants without the plasmid grow on lactose one-half as fast as the parent strain and contain only one-tenth to one-fifth as much β-galactosidase.     

beta-Galactosidases in Ripening Tomatoes

Tomatoes (Lycopersicon esculentum L.) contained a high level of β-galactosidase activity which was due to three forms of the enzyme. During tomato ripening, the sum of their activities remained relatively constant, but the levels of the individual forms of β-galactosidase changed markedly. The three enzymes were separated by a combination of chromatography of DEAE-Sephadex A-50 and Sephadex G-100. During ripening of tomatoes, β-galactosidases I and III levels decreased but the β-galactosidase II level increased more than 3-fold. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. However, the enzymes differed in molecular weight, K_m value with p-nitrophenyl-β-galactoside, and stability with respect to pH and temperature. β-Galactosidase II was the only enzyme capable of hydrolyzing a polysaccharide that was isolated from tomatoes and that consisted primarily of β-1, 4-linked galactose. The ability of β-galactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening.     

Genetic Construction of Lactose-Utilizing Xanthomonas campestris

Xanthomonas campestris, the producer of xanthan gum, possesses a β-galactosidase of very low specific activity. Plasmid pGC9114 (RP1::Tn951), generated by the transposition of the lactose transposon Tn951 to RP1, was conjugally transferred into XN1, a nalidixic acid-resistant derivative of X. campestris NRRL B-1459S-4L. Transfer occurred on membrane filters and in broth. The β-galactosidase gene of Tn951 was expressed in X. campestris. The specific activity of β-galactosidase in transconjugants was over 200-fold higher than that in XN1, and transconjugants grew as well in lactose-based media as in glucose-based media. The lactose-utilizing transconjugants could potentially be used to produce xanthan gum from cheese whey.     

Microbial Production of Poly-β-Hydroxybutyric Acid from d-Xylose and Lactose by Pseudomonas cepacia

Poly-β-hydroxybutyric acid (PHB) was produced from xylose and lactose by using Pseudomonas cepacia. Approximately 50% PHB (grams of PHB total/grams of biomass total) was produced. With a laser-based fluorescent probe, β-galactosidase activity was shown to be induced in P. cepacia cells grown on lactose but not in those grown on glucose or xylose. P. cepacia has the potential to produce biodegradable thermoplastics from hemicellulosic hydrolysates and cheese whey.     

Enzymic hydrolysis of sphingolipids: Hydrolysis of ceramide lactoside by an enzyme from rat brain

Ceramide lactoside [1-O-(galactosido-4-β-glucosido)-2-N-acyl-sphingosine] was hydrolysed to ceramide glucoside and galactose by β-galactosidase of rat brain. The reaction was not reversible, required cholate or taurocholate, had optimum pH5·0 and K_m 2·2×10⁻⁵m. It was inhibited by γ-galactonolactone and galactose as well as by ceramide, sphingosine and fatty acid. Ceramide lactoside could be degraded to ceramide, galactose and glucose by mixtures of rat-brain β-galactosidase and ox-brain β-glucosidase.     

Cold-Adapted β-Galactosidase from the Antarctic Psychrophile Pseudoalteromonas haloplanktis

The β-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was purified to homogeneity. The nucleotide sequence and the NH₂-terminal amino acid sequence of the purified enzyme indicate that the β-galactosidase subunit is composed of 1,038 amino acids with a calculated M_r of 118,068. This β-galactosidase shares structural properties with Escherichia coli β-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters. P. haloplanktis β-galactosidase was expressed in E. coli, and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both P. haloplanktis wild-type and recombinant β-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis β-galactosidase can outperform the current commercial β-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted β-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.     

Characterization of a Novel β-Galactosidase from Bifidobacterium adolescentis DSM 20083 Active towards Transgalactooligosaccharides

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highest V_max values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.