MX2008008117A - Alpha-galactosidase from bifidobacterium bifidum and its use - Google Patents

Abstract

The present invention concerns a newα-galactosidase with transgalactosylating activity isolated from Bifidobacterium bifidum. Theα-galactosidase is capable of converting mellibiose toα-galactobiose disaccharides which may be incorporated into numerous food products or animal feeds for improving gut health by promoting the growth of bifidobacteria in the gut, and repressing the growth of the pathogenic microflora.

Description

ALPHA-GALACTOSIDASE FROM BIFIDOBACTERIUM BIFIDUM AND ITS USE FIELD OF THE INVENTION The present invention concerns a new α-galactosidase with transgalactosylating activity capable of converting melibiose to disaccharides of α-galactobiose. In particular, it concerns an isolated β-galactosidase from a recently discovered Bifidoba cteri um bifidum strain. The invention particularly concerns DNA sequences encoding the isolated a-galactosidase enzyme, the enzyme encoded by such a DNA sequence and a host cell comprising a DNA sequence or containing a recombinant vector that incorporates the DNA sequence . The invention also concerns the use of the enzyme encoded by the DNA sequence, or of the host cell containing a DNA sequence or the recombinant vector, to produce α-galactobiose.

BACKGROUND OF THE INVENTION Bifidobacteria naturally colonizes the lower intestinal tract, an environment that is poor in mono and di-saccharides, therefore said sugars are preferably consumed by the host and microbes present in the upper intestinal tract. In order to survive in the lower intestinal tract, the bifidobacteria produce several classes of exo- and endo-glycosidases in extracellular and / or surface-bound forms, which is why they can use various carbohydrates. In addition to the hydrolase activity, some enzymes of bifidobacteria show transferase activity. This activity of transglycosylation of glycosidases is used extensively for the enzymatic synthesis of several oligosaccharides, which have proven to act as growth factors of bifidobacteria. It is known that members of bifidobacteria produce β-galactosidase enzymes that are involved in the bacterial metabolism of lactose. Moller, P. L. and collaborators in Appl. & Environ. Microbial , (2001), 62, (5), 2276-2283 describes the isolation and characterization of three ß-galactosidase genes from a strain of Bifidoba cteri um bifidum. They found that all three β-galactosidases were capable of forming galacto-oligosaccharide ligations in β by transglycosylation- It is known that some species of bifidobacteria, but not B. bifidum produce α-galactosidases as well as β-galactosidases belonging to the group of glycosyl hydrolases and can be classified into two groups based on their substrate specificity, that is, one group is specific for small saccharides such as p-nitrophenyl-aD-galactopyranoside, melibiose and raffinose, and the other group can release galactose from galactomannans such as guar gum, in addition to small substrates. A strain of Bifidoba cteri um bifidum has been found that is capable of producing an enzymatic activity galactosidase that converts lactose to a new mixture of galacto-oligosaccharides that unexpectedly contain up to 35% of disaccharides including galabiosa (Gal (a 1-6) -Gal ). This disaccharide is known (see Patón, JC &Patón, AW (1998), Microbial Clin., Rev., 11, 450-479; Carlsson, KA (1989), Ann. Reviews Biochem., 58, 309-350) for being an anti-adhesive capable of preventing the adhesion of toxins, for example the Shiga toxin and pathogens such as E. coli to the wall of the intestine. This strain of B. bifidum was deposited under Accession No. NCIMB 41171 in the National Collection of Industrial & Marine Bacteria, Aberdeen, UK on March 31, 2003. It is also described in UK Patent No. 2 412 It was found that this strain of Bifidum produces an α-galactosidase that is capable of converting melibiose to disaccharides of α-galactobiose.

SUMMARY OF THE INVENTION In accordance with the invention, there is provided a DNA sequence encoding a protein with an amino acid sequence as given in SEQ ID NO: 2 or hybridizing or stringency conditions to the DNA sequence encoding a this protein. The DNA sequence is given in SEQ ID NO: 1 or may comprise a fragment or degenerative thereof. The phrase "degenerative" was constructed to mean a DNA sequence that is at least 50% homologous to SEQ ID NO: 1, preferably from 50 to 98% homologous, more preferably from 75 to 95% homologous. Such a DNA sequence may comprise substitutions, additions or deletions of nucleotides that result in less than 60%, preferably less than 45%, more preferably less than 25% change in the amino acid sequence shown in SEQ ID NO: 2 , Nucleotide substitutions can result in conservative amino acid substitutions.

In accordance with a second aspect of the invention, an enzyme encoded by a DNA sequence was provided as defined above. Such an enzyme can comprise the amino acid sequence given in SEQ. ID. NO: 2 or a fragment of it. According to a third aspect of the invention there is provided a recombinant vector, preferably an expression vector, comprising a DNA sequence as defined above. Such a vector can be incorporated into a host cell such as a bacterial, yeast or fungal cell. Alternatively, the DNA sequence can be incorporated into such a host cell. A suitable host cell can be selected from Bifidoba cterium, La ctococcus, La ctoba cillus, Baci llus, for example Ba cil lus subti lis or Bacillus circulans, Escherichia and Aspergi l l us for example Aspergillus niger. Using melibiose as substrate, the enzyme encoded by the horn DNA sequence defined above produces a mixture of oligosaccharides, in particular a-galactobiose disaccharides. The enzyme or host cell as described above can be used to produce a-galactobiose disaccharides, which can be part of a product to improve intestinal health. A product such may be selected from the group consisting of dairy products (e.g., liquid milk, dry milk powder such as whole milk powder, skim milk powder, fat milk powder, whey powder, infant formula, ice cream, yogurts, cheese, fermented dairy products), beverages such as fruit juice, baby food, cereals, bread, pastries, confectionery, cakes, food supplements, dietary supplements, probiotic food products, animal foods, bird foods, or really any of other foods or beverages. Alternatively, the disaccharides thus produced can be used for the preparation of a medicament, for example in a tablet or capsule to prevent the adhesion of pathogens or toxins produced by pathogens to the intestinal walls. The medication can be administered to a patient, for example after a course of antibiotic treatment, which often alters or even destroys the healthy normal intestinal flora. In accordance with yet a further aspect of the invention there is provided a process for producing an enzyme as defined above which comprises culturing a host cell as defined above in a suitable culture medium ba or conditions that allow the expression of the enzyme and the recovery of the enzyme or enzymatic products resulting from the culture. The invention is also directed to a process for producing galactobiose disacchards comprising contacting the enzyme as defined above or a host cell as defined above with a material containing melibiose or conditions leading to the formation of the disaccharides. The material containing suitable melibiose can be selected from commercially available melibiose, raffinose, stachyose, or as a soybean extract. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) of Bifidoba cteri um bifidum a-galactosidase with the start and stop codons indicated in bold letters; Figure 2 shows the nucleotide sequence of Figure 1, with the amino acid sequence (SEQ ID NO: 2) of the Enzyme; Figure 3 shows the first 540 amino acids of the amino acid sequence (SEQ ID NO: 2) of Figure 2; Figure 4 is a graph showing the reaction over time during the synthesis of α-galacto-oligosaccharides with α-galactosidase and 40% (by weight) of melibiose in 0.1 M phosphate buffer at pH 6 as substratum; and Figure 5 shows a high resolution anion exchange chromatogram of the mixture of α-galacto-oligosaccharides synthesized by β-galactosidase of B. bifidum NCIMB 41171 using melibose at 40% by weight in 0.1 M phosphate buffer at pH 6.0 as substrate. (Glc = glucose, Gal = galactose, Mel = melibiose, DP = degree of polymerization). The darkened arrows denote the galacto-oligosacrylic products.

DETAILED DESCRIPTION OF THE INVENTION Genomic DNA was isolated from the strain Bifidoba cteri um bifidum (NCIMB 41171 using the method of Lawson et al. (1989) Fems Microbiol Letters, 65, (1-2), 41-45. was digested with restriction enzymes and fragments having a maximum size of 15 kbp were ligations with the vector pBluescript KS (+). E. coli cells were transformed with a vector containing inserts consisting of chromosomal DNA digested with PstI from of B. bifidum, clones were selected with a-galactosidase activity on Luna Bertam agar plates containing p-nitrophenyl a-D-galactopyranoside and isopropyl-β-D-thiogalactoside (IPTG). Two positive a-galactosidase clones (pMelAl and pMelA2) were identified. The two positive clones were digested with EcroRl, PstI and BamHI and showed a similar restriction pattern indicating that both contained the same inserted DNA fragment. The DNA sequence of the inserted MelAl DNA fragment was made, using Sanger's deoxid chain termination method (Russel P., 2002 Genetics, Pearson Education, Inc., San francisco, 187-189) using the equipment for BigDye Terminator V.3.0 cycle sequence formation (Applied Biosystems, USA). The MelAl DNA sequence is shown in Figure 1 (SEQ ID NO: 1). The Open Reading Frame (ORF) was located using the ORF discoverer from NCBI (National Center of Botechnology Information). The bacterial genetic code was used and the frame length was determined to be 300 bp. The nucleotide sequence of Figure 1 was translated in all six reading frames and an open reading frame of 759 amino acids encoding an α-galactosidase was identified. The translation is shown in Figure 2 (SEQ ID NO: 2).

The present invention will be further described by way of reference in the following examples. Example 1 Materials and Methods All chemicals and preparation media used in the course of this study were obtained from Sigma (Dorset, UK), Invitrogen (Paisley, UK), Oxoid (Basinbstoke, UK), Qiagen (West Sussex, UK ) and Promega (Southhampton, UK). Bacterial strains The strain of Bifidoba cteri um bifidum (NCIMB 41171) were kept on cryogenic beads in tubes of Microbank at - 70 ° C. For these experiments, the strain was revived on Wilkinson Chalgren (WC) agar (Oxoid, UK) and TPY medium (trypticase phytase yeast extract medium) and were anaerobically developed (composition of C02 and N2, 80% and 20%, respectively) a 37 ° C for 48 hours. The morphology of the colony and the sequence of contamination were tested by means of Gram stain. Strains of E. The strains of Escheri chia coli RAI Ir and DH5a used in this study were commonly incubated under aerobic conditions at 37 ° C in Luria Bertani's broth or agar (LB) (Sambrook J. and Russell, W. D. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York) and when necessary supplemented with antibiotics (100 μg / ml ampicillin and / or 15 μg / ml chloramphenicol) and 40 μl Xa-galactopyranoside (Xa-Gal), 7 μl of ( Isopropyl-ß-D-thiogalactoside) 20% IPTG which were applied on the surface of a pre-processed 90 mm agar plate. The strain of E. coli RAI Go deficient in α-galactosidase (Hanataní et al., 1983, J. Biol. Chem., 259, (3), 1807-18112 (genotype: MelA-B +, recA-, IacZ-Y -) is a derivative of E. col i K12 and was used in expression experiments E. coli strain DH5a (Invitrogen, Paisley, UK) (genotype: F. f80IacZ? M? (lacZYA-argF) U169 recAl e_ndAl hsdRl l (rk ", mk") p oA supE44 t? -1 gyrA96 relAl?) is a positive α-galactosidase strain and was used for all other genetic manipulations .The selection of E. coli strain RAllr, for expression experiments, it was done in accordance with its genotype.This strain does not code for an active a-galactosidase due to the melA mutation on its chromosomal DNA, however, this strain has an active melibiose transporter which is necessary for the transport of sugars (melibiose) in the cytoplasm and therefore the metabolism of them by means of active a-galactosides. It is not known if the a-galactosidase of Bifidoba cteri um bifidum was expressed intracellularly or extracellularly. Thus, the existence of an active melibiose transporter was essential for the identification of positive a-gal clones and consequently the isolation of genes encoding α-galactosidase. In addition this strain is a mutant recA, which minimizes the recombination of the introduced DNA with host DNA, thus increasing the stability of the inserts. Extraction of Genomic DNA from Bifidobactepum bifidum Genomic DNA was isolated from the strain Bifidoba ctep um bifidum (NCIMB 41171) using the method of Lawson et al. (1989). In accordance with this method, the cells were harvested from the plates in 0.5 ml of TES regulator in 1.5 ml Eppendorf tubes. 10 μl of mixture of lysozyme / mutanolysin (4: 1, lysozyme, 10 mg / ml, mutanolysin, 1 mg / ml) was added and the mixture was then mixed and incubated for 30 minutes at 37 ° C. The cells were then treated with 10 μl of proteinase K (at 20 mg / ml) and 10 μl of RNase (10 mg / ml), mixed and incubated for 1 hour.

Hour at 65 ° C. After incubation, 100 μl of 10% SDS was added and the cell lysates were gently mixed by inversion and incubated for an additional 15 minutes at 65 ° C, followed by addition of 0.62 ml of phenol / chloroform and mixed by inversion until an emulsion was formed. The cell lysate was centrifuged at 6,500 rpm for 10 minutes and the upper aqueous layer was transferred to a clean Eppendorf tube using a broad-bore, flamed blue pipette tip. The extraction (deprotemination step) was repeated until the cell debris was completely renewed. The DNA was precipitated by the addition of 1 ml of ice-cold ethanol followed by incubation for at least 30 minutes on ice or stored overnight in a freezer at 20 ° C. The genomic DNA was recovered by centrifugation at 13,000 rpm for 5 minutes and after drying it was resuspended in 50 μl of 10 mM sterile Tris-Cl at pH 8. The extracted DNA was analyzed by gel electrophoresis and the concentration was measured at 260 nm. It was stored at -20 ° C or - 70 ° C for long periods of time and defrosting and multiple freezing were avoided in order to reduce the possibility of degradation. Preparation of vector DNA The vector used in the course of this study was pBluescppt KS (+) (Stratagene, North Torrey Fines Road). This cloning vehicle was selected because of the promoter c, which codes for pBluescript KS (+), which is necessary for the initiation of transcription of genes lacking their own promoter.

The vector was digested with the following restriction enzymes: PstI, BaI7HI and EcoRI according to the manufacturer's instructions using a ten-fold excess of enzyme on the DNA (enzyme units: DNA equal to ten units of enzyme per one ug of plasmid DNA or ten enzyme units per 0.5 pmole of Plasmid DNA). After the thermal inactivation of the enzyme (20 minutes at 65 ° C) the restriction patterns were analyzed by horizontal gel electrophoresis analysis. The vectors were additionally dephosphorylated with CIAP (Calcium Alkaline Phosphatase of Veal (Promega, Southampton, UK) in accordance with the manufacturer's instructions.The efficiency of the treatment was tested by auto-ligation of the vector (with bacteriophage T4 DNA ligase according to manufacturer's instructions) followed by transformation into DH5a cells.

The presence of a single fragment in the gel indicated the complete digestion of the vector and the unique restriction digestion of it. Sufficient digestion of the vector was also proved by transformation of unbound molecules into DH5a cells of E. competent coli. The number of colonies formed on LB agar plates supplemented with ampicillin (100 μg / ml) was an indicator of the undigested molecules and the expected support during subsequent experiments. Construction of Genomic DNA Libraries Genomic DNA was partially digested with three restriction enzymes that frequently recognize the occurrence of hexa-nucleotide sequences in prokaryotic DNA. EcoRI, BamHI and PstI are type II restriction endonucleases that specifically recognize the 5'G / AATTC'3, 5'G / GATCC'3 and 5'CTGCA / G'3 sequences respectively, and elaborate double broken strands in these sequences generating 5 'suspensions of four nucleotides, AATT, GATC, by EcoRI and BamHI respectively, and 3' suspensions, ACGT by Pstl. All these enzymes were active and able to fragment DNA only in the presence of divalent magnesium ions. These ions were the only cofactors required. Digestion of DNA restriction All restriction digestions of the genomic DNA samples were incubated for 2 hours at 37 ° C and finally thermally inactivated at 65 ° C for 20 minutes. The reactions were then cooled to room temperature and the appropriate amount of charge regulator was added, followed by gentle mixing with a sealed glass capillary. The solution was then loaded into wells of a 0.6% agarose gel (energy delivered 4-5 volts / cm for 14-16 hours), and the size of the digested DNA was estimated to that of the DNA standards of 1 kbp ( Promega, UK) (Sambrook J. Molecular Cloning: A Laboratory Manual (2002)). Purification of the Generated Fragments After Restriction Digestion The purification of fragments of the reaction mixtures and the agarose gels were done using the QIAGEN gel extraction equipment from Qiagen (West Sussex, UK). The protocols are described in detail in the manufacturer's manual. Transformation and DNA Ligation After purification of the DNA fragments with the Qiagen gel extraction equipment, they were ligated with the pBluescript KS (+) vector treated with CIAP. For ligation, appropriate amounts of DNA were transferred to sterile 0.5 ml microfuge tubes as shown in Table 1. Table 1: Ligation mixtures. The A tube gives us the number of DNA ligands auto-ligations that must be subtracted from the total number of transformants after transformation. Tube B shows the ligation of the vector with the DNA fragments and Tube C shows the control in order to calculate the efficiency of the transformation. DNA A vector tube (15 fmoles [~ 29.7 ng]) B Vector (15 fmoles ~ 29.7 ng of DNA) plus insert (15 external moles ~ 69.3 ng) C pUC control (0.056 fmoles [~ 100 pgl]) The molar ratio of vector of plasmid DNA to inserted DNA fragment should be ~ 1: 1 in the ligation reaction. The final DNA concentration should be ~ 10 ng / μl. Before each ligation the DNA fragments were heated at 45 ° C for 5 minutes to melt any cohesive termini that was re-strengthened during the preparation of the fragment. A molar ratio of vector: insert DNA of 1: 1 was selected for all ligation reactions and the reaction assembly was made in accordance with Promega instructions.

To Pipes A and B were added 10 μl of lOx ligation buffer and 0.5 Weiss units of T4 DNA ligase (Promega, UK) and the ligation volume was adjusted to 10 μl with water of grade for molecular biology. To the tube C, 1.0 μl of lOx ligation buffer was added and the ligation volume was adjusted to 10 μl with water of grade for molecular biology. The DNA fragments were added to the tubes together with water and then heated at 45 ° C for 5 minutes to melt any cohesive termini that was re-strengthened during the preparation. The DNA was quenched at 0 ° C before the remaining ligation reagents were added and the reaction mixtures were incubated overnight at 16 ° C (Sambrook and Russell, 2001). After ethanol precipitation and purification of the ligation fragments (in order to remove the ligation mixture causing the reduction of transformation efficiency) the transformations were carried out in accordance with the Hanahan instructions. ~ 50 ng of DNA ligation was added in 5 μl of solution to 100 μl of RAI Ir cells from E. col i competent. After heat treatment and expression of the penicillin resistance gene, the cells were spread on the surface of LB plates containing ampicillin (100 μg / ml), X-a-Gal, (40 μl of 2% X-a-Gal) and IPTG (7 μl of 2% IPTG). The number of transformants of each ligation reaction was measured. The number of transformants commonly obtained from Tube C was 2x10 5 -lxlO6 cfu / μg while that of Tube A was 500-600 cfu / μg. The number of transformants in Tube A was an indication of efficient treatment of vector DNA. The number of transformants in Tube B was in the range of 2 -4xl04 cfu / μg. Number of transformants Mixtures of chromosomal DNA ligation with Pstl gave rise to two positive α-galactosidase clones (pMelAl and pMelA2) of approximately 2500 screened transformants, whereas chromosomal DNA treated with EcoRI and BamHI did not give any positive clones of approximately 4000 screened transformants. Digestion of the Positive Clone The two positive PstI clones were digested with the restriction enzymes EcroRI, PstI, Bamtil, HindIII, Smal, and Kpnl. The restriction enzymes EcroRI, PstI, and BamHI, showed similar restriction patterns, a fragment of ~ 5 kbp (gene of interest) and one of ~ 3 kbp (plasmid DNA) indicating that these enzymes were cut in the same position . HindIII, gave a fragment of 6.5 kbp and a fragment of 1.5 kbp while the enzymes Smal, and KpnX gave a fragment with size of ~ 8 kbp indicating that they were cut in only one position. Similar restriction patterns for both plasmids were an indication that both contained the same DNA fragment insert. Formation of DNA Sequences The formation of DNA sequences with the Sander dideoxy chain termination method was performed by using the BigDye Terminator v.3.0 cycle formation equipment (Applied Biosystems, USA) and analyzed with the ABI Ppsm 3100, a fluorescence-based DNA analysis system that incorporates capillary electrophoresis. The 5'- and 3'- ends of the inserted DNA fragments were sequenced with vector-specific primers. The inserts were further sequenced by using Genome Priming Systems (GPS-1) (New England Biolabs, UK), GPS-1 is a m vitro system based on the Transposon TN7, which uses the TnsABC Transposase to insert transposon randomly into the Target DNA The mass ratio of donor: target DNA of 1: 4 was used in accordance with the manufacturer's instructions. The number of isolated plasmids for sequence formation after Insertion of the Trans-primer in the target plasmid was 25. This number was calculated in accordance with the manufacturer's instructions and assumes a 5-fold depth of coverage. Unique priming sites at both ends of the Trans-primer element allowed sequencing of both strands of the target DNA in the position of the invention. The reaction mixture for sequence formation contained approximately 600-600 mg of plasmid DNA, 3.2 pmoles of primer solution and 4 μl of BigDye Terminator solution. Identification of the Open Reading Framework The open reading frame (ORF) was located using the ORF search engine from NCBI. The bacterial genetic code was used and the extension of the frame was determined to be 300 bp. The nucleotide sequence was transposed in the six possible frames and an open reading frame of 759 amino acids encoding putative a-galactosidase was identified (The translation is shown in Figure 2). The start and stop codons were confirmed. The a-galactosidase gene of Bi fidoba ctep um in the plasmid pMelAl was expressed in E. coll ba or developmental conditions that would normally repress the expression of the lacZ promoter from E. col i inducible located in the flanking region of the cloning vector. This observation indicated that internal bifidobacterial sequences, endogenous from the 5 'end to the 3' end in the sense of the duplex DNA strand of the a-galactosidase gene, can serve as a transcription initiation signal in E. coli The start of transcription with bold italics was indicated. The above results indicate that the gene is controlled from its own promoter for transcription. Example 2 Synthesis with the cloned enzyme α-galactosidase isolated from Bifidoba cteri um bifidum NCIMB 41171 in host E. coll (strain RA11) The following synthesis described, unless otherwise indicated, was carried out with E. coli RAll host cells after treatment of the E. coli biomass (collected by centrifugation at 10,000 g) with toluene at a concentration of 2000 rpm in order to increase cell permeability and also convert non-viable cells by destruction of their cytoplasmic membrane. The biomass of E. coli was prepared as described in Example 1 under "E.coli strain".

Synthesis with the cloned enzyme Synthesis of a-galactosidase at a substrate concentration of 40% (by weight) the initial melibiose concentration was performed. The synthesis solution was prepared in 0.1 M phosphate buffer at pH 6.0. The synthesis was carried out at 40 ° C in a water bath with shaking at 150 rpm. The optimum pH for the specific enzyme was selected on activity measurements (using p-mitrophenyl-α-D-galactopyranoside as a substrate) of an enzymatic preparation for varying pH values. For the synthesis of α-galactosidase 2 ml of a cellular suspension of RA11 of E. col i (with an activity of 0.3 U / ml) were centrifuged (at 10, 000 g) to collect the biomass and the supernatant was discarded. This biomass was re-suspended with 1 g of 40% melibiose substrate solution (by weight) in order to effect the synthesis. The concentrations of the different sugars present in the mixture during the synthesis are shown in Figure 4. High resolution ammonium exchange chromatography coupled with pulsed amperometric detection chromatograms (HPAEC-PAD) of mixtures of galacto-oligosaccharides synthesized by the α-galactosidase cloned from B. bifidum NCIMB 41171 are shown in Figure 5. Sugar concentrations of the mixture of galacto-oligosaccharides at the optimal time point of the synthesis are shown in Table 2. Table 2. Carbohydrate composition of the a-galacto-oligosaccharide synthesis at 40% (by weight of the initial melibiose concentration at the time point where the maximum concentration of oligosaccharides was observed Subst.Inic, GOS GOS Mel Glc Gal of Synthesis DP> 3 DP = 2 by weight Concentration of total sugars 40 13.93 6.61 38.06 24.1 17.29 Mel = Melibiose, Glc = Glucose, Gal = galactose, DP = degree of polymerization.

Claims (19)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as novelty, and therefore the content of the following is claimed as property: CLAIMS 1. - A DNA sequence characterized in that a) encodes a protein with an amino acid sequence as given in SEQ. ID. NO: 2, or b) hybridizes under severe hybridization conditions to the sequence of a), c or c) is a degenerative sequence of a) or b) 2. - The DNA sequence according to claim 1, characterized in that the sequence is given in the SEC. ID. NO: 1 or a fragment of it. 3. The DNA sequence according to claim 1 or claim 2, characterized in that said sequence comprises substitutions, additions or eliminations of nucleotides that result in less than 60%, preferably less than 45%, more preferably less than 25%. % change in the amino acid sequence according to SEC. ID. NO: 2 or a fragment of it. 4. - A DNA sequence in accordance with the Claim 1 or Claim 2, characterized in that said sequence comprises substitutions of nucleotides that result in conservative amino acid substitutions. 5. An enzyme encoded by a DNA sequence according to any one of Claims 1 to 4. 6. An enzyme characterized in that it comprises an amino acid sequence in accordance with SEQ. ID. NO: 2 or a fragment of it. 7. An a-galactosidase characterized in that it has the sequence according to SEC. ID. NO: 2. 8.- A recombinant vector characterized in that it comprises a DNA sequence according to any one of Claims 1 a. 9. The vector according to claim 8, characterized in that said vector is an expression vector. 10. A host cell characterized in that it comprises a DNA sequence according to any one of Claims I a. 11. A host cell characterized in that it comprises the vector according to claim 8 or claim 9. 12. - The host cell according to claim 10 or claim 11, characterized in that said cell is a bacterial cell, a yeast cell or a fungal cell. 13. The host cell according to claim 12, characterized in that it is selected from the group consisting of Bifidumbacterium, Lactococcus, Lactobacillus, Escherichia, Bacillus and Aspergillus. 14. The host cell according to claim 13, characterized in that the cell is selected from the group consisting of Bifidobacterium bifidum, Bacillus subtillus, Bacillus circulans and Aspergillus niger. 15. Use of an enzyme according to any one of Claims 5 to 7, or a cell according to any one of Claims 10 to 14, to produce a-galactosidase disacchards. 16. Use of an enzyme according to any one of Claims 5 to 7, or a cell according to Claims 10 to 14, to produce a-galactobiose disaccharides to be part of a product selected from the group consisting of : dairy products such as liquid milk, dry milk powder, baby milk, baby formula, ice cream, yogurt, cheese, fermented milk products, beverages such as fruit juice, baby food, cereals, bread, pastries, confectionery, cakes, food supplements, dietary supplements, probiotic food products, animal foods, poultry foods, and medications. 17. Use of a host cell according to any one of Claims 10 to 14, to produce a product selected from the group consisting of: dairy products such as liquid milk, dry milk powder, baby milk, baby formula , ice cream, yogurts, cheese, fermented milk products, beverages such as fruit juice, baby food, cereals, bread, pastries, confectionery, cakes, food supplements, dietary supplements, probiotic food products, animal feed, poultry feed, and medications. 18. A product for producing an enzyme according to any one of Claims 5 to 7, characterized in that it comprises culturing a host cell according to any one of Claims 10 to 14 in a suitable culture medium under conditions that allow the expression of said enzyme and recover the resulting enzyme, from the culture. 19. A process for producing a disaccharide, characterized in that it comprises contacting a enzyme according to any one of Claims 5 to 7 or a host cell according to any one of Claims 10 to 14 with a solution of melibiose.