JP2021087390A - β-galactosidase - Google Patents

Abstract

To provide a new β-galactosidase and a method for producing galactooligosaccharides using the same.SOLUTION: The present disclosure provides β-galactosidase that is derived from Streptomyces bacteria and has the following property (1). Action (1): transferring of a β-D-galactoside bond.SELECTED DRAWING: None

Description

The present invention relates to β-galactosidase derived from Streptomyces spp. And a method for producing galactooligosaccharide using the same.

Galactooligosaccharide (GOS) is one of the functional oligosaccharides that increases bifidobacteria in the digestive tract and exhibits an intestinal regulating effect. As the main components of galactooligosaccharides, trisaccharides in which galactose is glycosidic-bonded to the non-reducing end of lactose, and tetrasaccharides in which galactose is further bound are known. Transferred disaccharides are also included in it.

Galactooligosaccharides are industrially produced using lactose as a raw material by utilizing a sugar transfer reaction with β-galactosidase (EC3.2.1.23). As microorganisms that produce such β-galactosidase, Bacillus circulans, Aspergillus oryzae, Kluyveromyces lactis, and the like have been reported. Among them, β-galactosidase derived from Bacillus circulans is widely used due to its high transglycosylation activity (for example, Patent Document 1).

On the other hand, actinomycetes represented by Streptomyces spp. Are bacteria that have both the physical strength of the cell wall and the chemical strength of DNA, and are various secondary metabolites such as antibiotics. It is industrially used as a group of useful bacteria that produce. However, β-galactosidase, which is derived from Streptomyces spp. And has high transglycosylation activity, is not known.

International Publication No. 2016/027747

The present invention is intended to provide a novel β-galactosidase and a method for producing a galactooligosaccharide using the same β-galactosidase.

The present inventor has found two types of β-galactosidase having high transglycosylation activity from Streptomyces olivaceoviridis belonging to the genus Streptomyces. Furthermore, they have found that galactooligosaccharides can be obtained with high productivity by using this β-galactosidase, and completed the present invention.

That is, the present invention provides the following [1] to [12].
[1] A β-galactosidase derived from the genus Streptomyces and having the following property (1).
(1) Action: The β-galactosidase according to [1], which transfers the β-D-galactoside bond [2] and has the following properties (2).
(2) Action: β-galactosidase activity [3] The β-galactosidase according to [1] or [2], which further has the following property (3).
(3) Molecular weight by SDS polyacrylic gel electrophoresis: 50 to 55 kDa
[4] The β-galactosidase according to [1] to [3], which further has the following properties (4) to (5).
(4) Optimal pH; pH 5.5
(5) Optimal temperature: 50 ° C
[5] The β-galactosidase according to [1] to [3], which further has the following properties (4) to (5).
(4) Optimal pH; pH 6.5-7.0
(5) Optimal temperature: 40 ° C
[6] The β-galactosidase according to [1] to [5], wherein the genus Streptomyces is Streptomyces olivaceoviridis.
[7] The β-galactosidase according to [1] to [6], which comprises the amino acid sequence of any of the following (i) to (iii).
(I) Amino acid sequence represented by SEQ ID NO: 1 or 2 (ii) Amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 1 or 2 (iii) ) A gene encoding β-galactosidase according to the amino acid sequences [8] [1] to [7] having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 2.
[9] The gene according to [8], which is a gene consisting of any of the following DNAs (a) to (d).
(A) DNA consisting of the base sequence represented by SEQ ID NO: 3 or 4.
(B) DNA encoding a protein consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 3 or 4 and having β-galactosidase activity.
(C) A DNA encoding a protein having a base sequence having 80% or more sequence identity with respect to the base sequence represented by SEQ ID NO: 3 or 4 and having β-galactosidase activity.
(D) DNA encoding a protein that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 4 and has β-galactosidase activity.
[10] A recombinant vector having the gene according to [8] or [9].
[11] A transformant transformed with the recombinant vector according to [10].
[12] A method for producing a galactooligosaccharide, which comprises a step of allowing the β-galactosidase described in [1] to [7] to act on a substrate containing lactose.

The β-galactosidase of the present invention is useful as an enzyme for producing galactooligosaccharides because of its high transglycosylation activity. By using the β-galactosidase, galactooligosaccharides can be industrially advantageously produced from a substrate such as lactose.

It is a figure which shows the TLC analysis result of a reaction product. It is a figure which shows the pH dependence of β-galactosidase. It is a figure which shows the temperature dependence of β-galactosidase. It is a figure which shows the TLC analysis result of the reaction product of enzyme 3-3. Lac: lactose, Gal: galactose, Glc: glucose. It is a figure which shows the TLC analysis result of the reaction product of enzyme 4-1. Lac: lactose, Gal: galactose, Glc: glucose. It is a figure which shows the TLC analysis result of the reaction product of FN5. Lac: lactose, Gal: galactose, Glc: glucose. It is a figure which shows the HPLC analysis result of a reaction product. It is a figure which shows the HPAEC-PAD analysis result of a reaction product.

The β-galactosidase of the present invention is derived from the genus Streptomyces and has the following property (1).
(1) Action: Transfer β-D-galactoside bond In the present specification, this action is referred to as glycosyl transfer activity. The transglycosylation activity of a protein (β-galactosidase) can be determined, for example, by reacting lactose with the protein as a substrate and measuring the transglycosylation product.
The β-galactosidase of the present invention further has the following property (2).
(2) Action: β-galactosidase activity The β-galactosidase activity can be measured by the PNPG method.

The suitable β-galactosidase of the present invention further has the following properties (3) to (5).
(3) Molecular weight by SDS polyacrylic gel electrophoresis: 50 to 55 kDa
(4) Optimal pH; pH 5.5
(5) Optimal temperature: 50 ° C
The molecular weight of this enzyme indicates the molecular weight measured by SDS-PAGE.
The optimum pH of this enzyme is pH 5.5, and it exhibits high transglycosylation activity at pH 5-7. McIlvaine buffer can be used to measure the optimum pH, and the pH can be measured by the method described in Examples described later.
The optimum temperature of this enzyme is 50 ° C, and it exhibits high activity at 40 to 50 ° C. The optimum temperature can be measured by the method described in Examples described later (pH 6.0 during reaction).

Further, the suitable β-galactosidase of the present invention further has the following properties (3) to (5).
(3) Molecular weight by SDS polyacrylic gel electrophoresis: 50 to 55 kDa
(4) Optimal pH; pH 6.5-7.0
(5) Optimal temperature: 40 ° C
The molecular weight of this enzyme indicates the molecular weight measured by SDS-PAGE.
The optimum pH of this enzyme is pH 6.5-7.0, and it exhibits high transglycosylation activity at pH 3-8. McIlvaine buffer and Athkins-Pantin buffer can be used to measure the optimum pH, and the pH can be measured by the method described in Examples described later.
The optimum temperature of this enzyme is 40 ° C, and it shows high activity at 20 to 45 ° C. The optimum temperature can be measured by the method described in Examples described later (pH 6.0 during reaction).

The genus Streptomyces that produces β-galactosidase of the present invention is preferably Streptomyces olivaceoviridis. Streptomyces can be isolated mainly from soil. Although Streptomyces is known to produce antibiotics, it was surprising that it produced β-galactosidase and had glycosyltransferase activity. It is difficult to predict the presence or absence of glycosyltransferase activity and its level from the base sequence of the bacterium, and it cannot be evaluated without actual testing.

The β-galactosidase of the present invention preferably comprises any of the following amino acid sequences (i) to (iii).
(I) Amino acid sequence represented by SEQ ID NO: 1 or 2 (ii) Amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 1 or 2 (iii) ) An amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 2.

As the β-galactosidase consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2, Streptomyces olivaceoviridis-derived β-galactosidase consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2 is a preferable example.
The number of amino acid residue deletions, substitutions or insertions in the amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 1 or 2 is SEQ ID NO: 1. Alternatively, it is not limited as long as it exhibits an enzymatic activity equivalent to that of β-galactosidase consisting of the amino acid sequence represented by 2, but 1 to 20 is preferable, 1 to 10 is more preferable, and 1 to 8 is further preferable. ..
Further, in the present specification, the sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 2 is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more. , 99% or more is more preferable. The identity percentage of such sequences can be calculated using publicly available or commercially available software with an algorithm that compares the reference sequence as a query sequence. As an example, BLAST, FASTA, GENETYX (manufactured by Genetics) and the like can be used.

The gene encoding β-galactosidase of the present invention is preferably a gene encoding any of the above amino acid sequences (i) to (iii), and more preferably any of the following (a) to (d). It is a gene consisting of DNA.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 3 or 4.
(B) DNA encoding a protein consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 3 or 4 and having β-galactosidase activity.
(C) A DNA encoding a protein having a base sequence having 80% or more sequence identity with respect to the base sequence represented by SEQ ID NO: 3 or 4 and having β-galactosidase activity.
(D) DNA encoding a protein that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 4 and has β-galactosidase activity.

In the base sequence in which 1 to several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 3 or 4, the number of 1 to several bases is preferably 1 to 10, preferably 1 to 5. Is more preferable, 1 to 3 is more preferable, and 1 or 2 is further preferable. In addition, deletion of a base means loss or disappearance of a base, substitution of a base means that a base has been replaced with another base, and addition of a base means that a base has been added. means. "Addition" includes the addition of a base to one or both ends of a sequence and the insertion of another base between the bases in the sequence.

In the present specification, the sequence identity of the base sequence is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, still more preferably 99% or more.
Nucleotide sequence identity can be determined using the algorithm BLAST (Pro. Natl. Acad. Sci. USA, 1993, 90: 587-5877) by Karlin and Altschul. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed (J.Mol.Biol., 1990,215, p.403-410). You may also use the Search homology program of the genetic information processing software Geneticx. Specific methods for these analysis methods are known (see www.ncbi.nlm.nih.gov).

Further, in the present specification, the stringent condition means a condition in which base sequences having high identity hybridize with each other and base sequences having lower identity do not hybridize with each other. The "stringent condition" can be appropriately changed depending on the level of identity required. The higher the stringent conditions, the more identical sequences will hybridize. For example, as stringent conditions, the conditions described in Molecular Cloning: A Laboratory Manual (Second Edition, J. Sambrook et.al, 1989) can be mentioned. That is, in a solution containing 6 × SSC (composition of 1 × SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 × denhart and 100 mg / mL hercin sperm DNA. Conditions include conditions for constant temperature at 65 ° C. for 8 to 16 hours together with the probe for hybridization.

The β-galactosidase of the present invention can be obtained by a transformant obtained by introducing a vector containing a gene encoding β-galactosidase into a microorganism according to a known method. The transformant may be retained in the vector state, or the gene may be retained in the genome. That is, when the transformant is cultured in an appropriate medium, the gene encoded on the vector contained in the transformant is expressed and the β-galactosidase of the present invention is produced. The β-galactosidase can be obtained by isolating and purifying the produced β-galactosidase from the culture.

The gene encoding β-galactosidase can be isolated from Streptomyces spp. Using any method used in the art. For example, for the gene, after extracting the whole genomic DNA of Streptomyces spp., The target gene is selectively amplified by PCR using a primer designed based on the nucleotide sequence of SEQ ID NO: 3 or 4. , Can be obtained by purifying the amplified gene.

The type of vector containing the gene encoding β-galactosidase is not particularly limited, and examples thereof include vectors usually used for protein production, such as plasmids, cosmids, phages, viruses, YAC, and BAC. Among them, a plasmid vector is preferable, and a commercially available plasmid vector for protein expression, for example, pET or pBIC can be preferably used. Procedures for introducing genes into plasmid vectors are well known in the art.

In addition, as the microorganisms for introducing the constructed vector, Streptomyces, Brevibacillus, Staphylococcus, Enterococcus, Listeria, Bacillus ( Bacillus, Escherichia, Saccharomyces, Shizosaccharomyces, Cribellomyces, Aspergillus, Penicillium and Trichoderma Bacteria can be mentioned. As a method of introduction, a method usually used in the art can be used.

When the obtained transformant is cultured in an appropriate medium, the introduced gene contained in the transformant is expressed and the β-galactosidase of the present invention is produced. The medium used for culturing can be appropriately selected by those skilled in the art according to the type of transformant. The produced β-galactosidase of interest can be isolated from the culture by a conventional method and purified by a known purification method.

Β-galactosidase derived from Streptomyces spp. Has high transglycosylation activity, as shown in Examples described later. Therefore, the β-galactosidase of the present invention is useful for the production of galactooligosaccharides. Galactooligosaccharide is an oligosaccharide having a galactose residue and is represented by Gal- (Gal) n-Glc (Gal: galactose residue, Glc: glucose residue, n = 0 to 5).
The β-galactosidase of the present invention can be provided in the form of an enzyme composition for producing a galactooligosaccharide containing the β-galactosidase. The enzyme composition for producing galactooligosaccharide may contain, for example, an excipient, a suspending agent, a buffer, a stabilizer, a preservative, a physiological saline solution, etc., in addition to the active ingredient (β-galactosidase).

The production of galactooligosaccharide includes a step of allowing the β-galactosidase of the present invention to act on a substrate containing lactose. The conditions under which β-galactosidase acts on the substrate are not particularly limited as long as β-galactosidase is not inactivated.
Appropriate conditions can be determined by those skilled in the art depending on the type of substrate and the like, but for example, the reaction pH is preferably pH 1 to 15, and more preferably pH 3 to 8. The β-galactosidase represented by SEQ ID NO: 1 (enzyme 3-3) preferably has a reaction pH of 4 to 8, more preferably 5 to 7, and even more preferably 5 to 6. The β-galactosidase represented by SEQ ID NO: 2 (enzyme 4-1) preferably has a reaction pH of 4 to 9, more preferably 5 to 8, and even more preferably 6 to 7.
The reaction temperature is preferably 80 ° C. or lower, more preferably 70 ° C. or lower, and even more preferably 60 ° C. or lower. The β-galactosidase shown in SEQ ID NO: 1 preferably has a reaction temperature of 20 to 70 ° C, more preferably 30 to 60 ° C, and even more preferably 40 to 50 ° C. The β-galactosidase shown in SEQ ID NO: 2 preferably has a reaction temperature of 1 to 70 ° C, more preferably 10 ° C to 50 ° C, and even more preferably 30 to 45 ° C.
The reaction time is preferably 96 hours or less, more preferably 72 hours or less, still more preferably 48 hours or less.
The obtained galactooligosaccharide may be purified as it is or if necessary. Galactooligosaccharides can be mixed with milk powder and used as milk powder for childcare. It may be used as a syrup containing galactooligosaccharides.

The galactooligosaccharide obtained by the β-galactosidase of the present invention contains a large amount of metastatic disaccharides and trisaccharides. Since the galactooligosaccharide has a different composition from the existing β-galactosidase sugar metastasis product, a new function of the galactooligosaccharide is expected, and it is considered to be useful in various fields.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1
1. 1. Production of β-galactosidase A β-galactosidase-like sequence on the Streptomyces olivaceoviridis strain genome was extracted by bioinformatics analysis. A PCR reaction was carried out using the following primers designed based on this, and genes 3-3 and 4-1 encoding β-galactosidase-like protein were amplified. The base sequence of the gene 3-3 is shown in SEQ ID NO: 3, the base sequence of the gene 4-1 is shown in SEQ ID NO: 4, and the amino acid sequence of β-galactosidase encoded by the gene 3-3 is shown in SEQ ID NO: 1, gene 4-1. The amino acid sequence of β-galactosidase encoded by is shown in SEQ ID NO: 2.

The expression plasmid and β-galactosidase enzyme solution were obtained using the Brevibacillus Expression System (Takara HB300 product name BIC System). First, Brevibacillus transformants were obtained by adding the obtained genes 3-3 and 4-1 to the pBIC4 vector according to the attached manual. This transformant was cultured according to the attached manual. After completion of the culture, β-galactosidase enzyme solution (hereinafter referred to as enzyme 3-3 and enzyme 4-1) was obtained by removing the bacterial cells by centrifugation (15,000 r / min).

2. Enzyme activity The β-galactosidase activity of β-galactosidase enzyme 3-3 and enzyme 4-1 prepared above and the transglycosylation activity were measured.
(Measurement of β-galactosidase activity)
2 mM paranitrophenyl-β-D-galactopyranoside and 40 μL of McIlvaine buffer (pH 6.0) were mixed and preheated at 40 ° C. for 5 minutes. 10 μL of the enzyme solution was added to start the reaction, and after 10 minutes, 100 μL of 0.2 M sodium carbonate was added to stop the reaction. The absorbance of para-nitrophenol liberated in the reaction solution at 400 nm was defined as the enzymatic activity.
(Measurement of glycosyl transfer activity)
Preheat 450 μL of McIlvaine buffer (pH 6.0) containing 62.5% lactose (final concentration 50%) at 40 ° C. for 5 minutes, add 50 μL of enzyme solution thereto, react at 40 ° C. for 16 hours, and then 100. The reaction was stopped by heating at ° C. for 5 minutes.
The reaction solution was appropriately diluted, desalted with an ion exchange resin, and then analyzed by thin layer chromatography (hereinafter referred to as TLC). TLC was developed with a solvent of chloroform: methanol: water = 90:65:15. Detection was performed by spraying N-1-naphthylethylenediamine dihydrochloric acid: ethanol: sulfuric acid = 0.9 g: 100 mL: 10 mL on a TLC plate and then heating.
As a result, as shown in FIG. 1, the production of glycosyltransferase was confirmed as spots other than lactose as a substrate, glucose as a decomposition product, and galactose, and enzyme 3-3 and enzyme 4-1 have glycosyltransferase activity. It was confirmed that.

3. 3. Enzymatic properties β-galactosidase activity at different pH and temperature was measured. McIlvaine buffer (pH 2.2-8.0) or Athkins-Pantin buffer under different pH conditions (pH 2.2-9.0) at 40 ° C or different temperature conditions (10-70 ° C) at pH 6.0. Except for using a liquid (pH 7.0-9.0), the above 2. The β-galactosidase activity was measured by the same method as in the above, and the relative activity under each condition was determined when the condition showing the maximum activity was set to 100%.
As a result, as shown in FIG. 2, the optimum pH was pH 5.5 for enzyme 3-3 and pH 6.5-7.0 for enzyme 4-1. Further, as shown in FIG. 3, the optimum temperature was 50 ° C. for enzyme 3-3 and 40 ° C. for enzyme 4-1.

Example 2
1. 1. Production of galactooligosaccharide 200 μL of phosphate buffer (pH 3.0 to 8.0) is added to 0.2 g of lactose, distilled water is added so that the substrate concentration becomes 40%, and then prepared in Example 1 above. 50 μL of the above enzyme solution was added to prepare a reaction solution. For comparison, a reaction solution containing β-galactosidase (Biolacta FN5, manufactured by Amano Enzyme) derived from Bacillus circulans was prepared. Each reaction solution was reacted at 50 ° C. for 6 hours or 24 hours, and then heated at 100 ° C. for 10 minutes to stop the reaction.

2. TLC analysis After completion of the reaction, the obtained reaction product was analyzed by TLC. TLC was developed with a solvent of chloroform: methanol: water = 90:65:15. Detection was performed by spraying N-1-naphthylethylenediamine dihydrochloric acid: ethanol: sulfuric acid = 0.9 g: 100 mL: 10 mL on a TLC plate and then heating.
As a result, as shown in FIGS. 4 to 6, spots (Rf value = 0 to 0.22), which are considered to be glycosyl metastases, were confirmed. With enzyme 4-1 the glycosyltransferase could be confirmed at all pHs of 3.0 to 8.0 (Fig. 5), and it was considered that it could be used in a wide pH range, such as when using acidic whey as a substrate.

Example 3
1. 1. Production of galactooligosaccharide 80 μL of phosphate buffer (pH 6.0) is added to 0.1 g of lactose, distilled water is added so that the substrate concentration becomes 50%, and then 20 μL of the enzyme solution prepared in Example 1 above is added. Was added to prepare a reaction solution. For comparison, a reaction solution to which β-galactosidase (Biolacta FN5) derived from Bacillus circulans was added was prepared. Each reaction solution was reacted at 50 ° C. for 24 hours and then heated at 100 ° C. for 10 minutes to stop the reaction.

2. HPLC and HPAEC-PAD analysis In the HPLC analysis, Alliance HPLC Waters2629 (manufactured by Waters) was used as the apparatus, and CARBO Sep CHO-620 (φ6.5 × 300 mm, manufactured by Concise Separations) was used as the column. Water was used as the mobile phase to separate sugars. The sugar concentration of the reaction solution was adjusted to 5.25 mg / ml and used as a sample for analysis. This was analyzed with a Waters 2414 RI detector at a flow rate of 0.4 mL / min, an injection volume of 5 μL, and a column temperature of 85 ° C.
In HPAEC-PAD, an ion chromatograph ICS-3000 (manufactured by Thermo Scientific) was used as an apparatus, and CarboPac PA1 (φ4 mm x 250 mm, manufactured by Thermo Scientific) was used as a column. As the mobile phase, 100 mM sodium hydroxide solution (A), 100 mM sodium hydroxide-containing 600 mM sodium acetate solution (B), water (C), and 50 mM sodium acetate solution (D) were used, and sugars were separated by gradient. did. The conditions are shown in Table 2. The sugar concentration of the reaction solution was adjusted to 0.5 mg / mL and used as a sample for analysis. This was analyzed with a flow rate of 1.0 mL / min, an injection volume of 25 μL, a column temperature of 20 ° C, and a detector PAD.

As a result, as shown in FIGS. 7 and 8, enzyme 3-3 produced a large amount of transferred disaccharides, and enzyme 3-3 and enzyme 4-1 produced galactooligosaccharides completely different from FN5 with high productivity. It was confirmed that. From existing reports, it was inferred that the main component of the transferred disaccharide is a disaccharide in which galactose and glucose are β-1,2 or β-1,3-glycosidic bonded. The transferred disaccharide has the same molecular weight as lactose, which is a substrate, and can be used without changing physical properties such as osmotic pressure when used in food applications.

Claims (12)

A β-galactosidase derived from the genus Streptomyces and having the following property (1).
(1) Action: Transfer β-D-galactoside bond The β-galactosidase according to claim 1, further having the following property (2).
(2) Action: β-galactosidase activity Furthermore, the β-galactosidase according to claim 1 or 2, which has the following property (3).
(3) Molecular weight by SDS polyacrylic gel electrophoresis: 50 to 55 kDa The β-galactosidase according to any one of claims 1 to 3, further having the following properties (4) to (5).
(4) Optimal pH; pH 5.5
(5) Optimal temperature: 50 ° C The β-galactosidase according to any one of claims 1 to 3, further having the following properties (4) to (5).
(4) Optimal pH; pH 6.5-7.0
(5) Optimal temperature: 40 ° C The β-galactosidase according to any one of claims 1 to 5, wherein the genus Streptomyces is Streptomyces olivaceoviridis. The β-galactosidase according to any one of claims 1 to 6, which comprises the amino acid sequence of any of the following (i) to (iii).
(I) Amino acid sequence represented by SEQ ID NO: 1 or 2 (ii) Amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 1 or 2 (iii) ) An amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 2. The gene encoding β-galactosidase according to any one of claims 1 to 7. The gene according to claim 8, which is a gene consisting of any of the following DNAs (a) to (d).
(A) DNA consisting of the base sequence represented by SEQ ID NO: 3 or 4.
(B) DNA encoding a protein consisting of a base sequence in which one to several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 3 or 4 and having β-galactosidase activity.
(C) A DNA encoding a protein having a base sequence having 80% or more sequence identity with respect to the base sequence represented by SEQ ID NO: 3 or 4 and having β-galactosidase activity.
(D) DNA encoding a protein that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 4 and has β-galactosidase activity. A recombinant vector comprising the gene according to claim 8 or 9. A transformant transformed with the recombinant vector according to claim 10. A method for producing a galactooligosaccharide, which comprises the step of allowing the β-galactosidase according to any one of claims 1 to 7 to act on a substrate containing lactose.

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