JP5006380B2 - Novel α-galactosidase - Google Patents

Description

  The present invention relates to a novel α-galactosidase and a method for producing raffinose using them.

  In recent years, with the diversification of eating habits and social life, consumers' interest in foods, food ingredients, etc. is increasing due to the improvement of health awareness. Among them, raffinose is recognized to have functions such as improving the intestinal microflora, and is attracting attention as a food and drink, pharmaceuticals, cosmetics, and the like, or a raw material thereof. More recently, it has become clear that raffinose is also useful for immune stimulating action and atopic dermatitis.

  Currently, raffinose is recovered as a by-product in the production of beet sugar, but the raffinose content in beet is only about 0.1%, and the production amount is also the production of sugar, which is the main product. Because of this, there is a limit to increasing raffinose production.

  Therefore, in order to supply raffinose having such useful properties to the market at a low cost and stably, not only an extract from a natural product but also a synthetic product from an inexpensive raw material is required.

  Examples of methods for synthesizing raffinose that have been attempted so far include methods utilizing the catalytic action of α-galactosidase. Methods for obtaining raffinose using sucrose as a galactose acceptor and melibiose as a galactose donor (for example, Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2) have been reported. In addition to this, as a galactose donor, a method using p-nitrophenyl-α-D-galactopyranoside (for example, see Non-Patent Document 3), a method using galactinol (for example, see Patent Document 2), A method using galactobiose (see, for example, Patent Document 3) has been reported.

  In addition, some examples in which inexpensive sucrose and galactose can be used as raw materials have been reported. For example, a method for obtaining raffinose using α-galactosidase derived from Pycnoporus cinnabarinus (for example, see Non-Patent Document 4) and a method for synthesizing raffinose using α-galactosidase derived from Mortierella vinacea are disclosed. (For example, refer nonpatent literature 5).

Japanese Patent No. 2688854 JP 10-84973 A Japanese Patent Publication 8-24592 Agric. Biol. Chem., 52 (9), 2305-2311, 1988 Biosci. Biotech. Biochem., 59 (4), 619-623, 1995 Phytochemistry, 18, 35-38, 1979 Nippon Shokuhin Kogyo Gakkaishi, 38 (8), 722-728, 1991 Carbohydr. Res., 185, 139-146, 1989

  Since raffinose production efficiency by α-galactosidase is good, examples using melibiose, p-nitrophenyl-α-D-galactopyranoside, galactinol and galactobiose as raffinose synthesis raw materials have been reported. When this method was used, the raw materials used were expensive, and industrial production of raffinose was difficult.

  On the other hand, the method of obtaining raffinose using α-galactosidase derived from Pycnoporus cinnabarinus and the method of synthesizing raffinose using α-galactosidase derived from Mortierella vinacea can use sucrose and galactose which are inexpensive raw materials. Therefore, it is a very advantageous method in consideration of industrial properties. However, when these α-galactosidases are used, contaminated trisaccharides other than the target raffinose are produced in the product. Therefore, it is necessary to separate contaminated trisaccharides from the final product, and further waste of raw materials Such a problem will occur. When α-galactosidase derived from Pycnoporus cinnabarinus is used for the raffinose synthesis reaction, the raffinose content in the resulting oligosaccharide is 40%, and when α-galactosidase derived from Mortierella vinacea is used for the raffinose synthesis reaction, raffinose in the generated oligosaccharide The content is as low as 60%, and it has been impossible to selectively produce raffinose by an enzymatic method using conventional techniques.

  The present invention provides a novel α-galactosidase capable of selectively synthesizing raffinose using an inexpensive raw material under such circumstances, and an innovative method for producing raffinose using this enzyme Is intended to provide.

  As a result of intensive studies to solve these problems, the present inventors have discovered a novel enzyme capable of selectively synthesizing raffinose, and a method for producing raffinose that suppresses the production of contaminating oligosaccharides other than the target raffinose. As a result, the present invention has been completed.

That is, the present invention relates to a novel α-galactosidase and raffinose production method shown in the following [1] to [20].
[1] α-Galactosidase having the following characteristics.
(1) Action: This enzyme has a property that the raffinose synthesis selectivity is 65% or more when the raffinose content in the resulting oligosaccharide is 0.5% or more in the dehydration condensation reaction using sucrose and galactose as raw materials. .
(2) Optimal pH range: 3.5-5.0
(3) Stable pH range: 3.5 to 10.0
(4) Molecular weight: about 80,000
[2] The α-galactosidase according to [1], which is derived from a microorganism belonging to Bacillus coagulans.
[3] The α-galactosidase according to [2], wherein Bacillus coagulans is any of Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, and AK006 strain.
[4] Bacillus coagulans belonging to any of Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, and AKC006 strain and mutants thereof.

[5] α-galactosidase comprising the amino acid sequence of any one of (a), (b) and (c) below.
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having an α-galactosidase activity:
(C) an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an α-galactosidase activity:
[6] An α-galactosidase gene encoding α-galactosidase having the amino acid sequence of any one of (a), (b) and (c) below.
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having an α-galactosidase activity:
(C) an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an α-galactosidase activity:
[7] An α-galactosidase gene comprising the following base sequence (a) or (b):
(A) the base sequence represented by SEQ ID NO: 1;
(B) The base sequence represented by SEQ ID NO: 1 is a base sequence in which one or several bases are deleted, substituted, and / or added, and encodes a protein having α-galactosidase activity. Base sequence:

[8] A recombinant vector comprising the α-galactosidase gene according to [6] or [7].
[9] A transformant introduced with the α-galactosidase gene according to [6] or [7] or the recombinant vector according to [8].
[10] α-galactosidase obtained by culturing the transformant according to [9].
[11] An enzyme composition comprising α-galactosidase according to any one of [1] to [3], [5], or [10].
[12] α-glucosidase, β-glucosidase, β-galactosidase, cellulase, xylanase, protease, galactanase, arabinanase, mannanase, rhamnogalacturonase, polygalacturonase, pectin methylesterase, pectin lyase, and polygalacturonic acid The enzyme composition according to [11], further comprising at least one component selected from lyase.
[13] A raffinose synthesis reagent comprising the enzyme composition according to any one of [11] and [12].
[14] The α-galactosidase according to any one of [1] to [3], [5], or [10], the enzyme composition according to [11] or [12], or [13] A method for producing raffinose, comprising using the described raffinose synthesis reagent.

[15] A method for producing raffinose, comprising using a microbial catalyst obtained by culturing a microorganism belonging to Bacillus coagulans.
[16] A microbial catalyst obtained by culturing Bacillus coagulans and / or a mutant thereof belonging to any one of Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, and AK006 strain is used. A method for producing raffinose.
[17] A method for producing raffinose, comprising using a microbial catalyst obtained by culturing the transformant according to [9].

[18] The method for producing raffinose according to any one of [14] to [17], wherein the raffinose content in the generated oligosaccharide is 65% or more.
[19] The method for producing raffinose according to any one of [14] to [18], wherein sucrose and galactose are used as raw materials.
[20] The sucrose concentration in the raw material is 30% (w / v) to 90% (w / v), and the galactose concentration in the raw material is 2% (w / v) to 45% (w / v). [19] The method for producing raffinose according to [19].

  By using the present invention, raffinose can be selectively produced.

Hereinafter, the present invention will be specifically described.
The α-galactosidase of the present invention is derived from a microorganism belonging to Bacillus coagulans, and any microorganism belonging to Bacillus coagulans may be used as the microorganism, and raffinose can be selectively synthesized. Any microorganism that expresses any α-galactosidase can be used. Preferably, Bacillus coagulans AKC-003 strain, AKC-004 strain, AKC-005 strain, and AKC-006 strain are mentioned. In addition, the microorganism in the present invention may be a mutant obtained by using a microorganism belonging to Bacillus coagulans as a parent strain. The Bacillus coagulans AKC-003, AKC-004, AKC-005, and AKC-006 strains were registered on November 14, 2006 (National Institute of Advanced Industrial Science and Technology, Japan) It is deposited in 1st, 1-chome, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture. The accession numbers are as follows. The AKC-004 strain was transferred to the international deposit on January 30, 2008 as receipt number FERM-ABP10948 and deposit number FERM-BP10948.
AKC-003 strain (FERM P-21091)
AKC-004 strain (FERM P-21092; FERM-ABP10948; FERM-BP10948)
AKC-005 strain (FERM P-21093)
AKC-006 strain (FERM P-21094)

The α-galactosidase of the present invention has the following characteristics.
(1) Action: This enzyme has a property that the raffinose synthesis selectivity is 65% or more when the raffinose content in the resulting oligosaccharide is 0.5% or more in the dehydration condensation reaction using sucrose and galactose as raw materials. .
(2) Optimal pH range: 3.5-5.0
(3) Stable pH range: 3.5 to 10.0
(4) Molecular weight: about 80,000

Specific examples of the enzyme of the present invention include those having the following properties due to substrate specificity and influence of metal ions.
Substrate specificity: When p-nitrophenyl-α-D-galactopyranoside is used as a substrate, the degradation activity is the highest, followed by melibiose and then raffinose. On the other hand, p-nitrophenyl-β-D-galactopyranoside, lactose and sucrose are not decomposed.
Effect of metal ions: When potassium, calcium, magnesium, chromium, manganese, cobalt, iron (II), and iron (III) ions are added, no decrease in activity is observed. On the other hand, when nickel and zinc ions are added, the activity decreases, and when copper ions are added, the activity decreases most.
Further, specific examples of the enzyme of the present invention may have the following characteristics.
(5) Optimal temperature range (for positive reaction): 35-50 ° C
(6) Stable temperature range: Stable up to 45 ° C.

  As a method for culturing microorganisms used in the present invention, ordinary aeration and agitation cultures or solid cultures are used, and generally used microorganism cultivation methods can be applied. As the medium, a synthetic medium or a natural medium containing the carbon source, nitrogen source, inorganic salt, necessary nutrient source, etc. necessary for the microorganism to grow well and to smoothly produce α-galactosidase in the microorganism. Is mentioned. For example, as a carbon source, glucose, glycerol, sucrose, galactose, lactose, melibiose, raffinose, stachyose, cellobiose, erulose, organic acid, starch, olive oil, soybean oil and the like can be used. Examples of nitrogen sources include ammonium sulfate, ammonium nitrate, urea, amino acids, amines, ammonia, ammonium salts of various inorganic and organic acids, other nitrogen-containing compounds, peptone, tryptone, polypeptone, meat extract, yeast extract, cottonseed meal , Corn steep liquor, and soybean meal. Moreover, as inorganic salts, primary potassium phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, manganese sulfate, copper sulfate, iron sulfate, calcium carbonate and the like are used. From the viewpoint of the growth of microorganisms, the culture temperature is preferably 25 to 80 ° C, more preferably 40 to 65 ° C, still more preferably 40 to 55 ° C. The pH of the medium can be selected in a wide range, and is preferably pH 3.0 to 9.0, more preferably pH 3.5 to 8.5, and still more preferably pH 4.0 to 8. from the viewpoint of the growth of microorganisms. 0.

  Separation and purification of the α-galactosidase of the present invention can be carried out, for example, as follows.

Bacillus coagulans AKC-004 strain is cultured in the above medium, and the obtained culture solution is separated into cells and filtrate by known means such as centrifugation and filtration.

  The bacterial cells thus obtained contain α-galactosidase, and a crude extract of α-galactosidase is obtained by crushing the bacterial cells using lysozyme, an ultrasonic crusher, a French press or the like. When the activity of α-galactosidase is contained in the culture supernatant depending on the culture conditions or the like, the culture supernatant can be used as it is or after being subjected to an operation such as concentration as a crude extract of α-galactosidase for the next purification.

  The crude extract obtained as described above is fractionated by combining ordinary protein purification methods such as ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, hydroxyapatite chromatography, and salting out. Thus, α-galactosidase can be purified.

  The order of the above operations is not particularly limited, and each operation may be performed once or twice or more. In addition, it is desirable to exchange the sample solution with an appropriate buffer by dialysis or the like before passing the sample through each column. Further, the sample solution may be concentrated at each stage.

  In each step of purification, it is desirable to measure α-galactosidase activity contained in each fractionated fraction, collect fractions with high activity, and use them in the next step.

  As a method for measuring α-galactosidase activity, for example, 150 μL of an enzyme solution is mixed with 450 μL of 100 mM sodium acetate buffer having a pH of 5.0 containing 6 mM p-nitrophenyl-α-D-galactopyranoside, and 40 ° C. Then, the reaction is stopped by adding to 1 mL of 1 M aqueous sodium carbonate solution to deactivate the enzyme. The degree of coloring of the obtained solution is measured for absorption at a wavelength of 420 nm, and the concentration is calculated using a calibration curve prepared with p-nitrophenol at each concentration. The unit of enzyme activity is expressed as 1 U of the amount of enzyme that liberates 1 μmol of p-nitrophenol per minute under the above conditions.

Confirmation of the degree of purification and molecular weight measurement of the purified α-galactosidase can be performed by electrophoresis, gel filtration chromatography, or the like. Enzymatic properties can be examined by measuring the enzyme activity by changing the reaction temperature or pH, or by adding various enzyme inhibitors or metal ions to the reaction solution and measuring the residual activity. Good. Furthermore, the stable pH range and the stable temperature range can be examined by measuring the enzyme activity after exposing α-galactosidase to various pH conditions or temperature conditions for a certain period of time. Further, by carrying out the reaction while changing the substrate concentration, the Michaelis constant (K _m ) and the maximum velocity (V _max ) of α-galactosidase for each substrate can be obtained.

The α-galactosidase gene of the present invention can be obtained, for example, as follows.
Bacillus coagulans AKC-004 strain is cultured in the above medium, and the obtained culture solution is separated into cells and filtrate by known means such as centrifugation and filtration. The microbial cells obtained as described above are crushed using lysozyme, an ultrasonic crusher, a French press or the like, and chromosomal DNA is isolated. The chromosomal DNA obtained as described above is digested with various restriction enzymes to obtain DNA fragments. Using the chromosomal DNA fragment obtained as described above, a DNA fragment containing the full-length α-galactosidase gene or a part thereof is obtained by known means using shotgun cloning, inverse PCR, or the like. The base sequence of the DNA fragment containing the α-galactosidase gene obtained here is analyzed by a known method using a DNA sequencer or the like to clarify the base sequence. Further, when only a partial base sequence of the α-galactosidase gene is clarified, a DNA fragment containing the α-galactosidase gene can be obtained again based on the base sequence. It is also possible to clarify the full-length base sequence of the α-galactosidase gene by repeating this operation. Based on the base sequence of the α-galactosidase gene decoded as described above, a DNA fragment containing the full length of the α-galactosidase gene can be obtained by a known method using a PCR method or a restriction enzyme. The amino acid sequence of α-galactosidase can be determined from the base sequence of the α-galactosidase gene decoded as described above.

The amino acid sequence of the α-galactosidase of the present invention is exemplified in SEQ ID NO: 2, but as long as the protein comprising this amino acid sequence has α-galactosidase activity, at least one amino acid in the amino acid sequence is deleted, substituted, added, etc. Or an amino acid sequence having 60% or more homology with the amino acid sequence. That is, the α-galactosidase of the present invention is an α-galactosidase consisting of any of the following amino acid sequences (a), (b) or (c).
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having an α-galactosidase activity:
(C) an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an α-galactosidase activity:

Furthermore, according to this invention, the alpha-galactosidase gene which codes the alpha-galactosidase which consists of an amino acid sequence in any one of following (a), (b) or (c).
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having an α-galactosidase activity:
(C) an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having an α-galactosidase activity:

  The “one or several amino acids” in the above (b) is generally 1 to 50 amino acids, preferably 1 to 30 amino acids, more preferably 1 to 20 amino acids, still more preferably 1 Means 10 to 10 amino acids, particularly preferably 1 to 5 amino acids.

  The “amino acid sequence having 60% or more homology” in the above (c) is generally 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, Preferably, it means an amino acid sequence having a homology of 95% or more, particularly preferably 99% or more.

Specific examples of the α-galactosidase gene of the present invention include the α-galactosidase gene comprising the following base sequence (a) or (b).
(A) the base sequence represented by SEQ ID NO: 1;
(B) The base sequence represented by SEQ ID NO: 1 is a base sequence in which one or several bases are deleted, substituted, and / or added, and encodes a protein having α-galactosidase activity. Base sequence:

  The “one or several bases” in the above (b) is generally 1 to 150 bases, preferably 1 to 90 bases, more preferably 1 to 60 bases, still more preferably 1 -30 bases, more preferably 1-20 bases, more preferably 1-15 bases, more preferably 1-10 bases, particularly preferably 1-5 bases.

  The α-galactosidase gene of the present invention may be mutated in a peptide that does not impair the property of catalyzing the original reaction by known genetic manipulation means. Such a mutant gene is derived from the α-galactosidase gene of the present invention. Means an artificially mutated gene created by genetic engineering techniques. This artificially mutated gene uses site-specific mutation methods and various genetic engineering methods such as replacing specific DNA fragments of the target gene with artificially mutated DNA. Is obtained. That is, known methods such as PCR, error-prone PCR, DNA shuffling, and methods for producing chimeric enzymes are used as methods for generating mutations such as deletion, substitution, and addition in amino acids in α-galactosidase. it can.

  The DNA fragment completely containing the α-galactosidase structural gene obtained as described above is inserted into an expression vector for E. coli, for example, pBluescriptII KS (+), and ligated to construct a new recombinant plasmid. can do. In this plasmid vector, a lac promoter capable of efficiently expressing a gene linked as a foreign gene in E. coli is introduced, and α-galactosidase is obtained by culturing a transformant obtained by introducing the recombinant plasmid into E. coli. Can be expressed in large quantities.

  In addition to the above, a large amount of α-galactosidase can be expressed using various host microorganisms and vectors. For example, a large amount of α-galactosidase can be expressed by culturing a transformant of Brevibacillus choshinensis. More specifically, as the vector incorporating the α-galactosidase gene of the present invention, those constructed for gene recombination from phages or plasmids that can autonomously grow in the host microorganism are suitable. For example, when a microorganism belonging to Escherichia coli is used as a host microorganism, λgt · λC, λgt · λB, and the like can be used. Moreover, as a plasmid vector, for example, when Escherichia coli is used as a host microorganism, pET vectors (Novagen) such as plasmids pET-3a, pET-11a, and pET-32a, or pBR322, pBR325, pACYC184, pUC12, pUC13 , PUC18, pUC19, pUC118, pIN I, BluescriptKS +, pWH1520, pUB110, pKH300PLK when Bacillus subtilis is the host, pIJ680, pIJ702, when using actinomycetes as the host, and yeast, especially Saccharomyces cerevisiae YRp7, pYC1, YEp13, etc. can be used. Such a vector is cleaved with a restriction enzyme that produces the same end as the DNA end produced by the restriction enzyme used for cleaving the α-galactosidase gene of the present invention to produce a vector fragment, and the α-galactosidase of the present invention is prepared. The gene fragment and the vector fragment can be combined with a DNA ligase enzyme according to a conventional method to incorporate the DNA encoding the α-galactosidase gene of the present invention into the target vector.

  As the host microorganism for transferring the plasmid, it is sufficient that the recombinant DNA can be stably and autonomously propagated. For example, when the host microorganism is a microorganism belonging to Escherichia coli, Escherichia coli BL21, Escherichia coli BL21 (DE3), Escherichia coli BL21trxB, Escherichia coli Rosetta (DE3), Escherichia coli Rosetta, Escherichia coli Rosetta (DE3) pLysS, Escherichia coli Rosetta (DE3) pLac, Escherichia coli Luis et Kori Origami, Escherichia coli Origami, Escherichia coli Tuner, Escherichia coli DH1, Escherry Hia coli JM109, Escherichia coli W3110, Escherichia coli C600, etc. can be used. Further, when the microorganism host is a microorganism belonging to the genus Bacillus, Bacillus subtilis, Bacillus megaterium, etc., a microorganism belonging to actinomycetes, Streptomyces lividans TK24, etc., a microorganism belonging to Saccharomyces cerevisiae, Saccharomyces cerevisiae INVSC1 can be used.

  In producing the α-galactosidase of the present invention using a transformed microorganism, the transformed microorganism is cultured in a nutrient medium to produce the α-galactosidase of the present invention in the cells or in the culture solution. The obtained culture is collected by means of filtration or centrifugation, and then the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, EDTA and / or appropriate The aqueous solution of the α-galactosidase of the present invention is concentrated by adding a suitable surfactant or the like, or it is processed by adsorption chromatography such as ammonium sulfate fractionation, gel filtration, affinity chromatography, or ion exchange chromatography without concentration. Thus, the α-galactosidase of the present invention having good purity can be obtained.

  Culture conditions for transformed microorganisms may be selected in consideration of their nutritional physiological properties. Usually, liquid culture is used in many cases, but industrially, deep aeration and agitation culture is advantageous. is there. As a nutrient source for the medium, those commonly used for culturing microorganisms can be widely used. The culture temperature can be appropriately changed within the range in which microorganisms grow and produce the α-galactosidase of the present invention. In the case of Escherichia coli, it is preferably about 10 to 45 ° C, more preferably about 20 to 30 ° C. . The culture conditions vary somewhat depending on the conditions, but the culture should be terminated at an appropriate time in anticipation of the time when the α-galactosidase of the present invention reaches the maximum yield. In the case of Escherichia coli, it is usually about 12 to 48 hours. is there. The pH of the medium can be appropriately changed within the range in which the bacteria grow and produce the α-galactosidase of the present invention, but in the case of Escherichia coli, the pH is preferably about 6 to 8.

  According to the present invention, when using the enzyme described above, the degree of purification is not particularly limited unless the action of the enzyme of the present invention is inhibited. It may be used.

  According to the present invention, an enzyme composition comprising the above-described α-galactosidase of the present invention is provided. Examples of the enzyme composition include α-glucosidase, β-glucosidase, β-galactosidase, cellulase, xylanase, protease, galactanase, arabinanase, mannanase, rhamnogalacturonase, polygalacturonase, pectin methylesterase, pectin It may further contain at least one component selected from lyase and polygalacturonic acid lyase.

  According to the present invention, a raffinose synthesis reagent comprising the enzyme composition described above is provided.

In the present invention, as the microbial catalyst, in addition to the Bacillus coagulans AKC-004 strain, the above transformant can be used. In the present invention, as a microbial catalyst used for producing raffinose, a microorganism itself obtained by a usual culture method can be used, and it is not necessary to purify α-galactosidase from the microorganism. In some cases, a microorganism culture solution or a microorganism culture supernatant can be used. On the other hand, the microorganisms obtained by the culturing method can be used after washing with water or a buffer as required. For example, a culture solution of a cultured microorganism, a microorganism suspension obtained by centrifugation, washing with a buffer, an aqueous solution in which a microorganism or a processed microorganism product (for example, crushed microorganisms) is suspended or dissolved, or a microorganism Alternatively, a product obtained by immobilizing a processed microorganism product by a comprehensive method, a crosslinking method, or a carrier binding method can be used. Examples of the immobilization carrier used for immobilization include, but are not limited to, glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid and the like.

  According to this invention, the manufacturing method of raffinose using the enzyme composition containing the alpha-galactosidase or alpha-galactosidase of the above-mentioned this invention, a raffinose synthesis reagent, or a microbial catalyst is provided. For example, sucrose and galactose can be used as the raw material. When sucrose and galactose are used as raw materials, for example, due to the property of utilizing a dehydration condensation reaction with α-galactosidase, the raw material concentration is preferably high, but if the galactose concentration is too high, condensation between galactose molecules Since the dehydration condensation reaction between sucrose and galactose is suppressed, the sucrose concentration is preferably 30% (w / v) to 90% (w / v), more preferably 35% (w / v) to 80 % (W / v), more preferably 40% (w / v) to 70% (w / v) is preferable. The galactose concentration is preferably 2% (w / v) to 45% (w / v), more preferably 5% (w / v) to 35% (w / v), Preferably, 7% (w / v) to 30% (w / v) is preferable. In addition, it is preferable to prepare such a concentration that both sucrose and galactose in the raw material are soluble.

  The reaction temperature when producing raffinose using the α-galactosidase or the enzyme composition containing α-galactosidase of the present invention, or a raffinose synthesis reagent containing the enzyme composition or a microbial catalyst is determined from the viewpoint of reaction rate and enzyme stability. The temperature is preferably 10 to 90 ° C, more preferably 20 to 70 ° C, and still more preferably 30 to 60 ° C. The reaction pH can be adjusted over a wide range, and preferably from pH 2.0 to 10.0, more preferably from pH 3.0 to 7.5, still more preferably from 3.5 to 6.0 from the viewpoint of enzyme stability. is there. Although reaction time changes also with the usage-amount of an enzyme, when industrial utilization is considered, Preferably it is 20 minutes-200 hours, More preferably, it is 6-80 hours. However, the present invention is not limited to the above reaction conditions and reaction forms, and can be appropriately selected.

  In the present invention, α-galactosidase or an enzyme composition containing α-galactosidase, or a raffinose synthesis reaction using a raffinose synthesis reagent containing an enzyme composition, the production at the time when 0.5% or more of raffinose is accumulated in the reaction solution The raffinose content in the oligosaccharide can be improved to 65% or more, and can be improved to 80% or more under more preferable conditions. The raffinose content in the high production oligosaccharide in the present invention can be achieved even when raffinose is accumulated in the reaction solution by 0.75% or more, further 1.0% or more.

  In general, in the raffinose synthesis reaction using microorganisms itself, the raffinose content in the produced oligosaccharide is low due to the influence of contaminating enzymes, whereas in the raffinose synthesis reaction using the microbial catalyst of the present invention, the produced oligosaccharide is produced. The raffinose content in it is high.

The raffinose content in the product oligosaccharide obtained in the present invention is measured by the following method.
After completion of the raffinose synthesis reaction, the reaction solution was diluted 25 times and held at 99 ° C. for 10 minutes to stop the reaction. After the reaction was stopped, microorganisms were removed by centrifugation, and the resulting reaction solution was quantified using high performance liquid chromatography (HPLC). For measurement, a Hypercarb column manufactured by Thermoelectron was used, and RI was used as a detector. The raffinose content in the produced oligosaccharide was calculated from (the peak area of raffinose) / (the peak area of the produced oligosaccharide) × 100 from the ratio of each peak area detected on the HPLC analysis chart.

  As a method for purifying and separating raffinose produced by the method of the present invention, a commonly used purification method can be used. That is, for example, centrifugation, MF membrane or UF membrane treatment, filter press etc. removes the microbial catalyst, and buffering by chromatographic treatment such as cation exchange chromatography or anion exchange chromatography or desalting treatment such as dialysis. Removes salts, etc. brought in from liquids and culture media, and further uses cation exchange chromatography, anion exchange chromatography, high performance liquid chromatography, activated carbon chromatography, etc., and crystallization treatment using differences in solubility, etc. The raffinose can be separated and purified according to other conventional methods. For chromatographic treatment, these methods may be used alone or in combination, and a moving bed method, a pseudo moving bed method, a multi-component separation pseudo moving bed method, a multi-component separation and circulation method, etc. may be used appropriately Can do. These raffinose purification and separation treatment methods may be carried out batchwise or continuously using a column.

  Hereinafter, although an example explains concretely, the present invention is not limited at all by these examples.

Example 1
Bacillus coagulans AKC-004 strain (Accession No. FERM P-21092 (transferred to FERM-ABP10948): Depository Organization; National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center) on TBAB (Tryptose Blood Agar Base) plate (Difco) The cells were cultured at 55 ° C. for 1 day to form colonies.

  Place 30 mL of the medium shown in Table 1 adjusted to pH 7.2 into a 150 mL Erlenmeyer flask, inoculate the colony from the plate with a platinum loop, and perform rotary shaking culture at 180 rpm at 50 ° C. for 25 hours. It was used as a seed for jar culture.

  Next, 6 L of the medium shown in Table 1 adjusted to pH 7.2 was placed in a 10 L jar fermenter, and 15 mL of the jar culture seed was transplanted, and aerated at 50 ° C. for 48 hours at 200 rpm and 0.2 vvm. Stirring culture was performed.

  Subsequently, this culture broth was centrifuged at 10,000 g × 30 minutes at 4 ° C. to remove the supernatant, and the cells were collected. It was 261U as a result of measuring the activity of (alpha) -galactosidase of the obtained microbial cell.

  This microbial cell was suspended in a Lysis buffer (pH 8.0) containing 50 mM Tris-HCl, 20 mM EDTA, and 50 mM glucose to make 150 mL. The suspension was centrifuged at 10,000 g × 30 minutes at 4 ° C., the supernatant was removed, and the cells were collected.

  The cells after washing with the Lysis buffer were resuspended in the Lysis buffer to 150 mL. 0.02% lysozyme (derived from Sigma egg white) was added thereto, and the cells were shaken at 37 ° C. for 13 hours at 120 rpm to disrupt the cells.

  The solution after disrupting the cells was centrifuged at 10,000 g × 30 minutes at 4 ° C. to remove cell residues, and the supernatant was collected.

  Ammonium sulfate was added to the supernatant to achieve 37.5% saturation, and the mixture was left at 4 ° C. overnight to cause precipitation. This precipitate was removed by centrifugation at 10,000 g × 30 minutes at 4 ° C., and the supernatant was recovered.

  Ammonium sulfate was added to the supernatant to obtain 54.5% saturation, and the mixture was left at 4 ° C. overnight to cause precipitation. This precipitate was collected by centrifugation at 10,000 g × 30 minutes at 4 ° C., dissolved in 20 mM phosphate buffer at pH 7.0, and overnight at 4 ° C. against the same phosphate buffer. Dialyzed.

  The supernatant obtained by dialysis is adsorbed to “DEAE Sepharose FF” (GE Healthcare Bioscience Co., Ltd.) equilibrated with the same phosphate buffer as described above, and contains 0 to 0.4 M sodium chloride. The enzyme was eluted by a gradient method of 20 mM phosphate buffer at pH 7.0.

  The active fractions eluted above were collected, concentrated using an ultrafiltration membrane with an average molecular weight cut-off of 10,000, and equilibrated with 20 mM phosphate buffer at pH 7.0 containing 2 M sodium chloride. After adsorbing to Phenyl FF (high sub) "(GE Healthcare Bioscience Co., Ltd.), the enzyme was eluted by a concentration gradient method of 20 mM phosphate buffer with pH 7.0 containing 2.0 to 0 M sodium chloride. I let you.

  The active fraction eluted above was collected, concentrated using an ultrafiltration membrane having an average molecular weight cut off of 10,000, and equilibrated with 20 mM phosphate buffer having a pH of 7.0, “MonoQ 5/50 GL” (GE Then, the enzyme was eluted by a concentration gradient method of 20 mM phosphate buffer having a pH of 7.0 containing 0 to 0.4 M sodium chloride.

  The active fractions eluted above were collected, concentrated using an ultrafiltration membrane with an average molecular weight cut off of 10,000, and equilibrated with 20 mM phosphate buffer having a pH of 7.0, “HiLoad 16/60 Superdex 200” (GE (Healthcare Bioscience Co., Ltd.) was used for elution with the same buffer.

  The active fractions eluted above were collected and concentrated using an ultrafiltration membrane having an average fractional molecular weight of 10,000 to obtain purified α-galactosidase. The activity yield was 15% and the activity was 270 U / mL.

Example 2
Using the purified α-galactosidase obtained in Example 1, an experiment on its action was performed.

(1) Optimum pH range 50 μL of the purified enzyme solution of the present invention diluted 100 times is mixed with 150 μL of 6 mM p-nitrophenyl-α-D-galactopyranoside dissolved in 100 mM buffer at each pH, The reaction was carried out at 40 ° C. for 5 minutes. After the reaction, the activity was measured by quantifying the liberated p-nitrophenol, and the relative activity with the maximum activity of 100 was determined. The result is shown in FIG. The buffers used were glycine-HCl buffer (pH 2.5 to 3.5), sodium acetate buffer (pH 3.5 to 6.0), and sodium phosphate buffer (pH 6.0 to 8.5). ), Glycine-NaOH buffer (pH 8.5 to 10.0).

(2) Stable pH range Using a 10 mM buffer in the range of pH 4.0 to 10.0, heat treatment at 45 ° C. for 140 minutes at each pH, and measuring the residual activity, Relative activity with activity set to 100 was determined. The result is shown in FIG. In addition, the kind of buffer solution of each pH used is the same as the above-mentioned thing.

(3) Optimal temperature range 10 μL of the purified enzyme solution of the present invention diluted 20-fold in 190 μL of 4.7 mM p-nitrophenyl-α-D-galactopyranoside dissolved in a sodium acetate buffer solution of pH 4.5 Were mixed and reacted in the range of 20 to 60 ° C. for 5 minutes. After the reaction, the activity was measured by quantifying the liberated p-nitrophenol, and the relative activity with the maximum activity of 100 was determined. The result is shown in FIG.

(4) Stable temperature range Using a 100 mM sodium acetate buffer with a pH of 4.5, heat treatment is carried out at each temperature of 30 to 55 ° C. for 20 minutes, the residual activity is measured, and the maximum activity is set to 100 as described above. Relative activity was determined. The result is shown in FIG.

(5) Molecular Weight Molecular weight was determined by SDS-polyacrylamide gel electrophoresis using “Ready Gel J” (Nippon Bio-Rad Laboratories, Inc.) having a separation gel concentration of 10%. The result is shown in FIG. The molecular weight was about 80,000, which almost coincided with the molecular weight 83,122 estimated from the amino acid sequence.

(7) Substrate specificity 50 μL of the purified enzyme solution of the present invention diluted 100-fold was mixed with 150 μL of pH 4.5 100 mM sodium acetate buffer containing 10 mM of various substrates, and reacted at 40 ° C. for 20 minutes. However, in the reaction using p-nitrophenyl-α-D-galactopyranoside as a substrate, the reaction time was 5 minutes. After the reaction, the released p-nitrophenol was quantified, or the substrate decomposed from the analysis by high-performance liquid chromatography using a “Shodex SUGAR” (Showa Denko) column, and the maximum activity was set to 100. The relative activity for each substrate was determined. The results are shown in Table 2. In addition, Table 3 shows the results of measuring the reaction rate of those that could serve as substrates.

(8) Inhibitory factor Various metal ions were added to 6 mM p-nitrophenyl-α-D-galactopyranoside dissolved in pH 4.5 sodium acetate buffer to a final concentration of 1 mM to 150 μL, 50 μL of the purified enzyme solution of the present invention diluted 100 times was mixed and reacted at 40 ° C. for 20 minutes. After the reaction, the activity was measured by quantifying the liberated p-nitrophenol, and the relative activity with the maximum activity of 100 was determined. The results are shown in Table 4.

Example 3
The purified α-galactosidase 0.5 U described in Example 1 was collected, and 200 μL of sugar solution (pH 5.0, 100 mM sodium acetate buffer solution containing 73% sucrose and 12% galactose in a polypropylene tube). The mixture was added and mixed, and the reaction was performed at 60 ° C. and 1,200 rpm. After 45 hours from the start of the reaction, 20 μL of the reaction solution was collected, diluted with 480 μL distilled water, and then treated at 99 ° C. for 10 minutes to inactivate the enzyme. The diluted sugar solution after heat inactivation was returned to room temperature and then analyzed by high performance liquid chromatography using a “Hypercarb” (Thermoelectron) column. The result is shown in FIG. The raffinose content in the produced oligosaccharide was 82%, and the raffinose concentration in the reaction solution was 1.67% by weight.

Example 4
(1) Preparation of chromosomal DNA Chromosomal DNA of Bacillus coagulans AKC-004 strain was prepared according to a conventional method. 60 mL of the culture solution of Bacillus coagulans AKC-004 obtained by the method described in Example 1 was centrifuged to recover the cells. The obtained cells were suspended in Lysis buffer (50 mM Tris-HCl (pH 8.0), 20 mM EDTA, 50 mM glucose) and washed thoroughly. The cells were collected by centrifugation, then resuspended in Lysis buffer, added with lysozyme, and incubated at 37 ° C. for 45 minutes. Then SDS and RNase were added and incubated at 37 ° C. for 45 minutes. Proteinase K was then added and gently shaken at 50 ° C. for 60 minutes. The solution obtained here was treated with phenol-chloroform and chloroform and then precipitated with ethanol, and the precipitated nucleic acid was collected by winding it around a glass pipette. The nucleic acid was washed with 70% ethanol, dried and redissolved in TE. By this operation, about 1 mg of chromosomal DNA was prepared.

(2) Isolation of α-galactosidase gene PCR primers for amplifying an α-galactosidase gene fragment were designed and synthesized from the chromosomal DNA prepared in (1) above. The primer was designed based on the alignment results of several known microorganism-derived α-galactosidase genes. The sense primer was an oligo DNA having a sequence of 5′-GAAGTITACGGITTYAGYYTTGTITACAGYGG-3 ′ (SEQ ID NO: 3), and the antisense primer was Oligo DNAs having the sequence of 5′-CCAAACCAICCRTCRTCIARIACRAA-3 ′ (SEQ ID NO: 4) were respectively synthesized. In the sequences, I represents inosine, Y represents C or T, and R represents A or G. Using the PCR primer obtained here, the α-galactosidase gene fragment was amplified by PCR using the chromosomal DNA prepared in (1) above as a template to obtain an α-galactosidase gene fragment consisting of 380 base pairs. The base sequence of the gene fragment obtained here was analyzed using a DNA sequencer.

  Based on the base sequence of the α-galactosidase gene fragment obtained as a result of the analysis, an inverse PCR PCR primer was designed and synthesized in order to obtain a DNA fragment containing the full length of the α-galactosidase gene. An oligo DNA having the sequence 5′-TGATCAACAACTGGGAAAGCGACCT-3 ′ (SEQ ID NO: 5) was synthesized as the sense primer, and an oligo DNA having the sequence 5′-GAACTGGTCCTGCTGCACAATTCC-3 ′ (SEQ ID NO: 6) was synthesized. Next, a template used for inverse PCR was prepared. After digesting the chromosomal DNA prepared in (1) above with the restriction enzyme HindIII, the obtained DNA fragment was self-circulated using T4 DNA ligase and used as a template for inverse PCR. A PCR fragment consisting of 4500 base pairs including the entire sequence of the α-galactosidase gene is obtained by amplifying a DNA fragment containing the α-galactosidase gene by PCR using the inverse PCR primer synthesized as described above. Acquired the product.

(3) Analysis of base sequence The base sequence of the α-galactosidase gene was determined from the PCR product obtained in (2) using a DNA sequencer. As a result, the 2190 base pair α-galactosidase structural gene having the DNA base sequence from the 5 ′ end shown in SEQ ID NO: 1 was decoded. This sequence was a novel gene not found so far. The α-galactosidase produced by the Bacillus coagulans AKC-004 strain inferred from this DNA base sequence was composed of 730 amino acids and had an amino acid sequence from the N-terminus as shown in SEQ ID NO: 2. As a result of database search, this amino acid sequence was found to be 57% of α-galactosidase (AgaN) derived from Geobacillus stearothermophilus, α-galactosidase (AgaA) and 56% derived from Geobacillus stearothermophilus (AgaN), 56%, It was found that it has 56% homology with the derived α-galactosidase and encodes a novel α-galactosidase.

(4) Construction and transformation of expression plasmid vector of α-galactosidase gene Based on the sequence of α-galactosidase gene obtained in (3), a region containing the SD sequence of α-galactosidase gene, the structural gene, and the start codon. PCR primers for amplification were designed and synthesized. The PCR primer was designed based on the base sequence of the PCR product consisting of 4500 base pairs obtained in (2), and the sense primer was an oligo DNA having the sequence of 5′-TAAGGTAAAGCAGATGTGCCATT-3 ′ (SEQ ID NO: 7). As antisense primers, oligo DNAs having a sequence of 5′-TTACTCGTACACCGCCTC-3 ′ (SEQ ID NO: 8) were respectively synthesized. Using the PCR product consisting of 4500 base pairs obtained in (2) as a template, amplification by the PCR method was performed using the synthesized PCR primers, and a PCR product consisting of 2325 base pairs was obtained. The PCR product obtained here was blunt-ended and phosphorylated.
The blunt-ended and phosphorylated PCR product obtained above was ligated to the pBluescriptII KS (+) vector that had been digested with the restriction enzyme EcoRV and dephosphorylated to construct a new plasmid vector pBlue / agaA. This plasmid vector is introduced with a lac promoter capable of efficiently transcribing a gene linked as a foreign gene in E. coli, so that α-galactosidase can be efficiently expressed and produced. The obtained plasmid vector was transformed into a competent cell of E. coli strain JM109 prepared by the calcium chloride method by the heat shock method to produce a recombinant microorganism.

(5) Culture of transformant and expression of α-galactosidase Recombinant Escherichia coli JM109-pBlue / agaA prepared in (4) was subjected to shaking culture at 37 ° C. for 24 hours in 30 mL LB medium containing 100 mg / L ampicillin. . After culturing, the recombinant E. coli was collected by centrifugation. The cells were washed with 100 mM sodium acetate buffer (pH 5.0), resuspended in the same buffer, and crushed with an ultrasonic crusher. The α-galactosidase activity of this crushed liquid was measured by the above-mentioned method and found to be 19.9 U / culture liquid (mL).

Example 5
The α-galactosidase 1.0 U obtained by culturing the recombinant described in Example 4 was fractionated, and 200 μL of sugar solution (100 mM sodium acetate buffer solution, pH 5.0) placed in a 1.5 mL polypropylene tube. And 73% sucrose and 12% galactose), and the mixture was reacted at 60 ° C. and 1,200 rpm. After 16 hours from the start of the reaction, 20 μL of the reaction solution was collected, diluted with 480 μL distilled water, and then treated at 99 ° C. for 10 minutes to inactivate the enzyme. The diluted sugar solution after heat inactivation was returned to room temperature and then analyzed by high performance liquid chromatography using a “Hypercarb” (Thermoelectron) column. The result is shown in FIG. The raffinose content in the produced oligosaccharide was 82%, and the raffinose concentration in the reaction solution was 1.20% by weight.

Example 6
Bacillus coagulans AKC-004 strain (Accession No. FERM P-21092 (transferred to FERM-ABP10948): Depository Organization; National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) on TBAB (Tryptose Blood Agar Base) plate (Difco) Incubate at 55 ° C for 1 day to form colonies. One platinum loop was inoculated into a 30 mL medium flask of Table 1 dispensed into a 150 mL Erlenmeyer flask and cultured at 55 ° C. and 150 rpm for 2 days.

  Two days after the main culture, 10 mL of cultured cells were collected in a 15 mL tube. The 15 mL tube from which the culture solution was collected was centrifuged at 10,000 rpm, and the supernatant was removed. Next, 1 mL of 100 mM Na acetate buffer (pH 5.0) was added and resuspended, and then the suspension was transferred to a 2 mL polypropylene tube. After centrifuging the tube again and removing the supernatant, 300 μL of sugar solution (containing 62.5% sucrose and 12.5% galactose in 100 mM sodium acetate buffer at pH 5.0) was added, and the cells were vortexed. Suspended well and started the sugar synthesis reaction. The sugar synthesis reaction was performed at a reaction temperature of 60 ° C. and a rotation speed of 1200 rpm. After 16 hours from the start of the sugar synthesis reaction, 40 μL of the reaction solution was recovered, mixed well with 960 μL of distilled water, and the enzyme was heat-inactivated at 99 ° C. for 10 minutes. FIG. 8 shows the results of analysis by high performance liquid chromatography using a “Hypercarb” (manufactured by Thermoelectron) column after returning the diluted sugar solution to room temperature. The raffinose content in the produced oligosaccharide was 80% (the raffinose concentration in the reaction solution was 0.70% by weight).

Example 7
Bacillus coagulans AKC-003 strain (Accession number FERM P-21091: Depositary institution; National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) is cultured on a TBAB (Tryptose Blood Agar Base) plate (Difco) at 55 ° C for 1 day To form colonies. One platinum loop was inoculated into a 30 mL medium flask of Table 1 dispensed into a 150 mL Erlenmeyer flask and cultured at 55 ° C. and 150 rpm for 2 days.

  Two days after the main culture, 10 mL of cultured cells were collected in a 15 mL tube. The 15 mL tube from which the culture solution was collected was centrifuged at 10,000 rpm, and the supernatant was removed. Next, 1 mL of 100 mM Na acetate buffer (pH 5.0) was added and resuspended, and then the suspension was transferred to a 2 mL polypropylene tube. After centrifuging the tube again and removing the supernatant, 300 μL of sugar solution (containing 62.5% sucrose and 12.5% galactose in 100 mM sodium acetate buffer at pH 5.0) was added, and the cells were vortexed. Suspended well and started the sugar synthesis reaction. The sugar synthesis reaction was performed at a reaction temperature of 60 ° C. and a rotation speed of 1200 rpm. After 38 hours from the start of the sugar synthesis reaction, 40 μL of the reaction solution was collected and mixed well with 960 μL of distilled water, and the enzyme was heat-inactivated at 99 ° C. for 10 minutes. After returning the diluted sugar solution to room temperature, analysis was performed by high performance liquid chromatography using a “Hypercarb” (Thermoelectron) column, and the raffinose content in the resulting oligosaccharide was 80% (reaction). The raffinose concentration in the liquid is 0.76% by weight).

Example 8
Bacillus coagulans AKC-005 strain (Accession number FERM P-21093: Depositary institution; National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) was cultured on TBAB (Tryptose Blood Agar Base) plate (Difco) at 55 ° C for 1 day To form colonies. One platinum loop was inoculated into a 30 mL medium flask of Table 1 dispensed into a 150 mL Erlenmeyer flask and cultured at 55 ° C. and 150 rpm for 2 days.

  Two days after the main culture, 10 mL of cultured cells were collected in a 15 mL tube. The 15 mL tube from which the culture solution was collected was centrifuged at 10,000 rpm, and the supernatant was removed. Next, 1 mL of 100 mM Na acetate buffer (pH 5.0) was added and resuspended, and then the suspension was transferred to a 2 mL polypropylene tube. After centrifuging the tube again and removing the supernatant, 300 μL of sugar solution (containing 62.5% sucrose and 12.5% galactose in 100 mM sodium acetate buffer at pH 5.0) was added, and the cells were vortexed. Suspended well and started the sugar synthesis reaction. The sugar synthesis reaction was performed at a reaction temperature of 60 ° C. and a rotation speed of 1200 rpm. After 38 hours from the start of the sugar synthesis reaction, 40 μL of the reaction solution was collected and mixed well with 960 μL of distilled water, and the enzyme was heat-inactivated at 99 ° C. for 10 minutes. After returning the diluted sugar solution to room temperature, analysis was performed by high performance liquid chromatography using a “Hypercarb” (Thermoelectron) column. As a result, the raffinose content in the resulting oligosaccharide was 91% (reaction). The raffinose concentration in the liquid is 0.32% by weight).

Example 9
Bacillus coagulans AKC-006 strain (Accession No. FERM P-21094: Depositary Institution; National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) is cultured on TBAB (Tryptose Blood Agar Base) plate (Difco) at 55 ° C for 1 day To form colonies. One platinum loop was inoculated into a 30 mL medium flask of Table 1 dispensed into a 150 mL Erlenmeyer flask and cultured at 55 ° C. and 150 rpm for 2 days.

  Two days after the main culture, 10 mL of cultured cells were collected in a 15 mL tube. The 15 mL tube from which the culture solution was collected was centrifuged at 10,000 rpm, and the supernatant was removed. Next, 1 mL of 100 mM Na acetate buffer (pH 5.0) was added and resuspended, and then the suspension was transferred to a 2 mL polypropylene tube. After centrifuging the tube again and removing the supernatant, 300 μL of sugar solution (containing 62.5% sucrose and 12.5% galactose in 100 mM sodium acetate buffer at pH 5.0) was added, and the cells were vortexed. Suspended well and started the sugar synthesis reaction. The sugar synthesis reaction was performed at a reaction temperature of 60 ° C. and a rotation speed of 1200 rpm. After 38 hours from the start of the sugar synthesis reaction, 40 μL of the reaction solution was collected and mixed well with 960 μL of distilled water, and the enzyme was heat-inactivated at 99 ° C. for 10 minutes. After returning the diluted sugar solution to room temperature, analysis was performed by high performance liquid chromatography using a “Hypercarb” (Thermoelectron) column, and the raffinose content in the resulting oligosaccharide was 76% (reaction). The raffinose concentration in the liquid is 0.86% by weight).

  By using the present invention, a method for selectively producing raffinose using an inexpensive raw material can be provided.

FIG. 1 shows the optimum pH range (Example 2) of α-galactosidase purified from Bacillus coagulans AKC-004 cells. FIG. 2 shows the stable pH range of α-galactosidase purified from Bacillus coagulans AKC-004 cells (Example 2). FIG. 3 shows the optimum temperature range of α-galactosidase purified from Bacillus coagulans AKC-004 cells (Example 2). FIG. 4 shows the stable temperature range of α-galactosidase purified from Bacillus coagulans AKC-004 cells (Example 2). FIG. 5 shows the results of SDS-PAGE (Example 2) of α-galactosidase purified from Bacillus coagulans AKC-004 cells. FIG. 6 shows the results of HPLC analysis of a reaction solution for sugar synthesis (Example 3) using α-galactosidase purified from Bacillus coagulans AKC-004. FIG. 7 shows the result of HPLC analysis of a reaction solution for sugar synthesis (Example 5) using α-galactosidase obtained from recombinant Escherichia coli JM109 strain. FIG. 8 shows the results of HPLC analysis of a reaction solution for sugar synthesis (Example 3) using a microbial catalyst obtained by culturing Bacillus coagulans AKC-004 strain.

Claims (17)

An α-galactosidase derived from a microorganism belonging to Bacillus coagulans having the following characteristics.
(1) Action: This enzyme has a property that the raffinose synthesis selectivity is 65% or more when the raffinose content in the resulting oligosaccharide is 0.5% or more in the dehydration condensation reaction using sucrose and galactose as raw materials. .
(2) Optimal pH range: 3.5-5.0
(3) Stable pH range: 3.5 to 10.0
(4) Molecular weight: about 80,000 Bacillus coagulans are Bacillus coagulans AK003 (FERM P-21091) , AK004 (Ferm BP-10948 ), AKC005 (FERM P-21093) , and AK006 ( strain FERM P-21094) . α-galactosidase. Α-galactosidase comprising the amino acid sequence of any of the following (a), (b) or (c).
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having an α-galactosidase activity:
(C) an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 2, and having an α-galactosidase activity: The alpha-galactosidase gene which codes the alpha-galactosidase which consists of an amino acid sequence in any one of following (a), (b) or (c).
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having an α-galactosidase activity:
(C) an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 2, and having an α-galactosidase activity: An α-galactosidase gene comprising the following base sequence (a) or (b):
(A) the base sequence represented by SEQ ID NO: 1;
(B) The base sequence represented by SEQ ID NO: 1 is a base sequence in which one or several bases are deleted, substituted, and / or added, and encodes a protein having α-galactosidase activity. Base sequence: A recombinant vector comprising the α-galactosidase gene according to claim 4 or 5 . A transformant into which the α-galactosidase gene according to claim 4 or 5 or the recombinant vector according to claim 6 has been introduced. Α-galactosidase obtained by culturing the transformant according to claim 7 . An enzyme composition comprising the α-galactosidase according to any one of claims 1, 2, 3, and 8 . Selected from α-glucosidase, β-glucosidase, β-galactosidase, cellulase, xylanase, protease, galactanase, arabinanase, mannanase, rhamnogalacturonase, polygalacturonase, pectin methylesterase, pectin lyase, and polygalacturonic acid lyase The enzyme composition according to claim 9 , further comprising at least one component. A raffinose synthesis reagent comprising the enzyme composition according to claim 9 . The sucrose and galactose are used as a raw material, The α-galactosidase according to any one of claims 1, 2, 3, or 8 , the enzyme composition according to claim 9 or 10 , or the claim 11 A method for producing raffinose, which comprises using a raffinose synthesis reagent. A method for producing raffinose, characterized by using a microbial catalyst obtained by culturing microorganisms belonging to Bacillus coagulans using sucrose and galactose as raw materials. Using sucrose and galactose as raw materials, Bacillus coagulans AKC003 strain (FERM P-21091) , AKC004 strain ( FERM BP-10948 ), AKC005 strain (FERM P-21093) , AK006 strain (FERM P-21094) A method for producing raffinose, comprising using a microbial catalyst obtained by culturing Bacillus coagulans belonging to the above. A method for producing raffinose, characterized in that sucrose and galactose are used as raw materials and a microbial catalyst obtained by culturing the transformant according to claim 7 is used. The method for producing raffinose according to any one of claims 12 to 15 , wherein the raffinose content in the produced oligosaccharide is 65% or more. The sucrose concentration in the raw material is 30% (w / v) to 90% (w / v), and the galactose concentration in the raw material is 2% (w / v) to 45% (w / v). The manufacturing method of raffinose in any one of 12-16 .

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