TW200846472A - Novel a-galactosidase - Google Patents

Abstract

A novel a-galactosidase with which raffinose can be selectively synthesized. The a-galactosidase has the property of attaining a selectivity to raffinose of 65% or higher when the dehydrating condensation of sucrose and galactose as raw materials yields oligosaccharides having a raffinose content of 0.5% or higher.

Description

200846472 IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to a novel α-galactosidase, and a method of producing the raffinose using the same. [Prior Art] In recent years, with the diversification of dietary life and social life, consumers' health awareness has increased, so the concern for food or food ingredients has increased. Among them, raffinose is considered to have functions such as improving bacterial communities in the intestines, and it has been closely taken as a food or beverage, a pharmaceutical, a cosmetic, or the like, and it has recently been found that raffinose has an effect on immunity or ectopic Sexual dermatitis is also useful. Second, raffinose can be recovered as a by-product of the production of beet sugar, but the content of raffinose in sweetness is only about 1%, and the production amount is also related to the production of the main product, sugar. There is a limit to the increase in the production of raffinose. This is a cheap and stable supply to the market.

The raffinose does not only require extracts from natural substances, but also synthetic products from inexpensive raw materials. As a catalyst for galactosidase so far; == method for forming 'may use saccharide as galactose receptor L. "There is the following method. Using cane to obtain raffinose (cardioose as a galactose donor, Obtained non-patent literature 2) 1 reported 1 reference patent document 1, material literature bud as a semi-substitute; tanning (tetra) phenyl benzene · α · galacto-galactose galactose galactose is a semi-reference material a method of using a bovine sugar donor (for example, refer to Patent Document 129208.doc 200846472 2), a method using galactose as a galactose donor (for example, refer to Patent Document 3), etc. There are several examples in which low-cost sucrose and galactose can be used as raw materials. For example, there is disclosed a method of obtaining raffinose using heart galactosidase from Pycn〇p〇rus einnabarinus (for example, reference non- Patent Document 4) or a method of synthesizing raffinose using α-galactosidase derived from Mortierella sinensis (-"Vinacea" (for example, refer to Non-Patent Document 5). [Patent Document 1] Japanese Patent No. 2688854 [Patent Document 2] Japanese Patent Laid-Open Publication No. M4973 [Patent Document 3] Japanese Patent Special Publication No. 8_24592 [Non-Patent Document 1] Agdc. Biol. Chem,, 52(9), 23〇5_2311, 1988 ' [Non-Patent Document 2] Biosci Biotech· Bi〇chem, 59(4), 619_623, 1995 [Non-Patent Document 3] Phytochemistry, 18, 35-38., 1979 [Non-Patent Document 4] Nippon Shokuhin K 〇 〇 〇 38 (8), 722-728, 1991 [Non-Patent Document 5] Carbohydr. Res., 185, 139_146, 1989 [Summary of the Invention] [Problem to be solved by the invention] Due to the use of α-galactosidase The production efficiency of raffinose is good, so the use of melibiose, p-nitrophenyl pyridinium galactose, inositol galactose or galactobiose as an example of raw material for raffinose has been reported, but such use is used. In the case of the method, the raw materials used are expensive, and it is difficult to manufacture raffinose in the water of 129208.doc 200846472.

Sugar: square, noodles', using the method of obtaining cottonseed from the galactose 8. galactosidase or using the α-galactosidase from the genus Mortierella sinensis to synthesize = sub-^ can be used as an inexpensive raw material for sucrose And galactose, because of the industrial considerations, the method of feeding is the (four) favorable side. However, in the case of using the #α_galactosidase, it is possible to form a trisaccharide in addition to the raffinose in the product, and therefore must be from the final product: from the dry trisaccharide, which in turn produces wasteful raw materials. problem. In the case of synthesizing the lactose heart raffinose from the red ginseng, the raw == raffinose content is 4G%; in the case of the succulent succulent sucrose from the succulent succulent succulent The cotton in the oligosaccharide is produced: the sugar content is 峨, and the content rate is low, so the cutting technique cannot selectively produce raffinose by the transfer method. In the above circumstances, the present invention provides a novel lysine α-galactosidase which can selectively synthesize raffinose using an inexpensive raw material, and provides an innovative cotton saccharide production using the novel α-galactosidase described above. method. [Technical means to solve the problem] The present inventors have repeatedly studied the problem to solve these problems, and found a novel enzyme that can selectively synthesize raffinose, and found a kind of inclusion of the target cotton. The production of raffinose which is produced by sugar collection has completed the present invention. In other words, the present invention relates to a novel cardiac galactosidase and a method for producing raffinose, which are shown in the following [1] to [20]. α] An α-galactosidase having the following characteristics: 129208.doc 200846472 (1) Action: In the dehydration condensation reaction using sucrose and galactose as raw materials, the content of raffinose in the oligosaccharide is 0.5. Above %, α-galactosidase has a carnosamine synthesis selectivity of 65% or more; (2) Optimal pH range: 3.5 to 5.0; (3) Stable pH range: 3.5 to 10.0; (4) ) Molecular weight: about 80,000. [2] The α-galactosidase of [1], which is derived from a microorganism belonging to Bacillus coagulans. [3] α-galactosidase such as [2], which is any one of Bacillus coagulans bacillus AKC003 strain, AKC004 strain (FERM-ABP10948) (Taiwan registration number: BCRC 910388), AKC005 strain, AKC006 strain. By. [4] A Bacillus coagulans and a variant thereof, the Bacillus coagulans belonging to any one of Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP 10948) (Taiwan registration number: BCRC 910388), AKC005 strain, and AKC006 strain . [5] an α-galactosidase comprising the amino acid sequence of any one of the following (a), (b) or (c): (a) an amino acid sequence represented by SEQ ID NO: 2; b) an amino acid sequence having one or several amino acids deleted, substituted and/or added, and having α-galactosidase activity in the amino acid sequence represented by SEQ ID NO: 2; (c) and sequence number The amino acid sequence represented by 2 has an amino acid sequence having a homology of 60% or more and having α-galactosidase activity. 129208.doc 200846472 [6] A kind of α-semi-season yoghurt _gene' encodes α-galactosidase containing any of the following amino acids (b): (b) SEQ ID NO: 2 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ; or an additional amino acid sequence having cardiac galactosidase activity; ()', the amino acid sequence represented by sequence 2 has more than 6% homology

And has the amino acid sequence of α _ 聋 千 千 it it activity. [] a semi-glycosidase gene comprising the following sequence of (4) or (b): (4) the base sequence represented by SEQ ID NO: 1; (8) in the sequence of the sequence represented by H H1, - or A number of assays are deleted, substituted and/or affixed and encode a sequence of a protein having cardiac galactosidase activity. [] A recombinant vector containing an α-galactosidase gene such as [6] or [7]. [9] A transformant into which an α-galactosidase gene of [6] or [7] or a recombinant vector such as [8] is introduced. [10] An α-galactosidase obtained by culturing a transformant such as [9]. [11] An enzyme composition comprising an α-galactosidase according to any one of [3], [5], or [1〇]. [12] The enzyme composition according to [11], which further comprises a selected from the group consisting of cardiac glucosidase, glucosidase, β-galactosidase, cellulase, xylanase, protease, galactanase, Arabinanase, mannose polyzyme, buckthorn 129208.doc -10- 200846472 . _ lactose enzyme, polygalactosidase, pectin A (tetra), pectin dissociation enzyme, and polygalacturonolytic enzyme At least one or more of the ingredients. [13] A raffinose synthesizing agent comprising the enzyme composition according to any one of the above. a method for producing a raffinose, which is characterized in that it uses a galactosase such as any one of [1] to [3], [5], or [1〇], such as (1)] or [12] The enzyme composition, or the raffinose synthesis reagent according to [13]. [15] A method for producing raffinose, which is characterized in that it is a microbial catalyst obtained by cultivating a microorganism belonging to Bacillus coagulans. [1 6] A method for producing a raffinose, characterized in that it is cultured to belong to Bacillus coagulans AKC〇〇3 strain, AKC〇〇4 strain (ferm_ABP1〇948) (Taiwan registered number: bcrc 9i A microbial catalyst obtained by Bacillus coagulans and/or a variant thereof of any of the AC strain and the AKC006 strain. Π7] A method for producing a raffinose, which is characterized in that it is a microbial catalyst obtained by cultivating a transformant such as [9]. The method for producing raffinose according to any one of [14] to [17] wherein the raffinose content in the oligosaccharide is 65% or more. [-19] The method for producing raffinose according to [14] to [18] towel, which uses sucrose and galactose as raw materials. [〇] The method for producing raffinose according to [19], wherein the concentration of the vegetable sugar in the raw material is 3〇% (w/v) to 90% (w/v), and the concentration of galactose in the raw material is 2% ( w/v) ~45% (w/v). [Effects of the Invention] 129208.doc -11 - 200846472 By using the present invention, raffinose can be selectively produced. [Embodiment] 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 as the microorganism, if it is a microorganism belonging to Bacillus coagulans, any one may be used, and α-which exhibits selective synthesis of raffinose may be used. Any microorganism of the galactosidase. Preferably, Bacillus coagulans AKC-003 strain, AKC '004 strain, AKC-005 strain, and AKC-006 strain are listed. Further, the microorganism of the present invention may be a mutant obtained by using a microorganism belonging to Bacillus coagulans as a mother strain. Bacillus coagulans AKC-003, AKC-004, AKC-005, and AKC-006 were deposited in the Patent Bioburden Center of the Institute of Industrial Technology, the Independent Administrative Corporation, on November 14, 2006 (Heisei 18). (1st, the central part of the 1st, 1st, 1st, 1st, 1st, 1st, 1st, 1st, 1st, 1st, The registration number is as follows. Furthermore, the AKC-004 strain was handed over to the International Depository Management (Taiwan Registration Number: BCRC 910388) on January 30, 2008 (2008) with the recipient number FERM-ABP10948 and the deposit number FERM-BP10948. AKC-003 strain (FERM P-21091) AKC-004 strain (FERM P-21092; FERM-ABP10948; FERM-BP 10948) (Taiwan registration number · BCRC 910388) 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: In the dehydration condensation reaction using sucrose and galactose as raw materials, when the content of raffinose in the oligosaccharide is 〇 5% or more, the carbohydrase of the present invention has The raffinose synthesis selectivity is 65% or more. (2) Optimum pH range: 3.5~5〇, (3) Stable pH range: 3.5~1〇.〇(4) Molecular weight: about 8〇, 〇〇〇 For the specific example of the enzyme of the present invention, It is classified as a handyman. It has the following properties as a result of the influence of the substance-specificity and the five-genus ion. When the benzene-α-containing lactose material is subjected to the f-shaped shape of the shell, the decomposition activity is the highest, followed by ",,,, and 樯. Fork + mites and sugar, once again, the cotton nectar will not break down. ... Sugar, lactose, sugar cane Metal ion effect: When potassium, dance, magnesium, chromium, fierce, 2, iron (9), iron (10) ions were added, no decrease in activity was observed. Another drop: When the nickel and zinc ions were added separately, the activity decreased, and the activity decreased most when copper ions were added. Further, as the enzyme of the present invention. J苟 has the following characteristics: (5) (positive reaction) optimum temperature range ·· 35~5〇 It (6) Stable temperature range: until the judgment is stable. As a helium gas culture or solid culture for use in the microorganism of the present invention, it is applicable to the use of a normal medium, which can be used as a medium, and the microorganism can be used as a medium. The microorganisms grow well, and: the biological medium and the galactosidase can produce the carbon source and nitrogen necessary for the smooth production; the synthetic medium of the organic salt, the essential nutrient source, etc. or 丄129208.doc -13- 200846472 For the source, glucose, glycerin, sucrose, galactose, lactose, melibiose, raffinose, stachyose, cellobiose, organic acid, starch, olive oil, soybean oil and the like can be used. As the gas source, for example, sulfuric acid, ammonium nitrate, urea, amino acid, amine, ammonium or organic acid ammonium salt, other occluding compounds, protein glands, polyprotein glands, gravy, Yeast extract, cottonseed meal, corn syrup, and cakes. As the inorganic salt, X' can be used: two-disc discharge of a dish, a hydrogen-discharge of a dish, a sulphuric acid lock, a magnesium sulfate, a sodium chloride, a sulfuric acid, a steel sulfate, an iron sulfate, and the like. In terms of the growth of microorganisms, it is preferred to carry out 25~, C, and more preferably 40~65. Hey, better 1 40~S5C. Further, the pH of the medium may be in the range of growth of microorganisms in a larger range, and the pH is preferably 3 〇 to 9 〇, more preferably 3 to 5 8.5, more preferably The pH is 4〇~8〇. The separation/purification of the α-galactosidase of the present invention can be carried out, for example, by culturing the Bacillus coagulans AKC-004 strain in the above medium, and separating the obtained culture solution by a known method such as centrifugation, filtration, or the like. For the bacteria and filtrate. The cells thus obtained contain ... galactosidase, and the cells can be pulverized using a lysozyme, an ultrasonic pulverizer, a French high-waste crusher, etc., thereby obtaining a crude extract of _galactosidase. When the culture supernatant is contained (for the activity of X-galactosidase), the culture supernatant may be directly used, and the culture supernatant after the operation such as contraction may be used as the cardiac galactosidase. The crude extract is used in the subsequent purification. 129208.doc -14 - 200846472 It is possible to combine the purification methods of common proteins, such as ion exchange* water chromatography, gel filtration chromatography: ', sparse The crude extract obtained in the above manner (4) is subjected to cardiac galactosidase. The order of the above operations is not limited to F. V > Hehui can be performed once or twice, so that the father is ideal, Before the sample is passed into each column, the sample liquid is concentrated by exchanging the sample solution with an appropriate buffer. The ideal part of each stage is the stage of 1 refining, and the separated components are determined. The 3C-half-glucosidase activity of the middle 3 is a group stage test with a higher collection activity. The method of supplying the 敎α·galactosidase activity, for example, has the following method for the nitro group containing 6 mM. Benzene (4) ♦ Southern galactose yin value _ 100 mM sodium acetate buffer In 45〇#, the mixed enzyme solution 15 is called, and after reacting for 5 to 30 minutes under the thief, it is added to the aqueous solution of im sodium carbonate 1 red to inactivate the enzyme, and the reaction is stopped. The chromaticity was measured at an absorption peak at a wavelength of 420 nm, and the concentration was calculated using a calibration curve prepared for each concentration of the osmotic benzene. Further, the unit of the enzyme activity was i μ m 〇i within 1 minute under the above conditions. The amount of the enzyme which is free of the phenyl group is expressed as 1 U. It is confirmed that the purified a-galactosidase system and the molecular weight are determined by electrophoresis or gel filtration chromatography. The nature can be studied by changing the reaction temperature or the reaction_value to measure the enzyme activity, or adding various enzyme inhibitors or metal ions to the reaction solution, and measuring the residual activity by 129208.doc -15-200846472. Under the conditions of exposure-timed-gate carbohydrase under the conditions of each bay or the range of steadiness and stable temperature; circumference:: chymase: sex, which can be used to study the steady response, Qishan error by changing the concentration of the drug (6) Maximum speed (Vm Shi (four) phase For example, the Bacillus coagulans AKC__ strain can be cultured in the above medium by the Michaelis constant of each of the M. sinensis constants, and the obtained Bacillus coagulans AKC__ strain can be obtained by the method of separation The culture medium is separated into a bacterial cell/[4], an enzyme, an ultrasonic pulverizer, and a French sorghum crusher, which are pulverized in the above manner to cause chromosomal segregation. Two kinds of enzymes are limited, and the chromosome obtained by the above method is digested. Job, : Obtaining a DNA fragment. Using the chromosomal fragment obtained in the above manner, the full length or part of the galactosidase-containing gene is obtained by using a well-known method such as a shotgun or a reverse pcR method. Job film,. The nucleotide sequence of the fragment of the ship fragment containing the "galactosidase gene" obtained here is determined by a well-known method such as a Weser Sequencer. Further, in the case where only a part of the nucleotide sequence of the cardiac galactosidase gene is specified, the DNA fragment containing the α-galactosidase gene can be obtained again based on the sequence of the test. In other words, by repeating this operation, the base sequence of the entire length of the α-galactosidase gene can be confirmed. Based on the base sequence of the α-galactosidase gene decoded in the above manner, a DNA fragment containing the full length of the α-galactosidase gene can be obtained by a well-known method using a PCR method or a restriction enzyme. The amino acid sequence of the α-galactosidase can be determined by the sequence of the α-galactosidase gene of the above method 129208.doc -16 - 200846472. The amino acid sequence of the α-galactosidase of the present invention is exemplified in SEQ ID NO: 2, but may be produced in the amino acid sequence as long as the protein comprising the amino acid sequence has α-galactosidase activity. The at least one amino acid is deleted, substituted, affixed or the like, or may be an amino acid sequence having 6% or more homology to the amino acid sequence. Namely, the α-galactosidase of the present invention is a cardiac galactase comprising the amino acid sequence of any one of the following (a), (b) or (c). (a) an amino acid sequence represented by SEQ ID NO: 2; () in the amino acid sequence represented by SEQ ID NO: 2, one or several amino hydrazines, which are missing, substituted and/or attached And an amino acid sequence having cardiac galactosidase activity; (C) an amino acid sequence having 60% or more homologous 1* and having cardiac galactosidase activity with the amino acid sequence represented by SEQ ID NO: 2 . Further, the present invention provides an α-galactosidase gene encoding an α-galactosidase comprising any one of the following amino acid sequences (a), (b) or (c): (a) The sequence is the amino acid sequence represented by Tiger 2; (b) one or more of the amino acid thione, blown, and/or attached in the amino acid sequence represented by SEQ ID NO: 2, and has The acid sequence of the α-galactosidase activity of the face; the makeup (e) and the amine & acid sequence represented by the sequence of the tiger 2 have more than 6 % homology, and the ancient, upper ', /, 3 Amino acid sequence of α_galactosidase activity. 129208.doc -17- 200846472 In the above (b), "one or several amino acids" usually means 丨~ brother amino acid, preferably a carboxylic acid, more preferably (10) an amino acid, more preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids. The "amino acid sequence with more than 60% homology" in (e) is more than 6〇%, preferably more than above, more preferably more than 8〇%. More than 90%, more preferably more than 95%, especially preferably more than 99% homologous amino acid sequence.

Specific examples of the cardiac galactosidase gene of the present invention include a cardiac galactosidase gene having the base sequence of (a) or (b) below. (a) the base sequence represented by the sequence number; (8) the sequence number 丨 indicates the code base sequence, one or several test groups are replaced and/or added, and the code has α_galactosidase activity The base sequence of the protein. The "one or several bases" in the above (b) usually means 15 bases, 'preferably 1 to 90 bases, and more preferably (9) bases, and more preferably 1 to 3 验 one test base, more preferably 1 to 20 test bases, more preferably 15 test bases, and more preferably 1 to 10 test bases, especially 5 bases. The α-galactosidase gene of the present invention can realize the variation of the peptide by a well-known genetic manipulation method without damaging the nature of the catalytic reaction, such a variant gene refers to a genetic engineering method. An artificially mutated gene produced by the alpha-galactosidase gene of Benjamin, which is a genetic engineering method by using a site-directed variation method or a specific two-fragment of a target gene as a human mutated DNA. And get. That is, as a method of deleting the amino acid in the α-galactosidase and replacing the 'additional 129208.doc -18-200846472, etc., $彳彳, , ^PCR method, error-prone PCR method, DNA miscellaneous method, A well-known method such as a method of producing a chimeric enzyme. The visceral fragment of the structure of the complete G 3 galactase obtained in the above manner can be inserted and ligated into a thiol-based expression vector, for example, the polymorphism of pBluescriptII KS(+) Constructing a new Cao Nguyen. = The plastid vector has a gene that efficiently expresses the genes linked to it as a foreign gene in E. coli, and the stalk is cultured to introduce the recombinant plastid into Escherichia coli. The obtained transformant can exhibit a large amount of CX-galactosidase. In addition to the above, 'a large amount of α-galactosidase can be expressed by various host microorganisms and vectors, and the column can be expressed as a large amount of "galactosidase" by culturing a transformant of BreV1bacillus ehoshinensis. More specifically, as a vector incorporating the galactosidase gene of the present invention, a phage which can be independently proliferated in a host microorganism or a plastid for gene recombination is preferred as a bacterium carrier. For example, when a microorganism belonging to Escherichia coli is used as a host microorganism, Xgt · λ (:, Xgt · λΒ, etc. can be used. Further, when a plastid vector, for example, Escherichia coli is used as a host microorganism, it can be used. PET vector (Novagen) such as plastids pET_3a, pET-lla, pET-32a, or pgR322, pBR325, PACYC184, PUC12, pUC13, PUC18, PUC19, f pUC118, PIN I, Bluescript KS+; in the case of Bacillus subtilis as a host PWH1520, pUBllO, PKH300PLK can be used; pIj680, pIJ7〇2 can be used when actinomycetes are used as hosts; yeast, especially brewer's yeast, can be used. In the case of a host, YRp7, pYCl, YEpl3, etc. may be used, and the DNA ends generated by the restriction enzyme produced by the α-galactosidase gene for the purpose of cutting the invention of 129208.doc -19-200846472 are used. The restriction enzyme, the vector is cleaved to prepare a vector fragment, and the α-galactosidase gene fragment of the present invention is bound to the vector fragment by a DNA ligase according to a conventional method, whereby the α-galactose encoding the present invention can be encoded. The enzyme gene 2 DNA is incorporated into the target vector.

As a host microorganism to be implanted into the plastid, if 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), and Escherichia coli BL21trxB can be used. Escherichia coli Rosetta (DE3), Escherichia coli Rosetta, Escherichia coli R〇setta (DE3) pLysS, Escherichia coli R〇Setta (DE3) PLaCl, Escherichia coli R〇settaBlue, Escherichia coli R〇setta_gann, Escherichia coli 〇rigami, large intestine Bacillus rig ji, E. coli Timer, E. coli Dm, E. coli jmi 〇 9, E. coli W3110, E. coli C6 〇〇 and the like. Further, when the microbial host is a microbe belonging to the genus Bacillus, when a microorganism such as Bacillus subtilis or Bacillus megaterium is used as the microorganism belonging to the actinomycete, the Streptomyces elegans TK24 or the like can be used; When the microbial host is a beer yeast U organism, Saccharomyces cerevisiae 1 or the like can be used. ^,, when the α-galactosidase of the present invention is produced by the transformed microorganism, the microbial culture (4) nutrient medium towel is used to collect the cells in the method of (4) or the culture medium or the enzyme such as lysozyme == Then, the mechanical solution or the appropriate boundary, 35 of the cells, and, if necessary, the addition of the coffee, the surfactant, etc., the aqueous solution of the α-galactosidase of the present invention 129208.doc -20-200846472 It is stretched, or not concentrated, and then processed by adsorption chromatography such as sulfuric acid fractionation, gel transition, affinity chromatography, or ion exchange chromatography, whereby α_half of the present invention having higher purity can be obtained. Lactase. The culture conditions of the transformed microorganisms can be selected in consideration of their nutritional and physiological properties, and in many cases, they are cultured in a liquid, but it is advantageous to carry out deep ventilation and disturbance culture on the worksheet. As a nutrient source of the culture medium, a general user in the culture of microorganisms can be widely used. The culture temperature can be appropriately changed within the range of micro-material development and production of the galactosidase of the present invention. In the case of Escherichia coli, it is preferably ίο to hungry, more preferably 20 to 3 〇 °c. about. The culture conditions are somewhat different depending on the conditions. = Estimated " 'This is the time when the galactose enzyme reaches the highest yield and ends at the appropriate time. (4) Yes, the condition of the cut (4) is usually the heart:: Right The pH of the medium can be appropriately changed in the development of bacteria and in the production of the present invention. In the case of Escherichia coli, the pH of the medium is preferably about 6 to 8. In the case of using the above-mentioned enzyme, the enzyme of the present invention is not particularly limited, and the degree of purification or the like is not particularly limited, and a substance containing the enzyme may be used in addition to the purified enzyme. Group = Shuming, an enzyme enzyme containing the above-mentioned α-galactosidase of the present invention, β grape: The composition may further comprise, for example, α-glucosidase galactosidase, cellulase, xylan Enzyme, galactose 2: = glycanase, mannanase, rhamnase, and heart..._enzyme, pectin brewing enzyme, pectin dissociation, at least one of the above-mentioned acid dissociation enzymes . 129208.doc -21 - 200846472 According to the present invention, a raffinose synthesis containing the above enzyme composition can be provided. In the present description, as a microbial catalyst, in addition to the Bacillus coagulans akcmhm strain, the above-mentioned transformant can also be used. . The microbial catalyst used in the production of the raffinose of the present invention can utilize the microorganism itself obtained by a usual culture method, and it is not necessary to purify the galactosidase from the microorganism. Further, depending on the case, a microbial culture solution or a microbial culture supernatant may be used. On the other hand, the microorganism obtained by the culture method may be washed with water or a buffer or the like as needed. For example, it can be obtained by using cultured microbial culture silkworm liquid ' or by centrifugation, buffering, etc., and the core W liquid is used to treat microorganisms or microorganisms (for example, microorganisms:: debris, etc.) An aqueous solution that is suspended or dissolved, or a microorganism or a microbial treatment is immobilized by an embedding method, a cross-linking method, or a carrier-binding method (carrier 2). As an example of an immobilized carrier during immobilization In addition, it can be mentioned, for example, glass, glass, yttrium, urethane, polypropylamine, polyglycolic acid, carrageenan, alginic acid, but it is not limited to these. An enzyme composition using the α-galactosidase or the package 2 galactosidase of the present invention, a raffinose synthesis reagent or a microbial catalyst, and a method for producing the sugar can be provided. As a raw material, for example, sucrose and a half can be used. Lactose. When using the sugar and the body to be used as a raw material, for example, from the nature of the dehydration condensation reaction using α-half enzyme, it is preferred that the concentration of the raw material =, but if the concentration of galactose is excessive High, because of the contraction between the galactose molecules 5 'There will inhibit the dehydration condensation reaction between sucrose and galactose, so 129208.doc -22. 200846472 The concentration of sugar is preferably 30% (w/v) ~ 9〇% (w / v), better It is 35% (w/v)~80% (w/v), more preferably 4〇0/.(w/v)~7〇% (w/v). 2% (w/v) ~ 45% (w/v), more preferably 5% (w/v) ~ 35% (w/v), more preferably 7% (w/v) ~ 3〇 % (w/v). Further, it is preferred to prepare a concentration in which the sucrose and galactose in the raw material are soluble. _ Use the α-galactosidase of the present invention or the enzyme composition containing cardiac galactosidase or The reaction temperature of the raffinose synthase or the microbial catalyst comprising the enzyme composition for producing cotton φ saccharide is preferably from 10 to 90 ° C, more preferably from the viewpoint of the reaction rate or the stability of the enzyme. 20~70. 〇, more preferably 3〇~6 (rC. The pH of the reaction can be adjusted in a wide range. From the aspect of stability of the enzyme, the pH is preferably 2·0~1〇 ·〇, better? The value of 11 is 3〇~7.5, and the better is 疋3·5~6·0. The reaction time also varies according to the amount of enzyme used, considering the industrial benefits. In general, it is preferably 2 to 2 hours, more preferably 6 to 80 hours. However, the present invention is not limited to the above reaction conditions or reaction forms, and can be appropriately selected. In the raffinose synthesis reaction using a galactase or an enzyme composition comprising α-galactosidase or a raffinose synthesis reagent comprising the enzyme composition, the raffinose is accumulated in the reaction solution to 〇. 5 % In the above case, the content of raffinose in the sugar-producing sugar can be increased to 65% or more, and can be increased to 80% or more under better conditions. The raffinose content in the higher oligosaccharide produced in the present invention can also be achieved when the raffinose is accumulated in the reaction solution to 〇·75〇/〇 or more and further accumulated to 1.0% or more. Further, in general, in the raffinose synthesis reaction using the microorganism itself, the raffinose content in the oligosaccharide produced by the influence of the inclusion enzyme is 129208.doc -23-200846472, and is used. In the raffinose synthesis reaction of the microbial catalyst of the present invention, the raffinose content in the oligosaccharide is high. The raffinose content in the resulting oligosaccharide obtained in the present invention can be determined by the following method. After completion of the raffinose synthesis reaction, the reaction was stopped by diluting the reaction solution by 25 times and maintaining at 99 C for 10 minutes. After the reaction was stopped, the microorganisms were removed by centrifugation, and the obtained reaction solution was quantified using a high performance liquid chromatograph. The measurement was carried out using Thermoelectr〇n.

Hypercarb column, detector system (4). The rate of the raffinose in the sugar collection was 3, and the area ratio of each peak detected in the HPLC analysis chart was calculated based on (the area of the peak of raffinose) / (the area of the peak of the sugar-producing sugar). As a method of purifying and separating raffinose produced by the method of the present invention, a purification treatment method generally used can be used. That is, ^ such as 'by centrifugation, membrane treatment using MF membrane or UF membrane = removal of microbial catalyst, by (4) ion exchange chromatography or anion exchange: = method such as chromatography or dialysis, etc., removal Self-buffering nurturing groups, etc., such as cation, chromatography, cation exchange chromatography, high-performance liquid chromatography, high-pressure liquid chromatography, or the difference in solubility, etc. The crystallization treatment, 4 = decantation chromatography can be used separately:,! Use t to use the movable layer or analog shift J ί cotton: 拟 moving layer type, multi-component separation cycle type, etc. = its branching < refining, separation processing method, can be carried out in batches, and can also be used & Columns and the like are continuously performed. The following is a detailed description by way of examples, but the invention is not limited by the examples. [Examples] Example 1 Coagulation of dental rod _ AKC-004 strain (registration number FERM P-21092 (transferred with FERM-ABP10948 as number) (Taiwan registration number: 'BCRC 9103 88); depository: independent The Administrative Biotechnology Industrial Technology Research Institute Patent Bioburden Center is on the Tryptose Blood Agar Base (TBAB) plate medium (Difco) at 55. (: cultured for 1 day, and colonies were formed. 30 mL of the medium shown in Table 1 adjusted to pH 7.2 was added to a 150 mL capacity Erlenmeyer flask, and the colony was implanted on the above plate medium with platinum ear to Rotary shaking culture was carried out at 50 ° C, 25 hours, and 180 rpm, and it was used as a seed bacterium cultured in a bottle. [Table 1] Table 1: Medium composition medium composition Trace metal element solution composition

Raffinose l〇g CH3COONa 12.8 g Glycerol log FeS04*7H20 Ig Yeast extract log MnCl2.4H20 0.5 g Protein gland l〇g CoC12.6H20 0.3 g MgS04*7H20 0.5 g ZnS04*7H20 0.4 g CaCl2 0.04 g CuC12*2H20 50 mg 0.01 拧 citric acid 0.5 mL NaMo04.2H20 50 mg Sodium acetate trihydrate 0.132 g H3BO4 20 mg ΚΗ2Ρ04 0.544 g NiCl2.6H20 20 mg Na2HP〇4*12H2〇2.14 g Distilled water 1 L Trace metal element solution 0.5 mL Distilled water 1 L 129208.doc -25 200846472 Then, adjust the pH to 7·2. Table 1斛-十拉总* 6 L of the culture medium of Heartcoat 1 is added to the cylinder acid 醏梓φ of 10 L capacity In the alcohol tank, 15 mL of the seed bacteria cultured in the above bottle was transplanted, and aeration agitation culture was carried out under the conditions of 50 ° C, 48 hours, 2 (10) Λ , ^ 200 rpm, and 0.2 vvm. Then, at 4. (:, the culture solution was centrifuged for 1 〇 χ χ 3 〇 3 minutes to remove the supernatant, and the cells were recovered. The activity of the luciferase was measured, and the result was 261 U. .

The cells were suspended in Lysis buffer (pH 8 Torr) containing 5 〇 mMiTris_Hcl, 2 mM EDTA, 50 mM glucose to obtain 150 mL. The suspension was centrifuged at 4 C for 1 〇, 〇〇〇 gx3 〇 minutes to remove the supernatant, and the cells were recovered. The cells which were washed with the buffer were resuspended in the above Lysis buffer to become 150 mL, and lysozyme (manufactured by Sigma, from Imprint) was added thereto to 37 〇. c, 13 hours, i2 rpm conditions to oscillate, and the bacteria are pulverized. At 4C, the solution after the pulverization of the cells is subjected to 1 〇, 〇〇〇gx3 〇 minutes of centrifugation to remove the steroid residue, The supernatant was recovered. Ammonium sulfate was added to the supernatant to form a 37.5% saturated solution, which was allowed to stand overnight at 4 ° C to produce a precipitate. At 4 ° C, 1 〇, 000 g x 30 was carried out. The supernatant was removed by centrifugation to remove the precipitate, and ammonium sulfate was added to the supernatant to form a saturated solution of 54 and 5%, which was allowed to stand overnight at 4 ° to produce a sag. (:, 1 〇, 〇〇〇 &\30 minutes of centrifugation to recover the precipitate, and then dissolved in a pH of 7.0 20 mM acid buffer, placed at 4 ° C One night, dialysis was performed on the same acid buffer as the above 12920B.doc -26 - 200846472. The supernatant was adsorbed to "DEAE Sepharose FF" (GE HEALTHCARE. BIO-SCIENCES Co., Ltd.) equilibrated with the same phosphate buffer as above, and then passed through a pH value of sodium chloride containing 0 to 0.4 Torr. The enzyme was eluted by a concentration gradient method of 7 mM 20 mM phosphate buffer. β The above-dissolved active component was collected, concentrated using an ultrafiltration membrane having an average molecular weight cut off of 10,000, and adsorbed to contain 2 Μ. "HiTrap" for the equilibration of 20 mM phosphate buffer with a pH of 7.0 of chlorinated sodium

Phenyl FF (high sub)" (GE HEALTHCARE. BIO-SCIENCES Co., Ltd.), followed by concentration gradient method using a concentration gradient of 2.0 mM phosphate buffer containing 2.0 to 0 M of sodium chloride Dissolution. The above-dissolved active components were collected, concentrated using an ultrafiltration membrane having an average molecular weight cut off of 10,000, and adsorbed to "MonoQ 5/50 GL" (GE) equilibrated with 20 mM phosphate buffer having a pH of 7.0. φ HEALTHCARE. BIO-SCIENCES Co., Ltd.), and then the enzyme was eluted by a concentration gradient method of 20 mM phosphate buffer containing pH 7.0 of 0 to 0.4 Μ. ^ Collect the above-dissolved active components, concentrate them with an ultrafiltration membrane with an average molecular weight cut off of 10,000, and fill them with "HiLoad 16/60" equilibrated with 20 mM acid buffer buffer at pH 7.0. Superdex 200" (GE HEALTHCARE. BIO-SCIENCES Co., Ltd.) was dissolved in the same buffer. The above-dissolved active component was collected and concentrated using an ultrafiltration membrane having an average molecular weight cut off of 129208.doc -27-200846472 10,000 to prepare a purified galactosease. The activity yield was 15% and the activity was 270 U/mL. Example 2 The user and the purified α-galactosidase obtained in % of Example 1 were subjected to an experiment on the action thereof. (1) The optimum pH range is 6 mM p-nitrobenzene _ φ heart 0-pyranose 150 ^ dissolved in 1 mM buffer of each pH value, mixed and diluted 100 times The purified enzyme solution of the invention was 50 pL and allowed to react at 4 ° C for 5 minutes. After the reaction, the amount of the free pair of schiff base was determined, and the activity was measured to determine the relative activity when the maximum activity was 100. This result is shown in FIG. Further, the buffer used is glycine _HC1 buffer (pH 2 5 to 3 · 5), sodium acetate buffer (pH 3·5 〜 6 · 〇), and sulphate buffer Liquid (ρΗ value is 6〇~8·5), glycine-NaOH buffer (ρΗ value is 8·5~1〇〇). (2) Stable pH range • Use 10 mM buffer at pH value of 4·0~10·〇, heat treatment at 45 ° C for 140 minutes at each pH value, and measure residual activity. The relative activity when the maximum activity was 1 求出 was determined in the same manner as above. The results are shown in Fig. 2. Further, the types of buffers used for each pH value are the same as described above. (3) The optimum temperature range is 47 mM p-nitrophenyl-aD-galactopyranoside 190吣 dissolved in a pH 4.5 sodium acetate buffer, mixed and diluted 2 times the purification of the present invention The enzyme solution was 10 Torr and allowed to react for 5 minutes in the range of 20 to 6 (TC 129208.doc -28 - 200846472. After the reaction, the free p-nitrobenzene was quantified, thereby measuring the activity and determining the maximum. The activity is set to the relative activity at 100 ° C. The results are shown in Figure 3 ° (4) Stable temperature range using 100 mM sodium acetate buffer at pH 4.5, at 20 ° C to 55 ° C for 20 minutes The heat treatment was carried out, and the residual activity was measured, and the relative activity when the maximum activity was 1 Torr was determined in the same manner as above. The results are shown in Fig. 4. (5) The molecular weight was 10% by using a separation gel. r Ready Gels J" (曰本

Bio-Rad Laboratories, Inc.) sdS-polyacrylamide gel electrophoresis to determine the molecular weight. The result is shown in Fig. 5. The molecular weight is about 80, 〇〇〇, which is substantially identical to the molecular weight 83,122 estimated from the amino acid sequence. (7) The specificity is 5 〇KL of the purified enzyme solution of the present invention diluted and diluted 1 〇〇 in a 150 mM mM sodium acetate buffer having a pH of 45 mM containing various contents of 10 mM. The reaction was allowed to proceed for 20 minutes at 40 C. Among them, p-nitrobenzene_a_D_ domain galactoside was used in the reaction of the host, and the reaction time was set to 5 minutes. After the reaction, (4) the amount of the p-nitrobenzene material is quantified, or according to the analysis of the high-performance liquid chromatograph by using the column of "Shodex SUGAR" (Showa Denko Co., Ltd.) The relative activity of each substrate when the maximum activity was set to 100. The results are shown in Table 2. Further, where the reaction rate was measured for the recipient of the formation, the results are shown in Table 3. 129208.doc -29- 200846472 [Table 2] Table 2: Relative activity of α-galactosidase-specific matrix ρΝΡ-α-° than galactoside 100 ρΝΡ-β-吼 galactose bitter 0 cottonseed Sugar 10 Honey disaccharide 38 Lactose 0 Sucrose 0 [Table 3] Table 3: Reaction rate constant of α-galactosidase Matrix Km (mM) Vmax (U/mg) ρΝΡ-α-σ galactoside 0.53 82 Cotton Glycogen 40 48 melibiose 8.4 106 (8) Inhibitory factor in 6 mM p-nitrophenyl-α-D-galactopyranoside dissolved in pH 4.5 sodium acetate buffer to final concentration Various metal ions were added so as to be 1 mM, and 150 μL was added, and 50 g 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 free p-nitrophenol was quantified to measure the activity, and the relative activity at which the maximum activity was set to 100 was determined. The results are shown in Table 4. [Table 4] Table 4: Metal salt inhibition of a-galactosidase metal ion relative activity control 100 potassium 97 calcium 97 129208.doc -30- 200846472 magnesium 95 chromium 99 violent 95 iron (II) 98 iron (III) 99 Cobalt 95 Nickel 88 Copper 33 Zinc 80 Example 3 The purified α-galactosidase 0.5 U described in Example 1 was separately taken out and added to a 200 pL sugar liquid in a 1.5 mL-capacity polypropylene test tube ( The pH 5.0 of 100 mM sodium acetate buffer contained 73% sucrose and 12% galactose, and the mixture was mixed, and the reaction was carried out at 60 ° C and 1,200 rpm. After 45 hours from the start of the reaction, 20 μM was taken out from the reaction solution, diluted with 480 μL of distilled water, and then treated at 99 ° C for 10 minutes to inactivate the enzyme heat. After the heat-inactivated diluted sugar liquid was returned to normal temperature, it was analyzed by a high performance liquid chromatography using a "Hyp ere arb" (manufactured by Thermo Electro) column. The result is shown in Fig. 6. The raffinose content in the oligosaccharide was 82%, and the raffinose concentration in the reaction solution was 1.67 wt%. Example 4 (1) Preparation of chromosomal DNA Chromosomal DNA of Bacillus coagulans AKC-004 strain was prepared according to a usual method. 60 mL of the culture solution of the B. coagulans AKC-004 strain obtained by the method described in Example 1 was centrifuged to collect the cells. The obtained cells were suspended in Lysis buffer (50 mM Tris-HCl (pH 129208.doc • 31 - 200846472: 8.0), 20 mM EDTA, 50 mM glucose), and then thoroughly washed. After centrifugation, the cells were recovered, suspended again in Lysis buffer, lysozyme was added thereto, and cultured at 37 ° C for 45 minutes. Then, SDS and RNase were added and cultured at 37 ° C for 45 minutes. Thereafter, proteinase K (Proteinase K) was added, and the mixture was stably shaken at 50 ° C for 60 minutes. After the solution obtained here was treated with phenol-chloroform and chloroform, ethanol precipitation was carried out, and the deposited nucleic acid was wound around a glass pipette and recovered. The nucleic acid was washed with 70% ethanol, dried, and dissolved in mash. By this operation, about 1 mg of chromosomal DNA was prepared. (2) Isolation of α-galactosidase gene The PCR primer for amplifying the α-galactosidase gene fragment was designed and synthesized from the chromosomal DNA prepared in the above (1). The design of the primer is based on the well-known results of the α-galactosidase gene from several microorganisms, and the oligo DNA having the sequence of 5'-GAAGTITACGGITTYAGYYTTGTITACAGYGG-3' (SEQ ID NO: 3) is synthesized by synonymous primer. The antisense primer synthesizes an oligo DNA having the sequence 歹 ij of 5'·CCAAACCAICCRTCRTCIARIACRAA-3, (Sequence 歹U number 4). Further, in the sequence, I represents inosine, Y represents C or T, and R represents A or G. Using the PCR primer obtained here, the chromosomal DNA prepared in the above (1) was used as a template, and the α-galactosidase gene fragment was amplified by PCR to obtain α-galactose containing 380 base pairs. Enzyme gene fragment. The base sequence of the gene fragment obtained here was analyzed using a DNA sequencer. In order to obtain a DNA fragment containing the full length of the α-galactosidase gene, PCR for reverse PCR was designed based on the sequence of the α-galactosidase gene fragment obtained from the analysis of 129208.doc -32-200846472. Introduction. Synthetic primers were used to synthesize oligo DNA with the sequence of 5'-TGATCAACAACTGGGAAAGCGACCT-3, (Sequence 歹丨 J No. 5), and antisense primers were used to synthesize the DNA of the sequence with 5'-GAACTGGTCCTGCTGCACAATTCC-3f (Sequence No. 6). . Then, a template for reverse PCR was prepared. The chromosomal DNA prepared in the above (1) was digested with the restriction enzyme Hindlll', and the resulting DNA fragment was itself cyclized using T4 DNA ligase (T4 DNA Ligase) to form a template for reverse PCR. For the template, the DNA fragment containing the α-galactosidase gene was amplified by PCR using the primer for reverse PCR synthesized as described above, and 4,500 assays containing the entire sequence of the α-galactosidase gene were obtained. Base pair PCR 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, a structural gene of 2190 base pairs of α-galactosidase having a DNA base sequence from the 5' end shown by SEQ ID NO: 1 was decoded. This sequence is a novel gene that has never been discovered. Further, the α-galactosidase produced by the B. coagulans AKC-004 strain derived from the DNA base sequence contains 730 amino acids, and has an amino acid sequence from the quinone terminal shown by SEQ ID NO: 2. The database was searched and it was found that the amino acid sequence has 57% homology with α-galactosidase (AgaN) from Geobacillus stearothermophilus, and α-half from Thermomyces sp. Lactase (AgaA) has 56% homology with 56% homology to a galactosidase (AgaB) from Bacillus thermophilus, and from 129208.doc •33-200846472 Lactococcus lactis ( The α-galactosidase of Lactococcus raffinolactis has 56% homology, which is clearly encoded by the novel α-galactosidase. (4) Expression of α-galactosidase gene Construction and transformation of the plastid vector based on the sequence of the α-galactosidase gene obtained in (3), designed and synthesized to increase the alpha-galactosidase gene PCR primers for the region of the SD sequence, structural gene, and stop codon. The design of the PCR primer was based on the base sequence of the PCR product containing 4,500 base pairs obtained in (2), and the synonymous primer was used to synthesize the sequence having the sequence of 5*-TAAGGTAAAGCAGATGTGCCATT-3' (SEQ ID NO: 7). Poly DNA, antisense primers were used to synthesize oligo DNA with 5f-TTACTCGTACACCGCCTC-3, (Sequence No. 8). The PCR product containing 4,500 base pairs obtained in (2) was used as a template, and the PCR product was amplified by PCR using a synthetic PCR primer to obtain a PCR product containing 2325 base pairs. The PCR product obtained at the end of the PCR product was smoothed and phosphorylated. After digestion with the restriction enzyme EcoRV, the de-acidified pBluescriptll KS(+) vector was ligated to the terminal smoothed and phosphorylated PCR product to construct a new plastid vector pBlue/agaA. . A lac promoter which can efficiently transcribe a gene which is ligated as a foreign gene in Escherichia coli is introduced into the plastid vector, and α-galactosidase can be efficiently expressed/manufactured. The resulting plasmid vector was transformed into a competent cell of Escherichia coli JM109 strain prepared by the calcium chloride method by thermal shock method to prepare a recombinant microorganism. (5) Culture of transformant and expression of α-galactosidase The recombinant Escherichia coli JM109-pBlue/agaA prepared in (4) has 100 mg/L of ampicillin in 129208.doc -34-200846472 In a 30 mL nutrient medium, culture under shaking for 24 hours. After the cultivation, the recombinant Escherichia coli was centrifuged and recovered. After the cells were washed with 100 mM sodium acetate buffer (pH 5 〇), they were again suspended in the same buffer and pulverized by an ultrasonic pulverizer. The galactose activity of the pulverized liquid was measured by the above method, and as a result, it was 1991; / culture solution (mL). Example 5 The cardiac galactosidase ι·ο u obtained by culturing the recombinant body described in Example 4 was separately taken and added to a 200 μί sugar liquid (pH) in a polypropylene tube filled with 丨5 mL capacity. The value was 5 〇 in 1 mM sodium acetate buffer containing 73% sucrose and 12% galactose, and mixed, and the reaction was carried out under the conditions of 6 Å, 〖, 2 〇〇卬 (5). After 16 hours from the start of the reaction, 20 pL was taken out from the reaction liquid, diluted with 480 pL of distilled water, and treated at 99 for 1 minute, whereby the enzyme was heat-inactivated. The diluted sugar solution after the heat inactivation was returned to the usual dish and analyzed by a high performance liquid chromatograph using a "Hypercarb" (manufactured by Thermoelectron) column. The result is shown in Fig. 7. The raffinose content in the produced sugar was 82%, and the raffinose concentration in the reaction solution was 1.20% by weight. Example 6 Bacillus coagulans AKC-004 (registered number FERM P-21〇92 (transferred by the number of FERM-ABPl〇948) (Taiwan registered number: BCRc 910388) The Institute's patented biological deposit center was placed on Tryp〇se B1〇〇d Agar Base (TBAB) plate medium (Difc®) at 55. Underarm cultivation i 129208.doc •35- 200846472 days, colonies formed. One platinum ear was inoculated to a trifluoro flask having a volume of 15 〇 mL, and the medium shown in Table 1 was dispensed and cultured at 55 C and 150 rpm for 2 days.

After 2 days of the culture, a 10 mL amount of the cultured cells was recovered into a test tube of i5 mL capacity. The supernatant was removed by centrifugation at 10,000 rpm in a test tube having a volume of 15 mL of the culture solution. Then, 1 mL of 1 mM sodium acetate buffer (pH 5.0) was added, and after resuspending, the suspension was transferred to a 2 mL polypropylene tube. Centrifuge the tube again, remove the supernatant, add 300 sugar solution (pH 5 〗 (10) mM sodium acetate buffer containing 62.5% sucrose, galactose 12 5%), by vortex The mixer allows the cells to be fully suspended, and the sugar synthesis reaction begins. The reaction temperature of the sugar synthesis reaction is 6 (^c, the rotation speed is 12 〇〇 printing (5). After 16 hours from the start of the sugar synthesis reaction, the reaction liquid is recovered 4 〇, and it is thoroughly mixed with 96 〇pL distilled water at 99 ° C. The heat inactivation of the enzyme was carried out for 1 minute. After the diluted sugar solution was returned to normal temperature, the sample was analyzed by a high performance liquid chromatography using a "Hypercarb" (manufactured by Thermoelectron) column, and the results are shown in the figure. 8. The raffinose content in the oligosaccharide was 80% (the raffinose concentration in the reaction solution was 〇·7 〇 weight 〇/〇). Example 7 Bacillus coagulans AKC-003 strain (registered number PERM) Ρ-21091 ; The depository authority: Patent Bioburden Center of the Institute of Industrial Technology, Independent Legal Corporation, is cultured on the Trypt〇Se Blood Agar Base (TBAB) plate medium (Difco) at 55t: Days, and formed & drop 1 inoculation to the medium shown in Table 1 3 〇 mL was dispensed into the 129208.doc -36-200846472 150 mL capacity flask, to 55. The culture was carried out for 2 days under conditions of 15 rpm.

Two days after the culture was carried out, 1 μmL of the cultured cells were recovered into a test tube of i5 mL capacity. The supernatant was removed by centrifuging the 15 mL-capacity tube in which the culture solution was recovered at loooo rpm. Then, 1 mL of 1 mM sodium acetate buffer (pH 5.00) was added, and after resuspending, the suspension was transferred to a 2 mL-capacity polypropylene tube. The tube was centrifuged again, and after removing the supernatant, 300 ounces of sugar solution (pH 51 〇〇 mM sodium acetate buffer containing 62.5% sucrose and galactose 12 5%) was added. The vortex mixer allows the cells to be fully suspended, and the sugar synthesis reaction begins. The reaction temperature of the sugar synthesis reaction is 6 (^c, the rotation speed is 12 〇〇邛 (5). After 38 hours from the start of the sugar synthesis reaction, the reaction liquid is recovered 4 〇, and it is thoroughly mixed with 96 〇 μί distilled water, under 99 ^ The thermal inactivation of the enzyme is carried out for 1 minute. After the diluted mash is returned to the normal temperature, the sample is analyzed by a high performance liquid chromatography using a "Hypercarb" (manufactured by Thermoelectron) column, and the result is generated. The raffinose content in the nourishing sugar was 8〇〇/0 (the raffinose concentration in the reaction solution was 0.76% by weight). Example 8 Bacillus anaterium strain ACK-005 (registered number fERM Ρ-21093; The depository institution: the Patent and Bioburden Center of the Institute of Industrial Technology and Technology, the Independent Administrative Corporation, is cultured on the Trypt〇Se B1〇〇d Agar Base (TBAB) plate medium (Difc〇) at 55. Into the formation of 囷洛. Inoculate 1 platinum ear to the table! The medium shown in the table 3〇mL is dispensed into the 15 glutinous rice dumplings, at 55 ° C, 150 rpm 129208.doc -37- 200846472 pieces cultured for 2 days.

Two days after the culture was carried out, a 10 mL amount of the cultured cells was recovered into a test tube of i5 melon capacity. The supernatant was removed by centrifugation at 10,000 rpm on a 15 mL tube in which the culture solution was recovered. Then, add 1 mL of 1 mM • sodium acetate buffer (pH 5.0), resuspend, and then transfer the suspension to a 2 mL polypropylene tube. The tube was centrifuged again, and after removing the supernatant, 300 pL of sugar solution (pH of 5. 〇1 mM vinegar-sodium citrate buffer containing 62.5% sucrose and galactose 12.5%) was added. The vortex is mixed to sufficiently suspend the cells, and the sugar synthesis reaction starts. The reaction temperature of the present sugar synthesis reaction was 6 Torr: and the rotation speed was 12 (10) rpm. After 38 hours from the start of the sugar synthesis reaction, the reaction solution was recovered for 4 Torr, and it was thoroughly mixed with 960 called rectified water. The heat inactivation of the enzyme was carried out at 99 ° C for 1 minute. After the diluted sugar liquid was returned to normal temperature, it was analyzed by a high performance liquid chromatography using a "Hypercarb" (manufactured by Thermoelectron) column, and the result was that the raffinose content in the sugar-producing sugar was 91% ( The concentration of the sweet raffinose in the reaction solution was 0.32% by weight). Example 9 Bacillus coagulans AKC_006 strain (registered number: FERM P-21094; depository: patented bio-registration center of the Institute of Industrial Technology, Independent Administrative Corporation) on Tryptose Blood Agar Base (TBAB) plate medium (Difco), cultured at 55 for 1 day to form colonies. One platinum ear was inoculated to a mixture of 30 mL of the medium shown in Table 1 in a 150 mL gluten-free flask to obtain 55. 〇, 150 rpm conditions for 2 days. 129208.doc •38- 200846472 After 2 days of this culture, 10 mL of the cultured cells were recovered into a 15 mL-capacity test tube. The supernatant was removed by centrifugation at 10,000 rpm on a 15 mL tube in which the culture solution was recovered. Then, 1 mL of 100 mM sodium acetate buffer (pH 5.0) was added, and after resuspending, the suspension was transferred to a 2 mL-capacity polypropylene tube. The tube was centrifuged again, and after removing the supernatant, 300 pL of sugar solution (pH 5, 100 mM sodium acetate buffer containing 62.5% sucrose, galactose 12.5%) was added, and the mixture was made by a vortex mixer. The cells are fully suspended, and the sugar synthesis reaction begins. The reaction temperature of the present sugar synthesis reaction was 60 ° C and the rotation speed was 1200 rpm. After 38 hours from the start of the sugar synthesis reaction, 40 pL of the reaction solution was recovered, and it was thoroughly mixed with 960 pL of distilled water, and heat-deactivated of the enzyme was carried out at 99 ° C for 10 minutes. After the diluted sugar liquid was returned to normal temperature, it was analyzed by a high performance liquid chromatography using a "Hypercarb" (manufactured by Thermoelectron) column, and as a result, the raffinose content in the sugar-producing sugar was 76°/ 〇 (The concentration of raffinose in the reaction liquid is (K86% by weight). [Industrial Applicability] By using the present invention, it is possible to provide a method for selectively producing raffinose by using an inexpensive raw material. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a view showing an optimum pH range of α-galactosidase purified from Bacillus coagulans AKC-004 (Example 2). Fig. 2 shows a self-coagulated Bacillus AKC-004 cell. The stable pH range of refined α-galactosidase (Example 2). Figure 3 shows the optimum temperature of the α-half 129208.doc -39- 200846472 carbohydrate purified from Bacillus coagulans AKC-004 Scope (Example 2) Figure 4 shows the stable temperature range of α-galactosidase purified from Bacillus coagulans AKC-004 (Example 2). Figure 5 shows the self-coagulated Bacillus AKC-004 cells. The result of SDS=PAGE (Example 2) of purified α-galactosidase. The result of HPLC analysis of the reaction mixture of the sugar synthesis (Example 3) of α-< galactosylase purified from Bacillus coagulans AKC-004 strain. Fig. 7 shows the results obtained by using the self-recombinant Escherichia coli JM109 strain. The result of HPLC analysis of the reaction liquid of the sugar synthesis of the α-galactosidase (Example 5). Fig. 8 shows the sugar synthesis of the microbial catalyst obtained by using the cultured Bacillus coagulans AKC-004 strain (Example 3) The reaction solution was subjected to HPLC analysis.

129208.doc •40-

Claims (1)

200846472 X. Patent application scope: 1 · An α-galactosidase which has the following characteristics: (1) Action · In the dehydration condensation reaction of sucrose and galactose as raw materials, the raffinose in the vouchers When the content is 〇, 50 〇 or more, the heart sucrose enzyme has a carnosamine synthesis selectivity of 65% or more; U) optimal pH range: 3.5 to 5 〇; (3) stable PH Value range: 3.5 to 10.0; (4) Molecular weight: about 80,000. 2. The alpha-galactosidase of claim 1 which is derived from a sacred substance belonging to the Bacillus coaguians. 3. The α-galactosidase of claim 2, wherein any one of Bacillus coagulans bacillus AKC003 strain, AKC004 strain (FERM_ABP10948) (Taiwan registration number: BCRC 910388), AKC005 strain, and AKC006 strain. 4. A Bacillus coagulans and a variant thereof, the Bacillus coagulans belonging to any one of Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948) (Taiwan deposit number: BCRC 910388), AKC005 strain, and AKC006 strain. An alpha-galactosidase comprising the amino acid sequence of any one of the following (a), (b) or (c): (a) an amino acid sequence represented by SEQ ID NO: 2; An amino acid sequence having one or several amino acids deleted, substituted and/or added, and having α-galactosidase activity in the amino acid sequence represented by SEQ ID NO: 2; 129208.doc 200846472 (c) The amino acid sequence having a galactose activity of 6 〇% or more with the amino acid sequence represented by SEQ ID NO: 2 is not more than 6 〇. An α-galactosidase gene encoding a "galactosidase encoding an amino acid sequence of any one of the following (4), (8) or (4): a) an amino acid represented by Khan 2 The sequence; the amino acid sequence represented by W SEQ ID NO: 2, the deletion or substitution and/or addition of one or several amino acids, and the ❿ amino acid sequence; + saccharide _ active (c) and sequence The amino acid sequence of No. 2 has an amino acid sequence of 60% or more and has α-galactosidase activity. A galactosidase gene, and _ eight packets of 3 or less (a) or (b) base sequence: (4) sequence, the sequence of the sequence represented by the flat #ul; W sequence code t to indicate the deletion, replacement A medium or a few base substitutions and/or additions, and encoding a base sequence of a protein or a protein. , a caro-enzyme activity of 8 kinds of recombinant vectors, the content of which. Such as "Item 6 or ~ galactosyl group 9. - morphine" which is introduced with the recombinant vector as requested or as claimed in item 8. The galactosidase gene 10'-species (X-galactosidase' Request U. An enzyme composition comprising α-galactosidase as claimed in claim 15 #. Any of 3, 5 or 1 12 12. The enzyme composition of claim U, the enzyme , β-glucosidase, galactosidase, glucosinolate, xylan 129208.doc 200846472 enzyme, protease, galactanase, arabinase, mannanase, rhamnogalacturonan a component of at least one of an enzyme, a polygalacturonase, a pectin methylesterase, a pectin dissociation enzyme, and a polygalacturonan dissociating enzyme. 13. A raffinose synthesis reagent, which contains the request The enzyme composition of any one of clauses or 12. A method for producing raffinose, which is characterized in that it uses a heart galactosidase according to any one of claims 1 to 3, 5, or 1 , an enzyme composition as claimed in claim 1} or 12, or The raffinose synthesis reagent of claim 13. 15. A method for producing raffinose, which is characterized in that it is a microbial catalyst obtained by culturing a microorganism belonging to Bacillus coagulans. A method for producing cotton scorpion scorpion, characterized in that it is cultured and belongs to Bacillus anaterium AKC003 strain, AKC004 strain (FERM-ABP1〇948) (Taiwan registration number: BCRC 91〇388), and akc〇〇5 strain A microbial catalyst obtained by Bacillus coagulans and/or a variant thereof of any of the AKC006 strains. A method for producing a raffinose, which is characterized in that it is a microbial catalyst obtained by cultivating the transformant of claim 9. The method for producing raffinose according to any one of claims 14 to 17, wherein the raffinose content in the production of the sugar is 65 % or more. The method for producing raffinose according to any one of claims 14 to 18, which uses sucrose and galactose as raw materials. The manufacturing method of the raffinose according to the item 19, wherein 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)~45% (w/v) 〇129208.doc