CN101657544A - 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

Novel a-galactosidase

Technical field

The present invention relates to novel a-galactosidase and use this enzyme to make the method for raffinose.

Background technology

In recent years, along with dietetic life, the diversified development of social life, for the raising of health perception, the human consumer also increases to the degree of attentiveness of food, foodstuffs material etc.Wherein, find that raffinose has functions such as the intestinal flora of improvement, therefore receive much attention as food, medicine, spices etc. or its raw material.Recently, found that raffinose has immunity effect effect, useful to atopic dermatitis.

Now, the by-product recovery when raffinose is conduct manufacturing beet sugar, and the raffinose content in the beet only is about 0.1%, the turnout of raffinose is relevant with the turnout of principal product granulated sugar, so the volume increase of raffinose is limited.

Therefore, in order stably to have the raffinose of such useful property to the market supply with low cost, not only need be from natural extraction product, also need be from the synthetics of low-cost raw material.

Synthetic method as the raffinose of attempting so far, can enumerate the method for the katalysis that utilizes alpha-galactosidase, and having reported with sucrose to be galactosylated acceptor, is the method (for example referring to patent documentation 1, non-patent literature 1, non-patent literature 2) that galactose donor obtains raffinose with the melibiose.Reported in addition and utilized p-nitrophenyl-α-D-galactopyranoside (for example as the method for galactose donor, referring to non-patent literature 3), utilize galactinol (for example as the method for galactose donor, referring to patent documentation 2), utilize galactobiose as method (for example, referring to patent documentation 3) of galactose donor etc.

In addition, reported that also some can utilize cheaply sucrose and semi-lactosi as the example of raw material.For example, disclose the method (for example referring to non-patent literature 4) of using the alpha-galactosidase that derives from bright red samguineus (Pycnoporus cinnabarinus) to obtain raffinose, be used to come from the method (for example referring to non-patent literature 5) of the alpha galactosides enzymic synthesis raffinose of Portugal's wine and women-sensual pursuits mortierella (Mortierella vinacea).

Patent documentation 1: No. the 2688854th, Japan's special permission

Patent documentation 2: Japanese kokai publication hei 10-84973

Patent documentation 3: the special fair 8-24592 of Japan

Non-patent literature 1:Agric.Biol.Chem., 52 (9), 2305-2311,1988

Non-patent literature 2:Biosci.Biotech.Biochem., 59 (4), 619-623,1995

Non-patent literature 3:Phytochemistry, 18,35-38,1979

Non-patent literature 4:Nippon Shokuhin Kogyo Gakkaishi, 38 (8), 722-728,1991

Non-patent literature 5:Carbohydr.Res., 185,139-146,1989

Summary of the invention

Owing to utilize alpha-galactosidase to generate the efficient height of raffinose, so reported and utilized melibiose, p-nitrophenyl-α-D-galactopyranoside, galactinol, galactobiose example as the raffinose synthesis material, but adopt under the situation of these methods, the price height of employed raw material is difficult at industrial manufacturing raffinose.

On the other hand, owing to can utilize low-cost raw material sucrose and semi-lactosi, so use the alpha-galactosidase that derives from bright red samguineus to obtain the method for raffinose or the method that is used to come from the alpha galactosides enzymic synthesis raffinose of Portugal's wine and women-sensual pursuits mortierella is a very favorable method when considering industrialness.But, utilize under the situation of these alpha-galactosidases, in resultant, generated purpose raffinose assorted trisaccharide in addition, thus need from the finished product, separate the impurity removal trisaccharide, and produced the problem of wastage of material.The alpha-galactosidase that derives from bright red samguineus is used under the situation of raffinose building-up reactions, the raffinose containing ratio in the oligose of generation is low to moderate 40%; The alpha-galactosidase that derives from Portugal's wine and women-sensual pursuits mortierella is used under the situation of raffinose building-up reactions, and the raffinose containing ratio in the oligose of generation is low to moderate 60%, and prior art can not optionally be made raffinose by enzyme process.

In view of the situation, the object of the present invention is to provide a kind of new alpha-galactosidase, it can use the optionally synthetic raffinose of low cost raw material, and the present invention also provides the innovative manufacture method of the raffinose that uses this enzyme.

In order to solve these problems, the present inventor furthers investigate repeatedly, found that a kind of can selectivity the new enzyme of synthetic raffinose, and found to suppress the raffinose manufacture method of the generation of the impurity oligose beyond the purpose raffinose, thereby finished the present invention.

That is, the present invention relates to the novel a-galactosidase shown in following [1]～[20] and the manufacture method of raffinose etc.

[1] a kind of alpha-galactosidase has following characteristic:

(1) effect: described enzyme has following character: be in the dehydration condensation of raw material with sucrose and semi-lactosi, the raffinose containing ratio in the oligose of generation is 0.5% when above, and the synthetic selection rate of raffinose is more than 65%;

(2) optimal pH scope: 3.5～5.0;

(3) stable pH range: 3.5～10.0;

(4) molecular weight: about 80,000.

[2] as [1] described alpha-galactosidase, it derives from the microorganism that belongs to Bacillus coagulans (Bacilluscoagulans).

[3] as [2] described alpha-galactosidase, wherein, described Bacillus coagulans is any one in Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP 10948), AKC005 strain, the AKC006 strain.

[4] a kind of Bacillus coagulans and varient thereof, described Bacillus coagulans belong in Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, the AKC006 strain any one.

[5] a kind of alpha-galactosidase, its contain following (a) and (b) or (c) in aminoacid sequence arbitrarily:

(a) aminoacid sequence of sequence numbering 2 expressions;

(b) disappearance in the aminoacid sequence of sequence numbering 2 expression, replace and/or added the aminoacid sequence of 1 or several amino acid, it has alpha-galactosidase activity;

(c) have the aminoacid sequence of the homogeny more than 60% with the aminoacid sequence of sequence numbering 2 expression, it has alpha-galactosidase activity.

[6] a kind of alpha-galactosidase gene, its coding contain following (a) and (b) or (c) in the alpha-galactosidase of aminoacid sequence arbitrarily;

(a) aminoacid sequence of sequence numbering 2 expressions;

(b) disappearance in the aminoacid sequence of sequence numbering 2 expression, replace and/or added the aminoacid sequence of 1 or several amino acid, it has alpha-galactosidase activity;

(c) have the aminoacid sequence of the homogeny more than 60% with the aminoacid sequence of sequence numbering 2 expression, it has alpha-galactosidase activity.

[7] a kind of alpha-galactosidase gene, it contains following (a) or base sequence (b):

(a) base sequence of sequence numbering 1 expression;

(b) disappearance in the base sequence of sequence numbering 1 expression, replace and/or added the base sequence of 1 or several bases, and its coding has the protein of alpha-galactosidase activity.

[8] a kind of recombinant vectors, it contains [6] or [7] described alpha-galactosidase gene.

[9] a kind of transformant, its importing have [6] or [7] described alpha-galactosidase gene or [8] described recombinant vectors.

[10] a kind of alpha-galactosidase, it is cultivated [9] described transformant and obtains.

[11] a kind of enzyme composition, it contains each described alpha-galactosidase of [1]～[3], [5] or [10].

[12] as [11] described enzyme composition, wherein, said composition also contains at least a above composition that is selected from alpha-glucosidase, beta-glucosidase enzyme, beta-galactosidase enzymes, cellulase, zytase, proteolytic enzyme, Galactanase, arabanase (arabinanase), mannase, rhamno-galacturonic acid enzyme, polygalacturonase, pectin methylesterase, pectin lyase and polygalacturonic acid lyase.

[13] a kind of raffinose synthetic agent, it contains [11] or [12] each described enzyme composition.

[14] a kind of manufacture method of raffinose is characterized in that, this manufacture method is used each described alpha-galactosidase of [1]～[3], [5] or [10]; [11] or [12] described enzyme composition; Perhaps [13] described raffinose synthetic agent.

[15] a kind of manufacture method of raffinose is characterized in that, this method is utilized microbial catalyst, and described microbial catalyst is cultivated the microorganism that belongs to Bacillus coagulans and obtained.

[16] a kind of manufacture method of raffinose, it is characterized in that, this method is utilized microbial catalyst, and described microbial catalyst is cultivated and belonged in Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, the AKC006 strain any one Bacillus coagulans and/or its varient and obtain.

[17] a kind of manufacture method of raffinose is characterized in that, this method is utilized microbial catalyst, and described microbial catalyst is cultivated [9] described transformant and obtained.

[18] as the manufacture method of each described raffinose in [14]～[17], wherein, the raffinose containing ratio in the oligose of generation is more than 65%.

[19] as the manufacture method of each described raffinose in [14]～[18], wherein, this method uses sucrose and semi-lactosi as raw material.

[20] as the manufacture method of [19] described raffinose, wherein, the sucrose concentration in the raw material is 30% (w/v)～90% (w/v), and the galactose concentration in the raw material is 2% (w/v)～45% (w/v).

By adopting the present invention, can optionally make raffinose.

Description of drawings

Fig. 1 has provided the optimal pH scope (embodiment 2) of the alpha-galactosidase of being purified by Bacillus coagulans AKC-004 thalline.

Fig. 2 has provided the stable pH range (embodiment 2) of the alpha-galactosidase of being purified by Bacillus coagulans AKC-004 thalline.

Fig. 3 has provided the optimum temperature range (embodiment 2) of the alpha-galactosidase of being purified by Bacillus coagulans AKC-004 thalline.

Fig. 4 has provided the equilibrium temperature scope (embodiment 2) of the alpha-galactosidase of being purified by Bacillus coagulans AKC-004 thalline.

Fig. 5 has provided the result of the SDS-PAGE (embodiment 2) of the alpha-galactosidase of being purified by Bacillus coagulans AKC-004 thalline.

The reaction solution that Fig. 6 has provided the sugar synthetic (embodiment 3) that uses the alpha-galactosidase of being purified by Bacillus coagulans AKC-004 strain carries out the result that HPLC analyzes.

The reaction solution that Fig. 7 has provided the sugar synthetic (embodiment 5) that uses the alpha-galactosidase that is obtained by recombination bacillus coli JM109 strain carries out the result that HPLC analyzes.

The reaction solution that Fig. 8 has provided the sugar synthetic (embodiment 3) that uses the microbial catalyst of cultivating Bacillus coagulans AKC-004 strain and obtaining carries out the result that HPLC analyzes.

Embodiment

Below the present invention is specifically described.

Alpha-galactosidase of the present invention derives from the microorganism that belongs to Bacillus coagulans, as this microorganism, can use any microorganism in the microorganism that belongs to Bacillus coagulans, and can use expression can selectivity any microorganism of alpha-galactosidase of synthetic raffinose.Can preferably enumerate Bacillus coagulans AKC-003 strain, AKC-004 strain, AKC-005 strain, AKC-006 strain.In addition, the microorganism among the present invention can also be to be the variant that maternal plant obtains with the microorganism that belongs to Bacillus coagulans.Bacillus coagulans AKC-003 strain, AKC-004 strain, AKC-005 strain, AKC-006 strain all are deposited in Independent Administrative Leged Industrial Technology Complex Inst (putting down into 18 years) November 14 in 2006 and specially permit biological preservation center (a kind of ground of one fourth order, east, ripple city, 1 central authorities the 6th are built in the Hitachinaka County, Japan).Deposit number is as follows.Need to prove that the AKC-004 strain hands over to international preservation (putting down into 20 years) January 30 in 2008 to accept numbering 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)

Alpha-galactosidase of the present invention has following characteristic.

(1) effect: described enzyme has following character: be in the dehydration condensation of raw material with sucrose and semi-lactosi, the raffinose containing ratio in the oligose of generation is 0.5% when above, and the synthetic selection rate of raffinose is more than 65%;

(2) optimal pH scope: 3.5～5.0;

(3) stable pH range: 3.5～10.0;

(4) molecular weight: about 80,000.

As the concrete example of enzyme of the present invention, can enumerate at the enzyme that has following character aspect the influence of substrate specificity and metal ion.

Substrate specificity: under the p-nitrophenyl-α-situation of D-galactopyranoside as substrate, degrading activity is the highest, is followed successively by melibiose, raffinose thereafter.On the other hand, p-nitrophenyl-β-D-galactopyranoside, lactose, sucrose do not decompose.

The influence of metal ion: add under potassium ion, calcium ion, magnesium ion, chromium ion, mn ion, cobalt ion, iron (II) ion or each situation of iron (III) ionic, do not see active reduction.On the other hand, add under each situation of nickel ion or zine ion, visible active reduction is added under the situation of cupric ion, the active maximum that reduces.

In addition, as the concrete example of enzyme of the present invention, can also enumerate enzyme with following characteristic.

(5) (positive reaction) optimum temperature range: 35～50 ℃;

(6) equilibrium temperature scope: stable below 45 ℃.

As being used for method for culturing microbes of the present invention, adopting conventional aeration-agitation cultivation or solid culture, and can adopt the microbial culture method that carries out usually.As substratum, can enumerate synthetic medium or natural medium, wherein contain and make the fertility of this microorganism well and swimmingly generate the necessary carbon source of alpha-galactosidase in the microorganism, nitrogenous source, inorganic salt, necessary nutrition source etc.For example, as carbon source, can use glucose, glycerine, sucrose, semi-lactosi, lactose, melibiose, raffinose, stachyose, cellobiose, erlose, organic acid, starch, sweet oil, soybean wet goods.As nitrogenous source, for example can enumerate sulphur ammonium, ammonium nitrate, urea, amino acid, amine, ammonia, various mineral acid or organic acid ammonium salt, other nitrogenous compounds, peptone, tryptone, poly-peptone, meat soup, yeast extract, cottonseed meal, corn steep liquor and soybean meal etc.In addition, as inorganic salts, can enumerate potassium primary phosphate, dipotassium hydrogen phosphate, trimagnesium phosphate, sal epsom, sodium-chlor, manganous sulfate, copper sulfate, ferric sulfate, lime carbonate etc.Consider that from the aspect of the fertility of microorganism culture temperature is preferably 25～80 ℃, more preferably 40～65 ℃, more preferably 40～55 ℃.In addition, the pH of substratum can select at relative broad range, but considers from the aspect of the fertility of microorganism, preferred pH3.0～9.0, more preferably pH3.5～8.5, further preferred pH4.0～8.0.

The separation of alpha-galactosidase of the present invention, purification for example can followingly be implemented.

Culture medium culturing Bacillus coagulans AKC-004 strain with above-mentioned is separated into thalline and filtrate by known method such as centrifugation, filtrations with resulting nutrient solution.

Contain alpha-galactosidase in the thalline that so obtains, carry out bacterial cell disruption, obtain the crude extract of alpha-galactosidase by using N,O-Diacetylmuramidase, ultrasonic disruption machine, French press etc.According to culture condition etc., sometimes the activity that in culture supernatant, contains alpha-galactosidase, in this case, culture supernatant directly can be used for following purification as the crude extract of alpha-galactosidase, also can be to operations such as culture supernatant concentrate, the crude extract as alpha-galactosidase is used for following purification then.

The crude extract that so obtains is separated the alpha-galactosidase of can purifying thus by conventional protein method of purification (for example ion exchange chromatography, hydrophobicity chromatography, gel filtration chromatography, hydroxylapatite chromatography method, the combination of saltouing etc.).

Regardless of the order of aforesaid operations, each operation all can be carried out more than 1 time or 2 times.In addition, preferably before with each post wash-out sample, make test liquid and suitable buffer-exchanged by dialysis etc.In addition, can also concentrate test liquid in each stage.

In preferred each stage of purifying, the alpha-galactosidase activity that is contained in isolating each fraction is measured, the fraction that activity is high puts together, and uses for next stage.

For the method for measuring alpha-galactosidase activity, for example containing 450 μ L sodium acetate buffer (100mM of 6mM p-nitrophenyl-α-D-galactopyranoside, pH5.0) mix 150 μ L enzyme liquid in, after 40 ℃ of reactions of carrying out about 5～30 minutes, reaction solution is added in the 1mL aqueous sodium carbonate (1M), make enzyme deactivation, stopped reaction.Measure of the absorption (degree of staining of resulting solution) of resulting solution, use the working curve made from the p-NP of each concentration to calculate concentration at wavelength 420nm.In addition, the unit of enzymic activity is as follows: the enzyme amount with the 1 μ mol p-NP that dissociated in 1 minute under these conditions is 1U.

The affirmation of the purification degree of the alpha-galactosidase through purifying and the mensuration of molecular weight can be undertaken by electrophoresis or gel filtration chromatography etc.In addition, zymologic property can followingly be studied: change temperature of reaction or reaction pH, measure enzymic activity; Perhaps various enzyme inhibitorss or metal ion etc. are added in the reaction solution, measure remaining activity.In addition, by alpha-galactosidase is measured enzymic activity again after placing certain hour under the various pH conditions or under the temperature condition, can investigate stable pH range and equilibrium temperature scope thus.In addition, react, can obtain the Michaelis-Menton constant (K of alpha-galactosidase each substrate by changing concentration of substrate _m), top speed (V _Max).

Alpha-galactosidase gene of the present invention can obtain by for example following method.

Culture medium culturing Bacillus coagulans AKC-004 strain with above-mentioned is separated into thalline and filtrate by known method such as centrifugation, filtrations with resulting nutrient solution.Use N,O-Diacetylmuramidase, ultrasonic disruption machine, French press etc. that the thalline that so obtains is carried out bacterial cell disruption, separate chromosomal DNA.With various restriction enzymes the chromosomal DNA that so obtains is digested, obtain the DNA fragment.Use the chromosomal DNA fragment that so obtains, contain the DNA fragment of the total length or the part of alpha-galactosidase gene by utilizing known means acquisition such as shotgun cloning method, inverse PCR method.The base sequence of the DNA fragment that contains alpha-galactosidase gene that herein obtains can be resolved by the known method that utilizes dna sequencing instrument etc., and clear and definite its base sequence.In addition, only know under the situation of part base sequence of alpha-galactosidase gene, can obtain to contain the DNA fragment of alpha-galactosidase gene once more based on this base sequence.And,, can separate the base sequence of bright alpha-galactosidase gene total length by repeating this operation.Can be based on the base sequence of the alpha-galactosidase gene as above understood, the known method by PCR method or use restriction enzyme obtains containing the DNA fragment of the total length of alpha-galactosidase gene.So, according to the base sequence of the alpha-galactosidase gene of being understood, can determine the aminoacid sequence of alpha-galactosidase.

Sequence numbering 2 has been enumerated the aminoacid sequence of alpha-galactosidase of the present invention, as long as but the protein that contains this aminoacid sequence has alpha-galactosidase activity, this aminoacid sequence can have the variation of disappearance, replacement, at least 1 amino acid of interpolation etc., perhaps can be the aminoacid sequence that has the homogeny more than 60% with this aminoacid sequence.That is, alpha-galactosidase of the present invention be contain following (a) and (b) or (c) in the alpha-galactosidase of aminoacid sequence arbitrarily.

(a) aminoacid sequence of sequence numbering 2 expressions;

(b) disappearance in the aminoacid sequence of sequence numbering 2 expression, replace and/or added the aminoacid sequence of 1 or several amino acid, it has alpha-galactosidase activity;

(c) have the aminoacid sequence of the homogeny more than 60% with the aminoacid sequence of sequence numbering 2 expression, it has alpha-galactosidase activity.

In addition, according to the present invention, provide a kind of coding contain following (a) and (b) or (c) in the alpha-galactosidase gene of the alpha-galactosidase of aminoacid sequence arbitrarily.

(a) aminoacid sequence of sequence numbering 2 expressions;

(b) disappearance in the aminoacid sequence of sequence numbering 2 expression, replace and/or added the aminoacid sequence of 1 or several amino acid, it has alpha-galactosidase activity;

(c) have the aminoacid sequence of the homogeny more than 60% with the aminoacid sequence of sequence numbering 2 expression, it has alpha-galactosidase activity.

" 1 or several amino acid " in above-mentioned (b) typically refers to 1～50 amino acid, is preferably 1～30 amino acid, 1～20 amino acid more preferably, and more preferably 1～10 amino acid is preferably 1～5 amino acid especially.

" aminoacid sequence with the homogeny more than 60% " in above-mentioned (c) typically refers to and has more than 60% the aminoacid sequence of the homogeny of (preferred more than 70%, more preferably more than 80%, more preferably more than 90%, more more preferably more than 95%, be preferably more than 99% especially).

As the concrete example of alpha-galactosidase gene of the present invention, can enumerate the alpha-galactosidase gene that contains following (a) or base sequence (b).

(a) base sequence of sequence numbering 1 expression;

(b) disappearance in the base sequence of sequence numbering 1 expression, replace and/or added the base sequence of 1 or several bases, and its coding has the protein of alpha-galactosidase activity.

" 1 or several base " in above-mentioned (b) typically refers to 1～150 base, preferred 1～90 base, 1～60 base more preferably, 1～30 base more preferably, 1～20 base more preferably, 1～15 base more preferably, more preferably 1～10 base is preferably 1～5 base especially.

Alpha-galactosidase gene of the present invention can not damage the variation of peptide of the character of catalyzed reaction originally by known genetic manipulation method, such mutant gene is meant artificial variation's gene that the method by the gene engineering is made by alpha-galactosidase gene of the present invention, and this artificial variation's gene can be by using the special alternative method in position, obtaining with the range gene engineering methods such as method of the specific DNA fragment of artificial abnormal dna replacement goal gene.That is, the method for variations such as amino acid in the alpha-galactosidase lacks, replaces as making, interpolation can be utilized PCR method, error-prone PCR method, DNA reorganization method, be made the known method such as method that merge enzyme.

The DNA fragment that contains complete alpha-galactosidase structure gene that so obtains can be inserted multiple clone site and the connection of intestinal bacteria, constitute new recombinant plasmid with expression vector (for example pBluescriptII KS (+)).For this plasmid vector, owing to importing the lac promotor that the gene effective expression that can be in intestinal bacteria will connect as alien gene is arranged, so in intestinal bacteria, import the resulting transformant of recombinant plasmid by cultivating, can the great expression alpha-galactosidase.

In addition, except that above-mentioned, can also utilize various host microorganisms, carrier to come the great expression alpha-galactosidase, for example by cultivating the transformant of bridge stone bacillus pumilus (Brevibacilluschoshinensis), can the great expression alpha-galactosidase.In more detail, as the carrier that imports alpha-galactosidase gene of the present invention, what be fit to has by the phage of the breeding of can restraining oneself in the host microorganism body or the carrier that is used for gene recombination that plasmid is made, as phage vector, for example, can use λ gt λ C, λ gt λ B etc. to belong under the situation of colibacillary microorganism as host microorganism.In addition, as plasmid vector, for example, can use pET carriers (Novagen) such as plasmid pET-3a, pET-11a, pET-32a or pBR322, pBR325, pACYC184, pUC12, pUC13, pUC18, pUC19, pUC118, pIN I, BluescriptKS+ with under the situation of intestinal bacteria as host microorganism; Under the situation of withered grass bacterium, can use pWH1520, pUB110, pKH300PLK as the host; With actinomycetes is under host's the situation, can use pIJ680, pIJ702; Particularly under the situation of yeast saccharomyces cerevisiae as the host, can use YRp7, pYC1, YEp13 etc. with yeast.Such carrier is cut off with restriction enzyme, the end that makes generation is identical with the DNA end that generates with the restriction enzyme that is used to cut off alpha-galactosidase gene of the present invention, thereby make the carrier fragment, utilize dna ligase to make alpha-galactosidase gene fragment of the present invention and the combination of carrier fragment, the DNA of coding alpha-galactosidase gene of the present invention can be imported the purpose carrier thus according to ordinary method.

As the host microorganism that imports plasmid, the ground propagation as long as recombinant DNA can be stablized and restrain oneself, for example host microorganism is to belong under the situation of colibacillary microorganism, can utilize e. coli bl21, e. coli bl21 (DE3), e. coli bl21 trxB, intestinal bacteria Rosetta (DE3), intestinal bacteria Rosetta, intestinal bacteria Rosetta (DE3) pLysS, intestinal bacteria Rosetta (DE3) pLacl, intestinal bacteria RosettaBlue, intestinal bacteria Rosetta-gami, intestinal bacteria Origami, intestinal bacteria Origami, intestinal bacteria Tuner, intestinal bacteria DH1, e. coli jm109, intestinal bacteria W3110, intestinal bacteria C600 etc.In addition, microorganism host is to belong under the situation of microorganism of Bacillus, can utilize Bacillus subtillis, Bacillus megatherium etc.; Microorganism host is to belong under the situation of actinomycetic microorganism, can utilize lead-changing penicillium chain enzyme bacteria TK24 etc.; Microorganism host is to belong under the situation of microorganism of yeast saccharomyces cerevisiae, can utilize yeast saccharomyces cerevisiae INVSCl etc.

In addition, when making alpha-galactosidase of the present invention by transforming microorganism, cultivate this conversion microorganism with nutritional medium, produce alpha-galactosidase of the present invention in thalline or in the nutrient solution, after cultivating end, gather thalline by methods such as filtration or centrifugations by resulting culture, next, destroy this thalline with the method for enzymes such as mechanical means or N,O-Diacetylmuramidase, perhaps add EDTA and/or suitable tensio-active agent etc. as required, concentrate the aqueous solution of alpha-galactosidase of the present invention or do not concentrate and directly pass through the classification of sulphur ammonium, gel-filtration, adsorption chromatographies such as affinity chromatography, ion exchange chromatography is handled, and can obtain the good alpha-galactosidase of the present invention of purity thus.

About transforming the culture condition of microorganism, consider that its nutritional-physiological character get final product when selecting culture condition, cultivate by liquid culture usually, it is industrial that to carry out deep aeration-agitation cultivation be favourable.As the nutrition source of substratum, can use normally used nutrition source in the microorganism culturing widely.Culture temperature can suitably change in the scope that microorganism can breed and can produce alpha-galactosidase of the present invention, is example with intestinal bacteria, and culture temperature is preferably about 10～45 ℃, more preferably about 20～30 ℃.According to circumstances, culture condition is more or less different, but only need expect that alpha-galactosidase of the present invention reaches the period of maximum output, finish to cultivate in suitable period to get final product, and with intestinal bacteria example, incubation time is about 12～48 hours usually.Medium pH can suitably change in the scope that bacterium can breed and can produce alpha-galactosidase of the present invention, is example with intestinal bacteria, and medium pH is preferably about pH6～8.

According to the present invention, when using described enzyme, only otherwise suppress the effect of enzyme of the present invention, extent of purification etc. is not particularly limited, except using enzyme of the present invention, also can use the mixture that contains this enzyme through purifying.

According to the present invention, provide the enzyme composition that contains above-mentioned alpha-galactosidase of the present invention.Above-mentioned enzyme composition can further contain at least a above composition that for example is selected from alpha-glucosidase, beta-glucosidase enzyme, beta-galactosidase enzymes, cellulase, zytase, proteolytic enzyme, Galactanase, arabanase, mannase, rhamno-galacturonic acid enzyme, polygalacturonase, pectin methylesterase, pectin lyase and polygalacturonic acid lyase.

According to the present invention, provide the raffinose that contains above-mentioned enzyme composition synthetic agent.

Among the present invention,, except Bacillus coagulans AKC-004 strain, can also use above-mentioned transformant as microbial catalyst.Among the present invention, as the microbial catalyst that is used to make raffinose, the microorganism itself that can utilize the cultural method that undertaken by routine to obtain, and need not from microorganism purification alpha-galactosidase.In addition, can utilize microbial culture medium, microorganism culturing supernatant liquor sometimes.On the other hand, also can after cleaning the microorganism that obtains by culture method, water or damping fluid etc. be used again as required.For example, can use nutrient solution through cultured microorganism; Or clean etc. the microbial suspension that obtains by centrifugation, with damping fluid; Suspend or be dissolved with the aqueous solution of the handled thing (for example the broken thing of microorganism etc.) of microorganism or microorganism; Fixed microorganism or microbiological treatment thing with entrapping method, crosslinking or carrier combined techniques.The example of the fixation support when carrying out immobilization can be enumerated granulated glass sphere, silica gel, urethane, polyacrylamide, polyvinyl alcohol, carrageenin, alginic acid etc., but be not limited to these.

According to the present invention, a kind of manufacture method of raffinose is provided, this method is used above-mentioned alpha-galactosidase of the present invention, the enzyme composition that contains alpha-galactosidase, raffinose synthetic agent or microbial catalyst.As raw material, for example can use sucrose and semi-lactosi.When utilizing sucrose and semi-lactosi as raw material, for example, from utilizing alpha-galactosidase to carry out the properties of dehydration condensation, preferred feedstock concentration height, but when galactose concentration was too high, the intermolecular condensation of semi-lactosi had suppressed the dehydration condensation between sucrose and the semi-lactosi, and sucrose concentration preferably sets at 30% (w/v)～90% (w/v), more preferably 35% (w/v)～80% (w/v), more preferably 40% (w/v)～70% (w/v).Galactose concentration is preferably set to 2% (w/v)～45% (w/v), more preferably 5% (w/v)～35% (w/v), more preferably 7% (w/v)～30% (w/v).In addition, preferred preparation becomes sucrose and all soluble concentration of semi-lactosi in the raw material.

Use alpha-galactosidase of the present invention, contain the enzyme composition of alpha-galactosidase, when the raffinose synthetic agent that contains enzyme composition or microbial catalyst are made raffinose, consider from the aspect of the stability of speed of response, enzyme, temperature of reaction is preferably 10～90 ℃, more preferably 20～70 ℃, more preferably 30～60 ℃.Reaction pH can adjust at relative broad range, considers preferred pH2.0～10.0, more preferably pH3.0～7.5, more preferably 3.5～6.0 from the aspect of the stability of enzyme.Reaction times is according to the consumption of enzyme and different, and when considering industrial the utilization, being preferably usually is 20 minutes～200 hours, more preferably 6 hours～80 hours.But the present invention is not limited to above reaction conditions and reaction form, can suitably select.

Among the present invention, using alpha-galactosidase, containing the enzyme composition of alpha-galactosidase or contain in the raffinose building-up reactions of raffinose synthetic agent of enzyme composition, raffinose accumulates 0.5% when above in reaction soln, raffinose containing ratio in the oligose that generates can be brought up to more than 65%, under preferred condition, can bring up to more than 80%.The accumulation raffinose reaches more than 0.75% and then 1.0% when above in reaction soln, the high containing ratio of raffinose in the oligose that can realize generating among the present invention.

In addition, use in the raffinose building-up reactions of microorganism itself, owing to the influence of assorted enzyme, the raffinose containing ratio in the oligose of generation is low usually, and use in the raffinose building-up reactions of microbial catalyst of the present invention raffinose containing ratio height in the oligose of generation.

Raffinose containing ratio in the oligose of the generation that the present invention obtains can be measured by following method.

After the raffinose building-up reactions finishes,, kept 10 minutes at 99 ℃, thus stopped reaction 25 times of reaction solution dilutions.After reaction stops, removing microorganism by centrifugation, (HPLC) carries out quantitatively resulting reaction soln with high speed liquid chromatography.Measure the Hypercarb post that uses Thermoelectron society to make, detector uses RI.The area at raffinose containing ratio basis detected each peak in the HPLC analysis of spectra in the oligose that generates is than calculating by (peak area of raffinose)/(peak area of the oligose of generation) * 100.

As to purify by the raffinose of method manufacturing of the present invention, isolating method, can utilize the purification processing method of common employing.That is, for example can remove microbial catalyst by centrifugation, the film processing that utilizes MF (micro-filtration) film or UF (ultrafiltration) film etc., pressure filter etc.; Handle or desalting treatment such as dialysis is removed the salt brought into by damping fluid, substratum etc. etc. by cation-exchange chromatography, anion-exchange chromatography isochromatic spectrum; And by cation-exchange chromatography, anion-exchange chromatography, high speed liquid chromatography, activated carbon chromatography isochromatic spectrum handle or utilize the crystallization of poor solubility etc. to handle, other ordinary methods carry out separation, the purification of alpha-galactooligosaccharide.Chromatography can be used these methods separately, also can be used in combination these methods, and can suitably utilize moving-bed mode, simulation moving-bed mode, multicomponent separation simulation moving-bed mode, multicomponent partitioning cycle mode etc.The purification of these raffinoses, method for separating and processing can batch-type carry out, and also can utilize post etc. to carry out continuously.

Specifically describe below by embodiment, but the present invention is not subjected to any restriction of these embodiment.

Embodiment

Embodiment 1

With Bacillus coagulans AKC-004 strain (deposit number FERM P-21092 (transfer and be FERM-ABP10948); Preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological preservation center) cultivated 1 day at 55 ℃ with TBAB (Tryptones blood agar basis, Tryptose Blood Agar Base) culture plate (Difco), form bacterium colony.

30mL is adjusted to the substratum shown in the table 1 of pH7.2 and pack in the Erlenmeyer flask of 150mL, from above-mentioned culture plate inoculation bacterium colony, carry out 25 hours rotational oscillations and cultivate, cultivate seed as jar with this at 50 ℃, 180rpm with transfering loop.

Then, 6L is adjusted to the fermentor tank that the substratum shown in the table 1 of pH7.2 is put into 10L, transplant the above-mentioned jar of 15mL and cultivate seed, cultivated 48 hours with 50 ℃, the condition aeration-agitation of 200rpm, 0.2vvm.

Then, this nutrient solution is carried out 10 at 4 ℃, supernatant liquor is removed in the centrifugation of 000g * 30 minute, reclaims thalline.Measure the activity of the alpha-galactosidase of resulting thalline, this activity is 261U.

This thalline is suspended with the Lysis damping fluid (pH8.0) that contains 50mM Tris-HCl, 20mM EDTA, 50mM glucose, and quantitatively arrive 150mL.This suspension is carried out 10 at 4 ℃, and supernatant liquor is removed in the centrifugation of 000g * 30 minute, reclaims thalline.

To be suspended in once more in the above-mentioned Lysis damping fluid with the thalline after the Lysis buffer solution for cleaning, quantitatively be 150mL.Add 0.02% N,O-Diacetylmuramidase (Sigma society makes, from albumen) therein,, carry out bacterial cell disruption with 37 ℃, the condition vibration of 120rpm 13 hours.

Solution behind the bacterial cell disruption is carried out 10 at 4 ℃, and the thalline residue is removed in the centrifugation of 000g * 30 minute, reclaims supernatant liquor.

In this supernatant liquor, add ammonium sulfate, and reach 37.5% saturated, place a night, generate precipitation at 4 ℃.Carry out 10 at 4 ℃, this precipitation is removed in the centrifugation of 000g * 30 minute, reclaims supernatant liquor.

In this supernatant liquor, add ammonium sulfate, and reach 54.5% saturated, place a night, generate precipitation at 4 ℃.Carry out 10 at 4 ℃, the centrifugation of 000g * 30 minute is reclaimed this precipitation, and it is dissolved in the 20mM phosphoric acid buffer of pH7.0, at 4 ℃ phosphoric acid buffer same as described above is carried out night dialysis.

After the supernatant that obtains by dialysis being adsorbed on go up with " DEAE Sepharose FF " after the phosphoric acid buffer equilibration same as described above (GE Healthcare Bio-Sciences Co., Ltd.), the E-test of the 20mM phosphoric acid buffer of the pH7.0 by containing 0～0.4M sodium-chlor comes out the enzyme wash-out.

Collect the active fraction that above-mentioned wash-out goes out, use average mark level molecular weight 10,000 ultra-filtration membrane concentrates, it is adsorbed on after " HiTrap Phenyl FF (high sub) " (the GE Healthcare Bio-Sciences Co., Ltd.) after the 20mM phosphoric acid buffer equilibration of pH7.0 that contains 2M sodium-chlor, and the E-test of the 20mM phosphoric acid buffer of the pH7.0 by containing 2.0～0M sodium-chlor comes out the enzyme wash-out.

Collect the active fraction that above-mentioned wash-out goes out, use average mark level molecular weight 10,000 ultra-filtration membrane concentrates, it is adsorbed on after " MonoQ5/50 GL " (the GE Healthcare Bio-Sciences Co., Ltd.) after the 20mM phosphoric acid buffer equilibration of pH7.0, and the E-test of the 20mM phosphoric acid buffer of the pH7.0 by containing 0～0.4M sodium-chlor comes out the enzyme wash-out.

Collect the active fraction that above-mentioned wash-out goes out, use average mark level molecular weight 10,000 ultra-filtration membrane concentrates, after in " HiLoad16/60 Superdex 200 " (GE Healthcare Bio-Sciences Co., Ltd.) after making it be filled in the usefulness 20mM phosphoric acid buffer equilibration of pH7.0, carry out wash-out with identical damping fluid.

Collect the active fraction that above-mentioned wash-out goes out, use the ultra-filtration membrane of average mark level molecular weight 10,000 to concentrate, make the purification alpha-galactosidase.Activity yield is 15%, and activity is 270U/mL.

Embodiment 2

The purification alpha-galactosidase that uses embodiment 1 to obtain carries out the experiment about its effect.

(1) optimal pH scope

In being dissolved with the 100mM damping fluid of each pH of 6mM p-nitrophenyl-α-D-galactopyranoside, 150 μ L mix the of the present invention enzyme purification liquid of 50 μ L, 40 ℃ of reactions 5 minutes through 100 times of dilutions.After the reaction, to establish maximum activity be 100 o'clock relative reactivity by the free p-NP quantitatively being measured activity, obtaining.It the results are shown in Figure 1.Need to prove that employed damping fluid is glycine-HCl damping fluid (pH2.5～3.5), sodium acetate buffer (pH3.5～6.0), sodium phosphate buffer (pH6.0～8.5), glycine-NaOH damping fluid (pH8.5～10.0).

(2) stable pH range

Use the 10mM damping fluid of the scope of pH4.0～10.0, carry out 140 minutes heat treated in 45 ℃, measure its remaining activity, obtain similarly with above-mentioned that to establish maximum activity be 100 o'clock relative reactivity at each pH.It the results are shown in Figure 2.Need to prove that the kind of the damping fluid of employed each pH is same as described above.

(3) optimum temperature range

In being dissolved with the sodium acetate buffer of pH4.5 of 4.7mM p-nitrophenyl-α-D-galactopyranoside, 190 μ L mix the of the present invention enzyme purification liquid of 10 μ L, 20～60 ℃ scopes reactions 5 minutes through 20 times of dilutions.After the reaction, the free p-NP is carried out quantitatively, measure actively thus, obtaining and establishing maximum activity is 100 o'clock relative reactivity.It the results are shown in Figure 3.

(4) equilibrium temperature scope

Use the 100mM sodium acetate buffer of pH4.5, carry out 20 minutes heat treated, measure its remaining activity, obtain similarly with above-mentioned that to establish maximum activity be 100 o'clock relative reactivity each temperature of 30～55 ℃.It the results are shown in Figure 4.

(5) molecular weight

Using separating gel concentration is 10% " レ デ イ one ゲ Le J " (Japanese Bio-RadLaboratories Co., Ltd.), obtains molecular weight by the SDS-polyacrylamide gel electrophoresis.It the results are shown in Figure 5.Molecular weight is about 80,000, and this is roughly consistent with the molecular weight 83,122 of being inferred by aminoacid sequence.

(7) substrate specificity

In 150 μ L contain the 100mM sodium acetate buffer of pH4.5 of the various substrates of 10mM, mix the of the present invention enzyme purification liquid of 50 μ L, carry out reaction in 20 minutes at 40 ℃ through 100 times of dilutions.But the reaction for using p-nitrophenyl-α-D-galactopyranoside as substrate will be set at 5 minutes the reaction times.After the reaction, to the free p-NP quantitatively or use " ShodexSUGAR " (Showa Denko K. K) post to analyze by high speed liquid chromatography, the substrate that decomposes is carried out quantitatively, and obtaining and establishing maximum activity is 100 o'clock relative reactivities to each substrate.This results are shown in Table 2.In addition, for wherein can be as the material of substrate, assaying reaction speed, it the results are shown in Table 3.

[table 2]

Substrate	Relative reactivity
		PNP-α-galactopyranoside	??100
PNP-β-galactopyranoside	??0
		Raffinose	??10
Melibiose	??38
		Lactose	??0
Sucrose	??0

The reaction velocity constant of [table 3] alpha-galactosidase

Substrate	??Km(mM)	??Vmax(U/mg)
			PNP-α-galactopyranoside	??0.53	??82
Raffinose	??40	??48
			Melibiose	??8.4	??106

(8) supressor

In the sodium acetate buffer of the pH4.5 that is dissolved with 6mM p-nitrophenyl-α-D-galactopyranoside, add each metal ion species, making ultimate density is 1mM, and, mix the of the present invention enzyme purification liquid of 50 μ L through 100 times of dilutions quantitatively to 150 μ L, 40 ℃ of reactions 20 minutes.After the reaction, the free p-NP is carried out quantitatively, measure actively thus, obtaining and establishing maximum activity is 100 o'clock relative reactivity.This results are shown in Table 4.

The inhibition of [table 4] metal pair alpha-galactosidase

Metal ion	Relative reactivity
		Blank	??100
Potassium	??97
		Calcium	??97
Magnesium	??95
		Chromium	??99
Manganese	??95
		Iron (II)	??98
Iron (III)	??99
		Cobalt	??95
Nickel	??88
		Copper	??33
Zinc	??80

Embodiment 3

Get 0.5U embodiment 1 described purification alpha-galactosidase, add in the 200 μ L liquid glucoses (containing 73% sucrose, 12% semi-lactosi in the 100mM sodium acetate buffer of pH5.0) that are contained in the 1.5mL polypropylene tubulation it and mixing, at 60 ℃, 1,200rpm reacts.After the reaction beginning 45 hours, gather 20 μ L among the reaction solution, after diluting with 480 μ L distilled water,, make the enzyme heat inactivation thus 99 ℃ of processing 10 minutes.Dilution liquid glucose behind the heat inactivation is cooled to normal temperature, use " Hypercarb " (manufacturing of Thermoelectron society) post, analyze by high speed liquid chromatography.It the results are shown in Figure 6.Raffinose containing ratio in the oligose that generates is 82%, and the raffinose concentration in the reaction solution is 1.67 weight %.

Embodiment 4

(1) preparation of chromosomal DNA

The chromosomal DNA for preparing Bacillus coagulans AKC-004 strain by ordinary method.The nutrient solution of the Bacillus coagulans AKC-004 strain that 60mL is obtained with embodiment 1 described method carries out centrifugation, reclaims thalline.Resulting thalline is suspended in the Lysis damping fluid (50mMTris-HCl (pH 8.0), 20mM EDTA, 50mM glucose), fully cleans.Carry out centrifugation, behind the recovery thalline, be suspended in once more in the Lysis damping fluid, add N,O-Diacetylmuramidase therein, cultivated 45 minutes at 37 ℃.Then, add SDS and RNase, cultivated 45 minutes at 37 ℃., add Proteinase K, slowly vibrated 60 minutes at 50 ℃ thereafter.Behind the solution usefulness phenol-chloroform and chloroform processing that obtain herein, use ethanol sedimentation, the nucleic acid of separating out is entangled on the glass pipet, reclaim.This nucleic acid carries out drying, and is dissolved among the TE once more after cleaning with 70% ethanol.By this operation, prepare the chromosomal DNA of about 1mg.

(2) separation of alpha-galactosidase gene

Be designed for the PCR primer of amplification alpha-galactosidase gene fragment and synthesize according to the chromosomal DNA of above-mentioned (1) preparation.Can carry out primer design based on the The sequencing results of the alpha-galactosidase gene that is derived from known several microorganisms, have 5 as upstream primer is synthetic '-oligomer DNA of GAAGTITACGGITTYAGYYTTGTITACAGYGG-3 ' (sequence numbering 3) sequence, have 5 as downstream primer is synthetic '-oligomer DNA of CCAAACCAICCRTCRTCIARIACRAA-3 ' (sequence numbering 4) sequence.Need to prove that in the sequence, I represents inosine, Y represents C or T, and R represents A or G.Using the PCR primer that obtains herein, is template with the chromosomal DNA for preparing in above-mentioned (1), carries out the amplification of alpha-galactosidase gene fragment by the PCR method, obtains containing the alpha-galactosidase gene fragment of 380 base pairs.Use the dna sequencing instrument, the base sequence of the gene fragment that herein obtains is resolved.

For the base sequence of the alpha-galactosidase gene fragment that obtains based on parsing obtains to contain the DNA fragment of alpha-galactosidase gene total length, designed and synthesized inverse PCR PCR primer.Have 5 as upstream primer is synthetic '-oligomer DNA of TGATCAACAACTGGGAAAGCGACCT-3 ' (sequence numbering 5) sequence, have 5 as downstream primer is synthetic '-oligomer DNA of GAACTGGTCCTGCTGCACAATTCC-3 ' (sequence numbering 6) sequence.Then, preparation is used for the template of inverse PCR.After chromosomal DNA usefulness restriction enzyme HindIII digestion with preparation in above-mentioned (1), resulting DNA fragment is carried out recirculation with T4 DNA Ligase, make the template of inverse PCR.For this template, use as above synthetic inverse PCR primer, contain the amplification of the DNA fragment of alpha-galactosidase gene by the PCR method, obtain containing the PCR product that constitutes by 4500 base pairs of the complete sequence of alpha-galactosidase gene.

(3) parsing of base sequence

Use the dna sequencing instrument, determine the base sequence of alpha-galactosidase gene by the PCR product that obtains in (2).The result separates and reads the structure gene that has from the alpha-galactosidase of 2190 base pairs of the DNA base sequence of the terminal beginning of 5 ' shown in the sequence numbering 1.This sequence is undiscovered so far novel gene.In addition, the alpha-galactosidase that the Bacillus coagulans AKC-004 strain of being analogized by this DNA base sequence is produced contains 730 amino acid, has such aminoacid sequence that begins from N-terminal shown in the sequence numbering 2.The result of database retrieval is as follows: this aminoacid sequence and the alpha-galactosidase (AgaN) that derives from stearothermophilus ground bacillus (Geobacillus stearothermophilus) have 57% homogeny, has 56% homogeny with the alpha-galactosidase that derives from the stearothermophilus ground bacillus (AgaA), has 56% homogeny with the alpha-galactosidase that derives from the stearothermophilus ground bacillus (AgaB), have 56% homogeny with the alpha-galactosidase that derives from lactococcus raffinolactis (Lactococcus raffinolactis), show its encoding novel alpha-galactosidase.

(4) structure and the conversion of the expression plasmid carrier of alpha-galactosidase gene

Based on the sequence of the alpha-galactosidase gene that obtains in (3), the PCR primer in the zone of the SD sequence that designing and synthesizing is used to increase contains alpha-galactosidase gene, structure gene, terminator codon.Base sequence based on the PCR product that contains 4500 base pairs that obtains in (2) carries out the PCR primer design, have 5 as upstream primer is synthetic '-oligomer DNA of TAAGGTAAAGCAGATGTGCCATT-3 ' (sequence numbering 7) sequence, have 5 as downstream primer is synthetic '-oligomer DNA of TTACTCGTACACCGCCTC-3 ' (sequence numbering 8) sequence.As template, use synthetic PCR primer with the PCR product that contains 4500 base pairs that obtains in (2), increase, obtain containing the PCR product of 2325 base pairs by the PCR method.The PCR product that herein obtains is carried out terminal smoothing, phosphorylation.

PBluescript II KS (+) carrier digested and carry out dephosphorylation with restriction enzyme EcoRV, on this carrier, connect above-mentioned obtain through the PCR of terminal smoothing, phosphorylation product, make new plasmid vector pBlue/agaA.Import in this plasmid vector have can the gene that effective expression connects as alien gene in intestinal bacteria the lac promotor, can express, make alpha-galactosidase effectively.By heat shock, resulting plasmid vector is transformed in the e. coli jm109 strain competent cell that utilizes the Calcium Chloride Method preparation, make recombinant microorganism.

(5) expression of the cultivation of transformant and alpha-galactosidase

With recombination bacillus coli JM109-pBlue/agaA of making in (4) with the 30mL LB substratum that contains the 100mg/L penbritin 37 ℃ of shaking culture 24 hours.After the cultivation, carry out centrifugation and reclaim recombination bacillus coli.Thalline is suspended in the same damping fluid after cleaning with 100mM sodium acetate buffer (pH5.0) once more, carries out fragmentation with the ultrasonic disruption machine.Measure the alpha-galactosidase activity of this fragmentation liquid by aforesaid method, the result is 19.9U/ nutrient solution (mL).

Embodiment 5

It is described by cultivating the resulting alpha-galactosidase of recombinant chou to get 1.0U embodiment 4, add in the 200 μ L liquid glucoses (the 100mM sodium acetate buffer of pH5.0 contains 73% sucrose, 12% semi-lactosi) that are contained in the 1.5mL polypropylene tubulation it and mixing, at 60 ℃, 1,200rpm reacts.After the reaction beginning 16 hours, gather 20 μ L among the reaction solution, after diluting with 480 μ L distilled water, carry out processing in 10 minutes, make the enzyme heat inactivation thus at 99 ℃.After dilution liquid glucose behind the heat inactivation cooled to normal temperature, use " Hypercarb " (manufacturing of Thermoelectron society) post, analyze by high speed liquid chromatography.It the results are shown in Figure 7.Raffinose containing ratio in the oligose that generates is 82%, and the raffinose concentration in the reaction solution is 1.20 weight %.

Embodiment 6

With Bacillus coagulans AKC-004 strain (deposit number FERM P-21092 (transfer and be FERM-ABP10948); Preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological preservation center) cultivated 1 day at 55 ℃ with TBAB (Tryptones blood agar basis) culture plate (Difco), form bacterium colony.1 transfering loop culture is inoculated in the described substratum of 30mL table 1 that is divided in the 150mL Erlenmeyer flask, cultivated 2 days with 150rpm at 55 ℃.

This was cultivated after 2 days, and the cultivation thalline that 10mL is measured is recovered in the 15mL pipe.The 15mL pipe that recovery is had a nutrient solution with 10000rpm centrifugal after, remove supernatant liquor.Then, add the 100mM sodium acetate buffer (pH5.0) of 1mL, after suspending once more, suspension is moved in the polypropylene tubulation of 2mL.Once more pipe is carried out centrifugal, remove supernatant liquor after, add 300 μ L liquid glucoses (the 100mM sodium acetate buffer (pH5.0) that contains 62.5% sucrose, 12.5% semi-lactosi), with vortex stirrer thalline is fully suspended, begin sugared building-up reactions.This sugar building-up reactions is carried out under the condition of 60 ℃ of temperature of reaction, rotating speed 1200rpm.Sugar reclaims 40 μ L reaction solutions after building-up reactions begins 16h, with itself and 960 μ L distilled water thorough mixing, at 99 ℃ of heat inactivations that enzyme carried out 10 minutes.After the temperature of this dilution liquid glucose dropped to normal temperature, use " Hypercarb " (manufacturing of Thermoelectron society) post, analyze by high speed liquid chromatography, it the results are shown in Figure 8.Raffinose containing ratio in the oligose that generates is 80% (the raffinose concentration in the reaction solution is 0.70 weight %).

Embodiment 7

With Bacillus coagulans AKC-003 strain (deposit number FERM P-21091; Preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological preservation center) cultivated 1 day at 55 ℃ with TBAB (Tryptones blood agar basis) culture plate (Difco), form bacterium colony.1 transfering loop culture is inoculated in the described substratum of 30mL table 1 that is divided in the 150mL Erlenmeyer flask, cultivated 2 days with 150rpm at 55 ℃.

This was cultivated after 2 days, and the cultivation thalline that 10mL is measured is recovered in the 15mL pipe.The 15mL pipe that recovery is had a nutrient solution with 10000rpm centrifugal after, remove supernatant liquor.Then, add the 100mM sodium acetate buffer (pH5.0) of 1mL, after suspending once more, suspension is moved in the polypropylene tubulation of 2mL.Once more pipe is carried out centrifugal, remove supernatant liquor after, add 300 μ L liquid glucoses (the 100mM sodium acetate buffer (pH5.0) that contains 62.5% sucrose, 12.5% semi-lactosi), with vortex stirrer thalline is fully suspended, begin sugared building-up reactions.This sugar building-up reactions is carried out under the condition of 60 ℃ of temperature of reaction, rotating speed 1200rpm.Sugar reclaims 40 μ L reaction solutions after building-up reactions begins 38h, with itself and 960 μ L distilled water thorough mixing, at 99 ℃ of heat inactivations that enzyme carried out 10 minutes.After the temperature of this dilution liquid glucose dropped to normal temperature, use " Hypercarb " (manufacturing of Thermoelectron society) post, analyze by high speed liquid chromatography, at this moment, the raffinose containing ratio in the oligose of generation is 80% (the raffinose concentration in the reaction solution is 0.76 weight %).

Embodiment 8

With Bacillus coagulans AKC-005 strain (deposit number FERM P-21093; Preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological preservation center) cultivated 1 day at 55 ℃ with TBAB (Tryptones blood agar basis) culture plate (Difco), form bacterium colony.1 transfering loop culture is inoculated in the described substratum of 30mL table 1 that is divided in the 150mL Erlenmeyer flask, cultivated 2 days with 150rpm at 55 ℃.

This was cultivated after 2 days, and the cultivation thalline that 10mL is measured is recovered in the 15mL pipe.The 15mL pipe that recovery is had a nutrient solution with 10000rpm centrifugal after, remove supernatant liquor.Then, add the 100mM sodium acetate buffer (pH5.0) of 1mL, after suspending once more, suspension is moved in the polypropylene tubulation of 2mL.Once more pipe is carried out centrifugal, remove supernatant liquor after, add 300 μ L liquid glucoses (the 100mM sodium acetate buffer (pH5.0) that contains 62.5% sucrose, 12.5% semi-lactosi), with vortex stirrer thalline is fully suspended, begin sugared building-up reactions.This sugar building-up reactions is carried out under the condition of 60 ℃ of temperature of reaction, rotating speed 1200rpm.Sugar reclaims 40 μ L reaction solutions after building-up reactions begins 38h, with itself and 960 μ L distilled water thorough mixing, at 99 ℃ of heat inactivations that enzyme carried out 10 minutes.After the temperature of this dilution liquid glucose dropped to normal temperature, use " Hypercarb " (manufacturing of Thermoelectron society) post, analyze by high speed liquid chromatography, at this moment, the raffinose containing ratio in the oligose of generation is 91% (the raffinose concentration in the reaction solution is 0.32 weight %).

Embodiment 9

With Bacillus coagulans AKC-006 strain (deposit number FERM P-21094; Preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological preservation center) cultivated 1 day at 55 ℃ with TBAB (Tryptones blood agar basis) culture plate (Difco), form bacterium colony.1 transfering loop culture is inoculated in the described substratum of 30mL table 1 that is divided in the 150mL Erlenmeyer flask, cultivated 2 days with 150rpm at 55 ℃.

This was cultivated after 2 days, and the cultivation thalline that 10mL is measured is recovered in the 15mL pipe.The 15mL pipe that recovery is had a nutrient solution with 10000rpm centrifugal after, remove supernatant liquor.Then, add the 100mM sodium acetate buffer (pH5.0) of 1mL, after suspending once more, suspension is moved in the polypropylene tubulation of 2mL.Once more pipe is carried out centrifugal, remove supernatant liquor after, add 300 μ L liquid glucoses (the 100mM sodium acetate buffer (pH5.0) that contains 62.5% sucrose, 12.5% semi-lactosi), with vortex stirrer thalline is fully suspended, begin sugared building-up reactions.This sugar building-up reactions is carried out under the condition of 60 ℃ of temperature of reaction, rotating speed 1200rpm.Sugar reclaims 40 μ L reaction solutions after building-up reactions begins 38h, with itself and 960 μ L distilled water thorough mixing, at 99 ℃ of heat inactivations that enzyme carried out 10 minutes.After the temperature of this dilution liquid glucose dropped to normal temperature, use " Hypercarb " (manufacturing of Thermoelectron society) post, analyze by high speed liquid chromatography, at this moment, the raffinose containing ratio in the oligose of generation is 76% (the raffinose concentration in the reaction solution is 0.86 weight %).

Utilizability on the industry

The application of the invention can provide the method for using raw material cheaply selectively to make gossypose.

Sequence table

＜110〉Asahi Chemical Corp

＜120〉novel a-galactosidase

<130>F108020-WO-00

<160>8

<170>PatentIn?version?3.1

<210>1

<211>2193

<212>DNA

＜213〉Bacillus coagulans (Bacillus coagulans)

<220>

<221>CDS

<222>(1)..(2190)

<223>

<400>1

atg?att?aca?ttt?gat?gaa?caa?acg?aaa?act?ttc?cat?ttg?caa?aat?gat?????48

Met?Ile?Thr?Phe?Asp?Glu?Gln?Thr?Lys?Thr?Phe?His?Leu?Gln?Asn?Asp

1???????????????5???????????????????10??????????????????15

gaa?gtg?agt?tac?ctc?tta?caa?gtg?gat?gca?ttc?ggc?tgc?gtt?gaa?cat?????96

Glu?Val?Ser?Tyr?Leu?Leu?Gln?Val?Asp?Ala?Phe?Gly?Cys?Val?Glu?His

20??????????????????25??????????????????30

ctg?tat?tgg?gga?gcg?ccg?gtc?agg?gcc?tac?cac?ggg?ggg?cgc?gcc?tat????144

Leu?Tyr?Trp?Gly?Ala?Pro?Val?Arg?Ala?Tyr?His?Gly?Gly?Arg?Ala?Tyr

35??????????????????40??????????????????45

ccc?cgc?atc?gcc?cgc?agt?ttt?tcc?ccg?aac?ccg?ccc?ggt?gca?aaa?gac????192

Pro?Arg?Ile?Ala?Arg?Ser?Phe?Ser?Pro?Asn?Pro?Pro?Gly?Ala?Lys?Asp

50??????????????????55??????????????????60

cgc?aaa?ttt?tca?ctg?gat?acg?gtg?ctg?cag?gaa?ttc?ccg?gga?tac?ggc????240

Arg?Lys?Phe?Ser?Leu?Asp?Thr?Val?Leu?Gln?Glu?Phe?Pro?Gly?Tyr?Gly

65??????????????????70??????????????????75??????????????????80

aac?ggc?gat?ttc?cgt?gag?ccg?gcg?cat?gtt?atc?cgg?cat?gcg?gac?ggg????288

Asn?Gly?Asp?Phe?Arg?Glu?Pro?Ala?His?Val?Ile?Arg?His?Ala?Asp?Gly

85??????????????????90??????????????????95

tca?acg?gtc?acg?gac?ttc?cgc?tac?cgg?gcg?tat?gac?att?tta?aaa?ggc????336

Ser?Thr?Val?Thr?Asp?Phe?Arg?Tyr?Arg?Ala?Tyr?Asp?Ile?Leu?Lys?Gly

100?????????????????105?????????????????110

aag?ccg?gcg?ctt?ccc?ggg?ctt?ccc?gct?act?ttt?gcc?aat?cca?aat?gaa????384

Lys?Pro?Ala?Leu?Pro?Gly?Leu?Pro?Ala?Thr?Phe?Ala?Asn?Pro?Asn?Glu

115?????????????????120?????????????????125

gcc?gaa?aca?ttg?aaa?atc?atg?ctg?gaa?gac?aaa?ctc?aca?ggc?ctt?caa????432

Ala?Glu?Thr?Leu?Lys?Ile?Met?Leu?Glu?Asp?Lys?Leu?Thr?Gly?Leu?Gln

130?????????????????135?????????????????140

tta?acg?ctt?tta?tac?acc?atc?ttt?cgc?gac?ttg?ccg?gcg?att?tcc?cgc????480

Leu?Thr?Leu?Leu?Tyr?Thr?Ile?Phe?Arg?Asp?Leu?Pro?Ala?Ile?Ser?Arg

145?????????????????150?????????????????155?????????????????160

gcg?gct?gtg?ctg?aca?aac?ggc?gga?aac?aag?cct?gtt?cgg?att?gaa?cgt????528

Ala?Ala?Val?Leu?Thr?Asn?Gly?Gly?Asn?Lys?Pro?Val?Arg?Ile?Glu?Arg

165?????????????????170?????????????????175

ttg?atg?agc?ctg?aat?gtc?gat?ttc?ccg?gcc?gga?cgt?ttt?gag?ctg?ttg????576

Leu?Met?Ser?Leu?Asn?Val?Asp?Phe?Pro?Ala?Gly?Arg?Phe?Glu?Leu?Leu

180?????????????????185?????????????????190

cat?ctc?ccc?ggc?gcc?cat?aaa?cgc?gaa?cgg?caa?atc?aaa?aga?gaa?acg????624

His?Leu?Pro?Gly?Ala?His?Lys?Arg?Glu?Arg?Gln?Ile?Lys?Arg?Glu?Thr

195?????????????????200?????????????????205

gta?acg?gac?ggg?atc?agg?cgt?gta?gac?agc?aaa?cgc?ggc?gcc?agc?agc????672

Val?Thr?Asp?Gly?Ile?Arg?Arg?Val?Asp?Ser?Lys?Arg?Gly?Ala?Ser?Ser

210?????????????????215?????????????????220

cac?cag?gaa?aat?cct?ttt?ttg?gcg?ctt?gtc?cgg?cct?gaa?aca?acg?gaa????720

His?Gln?Glu?Asn?Pro?Phe?Leu?Ala?Leu?Val?Arg?Pro?Glu?Thr?Thr?Glu

225?????????????????230?????????????????235?????????????????240

ttc?caa?ggt?gaa?gca?tat?gcg?gtg?aac?ctc?gtt?tac?agc?ggc?aat?ttt????768

Phe?Gln?Gly?Glu?Ala?Tyr?Ala?Val?Asn?Leu?Val?Tyr?Ser?Gly?Asn?Phe

245?????????????????250?????????????????255

gcc?gga?att?gtg?cag?cag?gac?cag?ttc?ggc?cag?gtg?cgt?ctc?ggg?atc????816

Ala?Gly?Ile?Val?Gln?Gln?Asp?Gln?Phe?Gly?Gln?Val?Arg?Leu?Gly?Ile

260?????????????????265?????????????????270

ggg?ctg?aat?gat?ttc?ggg?ttc?agc?tgg?gcg?ttg?cag?ccg?ggt?gac?act????864

Gly?Leu?Asn?Asp?Phe?Gly?Phe?Ser?Trp?Ala?Leu?Gln?Pro?Gly?Asp?Thr

275?????????????????280?????????????????285

ttt?tat?tcc?cct?gaa?gca?gtg?atg?gca?tac?agc?aga?gac?ggg?ctg?aac????912

Phe?Tyr?Ser?Pro?Glu?Ala?Val?Met?Ala?Tyr?Ser?Arg?Asp?Gly?Leu?Asn

290?????????????????295?????????????????300

ggg?atg?tcg?caa?acg?ttt?cac?acg?ctc?tac?cgc?cgc?cat?ctt?ttg?cgc????960

Gly?Met?Ser?Gln?Thr?Phe?His?Thr?Leu?Tyr?Arg?Arg?His?Leu?Leu?Arg

305?????????????????310?????????????????315?????????????????320

ggc?aaa?cat?aaa?gat?gcg?gaa?cgc?ccg?gtg?ttg?atc?aac?aac?tgg?gaa???1008

Gly?Lys?His?Lys?Asp?Ala?Glu?Arg?Pro?Val?Leu?Ile?Asn?Asn?Trp?Glu

325?????????????????330?????????????????335

gcg?acc?tat?ttc?cgg?ttt?cat?gat?caa?aaa?ttg?ctt?gaa?ctc?gcc?gac???1056

Ala?Thr?Tyr?Phe?Arg?Phe?His?Asp?Gln?Lys?Leu?Leu?Glu?Leu?Ala?Asp

340?????????????????345?????????????????350

gag?gcg?caa?aag?ctc?ggc?att?gag?ctg?ttt?gtg?ctg?gat?gac?ggc?tgg???1104

Glu?Ala?Gln?Lys?Leu?Gly?Ile?Glu?Leu?Phe?Val?Leu?Asp?Asp?Gly?Trp

355?????????????????360?????????????????365

ttc?ggc?cac?cgg?gac?aat?gac?cgc?agt?tct?tta?ggg?gac?tgg?tat?gaa???1152

Phe?Gly?His?Arg?Asp?Asn?Asp?Arg?Ser?Ser?Leu?Gly?Asp?Trp?Tyr?Glu

370?????????????????375?????????????????380

tac?gcc?gga?aaa?atc?cgg?atg?ggg?atc?aaa?aat?ctg?gcg?gaa?gaa?atc???1200

Tyr?Ala?Gly?Lys?Ile?Arg?Met?Gly?Ile?Lys?Asn?Leu?Ala?Glu?Glu?Ile

385?????????????????390?????????????????395?????????????400

cat?aaa?cgc?ggc?ttg?aaa?ttc?ggg?ctc?tgg?ttt?gaa?ccg?gaa?atg?gtt???1248

His?Lys?Arg?Gly?Leu?Lys?Phe?Gly?Leu?Trp?Phe?Glu?Pro?Glu?Met?Val

405?????????????????410?????????????415

tcg?cgg?gac?agt?gac?ctt?ttc?cgt?gaa?cat?ccg?gac?tgg?gca?ttg?caa???1296

Ser?Arg?Asp?Ser?Asp?Leu?Phe?Arg?Glu?His?Pro?Asp?Trp?Ala?Leu?Gln

420?????????????????425?????????????430

att?ccg?ggc?cgc?ggg?att?tcc?gaa?ggg?cgc?tcc?caa?tat?gtg?ctc?gat???1344

Ile?Pro?Gly?Arg?Gly?Ile?Ser?Glu?Gly?Arg?Ser?Gln?Tyr?Val?Leu?Asp

435?????????????????440?????????????445

ttt?agc?cgc?ggg?gat?gtg?cgc?gaa?aat?att?ttc?cgg?cag?atg?acg?gat???1392

Phe?Ser?Arg?Gly?Asp?Val?Arg?Glu?Asn?Ile?Phe?Arg?Gln?Met?Thr?Asp

450?????????????????455?????????????460

att?ctg?gat?cat?gtc?cat?gtc?gat?tat?atc?aaa?tgg?gat?atg?aac?cgc???1440

Ile?Leu?Asp?His?Val?His?Val?Asp?Tyr?Ile?Lys?Trp?Asp?Met?Asn?Arg

465?????????????????470?????????????????475?????????????????480

cat?atg?aca?gaa?gtc?cat?tcc?gcc?ctg?ctg?gaa?ccg?gaa?aac?cag?gga???1488

His?Met?Thr?Glu?Val?His?Ser?Ala?Leu?Leu?Glu?Pro?Glu?Asn?Gln?Gly

485?????????????????490?????????????????495

gaa?acc?gcc?cac?cgc?tat?atg?ctc?ggg?ctg?tat?gac?ttt?tta?gaa?aag???1536

Glu?Thr?Ala?His?Arg?Tyr?Met?Leu?Gly?Leu?Tyr?Asp?Phe?Leu?Glu?Lys

500?????????????????505?????????????????510

ttg?acg?tcg?cgt?tac?cct?gac?att?ttg?ttt?gaa?agc?tgt?tcc?ggc?ggc???1584

Leu?Thr?Ser?Arg?Tyr?Pro?Asp?Ile?Leu?Phe?Glu?Ser?Cys?Ser?Gly?Gly

515?????????????????520?????????????????525

ggc?ggg?cgt?ttt?gat?ccg?ggc?atg?ctg?tat?tat?atg?ccg?caa?acg?tgg???1632

Gly?Gly?Arg?Phe?Asp?Pro?Gly?Met?Leu?Tyr?Tyr?Met?Pro?Gln?Thr?Trp

530?????????????????535?????????????????540

acg?agc?gat?aat?acc?gat?gcg?gcc?gac?cgg?ctg?aaa?atc?cag?tac?ggc???1680

Thr?Ser?Asp?Asn?Thr?Asp?Ala?Ala?Asp?Arg?Leu?Lys?Ile?Gln?Tyr?Gly

545?????????????????550?????????????????555?????????????????560

aca?agc?ctc?gcc?tat?ccg?ccg?gtt?acc?atg?ggc?gcg?cat?gtt?tcg?gcc???1728

Thr?Ser?Leu?Ala?Tyr?Pro?Pro?Val?Thr?Met?Gly?Ala?His?Val?Ser?Ala

565?????????????????570?????????????????575

gtt?ccg?aac?cac?caa?acc?ggc?cgg?gtt?acg?ccg?cta?tcg?acg?cgc?ggt???1776

Val?Pro?Asn?His?Gln?Thr?Gly?Arg?Val?Thr?Pro?Leu?Ser?Thr?Arg?Gly

580?????????????????585?????????????????590

gac?gtg?gca?atg?ggc?ggc?aat?ttc?ggc?tat?gag?ttg?gat?ttg?aca?aaa???1824

Asp?Val?Ala?Met?Gly?Gly?Asn?Phe?Gly?Tyr?Glu?Leu?Asp?Leu?Thr?Lys

595?????????????????600?????????????????605

tgc?acg?gaa?gat?gaa?aaa?gca?gtg?atc?cgg?gag?cag?att?gca?ttt?tac???1872

Cys?Thr?Glu?Asp?Glu?Lys?Ala?Val?Ile?Arg?Glu?Gln?Ile?Ala?Phe?Tyr

610?????????????????615?????????????????620

aaa?gca?cac?cgt?caa?ttg?ttc?cag?ttt?ggc?cag?ttt?tac?cgc?ctg?ata????1920

Lys?Ala?His?Arg?Gln?Leu?Phe?Gln?Phe?Gly?Gln?Phe?Tyr?Arg?Leu?Ile

625?????????????????630?????????????????635?????????????????640

agc?ccg?ttt?gaa?ggc?aac?agc?gcc?gcc?tgg?caa?ttt?gtc?tcg?ccg?gac????1968

Ser?Pro?Phe?Glu?Gly?Asn?Ser?Ala?Ala?Trp?Gln?Phe?Val?Ser?Pro?Asp

645?????????????????650?????????????????655

cgg?aaa?cat?acc?att?gcc?tat?ttt?ttc?aac?gta?ctg?tct?gaa?gca?gcc????2016

Arg?Lys?His?Thr?Ile?Ala?Tyr?Phe?Phe?Asn?Val?Leu?Ser?Glu?Ala?Ala

660?????????????????665?????????????????670

ggc?ccg?gtg?aaa?ata?tta?aaa?ctg?gca?ggc?ctt?gac?cca?ggc?aaa?aac????2064

Gly?Pro?Val?Lys?Ile?Leu?Lys?Leu?Ala?Gly?Leu?Asp?Pro?Gly?Lys?Asn

675?????????????????680?????????????????685

tac?cgg?cat?att?gaa?aca?ggc?aac?att?tac?ggc?ggc?gat?gaa?ctg?atg????2112

Tyr?Arg?His?Ile?Glu?Thr?Gly?Asn?Ile?Tyr?Gly?Gly?Asp?Glu?Leu?Met

690?????????????????695?????????????????700

aac?atc?ggg?ctg?tat?ttg?cca?ttg?ttt?aac?aaa?cag?gac?ttt?gca?agc????2160

Asn?Ile?Gly?Leu?Tyr?Leu?Pro?Leu?Phe?Asn?Lys?Gln?Asp?Phe?Ala?Ser

705?????????????????710?????????????????715?????????????????720

tac?aaa?gca?gag?ttt?gag?gcg?gtg?tac?gag?taa????????????????????????2193

Tyr?Lys?Ala?Glu?Phe?Glu?Ala?Val?Tyr?Glu

725?????????????????730

<210>2

<211>730

<212>PRT

＜213〉Bacillus coagulans

<400>2

Met?Ile?Thr?Phe?Asp?Glu?Gln?Thr?Lys?Thr?Phe?His?Leu?Gln?Asn?Asp

1???????????????5???????????????????10??????????????????15

Glu?Val?Ser?Tyr?Leu?Leu?Gln?Val?Asp?Ala?Phe?Gly?Cys?Val?Glu?His

20??????????????????25??????????????????30

Leu?Tyr?Trp?Gly?Ala?Pro?Val?Arg?Ala?Tyr?His?Gly?Gly?Arg?Ala?Tyr

35??????????????????40??????????????????45

Pro?Arg?Ile?Ala?Arg?Ser?Phe?Ser?Pro?Ash?Pro?Pro?Gly?Ala?Lys?Asp

50??????????????????55??????????????????60

Arg?Lys?Phe?Ser?Leu?Asp?Thr?Val?Leu?Gln?Glu?Phe?Pro?Gly?Tyr?Gly

65??????????????????70??????????????????75??????????????????80

Asn?Gly?Asp?Phe?Arg?Glu?Pro?Ala?His?Val?Ile?Arg?His?Ala?Asp?Gly

85??????????????????90??????????????????95

Ser?Thr?Val?Thr?Asp?Phe?Arg?Tyr?Arg?Ala?Tyr?Asp?Ile?Leu?Lys?Gly

100?????????????????105?????????????????110

Lys?Pro?Ala?Leu?Pro?Gly?Leu?Pro?Ala?Thr?Phe?Ala?Asn?Pro?Asn?Glu

115?????????????????120?????????????????125

Ala?Glu?Thr?Leu?Lys?Ile?Met?Leu?Glu?Asp?Lys?Leu?Thr?Gly?Leu?Gln

130?????????????????135?????????????????140

Leu?Thr?Leu?Leu?Tyr?Thr?Ile?Phe?Arg?Asp?Leu?Pro?Ala?Ile?Ser?Arg

145?????????????????150?????????????????155?????????????????160

Ala?Ala?Val?Leu?Thr?Asn?Gly?Gly?Asn?Lys?Pro?Val?Arg?Ile?Glu?Arg

165?????????????????170?????????????????175

Leu?Met?Ser?Leu?Asn?Val?Asp?Phe?Pro?Ala?Gly?Arg?Phe?Glu?Leu?Leu

180?????????????????185?????????????????190

His?Leu?Pro?Gly?Ala?His?Lys?Arg?Glu?Arg?Gln?Ile?Lys?Arg?Glu?Thr

195?????????????????200?????????????????205

Val?Thr?Asp?Gly?Ile?Arg?Arg?Val?Asp?Ser?Lys?Arg?Gly?Ala?Ser?Ser

210?????????????????215?????????????????220

His?Gln?Glu?Asn?Pro?Phe?Leu?Ala?Leu?Val?Arg?Pro?Glu?Thr?Thr?Glu

225?????????????????230?????????????????235?????????????????240

Phe?Gln?Gly?Glu?Ala?Tyr?Ala?Val?Asn?Leu?Val?Tyr?Ser?Gly?Asn?Phe

245?????????????????250?????????????????255

Ala?Gly?Ile?Val?Gln?Gln?Asp?Gln?Phe?Gly?Gln?Val?Arg?Leu?Gly?Ile

260?????????????????265?????????????????270

Gly?Leu?Asn?Asp?Phe?Gly?Phe?Ser?Trp?Ala?Leu?Gln?Pro?Gly?Asp?Thr

275?????????????????280?????????????????285

Phe?Tyr?Ser?Pro?Glu?Ala?Val?Met?Ala?Tyr?Ser?Arg?Asp?Gly?Leu?Asn

290?????????????????295?????????????????300

Gly?Met?Ser?Gln?Thr?Phe?His?Thr?Leu?Tyr?Arg?Arg?His?Leu?Leu?Arg

305?????????????????310?????????????????315?????????????????320

Gly?Lys?His?Lys?Asp?Ala?Glu?Arg?Pro?Val?Leu?Ile?Asn?Asn?Trp?Glu

325?????????????????330?????????????????335

Ala?Thr?Tyr?Phe?Arg?Phe?His?Asp?Gln?Lys?Leu?Leu?Glu?Leu?Ala?Asp

340?????????????????345?????????????????350

Glu?Ala?Gln?Lys?Leu?Gly?Ile?Glu?Leu?Phe?Val?Leu?Asp?Asp?Gly?Trp

355?????????????????360?????????????????365

Phe?Gly?His?Arg?Asp?Asn?Asp?Arg?Ser?Ser?Leu?Gly?Asp?Trp?Tyr?Glu

370?????????????????375?????????????????380

Tyr?Ala?Gly?Lys?Ile?Arg?Met?Gly?Ile?Lys?Asn?Leu?Ala?Glu?Glu?Ile

385?????????????????390?????????????????395?????????????????400

His?Lys?Arg?Gly?Leu?Lys?Phe?Gly?Leu?Trp?Phe?Glu?Pro?Glu?Met?Val

405?????????????????410?????????????????415

Ser?Arg?Asp?Ser?Asp?Leu?Phe?Arg?Glu?His?Pro?Asp?Trp?Ala?Leu?Gln

420?????????????????425?????????????????430

Ile?Pro?Gly?Arg?Gly?Ile?Ser?Glu?Gly?Arg?Ser?Gln?Tyr?Val?Leu?Asp

435?????????????????440?????????????????445

Phe?Ser?Arg?Gly?Asp?Val?Arg?Glu?Asn?Ile?Phe?Arg?Gln?Met?Thr?Asp

450?????????????????455?????????????????460

Ile?Leu?Asp?His?Val?His?Val?Asp?Tyr?Ile?Lys?Trp?Asp?Met?Asn?Arg

465?????????????????470?????????????????475?????????????????480

His?Met?Thr?Glu?Val?His?Ser?Ala?Leu?Leu?Glu?Pro?Glu?Asn?Gln?Gly

485?????????????????490?????????????????495

Glu?Thr?Ala?His?Arg?Tyr?Met?Leu?Gly?Leu?Tyr?Asp?Phe?Leu?Glu?Lys

500?????????????????505?????????????????510

Leu?Thr?Ser?Arg?Tyr?Pro?Asp?Ile?Leu?Phe?Glu?Ser?Cys?Ser?Gly?Gly

515?????????????????520?????????????????525

Gly?Gly?Arg?Phe?Asp?Pro?Gly?Met?Leu?Tyr?Tyr?Met?Pro?Gln?Thr?Trp

530?????????????????535?????????????????540

Thr?Ser?Asp?Asn?Thr?Asp?Ala?Ala?Asp?Arg?Leu?Lys?Ile?Gln?Tyr?Gly

545?????????????????550?????????????????555?????????????????560

Thr?Ser?Leu?Ala?Tyr?Pro?Pro?Val?Thr?Met?Gly?Ala?His?Val?Ser?Ala

565?????????????????570?????????????????575

Val?Pro?Asn?His?Gln?Thr?Gly?Arg?Val?Thr?Pro?Leu?Ser?Thr?Arg?Gly

580?????????????????585?????????????????590

Asp?Val?Ala?Met?Gly?Gly?Asn?Phe?Gly?Tyr?Glu?Leu?Asp?Leu?Thr?Lys

595?????????????????600?????????????????605

Cys?Thr?Glu?Asp?Glu?Lys?Ala?Val?Ile?Arg?Glu?Gln?Ile?Ala?Phe?Tyr

610?????????????????615?????????????????620

Lys?Ala?His?Arg?Gln?Leu?Phe?Gln?Phe?Gly?Gln?Phe?Tyr?Arg?Leu?Ile

625?????????????????630?????????????????635?????????????????640

Ser?Pro?Phe?Glu?Gly?Asn?Ser?Ala?Ala?Trp?Gln?Phe?Val?Ser?Pro?Asp

645?????????????????650?????????????????655

Arg?Lys?His?Thr?Ile?Ala?Tyr?Phe?Phe?Asn?Val?Leu?Ser?Glu?Ala?Ala

660?????????????????665?????????????????670

Gly?Pro?Val?Lys?Ile?Leu?Lys?Leu?Ala?Gly?Leu?Asp?Pro?Gly?Lys?Asn

675?????????????????680?????????????????685

Tyr?Arg?His?Ile?Glu?Thr?Gly?Asn?Ile?Tyr?Gly?Gly?Asp?Glu?Leu?Met

690?????????????????695?????????????????700

Asn?Ile?Gly?Leu?Tyr?Leu?Pro?Leu?Phe?Asn?Lys?Gln?Asp?Phe?Ala?Ser

705?????????????????710?????????????????715?????????????????720

Tyr?Lys?Ala?Glu?Phe?Glu?Ala?Val?Tyr?Glu

725?????????????????730

<210>3

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: nucleic acid

<400>3

gaagtitacg?gittyagyyt?tgtitacagy?gg??????????????????????????????????32

<210>4

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: nucleic acid

<400>4

ccaaaccaic?crtcrtciar?iacraa?????????????????????????????????????????26

<210>5

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: nucleic acid

<400>5

tgatcaacaa?ctgggaaagc?gacct??????????????????????????????????????????25

<210>6

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: nucleic acid

<400>6

gaactggtcc?tgctgcacaa?ttcc???????????????????????????????????????????24

<210>7

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: nucleic acid

<400>7

taaggtaaag?cagatgtgcc?att????????????????????????????????????????????23

<210>8

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: nucleic acid

<400>8

ttactcgtac?accgcctc??????????????????????????????????????????????????18

Claims (20)

1, a kind of alpha-galactosidase, it has following characteristic:

(1) effect: described enzyme has following character: be in the dehydration condensation of raw material with sucrose and semi-lactosi, the raffinose containing ratio in the oligose that generates is 0.5% when above, and the synthetic selection rate of raffinose is more than 65%;

(2) optimal pH scope: 3.5～5.0;

(3) stable pH range: 3.5～10.0;

(4) molecular weight: about 80,000.

2, alpha-galactosidase as claimed in claim 1, it derives from the microorganism that belongs to Bacillus coagulans.

3, alpha-galactosidase as claimed in claim 2, wherein, described Bacillus coagulans is any one in Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, the AKC006 strain.

4, a kind of Bacillus coagulans and varient thereof, described Bacillus coagulans belong to any one in Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, the AKC006 strain.

5, a kind of alpha-galactosidase, its contain following (a) and (b) or (c) in aminoacid sequence arbitrarily:

(a) aminoacid sequence of sequence numbering 2 expressions;

(b) in the aminoacid sequence of sequence numbering 2 expression, lacked, replaced and/or added the aminoacid sequence of 1 or several amino acid, it has alpha-galactosidase activity;

(c) have the aminoacid sequence of the homogeny more than 60% with the aminoacid sequence of sequence numbering 2 expression, it has alpha-galactosidase activity.

6, a kind of alpha-galactosidase gene, its coding contain following (a) and (b) or (c) in the alpha-galactosidase of aminoacid sequence arbitrarily:

(a) aminoacid sequence of sequence numbering 2 expressions;

(b) in the aminoacid sequence of sequence numbering 2 expression, lacked, replaced and/or added the aminoacid sequence of 1 or several amino acid, it has alpha-galactosidase activity;

(c) have the aminoacid sequence of the homogeny more than 60% with the aminoacid sequence of sequence numbering 2 expression, it has alpha-galactosidase activity.

7, a kind of alpha-galactosidase gene, it contains following (a) or base sequence (b):

(a) base sequence of sequence numbering 1 expression;

(b) in the base sequence of sequence numbering 1 expression, lacked, replaced and/or added the base sequence of 1 or several bases, and its coding has the protein of alpha-galactosidase activity.

8, a kind of recombinant vectors, it contains claim 6 or 7 described alpha-galactosidase genes.

9, a kind of transformant, its importing have the right requirement 6 or 7 described alpha-galactosidase genes or the described recombinant vectors of claim 8.

10, a kind of alpha-galactosidase, it is cultivated the described transformant of claim 9 and obtains.

11, a kind of enzyme composition, it contains claim 1～3, each described alpha-galactosidase of 5 or 10.

12, enzyme composition as claimed in claim 11, wherein, said composition also contains at least a above composition that is selected from alpha-glucosidase, beta-glucosidase enzyme, beta-galactosidase enzymes, cellulase, zytase, proteolytic enzyme, Galactanase, arabanase, mannase, rhamno-galacturonic acid enzyme, polygalacturonase, pectin methylesterase, pectin lyase and polygalacturonic acid lyase.

13, a kind of raffinose synthetic agent, it contains claim 11 or 12 each described enzyme composition.

14, a kind of manufacture method of raffinose is characterized in that, this manufacture method is used claim 1～3, each described alpha-galactosidase of 5 or 10; Claim 11 or 12 described enzyme composition; The perhaps described raffinose synthetic agent of claim 13.

15, a kind of manufacture method of raffinose is characterized in that, this method is utilized microbial catalyst, and described microbial catalyst is cultivated the microorganism that belongs to Bacillus coagulans and obtained.

16, a kind of manufacture method of raffinose, it is characterized in that, this method is utilized microbial catalyst, and described microbial catalyst is cultivated and belonged in Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, the AKC006 strain any one Bacillus coagulans and/or its varient and obtain.

17, a kind of manufacture method of raffinose is characterized in that, this method is utilized microbial catalyst, and described microbial catalyst is cultivated the described transformant of claim 9 and obtained.

18, as the manufacture method of each described raffinose in the claim 14～17, wherein, the raffinose containing ratio in the oligose of generation is more than 65%.

19, as the manufacture method of each described raffinose in the claim 14～18, wherein, this method uses sucrose and semi-lactosi as raw material.

20, the manufacture method of raffinose as claimed in claim 19, wherein, the sucrose concentration in the raw material is 30% (w/v)～90% (w/v), the galactose concentration in the raw material is 2% (w/v)～45% (w/v).

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