CN101657544B - Novel a-galactosidase - Google Patents

Novel a-galactosidase Download PDF

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CN101657544B
CN101657544B CN200880005393.8A CN200880005393A CN101657544B CN 101657544 B CN101657544 B CN 101657544B CN 200880005393 A CN200880005393 A CN 200880005393A CN 101657544 B CN101657544 B CN 101657544B
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CN101657544A (en
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今津晋一
小西一诚
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Asahi Kasei Corp
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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 manufacture 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, human consumer also increases to the degree of attentiveness of food, foodstuffs material etc.Wherein, find that raffinose has the functions such as the intestinal flora of improvement, therefore as food, medicine, spices etc. or its raw material, receive much attention.Recently, found that raffinose has immunity effect effect, useful to atopic dermatitis.
Now, raffinose is by-product recovery when manufacturing beet sugar, and raffinose content in beet is only 0.1% left and right, and 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 there is the raffinose of such useful property to the market supply with low cost, not only need to be from natural extraction product, also need to 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 reported and take sucrose as galactosylated acceptor, the melibiose of take obtains the method (for example, referring to patent documentation 1, non-patent literature 1, non-patent literature 2) of raffinose as galactose donor.Reported in addition and utilized p-nitrophenyl-α-D-galactopyranoside as the method for galactose donor (for example, referring to non-patent literature 3), utilize galactinol as the method for galactose donor (for example, referring to patent documentation 2), utilize galactobiose as method (for example,, referring to patent documentation 3) of galactose donor etc.
In addition, also reported that 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. 2688854th, Japanese Patent
Patent documentation 2: Japanese kokai publication hei 10-84973
Patent documentation 3: Japanese Patent Publication 8-24592
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 utilizing, the efficiency of alpha-galactosidase generation raffinose is high, so reported and utilized melibiose, p-nitrophenyl-α-D-galactopyranoside, galactinol, galactobiose as the example of raffinose synthesis material, but adopt in the situation of these methods, the price of the raw material using is high, is difficult at industrial manufacture raffinose.
On the other hand, owing to can utilizing low-cost raw material sucrose and semi-lactosi, so use the alpha-galactosidase derive 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 very favorable method while considering industrialness.But, utilize in the situation of these alpha-galactosidases, in resultant, generated the assorted trisaccharide beyond object raffinose, so need to be from the finished product separated impurity removal trisaccharide, and produced the problem of wastage of material.In situation by the alpha-galactosidase that derives from bright red samguineus for raffinose building-up reactions, the raffinose containing ratio in the oligose of generation is low to moderate 40%; In situation by the alpha-galactosidase that derives from Portugal's wine and women-sensual pursuits mortierella for raffinose building-up reactions, the raffinose containing ratio in the oligose of generation is low to moderate 60%, and prior art can not optionally be manufactured 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 and synthesize to material choice cheaply raffinose, 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 conducts in-depth research 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 object raffinose, thereby completed 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.
, there is following characteristic:
(1) effect: described enzyme has following character: take in the dehydration condensation that sucrose and semi-lactosi be raw material, 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: approximately 80,000.
Alpha-galactosidase as described in [1], it derives from the microorganism that belongs to Bacillus coagulans (Bacilluscoagulans).
Alpha-galactosidase as described in [2], wherein, described Bacillus coagulans is any one in Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, AKC006 strain.
And a varient, described Bacillus coagulans belongs in Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, AKC006 strain any one.
, its contain following (a), (b) or (c) in aminoacid sequence arbitrarily:
(a) aminoacid sequence that sequence numbering 2 represents;
(b) in the aminoacid sequence that sequence numbering 2 represents, lack, replace and/or added 1 or several amino acid whose aminoacid sequence, it has alpha-galactosidase activity;
(c) have the aminoacid sequence of more than 60% homogeny with the aminoacid sequence of sequence numbering 2 expressions, it has alpha-galactosidase activity.
, its coding contain following (a), (b) or (c) in the alpha-galactosidase of aminoacid sequence arbitrarily;
(a) aminoacid sequence that sequence numbering 2 represents;
(b) in the aminoacid sequence that sequence numbering 2 represents, lack, replace and/or added 1 or several amino acid whose aminoacid sequence, it has alpha-galactosidase activity;
(c) have the aminoacid sequence of more than 60% homogeny with the aminoacid sequence of sequence numbering 2 expressions, it has alpha-galactosidase activity.
, it contains following (a) or base sequence (b):
(a) base sequence that sequence numbering 1 represents;
(b) disappearance in the base sequence that sequence numbering 1 represents, replace and/or added the base sequence of 1 or several bases, and its coding has the protein of alpha-galactosidase activity.
, it contains [6] or [7] described alpha-galactosidase gene.
, its importing has [6] or [7] described alpha-galactosidase gene or [8] described recombinant vectors.
, it is cultivated [9] described transformant and obtains.
, the alpha-galactosidase described in its any one that contains [1]～[3], [5] or [10].
Enzyme composition as described in [11], wherein, said composition also contains at least one the above composition that is selected from alpha-glucosidase, beta-glucosidase enzyme, beta-galactosidase enzymes, cellulase, zytase, proteolytic enzyme, Galactanase, arabanase (arabinanase), mannonase rhamno-galacturonic acid enzyme, polygalacturonase, pectin methylesterase, pectin lyase and polygalacturonic acid lyase.
A synthetic agent, it contains the enzyme composition described in [11] or [12] any one.
A manufacture method for raffinose, is characterized in that, this manufacture method is used the alpha-galactosidase described in any one of [1]～[3], [5] or [10]; [11] enzyme composition or described in [12]; Or [13] described raffinose synthetic agent.
A manufacture method for raffinose, is characterized in that, the method is utilized microbial catalyst, and the microorganism that described microbial catalyst cultivation belongs to Bacillus coagulans obtains.
A kind of manufacture method of raffinose, it is characterized in that, the method is utilized microbial catalyst, and described microbial catalyst is cultivated and belonged in Bacillus coagulans AKC003 strain, AKC004 strain (FERM-ABP10948), AKC005 strain, AKC006 strain any one Bacillus coagulans and/or its varient and obtain.
A manufacture method for raffinose, is characterized in that, the method is utilized microbial catalyst, and described microbial catalyst is cultivated [9] described transformant and obtained.
The manufacture method of the raffinose as described in any one in [14]～[17], wherein, the raffinose containing ratio in the oligose of generation is more than 65%.
The manufacture method of the raffinose as described in any one in [14]～[18], wherein, the method is used sucrose and semi-lactosi as raw material.
The manufacture method of the raffinose as described in [19], wherein, the sucrose concentration in raw material is 30% (w/v)～90% (w/v), the galactose concentration in raw material is 2% (w/v)～45% (w/v).
By adopting the present invention, can optionally manufacture raffinose.
Accompanying drawing explanation
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.
Fig. 6 has provided the result to using the reaction solution of the sugar synthetic (embodiment 3) of the alpha-galactosidase of being purified by Bacillus coagulans AKC-004 strain to carry out HPLC analysis.
Fig. 7 has provided the result to using the reaction solution of the sugar synthetic (embodiment 5) of the alpha-galactosidase being obtained by recombination bacillus coli JM109 strain to carry out HPLC analysis.
Fig. 8 has provided the result to using the reaction solution of the sugar synthetic (embodiment 3) of the microbial catalyst of cultivating Bacillus coagulans AKC-004 strain and obtaining to carry out HPLC analysis.
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 in the present invention can also be the microorganism that belongs to Bacillus coagulans be the variant that maternal plant obtains.Bacillus coagulans AKC-003 strain, AKC-004 strain, AKC-005 strain, AKC-006 strain are all 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, Zhu Bo city, Hitachinaka County, Japan, 1 central authorities the 6th).Deposit number is as follows.It should be noted that, 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: take in the dehydration condensation that sucrose and semi-lactosi be raw material, 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: approximately 80,000.
As the concrete example of enzyme of the present invention, can enumerate and aspect the impact of substrate specificity and metal ion, there is the enzyme of following character.
Substrate specificity: using in the situation of p-nitrophenyl-α-D-galactopyranoside as substrate, degrading activity is the highest, is followed successively by thereafter melibiose, raffinose.On the other hand, p-nitrophenyl-β-D-galactopyranoside, lactose, sucrose do not decompose.
The impact of metal ion: add in each situation of potassium ion, calcium ion, magnesium ion, chromium ion, mn ion, cobalt ion, iron (II) ion or iron (III) ion, have no active reduction.On the other hand, add in each situation of nickel ion or zine ion, visible active reduction, adds in the situation of cupric ion, and activity decreased is maximum.
In addition, as the concrete example of enzyme of the present invention, can also enumerate the enzyme with following characteristic.
(5) (positive reaction) optimum temperature range: 35～50 ℃;
(6) equilibrium temperature scope: 45 ℃ following stable.
As for method for culturing microbes of the present invention, adopt conventional aeration-agitation cultivation or solid culture, and can adopt the microbial culture method conventionally carrying out.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 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, such as enumerating 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, calcium carbonate etc.From the viewpoint of the fertility of microorganism, culture temperature is preferably 25～80 ℃, and more preferably 40～65 ℃, more preferably 40～55 ℃.In addition, the pH of substratum can select at relative broad range, but from the viewpoint 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 be implemented as follows.
Culture medium culturing Bacillus coagulans AKC-004 strain with above-mentioned, is separated into thalline and filtrate by known methods such as centrifugation, filtrations by resulting nutrient solution.
In the thalline so obtaining, contain alpha-galactosidase, by using N,O-Diacetylmuramidase, ultrasonic disruption machine, French press etc. to carry out bacterial cell disruption, obtain the crude extract of alpha-galactosidase.According to culture condition etc., sometimes in culture supernatant, contain the activity of alpha-galactosidase, in this case, can using culture supernatant directly as the crude extract of alpha-galactosidase for below purification, also can be to operations such as culture supernatant concentrate, then the crude extract as alpha-galactosidase is used for purification below.
The crude extract so obtaining is carried out to separation, the alpha-galactosidase of can purifying thus by conventional purify protein method (such as ion exchange chromatography, hydrophobic chromatography method, 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, by dialysis, wait and make test liquid and suitable buffer-exchanged.In addition, can also to test liquid, concentrate in each stage.
In each stage of preferably purifying, contained alpha-galactosidase activity in each fraction of separation is measured, the fraction that activity is high puts together, 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) in, mix 150 μ L enzyme liquid, after 40 ℃ of reactions of carrying out about 5～30 minutes, reaction solution is added in 1mL aqueous sodium carbonate (1M), make enzyme deactivation, stopped reaction.Measure resulting solution in the absorption (degree of staining of resulting solution) of wavelength 420nm, use the working curve made from the p-NP of each concentration to calculate concentration.In addition, the unit of enzymic activity is as follows: dissociate for the 1 minute under these conditions enzyme amount of 1 μ mol p-NP of take is 1U.
The confirmation of purification degree and the mensuration of molecular weight of the alpha-galactosidase through purifying can be undertaken by electrophoresis or gel filtration chromatography etc.In addition, zymologic property can be studied as follows: change temperature of reaction or reaction pH, measure enzymic activity; Or various enzyme inhibitorss or metal ion etc. are added in reaction solution, measure remaining activity.In addition, by alpha-galactosidase is measured to enzymic activity under various pH conditions or after placing certain hour under temperature condition again, can investigate thus stable pH range and equilibrium temperature scope.In addition, by changing concentration of substrate, react, can obtain the Michaelis-Menton constant (K of alpha-galactosidase to each substrate m), top speed (V max).
Alpha-galactosidase gene of the present invention can obtain as described below by example.
Culture medium culturing Bacillus coagulans AKC-004 strain with above-mentioned, is separated into thalline and filtrate by known methods such as centrifugation, filtrations by resulting nutrient solution.Use N,O-Diacetylmuramidase, ultrasonic disruption machine, French press etc. the thalline so obtaining to be carried out to bacterial cell disruption, separated chromosomal DNA.With various restriction enzymes, the chromosomal DNA so obtaining is digested, obtain DNA fragment.Use the chromosomal DNA fragment so obtaining, the total length or a part of DNA fragment that by utilizing the known means such as Shotgun cloning, inverse PCR method to obtain, contain alpha-galactosidase gene.The base sequence of the DNA fragment that contains alpha-galactosidase gene herein obtaining can be by utilizing the known method of DNA sequencer etc. to resolve, and clear and definite its base sequence.In addition, only know in the situation of part base sequence of alpha-galactosidase gene, can take this base sequence as basis, again obtain the DNA fragment that contains alpha-galactosidase gene.And, by repeating this operation, can separate the base sequence of bright alpha-galactosidase gene total length.The base sequence of the alpha-galactosidase gene as above understood of can take is basis, the DNA fragment of the total length that obtains by PCR method or with the known method of restriction enzyme containing alpha-galactosidase gene.So, according to the base sequence of understood alpha-galactosidase gene, 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., or can be to have the aminoacid sequence of more than 60% homogeny with this aminoacid sequence.That is, alpha-galactosidase of the present invention be contain following (a), (b) or (c) in the alpha-galactosidase of aminoacid sequence arbitrarily.
(a) aminoacid sequence that sequence numbering 2 represents;
(b) in the aminoacid sequence that sequence numbering 2 represents, lack, replace and/or added 1 or several amino acid whose aminoacid sequence, it has alpha-galactosidase activity;
(c) have the aminoacid sequence of more than 60% homogeny with the aminoacid sequence of sequence numbering 2 expressions, it has alpha-galactosidase activity.
In addition, according to the present invention, provide a kind of coding contain following (a), (b) or (c) in the alpha-galactosidase gene of the alpha-galactosidase of aminoacid sequence arbitrarily.
(a) aminoacid sequence that sequence numbering 2 represents;
(b) in the aminoacid sequence that sequence numbering 2 represents, lack, replace and/or added 1 or several amino acid whose aminoacid sequence, it has alpha-galactosidase activity;
(c) have the aminoacid sequence of more than 60% homogeny with the aminoacid sequence of sequence numbering 2 expressions, 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 particularly preferably 1～5 amino acid.
" aminoacid sequence with more than 60% homogeny " in above-mentioned (c) typically refers to and has more than 60% the aminoacid sequence of the homogeny of (preferably more than 70%, more preferably more than 80%, more preferably more than 90%, more more preferably more than 95%, being particularly preferably more than 99%).
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 that sequence numbering 1 represents;
(b) disappearance in the base sequence that sequence numbering 1 represents, 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 particularly preferably 1～5 base.
Alpha-galactosidase gene of the present invention can not damage by known genetic manipulation method the variation of peptide of the character of catalyzed reaction originally, such mutant gene refers to artificial variation's gene of being made by alpha-galactosidase gene of the present invention by the method for gene engineering, and this artificial variation's gene can be by with the special alternative method in position, obtain by the range gene engineering methods such as method that artificial abnormal dna replaces the specific DNA fragment of goal gene.That is, the method for the variations such as amino acid in alpha-galactosidase lacks, replaces as making, interpolation, can utilize PCR method, error-prone PCR method, DNA reorganization method, make the known methods such as method that merge enzyme.
The DNA fragment that contains complete alpha-galactosidase structure gene so obtaining can be inserted to the multiple clone site of expression vector (for example pBluescriptII KS (+)) for intestinal bacteria and connect, form new recombinant plasmid.For this plasmid vector, due to importing has can be using the lac promotor of the gene effective expression connecting as alien gene in intestinal bacteria, so by cultivating, in intestinal bacteria, import the resulting transformant of recombinant plasmid, can great expression alpha-galactosidase.
In addition, except above-mentioned, can also utilize various host microorganisms, carrier to carry out great expression alpha-galactosidase, for example, by cultivating the transformant of bridge stone bacillus pumilus (Brevibacilluschoshinensis), can great expression alpha-galactosidase.In more detail, as the carrier that imports alpha-galactosidase gene of the present invention, what be applicable to has by the phage of the breeding of can restraining oneself in host microorganism body or the carrier for gene recombination that plasmid is made, as phage vector, for example using and belong in the situation of colibacillary microorganism as host microorganism, can use λ gt λ C, λ gt λ B etc.In addition, as plasmid vector, for example using in the situation of intestinal bacteria as host microorganism, can use the pET carriers (Novagen) such as plasmid pET-3a, pET-11a, pET-32a or pBR322, pBR325, pACYC184, pUC12, pUC13, pUC18, pUC19, pUC118, pIN I, BluescriptKS+; Using in the situation of withered grass bacterium as host, can use pWH1520, pUB110, pKH300PLK; In the situation that the actinomycetes of take are host, can use pIJ680, pIJ702; Using yeast particularly in the situation of yeast saccharomyces cerevisiae as host, can use YRp7, pYC1, YEp13 etc.Such carrier is cut off with restriction enzyme, make the end that generates with identical for cutting off the DNA end of restriction enzyme generation of alpha-galactosidase gene of the present invention, thereby make carrier fragment, utilize DNA ligase to make alpha-galactosidase gene fragment of the present invention and the combination of carrier fragment according to ordinary method, the DNA of coding alpha-galactosidase gene of the present invention can be imported to object carrier thus.
As the host microorganism that imports plasmid, as long as recombinant DNA can be stablized and restrain oneself and breed, for example host microorganism is to belong in 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 in the situation of microorganism of Bacillus, can utilize Bacillus subtillis, Bacillus megatherium etc.; Microorganism host is to belong in the situation of actinomycetic microorganism, can utilize lead-changing penicillium chain enzyme bacteria TK24 etc.; Microorganism host is to belong in the situation of microorganism of yeast saccharomyces cerevisiae, can utilize yeast saccharomyces cerevisiae INVSC1 etc.
In addition, while manufacturing alpha-galactosidase of the present invention by microbial, with nutritional medium, cultivate this microbial, in thalline or in nutrient solution, produce alpha-galactosidase of the present invention, after cultivation finishes, by methods such as filtration or centrifugations, by resulting culture, gather thalline, next, by the method for the enzymes such as mechanical means or N,O-Diacetylmuramidase, destroy this thalline, or add as required EDTA and/or suitable tensio-active agent etc., the aqueous solution of concentrated alpha-galactosidase of the present invention or not concentrate and directly pass through the classification of sulphur ammonium, gel-filtration, the adsorption chromatographies such as affinity chromatography, ion exchange chromatography is processed, can obtain thus the alpha-galactosidase of the present invention that purity is good.
About the culture condition of microbial, while selecting culture condition, consider its nutritional-physiological character, conventionally by liquid culture, cultivate, it is industrial that to carry out deep aeration-agitation cultivation be favourable.As the nutrition source of substratum, can use widely normally used nutrition source in microorganism culturing.The scope that alpha-galactosidase of the present invention can be bred and can be produced to culture temperature can in microorganism suitably changes, and take intestinal bacteria as example, and culture temperature is preferably 10～45 ℃ of left and right, more preferably 20～30 ℃ of left and right.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, in suitable period, finishes to cultivate, and take intestinal bacteria as example, and incubation time is about 12～48 hours conventionally.The scope that alpha-galactosidase of the present invention can be bred and can be produced to medium pH can on bacterium suitably changes, and take intestinal bacteria as example, and medium pH is preferably left and right, pH6～8.
According to the present invention, while using described enzyme, only otherwise suppress the effect of enzyme of the present invention, extent of purification etc. is not particularly limited, except using the enzyme of the present invention through purifying, also can use the mixture that contains this enzyme.
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 one the above composition that is for example selected from alpha-glucosidase, beta-glucosidase enzyme, beta-galactosidase enzymes, cellulase, zytase, proteolytic enzyme, Galactanase, arabanase, mannonase 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.
In the present invention, as microbial catalyst, except Bacillus coagulans AKC-004 strain, can also use above-mentioned transformant.In the present invention, as the microbial catalyst for the manufacture of raffinose, the microorganism itself that can utilize the cultural method that undertaken by routine to obtain, and without from microorganism purification alpha-galactosidase.In addition, sometimes can utilize microbial culture medium, microorganism culturing supernatant liquor.On the other hand, also can after cleaning the microorganism obtaining by culture method, water or damping fluid etc. be used again as required.For example, can use the nutrient solution of the microorganism through cultivating; Or by centrifugation, with damping fluid, clean etc. the microbial suspension obtaining; Suspend or be dissolved with the aqueous solution of the handled thing (such as the broken thing of microorganism etc.) of microorganism or microorganism; Fixing microorganism or the microbiological treatment thing with entrapping method, crosslinking or carrier combined techniques.The example of the fixation support during as being fixed, can enumerate 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, the 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.While 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 is high, but when galactose concentration is too high, the intermolecular condensation of semi-lactosi has suppressed the dehydration condensation between sucrose and semi-lactosi, 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, be preferably prepared into all soluble concentration of sucrose in raw material and semi-lactosi.
When the enzyme composition that uses alpha-galactosidase of the present invention, contains alpha-galactosidase, the raffinose synthetic agent that contains enzyme composition or microbial catalyst are manufactured raffinose, from the viewpoint 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, from the viewpoint of the stability of enzyme, and preferred pH2.0～10.0, more preferably pH3.0～7.5, more preferably 3.5～6.0.Reaction times is according to the consumption of enzyme and different, and while considering industrial utilization, being conventionally preferably 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.
In the present invention, in the raffinose building-up reactions of the raffinose synthetic agent that uses alpha-galactosidase, the enzyme composition that contains alpha-galactosidase or contain enzyme composition, raffinose accumulates 0.5% when above in reaction soln, raffinose containing ratio in the oligose generating can be brought up to more than 65%, under preferred condition, can bring up to more than 80%.In reaction soln, accumulate raffinose and reach more than 0.75% and then when 1.0% is above, can realize the high containing ratio of raffinose in the oligose generating in the present invention.
In addition, use in the raffinose building-up reactions of microorganism itself, due to the impact of assorted enzyme, the raffinose containing ratio in the oligose of generation is low conventionally, and use in the raffinose building-up reactions of microbial catalyst of the present invention, the raffinose containing ratio in the oligose of generation is high.
Raffinose containing ratio in the oligose of the generation that the present invention obtains can be measured by following method.
After raffinose building-up reactions finishes, by 25 times of reaction solution dilutions, at 99 ℃, keep 10 minutes, thus stopped reaction.After reaction stops, by centrifugation, remove microorganism, by high speed liquid chromatography (HPLC), resulting reaction soln is carried out quantitatively.Measure the Hypercarb post that uses Thermoelectron society to manufacture, detector uses RI.Raffinose containing ratio in the oligose generating calculates by (peak area of raffinose)/(peak area of the oligose of generation) * 100 according to the Area Ratio at each peak detecting in HPLC analysis of spectra.
As the raffinose of manufacturing by method of the present invention is purified, separated method, can utilize the purification processing method of common employing.That is, such as removing microbial catalyst by centrifugation, the film processing that utilizes MF (micro-filtration) film or UF (ultrafiltration) film etc., pressure filter etc.; By cation-exchange chromatography, anion-exchange chromatography isochromatic spectrum, process or the desalting treatment such as dialysis is removed the salt brought into by damping fluid, substratum etc. etc.; And by cation-exchange chromatography, anion-exchange chromatography, high speed liquid chromatography, activated carbon chromatography isochromatic spectrum process or utilize the crystallization of poor solubility etc. to process, other ordinary methods carry out separation, the purification of alpha-galactooligosaccharide.Chromatography can be used separately these methods, 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.
Below by embodiment, specifically describe, but the present invention is not subject to any restriction of these embodiment.
Embodiment
Embodiment 1
By Bacillus coagulans AKC-004 strain (deposit number FERM P-21092 (transferring as FERM-ABP10948); Preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological preservation center) with TBAB (Tryptones blood agar basis, Tryptose Blood Agar Base) culture plate (Difco), at 55 ℃, cultivate 1 day, 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, with transfering loop, from above-mentioned culture plate inoculation bacterium colony, at 50 ℃, 180rpm, carry out 25 hours rotational oscillations and cultivate, using that this cultivates seed as tank.
[table 1] substratum forms
Raffinose glycerol yeast extracting solution peptone MgSO 4·7H 2O CaCl 20.01M citric acid sodium acetate trihydrate KH 3PO 4 Na 2HPO 4·12H 2O micro-metals solution distilled water 10g 10g 10g 10g 0.5g 0.04g 0.5mL 0.132g 0.544g 2.14g 0.5mL 1L
Micro-metals solution composition
CH 3COONa FeSO 4·7H 2O MnCl 2·4H 2O CoCl 2·6H 2O ZnSO 4·7H 2O CuCl 2·2H 2O NaMoO 4·2H 2O H 3BO 4 NiCl 2·6H 2O distilled water 12.8g 1g 0.5g 0.3g 0.4g 50mg 50mg 20mg 20mg 1L
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 tank of 15mL and cultivate seed, with 50 ℃, the condition aeration-agitation of 200rpm, 0.2vvm, cultivate 48 hours.
Then, this nutrient solution, 4 ℃ of centrifugations of carrying out 10,000g * 30 minute, is removed to supernatant liquor, reclaim 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, 4 ℃ of centrifugations of carrying out 10,000g * 30 minute, is removed to supernatant liquor, reclaim thalline.
Thalline with after Lysis buffer solution for cleaning is suspended in above-mentioned Lysis damping fluid again, is quantitatively 150mL.Add therein 0.02% N,O-Diacetylmuramidase (Sigma society manufactures, from albumen), with 37 ℃, the condition vibration of 120rpm 13 hours, carry out bacterial cell disruption.
Solution after bacterial cell disruption, 4 ℃ of centrifugations of carrying out 10,000g * 30 minute, is removed to thalline residue, reclaim supernatant liquor.
In this supernatant liquor, add ammonium sulfate, and reach 37.5% saturated, at 4 ℃, place a night, generate precipitation.4 ℃ of centrifugations of carrying out 10,000g * 30 minute, remove this precipitation, reclaim supernatant liquor.
In this supernatant liquor, add ammonium sulfate, and reach 54.5% saturated, at 4 ℃, place a night, generate precipitation.4 ℃ of centrifugations of carrying out 10,000g * 30 minute, reclaim this precipitation, and be dissolved in the 20mM phosphoric acid buffer of pH7.0, at 4 ℃, phosphoric acid buffer same as described above is carried out to night dialysis.
The supernatant that makes to obtain by dialysis be adsorbed on upper with " DEAE Sepharose FF " after phosphoric acid buffer equilibration same as described above (GE Healthcare Bio-Sciences Co., Ltd.) after, the E-test of the 20mM phosphoric acid buffer of the pH7.0 by containing 0～0.4M sodium-chlor is by enzyme wash-out out.
Collect the above-mentioned active fraction eluting, 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 the 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 by enzyme wash-out out.
Collect the above-mentioned active fraction eluting, use average mark level molecular weight 10,000 ultra-filtration membrane concentrates, it is adsorbed on after " MonoQ5/50GL " (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 by enzyme wash-out out.
Collect the above-mentioned active fraction eluting, use average mark level molecular weight 10,000 ultra-filtration membrane concentrates, after making it be filled in " HiLoad16/60 Superdex 200 " (the GE Healthcare Bio-Sciences Co., Ltd.) using after the 20mM phosphoric acid buffer equilibration of pH7.0, with identical damping fluid, carry out wash-out.
Collect the above-mentioned active fraction eluting, use the ultra-filtration membrane of average mark level molecular weight 10,000 to concentrate, make 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 50 μ L through the enzyme purification liquid of the present invention of 100 times of dilutions, 40 ℃ of reactions 5 minutes.After reaction, by the p-NP to free, quantitatively measure activity, obtaining and establishing maximum activity is the relative reactivity of 100 o'clock.It the results are shown in Figure 1.It should be noted that, the damping fluid using 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, at each pH, carry out 140 minutes heat treated in 45 ℃, measure its remaining activity, similarly obtain with above-mentioned that to establish maximum activity be the relative reactivity of 100 o'clock.It the results are shown in Figure 2.It should be noted that, the kind of the damping fluid of each pH using 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 10 μ L through the enzyme purification liquid of the present invention of 20 times of dilutions, the scopes reactions of 20～60 ℃ 5 minutes.After reaction, free p-NP is carried out quantitatively, measure thus actively, obtaining and establishing maximum activity is the relative reactivity of 100 o'clock.It the results are shown in Figure 3.
(4) equilibrium temperature scope
The 100mM sodium acetate buffer that uses pH4.5, carries out the heat treated of 20 minutes each temperature of 30～55 ℃, measure its remaining activity, similarly obtains with above-mentioned that to establish maximum activity be the relative reactivity of 100 o'clock.It the results are shown in Figure 4.
(5) molecular weight
Use " the レ デ イ mono-ゲ Le J " that separating gel concentration is 10% (Japanese Bio-RadLaboratories Co., Ltd.), by SDS-polyacrylamide gel electrophoresis, obtain molecular weight.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 the 100mM sodium acetate buffer of the pH4.5 that contains the various substrates of 10mM at 150 μ L, mix 50 μ L through the enzyme purification liquid of the present invention of 100 times of dilutions, at 40 ℃, carry out reaction in 20 minutes.But the reaction for using p-nitrophenyl-α-D-galactopyranoside as substrate, will be set as 5 minutes the reaction times.After reaction, to free p-NP quantitatively or use " ShodexSUGAR " (Showa Denko K. K) post to analyze by high speed liquid chromatography, the substrate decomposing 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 pH4.5 that is dissolved with 6mM p-nitrophenyl-α-D-galactopyranoside, add each metal ion species, making ultimate density is 1mM, and quantitatively to 150 μ L, mix 50 μ L through the enzyme purification liquid of the present invention of 100 times of dilutions, 40 ℃ of reactions 20 minutes.After reaction, free p-NP is carried out quantitatively, measure thus actively, obtaining and establishing maximum activity is the relative reactivity of 100 o'clock.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 the purification alpha-galactosidase described in 0.5U embodiment 1, added 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 1.5mL polypropylene tubulation and mixed, at 60 ℃, 1,200rpm reacts.Reaction started after 45 hours, and 20 μ L among collection reaction solution, after diluting with 480 μ L distilled water, process 10 minutes at 99 ℃, make thus enzyme heat inactivation.Dilution liquid glucose after heat inactivation is cooled to normal temperature, use " Hypercarb " (manufacture of Thermoelectron society) post, by high speed liquid chromatography, analyze.It the results are shown in Figure 6.Raffinose containing ratio in the oligose generating is 82%, and the raffinose concentration in reaction solution is 1.67 % by weight.
Embodiment 4
(1) preparation of chromosomal DNA
The chromosomal DNA of preparing Bacillus coagulans AKC-004 strain by ordinary method.The nutrient solution of the Bacillus coagulans AKC-004 strain that 60mL is obtained with the method described in embodiment 1 carries out centrifugation, reclaims thalline.Resulting thalline is suspended in Lysis damping fluid (50mMTris-HCl (pH 8.0), 20mM EDTA, 50mM glucose), fully cleans.Carry out centrifugation, reclaim after thalline, be again suspended in Lysis damping fluid, add therein N,O-Diacetylmuramidase, at 37 ℃, cultivate 45 minutes.Then, add SDS and RNase, at 37 ℃, cultivate 45 minutes., add Proteinase K, at 50 ℃, slowly vibrate 60 minutes thereafter.After the solution herein obtaining is processed with benzene phenol-chloroform and chloroform, by ethanol precipitation, the nucleic acid of separating out is entangled on glass pipet, reclaims.This nucleic acid is dried, and is again dissolved in TE after cleaning with 70% ethanol.By this operation, prepare the chromosomal DNA of about 1mg.
(2) separation of alpha-galactosidase gene
The chromosomal DNA of preparing according to above-mentioned (1) is designed for the PCR primer of amplification alpha-galactosidase gene fragment and synthesizes.Can the sequencing results based on being derived from the alpha-galactosidase gene of known several microorganisms carry out the design of primer, as the synthetic oligomer DNA with 5 '-GAAGTITACGGITTYAGYYTTGTITACAGYGG-3 ' (sequence numbering 3) sequence of upstream primer, as the synthetic oligomer DNA with 5 '-CCAAACCAICCRTCRTCIARIACRAA-3 ' (sequence numbering 4) sequence of downstream primer.It should be noted that, in sequence, I represents inosine, and Y represents C or T, and R represents A or G.Use the PCR primer obtaining herein, above-mentioned (1) the middle chromosomal DNA prepared of take is template, carries out the amplification of alpha-galactosidase gene fragment by PCR method, obtains the alpha-galactosidase gene fragment that contains 380 base pairs.Use DNA sequencer, the base sequence of the gene fragment herein obtaining is resolved.
For the base sequence of the alpha-galactosidase gene fragment that obtains based on parsing obtains the DNA fragment that contains alpha-galactosidase gene total length, designed and synthesized inverse PCR PCR primer.As the synthetic oligomer DNA with 5 '-TGATCAACAACTGGGAAAGCGACCT-3 ' (sequence numbering 5) sequence of upstream primer, as the synthetic oligomer DNA with 5 '-GAACTGGTCCTGCTGCACAATTCC-3 ' (sequence numbering 6) sequence of downstream primer.Then, for the preparation of the template of inverse PCR.The chromosomal DNA of preparation in above-mentioned (1), with after restriction enzyme HindIII digestion, is carried out to recirculation by resulting DNA fragment with T4 DNA Ligase, make the template of inverse PCR.For this template, use as above synthetic inverse PCR primer, the amplification of the DNA fragment that contains alpha-galactosidase gene by PCR method, the PCR product being formed by 4500 base pairs of the complete sequence that obtains containing alpha-galactosidase gene.
(3) parsing of base sequence
Use DNA sequencer, the PCR product obtaining in (2) is determined the base sequence of alpha-galactosidase gene.Result solution is read the structure gene of the alpha-galactosidase of 2190 base pairs with the DNA base sequence starting from 5 ' end shown in sequence numbering 1.This sequence is undiscovered novel gene so far.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 the aminoacid sequence starting from N-terminal such shown in sequence numbering 2.The result of database retrieval is as follows: this aminoacid sequence has 57% homogeny with the alpha-galactosidase (AgaN) that derives from stearothermophilus ground bacillus (Geobacillus stearothermophilus), there is 56% homogeny with the alpha-galactosidase (AgaA) that derives from stearothermophilus ground bacillus, there is 56% homogeny with the alpha-galactosidase (AgaB) that derives from stearothermophilus ground bacillus, there is 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
The sequence of the alpha-galactosidase gene based on obtaining in (3), designs and synthesizes the PCR primer in the region of the SD sequence that contains alpha-galactosidase gene for increasing, structure gene, terminator codon.The base sequence of the PCR product that contains 4500 base pairs based on obtaining in (2) carries out the design of PCR primer, as the synthetic oligomer DNA with 5 '-TAAGGTAAAGCAGATGTGCCATT-3 ' (sequence numbering 7) sequence of upstream primer, as the synthetic oligomer DNA with 5 '-TTACTCGTACACCGCCTC-3 ' (sequence numbering 8) sequence of downstream primer.Using the PCR product that contains 4500 base pairs that obtains in (2) as template, use synthetic PCR primer, by PCR method, increase, obtain the PCR product that contains 2325 base pairs.The PCR product herein obtaining is carried out to end smoothing, phosphorylation.
With restriction enzyme EcoRV, pBluescript II KS (+) carrier digested and carry out dephosphorylation, on this carrier, connecting obtained abovely through the PCR of end smoothing, phosphorylation product, making new plasmid vector pBlue/agaA.In this plasmid vector, import have can the gene that effective expression connects as alien gene in intestinal bacteria lac promotor, can effectively express, manufacture alpha-galactosidase.By heat shock, resulting plasmid vector is transformed into and is utilized in e. coli jm109 strain competent cell prepared by Calcium Chloride Method, make recombinant microorganism.
(5) cultivation of transformant and the expression of alpha-galactosidase
By the recombination bacillus coli JM109-pBlue/agaA making in (4) with the 30mL LB substratum that contains 100mg/L penbritin 37 ℃ of shaking culture 24 hours.After cultivation, carry out centrifugation and reclaim recombination bacillus coli.Thalline is suspended in same damping fluid after cleaning with 100mM sodium acetate buffer (pH5.0) again, with ultrasonic disruption machine, carries out fragmentation.By aforesaid method, measure the alpha-galactosidase activity of this fragmentation liquid, result is 19.9U/ nutrient solution (mL).
Embodiment 5
Get passing through described in 1.0U embodiment 4 and cultivate the resulting alpha-galactosidase of recombinant chou, added 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 1.5mL polypropylene tubulation and mixed, at 60 ℃, 1,200rpm reacts.Reaction started after 16 hours, and 20 μ L among collection reaction solution, after diluting with 480 μ L distilled water, carry out processing for 10 minutes at 99 ℃, make thus enzyme heat inactivation.Dilution liquid glucose after heat inactivation is cooled to after normal temperature, use " Hypercarb " (manufacture of Thermoelectron society) post, by high speed liquid chromatography, analyze.It the results are shown in Figure 7.Raffinose containing ratio in the oligose generating is 82%, and the raffinose concentration in reaction solution is 1.20 % by weight.
Embodiment 6
By Bacillus coagulans AKC-004 strain (deposit number FERM P-21092 (transferring as FERM-ABP10948); Preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological preservation center) with TBAB (Tryptones blood agar basis) culture plate (Difco), at 55 ℃, cultivate 1 day, form bacterium colony.1 transfering loop culture is inoculated in the substratum described in the 30mL table 1 being divided in 150mL Erlenmeyer flask, at 55 ℃, with 150rpm, cultivates 2 days.
This was cultivated after 2 days, and the cultivation thalline of 10mL amount is recovered in 15mL pipe.After having the 15mL pipe of nutrient solution centrifugal with 10000rpm recovery, remove supernatant liquor.Then, add the 100mM sodium acetate buffer (pH5.0) of 1mL, after again suspending, suspension is moved in the polypropylene tubulation of 2mL.Again pipe is carried out centrifugally, remove after supernatant liquor, add 300 μ L liquid glucoses (containing the 100mM sodium acetate buffer (pH5.0) of 62.5% sucrose, 12.5% semi-lactosi), with vortex stirrer, thalline is fully suspended, start 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 building-up reactions starts after 16h, reclaims 40 μ L reaction solutions, and it is fully mixed with 960 μ L distilled water, at 99 ℃, enzyme is carried out the heat inactivation of 10 minutes.The temperature of this dilution liquid glucose is dropped to after normal temperature, use " Hypercarb " (manufacture of Thermoelectron society) post, by high speed liquid chromatography, analyze, it the results are shown in Figure 8.Raffinose containing ratio in the oligose generating is 80% (the raffinose concentration in reaction solution is 0.70 % by weight).
Embodiment 7
By Bacillus coagulans AKC-003 strain (deposit number FERM P-21091; Preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological preservation center) with TBAB (Tryptones blood agar basis) culture plate (Difco), at 55 ℃, cultivate 1 day, form bacterium colony.1 transfering loop culture is inoculated in the substratum described in the 30mL table 1 being divided in 150mL Erlenmeyer flask, at 55 ℃, with 150rpm, cultivates 2 days.
This was cultivated after 2 days, and the cultivation thalline of 10mL amount is recovered in 15mL pipe.After having the 15mL pipe of nutrient solution centrifugal with 10000rpm recovery, remove supernatant liquor.Then, add the 100mM sodium acetate buffer (pH5.0) of 1mL, after again suspending, suspension is moved in the polypropylene tubulation of 2mL.Again pipe is carried out centrifugally, remove after supernatant liquor, add 300 μ L liquid glucoses (containing the 100mM sodium acetate buffer (pH5.0) of 62.5% sucrose, 12.5% semi-lactosi), with vortex stirrer, thalline is fully suspended, start 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 building-up reactions starts after 38h, reclaims 40 μ L reaction solutions, and it is fully mixed with 960 μ L distilled water, at 99 ℃, enzyme is carried out the heat inactivation of 10 minutes.The temperature of this dilution liquid glucose is dropped to after normal temperature, use " Hypercarb " (manufacture of Thermoelectron society) post, by high speed liquid chromatography, analyze, now, the raffinose containing ratio in the oligose of generation is 80% (the raffinose concentration in reaction solution is 0.76 % by weight).
Embodiment 8
By Bacillus coagulans AKC-005 strain (deposit number FERM P-21093; Preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological preservation center) with TBAB (Tryptones blood agar basis) culture plate (Difco), at 55 ℃, cultivate 1 day, form bacterium colony.1 transfering loop culture is inoculated in the substratum described in the 30mL table 1 being divided in 150mL Erlenmeyer flask, at 55 ℃, with 150rpm, cultivates 2 days.
This was cultivated after 2 days, and the cultivation thalline of 10mL amount is recovered in 15mL pipe.After having the 15mL pipe of nutrient solution centrifugal with 10000rpm recovery, remove supernatant liquor.Then, add the 100mM sodium acetate buffer (pH5.0) of 1mL, after again suspending, suspension is moved in the polypropylene tubulation of 2mL.Again pipe is carried out centrifugally, remove after supernatant liquor, add 300 μ L liquid glucoses (containing the 100mM sodium acetate buffer (pH5.0) of 62.5% sucrose, 12.5% semi-lactosi), with vortex stirrer, thalline is fully suspended, start 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 building-up reactions starts after 38h, reclaims 40 μ L reaction solutions, and it is fully mixed with 960 μ L distilled water, at 99 ℃, enzyme is carried out the heat inactivation of 10 minutes.The temperature of this dilution liquid glucose is dropped to after normal temperature, use " Hypercarb " (manufacture of Thermoelectron society) post, by high speed liquid chromatography, analyze, now, the raffinose containing ratio in the oligose of generation is 91% (the raffinose concentration in reaction solution is 0.32 % by weight).
Embodiment 9
By Bacillus coagulans AKC-006 strain (deposit number FERM P-21094; Preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological preservation center) with TBAB (Tryptones blood agar basis) culture plate (Difco), at 55 ℃, cultivate 1 day, form bacterium colony.1 transfering loop culture is inoculated in the substratum described in the 30mL table 1 being divided in 150mL Erlenmeyer flask, at 55 ℃, with 150rpm, cultivates 2 days.
This was cultivated after 2 days, and the cultivation thalline of 10mL amount is recovered in 15mL pipe.After having the 15mL pipe of nutrient solution centrifugal with 10000rpm recovery, remove supernatant liquor.Then, add the 100mM sodium acetate buffer (pH5.0) of 1mL, after again suspending, suspension is moved in the polypropylene tubulation of 2mL.Again pipe is carried out centrifugally, remove after supernatant liquor, add 300 μ L liquid glucoses (containing the 100mM sodium acetate buffer (pH5.0) of 62.5% sucrose, 12.5% semi-lactosi), with vortex stirrer, thalline is fully suspended, start 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 building-up reactions starts after 38h, reclaims 40 μ L reaction solutions, and it is fully mixed with 960 μ L distilled water, at 99 ℃, enzyme is carried out the heat inactivation of 10 minutes.The temperature of this dilution liquid glucose is dropped to after normal temperature, use " Hypercarb " (manufacture of Thermoelectron society) post, by high speed liquid chromatography, analyze, now, the raffinose containing ratio in the oligose of generation is 76% (the raffinose concentration in reaction solution is 0.86 % by weight).
Utilizability in industry
The application of the invention, can provide the method for using the raffinose of material choice manufacture cheaply.
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 Asn 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 (16)

1. a Bacillus coagulans, described Bacillus coagulans belongs to any one in Bacillus coagulans AKC003 strain, AKC004 strain, AKC005 strain, AKC006 strain, the preserving number of described AKC003 strain is FERMBP-11192, and the preserving number of described AKC004 strain is that the preserving number of FERMBP-10948, described AKC005 strain is that the preserving number of FERMBP-11193, described AKC006 strain is FERMBP-11194.
2. an alpha-galactosidase, its aminoacid sequence is the aminoacid sequence of following (a):
(a) aminoacid sequence that sequence numbering 2 represents.
3. an alpha-galactosidase gene, the alpha-galactosidase of the aminoacid sequence of its coding following (a):
(a) aminoacid sequence that sequence numbering 2 represents.
4. an alpha-galactosidase gene, its base sequence is the base sequence of following (a):
(a) base sequence that sequence numbering 1 represents.
5. a recombinant vectors, it contains the alpha-galactosidase gene described in claim 3 or 4.
6. a transformant, its importing have the right alpha-galactosidase gene or recombinant vectors claimed in claim 5 described in requirement 3 or 4.
7. an alpha-galactosidase, it is cultivated transformant claimed in claim 6 and obtains.
8. an enzyme composition, it contains the alpha-galactosidase described in claim 2 or 7.
9. enzyme composition as claimed in claim 8, wherein, said composition also contains at least one the above composition that is selected from alpha-glucosidase, beta-glucosidase enzyme, beta-galactosidase enzymes, cellulase, zytase, proteolytic enzyme, Galactanase, arabanase, mannonase rhamno-galacturonic acid enzyme, polygalacturonase, pectin methylesterase, pectin lyase and polygalacturonic acid lyase.
10. a raffinose synthetic agent, it contains the enzyme composition described in claim 8 or 9.
The manufacture method of 11. 1 kinds of raffinoses, is characterized in that, this manufacture method right to use requires the alpha-galactosidase described in 2 or 7; Enzyme composition described in claim 8 or 9; Or raffinose synthetic agent claimed in claim 10.
The manufacture method of 12. 1 kinds of raffinoses, it is characterized in that, the method is utilized microbial catalyst, the microorganism that described microbial catalyst cultivation belongs to Bacillus coagulans obtains, described Bacillus coagulans is any one in Bacillus coagulans AKC003 strain, AKC004 strain, AKC005 strain, AKC006 strain, the preserving number of described AKC003 strain is FERMBP-11192, and the preserving number of described AKC004 strain is that the preserving number of FERMBP-10948, described AKC005 strain is that the preserving number of FERMBP-11193, described AKC006 strain is FERMBP-11194.
The manufacture method of 13. 1 kinds of raffinoses, is characterized in that, the method is utilized microbial catalyst, and described microbial catalyst is cultivated transformant claimed in claim 6 and obtained.
The manufacture method of 14. raffinoses as described in any one in claim 11～13, wherein, the raffinose containing ratio in the oligose of generation is more than 65%.
The manufacture method of 15. raffinoses as described in any one in claim 11～13, wherein, the method is used sucrose and semi-lactosi as raw material.
The manufacture method of 16. raffinoses as claimed in claim 15, wherein, the sucrose concentration in raw material is 30% (w/v)～90% (w/v), the galactose concentration in raw material is 2% (w/v)～45% (w/v).
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CN101974583A (en) * 2010-09-20 2011-02-16 南京农业大学 Method for synthesizing alpha-galacto-oligosaccharide by using alpha-galactosidase
EP3464580A1 (en) 2016-05-24 2019-04-10 Novozymes A/S Compositions comprising polypeptides having galactanase activity and polypeptides having beta-galactosidase activity
AU2017270269A1 (en) 2016-05-24 2018-11-15 Novozymes A/S Compositions comprising polypeptides having galactanase activity and polypeptides having beta-galactosidase activity
MX2018014234A (en) * 2016-05-24 2019-03-28 Novozymes As Polypeptides having alpha-galactosidase activity and polynucleotides encoding same.
CN106282142B (en) * 2016-08-05 2019-05-31 南京林业大学 A kind of preparation method for the alpha-galactosidase that beta-Mannosidase content is low
CN110066780A (en) * 2019-03-29 2019-07-30 上海国龙生物技术集团有限公司 A kind of feeding alpha-galactosidase solid fermenting production technology
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