CN103614322B - The streptomycete producing Glycosylase and the application prepared in bio-transformation in Cucurbitacin B thereof - Google Patents
The streptomycete producing Glycosylase and the application prepared in bio-transformation in Cucurbitacin B thereof Download PDFInfo
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- CN103614322B CN103614322B CN201310597855.2A CN201310597855A CN103614322B CN 103614322 B CN103614322 B CN 103614322B CN 201310597855 A CN201310597855 A CN 201310597855A CN 103614322 B CN103614322 B CN 103614322B
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- CVKKIVYBGGDJCR-SXDZHWHFSA-N Cucurbitacin B Natural products CC(=O)OC(C)(C)C=CC(=O)[C@@](C)(O)[C@@H]1[C@@H](O)C[C@]2(C)C3=CC[C@@H]4C(C)(C)C(=O)[C@H](O)C[C@@]4(C)[C@@H]3CC(=O)[C@@]12C CVKKIVYBGGDJCR-SXDZHWHFSA-N 0.000 title claims abstract description 84
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- IXQKXEUSCPEQRD-NRNCYQGDSA-N (2S,4R,23E)-2,16beta,20-trihydroxy-9beta,10,14-trimethyl-1,11,22-trioxo-4,9-cyclo-9,10-secocholesta-5,23-dien-25-yl acetate Chemical compound C([C@H]1[C@]2(C)C[C@@H](O)[C@@H]([C@]2(CC(=O)[C@]11C)C)[C@@](C)(O)C(=O)C=CC(C)(C)OC(=O)C)C=C2[C@H]1C[C@H](O)C(=O)C2(C)C IXQKXEUSCPEQRD-NRNCYQGDSA-N 0.000 title claims abstract description 41
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- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a strain Glycosylase producing strains---streptomycete (Streptomyces sp.) RW-2, and the application in Cucurbitacin B is prepared at bio-transformation Cucurbitacin B-2-O-glucoside.This bacterial strain is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode: 430072, deposit number: CCTCC No:M2013330, preservation date on July 17th, 2013.Beneficial effect of the present invention is mainly reflected in: (1) streptomycete is conventional bacterial classification in fermentation industry, nontoxic, use safety; (2) simple, the growth of streptomycete RW-2 nutritional requirement is cultivated rapidly, easily; (3) Glycosylase that streptomycete RW-2 produces is extracellular enzyme, be separated removing thalline, crude enzyme liquid or through dilution after, namely can be used as bio-transformation system, (4) from Muskmelon Base extract total cucurbitacin after bio-conversion processes, Cucurbitacin B-2-O-glucoside wherein almost all can be converted into Cucurbitacin B, and the content of Cucurbitacin B can improve 1 ~ 2.7 times.
Description
(1) technical field
The present invention relates to the streptomycete that Glycosylase is produced in a strain and the application prepared in bio-transformation in Cucurbitacin B thereof.
(2) background technology
Muskmelon Base is the dry carpopodium after Curcurbitaceae annual herbaceous species plant muskmelon (Cucumis melo) maturation, another name is also named muskmelon pedicel, melon fourth, Pedicellus Melo, begin to be loaded in Shennong's Herbal, for top grade medicine, system's " Chinese Pharmacopoeia " version in 1977 records kind, bitter, cold in nature, enter spleen, stomach warp.There is the merit of vomiting phlegm and indigested food by emesis, clearing damp removing jaundice.Modern clinic is used for treating accumulation of food in the stomach and intes tine due to indigestion, food poisoning, and epilepsy phlegm is contained and acute hepatitis, chronic hepatitis and liver cirrhosis etc.
The effective component of Muskmelon Base is cucurbitacin (cucurbitacins), and cucurbitacin is a class tetracyclic triterpenoid, has now found that more than 40 plant, and as Cucurbitacin B, cucurbitacin D and Cucurbitacin E etc., they have protects liver anti-inflammatory and the various biological such as antitumor is active.In Muskmelon Base, the ratio that Cucurbitacin B accounts for total cucurbitacin is the highest, and active best, being the most effective monomer of anti-hepatitis, liver cancer, having good adjuvant treatment effect to containing caused chronic persistent hepatitis, chronic hepatitis and primary hepatocarcinoma because of damp and hot poison.
70 ~ eighties of last century, domesticly develop medicine cucurbitacin sheet, the clinical assisting therapy for hepatitis and primary hepatocarcinoma.Cucurbitacin sheet be total cucurbitacin of extracting from Muskmelon Base with ethanol purified after add auxiliary material compressing tablet and form, Cucurbitacin B is main pharmacodynamics composition, and be also the index of this medicine effective constituent inspection, its content of pharmacopoeial requirements is not less than 60%.
Cucurbitacin B-2-O-glucoside (hereinafter referred to as Cucurbitacin B glucosides) is there is from total cucurbitacin that Muskmelon Base extracts, its content is close to the content being even greater than Cucurbitacin B, but biological activity is nothing like Cucurbitacin B, as being only 1.1% of Cucurbitacin B to the inhibiting of people's HepG-2 cell line.The compound that glycosidic link connects needs to be first that aglycon just can be absorbed by the body through metabolic conversion, but is hydrolyzed difficulty because human body lacks corresponding enzyme, almost can not be absorbed by the body.
(3) summary of the invention
The object of the invention is to provide a strain Glycosylase producing strains---streptomycete (Streptomyces sp.) RW-2, and prepare the application in Cucurbitacin B glucosides in bio-transformation, Cucurbitacin B content the total cucurbitacin extracted from Muskmelon Base can be significantly improved, have that cost is low, technique is simple, efficiency advantages of higher.
The technical solution used in the present invention is:
Streptomycete (Streptomyces sp.) RW-2, is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode: 430072, deposit number: CCTCC No:M2013330, preservation date on July 11st, 2013.
Streptomycete RW-2 of the present invention, is mix with Muskmelon Base powder the strain excellent that in enrichment culture thing, Isolation and screening obtains by river sewage, then obtains through ultraviolet radiation mutagenesis process.
The colony characteristics of described streptomycete RW-2 is as follows: on plate culture medium, under 30 DEG C of conditions, cultivates 3 days, and the bacterium colony initial stage is white, little and fine and close, dry and opaque, not easily provoke, become large afterwards gradually, in greyish-green, surface is in powdery, and have fold, the back side is grey black.Examine under a microscope, substrate mycelium is elongated, multi-branched; Diameter is 0.8 ~ 1.0 μm; Aerial hyphae is slightly thick; Conidium ovalize, smooth surface.
The partial nucleotide sequence of the 16s rDNA of described streptomycete RW-2 is as shown in SEQ ID No.1:
GCTTACCATGCAAGTCGAACGATGAACCACTTCGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATACTGAGCCACTTGGGCATCCAAGTGGTTCGAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAACGTCCAGAGATGGGCGCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTGGACGAAGTCGAACAA。
The invention still further relates to described streptomycete RW-2 and prepare application in Glycosylase at fermentable.
Concrete, described application is as follows: by streptomycete RW-2 bacterial classification spore or be seeded in culture medium through the seed of enlarged culturing, cultivates 3 ~ 6 days for 25 ~ 35 DEG C, obtains the fermented liquid containing Glycosylase.Gained fermentation liquor is filtered or the clear liquid of centrifugal removing thalline is Glycosylase crude enzyme liquid, can directly apply to bio-transformation Cucurbitacin B glucosides; Also can be preserved in 4 DEG C of refrigerators 15 days and use; Also through lyophilize, thick enzyme powder can be made and is preserved in 4 DEG C of refrigerator life-time service.
Described culture medium is composed as follows: potato 100 ~ 200g/L, sucrose 20 ~ 50g/L, soyflour 10 ~ 20g/L, and solvent is water, pH6.0 ~ 8.0.
The invention still further relates to described streptomycete RW-2 and prepare application in Cucurbitacin B at microbial transformation Cucurbitacin B glucosides, Cucurbitacin B content in total cucurbitacin of Muskmelon Base extraction using alcohol can be improved.
Concrete, describedly to be applied as: by streptomycete RW-2 bacterial classification spore or be seeded in culture medium through the seed liquor of enlarged culturing, cultivate 3 ~ 6 days for 25 ~ 35 DEG C, the centrifugal removing thalline of fermentation liquor obtains Glycosylase crude enzyme liquid, crude enzyme liquid is after 1 ~ 5 times of water dilution, add total cucurbitacin solution (wherein Cucurbitacin B content is 1 ~ 2mg/mL) of the Muskmelon Base extraction using alcohol of 5 ~ 20% percent by volumes, in 25 ~ 35 DEG C, carry out conversion 12 ~ 48h under 150 ~ 250r/min oscillating condition, conversion fluid reclaims total cucurbitacin through extraction into ethyl acetate.
Described culture medium is composed as follows: potato 100 ~ 200g/L, sucrose 20 ~ 50g/L, soyflour 10 ~ 20g/L, and solvent is water, pH6.0 ~ 8.0.
Described bacterial strain, before product enzyme is cultivated, needs first through slant medium activation culture usually, or through seed enlarged culturing, then carries out product enzyme with spore or seed liquor access culture medium and cultivate.
Concrete, described activation, seed enlarged culturing and product enzyme cultural method are as follows:
(1) by streptomycete RW-2 bacterial classification spore inoculating in slant medium, in 25 ~ 35 DEG C of constant incubators cultivate 2 ~ 3 days, obtain activate after streptomycete RW-2 bacterial classification; Described slant medium consists of: potato 100 ~ 200g/L(potato cleans peeling, be cut into small pieces, add 5 times of quality water boil 20 ~ 30min, 4 layers of filtered through gauze remove potato balls), sucrose 10 ~ 20g/L, agar 15 ~ 20g/L, solvent is water, pH6.0 ~ 8.0, high pressure steam 121 DEG C of sterilizing 20min;
(2) streptomycete RW-2 slant pore after step (1) activation culture is seeded in seed culture medium, in 25 ~ 35 DEG C, cultivate 1 ~ 2 day under 150 ~ 250r/min oscillating condition, obtain seed liquor; Described seed culture medium does not contain agar, other compositions and the same slant medium of compound method;
(3) by the streptomycete RW-2 slant pore of step (1), or seed liquor access culture medium prepared by step (2), culture medium in 25 ~ 35 DEG C, cultivate 3 ~ 6 days under 150 ~ 250r/min oscillating condition, obtain the fermented liquid of streptomycete RW-2; Described culture medium consists of: the same slant medium of potato 100 ~ 200g/L(treatment process), sucrose 20 ~ 50g/L, soyflour 10 ~ 20g/L, solvent is water, pH6.0 ~ 8.0, high pressure steam 121 DEG C of sterilizing 20min.
Microorganism has the ability of very powerful enzyme system Sum decomposition transformation substance, and the hydrolysis of the glucosyl residue of Cucurbitacin B glucosides can make it be converted into Cucurbitacin B by some glucoside enzyme.Enzyme hydrolysis method more has specificity than acid and alkali hydrolysis method, can not damage, have the advantages such as transformation efficiency is high, mild condition to product Cucurbitacin B structure.Total cucurbitacin that Muskmelon Base extracts by the present invention, to be hydrolyzed process with specific glycosidase prepared by fermentable, Cucurbitacin B glucosides is converted into Cucurbitacin B, but hydrolytic action is not produced to the composition such as Cucurbitacin B and Cucurbitacin E, improve the content of Cucurbitacin B in total cucurbitacin, thus improve the active constituent content of this medicine, reduce the production cost of medicine.
Beneficial effect of the present invention is mainly reflected in: (1) streptomycete is conventional bacterial classification in fermentation industry, nontoxic, use safety; (2) simple, the growth of streptomycete RW-2 nutritional requirement is cultivated rapidly, easily; (3) Glycosylase that streptomycete RW-2 produces is extracellular enzyme, be separated removing thalline, crude enzyme liquid or through dilution after, namely can be used as bio-transformation system, (4) from Muskmelon Base extract total cucurbitacin after bio-conversion processes, Cucurbitacin B glucosides wherein almost all can be converted into Cucurbitacin B, and the content of Cucurbitacin B can improve 1 ~ 2.7 times.
(4) accompanying drawing explanation
Fig. 1 is standard substance Cucurbitacin B (concentration is 0.2g/L) high-efficient liquid phase chromatogram;
Fig. 2 is the high-efficient liquid phase chromatogram of the total cucurbitacin without bio-conversion processes;
Fig. 3 is the high-efficient liquid phase chromatogram of the total cucurbitacin through bio-conversion processes.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: produce the enrichment of Glycosylase microorganism, separation, screening and mutagenesis
Add about 2g Muskmelon Base in 250mL triangular flask, add the sewage in appropriate river course, stir, the thermostat container being placed in 30 DEG C cultivates 7 days, carries out the enrichment of producing Glycosylase microorganism.
Coat on plate culture medium after the above-mentioned enriched substance sterilized water covering with mycelia is diluted, cultivate 3 days in the biochemical cultivation case of 30 DEG C.The picking form bacterium colony spore inoculating different with color is on slant medium, and inclined-plane is placed in 30 DEG C of constant temperature culture 3 days, obtains the slant strains that spore is abundant.
With the spore of transfering loop difference picking each bacterial strain slant strains above-mentioned, in access 50mL culture medium (250mL triangle is bottled), in 28 DEG C, after 180r/min oscillating condition bottom fermentation cultivates 5 days, by fermented liquid 4 layers of filtered through gauze, get 9mL filtrate (i.e. crude enzyme liquid) to proceed in the triangular flask of a 50mL, add total cucurbitacin solution (wherein Cucurbitacin B content is 1.42g/L) of 1mL Muskmelon Base extraction using alcohol.Triangular flask in 28 DEG C, in the water-bath of 180r/min vibration case after bio-transformation 24h, with the extraction into ethyl acetate 2 times of 10mL, combined ethyl acetate, uses 10mL dissolve with methanol after evaporated under reduced pressure, 0.45 μm of membrane filtration, as Cucurbitacin B content analysis.
High-efficient liquid phase chromatogram technique analysis Cucurbitacin B content in total cucurbitacin of bio-conversion processes, compare thus different strains produce the capacity of water that Cucurbitacin B glucosides is converted into Cucurbitacin B by Glycosylase, through comparing, one be numbered the bacterial strain of R-2 to produce the transformation efficiency that Cucurbitacin B glucosides is converted into cucurbitacin by Glycosylase the highest, Cucurbitacin B content can improve 1.43 times.
Tentatively be judged as actinomycetes according to the bacterial strain colonial morphology of R-2, mycelia size and spore shape, adopt 16S rDNA sequence analysis method to identify bacterial strain R-2, determine that it is a streptomycete (Streptomyces sp.).
Uv irradiating mutagenic and breeding has been carried out to wild strain streptomycete R-2, obtaining product enzymatic conversion Cucurbitacin B glucosides through screening is the bacterial strain RW-2 that Cucurbitacin B ability increases, Cucurbitacin B content can improve 1.86 times, comparatively transform with wild strain and improve 30.1%, submit this bacterial strain to China typical culture collection center preservation, numbering: CCTCC No:M2013330, preservation date: on July 11st, 2013.
Described plate culture medium is identical with the composition of slant medium, by following composition and method preparation: potato cleans peeling, be cut into small pieces, take 200g, add tap water 1000mL and boil 30min, 4 layers of filtered through gauze remove potato ball, filtrate supplies 1000mL, then adds sucrose 20g, agar 18g, pH nature, heating dissolves rear packing test tube, high pressure steam 121 DEG C of sterilizing 20min.
Described culture medium is by forming preparation as follows: the same plate culture medium of potato 200g/L(treatment process), sucrose 20g/L, soyflour 10g/L, solvent is water, pH nature (measured value is 6.8).
Embodiment 2: the Glycosylase for biotransformation ferments
With streptomycete RW-2 for zymogenic bacteria kind, after substratum composition and fermentation condition optimization, conversion Cucurbitacin B glucosides is significantly improve before the ability of Cucurbitacin B is not optimized, and Cucurbitacin B content can improve 2.71 times, improve 45.7% before not optimizing, preferred preparation method is as follows:
(1) by the streptomycete RW-2 strain inoculation of test tube slant preservation in slant medium, inclined-plane is cultivated 3 days in 30 DEG C of biochemical cultivation cases.Described slant medium is by following composition and method preparation: potato cleans peeling, be cut into small pieces, take 200g, add tap water 1000mL and boil 30min, 4 layers of gauze elimination potato ball, filtrate supplies 1000mL, add sucrose 20g, agar 18g again, pH nature, heating dissolves rear packing test tube, high pressure steam 121 DEG C of sterilizing 20min.
(2) with the streptomycete RW-2 spore after transfering loop picking step (1) activation culture in culture medium, in 28 DEG C, cultivate 5 days under 180r/min oscillating condition, obtain the fermented liquid containing Glycosylase; Described culture medium is by forming preparation as follows: potato 200g/L, sucrose 30g/L, soyflour 14g/L, and solvent is water, pH7.5.
(3) by the fermented liquid of step (2) streptomycete RW-2, with 4 layers of filtered through gauze removing thalline, gained clear liquid is Glycosylase crude enzyme liquid.
Gained Glycosylase crude enzyme liquid may be used for biotransformation method and improves Cucurbitacin B content in total cucurbitacin, can use immediately, also can be preserved in 4 DEG C of refrigerators 15 days and use, also can prepare enzyme dry powder after lyophilize, be preserved in 4 DEG C of refrigerator long term storage and use.
Embodiment 3: biotransformation method improves the content of Cucurbitacin B in total cucurbitacin
Commercially available Chinese medicinal materials Muskmelon Base is dried 24h in the baking oven of 85 DEG C, is pulverized with pulverizer.Add 70% ethanol of 200mL in 10g Muskmelon Base powder, 40 DEG C of water-bath lixiviate 2.5h, more ultrasonic lixiviate 0.5h, 4 layers of filtered through gauze, collect filtrate.Use 40mL dissolve with methanol after filtrate decompression evaporate to dryness, obtain total cucurbitacin solution (Cucurbitacin B content is 1.68g/L).
In Example 2, the Glycosylase crude enzyme liquid 9mL of preparation is in 50mL triangular flask, adds above-mentioned total cucurbitacin solution of 1mL, triangular flask with after preservative film sealing, in 32 DEG C, bio-transformation 16h in the water-bath of 180r/min vibration case.Conversion fluid 10mL extraction into ethyl acetate 2 times, combined ethyl acetate, namely evaporated under reduced pressure ethyl acetate obtains the total cucurbitacin product through bio-conversion processes.
Embodiment 4: the content analysis of Cucurbitacin B in total cucurbitacin of biotransformation method process
By the total cucurbitacin sample 10mL dissolve with methanol through bio-conversion processes in embodiment 3,0.45 μm of membrane filtration, adopt the content of high-efficient liquid phase chromatogram technique analysis wherein Cucurbitacin B, result shows, in total cucurbitacin of bio-conversion processes, Cucurbitacin B content reaches 0.46g/L, and be only 0.17g/L without Cucurbitacin B content in bio-conversion processes reference substance, visible, after bio-conversion processes, Cucurbitacin B content improves 2.71 times.
Claims (3)
1. streptomycete (Streptomyces sp.) RW-2, is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode: 430072, deposit number: CCTCC No:M 2013330, preservation date on July 11st, 2013.
2. streptomycete RW-2 according to claim 1 prepares the application in Glycosylase at fermentable.
3. streptomycete RW-2 according to claim 1 prepares the application in Cucurbitacin B at bio-transformation Cucurbitacin B-2-O-glucoside.
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| CN101838618A (en) * | 2009-07-02 | 2010-09-22 | 江苏奕农生物工程有限公司 | Neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide, and genes and application thereof |
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| CN101838618A (en) * | 2009-07-02 | 2010-09-22 | 江苏奕农生物工程有限公司 | Neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide, and genes and application thereof |
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