CN101838618A - Neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide, and genes and application thereof - Google Patents
Neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide, and genes and application thereof Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering and particularly provides neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide, and genes and application thereof. The neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide can be obtained from a bacterial strain, i.e., novel streptomyces sp.S27 (CGMCC No.3146) screened by the invention, and the genes thereof and recombinant vector containing the genes can be further obtained, wherein the amino acid sequences of alpha-galactosidase Aga-S27 are shown in SEQ ID No.1. The alpha-galactosidase Aga-S27 has the advantages of appropriate operating temperature and pH value, higher protease resistance and higher ability to hydrolyze various substrates. Therefore, the alpha-galactosidase Aga-S27 is applicable as feed or food additives in the industries of feed and food.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of neutral alpha-galactosidase A ga-S27 and gene, the recombinant vectors that comprises this gene and application of high alpha galactosides oligosaccharides degradation capability.
Background technology
Alpha-galactosidase (α-galactosidase, EC3.2.1.22) be melibiose, belong to the exoglycosidase class, hydrolysis that can the non-reduced terminal alpha galactosides key of specificity catalysis sugar chain, it not only can hydrolysis contains the oligosaccharides of alpha galactosides key, but also can catalysis contains the polysaccharide of this key.Raffinose, stachyose, verbascose are the oligosaccharides that extensively exists in the bean, and under the effect of alpha-galactosidase, these oligosaccharides can be broken down into D-semi-lactosi and other corresponding monose and oligosaccharides.
Dregs of beans is very outstanding vegetable protein feed raw material, and general consumption in daily ration is 25～30%.But the oligosaccharide that in dregs of beans, contains the alpha galactosides class that 5～6% monogastric animal can not digest, as raffinose, stachyose etc., these oligosaccharides can cause the flatulence disease of animal on the one hand, can increase the viscosity of chyme on the other hand, thereby reduce digestion and the absorption of animal nutritive substance.And the digestion of oligosaccharide in the interpolation alpha galactosides endonuclease capable increase feed in legume forage, reduce the anti-oxidant action of this type of oligosaccharide, alleviate or eliminate the digestive disorders of monogastric animal, thereby the utilization ratio of nutritive substance and metabolizable energy in the raising feed (Ghazi S, et al.British Poultry Science.44 (3): 410-418).
In in the food of soybean and goods thereof, contain 1% raffinose, 4% stachyose and micro-verbascose.These oligosaccharides hinder human digesting and assimilating beans, can produce diseases such as flatulence, and the human gastrointestinal tract does not secrete alpha-galactosidase, therefore in bean product, add alpha-galactosidase, can decompose these oligosaccharides, increase human absorption (Prashanth S J and Mulimani V H, Process Biochemistry.40:1199-1205) to beans nutrition.
In sugar industry,, candy output is reduced because the existence of raffinose in the beet causes increase of molasses viscosity and obstruction crystallization of sucrose to separate out, so that produces a large amount of waste molasses.The application alpha-galactosidase is handled the raffinose removing that waste molasses can hinder crystallization of sucrose, when decomposing a part raffinose, also produce the sucrose of a part, thereby improve yield (Ganter C, the et al.Journal of Biotechnology.8 (4): 301-310) of sucrose.Enzyme process sugaring simultaneously can make homogeneity, color and luster and the purity of the granular crystal of sucrose improve.
In addition, alpha-galactosidase can also be used for sick treatment (the Tsuboi K of Fa Bulishi (Fabry ' s) in medical treatment, The Journal of International Medical Research.35 (4): 574-581) and the transformation of B → O blood group (ZhuA and Goldstein J, Gene.140 (2): 227-231) etc.
Alpha-galactosidase extensively is present in microorganism, plant, the animal and human's body.In microorganism, bacterium, actinomycetes, filamentous fungus, yeast etc. can both synthesize alpha-galactosidase.At present, separated the microorganism of many product alpha-galactosidases, wrapped mould, Mucor, head mold, flavus, cereuisiae fermentum etc. as intestinal bacteria, genus bacillus, streptomycete, mould, monascus, aspergillus oryzae, aspergillus niger, Aspergillus ustus, Aspergillus awamori, Portugal's wine and women-sensual pursuits.This difference according to its character characteristic of the microorganism alpha-galactosidase of different sources is applied to different fields.The alpha-galactosidase that then requires to add in food and fodder industry all will have higher hydrolysis ability to various alpha galactosides class oligosaccharides, and in the past its substrate specificity of alpha-galactosidase of report can not the multiple substrate of hydrolysis, respectively had its limitation on using.
The invention discloses a new alpha-galactosidase gene, the alpha-galactosidase of its coding has suitable action pH value, the stronger protease resistant and the ability of the various substrates of hydrolysis preferably can be used as a kind of feed or foodstuff additive and are applied to feed and food service industry.
Summary of the invention
One of purpose of the present invention provides a kind of streptomycete.
One of purpose of the present invention provides a kind of neutral alpha-galactosidase A ga-S27 of high alpha galactosides oligosaccharides degradation capability.
A further object of the present invention provides the gene of the above-mentioned alpha-galactosidase A ga-S27 of coding.
Another object of the present invention provides the recombinant vectors that comprises said gene.
Another object of the present invention provides the recombinant bacterial strain that comprises said gene.
Another object of the present invention provides a kind of gene engineering method for preparing above-mentioned alpha-galactosidase A ga-S27.
Another object of the present invention provides the application of above-mentioned alpha-galactosidase A ga-S27.
The invention provides a kind of streptomycete streptomycete Streptomyces sp.S27, its preserving number is: CGMCC No.3146.
The invention provides a kind of alpha-galactosidase A ga-S27 that derives from above-mentioned bacterial strains, its aminoacid sequence is shown in SEQ ID NO.1:
VPVWPGRRHTVASRSAPLPLDVTHLRAAGVSLVLDLTGGTLPRVLHWGADLGTLGPEELG?60
SLRLAGAPQPIGFSVDGPVEVSVLPEQSAGWLGTPGLVGNRGGRDFSTAFAVREAALTGP?120
VEGSRSPGGGGGAIVTVLARDEAAALDLELVIELTASGLVRQRATVANRGSSPFTVDAVN?180
LTLPVPAEAVELLDFTGHHLRERSPQRTAFTQGLRMRENRTGRTGYDSAYLLAAGTAGFG?240
NRSGEVWAVHTAWSGNHRTFAERTFHSVSLLGSGELLLSGEVVLEPGESYASPWQYGSYG?300
RHGLDEVSARFHRWLRSRPHHPSTPRPVTLNTWEAVYFDHDLDRLRALADAAAAVGAERF?360
VLDDGWFGSRRDDRRALGDWYVSDEVWPDGLGPLTGHVTGLGMQFGLWVEPEMVNEDSDL?420
ARAHPDWVMAAGDRLPGAARHQQVLDLARPEAFAYILGRLDELLEAYPIAYLKWDHNRDL?480
VDAGHRPTGRAGVHGQTRAVYRLMDELRRRHPGVEIESCSSGGGRVDLEILQRTDRVWVS?540
DCIDALERQTIQRWTNALIPLELMGTHVGSDVAHTTGRRHPVDFRAGTALFGHFGIEWDL?600
TAAAPEELARLAQWVDLYKELRGLLHTGVRVHADHPDPAHRLHGVVAEDGSDAVYALVAT?660
ASSAMYPAGAIRLPGLDADAVYRVRPQAPGDLPDGNAHHWGVRLPWWTPEGVRLPGRVLA?720
AAGLQAPVLHPERLVLLRATRV?742
Its molecular weight of alpha-galactosidase A ga-S27 according to the present invention is 80kDa, is 11.26U/mg than living, and optimal pH is 7.4, and optimum temperature is 35 ℃, has Chymetin, Validase TSP Concentrate II A, Collagenase resistance preferably.
The invention provides the gene of the above-mentioned alpha-galactosidase A ga-S27 of coding, its complete genome sequence is shown in SEQ IDNO.2.
GTGCCGGTTTGGCCAGGACGGAGACACACCGTGGCATCACGTTCCGCTCCCCTTCCCCTC?60
GATGTGACGCACCTGCGGGCGGCCGGCGTCAGCCTCGTACTGGACCTGACCGGCGGCACC?120
CTCCCCCGGGTCCTGCACTGGGGTGCCGACCTCGGCACGCTCGGCCCCGAGGAGCTGGGC?180
TCCCTGCGGCTGGCCGGGGCGCCGCAGCCCATCGGCTTCTCCGTGGACGGCCCGGTCGAG?240
GTCTCGGTGCTGCCCGAGCAGTCGGCCGGCTGGCTGGGCACCCCCGGCCTGGTGGGCAAC?300
CGCGGCGGCCGGGACTTCTCCACCGCCTTCGCCGTCCGCGAGGCCGCCCTCACCGGGCCC?360
GTGGAGGGGTCCCGCTCCCCCGGCGGCGGAGGCGGCGCCATCGTCACCGTCCTCGCCCGC?420
GACGAGGCCGCGGCCCTGGATCTGGAGCTGGTGATCGAGCTGACCGCGTCCGGACTGGTG?480
CGCCAGCGCGCCACGGTCGCCAACCGCGGCTCCTCCCCCTTCACCGTCGACGCCGTGAAC?540
CTCACGCTGCCGGTGCCCGCCGAAGCCGTCGAGCTGCTGGACTTCACCGGGCACCACCTG 600
CGCGAACGCAGCCCGCAGCGCACCGCCTTCACCCAGGGGCTGCGGATGCGGGAGAACCGC 660
ACCGGCCGCACCGGCTACGACAGCGCCTACCTGCTGGCCGCGGGCACCGCGGGCTTCGGC 720
AACCGCTCGGGCGAGGTGTGGGCCGTGCACACCGCGTGGTCCGGCAACCACCGCACCTTC 780
GCCGAGCGCACCTTCCACTCGGTGTCGCTGCTGGGCTCGGGCGAGCTGCTGCTCTCCGGC 840
GAGGTGGTGCTGGAGCCCGGGGAGTCCTACGCCTCCCCCTGGCAGTACGGCTCCTACGGG 900
CGGCACGGCCTGGACGAGGTCTCCGCCCGCTTCCACCGCTGGCTGCGCTCCCGCCCGCAC 960
CACCCCTCCACGCCCCGGCCGGTCACCCTCAACACCTGGGAGGCGGTCTACTTCGACCAC 1020
GATCTGGACCGGCTGCGCGCCCTGGCCGACGCGGCCGCCGCGGTGGGCGCGGAGCGCTTC 1080
GTCCTGGACGACGGCTGGTTCGGCTCGCGCCGCGACGACCGGCGCGCCCTGGGCGACTGG 1140
TACGTCTCGGACGAGGTGTGGCCGGACGGACTGGGCCCGCTGACCGGTCATGTCACCGGC 1200
CTGGGCATGCAGTTCGGCCTCTGGGTCGAGCCGGAGATGGTCAACGAGGACTCCGACCTG 1260
GCCCGCGCCCACCCCGACTGGGTCATGGCCGCCGGGGACCGGCTGCCCGGCGCGGCCCGC 1320
CACCAGCAGGTCCTCGACCTGGCCCGCCCGGAGGCGTTCGCGTACATCCTCGGGCGCCTG 1380
GACGAGCTGCTGGAGGCGTACCCCATCGCGTATCTGAAGTGGGACCACAACCGGGACCTG 1440
GTGGACGCCGGGCACCGGCCCACCGGCCGCGCCGGCGTGCACGGCCAGACCCGGGCGGTC 1500
TACCGGCTGATGGACGAGCTGCGCCGCCGCCACCCCGGGGTGGAGATCGAGTCCTGCTCC 1560
TCCGGCGGCGGACGGGTGGACCTGGAGATCCTCCAGCGCACCGACCGGGTGTGGGTCTCG 1620
GACTGCATCGACGCCCTGGAGCGCCAGACCATCCAGCGCTGGACGAACGCCCTGATCCCC 1680
CTCGAACTCATGGGCACCCACGTGGGGTCGGACGTCGCGCACACCACCGGGCGCCGCCAC 1740
CCGGTCGACTTCCGGGCGGGCACGGCGCTGTTCGGGCACTTCGGCATCGAGTGGGACCTG 1800
ACCGCCGCCGCGCCGGAGGAGCTGGCGCGGCTGGCGCAGTGGGTGGACCTCTACAAGGAG 1860
CTGCGCGGCCTGCTCCACACCGGCGTCCGTGTCCACGCCGACCACCCGGACCCGGCCCAC 1920
CGGCTGCACGGGGTGGTCGCCGAGGACGGCTCGGACGCGGTGTACGCCCTCGTCGCCACC 1980
GCCTCCTCCGCGATGTACCCGGCCGGTGCGATCCGGCTGCCGGGGCTGGACGCGGACGCC?2040
GTCTACCGGGTGCGGCCGCAGGCCCCCGGCGACCTGCCGGACGGCAACGCCCACCACTGG?2100
GGCGTCCGCCTGCCCTGGTGGACGCCGGAGGGTGTGCGGCTGCCGGGCCGGGTGCTGGCG?2160
GCCGCGGGGCTCCAGGCCCCGGTGCTCCACCCCGAGCGGCTGGTGCTGCTGCGCGCGACC?2220
CGGGTGTGA?2229
The present invention also provides the recombinant vectors that comprises above-mentioned alpha-galactosidase gene aga-S27.
The present invention also provides the recombinant bacterial strain that comprises above-mentioned alpha-galactosidase gene aga-S27, and preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus.
The present invention also provides a kind of preparation to have the method for the neutral alpha-galactosidase A ga-S27 of high alpha galactosides oligosaccharides degradation capability, it is characterized in that, may further comprise the steps:
1), gets recombinant bacterial strain with above-mentioned recombinant vectors transformed host cell;
2) cultivate recombinant bacterial strain, induce the reorganization alpha-galactosidase to express; And
3) reclaim the also expressed alpha-galactosidase A ga-S27 of purifying.
The present invention also provides the application of alpha-galactosidase A ga-S27.
The invention discloses a new alpha-galactosidase gene, the alpha-galactosidase of its coding has suitable action pH value, the stronger protease resistant and the ability of the various substrates of hydrolysis preferably can be used as a kind of feed or foodstuff additive and are applied to feed and food service industry.
Description of drawings
Streptomycete Streptomyces sp.S27 is stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCCNo.3146, preservation date is: on June 29th, 2009.
The recombinate SDS-PAGE of alpha-galactosidase of Fig. 1 analyzes M: low molecular weight protein Marker; 1: do not induce contrast, the CTP after contrast empty carrier pET-28a (+) Transformed E .coli BL21 induces; 2: the CTP after carrier pET-28a (+)/aga-S27 Transformed E .coli BL21 induces; 3: the alpha-galactosidase A ga-S27 of purifying.
The optimal pH of Fig. 2 alpha-galactosidase.
Fig. 3 alpha-galactosidase pH stability.
The optimum temperuture of Fig. 4 alpha-galactosidase effect.
The thermostability of Fig. 5 alpha-galactosidase.
The protease resistant analysis of Fig. 6 alpha-galactosidase A ga-S27.
Embodiment
Experiment condition:
1, bacterial strain and carrier: streptomycete (Streptomyces sp.S27) is by contriver screening, and host e. coli (Escherichia coli) BL21 (DE3), carrier pET-28a (+) are all available from Invitrogen company.
2, enzyme and other biochemical reagents: restriction endonuclease is available from TaKaRa company, and ligase enzyme is available from Invitrogen company.Trypsin Trypsin), (α-Chymotrypsin), Proteinase K (Proteinase K), subtilopeptidase A (Subtilisin A), Collagenase (Collagenase) are Sigma (USA) product to Chymetin; Proteolytic enzyme proleather is Amano Enzyme Inc. (Japan) product; IPTG is a Promega company product; Melibiose, raffinose, stachyose and bovine serum albumin are Sigma (USA) product; Other all is a domestic reagent.
3, No. 1 substratum: 0.1%KNO of Gao Shi
3, 0.05% K
2HPO
4, 2% Zulkovsky starch, 0.05%MgSO
47H
2O, 0.05%NaCl, 0.001%FeSO
47H
2O, pH7.2-7.4.
Cha Shi inducing culture: 0.1%K
2HPO
4, 0.3%NaNO
3, 0.05%KCl, 0.05%MgSO
47H
2O, 0.05%FeSO
47H
2O, 3% dregs of beans is a carbon source, pH is natural.
The intestinal bacteria substratum be LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
Illustrate: the genetic recombination of using learns a skill and is routine techniques in this area among the present invention.The technology that does not describe in detail in following examples is all carried out according to related Sections in following laboratory manual or the document or part, comprising: people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition .2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); With Current Protocols in Molecular Biology (people such as Ausubel compiles, 1994).
Bacterial strain Streptomyces sp.S27 separates from flame hill, Xinjiang soil, and after 2 days, it is white bead that thalline is assembled in growth on No. 1 substratum of Gao Shi.According to the primer of the conserved sequence of bacterial 16 S rDNA design (27F:5 '-AGAGTTTGATCMTGGCTCAG-3 ' and 1492R:5 '-TACGGHTACCTTACGACTT-3 ') the 16S rDNA of bacterial strain is carried out pcr amplification, nucleotide sequence comparison in sequencing result and the Genbank database finds that the 16S rDNA nucleotide sequence of S27 and Streptomyces radiopugnans strain HBUM174051 (Genbank accession No.FJ486333) are 100% according to the highest similarity is arranged.Combining form is observed, and provable this bacterial strain is a streptomycete, called after Streptomyces sp.S27.Dregs of beans is added (0.1%K in the product enzyme substratum as inductor and carbon source
2HPO
4, 0.3%NaNO
3, 0.05%KCl, 0.05%MgSO
47H
2O, 0.05%FeSO
47H
2O, 3% dregs of beans are carbon source), 30 ℃, 250rpm shake were cultivated after 2-5 days, and bacterium liquid is centrifugal, got supernatant and surveyed enzyme and live.Go out to reveal higher alpha-galactosidase activity behind the 48h (about 2d) as a result, about 0.17U/mL (is substrate with pNPG).
The clone of embodiment 2 streptomycete alpha galactosides enzyme coding gene aga-S27
Extract streptomycete (Streptomyces sp.S27) genomic dna: will be in No. 1 substratum of Gao Shi 30 ℃ of centrifugal 10min of bacterium liquid 10000rpm that cultivated 2d, take by weighing 50mg bacterium mud and add 500 μ L sterile water wash, the sedimentary thalline of centrifuging and taking.Sedimentary thalline is resuspended in 500 μ L N,O-Diacetylmuramidase mixed solutions, and 37 ℃ of incubation 1h add enzyme liquid 100 μ L again and continue insulation 30min in 45 ℃, to bacterium liquid transparent after, add 10%SDS to final concentration 2%, stir about 5min significantly descends to the bacterium fluid viscosity, and 15000rpm is centrifugal, and 10min removes fragment.Supernatant liquor is used equal-volume phenol respectively, phenol: chloroform, chloroform is extracting successively.Get the Virahol normal temperature precipitation 10min that upper solution adds 0.6-1 times of volume.The centrifugal 15min of 16000rpm.Precipitation is cleaned with 70% ethanol, and is centrifugal slightly, precipitation drained the back dissolve with 30 μ L sterilized waters, and-20 ℃ of preservations are standby.
Degenerated primer P4 has been synthesized in conserved sequence design according to the alpha-galactosidase of delivering, 5 '-GAYGAYGGNTGGTTYGGN-3 ' and Pr8,5 '-(Y represents C/T to GACCATYTCNGGYTCNAMCC-3 ', N represents A/T/C/C, M represents A/G), the total DNA of streptomycete S27 is that template is carried out pcr amplification.The PCR reaction parameter is: 95 ℃ of 5min; 94 ℃ of 30sec, 58-48 ℃ of 30sec (wherein each circulation back renaturation temperature descends 1 ℃), 72 ℃ of 1min, 10 circulations enter second cycling program then: 94 ℃ of 30sec, 48 ℃ of 30sec, 72 ℃ of 1min are after 25 circulations; 72 ℃ of 10min, agarose electrophoresis detects.The sequencing fragment that obtains.
Adopt half polishing method to make up streptomycete S27 gene library, sieve storehouse primer S27sp1 according to the nucleotide sequence design that order-checking obtains, 5 '-CCATCTCCGGCTCGACCCAGAGGCCG-3 ' and S27rsp1,5 '-GACGACGGCTGGTTCGGCTCGCGC-3 ', in gene library, screen the positive colony that contains the aga-S27 full length gene by PCR.The PCR reaction parameter is: 95 ℃ of 5min; 94 ℃ of 30sec, 65 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations; 72 ℃ of 5min, agarose electrophoresis detects.Check order and obtain the aga-S27 full-length gene inserting fragment.The result shows that this gene is the gene fragment of a long 2229bp, encode 742 amino acid and a terminator codon.If it is 55% that the alpha-galactosidase of zymoprotein and Clavibacter michiganensis subsp.sepedonicus (Genbank accession No.YP_001709642) has the highest amino acid sequence similarity, verify that with Bifidobacterium bifidum NCIMB 41171 active alpha-galactosidase (Genbank accession No.ABD96085) similaritys are 46%, verify that with Escherichia coli K12 active alpha-galactosidase (Genbank accession No.AAA24497) similarity is 40%.This encoding gene is a new gene.
The activation analysis of embodiment 3 alpha-galactosidases
Activity determination method adopts the pNPG method.PNPG is dissolved in the 0.1mol/L McIlvaine damping fluid, and making its final concentration is 2mmol/L.With 20 μ L enzyme liquid, the pNPG of the McIlvaine damping fluid of 230 μ L and 250 μ L 2mmol/L mixes, and shakes up.Behind 37 ℃ of incubation 5min, add the Na of 1.5mL 1mol/L in the reaction solution
2CO
3Solution comes termination reaction.Survey its OD value at the 405nm place, represent enzyme activity with the growing amount of p-nitrophenol (pNP).After enzyme-added liquid of control tube and the damping fluid, add Na earlier
2CO
3Solution adds pNPG solution again.
Enzyme (U/mL) unit definition of living: decompose enzyme amount that pNPG discharge 1 μ mol pNP and need at 37 ℃ of following per minutes and be defined as the enzyme unit that lives.
The recombinase that embodiment 4 preparation alpha galactosides enzyme coding gene aga-S27 express
Design primer according to sequencing result:
S27-AG-HS (5 '-CTG
GAATTCATGCCGGTTTGGCCAGGACGGAGACACAC-3 ', underscore are the EcoRI site) and S27AG-2 (5 '-CTT
AAGCTTTCACACCCGGGTCGCGCGC-3 ', underscore are Hind III site), carry out pcr amplification.Plasmid pET28a (+) and gene segment carry out being connected to form carrier pET28a (+)/aga-S27 behind the double digestion, and preparation BL21 (DE3) competent cell transforms the host bacterium with recombinant plasmid pET28a (+)/aga-S27 electric shock.Identify positive recombinant, and induce the detection enzyme to live.Get BL21 (DE3) bacterial strain that contains plasmid recombinant and BL21 (DE3) bacterial strain (comparing) that contains pET-28a (+) empty plasmid, be inoculated in 3mL LB (being added with the kantlex of the 50 μ g/mL) nutrient solution 37 ℃ of quick oscillation overnight incubation respectively.Get the adding of 100 μ L incubated overnight liquid respectively and contain in the 10mL LB nutrient solution (1% inoculum size) of 50 μ g/mL kantlex 37 ℃ of about 2-3h (OD of shaking culture
600Reach 0.6-0.8) the back inductor IPTG that adds final concentration 1mmol/L, 12h are cultivated in 18 ℃ of 180rpm concussions.Nutrient solution 12, the centrifugal 5min of 000rpm collects cleer and peaceful precipitation on the substratum respectively, and cell precipitation is resuspended with the McIlvaine damping fluid of the 0.1mol/L of pH7.4, and after the ultrasonication 12, the centrifugal 10min of 000rpm, collection supernatant are total protein of cell (CTP).Detect cleer and peaceful CTP enzyme work on the substratum respectively by above-mentioned enzyme activity determination method.Recombinase is mainly expressed in Escherichia coli cell as a result, and enzymic activity is 22.97U/ml among the CTP.Through the SDS-PAGE electrophoresis detection, can see a tangible specific band (Fig. 1, LANE2).
Intestinal bacteria induce the alpha-galactosidase A ga-S27 of generation through a step Ni-NTA column purification (NEB), are increased to 11.26U/mg than living.Purifying Aga-S27 obtains electrophoretically pure single band through SDS-PAGE, and molecular weight is about 80kDa close with predicted molecular weight (Fig. 1, LANE 3).
The zymologic property of embodiment 5 alpha-galactosidase A ga-S27
Purified alpha-galactosidase A ga-S27 carries out enzymatic reaction to measure its optimal pH under different pH.Used damping fluid is glycine-NaOH damping fluid of the 0.1mol/L of the McIlvaine damping fluid of 0.1mol/L of pH 2.0～8.0 and pH 9.0～11.0.The alpha-galactosidase A ga-S27 of purifying is in the buffer system of different pH, 37 ℃ of suitable property results (Fig. 2) of the pH that measures down show: the suitableeest action pH of Aga-S27 is 7.4, keep relative activity 80% or more, pH to be lower than 5 or be higher than 10.0 and almost lose all enzymes work at pH7.0-7.4.
Enzyme liquid is handled 0.5h in the damping fluid of different pH values, measure the pH stability of enzymic activity again under 37 ℃ with the research enzyme.The result shows (Fig. 3), and Aga-S27 is very stable between 7.0-10.0 in the pH scope, keeps the enzyme more than 80% to live.
Carry out enzymatic reaction under the McIlvaine buffer solution system of the 0.1mol/L that is determined at pH7.4 of optimum temperuture and the different temperature (0～70 ℃).Enzyme reaction optimum temperuture measurement result (Fig. 4) shows, 35 ℃ of the optimum temperatures of Aga-S27, and residue was lived less than 2% enzyme when temperature was higher than 60 ℃.
Measure alpha-galactosidase and under different temperature (50 ℃), be incubated 2,5,10,15,20,30 o'clock relative enzyme activity respectively, draw the thermostability curve of enzyme.50 ℃ down handle 30min after the enzyme residue of living about 50%, better heat stability.(Fig. 5).
Proteolytic enzyme is mixed with the solution of 1mg/mL respectively with following solution: with pH 7.00.1mol/L Tris-HCl preparation trypsinase, Chymetin solution; With pH7.50.1mol/L Tris-HCl preparation Proteinase K, subtilopeptidase A, collagen protein enzyme solution; With pH10.00.1mol/L glycine-NaOH preparation proleather, alkaline protease solution.The alpha-galactosidase of purifying and proteolytic enzyme handled 30min in 10: 1 (w/w) ratios in this proteolytic enzyme damping fluid after, detect the residual enzyme of treat enzyme by standard method and live.The result shows, residual enzyme work with Aga-S27-H after the different protease treatment is respectively 49.41% (trypsinase), 100.87% (Chymetin), 14.52% (Proteinase K), 95.31% (Validase TSP Concentrate II A), 101.84% (Collagenase) and 28.58% (proleather) this shows that Aga-S27-H is more inconsistent to various resistance towards proteases.For Chymetin, Validase TSP Concentrate II A, Collagenase, Aga-S27-H has resistance preferably; And it is general for trypsinase, Proteinase K and proleather resistance; Then there is not resistance (Fig. 6) for Sumizyme MP.
The substrate specificity of embodiment 7 alpha-galactosidase A ga-S27
Melibiose, raffinose, stachyose are dissolved in 0.1mol/L, and in the McIlvaine damping fluid of pH 7.4, each 1mg/mL of concentration uses the alpha-galactosidase of 1U/mL and 3U/mL to act on 24h with these substrates down at 37 ℃ respectively.(Dionex Corporation USA) measures with ion-exchange chromatography instrument CARBOPAC PA10 in the release of semi-lactosi.The result shows (table 1), and the percent hydrolysis that Aga-S27 decomposes melibiose is up to 37.91%, and its hydrolysis ability to natural substrate is melibiose＞raffinose＞stachyose.And the enzyme of 3 units is stronger than the enzyme capacity of decomposition of 1 unit.Illustrate that this enzyme has substrate specificity and higher hydrolysis ability preferably.
Table 1Aga-S27 is to the degradation capability of different substrates
Sequence table
＜110〉Jiangsu Yi Nong biotechnology company limited
＜120〉a kind of neutral alpha-galactosidase A ga-S27 and gene and application of high alpha galactosides class oligosaccharides degradation capability
<160>2
<210>1
<211>742
<212>PRT
＜213〉streptomycete (Streptomyces sp.S27)
<400>1
VPVWPGRRHTVASRSAPLPLDVTHLRAAGVSLVLDLTGGTLPRVLHWGADLGTLGPEELG?60
SLRLAGAPQPIGFSVDGPVEVSVLPEQSAGWLGTPGLVGNRGGRDFSTAFAVREAALTGP?120
VEGSRSPGGGGGAIVTVLARDEAAALDLELVIELTASGLVRQRATVANRGSSPFTVDAVN?180
LTLPVPAEAVELLDFTGHHLRERSPQRTAFTQGLRMRENRTGRTGYDSAYLLAAGTAGFG?240
NRSGEVWAVHTAWSGNHRTFAERTFHSVSLLGSGELLLSGEVVLEPGESYASPWQYGSYG?300
RHGLDEVSARFHRWLRSRPHHPSTPRPVTLNTWEAVYFDHDLDRLRALADAAAAVGAERF?360
VLDDGWFGSRRDDRRALGDWYVSDEVWPDGLGPLTGHVTGLGMQFGLWVEPEMVNEDSDL?420
ARAHPDWVMAAGDRLPGAARHQQVLDLARPEAFAYILGRLDELLEAYPIAYLKWDHNRDL?480
VDAGHRPTGRAGVHGQTRAVYRLMDELRRRHPGVEIESCSSGGGRVDLEILQRTDRVWVS?540
DCIDALERQTIQRWTNALIPLELMGTHVGSDVAHTTGRRHPVDFRAGTALFGHFGIEWDL?600
TAAAPEELARLAQWVDLYKELRGLLHTGVRVHADHPDPAHRLHGVVAEDGSDAVYALVAT?660
ASSAMYPAGAIRLPGLDADAVYRVRPQAPGDLPDGNAHHWGVRLPWWTPEGVRLPGRVLA?720
AAGLQAPVLHPERLVLLRATRV?742
<210>2
<211>2229
<212>DNA
＜213〉streptomycete (Streptomyces sp.S27)
<400>2
GTGCCGGTTTGGCCAGGACGGAGACACACCGTGGCATCACGTTCCGCTCCCCTTCCCCTC?60
GATGTGACGCACCTGCGGGCGGCCGGCGTCAGCCTCGTACTGGACCTGACCGGCGGCACC?120
CTCCCCCGGGTCCTGCACTGGGGTGCCGACCTCGGCACGCTCGGCCCCGAGGAGCTGGGC?180
TCCCTGCGGCTGGCCGGGGCGCCGCAGCCCATCGGCTTCTCCGTGGACGGCCCGGTCGAG?240
GTCTCGGTGCTGCCCGAGCAGTCGGCCGGCTGGCTGGGCACCCCCGGCCTGGTGGGCAAC?300
CGCGGCGGCCGGGACTTCTCCACCGCCTTCGCCGTCCGCGAGGCCGCCCTCACCGGGCCC?360
GTGGAGGGGTCCCGCTCCCCCGGCGGCGGAGGCGGCGCCATCGTCACCGTCCTCGCCCGC?420
GACGAGGCCGCGGCCCTGGATCTGGAGCTGGTGATCGAGCTGACCGCGTCCGGACTGGTG?480
CGCCAGCGCGCCACGGTCGCCAACCGCGGCTCCTCCCCCTTCACCGTCGACGCCGTGAAC?540
CTCACGCTGCCGGTGCCCGCCGAAGCCGTCGAGCTGCTGGACTTCACCGGGCACCACCTG?600
CGCGAACGCAGCCCGCAGCGCACCGCCTTCACCCAGGGGCTGCGGATGCGGGAGAACCGC?660
ACCGGCCGCACCGGCTACGACAGCGCCTACCTGCTGGCCGCGGGCACCGCGGGCTTCGGC?720
AACCGCTCGGGCGAGGTGTGGGCCGTGCACACCGCGTGGTCCGGCAACCACCGCACCTTC?780
GCCGAGCGCACCTTCCACTCGGTGTCGCTGCTGGGCTCGGGCGAGCTGCTGCTCTCCGGC?840
GAGGTGGTGCTGGAGCCCGGGGAGTCCTACGCCTCCCCCTGGCAGTACGGCTCCTACGGG?900
CGGCACGGCCTGGACGAGGTCTCCGCCCGCTTCCACCGCTGGCTGCGCTCCCGCCCGCAC?960
CACCCCTCCACGCCCCGGCCGGTCACCCTCAACACCTGGGAGGCGGTCTACTTCGACCAC?1020
GATCTGGACCGGCTGCGCGCCCTGGCCGACGCGGCCGCCGCGGTGGGCGCGGAGCGCTTC?1080
GTCCTGGACGACGGCTGGTTCGGCTCGCGCCGCGACGACCGGCGCGCCCTGGGCGACTGG?1140
TACGTCTCGGACGAGGTGTGGCCGGACGGACTGGGCCCGCTGACCGGTCATGTCACCGGC?1200
CTGGGCATGCAGTTCGGCCTCTGGGTCGAGCCGGAGATGGTCAACGAGGACTCCGACCTG?1260
GCCCGCGCCCACCCCGACTGGGTCATGGCCGCCGGGGACCGGCTGCCCGGCGCGGCCCGC?1320
CACCAGCAGGTCCTCGACCTGGCCCGCCCGGAGGCGTTCGCGTACATCCTCGGGCGCCTG?1380
GACGAGCTGCTGGAGGCGTACCCCATCGCGTATCTGAAGTGGGACCACAACCGGGACCTG?1440
GTGGACGCCGGGCACCGGCCCACCGGCCGCGCCGGCGTGCACGGCCAGACCCGGGCGGTC?1500
TACCGGCTGATGGACGAGCTGCGCCGCCGCCACCCCGGGGTGGAGATCGAGTCCTGCTCC?1560
TCCGGCGGCGGACGGGTGGACCTGGAGATCCTCCAGCGCACCGACCGGGTGTGGGTCTCG?1620
GACTGCATCGACGCCCTGGAGCGCCAGACCATCCAGCGCTGGACGAACGCCCTGATCCCC?1680
CTCGAACTCATGGGCACCCACGTGGGGTCGGACGTCGCGCACACCACCGGGCGCCGCCAC?1740
CCGGTCGACTTCCGGGCGGGCACGGCGCTGTTCGGGCACTTCGGCATCGAGTGGGACCTG?1800
ACCGCCGCCGCGCCGGAGGAGCTGGCGCGGCTGGCGCAGTGGGTGGACCTCTACAAGGAG?1860
CTGCGCGGCCTGCTCCACACCGGCGTCCGTGTCCACGCCGACCACCCGGACCCGGCCCAC?1920
CGGCTGCACGGGGTGGTCGCCGAGGACGGCTCGGACGCGGTGTACGCCCTCGTCGCCACC?1980
GCCTCCTCCGCGATGTACCCGGCCGGTGCGATCCGGCTGCCGGGGCTGGACGCGGACGCC?2040
GTCTACCGGGTGCGGCCGCAGGCCCCCGGCGACCTGCCGGACGGCAACGCCCACCACTGG?2100
GGCGTCCGCCTGCCCTGGTGGACGCCGGAGGGTGTGCGGCTGCCGGGCCGGGTGCTGGCG?2160
GCCGCGGGGCTCCAGGCCCCGGTGCTCCACCCCGAGCGGCTGGTGCTGCTGCGCGCGACC?2220
CGGGTGTGA?2229
Claims (9)
1. streptomycete Streptomyces sp.S27, its preserving number is CGMCC No.3146.
2. the neutral alpha-galactosidase A ga-S27 of one kind high alpha galactosides class oligosaccharides degradation capability is characterized in that, its aminoacid sequence is shown in SEQ ID NO.1.
3. the neutral alpha-galactosidase gene aga-S27 of one kind high alpha galactosides class oligosaccharides degradation capability is characterized in that, the described alpha-galactosidase A ga-S27 of coding claim 2.
4. alpha galactosides enzyme coding gene aga-S27 as claimed in claim 3 is characterized in that its base sequence is shown in SEQ ID NO.2.
5. the recombinant vectors that comprises claim 3 or 4 described alpha-galactosidase gene aga-S27.
6. the recombinant bacterial strain that comprises claim 3 or 4 described alpha-galactosidase gene aga-S27.
7. recombinant bacterial strain as claimed in claim 6 is characterized in that, described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus.
8. a method for preparing the neutral alpha-galactosidase A ga-S27 of high alpha galactosides class oligosaccharides degradation capability is characterized in that, may further comprise the steps:
1) with the recombinant vectors transformed host cell of claim 6, gets recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induce the reorganization alpha-galactosidase to express; And
3) reclaim the also expressed alpha-galactosidase A ga-S27 of purifying.
9. the application of the neutral alpha-galactosidase A ga-S27 of the described high alpha galactosides oligosaccharides degradation capability of claim 2.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399764A (en) * | 2011-11-04 | 2012-04-04 | 中国农业科学院饲料研究所 | Alkaline pectinase PL-STR as well as gene and application thereof |
| CN103614322A (en) * | 2013-11-22 | 2014-03-05 | 浙江工业大学 | Streptomycete for producing glycosidase and application of streptomycete in preparing cucurbitacin B by virtue of biotransformation |
| CN108179114A (en) * | 2017-11-27 | 2018-06-19 | 南京晓庄学院 | Produce bacterial strain and fermentation process, the anaerobe resistant compound methods of extraction and preparation and application method of anaerobe resistant compound |
| CN109153981A (en) * | 2016-05-24 | 2019-01-04 | 诺维信公司 | Polypeptide with alpha-galactosidase activity and the polynucleotides for encoding it |
| CN114404667A (en) * | 2021-12-07 | 2022-04-29 | 尚诚怡美(成都)生物科技有限公司 | Long-acting recombinant human-derived collagen implant and application thereof |
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2009
- 2009-07-02 CN CN200910087938A patent/CN101838618A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399764A (en) * | 2011-11-04 | 2012-04-04 | 中国农业科学院饲料研究所 | Alkaline pectinase PL-STR as well as gene and application thereof |
| CN103614322A (en) * | 2013-11-22 | 2014-03-05 | 浙江工业大学 | Streptomycete for producing glycosidase and application of streptomycete in preparing cucurbitacin B by virtue of biotransformation |
| CN103614322B (en) * | 2013-11-22 | 2015-08-05 | 浙江工业大学 | The streptomycete producing Glycosylase and the application prepared in bio-transformation in Cucurbitacin B thereof |
| CN109153981A (en) * | 2016-05-24 | 2019-01-04 | 诺维信公司 | Polypeptide with alpha-galactosidase activity and the polynucleotides for encoding it |
| CN108179114A (en) * | 2017-11-27 | 2018-06-19 | 南京晓庄学院 | Produce bacterial strain and fermentation process, the anaerobe resistant compound methods of extraction and preparation and application method of anaerobe resistant compound |
| CN108179114B (en) * | 2017-11-27 | 2021-08-20 | 南京晓庄学院 | Strain for producing anti-anaerobic bacteria compound, fermentation method, extraction and preparation method of anti-anaerobic bacteria compound and use method |
| CN114404667A (en) * | 2021-12-07 | 2022-04-29 | 尚诚怡美(成都)生物科技有限公司 | Long-acting recombinant human-derived collagen implant and application thereof |
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