(3) summary of the invention
The object of the invention provides a kind of beta-glucosidase, encoding sox, carrier, engineering bacteria and application thereof, and this beta-glucoside endonuclease capable hydrolysis polygonin prepares trans-resveratrol.
The technical scheme that the present invention adopts is:
A kind of beta-glucosidase gene (Y1), said gene have the homology with the polynucleotide sequence 70-100% shown in the SEQ ID NO:2, and concrete preferred said gene has the polynucleotide sequence shown in the SEQ ID NO:2.
atgaaaatct taagtacgaa tattctcatt gttagtctgg tccttttggt tggatgtagt 60
cagccggaat cttcacagca agcctccaat cctaaaatgg atgcctttat taatgacttg 120
atgctgaaga tgacgctgga agaaaaaatc ggacagctaa accttcccgt tgcgggtggc 180
cctgccaccg gcattgccgt aaataaaggt cttgaagata aaatacgggc agggcaagta 240
ggtggaatct ttggtgtgtg ggggcccgat aaagttagaa aggtgcagga gattgcagtg 300
aatgaatcac gactgaagat ccctctgttt tttggattgg atgtaattca cgggcacaaa 360
actatctacc cgataccact gggtatggcc gccacgtggg acatgtcgtt aattgaacgt 420
ggtgcccagt tggctgcgca ggaagcatca gccgaaggat tgaactggac cttctcaccc 480
atggtggata tttcccgcga ccctcgctgg ggtcgtattt cagaaggctc tggtgaagat 540
ccctatctcg gttcgctcat cgcgcaagcc atggtaagag gctatcaggg taatgatctg 600
gcggccacca ataccattat ggcatgtgtg aaacactttg ctttatatgg cgctgccgaa 660
gccggcagag attataacac agtggatatg agtcgtgcta cgatgtataa tttttttctt 720
cctccttata aagctgcgct ggatgctggt gcggccagta tcatgagttc atttaatgta 780
gtggatggaa tcccggcttc gggtaacaaa tggctcttaa ccgatgtgct gcgaaagcaa 840
tggggcttca agggctttgt ggtgtcagat tacaccagta ttaacgaaat gatcaatcac 900
ggcatgggcg atttgaaaac ggtttcatca ctcgcgctga atgcaggcat ggacatggac 960
atggtgggag aaggtttttt aactacgttg aagaaatcaa tagaagagaa aacggttacc 1020
gaagaacaaa tcgatcaggc atgccgcaga atattggaag ccaaatacaa attgggattg 1080
ttcgatgatc cttacaaata tatcagcgaa gaacgtgctg ctaaagaagt tttcaataac 1140
gatactcgcc aggtagcacg ccagttggct gcccattctt ttgtgttgct gaaaaacaag 1200
gatcaacttt tgcctctcaa caaaaccaaa acaatggctt tcattgggcc gttggccaat 1260
aatcaacgcg atatgttggg tacgtgggtg attggtggtg aatgggataa atctgtttcg 1320
gtgctggaag gtgttaaaaa tgcactgggc gaaaaaggca aagtgttgca tgccaaaggt 1380
gccaacatta ccaacgatcc ggagatgatt aagcggttga atttctttgg tcaacctaac 1440
gtggtgctcg atgagcgctc ctcacaagca atgttgcagg aggcagtagc cactgcatct 1500
cgtgcagata ttattgtagc ggtgttgggc gaatcgcaaa gcatgtcggg tgagtcgtcc 1560
agccgtacac agttagatct cccggaaagc caaaaagaac tattgaaggc actcgtaaaa 1620
actggtaaga aggtagtgct tgtattgttt accggtcgcc cgttaacatt aacctgggaa 1680
gatgaaaatg tggatgctat tctgaatgta tgggcacccg gacatgaagc aggtaatgcc 1740
attgccgatg tgttgtttgg taactacaat ccttccggta aactgcctgc aacgtttccg 1800
cgaagcgtgg gccaggttcc gttgtattac aatcacttga atacaggccg cccctggaat 1860
ggtattgatg acaccaagtt taaatccaac tacctggacg aagccaatgt gcccttgtat 1920
cctttcggat ttggacttag ttacactacg tttggttttt ctgatataac cctcagtaag 1980
caagagttga aaggcaatga aacattaacg gccacggtaa cggtaacgaa taccggaaac 2040
tacgaaggcg aggaaactgt gcagttatac attcgcgatg tggtgggatc ggtgtctcgc 2100
ccgatgaaag agctgaaagg cttttcaaag atcaatctta agcccggaga aagtaagcaa 2160
gtgagtttcg aaatcacacc caatgatctt aagttttata attacgatct gaagtatgag 2220
tgggaaccgg gcgattttga aatcatgatt ggctccaatt cccgcgatgt aaaaatggct 2280
tctataaact ggaagaaata a 2301
Because the singularity of nucleotide sequence, the variant of polynucleotide shown in any SEQ ID NO:2 as long as itself and this polynucleotide have 70% above homology, all belongs to the row of protection domain of the present invention.The variant of said polynucleotide is meant a kind of polynucleotide sequence that one or more Nucleotide change that has.The variant of these polynucleotide can make the living displacement varient or the varient of non-life, comprises replacing varient, deletion mutation body and inserting varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it possibly be replacement, disappearance or the insertion of polynucleotide, but can be from the function of the peptide protein that changes its coding in fact.
In addition; Can (have 50% homology at least with the polynucleotide of the hybridization of polynucleotide sequence shown in the SEQ ID NO:2; Preferably have 70% homology at least), also at the row of protection domain of the present invention, particularly under stringent condition can with the polynucleotide of nucleotide sequence hybridization according to the invention.Said " stringent condition " is meant: (1) than hybridization under LIS and the comparatively high temps and wash-out, like 0.2SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time adds and to use denaturing agent, like 50% (v/v) methane amide, 0.1% calf serum, 0.1%Ficoll, 42 ℃; Or (3) only in the homology between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the peptide protein of interfertile polynucleotide encoding has identical biological function and activity with the peptide protein shown in the SEQ ID NO:1.
The polynucleotide sequence of beta-glucosidase according to the invention of encoding can obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: (1) with probe and gene or the hybridization of cDNA library with the screening active ingredients of the polynucleotide sequence that detects homology and (2) expression library to detect the clone's with common structure characteristic polynucleotide passage, this expression library can comprise the environment macro genomic library.Sequence dna fragment of the present invention also can use following method to obtain: double chain DNA sequence is separated from genomic dna in (1); (2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of said peptide protein.
Available ordinary method is screened gene of the present invention from these cNDA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) through measuring BA, detect the protein product of genetic expression.Aforesaid method can singly be used, but also several different methods combined utilization.
The beta-glucosidase gene of the present invention that obtains as stated, perhaps the polynucleotide sequence of various dna fragmentations etc. can be used the ordinary method double deoxidating chain termination measuring.This type polynucleotide sequence is measured also available commercial sequencing kit etc.For the cDNA sequence order-checking that obtains total length need be carried out repeatedly.Sometimes a plurality of clones' cDNA sequence need be measured, the cDNA sequence of total length could be spliced.
A kind of beta-glucosidase (peptide protein) of beta-glucosidase gene coding; Said beta-glucosidase has the homology with aminoacid sequence 95%-100% shown in the SEQ ID NO.1, and concrete preferred described beta-glucosidase has the aminoacid sequence shown in the SEQ ID NO.1.
Met Lys Ile Leu Ser Thr Asn Ile Leu Ile Val Ser Leu Val Leu Leu
1 5 10 15
Val Gly Cys Ser Gln Pro Glu Ser Ser Gln Gln Ala Ser Asn Pro Lys
20 25 30
Met Asp Ala Phe Ile Asn Asp Leu Met Leu Lys Met Thr Leu Glu Glu
35 40 45
Lys Ile Gly Gln Leu Asn Leu Pro Val Ala Gly Gly Pro Ala Thr Gly
50 55 60
Ile Ala Val Asn Lys Gly Leu Glu Asp Lys Ile Arg Ala Gly Gln Val
65 70 75 80
Gly Gly Ile Phe Gly Val Trp Gly Pro Asp Lys Val Arg Lys Val Gln
85 90 95
Glu Ile Ala Val Asn Glu Ser Arg Leu Lys Ile Pro Leu Phe Phe Gly
100 105 110
Leu Asp Val Ile His Gly His Lys Thr Ile Tyr Pro Ile Pro Leu Gly
115 120 125
Met Ala Ala Thr Trp Asp Met Ser Leu Ile Glu Arg Gly Ala Gln Leu
130 135 140
Ala Ala Gln Glu Ala Ser Ala Glu Gly Leu Asn Trp Thr Phe Ser Pro
145 150 155 160
Met Val Asp Ile Ser Arg Asp Pro Arg Trp Gly Arg Ile Ser Glu Gly
165 170 175
Ser Gly Glu Asp Pro Tyr Leu Gly Ser Leu Ile Ala Gln Ala Met Val
180 185 190
Arg Gly Tyr Gln Gly Asn Asp Leu Ala Ala Thr Asn Thr Ile Met Ala
195 200 205
Cys Val Lys His Phe Ala Leu Tyr Gly Ala Ala Glu Ala Gly Arg Asp
210 215 220
Tyr Asn Thr Val Asp Met Ser Arg Ala Thr Met Tyr Asn Phe Phe Leu
225 230 235 240
Pro Pro Tyr Lys Ala Ala Leu Asp Ala Gly Ala Ala Ser Ile Met Ser
245 250 255
Ser Phe Asn Val Val Asp Gly Ile Pro Ala Ser Gly Asn Lys Trp Leu
260 265 270
Leu Thr Asp Val Leu Arg Lys Gln Trp Gly Phe Lys Gly Phe Val Val
275 280 285
Ser Asp Tyr Thr Ser Ile Asn Glu Met Ile Asn His Gly Met Gly Asp
290 295 300
Leu Lys Thr Val Ser Ser Leu Ala Leu Asn Ala Gly Met Asp Met Asp
305 310 315 320
Met Val Gly Glu Gly Phe Leu Thr Thr Leu Lys Lys Ser Ile Glu Glu
325 330 335
Lys Thr Val Thr Glu Glu Gln Ile Asp Gln Ala Cys Arg Arg Ile Leu
340 345 350
Glu Ala Lys Tyr Lys Leu Gly Leu Phe Asp Asp Pro Tyr Lys Tyr Ile
355 360 365
Ser Glu Glu Arg Ala Ala Lys Glu Val Phe Asn Asn Asp Thr Arg Gln
370 375 380
Val Ala Arg Gln Leu Ala Ala His Ser Phe Val Leu Leu Lys Asn Lys
385 390 395 400
Asp Gln Leu Leu Pro Leu Asn Lys Thr Lys Thr Met Ala Phe Ile Gly
405 410 415
Pro Leu Ala Asn Asn Gln Arg Asp Met Leu Gly Thr Trp Val Ile Gly
420 425 430
Gly Glu Trp Asp Lys Ser Val Ser Val Leu Glu Gly Val Lys Asn Ala
435 440 445
Leu Gly Glu Lys Gly Lys Val Leu His Ala Lys Gly Ala Asn Ile Thr
450 455 460
Asn Asp Pro Glu Met Ile Lys Arg Leu Asn Phe Phe Gly Gln Pro Asn
465 470 475 480
Val Val Leu Asp Glu Arg Ser Ser Gln Ala Met Leu Gln Glu Ala Val
485 490 495
Ala Thr Ala Ser Arg Ala Asp Ile Ile Val Ala Val Leu Gly Glu Ser
500 505 510
Gln Ser Met Ser Gly Glu Ser Ser Ser Arg Thr Gln Leu Asp Leu Pro
515 520 525
Glu Ser Gln Lys Glu Leu Leu Lys Ala Leu Val Lys Thr Gly Lys Lys
530 535 540
Val Val Leu Val Leu Phe Thr Gly Arg Pro Leu Thr Leu Thr Trp Glu
545 550 555 560
Asp Glu Asn Val Asp Ala Ile Leu Asn Val Trp Ala Pro Gly His Glu
565 570 575
Ala Gly Asn Ala Ile Ala Asp Val Leu Phe Gly Asn Tyr Asn Pro Ser
580 585 590
Gly Lys Leu Pro Ala Thr Phe Pro Arg Ser Val Gly Gln Val Pro Leu
595 600 605
Tyr Tyr Asn His Leu Asn Thr Gly Arg Pro Trp Asn Gly Ile Asp Asp
610 615 620
Thr Lys Phe Lys Ser Asn Tyr Leu Asp Glu Ala Asn Val Pro Leu Tyr
625 630 635 640
Pro Phe Gly Phe Gly Leu Ser Tyr Thr Thr Phe Gly Phe Ser Asp Ile
645 650 655
Thr Leu Ser Lys Gln Glu Leu Lys Gly Asn Glu Thr Leu Thr Ala Thr
660 665 670
Val Thr Val Thr Asn Thr Gly Asn Tyr Glu Gly Glu Glu Thr Val Gln
675 680 685
Leu Tyr Ile Arg Asp Val Val Gly Ser Val Ser Arg Pro Met Lys Glu
690 695 700
Leu Lys Gly Phe Ser Lys Ile Asn Leu Lys Pro Gly Glu Ser Lys Gln
705 710 715 720
Val Ser Phe Glu Ile Thr Pro Asn Asp Leu Lys Phe Tyr Asn Tyr Asp
725 730 735
Leu Lys Tyr Glu Trp Glu Pro Gly Asp Phe Glu Ile Met Ile Gly Ser
740 745 750
Asn Ser Arg Asp Val Lys Met Ala Ser Ile Asn Trp Lys Lys
755 760 765
Beta-glucoside enzyme sequence of the present invention is searched comparison respectively through Protein Data Bank and Nucleotide DB; Do not find to have any identical peptide protein sequence and nucleotide sequence; Belong to novel beta-glucosidase, called after beta-glucosidase (Y1).
Because the singularity of aminoacid sequence; Any fragment or its variant that contains the peptide protein of aminoacid sequence shown in the SEO ID NO.1; Like its examples of conservative variations, bioactive fragment or verivate; As long as the fragment of this peptide protein or peptide protein variant and aforementioned amino acid sequence homology all belong to the row of protection domain of the present invention more than 95%.Concrete said change can comprise amino acid whose disappearance, insertion or replacement in the aminoacid sequence; Wherein, for the conservative property change of variant, the amino acid of being replaced has structure similar with original acid or chemical property, and as replacing Isoleucine with leucine, variant also can have non-conservation and change, as replacing glycocoll with tryptophane.The fragment of peptide protein according to the invention, verivate or analogue are meant and keep identical biological function of beta-glucosidase of the present invention or active peptide protein basically; Can be under conditions: (I) one or more amino-acid residues be replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and substituted amino acid can be also can not encoded by genetic codon; (II) certain group on one or more amino-acid residues is replaced by other group; (III) mature peptide albumen and another kind of compound (such as the compound that prolongs the peptide protein transformation period, for example polyoxyethylene glycol) merge; (IV) additional aminoacid sequence is integrated into sophisticated peptide protein and the peptide protein sequence that the forms sequence or the proteinogen sequence of this peptide protein of purifying (as be used for).
Said peptide protein can be recombinant protein, native protein or synthetic proteins; It can be the product of pure natural purifying; Or the product of chemosynthesis, or use recombinant technology from protokaryon or eucaryon host (for example: bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, peptide protein of the present invention can be glycosylated.Peptide protein of the present invention can also comprise or not comprise initial methionine residues.
The invention still further relates to a kind of recombinant vectors that contains described beta-glucosidase gene, and said recombinant vectors transforms the recombination engineering bacteria that obtains.
The invention still further relates to the application of said beta-glucosidase gene in preparation reorganization beta-glucosidase; Concrete described being applied as: make up the recombinant vectors that contains beta-glucosidase gene; Said recombinant vectors is converted in the host; The recombination engineering bacteria that obtains is carried out inducing culture, and the nutrient solution separation obtains containing the reorganization beta-glucosidase gene.
Beta-glucosidase gene according to the invention can be inserted in the carrier, contains the recombinant vectors of beta-glucosidase gene according to the invention with formation." carrier " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral like adenovirus, retrovirus or other carrier.The carrier that is suitable in the present invention also includes but not limited to: the expression vector based on the T7 promotor of in bacterium, expressing; The pcDNA3.1 carrier of in mammalian cell, expressing and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can in host, duplicate and stablize, any plasmid and carrier may be used to make up recombinant expression vector, and preferred pET carrier series is with its other prokaryotic expression carrier series.Key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.
Method well-known to those having ordinary skill in the art can be used to make up the expression vector that contains beta-glucosidase gene and suitable transcribing/translational control element.These methods comprise extracorporeal recombinant DNA sequence, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA's.The representative example of these promotors has: colibacillary lac or trp promotor; The PL promotor of phage; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte or eukaryotic cell or its virus of CMV early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site that translation initiation is used and transcription terminator etc.In carrier, inserting enhancer sequence will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is that DNA expresses the cis acting factor, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.The example that can take is included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
In addition; Expression vector preferably comprises one or more selected markers; To be provided for selecting the phenotypic character of transformed host cells; Cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein of usefulness like eukaryotic cell, or be used for colibacillary kantlex or penbritin etc.
The recombinant vectors that contains beta-glucosidase gene or contain beta-glucosidase gene according to the invention can transform or transduce into host cell, contains the genetic engineering bacterium of this gene or recombinant vectors with formation." host cell " refers to prokaryotic cell prokaryocyte, like bacterial cell; Or eukaryotic cell such as low, like yeast cell; Or higher eucaryotic cells, like mammalian cell.Representation has: intestinal bacteria, streptomyces; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.
With gene of the present invention (dna sequence dna) or contain the recombinant vectors transformed host cell of said gene (dna sequence dna), the routine techniques that available art technology is known carries out.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used method is that this area is well-known, and alternative is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, perhaps conventional mechanical method such as microinjection, electroporation, liposome packing etc.
Through the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or produce the beta-glucosidase of reorganization.In general following steps are arranged:
(1) with beta-glucosidase gene of the present invention (or varient), or with the recombinant vectors conversion or the transfection proper host cell that contain this gene;
(2) in suitable medium, cultivate host cell;
(3) separation, protein purification from substratum or cell.
In step (2), according to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Cultivate under the condition of host cell being suitable for.After host cell is grown in suitable cell density, induce the promotor of selection with suitable method, cell is cultivated for some time again.
In step (3), recombinant peptide albumen can encapsulate expresses or is secreted into the extracellular in cell or on cytolemma.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.These methods include but not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, ultrasonication, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.
Main points of the present invention have been to provide the nucleotide sequence shown in aminoacid sequence shown in the SEQ ID NO:1 and the SEQ ID NO:2; Under the situation of known this aminoacid sequence and nucleotide sequence; The acquisition of this aminoacid sequence and nucleotide sequence; And the acquisition of related vector, host cell, all be conspicuous to those skilled in the art.
The invention still further relates to described beta-glucosidase and prepare the application in the trans-resveratrol, described being applied as: the Na that at pH is 0.1~0.5mol/L of 3~7 at the hydrolysis polygonin
2HPO
4In-the citrate buffer solution, add isopyknic 0.5~5mg/ml polygonin aqueous solution, mix; Beta-glucosidase with 0.5~5mg/ml is a catalyzer; The final concentration of described beta-glucosidase is 0.01~0.1mg/ml, in 25~40 ℃ of shaking table reactions 10~20 hours, gets reaction solution 3000~6000rpm centrifugal 5 minutes behind the mixing; Throw out is the trans-resveratrol raw product, processes trans-resveratrol after aftertreatment is refining.
Concrete, described being applied as: the Na of the 0.2mol/L of (1) preparation pH=4
2HPO
4-citrate buffer solution; (2) get a test tube, add above-mentioned damping fluid 20ml, add 1mg/ml polygonin solution 20ml; Mix, add the beta-glucosidase 3ml of 1mg/ml, reacted 12 hours in 37 ℃ of shaking tables behind the mixing; Got reaction solution 5000rpm centrifugal 5 minutes, throw out is the trans-resveratrol raw product, and polygonin and trans-resveratrol bullion are analyzed with liquid chromatograph respectively; Liquid phase chromatogram condition: Irregular C18 post (250mm * 4.6mm, 5 μ m); Moving phase: acetonitrile-water (25: 75); Volumetric flow rate: 1.0mL/min; Column temperature: 35 ℃; Detect wavelength: 306nm; Sample size: 20 μ L.
Compared with prior art; Beneficial effect of the present invention is mainly reflected in: the invention provides a kind of beta-glucosidase, encoding sox, carrier, engineering bacteria and application thereof, this beta-glucoside endonuclease capable hydrolysis polygonin prepares trans-resveratrol, environmental friendliness; Productive rate is high, has a extensive future.
(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: the Vitamin C2 screening method of beta-glucosidase Y1 gene
Preparation LB solid medium.The final concentration of LB solid medium consists of: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar powder 15g/L, transfer pH to 7.0 with 5mol/L NaOH solution, solvent is a water, at 15psi (1.05kg/cm
2) steam sterilizing 20min under the high pressure.The cooling substratum;, substratum adds final concentration before not solidifying and is 2.5% ferric ammonium citrate (filtration sterilization) and 0.1% Vitamin C2 (filtration sterilization); After the substratum cooling; Microbial inoculant to be screened in this substratum, was cultivated 24 hours for 37 ℃, promptly positive (Fig. 1) of black splotch arranged around the microbe colony.
The cDNA screening and the clone of embodiment 2:Y1 full length gene
Make up the soil genomic library.According to embodiment 1 method screening positive clone and make up Ya Wenku,, obtain nucleotide sequence shown in the SEQ ID NO.1 through gene sequencing (measuring) by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.According to nucleotide sequence shown in the SEQ ID NO.1; Design amplifies the primer that complete coding is read frame; And on the upstream and downstream primer, introduce restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively; Beta-glucosidase gene through the nucleotide sequence shown in the amplification in vitro technology acquisition SEQ ID NO.1 with this gene clone to pET 28a intermediate carrier (Hangzhou Pedagogic University's biological medicine and health center preserve), is guaranteeing to identify good expression vector under the correct prerequisite of reading frame; Again it is changed among the E.Coil BL21, obtain engineering bacteria E.Coil BL21/pET 28a/Y1.
The proteic expression of embodiment 3:Y1
E.Coil BL21/pET 28a/Y1 shaking table incubated overnight in the LB liquid nutrient medium that contains 100mL 50 μ g/mL kantlex with embodiment 2 acquisitions.Bacterium liquid after the 10ml incubated overnight is poured into the LB liquid nutrient medium of 1L 50 μ g/mL kantlex and cultivated, up to bacterium liquid OD
600Adding the IPTG final concentration when reaching 0.6-0.8 is 1.0mmol/L, 28 ℃ induce 5h after, centrifugal collection thalline, thalline makes its resuspension with 25ml phosphoric acid buffer pH7.4, the adding N,O-Diacetylmuramidase, the N,O-Diacetylmuramidase final concentration is 1mg/ml, and multigelation makes cytoclasis.The centrifuging and taking supernatant, contain the proteic supernatant of Y1 with 0.22 μ m FM membrane filtration after nickel post affinity chromatography carry out purifying and obtain the pure enzyme of Y1.
Embodiment 4:Y1 catalysis p-nitrophenyl-beta-glucoside is measured the righttest catalytic condition
The present invention is a substrate with p-nitrophenyl-beta-glucoside (ONPG); Y1 catalytic pyrolysis a part ONPG obtains an one's share of expenses for a joint undertaking p-NP, absorbs at the UV of 450nm wavelength through the mensuration p-NP and measures kinetic curve with the righttest catalytic condition of assay determination Y1.
The pure enzyme of Y1 of using pH4.94,5.39,5.91,6.24,6.47,6.81,6.98,7.38,7.5,7.73,8.04,8.34,8.69,9.18 phosphoric acid buffers (0.15mol/L) that embodiment 3 is obtained respectively is made into the enzyme liquid of 1mg/ml; Get the phosphoric acid buffer 3ml of each pH value and the enzyme liquid 1ml of corresponding pH value again; Add 3mlONPG (1mg/ml) more respectively; 30 ℃ of insulations, the kinetic curve at mensuration wavelength 450nm place draws the Y1 optimum pH and is about 7.5 (Fig. 2).
The Y1 enzyme of embodiment 3 being obtained with the phosphoric acid buffer (pH7.5) of 0.15mol/L is made into the enzyme liquid of 1mg/ml; Get this enzyme liquid 1ml respectively; And add 3ml phosphoric acid buffer (pH7.5) and 3mlONPG (1mg/ml) respectively; Under 10,15,20,25,30,35,45,50 ℃ of temperature condition (10 ℃-50 ℃) respectively, the kinetic curve (Fig. 3) at mensuration wavelength 450nm place draws the Y1 optimum temperuture and is about 35 ℃.
Embodiment 6:Y1 catalysis polygonin method
Use the Na of pH4.0 and pH5.0 respectively
2HPO
4-citrate buffer solution (0.2mol/L) is mixed with the Y1 enzyme liquid of 1mg/ml; Respectively pH4.0 and the above-mentioned damping fluid 20ml of pH5.0 and 1mg/ml polygonin solution 20ml are mixed, add the Y1 enzyme liquid 3ml of above-mentioned pH4.0 and pH5.0 more respectively, behind the mixing in 37 ℃ of shaking tables reactions 12 hours; Reaction finishes; Got reaction solution 5000rpm centrifugal 5 minutes, abandoning supernatant is precipitated as the trans-resveratrol bullion.Polygonin standard substance, trans-resveratrol standard substance and trans-resveratrol bullion are analyzed (Fig. 4-7) with liquid chromatograph respectively.Liquid phase chromatogram condition: Irregular C18 post (250mm * 4.6mm, 5 μ m); Moving phase: acetonitrile-water (25: 75); Volumetric flow rate: 1.0mL/min; Column temperature: 35 ℃; Detect wavelength: 306nm; Sample size: 20 μ L.Conclusion: this Y1 enzyme can the catalysis polygonin be a trans-resveratrol, and transformation efficiency is about 52%.
SEQUENCE LISTING
< 110>Hangzhou Pedagogic University
< 120>a kind of beta-glucosidase, encoding sox, carrier, engineering bacteria and application thereof
<130>
<160> 2
<170> PatentIn version 3.4
<210> 1
<211> 766
<212> PRT
<213> Unknown
<220>
< 223>artificial sequence
<400> 1
Met Lys Ile Leu Ser Thr Asn Ile Leu Ile Val Ser Leu Val Leu Leu
1 5 10 15
Val Gly Cys Ser Gln Pro Glu Ser Ser Gln Gln Ala Ser Asn Pro Lys
20 25 30
Met Asp Ala Phe Ile Asn Asp Leu Met Leu Lys Met Thr Leu Glu Glu
35 40 45
Lys Ile Gly Gln Leu Asn Leu Pro Val Ala Gly Gly Pro Ala Thr Gly
50 55 60
Ile Ala Val Asn Lys Gly Leu Glu Asp Lys Ile Arg Ala Gly Gln Val
65 70 75 80
Gly Gly Ile Phe Gly Val Trp Gly Pro Asp Lys Val Arg Lys Val Gln
85 90 95
Glu Ile Ala Val Asn Glu Ser Arg Leu Lys Ile Pro Leu Phe Phe Gly
100 105 110
Leu Asp Val Ile His Gly His Lys Thr Ile Tyr Pro Ile Pro Leu Gly
115 120 125
Met Ala Ala Thr Trp Asp Met Ser Leu Ile Glu Arg Gly Ala Gln Leu
130 135 140
Ala Ala Gln Glu Ala Ser Ala Glu Gly Leu Asn Trp Thr Phe Ser Pro
145 150 155 160
Met Val Asp Ile Ser Arg Asp Pro Arg Trp Gly Arg Ile Ser Glu Gly
165 170 175
Ser Gly Glu Asp Pro Tyr Leu Gly Ser Leu Ile Ala Gln Ala Met Val
180 185 190
Arg Gly Tyr Gln Gly Asn Asp Leu Ala Ala Thr Asn Thr Ile Met Ala
195 200 205
Cys Val Lys His Phe Ala Leu Tyr Gly Ala Ala Glu Ala Gly Arg Asp
210 215 220
Tyr Asn Thr Val Asp Met Ser Arg Ala Thr Met Tyr Asn Phe Phe Leu
225 230 235 240
Pro Pro Tyr Lys Ala Ala Leu Asp Ala Gly Ala Ala Ser Ile Met Ser
245 250 255
Ser Phe Asn Val Val Asp Gly Ile Pro Ala Ser Gly Asn Lys Trp Leu
260 265 270
Leu Thr Asp Val Leu Arg Lys Gln Trp Gly Phe Lys Gly Phe Val Val
275 280 285
Ser Asp Tyr Thr Ser Ile Asn Glu Met Ile Asn His Gly Met Gly Asp
290 295 300
Leu Lys Thr Val Ser Ser Leu Ala Leu Asn Ala Gly Met Asp Met Asp
305 310 315 320
Met Val Gly Glu Gly Phe Leu Thr Thr Leu Lys Lys Ser Ile Glu Glu
325 330 335
Lys Thr Val Thr Glu Glu Gln Ile Asp Gln Ala Cys Arg Arg Ile Leu
340 345 350
Glu Ala Lys Tyr Lys Leu Gly Leu Phe Asp Asp Pro Tyr Lys Tyr Ile
355 360 365
Ser Glu Glu Arg Ala Ala Lys Glu Val Phe Asn Asn Asp Thr Arg Gln
370 375 380
Val Ala Arg Gln Leu Ala Ala His Ser Phe Val Leu Leu Lys Asn Lys
385 390 395 400
Asp Gln Leu Leu Pro Leu Asn Lys Thr Lys Thr Met Ala Phe Ile Gly
405 410 415
Pro Leu Ala Asn Asn Gln Arg Asp Met Leu Gly Thr Trp Val Ile Gly
420 425 430
Gly Glu Trp Asp Lys Ser Val Ser Val Leu Glu Gly Val Lys Asn Ala
435 440 445
Leu Gly Glu Lys Gly Lys Val Leu His Ala Lys Gly Ala Asn Ile Thr
450 455 460
Asn Asp Pro Glu Met Ile Lys Arg Leu Asn Phe Phe Gly Gln Pro Asn
465 470 475 480
Val Val Leu Asp Glu Arg Ser Ser Gln Ala Met Leu Gln Glu Ala Val
485 490 495
Ala Thr Ala Ser Arg Ala Asp Ile Ile Val Ala Val Leu Gly Glu Ser
500 505 510
Gln Ser Met Ser Gly Glu Ser Ser Ser Arg Thr Gln Leu Asp Leu Pro
515 520 525
Glu Ser Gln Lys Glu Leu Leu Lys Ala Leu Val Lys Thr Gly Lys Lys
530 535 540
Val Val Leu Val Leu Phe Thr Gly Arg Pro Leu Thr Leu Thr Trp Glu
545 550 555 560
Asp Glu Asn Val Asp Ala Ile Leu Asn Val Trp Ala Pro Gly His Glu
565 570 575
Ala Gly Asn Ala Ile Ala Asp Val Leu Phe Gly Asn Tyr Asn Pro Ser
580 585 590
Gly Lys Leu Pro Ala Thr Phe Pro Arg Ser Val Gly Gln Val Pro Leu
595 600 605
Tyr Tyr Asn His Leu Asn Thr Gly Arg Pro Trp Asn Gly Ile Asp Asp
610 615 620
Thr Lys Phe Lys Ser Asn Tyr Leu Asp Glu Ala Asn Val Pro Leu Tyr
625 630 635 640
Pro Phe Gly Phe Gly Leu Ser Tyr Thr Thr Phe Gly Phe Ser Asp Ile
645 650 655
Thr Leu Ser Lys Gln Glu Leu Lys Gly Asn Glu Thr Leu Thr Ala Thr
660 665 670
Val Thr Val Thr Asn Thr Gly Asn Tyr Glu Gly Glu Glu Thr Val Gln
675 680 685
Leu Tyr Ile Arg Asp Val Val Gly Ser Val Ser Arg Pro Met Lys Glu
690 695 700
Leu Lys Gly Phe Ser Lys Ile Asn Leu Lys Pro Gly Glu Ser Lys Gln
705 710 715 720
Val Ser Phe Glu Ile Thr Pro Asn Asp Leu Lys Phe Tyr Asn Tyr Asp
725 730 735
Leu Lys Tyr Glu Trp Glu Pro Gly Asp Phe Glu Ile Met Ile Gly Ser
740 745 750
Asn Ser Arg Asp Val Lys Met Ala Ser Ile Asn Trp Lys Lys
755 760 765
<210> 2
<211> 2301
<212> DNA
<213> Unknown
<220>
< 223>artificial sequence
<400> 2
atgaaaatct taagtacgaa tattctcatt gttagtctgg tccttttggt tggatgtagt 60
cagccggaat cttcacagca agcctccaat cctaaaatgg atgcctttat taatgacttg 120
atgctgaaga tgacgctgga agaaaaaatc ggacagctaa accttcccgt tgcgggtggc 180
cctgccaccg gcattgccgt aaataaaggt cttgaagata aaatacgggc agggcaagta 240
ggtggaatct ttggtgtgtg ggggcccgat aaagttagaa aggtgcagga gattgcagtg 300
aatgaatcac gactgaagat ccctctgttt tttggattgg atgtaattca cgggcacaaa 360
actatctacc cgataccact gggtatggcc gccacgtggg acatgtcgtt aattgaacgt 420
ggtgcccagt tggctgcgca ggaagcatca gccgaaggat tgaactggac cttctcaccc 480
atggtggata tttcccgcga ccctcgctgg ggtcgtattt cagaaggctc tggtgaagat 540
ccctatctcg gttcgctcat cgcgcaagcc atggtaagag gctatcaggg taatgatctg 600
gcggccacca ataccattat ggcatgtgtg aaacactttg ctttatatgg cgctgccgaa 660
gccggcagag attataacac agtggatatg agtcgtgcta cgatgtataa tttttttctt 720
cctccttata aagctgcgct ggatgctggt gcggccagta tcatgagttc atttaatgta 780
gtggatggaa tcccggcttc gggtaacaaa tggctcttaa ccgatgtgct gcgaaagcaa 840
tggggcttca agggctttgt ggtgtcagat tacaccagta ttaacgaaat gatcaatcac 900
ggcatgggcg atttgaaaac ggtttcatca ctcgcgctga atgcaggcat ggacatggac 960
atggtgggag aaggtttttt aactacgttg aagaaatcaa tagaagagaa aacggttacc 1020
gaagaacaaa tcgatcaggc atgccgcaga atattggaag ccaaatacaa attgggattg 1080
ttcgatgatc cttacaaata tatcagcgaa gaacgtgctg ctaaagaagt tttcaataac 1140
gatactcgcc aggtagcacg ccagttggct gcccattctt ttgtgttgct gaaaaacaag 1200
gatcaacttt tgcctctcaa caaaaccaaa acaatggctt tcattgggcc gttggccaat 1260
aatcaacgcg atatgttggg tacgtgggtg attggtggtg aatgggataa atctgtttcg 1320
gtgctggaag gtgttaaaaa tgcactgggc gaaaaaggca aagtgttgca tgccaaaggt 1380
gccaacatta ccaacgatcc ggagatgatt aagcggttga atttctttgg tcaacctaac 1440
gtggtgctcg atgagcgctc ctcacaagca atgttgcagg aggcagtagc cactgcatct 1500
cgtgcagata ttattgtagc ggtgttgggc gaatcgcaaa gcatgtcggg tgagtcgtcc 1560
agccgtacac agttagatct cccggaaagc caaaaagaac tattgaaggc actcgtaaaa 1620
actggtaaga aggtagtgct tgtattgttt accggtcgcc cgttaacatt aacctgggaa 1680
gatgaaaatg tggatgctat tctgaatgta tgggcacccg gacatgaagc aggtaatgcc 1740
attgccgatg tgttgtttgg taactacaat ccttccggta aactgcctgc aacgtttccg 1800
cgaagcgtgg gccaggttcc gttgtattac aatcacttga atacaggccg cccctggaat 1860
ggtattgatg acaccaagtt taaatccaac tacctggacg aagccaatgt gcccttgtat 1920
cctttcggat ttggacttag ttacactacg tttggttttt ctgatataac cctcagtaag 1980
caagagttga aaggcaatga aacattaacg gccacggtaa cggtaacgaa taccggaaac 2040
tacgaaggcg aggaaactgt gcagttatac attcgcgatg tggtgggatc ggtgtctcgc 2100
ccgatgaaag agctgaaagg cttttcaaag atcaatctta agcccggaga aagtaagcaa 2160
gtgagtttcg aaatcacacc caatgatctt aagttttata attacgatct gaagtatgag 2220
tgggaaccgg gcgattttga aatcatgatt ggctccaatt cccgcgatgt aaaaatggct 2280
tctataaact ggaagaaata a 2301