CN102653739A - Method for producing inulin fructose transferase by use of yeast expression system - Google Patents
Method for producing inulin fructose transferase by use of yeast expression system Download PDFInfo
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- CN102653739A CN102653739A CN2012101870690A CN201210187069A CN102653739A CN 102653739 A CN102653739 A CN 102653739A CN 2012101870690 A CN2012101870690 A CN 2012101870690A CN 201210187069 A CN201210187069 A CN 201210187069A CN 102653739 A CN102653739 A CN 102653739A
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Abstract
The invention discloses a method for producing inulin fructose transferase by use of a yeast expression system, belonging to the technical field of genetic engineering. The method comprises the following steps of: (1) cloning the inulin fructose transferase gene (ift), and reconstructing a recombinant vector X-IFTase through double digests; (2) transforming the recombinant vector X-IFTase into pichia yeast competent cell, and performing auxotroph screening and antibiotic resistance screening of positive transformant to obtain the yeast engineering bacteria of inulin fructose transferase; and (3) fermenting the liquid of the engineering bacteria to secrete active inulin fructose transferase. The method disclosed by the invention realizes the yeast secretion expression of the inulin fructose transferase, obtains the inulin fructose transferase with activity, and has a broad application prospect and significance in the enzymic-method industrial production of a novel functional sweetener twintose III.
Description
Technical field
The present invention relates to a kind of Yeast engineering bacteria that is used to produce inulin ftructotransferase, the invention still further relates to the preparation method of engineering bacillus and use the method that engineering bacillus is produced inulin ftructotransferase, belong to gene engineering technology field.
Background technology
(Difructose anhydride is a kind of novel irreducibility disaccharide DFAIII) to difructose anhydride III, is present on a small quantity at occurring in nature in the plants such as witloof, jerusalem artichoke, comes across on a small quantity in the course of processing of honey, coffee etc.Its relative sweetness is 52% of a sucrose, and caloric value has only 1/15 (0.263kcal/g) of sucrose; Character is very stable, and is non-hygroscopic under 74% relative humidity, than the more difficult moisture absorption of sucrose, has the potential that replaces traditional sweeting agent sucrose.Difructose anhydride III is very stable to heat and acid, under the normal food processing conditions, brown stain or decomposing phenomenon can occur hardly, the high temperature infusion when the ability hard candy is produced and not brown stain.Simultaneously, difructose anhydride III also has good functional characteristics, as not absorbing at digestive tube, and generate energy not, can be used as a sweeting agent and be used as the fat-reducing assisting therapy; Increase the divergent factor as a kind of, can promote the growth of enteric microorganism, obviously improve defecation, diuresis function; As the functional sugar that promotes that mineral element absorbs, can make mineral elements such as Ca, Mg, Zn, the Cu degree of utilizing that is absorbed by the body improve greatly, promote bone growth; Aspect dental caries, its character can compare favourably with Xylitol, sorbyl alcohol, is not brought out the Streptococcus oralis utilization of decayed tooth, does not also produce acid.These advantages have broad application prospects it in food, health care, medical field.
Yet difructose anhydride III is extremely low at natural content, and the natural extract separation costs is high; The demand of the suitability for industrialized production that do not suit; And chemical catalysis has many unfavorable factors, for example forms many by products and chemical pollutant, and the many by products that form make purification step become complicated.And biological process prepares the raw material of difructose anhydride III is synanthrin, and its wide material sources are cheap; Transformation efficiency is high, can reach more than 75%, belongs to natural product; Meet the consumer psychology that the human consumer pursues natural product, therefore adopt conversion technology to produce the focus that difructose anhydride III becomes research.
From produce the urea Arthrobacter, find inulin ftructotransferase (Inulin fructotransferase first from Uchiyama I in 1973; IFTase; EC 2.4.1.93); The history that the research for preparing difructose anhydride III with biotransformation method has had surplus in the of 30 year, however domesticly sieve to such an extent that a strain IFTase produces bacterial strain and it is carried out the correlative study except that this seminar, and domestic still do not have a correlative study report.Abroad the molecular biology research about inulin ftructotransferase starts from 1997, people such as Sakurai take the lead in
Arthrobacter sp.The inulin ftructotransferase gene of H65-7 changes in the intestinal bacteria, has obtained first inulin ftructotransferase gene ORFs, and its enzyme work is brought up to intracellular enzyme 170 U/mL alive by outer 80 U/mL of the born of the same parents of initial wild bacterium.Only there are 5 strain bacterium to realize the activity expression of the prokaryotic expression system of IFTase up to now, are respectively
Arthrobacter sp.H65-7,
Arthrobacter sp.A-6,
Arthrobacter globiformisC11-1,
Arthrobacter sp.Buo141,
Bacillus sp.Snu-7.And only
A. globiformisThe C11-1 inulin ftructotransferase has the enzyme biopsy to go out outside born of the same parents, and enzyme very low (1.5 U/mL) [document Haraguchi K, Mori S, Hayashi K. Cloning of inulin fructotransferase (DFA III-Producing) gene from alive
Arthrobacter globiformisC11-1 [J]. Journal of Bioscience and Bioengineering, 2000,89:590-595.], other these proenzyme nuclear expressions nearly all are to express in the born of the same parents, this makes the later stage purifying process of IFTase enzyme comparatively loaded down with trivial details.Simultaneously,
IftOwing to be mostly to express in the born of the same parents, signal peptide sequence exists can not be by the problem of effective identification and cutting, according to inferring that this also is one of lower reason of this proenzyme nuclear expression activity in prokaryotic expression for gene.In addition, intestinal bacteria are prokaryotic organism, and its expression process often produces toxin, and there is certain problem in its security.
The pichia spp gene expression system becomes more perfect heterologous gene expression system basically through development in nearly ten years, has the high density fermentation of being easy to, and the expressing gene stable integration can make product effectively secrete in host genome, cultivates the economic dispatch characteristics that make things convenient for.Compare with intestinal bacteria and can carry out proteic posttranslational modification processing; Comprise signal peptide processing, protein folding, disulfide linkage form, glycosylation modified etc.; Can effectively solve inclusion body formation problem in some albumen pronucleus expression signal peptide identification problems and the born of the same parents; And can increase the stability of enzyme to a certain extent, also have higher security.At present, U.S. FDA can have been estimated the gene engineering product from this system, and the Cephelon preparation from this system has obtained the FDA approval recently, so this system is considered to safe.Simultaneously, pichia yeast expression system can adopt the basic medium fermentation, only provides the yeast growth desired nutritional to get final product high density fermentation, and dried cell weight can reach 100 g/L.In addition, yeast cell self excretory albumen seldom can effectively be simplified separation purifying technique.
The present invention has realized the secreting, expressing of inulin ftructotransferase gene in yeast first; Obtain the engineering bacteria that the ability fermentative prodn has higher-security and bioactive inulin ftructotransferase; Has important significance for theories, for the domestic and international eukaryotic expression of inulin ftructotransferase has carried out successful pre-test.The present invention simultaneously also has actual application value, for inulin ftructotransferase and functional sweetener DFA III suitability for industrialized production provide theoretical foundation and application potential, also for the R and D of functional sweetener the broad space is provided.
Summary of the invention
The purpose of this invention is to provide the method that makes up the inulin ftructotransferase Yeast engineering bacteria.
Technical scheme of the present invention: a kind of method of utilizing yeast expression system to produce inulin ftructotransferase, step is:
(1) with golden yellow Arthrobacter (
Arthrobacter aurescens) SK8.001 is starting strain, the PCR method obtains the inulin ftructotransferase gene
Ift, make up recombinant vectors X-IFTase through double digestion;
Said recombinant vectors X-IFTase is: pPIC9K – IFTase, pPIC9 – IFTase, pPICZaA/B/C – IFTase or pGAPZaA/B/C – IFTase;
(2) recombinant vectors X-IFTase is transformed into the pichia spp competent cell after linearizing, and the Screening and Identification positive transformant, promptly make up and obtain producing the inulin ftructotransferase Yeast engineering bacteria;
Said Pichi strain is: GS115, X-33, KM71 or SMD1168;
(3) the engineering bacteria liquid fermenting is produced active inulin ftructotransferase.
The inulin ftructotransferase Yeast engineering bacteria is expressed through methanol induction; The HPLC method of reacting fermented supernatant fluid and synanthrin detects difructose anhydride III and generates situation to carry out the inulin ftructotransferase activity identification, and the Yeast engineering bacteria that shows acquisition has secretion and produces and have the ability of active inulin ftructotransferase;
The single colony inoculation of reorganization bacterium is cultured to OD in 28-30 ℃ of BMGY substratum, 280 rpm
600=2-6, centrifugal collection thalline makes OD with the resuspended thalline of BMMY substratum
600About=1, double gauze seals 30 ℃, 280 rpm shake flask fermentations, and it is 0.5%-1.0% that every 24h adds 100% methyl alcohol to final concentration to substratum.
The positive recombinant bacterial strain 60h of the 4 mg/mL G418-YPD that obtain fermented liquid produces the thick enzyme of the active inulin ftructotransferase 10.3U/mL of being alive.
Inulin ftructotransferase gene described in the step (1) is full gene of inulin ftructotransferase or the inulin ftructotransferase gene transformed according to the yeast codon preference.
Recombinant vectors linearizing described in the step (2) utilizes Sac I or Sal I to carry out enzyme and cuts.
The method that transforms the Pichi strain cell described in the step (2) adopts electric shock to transform or chemical conversion.
The engineering bacteria liquid fermentation medium is BMGY/BMMY, BMG/BMM or YPD described in the step (3).
From golden yellow Arthrobacter (
Arthrobacter aurescens) SK8.001 extracts the inulin ftructotransferase genomic dna, obtains not comprise signal peptide inulin ftructotransferase gene partly with the method amplification of Auele Specific Primer PCR
Ift, with this gene with
EcoRI with
NotI is as the restriction enzyme site construction of expression vector; Recombinant vectors is identified through double digestion and gene sequencing, is transformed into the yeast competent cell, through histidine defect and resistance screening and PCR checking acquisition positive colony; Then; Utilize methanol induction secreting, expressing inulin ftructotransferase, thereby realize that synanthrin is the enzyme process preparation of new type functional sweeting agent difructose anhydride III, has better industrialized production.
This golden yellow Arthrobacter (
Arthrobacter aurescens) SK8.001 carried out preservation and disclose in Chinese patent 200810196709.8 " strain is produced the bacterial strain of inulin ftructotransferase and produced the method for difructose anhydride III with this enzyme ", deposit number is CCTCC NO:M 208120.
Beneficial effect of the present invention: the present invention has realized the yeast secreted expression of inulin ftructotransferase; Acquisition has active inulin ftructotransferase, is with a wide range of applications and meaning for the enzyme process suitability for industrialized production of new type functional sweeting agent difructose anhydride III.
Description of drawings
The structure synoptic diagram of Fig. 1 recombinant plasmid pPPIC9K-IFTase.Be used for yeast and produce the PPIC9K-IFTase expression vector figure of IFTase
Fig. 2 recombination yeast abduction delivering crude enzyme liquid SDS-PAGE.Yeast reorganization bacterium is through the supernatant SDS-PAGE of each time point of methanol induction figure.
Fig. 3 HPLC collection of illustrative plates that the thick enzymic activity of bacterium IFTase detects of recombinating.The HPLC method detects the HPLC collection of illustrative plates of the reorganization thick enzymic activity of bacterium IFTase (a) difructose anhydride III standard substance; (b) the reacted HPLC collection of illustrative plates of recombination yeast fermented supernatant fluid and synanthrin.
Fig. 4 bacterium of recombinating produces inulin ftructotransferase fermented supernatant fluid enzyme and lives and the thalli growth situation.
Embodiment
The preparation of template: get the golden yellow Arthrobacter of a ring
Arthrobacter aurescensThe single bacterium colony of SK8.001 is dissolved in the 100 μ L aqua sterilisas, with bacteria suspension-20 ℃ freezing 10min, and boiling water bath 10min again, room temperature leaves standstill.
Get supernatant 3 μ L as the target gene PCR template, to comprise
EcoR I with
NotThe following nucleotide sequences of I restriction enzyme site is as primer, do not contained the inulin ftructotransferase dna fragmentation of signal peptide sequence through pcr amplification, promptly
Ift
IftThe gene amplification primer:
Upstream primer P1:5 '-CCGGAATTCGCCGAAGGCGCGAAGG-3 ',
Downstream primer P2:5 '-ATAAGAATGCGGCCGCTCAGGGCGTGGGCCGA-3 ',
IftGene amplification system (50 μ L)
| dd H 2O | 22 |
| Template | |
| 3 μL | |
| 50 μ M upstream primers | 1 μL |
| 50 μ M downstream primers | 1 μL |
| Premix TaqEnzyme | 23 μL |
PCR reaction conditions: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 1min, 60 ℃ of annealing 1min, 72 ℃ are extended 90s, carry out 35 circulations; Last 72 ℃ are extended 10min.
Promptly with golden yellow Arthrobacter (
Arthrobacter aurescens) SK8.001 is starting strain, the PCR method obtains goal gene
Ift, it contains 1233 bp, and coding has 410 amino acid whose sequences, and nucleotides sequence is classified SEQ ID NO:1 as.
The structure of embodiment 2 recombinant vectors pPIC9K-IFTase
1, makes up the pPIC9K-IFTase recombinant vectors: the PCR product of embodiment 1 is carried out carrying out the DNA recovery behind the agarose gel electrophoresis, to reclaiming product through restriction enzyme
EcoR I with
NotI carries out double digestion, and spending the night under the effect of T4 ligase enzyme with the plasmid pPIC9K of the same double digestion of process is connected, and realizes the Primary Construction of recombinant vectors pPIC9K-IFTase.
2, the preparation of DH5 α competent cell: picking bacillus coli DH 5 alpha list bacterium colony is in the LB liquid nutrient medium; 37 ℃, 200 rpm incubated overnight; Be inoculated into 37 ℃ of new 100mL LB substratum, 200 rpm shaking culture by 3% inoculum size, be interrupted sampling behind about 1h, make OD
600=0.35-0.4.Culture cooled on ice 1h, centrifugal supernatant discarded, deposition is ice-cold 100mM MgCl respectively
2And 100mM CaCl
2The suspension washed cell is finally used 85mM CaCl
2(being dissolved in 15% glycerine) re-suspended cell, packing obtains the DH5a competent cell, and-80 ℃ of freezing preservations are subsequent use.
3, recombinant vectors pPIC9K-IFTase is converted among the DH5 α: the recombinant vectors pPIC9K-IFTase that will spend the night after connecting is transformed in the DH5 α competent cell; Coating contains the LB solid medium of 50 μ g/ mL ammonia benzyl mycin resistances, cultivates 18-24h for 37 ℃ and tentatively obtains positive colony.
4, the double digestion of recombinant vectors checking: obtain positive colony through the resistance screening of medium: the preliminary positive colony of picking is in containing the antibiotic LB liquid nutrient medium of 50 μ g/mL ammonia benzyls respectively; 37 ℃, 200 rpm overnight cultures; Extract plasmid, through restriction enzyme
EcoR I with
NotI double digestion plasmid is tentatively judged positive colony according to the double digestion result behind the electrophoresis.
5, the recombinant vectors pPIC9K-IFTase to preliminary acquisition carries out gene sequencing; The result shows that pPC9K insertion fragment is one and contains 1233 bp; Coding has 410 amino acid whose sequences; Success makes up recombinant vectors pPIC9K-IFTase, and it contains 10503 bp, and nucleotides sequence is classified SEQ ID NO:2 as.
The screening of embodiment 3 inulin ftructotransferase pichia spp reorganization bacterium
1, the preparation of pichia spp GS115 competent cell: picking GS115 yeast list bacterium colony is pressed 1% inoculum size enlarged culturing to OD in 30 ℃ of YPD liquid nutrient mediums, 200 rpm incubated overnight
600=1.3-1.5, ice-cold aqua sterilisa and 1M Sorbitol Solution USP washing respectively finally obtains pichia spp GS115 competent cell with the packing of 1M sorbyl alcohol.
2, recombinant vectors pPIC9K-IFTase electricity is transformed into pichia spp GS115 competent cell: with pPIC9K-IFTase plasmid warp
SalElectricity is transformed into pichia spp GS115 competent cell (electric conversion condition: voltage 1500 V after the I linearizing; Electric capacity 25 μ F; Resistance 200 Europe, transformation time 4-10 msec), coat the MD flat board; Cultivate 2d to single bacterium colony occurring, carry out the possible positive reorganization bacterium of preliminary screening for 30 ℃ with the His defective type.
3, G418 gradient resistance reorganization bacterium screening: the MD flat board is cultivated the dull and stereotyped pPIC9K-IFTase pichia spp reorganization bacterium that obtains different copy numbers of YPD that the bacterium point of possibly positively recombinating that obtains is inoculated in 10 concentration gradients of G418; Each Geneticin concentration is 0,0.25,0.5,0.75,1.0,1.5,1.75,2.0,3.0,4.0 mg/mL, cultivates 2d to single bacterium colony occurring for 30 ℃.
4, bacterium colony PCR method is identified recombination yeast: the supposition reorganization bacterium that will pass through the G418 resistance screening through Lyticase enzymic digestion ,-20 ℃ freezing and room temperature thaw; The thalline supernatant is as treating that PCR identifies the template of reorganization bacterium; With universal primer 5 ' AOX and 3 ' AOX is that the upstream and downstream primer carries out bacterium colony PCR, and the result identifies through agarose gel electrophoresis and obtains positive inulin ftructotransferase pichia spp reorganization bacterium.
PCR identification system (20 μ L):
| dd H 2O | 8 μL |
| Template | 1.2 μL |
| 50 μ M5 ' AOX upstream primers | 0.4 μL |
| 50 μ M3 ' AOX downstream primers | 0.4 μL |
| Premix TaqEnzyme | 10 μL |
PCR reaction conditions: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 1min, 53 ℃ of annealing 1min, 72 ℃ are extended 90s, carry out 35 circulations; Last 72 ℃ are extended 10min.
Abduction delivering of embodiment 4 reorganization bacterium and active the detection
The single colony inoculation of reorganization bacterium that embodiment 3 is obtained is cultured to OD in 30 ℃ of BMGY substratum, 280 rpm
600=2-6, centrifugal collection thalline makes OD with the resuspended thalline of BMMY substratum
600About=1, double gauze seals 30 ℃, 280 rpm shake flask fermentations, and it is 0.5%-1.0% that every 24h adds 100% methyl alcohol to final concentration to substratum.Different time is collected thalline, carries out SDS-PAGE and identifies albumen and the enzyme assay of HPLC method.Under unexecuted culture condition optimization and suitable copy screening conditions, the thick enzyme of the positive recombinant bacterial strain fermented liquid of 4 mg/mL G418-YPD of acquisition 60h alive is 10.3U/mL.
Inulin ftructotransferase vigor definition: to produce the required enzyme amount of 1.0 μ M difructose anhydride IIIs be 1 enzyme activity unit (U) to PM under 60 ℃, pH5.5 condition.
<210> SEQ?ID?NO:?1
<211> 1233
<212> DNA
< 213>golden yellow Arthrobacter Arthrobacter aurescens SK8.001
<400> 1
gccgaaggcg?cgaaggccag?cccgttgaac?tcgccgaatg?tctatgacgt?cacggcctgg 60
cgaatcaagg?gccagcccaa?ggtcacggca?gagtcggata?taggcgccgt?catcaacgac 120
atcatcgcgg?acatcaagaa?acgccaatcc?acaccggaaa?ccagaccggg?agccgtggtg 180
atcatcccgc?cgggggacta?cgacctccac?actcaagtgg?tggtggacgt?ggattacctc 240
accatcgccg?gttttggcca?tggtttcttc?tcccgcagca?tcaaagacaa?cgtggacacc 300
acgggctggc?tgaaccttca?gccgggtggc?agccacatcc?gcgtcctcac?gcccccaacc 360
tcgccccagg?cattccttgt?gcggcgggac?gggagtccgc?gcctgtccgg?gattgtgttc 420
aaggacttct?gccttgacgg?agtcagcttc?gtcccggacg?gaaacagcta?caagaacggt 480
aagaccggta?ttgacgtcgc?ctccgacaat?gactccatcc?acatcaccgg?aatggggttc 540
gtctatcttg?aacacgctct?gatagttcga?ggtgcggacg?ccctccgggt?ccacgacaac 600
atggttgccg?agtgcgggaa?ctgcgttgaa?ctgaccggtg?ccggacaagc?aacaattgtc 660
agcgacaacc?tgatgggtgc?cggccccgaa?ggtgtgacgc?ttctcgccga?aaaccacgag 720
ggcctcctgg?tcacaggaaa?caacttcttt?cctcgaggac?gcagtctcct?tgagttcacc 780
ggctgcaacc?gctgcagcgt?gacatcaaac?aggttccagg?gcttctaccc?cggcatgatg 840
cggctgctga?atggctgcaa?ggaaaacctc?atcacgtcca?accatttccg?tcggggcacg 900
gaagggttcc?ctccgttcat?tgaccgcact?aacggcctgg?atgacctcta?cggtgtcatc 960
cacgcgatgg?gggacaacaa?cctgatctcg?aacaacctgt?ttgcctacga?cgtcccgccg 1020
ggcaagattg?cccccgccgg?agcccagccc?accatcatgt?tgatcgcagg?cggggacggc 1080
aacgtcatag?ccaccaatca?cgtcatcagc?aacgtggaca?cccaacacgt?ggttcttgat 1140
ggatcggcaa?cccggtcaaa?ggttttggac?agcggcgccg?cctccaccat?cacgtcctac 1200
agcccggaca?cagccattcg?gcccacgccc?tga 1233
<210> SEQ?ID?NO:?2
<211> 10503
<212> DNA
< 213>engineering bacteria pPIC9K-IFTase
<400> 2
agatctaaca?tccaaagacg?aaaggttgaa?tgaaaccttt?ttgccatccg?acatccacag 60
gtccattctca?cacataagtg?ccaaacgca?acaggagggg?atacactagc?agcagaccgt 120
tgcaaacgcag?gacctccact?cctcttctc?ctcaacaccc?acttttgcca?tcgaaaaacc 180
agcccagtta?ttgggcttga?ttggagctcg?ctcattccaa?ttccttctat?taggctacta 240
acaccatgac?tttattagcc?tgtctatcct?ggcccccctg?gcgaggttca?tgtttgttta 300
tttccgaatg?caacaagctc?cgcattacac?ccgaacatca?ctccagatga?gggctttctg 360
agtgtggggt?caaatagttt?catgttcccc?aaatggccca?aaactgacag?tttaaacgct 420
gtcttggaac?ctaatatgac?aaaagcgtga?tctcatccaa?gatgaactaa?gtttggttcg 480
ttgaaatgcta?acggccagt?tggtcaaaaa?gaaacttcca?aaagtcgcca?taccgtttgt 540
cttgtttggta?ttgattgac?gaatgctcaa?aaataatctc?attaatgctt?agcgcagtct 600
ctctatcgctt?ctgaaccccg?gtgcacctg?tgccgaaacg?caaatgggga?aacacccgct 660
ttttggatgat?tatgcattgt?ctccacatt?gtatgcttcc?aagattctgg?tgggaatact 720
gctgatagcct?aacgttcatg?atcaaaatt?taactgttct?aacccctact?tgacagcaat 780
atataaacaga?aggaagctgc?cctgtctta?aacctttttt?tttatcatca?ttattagctt 840
actttcataat?tgcgactggt?tccaattga?caagcttttg?attttaacgac?ttttaacga 900
caacttgaga?agatcaaaaa?acaactaatt?attcgaagga?tccaaacgatg?agatttcct 960
tcaattttta?ctgcagtttt?attcgcagca?tcctccgcat?tagctgctcc?agtcaacact 1020
acaacagaag?atgaaacggca?caaattccg?gctgaagctg?tcatcggtta?ctcagattta 1080
gaaggggatt?tcgatgttgc?tgttttgcca?ttttccaaca?gcacaaataa?cgggttattg 1140
tttataaata?ctactattgc?cagcattgct?gctaaagaag?aaggggtatc?tctcgagaaa 1200
agagaggctg?aagcttacgt?agaattcgcc?gaaggcgcga?aggccagccc?gttgaactcg 1260
ccgaatgtct?atgacgtcac?ggcctggcga?atcaagggcc?agcccaaggtc?acggcagag 1320
tcggatatag?gcgccgtcat?caacgacatc?atcgcggaca?tcaagaaacg?ccaatccaca 1380
ccggaaacca?gaccgggagc?cgtggtgatc?atcccgccgg?gggactacga?cctccacact 1440
caagtggtggt?ggacgtgga?ttacctcacc?atcgccggtt?ttggccatgg?tttcttctcc 1500
cgcagcatca?aagacaacgt?ggacaccacg?ggctggctga?accttcagcc?gggtggcagc 1560
cacatccgcg?tcctcacgcc?cccaacctcg?ccccaggcat?tccttgtgcg?gcgggacggg 1620
agtccgcgcc?tgtccgggat?tgtgttcaag?gacttctgcc?ttgacggagt?cagcttcgtc 1680
ccggacggaa?acagctacaa?gaacggtaag?accggtattg?acgtcgcctc?cgacaatgac 1740
tccatccaca?tcaccggaat?ggggttcgtc?tatcttgaac?acgctctgat?agttcgaggt 1800
gcggacgccc?tccgggtcca?cgacaacatg?gttgccgagt?gcgggaactg?cgttgaactg 1860
accggtgccg?gacaagcaac?aattgtcagc?gacaacctga?tgggtgccgg?ccccgaaggt 1920
gtgacgcttc?tcgccgaaaa?ccacgagggc?ctcctggtca?caggaaacaa?cttctttcct 1980
cgaggacgca?gtctccttga?gttcaccggc?tgcaaccgct?gcagcgtgac?atcaaacagg 2040
ttccagggct?tctaccccgg?catgatgcgg?ctgctgaatg?gctgcaagga?aaacctcatc 2100
acgtccaacc?atttccgtcg?gggcacggaa?gggttccctc?cgttcattga?ccgcactaac 2160
ggcctggatg?acctctacgg?tgtcatccac?gcgatggggg?acaacaacct?gatctcgaac 2220
aacctgtttg?cctacgacgt?cccgccgggc?aagattgccc?ccgccggagc?ccagcccacc 2280
atcatgttga?tcgcaggcgg?ggacggcaac?gtcatagcca?ccaatcacgt?catcagcaac 2340
gtggacaccc?aacacgtggt?tcttgatgga?tcggcaaccc?ggtcaaaggt?tttggacagc 2400
ggcgccgcct?ccaccatcac?gtcctacagc?ccggacacag?ccattcggcc?cacgccctga 2460
gcggccgcga?attaattcgc?cttagacatg?actgttcctc?agttcaagtt?gggcacttac 2520
gagaagaccg?gtcttgctag?attctaatca?agaggatgtc?agaatgccat?ttgcctgaga 2580
gatgcaggct?tcatttttga?tactttttta?tttgtaacct?atatagtata?ggattttttt 2640
tgtcattttg?tttcttctcg?tacgagcttg?ctcctgatca?gcctatctcg?cagctgatga 2700
atatcttgtg?gtaggggttt?gggaaaatca?ttcgagtttg?atgtttttct?tggtatttcc 2760
cactcctctt?cagagtacag?aagattaagt?gagaagttcg?tttgtgcaag?cttatcgata 2820
agctttaatg?cggtagttta?tcacagttaa?attgctaacg?cagtcaggca?ccgtgtatga 2880
aatctaacaa?tgcgctcatc?gtcatcctcg?gcaccgtcac?cctggatgct?gtaggcatag 2940
gcttggttat?gccggtactg?ccgggcctct?tgcgggatat?cgtccattcc?gacagcatcg 3000
ccagtcacta?tggcgtgctg?ctagcgctat?atgcgttgat?gcaatttcta?tgcgcacccg 3060
ttctcggagc?actgtccgac?cgctttggcc?gccgcccagt?cctgctcgct?tcgctacttg 3120
gagccactat?cgactacgcg?atcatggcga?ccacacccgt?cctgtggatc?tatcgaatct 3180
aaatgtaagt?taaaatctct?aaataattaa?ataagtccca?gtttctccat?acgaacctta 3240
acagcattgc?ggtgagcatc?tagaccttca?acagcagcca?gatccatcac?tgcttggcca 3300
atatgtttca?gtccctcagg?agttacgtct?tgtgaagtga?tgaacttctg?gaaggttgca 3360
gtgttaactc?cgctgtattg?acgggcatat?ccgtacgttg?gcaaagtgtg?gttggtaccg 3420
gaggagtaat?ctccacaact?ctctggagag?taggcaccaa?caaacacaga?tccagcgtgt 3480
tgtacttgat?caacataaga?agaagcattc?tcgatttgca?ggatcaagtg?ttcaggagcg 3540
tactgattgg?acatttccaa?agcctgctcg?taggttgcaa?ccgatagggt?tgtagagtgt 3600
gcaatacact?tgcgtacaat?ttcaaccctt?ggcaactgca?cagcttggtt?gtgaacagca 3660
tcttcaattc?tggcaagctc?cttgtctgtc?atatcgacag?ccaacagaat?cacctgggaa 3720
tcaataccat?gttcagcttg?agacagaagg?tctgaggcaa?cgaaatctgg?atcagcgtat 3780
ttatcagcaa?taactagaac?ttcagaaggc?ccagcaggca?tgtcaatact?acacagggct 3840
gatgtgtcat?tttgaaccat?catcttggca?gcagtaacga?actggtttcc?tggaccaaat 3900
attttgtcac?acttaggaac?agtttctgtt?ccgtaagcca?tagcagctac?tgcctgggcg 3960
cctcctgcta?gcacgataca?cttagcacca?accttgtggg?caacgtagat?gacttctggg 4020
gtaagggtac?catccttctt?aggtggagat?gcaaaaacaa?tttctttgca?accagcaact 4080
ttggcaggaa?cacccagcat?cagggaagtg?gaaggcagaa?ttgcggttcc?accaggaata 4140
tagaggccaa?ctttctcaat?aggtcttgca?aaacgagagc?agactacacc?agggcaagtc 4200
tcaacttgca?acgtctccgt?tagttgagct?tcatggaatt?tcctgacgtt?atctatagag 4260
agatcaatgg?ctctcttaac?gttatctggc?aattgcataa?gttcctctgg?gaaaggagct 4320
tctaacacag?gtgtcttcaa?agcgactcca?tcaaacttgg?cagttagttc?taaaagggct 4380
ttgtcaccat?tttgacgaac?attgtcgaca?attggtttga?ctaattccat?aatctgttcc 4440
gttttctgga?taggacgacg?aagggcatct?tcaatttctt?gtgaggaggc?cttagaaacg 4500
tcaattttgc?acaattcaat?acgaccttca?gaagggactt?ctttaggttt?ggattcttct 4560
ttaggttgtt?ccttggtgta?tcctggcttg?gcatctcctt?tccttctagt?gacctttagg 4620
gacttcatat?ccaggtttct?ctccacctcg?tccaacgtca?caccgtactt?ggcacatcta 4680
actaatgcaa?aataaaataa?gtcagcacat?tcccaggcta?tatcttcctt?ggatttagct 4740
tctgcaagtt?catcagcttc?ctccctaatt?ttagcgttca?acaaaacttc?gtcgtcaaat 4800
aaccgtttgg?tataagaacc?ttctggagca?ttgctcttac?gatcccacaa?ggtggcttcc 4860
atggctctaa?gaccctttga?ttggccaaaa?caggaagtgc?gttccaagtg?acagaaacca 4920
acacctgttt?gttcaaccac?aaatttcaag?cagtctccat?cacaatccaa?ttcgataccc 4980
agcaactttt?gagttgctcc?agatgtagca?cctttatacc?acaaaccgtg?acgacgagat 5040
tggtagactc?cagtttgtgt?ccttatagcc?tccggaatag?actttttgga?cgagtacacc 5100
aggcccaacg?agtaattaga?agagtcagcc?accaaagtag?tgaatagacc?atcggggcgg 5160
tcagtagtca?aagacgccaa?caaaatttca?ctgacaggga?actttttgac?atcttcagaa 5220
agttcgtatt?cagtagtcaa?ttgccgagca?tcaataatgg?ggattatacc?agaagcaaca 5280
gtggaagtca?catctaccaa?ctttgcggtc?tcagaaaaag?cataaacagt?tctactaccg 5340
ccattagtga?aacttttcaa?atcgcccagt?ggagaagaaa?aaggcacagc?gatactagca 5400
ttagcgggca?aggatgcaac?tttatcaacc?agggtcctat?agataaccct?agcgcctggg 5460
atcatccttt?ggacaactct?ttctgccaaa?tctaggtcca?aaatcacttc?attgatacca 5520
ttattgtaca?acttgagcaa?gttgtcgatc?agctcctcaa?attggtcctc?tgtaacggat 5580
gactcaactt?gcacattaac?ttgaagctca?gtcgattgag?tgaacttgat?caggttgtgc 5640
agctggtcag?cagcataggg?aaacacggct?tttcctacca?aactcaagga?attatcaaac 5700
tctgcaacac?ttgcgtatgc?aggtagcaag?ggaaatgtca?tacttgaagt?cggacagtga 5760
gtgtagtctt?gagaaattct?gaagccgtat?ttttattatc?agtgagtcag?tcatcaggag 5820
atcctctacg?ccggacgcat?cgtggccgac?ctgcaggggg?ggggggggcg?ctgaggtctg 5880
cctcgtgaag?aaggtgttgc?tgactcatac?caggcctgaa?tcgccccatc?atccagccag 5940
aaagtgaggg?agccacggtt?gatgagagct?ttgttgtagg?tggaccagtt?ggtgattttg 6000
aacttttgct?ttgccacgga?acggtctgcg?ttgtcgggaa?gatgcgtgat?ctgatccttc 6060
aactcagcaa?aagttcgatt?tattcaacaa?agccgccgtc?ccgtcaagtc?agcgtaatgc 6120
tctgccagtg?ttacaaccaa?ttaaccaatt?ctgattagaa?aaactcatcg?agcatcaaat 6180
gaaactgcaa?tttattcata?tcaggattat?caataccata?tttttgaaaa?agccgtttct 6240
gtaatgaagg?agaaaactca?ccgaggcagt?tccataggat?ggcaagatcc?tggtatcggt 6300
ctgcgattcc?gactcgtcca?acatcaatac?aacctattaa?tttcccctcg?tcaaaaataa 6360
ggttatcaag?tgagaaatca?ccatgagtga?cgactgaatc?cggtgagaat?ggcaaaagct 6420
tatgcatttc?tttccagact?tgttcaacag?gccagccatt?acgctcgtca?tcaaaatcac 6480
tcgcatcaac?caaaccgtta?ttcattcgtg?attgcgcctg?agcgagacga?aatacgcgat 6540
cgctgttaaa?aggacaatta?caaacaggaa?tcgaatgcaa?ccggcgcagg?aacactgcca 6600
gcgcatcaac?aatattttca?cctgaatcag?gatattcttc?taatacctgg?aatgctgttt 6660
tcccggggat?cgcagtggtg?agtaaccatg?catcatcagg?agtacggata?aaatgcttga 6720
tggtcggaag?aggcataaat?tccgtcagcc?agtttagtct?gaccatctca?tctgtaacat 6780
cattggcaac?gctacctttg?ccatgtttca?gaaacaactc?tggcgcatcg?ggcttcccat 6840
acaatcgata?gattgtcgca?cctgattgcc?cgacattatc?gcgagcccat?ttatacccat 6900
ataaatcagc?atccatgttg?gaatttaatc?gcggcctcga?gcaagacgtt?tcccgttgaa 6960
tatggctcat?aacacccctt?gtattactgt?ttatgtaagc?agacagtttt?attgttcatg 7020
atgatatatt?tttatcttgt?gcaatgtaac?atcagagatt?ttgagacaca?acgtggcttt 7080
cccccccccc?cctgcaggtc?ggcatcaccg?gcgccacagg?tgcggttgct?ggcgcctata 7140
tcgccgacat?caccgatggg?gaagatcggg?ctcgccactt?cgggctcatg?agcgcttgtt 7200
tcggcgtggg?tatggtggca?ggccccgtgg?ccgggggact?gttgggcgcc?atctccttgc 7260
atgcaccatt?ccttgcggcg?gcggtgctca?acggcctcaa?cctactactg?ggctgcttcc 7320
taatgcagga?gtcgcataag?ggagagcgtc?gagtatctat?gattggaagt?atgggaatgg 7380
tgatacccgc?attcttcagt?gtcttgaggt?ctcctatcag?attatgccca?actaaagcaa 7440
ccggaggagg?agatttcatg?gtaaatttct?ctgacttttg?gtcatcagta?gactcgaact 7500
gtgagactat?ctcggttatg?acagcagaaa?tgtccttctt?ggagacagta?aatgaagtcc 7560
caccaataaa?gaaatccttg?ttatcaggaa?caaacttctt?gtttcgaact?ttttcggtgc 7620
cttgaactat?aaaatgtaga?gtggatatgt?cgggtaggaa?tggagcgggc?aaatgcttac 7680
cttctggacc?ttcaagaggt?atgtagggtt?tgtagatact?gatgccaact?tcagtgacaa 7740
cgttgctatt?tcgttcaaac?cattccgaat?ccagagaaat?caaagttgtt?tgtctactat 7800
tgatccaagc?cagtgcggtc?ttgaaactga?caatagtgtg?ctcgtgtttt?gaggtcatct 7860
ttgtatgaat?aaatctagtc?tttgatctaa?ataatcttga?cgagccaagg?cgataaatac 7920
ccaaatctaa?aactctttta?aaacgttaaa?aggacaagta?tgtctgcctg?tattaaaccc 7980
caaatcagct?cgtagtctga?tcctcatcaa?cttgaggggc?actatcttgt?tttagagaaa 8040
tttgcggaga?tgcgatatcg?agaaaaaggt?acgctgattt?taaacgtgaa?atttatctca 8100
agatctctgc?ctcgcgcgtt?tcggtgatga?cggtgaaaac?ctctgacaca?tgcagctccc 8160
ggagacggtc?acagcttgtc?tgtaagcgga?tgccgggagc?agacaagccc?gtcagggcgc 8220
gtcagcgggt?gttggcgggt?gtcggggcgc?agccatgacc?cagtcacgta?gcgatagcgg 8280
agtgtatact?ggcttaacta?tgcggcatca?gagcagattg?tactgagagt?gcaccatatg 8340
cggtgtgaaa?taccgcacag?atgcgtaagg?agaaaatacc?gcatcaggcg?ctcttccgct 8400
tcctcgctca?ctgactcgct?gcgctcggtc?gttcggctgc?ggcgagcggt?atcagctcac 8460
tcaaaggcgg?taatacggtt?atccacagaa?tcaggggata?acgcaggaaa?gaacatgtga 8520
gcaaaaggcc?agcaaaaggc?caggaaccgt?aaaaaggccg?cgttgctggc?gtttttccat 8580
aggctccgcc?cccctgacga?gcatcacaaa?aatcgacgct?caagtcagag?gtggcgaaac 8640
ccgacaggac?tataaagata?ccaggcgttt?ccccctggaa?gctccctcgt?gcgctctcct 8700
gttccgaccc?tgccgcttac?cggatacctg?tccgcctttc?tcccttcggg?aagcgtggcg 8760
ctttctcaat?gctcacgctg?taggtatctc?agttcggtgt?aggtcgttcg?ctccaagctg 8820
ggctgtgtgc?acgaaccccc?cgttcagccc?gaccgctgcg?ccttatccgg?taactatcgt 8880
cttgagtcca?acccggtaag?acacgactta?tcgccactgg?cagcagccac?tggtaacagg 8940
attagcagag?cgaggtatgt?aggcggtgct?acagagttct?tgaagtggtg?gcctaactac 9000
ggctacacta?gaaggacagt?atttggtatc?tgcgctctgc?tgaagccagt?taccttcgga 9060
aaaagagttg?gtagctcttg?atccggcaaa?caaaccaccg?ctggtagcgg?tggttttttt 9120
gtttgcaagc?agcagattac?gcgcagaaaa?aaaggatctc?aagaagatcc?tttgatcttt 9180
tctacggggt?ctgacgctca?gtggaacgaa?aactcacgtt?aagggatttt?ggtcatgaga 9240
ttatcaaaaa?ggatcttcac?ctagatcctt?ttaaattaaa?aatgaagttt?taaatcaatc 9300
taaagtatat?atgagtaaac?ttggtctgac?agttaccaat?gcttaatcag?tgaggcacct 9360
atctcagcga?tctgtctatt?tcgttcatcc?atagttgcct?gactccccgt?cgtgtagata 9420
actacgatac?gggagggctt?accatctggc?cccagtgctg?caatgatacc?gcgagaccca 9480
cgctcaccgg?ctccagattt?atcagcaata?aaccagccag?ccggaagggc?cgagcgcaga 9540
agtggtcctg?caactttatc?cgcctccatc?cagtctatta?attgttgccg?ggaagctaga 9600
gtaagtagtt?cgccagttaa?tagtttgcgc?aacgttgttg?ccattgctgc?aggcatcgtg 9660
gtgtcacgct?cgtcgtttgg?tatggcttca?ttcagctccg?gttcccaacg?atcaaggcga 9720
gttacatgat?cccccatgtt?gtgcaaaaaa?gcggttagct?ccttcggtcc?tccgatcgtt 9780
gtcagaagta?agttggccgc?agtgttatca?ctcatggtta?tggcagcact?gcataattct 9840
cttactgtca?tgccatccgt?aagatgcttt?tctgtgactg?gtgagtactc?aaccaagtca 9900
ttctgagaat?agtgtatgcg?gcgaccgagt?tgctcttgcc?cggcgtcaac?acgggataat 9960
accgcgccac?atagcagaac?tttaaaagtg?ctcatcattg?gaaaacgttc?ttcggggcga 10020
aaactctcaa?ggatcttacc?gctgttgaga?tccagttcga?tgtaacccac?tcgtgcaccc 10080
aactgatctt?cagcatcttt?tactttcacc?agcgtttctg?ggtgagcaaa?aacaggaagg 10140
caaaatgccg?caaaaaaggg?aataagggcg?acacggaaat?gttgaatact?catactcttc 10200
ctttttcaat?attattgaag?catttatcag?ggttattgtc?tcatgagcgg?atacatattt 10260
gaatgtattt?agaaaaataa?acaaataggg?gttccgcgca?catttccccg?aaaagtgcca 10320
cctgacgtct?aagaaaccat?tattatcatg?acattaacct?ataaaaatag?gcgtatcacg 10380
aggccctttc?gtcttcaaga?attaattctc?atgtttgaca?gcttatcatc?gataagctga 10440
ctcatgttgg?tattgtgaaa?tagacgcaga?tcgggaacac?tgaaaaataa?cagttattat 10500
tcg
Claims (5)
1. method of utilizing yeast expression system to produce inulin ftructotransferase is characterized in that step is:
(1) with golden yellow Arthrobacter (
Arthrobacter aurescens) SK8.001 is starting strain, the PCR method obtains the inulin ftructotransferase gene
Ift, make up recombinant vectors X-IFTase through double digestion;
Said recombinant vectors X-IFTase is: pPIC9K – IFTase, pPIC9 – IFTase, pPICZaA/B/C – IFTase or pGAPZaA/B/C – IFTase;
(2) recombinant vectors X-IFTase is transformed into the pichia spp competent cell after linearizing, and the Screening and Identification positive transformant, promptly make up and obtain producing the inulin ftructotransferase Yeast engineering bacteria;
Said Pichi strain is: GS115, X-33, KM71 or SMD1168;
(3) the engineering bacteria liquid fermenting is produced active inulin ftructotransferase
The inulin ftructotransferase Yeast engineering bacteria is expressed through methanol induction; The HPLC method of reacting fermented supernatant fluid and synanthrin detects difructose anhydride III and generates situation to carry out the inulin ftructotransferase activity identification, and the Yeast engineering bacteria that shows acquisition has secretion and produces and have the ability of active inulin ftructotransferase;
The single colony inoculation of reorganization bacterium is cultured to OD in 28-30 ℃ of BMGY substratum, 280 rpm
600=2-6, centrifugal collection thalline makes OD with the resuspended thalline control of BMMY substratum
600=1, double gauze seals 30 ℃, 280 rpm shake flask fermentations, and it is 0.5%-1.0% that every 24h adds 100% methyl alcohol to final concentration to substratum;
The positive recombinant bacterial strain 60h of the 4 mg/mL G418-YPD that obtain fermented liquid produces the thick enzyme of the active inulin ftructotransferase 10.3U/mL of being alive.
2. method according to claim 1 is characterized in that inulin ftructotransferase gene described in the step (1) is full gene of inulin ftructotransferase or the inulin ftructotransferase gene transformed according to the yeast codon preference.
3. method according to claim 1 is characterized in that recombinant vectors linearizing described in the step (2) utilizes Sac I or Sal I to carry out enzyme and cuts.
4. method according to claim 1 is characterized in that the method that transforms the Pichi strain cell described in the step (2) adopts electric shock to transform or chemical conversion.
5. method according to claim 1 is characterized in that the engineering bacteria liquid fermentation medium is BMGY/BMMY, BMG/BMM or YPD described in the step (3).
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004305125A (en) * | 2003-04-08 | 2004-11-04 | Nippon Beet Sugar Mfg Co Ltd | Method for mass-producing enzyme |
| CN101363008A (en) * | 2008-09-17 | 2009-02-11 | 江南大学 | Bacterial strain for producing inulin fructose transferase and method for producing difructose anhydride III using said transferase |
| CN101492661A (en) * | 2009-01-16 | 2009-07-29 | 江南大学 | Clone, expression of beta-glucosidase gene, and preparation for gentian oligose |
| CN101906405A (en) * | 2010-07-15 | 2010-12-08 | 江南大学 | Cloning of inulin ftructotransferase and efficient expression thereof |
-
2012
- 2012-06-08 CN CN2012101870690A patent/CN102653739A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004305125A (en) * | 2003-04-08 | 2004-11-04 | Nippon Beet Sugar Mfg Co Ltd | Method for mass-producing enzyme |
| CN101363008A (en) * | 2008-09-17 | 2009-02-11 | 江南大学 | Bacterial strain for producing inulin fructose transferase and method for producing difructose anhydride III using said transferase |
| CN101492661A (en) * | 2009-01-16 | 2009-07-29 | 江南大学 | Clone, expression of beta-glucosidase gene, and preparation for gentian oligose |
| CN101906405A (en) * | 2010-07-15 | 2010-12-08 | 江南大学 | Cloning of inulin ftructotransferase and efficient expression thereof |
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| MENG ZHAO等人: "Cloning and extracellular expression of inulin", 《J SCI FOOD AGRIC》, no. 91, 30 August 2011 (2011-08-30) * |
| 张显升等: "巴斯德毕赤酵母表达***及其在医学领域的应用", 《山东科学》, vol. 18, no. 4, 31 October 2005 (2005-10-31) * |
| 谢涛等: "外源基因在巴斯德毕赤酵母中多拷贝整合的研究进展", 《生物技术通讯》, vol. 17, no. 3, 31 May 2006 (2006-05-31) * |
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