CN1465699A - Gene engineering of producing fructose by inulase hydrolyzing helianthus tuberosus - Google Patents
Gene engineering of producing fructose by inulase hydrolyzing helianthus tuberosus Download PDFInfo
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- CN1465699A CN1465699A CNA021324468A CN02132446A CN1465699A CN 1465699 A CN1465699 A CN 1465699A CN A021324468 A CNA021324468 A CN A021324468A CN 02132446 A CN02132446 A CN 02132446A CN 1465699 A CN1465699 A CN 1465699A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The present invention relates to a method for producing fructose by using gene engineering inulase to hydrolyze girasole. Said invention provides a structure of inulase gene inuA1 from aspergillus nigar AF10 and amino acid sequence of inulae protein INUA1, provides gene engineering microzyme GS115/inuA1 for expressing INUA1, also provides the method for producing frustose by using GS115/inuA1 inulase to hydrolyze inulin and provides the method for producing fructose by using GS115/inuA1 inulase to hydrolyze cane sugar. Said invention also can be used for producing fructose by using cane sugar, beet or cane extract and treacle as raw material.
Description
The invention relates to genetically engineered inulinase hydrolyzing helianthus tuberosus or sucrose and produce the method for fructose, belong to bioengineering field, specially refer to a kind of structure and the aminoacid sequence of inulinase protein I NUA1, the gene engineering microzyme GS115/inuA1 of expression INUA1 and method of producing fructose by INUA1 that comes from the inulinase gene inuA1 of aspergillus niger (Aspergillusniger) AF10.
Fructose sweetness value height, heat be low, do not fermented by oral microorganism, fructose changes glycogen into not need Regular Insulin is transduction signal in blood, fructose has superior food processing properties in addition, good to eat as sweet taste, that coking temperature is low, moisture retention reaches freezing point temperature by force is low etc., therefore as sweeting agent and foodstuff additive, fructose can be avoided the side effects such as carious tooth, hypertension and diabetes that caused by sucrose and glucose, and is the first-selection of sweetening agent.Fructose has broad application prospects in food_beverage industry and medicine industry.Existing fructose production is to be raw material with starch, and through the catalytic multistep processes technological process of three kinds of enzymes (amylase, saccharifying enzyme and glucose isomerase), the fructose content of its product is up to 55%, complex technical process, and raw material availability and productive rate are all lower.With jerusalem artichoke (or inulin) is raw material, produces fructose with the inulinase hydrolysis method, become the research focus in recent years, and gordian technique is to make inulinase have high reactivity.Genetic engineering means is to improve one of active effective way of inulinase.The invention provides the structure of a kind of inulinase gene inuA1 that comes from aspergillus niger (Aspergillus niger) AF10 and the aminoacid sequence of inulinase protein I NUA1, the gene engineering microzyme GS115/inuA1 that expresses INUA1 is provided, the method for producing fructose by INUA1 hydrolytic inulin or sucrose also is provided.
The present invention screens and has identified strain inulinase enzyme source bacterial strain aspergillus niger (Aspergillus niger) AF10 from soil, the PCR method inulinase gene inuA1 that from the genomic dna of AF10, increases, and measure PCR product nucleotide sequence, obtained to have the inulinase gene inuA1 that forms by 1515 Nucleotide of entire reading frame frame.Cutting by restriction enzyme is connected with archaeal dna polymerase, and inuA1 is integrated among the cloning vector pUC118, obtains the cloning vector pUC118/inuA1 of inuA1.The thermal shock method is converted into this cloning vector among intestinal bacteria (E.coli) JM109, becomes the e. coli jm109/inuA1 that contains pUC118/inuA1.
Pcr amplification inuA1 from JM109/inuA1 with yeast cloning vector pPIC9K reorganization, forms pPIC9K/inuA1, and pPIC9K/inuA1 electricimpulse method transforms pichia spp (Pichia Pastoris) GS115 (His, Mut
+), the screening positive transformant is also done molecular biology identification, obtains inulinase genetic engineering bacterium GS115/inuA1.The outer inulinase of GS115/inuA1 methanol induction fermentation secretion born of the same parents, the separation and purification inulinase is measured its zymologic property.
With the enzyme addition of every gram substrate 25U, hydrolytic inulin (15%) under 55 ℃, pH5.5 condition, the highest percent hydrolysis 92.3%, fructose content are 92.1%.
With the enzyme addition of every gram substrate 25U, sucrose hydrolysis (15%) under 55 ℃, pH5.5 condition, percent hydrolysis reached 100% in 1 hour, generated 46.3% glucose and 47.6% fructose.
InuA1 is the inulinase gene that is extracted first, checks order and clone, and the inulinase that GS115/inuA1 expresses has higher hydrolytic activity, and transformation efficiency height, product purity height, production cost that hydrolytic inulin is produced fructose are low.It is raw material production fructose that this inulinase also can be used for sucrose, beet or sugarcane immersion liquid, molasses.
It below is most preferred embodiment of the present invention.
Embodiment 1. inulinase gene inuA1 clone
(1) extracts aspergillus niger AF10 genomic dna
Be inoculated in the 200ml potato liquid nutrient medium 28 ℃, 135r/min shaking table cultivation 24hr, centrifugal and washing mycelium after the AF10 activation.In the aseptic mortar, mycelium is ground into powder rapidly with liquid nitrogen.Add about 0.1g thalline and 500 μ L DNA extraction liquid in the 1.5mL centrifuge tube, vibration makes it thorough mixing, adds 100 μ L 10%SDS, Benzyl Chloride 300 μ L again, and thermal agitation makes the interior mixture of pipe become emulsus, 50 ℃ of insulation 1hr.Add 300 μ lL 3MNaAC (PH5.2) mixings then, the centrifugal 15min of 6000r/min collects supernatant liquor.Add 2 times of volume dehydrated alcohols, 4 ℃ of precipitation 20min.10, the centrifugal 15min of 000r/min room temperature, precipitation is washed once the trace ethanol that room temperature is volatilized residual as far as possible, TE damping fluid dissolving DNA with 70% ethanol.The RNase enzyme that in each centrifuge tube, adds 1 μ g/ μ l, 37 ℃ of constant temperature 30min.Identify with 1% agarose gel electrophoresis, with λ-HindIII as dna molecular amount mark, electrophoresis 30min under the 10V/cm voltage.
(2) pcr amplification inulinase gene inuA1
With the AF10 genomic dna is template, is reference design and synthetic pcr primer thing with A.niger inulinase gene (INUB) sequence, uses the Pyrobest archaeal dna polymerase, amplification inuA1.Primer: F
1: 5 '-ATGTTGAATCCGAAGGTTGC-3 '; R
1: 5 '-TCATTCAAGTGAAACACTCC-3 '.PCR reaction: Buffer 5 μ L, dNTP 4 μ L, F
10.5 μ L, R
10.5 μ L, AF10DNA 2 μ L, PyrobestDNA polysaccharase 0.25 μ L, dH
2Conditions:94 ℃ of of of of 98 ℃ of of of of of of of of of 15s of 1min of O, 37.75 μ L.Reaction are a circulation with, 55 ℃ of of of of of 30s, 72 ℃ of of of of of 2min then, move 35 circulations altogether.The PCR product is done the agarose gel electrophoresis analysis, present specific band at about 1.6kb place. are, recovery refining to 3 PCR products respectively; .3,：1 ATGTTGAATC CGAAGGTTGC CTACATGGTC TGGATGACCT GCCTGGGTTT51 AACGTTGCCC AGCCAGGCGC AGTCTAATGA TTACCGTCCA TCATACCACT101 TCACACCGGA CCAGTACTGG AACGAGCCAA ACGGCCTGAT TAAAATCGGA151 TCCACCTGGC ACCTGTTCTT TCAACACAAT CCGACGGCCA ATGTGTGGGG201 CAACATATGC TGGGGGCACG CTACGAGCAC CGATCTGATG CACTGGGCCT251 ACAAACCCAC TGCCATTGCG GATGAGAACG GAGTCGAAGC GTTTACCGGT301 ACAGCCTATT ATGATCCAAA CAATACCTCT GGCCTTGGGG ATTCGGCAAA351 CCCACCCTAT CTGGCCTGGT TCACAGGTTA TACCACTTCA AGCCAAACAC401 AGGACCAGCG CCTGGCTTTC AGTGTGGATA ACGGGGCGAC GTGGACCAAA451 TTTCAAGGCA ACCCCATCAT ATCAACTAGC CAGGAAGCAC CACATGATAT501 AACGGGCGGC CTCGAGAGTC GGGATCCAAA GGTATTCTTC CATCGCCAAT551 CGGGGAATTG GATCATGGTT CTCGCCCATG GCGGGCAGGA CAAGCTGTCT601 TTCTGGACGT CTGCAGACAC CATACACTGG ACATGGCAGA GTGACCTGAA651 GTCCACCTCG ATCAACGGCC TATCGTCCGA TATTACAGGG TGGGAAGTCC701 CCGACATGTT TGAACTCCCG GTTGAAGGCA CTGGGGAGAC CACTGGGGTG751 GTGATGATGA CGCCGGCTGA AGGATCTCCT GCCGGTGGTA ACGGGGTCTT801 AGCTATCACC GGTTCTTTTG ACGGGAAAAC TTTTACGGCA GATCCCGTCG851 ATGCTTCGAC CATGTGGCTG GACAATGGGC GTGATTTCGA TGGCGCTCTG901 AGCTGGGTGA ACGTGCCTGC GTCCGATGGA CGGCGGATTA TCGCCGCCGT951 CATGAATAGC TACGGTTCCA ACCCGCCTAC AACCACCTGG AAAGGGATGC1001 TCTCCTTTCC CCGGACGCTG TCGCTCAAGA AAGTTGGCAC GCAGCAGCAC1051 TTTGTTCAAC AGCCGATCAC AGAGTTGGAT ATGACAATTA GTACCAGTAT1101 GCAAACACTA GCAAACCAGA CCATTACCCC TGGCCAAACA TTGCTGTCAT1151 CGATTCGGGG AACTGCTCTC GATGTTCGAG TTGCTTTTTA CCCTGATGCT1201 GGCTCGGTTC TGTCCCTCAC CGTCCGAAAG GGTGCTTCGG AGCAAACAGT1251 CATTAATTAC ACCCAGTCAA ATGCCACATT GTCGGTTGAT CGAACAGAGA1301 GTGGAGATAT CTCGTATGAC ACGGCCGCAG GTGGCGTCCA TACCGCCAAG1351 TTGGAAGAGG ACGGCACCGG ACTGGTTTCC ATCCGGGTGT TGGTGGATAC1401 GTGTTCTGTA GAGGTTTTTG GCGGACAAGG AGAGGCCGTC ATTTCCGACC1451 TCATCTTCCC GAGTGACAGC TCTGACGGCC TGGCCTTGGA GGTAACTGGC1501 GGGAATGCAG TGCTGCAGTC GGTGGACGTA CGGAGTGTTT CACTTGAATG1551 A
Reading frame total length 1551bp, wherein G+C content is 54.3%, does not have intron sequences, with the inulinase gene nucleotide of having reported in various degree homology is arranged.
inuA1INUA1：1 MLNPKVAYMA WMTCLGLTLP SQAQSNDYRP SYHFTPDQYW MNEPNGLIKI51 GSTWHLFFQH NPTANVWGNI CWGHATSTDL MHWAYKPTAI ADENGVEAFT101 GTAYYDPNNT SGLGDSANPP YLAWFTGYTT SSQTQDQRLA FSVDNGATWT151 KFQGNPIIST SQEAPHDITG GLESRDPKVF FHRQSGNWIM VLAGHGQDKL201 SFWTSADTIH WTWQSDLKST SINGLSSDIT GWEVPDMFEL PVEGTGETTW251 VVMMTPAEGS PAGGNGVLAI TGSFDGKTFT ADPVDASTMW LDNGRDFDGA301 LSWVNVPASD GRRIIAAVMN SYGSNPPTTT WKGMLSFPRT LSLKKVGTQQ351 HFVQQPITEL DTISTSMQTL ANQTITPGQT LLSSIRGTAL DVRVAFYPDA401 DSVLSLTVRK GASEQTVINY TQSNATLSVD RTESGDISYD PAAGGVHTAK451 LEEDGTGLVS IRVLVDTCSV EVFGGQGEAV ISTLIFPSDS SDGLALEVTG501 GNAVLQSVDV RSVSLE
A.niger AF10 inulinase INUA1 is made up of 516 amino acid, wherein has 4 potential N-glycosylation sites, and the 40th～45 amino acids is the conserved sequence WMNEPN of inulinase.Acidic amino acid has 56, and basic aminoacids has 39.Molecular weight is 55851.9, and molecular formula is C
2474H
3770N
658O
790S
15. theoretical pI is 4.58.Negative charge amino-acid residue (Asp+Glu) has 56, and positive charge amino-acid residue (Arg+Lys) has 28.
(3) clone of inulinase gene inuA1
With above-mentioned PCR product and cloning vector pUC118 reorganization.At first use " TaKaRa BKLKit " the PCR product to be carried out the phosphorylation of smoothing and the 5 ' end of 3 ' end.In Eppendorf, add Blunting Kination Enzyme Mix 1 μ L, 10 * BluntingKination Buffer, 2 μ L, PCR reaction solution 7 μ L, dH
2O 10 μ L, 37 ℃, 10min.Refining, the recovery of product.Add the carrier pUC118 that 1 μ L handles through " TaKaRa BAP; CIAP " dephosphorylation with smooth end, ligation Solution A 6 μ L, thorough mixing, 16 ℃, spend the night, total overall reaction liquid thermal shocking method Transformed E .coli JM109, coat on the LB culture medium flat plate that contains Amp and X-gal/IPTG, 30 ℃, cultivated 24 hours, select white colony and be PCR by preceding method, the PCR product is done the agarose gel electrophoresis analysis, presents specific band at the 1.6kb place, has obtained A.niger AF10 inulinase gene inuA1 clone JM109/inuA1.
Make up the yeast conversion carrier pPIC9K/inuA1 of inuA1.Primers F
2: 5 '-CCGAATTCCAGTCTAATGATTACCGTCCT-3 ', R
2: 5 '-CAGGCGGCCGCTCATTCAAGTGAAACACTCCG-3 '.PCR reaction system: PyrobestDNA polysaccharase 0.25 μ L, Buffer 5 μ L, dNTP 4 μ L, F
20.5 μ L, R
20.5 μ L, template (the inuA1 recombinant plasmid pUC118 that from JM109/inuA1, extracts) 2 μ L, dH
2O 37.75 μ L.Reaction conditions: 94 ℃ of 1min are a circulation with 98 ℃ of 15s, 55 ℃ of 30s, 72 ℃ of 1m30s then, move 35 circulations altogether.The PCR product is done the agarose gel electrophoresis analysis, present specific band at about 1.5kb place.PCR product and Yeast expression carrier pPIC9K cut (the pPIC9K dephosphorylation after the cutting is handled) with restriction enzyme EcoRI and NotI respectively, connect with dna ligase: pPIC9K1 μ L (50ng), inuA9 μ L (10ng), SolnI10 μ L, 16 ℃, 12h.Obtain the yeast conversion carrier pPIC9K/inuA1 of inuA1, pPIC9K/inuA1 thermal shocking method Transformed E .coli JM109 recipient cell.Extract pPIC9K/inuA1 from E.coli JM109, and do linearization process with BPU1102I, the electricimpulse method transforms pichia spp (Pichia Pastoris) GS115 (His, Mut
+).Conversion product is coated with recombination yeast screening culture medium { w/w%, YNB (Yeast Nitrogen Base aminoacids) 1.3, Biotin0.00004; Glucose 2.0, agar 1.5} therefrom selects His
+Bacterium colony is that template is PCR with the genomic dna of this bacterial strain, and the agarose gel electrophoresis of PCR product presents specific band at the 1.5kb place.Obtain inulinase gene engineering microzyme GS115/inuA1.
The fermentation of embodiment 3.GS115/inuA1 inulinase
(1) inulinase activity determination method
The active definition of inulinase: 55 ℃, it is 1 enzyme U of unit alive that following 1 minute hydrolysis substrate of pH5.5 condition generates the required enzyme amount of 1 μ mol hexose.
Make the enzyme assay typical curve, the glucose that adds 0mg, 0.2mg, 0.4mg, 0.6mg, 0.8mg, 1.0mg, 1.2mg, 1.4mg, 1.6mg, 1.8mg and 2.0mg in the colorimetric cylinder respectively, aquae destillata is settled to 2mL, add film reagent A, each 0.5mL of B liquid, in boiling water bath behind the constant temperature 15min, the 590nm wavelength is measured the OD value down, draws glucose amount-OD value typical curve.
Sample enzyme assay: get liquid 20 μ L to be measured (control sample boiling water 5min deactivation), add 180 μ L aquae destillatas, the inulin (using the 0.1MpH5.5HAC-NaAC buffer preparation) that adds 800 μ L, 4% concentration, 55 ℃, hydrolysis 10min puts in the boiling water 5min with termination reaction, add film reagent A, each 0.5mL of B liquid, place the 15min colour developing in boiling water bath, 8000rpm10min is centrifugal, gets supernatant liquor colorimetric under the 590nm wavelength.
(2) GS115/inuA1 inulinase fermentation
Be inoculated in { w/w%, glucose 4.0, peptone 1.0, yeast extract paste 0.2, KH in the 50mL/300mL recombination yeast growth medium behind the actication of culture
2PO
40.05, Na
2HPO
40.05, MgSO
47H
2O0.05, MnSO
47H
2O0.003, FeSO
47H
2O0.003, pH5.5}, 30 ℃, 200rpm shaking culture 36 hours.Centrifugal collection thalline adds 50mL recombination yeast fermention medium (do not contain glucose in the growth medium, all the other are identical), and 30 ℃, the 200rpm shaking culture added 0.5mL methyl alcohol every 24 hours, stopped fermenting in 72 hours.Fermenting enzyme activity under the different time is seen Fig. 1.Produce the enzyme peak at 72 hours, enzymic activity is 50.6U/mL, and its fermentation broth enzyme specific activity gene source bacterium AF10 has improved about 10 times.
Embodiment 4.GS115/inuA1 inulinase character
(1) purifying GS115/inuA1 inulinase
GS115/inuA1 fermentation fermented liquid 5000rpm, 10mim are centrifugal, get supernatant liquor, 80% saturation ratio (NH
4)
2SO
4Sedimentation is spent the night, and the centrifugal collection sediment of 18000rpm, 20min is with the dialysis of 0.1MpH5.0HAC-NaAC damping fluid.Dialyzate separates with protein preparing instrument (Pharmacia Biotech KTA explorer 10s), use anion-exchange column hitrapsp 5mL (Pharmacia), use buffered soln A (0.1MpH5.0HAC-NaAC) wash-out of 25mL earlier, use buffered soln A and B (HAC-NaAC that contains the 0.1MpH5.0 of 0.5MNaCl) the solution gradient wash-out of 100mL then, use 25mL buffered soln B wash-out at last, eluent flow rate 2.0mL/min.Effluent liquid is with the determination of ultra-violet OD value of 280nm wavelength.(PIERCE, Rockford U.S.A) measure protein content with BC A Protein Assay ReagentKit.Protein purification electrophoretic analysis polyacrylamide gel electrophoresis is gone up (layer) gum concentration 4.5%, and (layer) gum concentration 10% 100v hour, with the dyeing of Bromo phenol staining fluid, decolours with ethanol decolorization liquid down.Do polyacrylamide gel electrophoresis, present single band at the 90KD place, the GS115/inuA1 inulinase of purifying is designated as INUA1, and the ratio enzymic activity of INUA1 is 16.3 μ mol/mg.
(2) inulinase property testing
1. the suitableeest action pH: the enzymic activity of under the buffer system of pH3.0, pH3.5, pH4.0, pH4.5, pH5.0, pH5.5, pH6.0, pH6.5 and pH7.0, measuring testing sample respectively, the suitableeest action pH value of GS115/inuA1 fermented liquid inulinase is 5.5, and the suitableeest action pH of purifying inulinase is 5.0 (Fig. 2).
2. optimum temperature: measure the inulinase activity respectively under the temperature of 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃, the optimum temperature of GS115/inuB1 fermented liquid and INUA1 is 55 ℃ (Fig. 3).
4. metal ion and chemical reagent are to the influence of enzymic activity
Adding metal ion and the chemical reagent of 5mM in hydrolyzation system, measure enzymic activity behind 55 ℃ of hydrolysis 10min, is 100% enzymic activity with the enzymic activity that does not add as the metal ion sample, and the relative activity of each working sample is as shown in the table
Table. metal ion and chemical reagent are to the influence of enzymic activity
| Metal ion and chemical reagent | Relative activity (%) 5mM | Metal ion and chemical reagent | Relative activity (%) 5mM |
| ????KCl ????NaCl ????MgCl 2????MnSO 4????ZnCl 2????FeSO 4 | ????175 ????280 ????150 ????160 ????0 ????50 | ????CuSO 4????CaCl 2????BaCl 2????AlCl 3????FeCl 3 | ????0 ????70 ????200 ????200 ????300 |
Embodiment 5.GS115/inuA1 inulinase hydrolytic inulin
Preparation 15% inulin aqueous solution 100mL, with the enzyme addition of every gram substrate 25U, 55 ℃, pH5.5, the hydrolysis of 40 times/min reciprocating vibration.High performance liquid chromatography (L-6000 PumpHITACHI) is regularly measured substrate inulin and hydrolysis resultant fructose and glucose amount, (GL-C 620 for the molecular sieve gel post, GL Science Co., Tokyo), water is done moving phase, 60 ℃ of column temperature, flow velocity 0.6mL/min, Refractive index detector detects.Bigger hydrolysis rate was arranged in preceding 3 hours, and the hydrolysis of inulin rate was 80% in 3 hours, and fructose content is to reach the highest percent hydrolysis 92.3% in 77.6%, 24 hour, and fructose content is 92.1%.Glucose content is less than 0.1% in the hydrolyzation system.Substrate inulin and product fructose and glucose change in time and see Fig. 4 in the hydrolyzation system.
Embodiment 6.GS115/inuA1 inulinase sucrose hydrolysis
Sucrose with 15% is substrate, and by two, 55 ℃ of enzyme interpolations, the pH5.5 hydrolysis 1 hour of every gram substrate 25U, percent hydrolysis reaches 100%, the glucose of generation 46.3% and 47.6% fructose.
Description of drawings:
Fig. 1: recombination yeast GS115/inuA1 fermentation generates inulinase, and different time is measured fermented liquid inulinase activity down, X-coordinate represent fermentation time (hour), ordinate zou is represented enzymic activity (μ mol/mL).
Fig. 2: the suitableeest action pH of recombination yeast GS115/inuA1 inulinase, X-coordinate is represented the enzyme assay pH value of buffer solution, 2 ordinate zous are represented fermented liquid inulinase activity (left side) and purified inulinase INUA1 enzymic activity (right side) respectively.
Fig. 3: recombination yeast GS115/inuA1 inulinase optimum temperature, X-coordinate represent the enzyme assay temperature (℃), 2 ordinate zous are represented fermented liquid inulinase activity (left side) and purified inulinase INUA1 enzymic activity (right side) respectively.
Fig. 4: the inulinase hydrolytic inulin, substrate inulin and product fructose and glucose change in time in the hydrolyzation system.X-coordinate represent hydrolysis time (hour), ordinate zou is represented inulin, fructose and glucose quality per-cent.
Claims (6)
1. the structure of an inulinase gene inuA1 who comes from aspergillus niger (Aspergillus niger) AF10 and the aminoacid sequence of inulinase protein I NUA1.
2.1：1 ATGTTGAATC CGAAGGTTGC CTACATGGTC TGGATGACCT GCCTGGGTTT51 AACGTTGCCC AGCCAGGCGC AGTCTAATGA TTACCGTCCA TCATACCACT101 TCACACCGGA CCAGTACTGG AACGAGCCAA ACGGCCTGAT TAAAATCGGA151 TCCACCTGGC ACCTGTTCTT TCAACACAAT CCGACGGCCA ATGTGTGGGG201 CAACATATGC TGGGGGCACG CTACGAGCAC CGATCTGATG CACTGGGCCT251 ACAAACCCAC TGCCATTGCG GATGAGAACG GAGTCGAAGC GTTTACCGGT301 ACAGCCTATT ATGATCCAAA CAATACCTCT GGCCTTGGGG ATTCGGCAAA351 CCCACCCTAT CTGGCCTGGT TCACAGGTTA TACCACTTCA AGCCAAACAC401 AGGACCAGCG CCTGGCTTTC AGTGTGGATA ACGGGGCGAC GTGGACCAAA451 TTTCAAGGCA ACCCCATCAT ATCAACTAGC CAGGAAGCAC CACATGATAT501 AACGGGCGGC CTCGAGAGTC GGGATCCAAA GGTATTCTTC CATCGCCAAT551 CGGGGAATTG GATCATGGTT CTCGCCCATG GCGGGCAGGA CAAGCTGTCT601 TTCTGGACGT CTGCAGACAC CATACACTGG ACATGGCAGA GTGACCTGAA651 GTCCACCTCG ATCAACGGCC TATCGTCCGA TATTACAGGG TGGGAAGTCC701 CCGACATGTT TGAACTCCCG GTTGAAGGCA CTGGGGAGAC CACTGGGGTG751 GTGATGATGA CGCCGGCTGA AGGATCTCCT GCCGGTGGTA ACGGGGTCTT801 AGCTATCACC GGTTCTTTTG ACGGGAAAAC TTTTACGGCA GATCCCGTCG851 ATGCTTCGAC CATGTGGCTG GACAATGGGC GTGATTTCGA TGGCGCTCTG901 AGCTGGGTGA ACGTGCCTGC GTCCGATGGA CGGCGGATTA TCGCCGCCGT951 CATGAATAGC TACGGTTCCA ACCCGCCTAC AACCACCTGG AAAGGGATGC1001 TCTCCTTTCC CCGGACGCTG TCGCTCAAGA AAGTTGGCAC GCAGCAGCAC1051 TTTGTTCAAC AGCCGATCAC AGAGTTGGAT ATGACAATTA GTACCAGTAT1101 GCAAACACTA GCAAACCAGA CCATTACCCC TGGCCAAACA TTGCTGTCAT1151 CGATTCGGGG AACTGCTCTC GATGTTCGAG TTGCTTTTTA CCCTGATGCT1201 GGCTCGGTTC TGTCCCTCAC CGTCCGAAAG GGTGCTTCGG AGCAAACAGT1251 CATTAATTAC ACCCAGTCAA ATGCCACATT GTCGGTTGAT CGAACAGAGA1301 GTGGAGATAT CTCGTATGAC ACGGCCGCAG GTGGCGTCCA TACCGCCAAG1351 TTGGAAGAGG ACGGCACCGG ACTGGTTTCC ATCCGGGTGT TGGTGGATAC1401 GTGTTCTGTA GAGGTTTTTG GCGGACAAGG AGAGGCCGTC ATTTCCGACC1451 TCATCTTCCC GAGTGACAGC TCTGACGGCC TGGCCTTGGA GGTAACTGGC1501 GGGAATGCAG TGCTGCAGTC GGTGGACGTA CGGAGTGTTT CACTTGAATG1551 A
3.1INUA1：1 MLNPKVAYMA MMTCLGLTLP SQAQSNDYRP SYHFTPDQYW MNEPNGLIKI51 GSTWHLFFQH NPTANVWGNI CWGHATSTDL MHWAYKPTAI ADENGVEAFT101 GTAYYDPNNT SGLGDSANPP YLAWFTGYTT SSQTQDQRLA FSVDNGATWT151 KFQGNPIIST SQEAPHDITG GLESRDPKVF FHRQSGNWIM VLAGHGQDKL201 SFWTSADTIH WTWQSDLKST SINGLSSDIT GWEVPDMFEL PVEGTGETTW251 VVMMTPAEGS PAGGNGVLAI TGSFDGKTFT ADPVDASTMW LDNGRDFDGA301 LSWVNVPASD GRRIIAAVMN SYGSNPPTTT WKGMLSFPRT LSLKKVGTQQ351 HFVQQPITEL DTISTSMQTL ANQTITPGQT LLSSIRGTAL DVRVAFYPDA401 DSVLSLTVRK GASEQTVINY TQSNATLSVD RTESGDISYD PAAGGVHTAK451 LEEDGTGLVS IRVLVDTCSV EVFGGQGEAV ISTLIFPSDS SDGLALEVTG501 GNAVLQSVDV RSVSLE
4. express the gene engineering microzyme GS115/inuA1 of INUA1.
5. the method for using GS115/inuA1 inulinase hydrolytic inulin to produce fructose.
6. the method for using GS115/inuA1 inulinase sucrose hydrolysis, beet or sugarcane immersion liquid, molasses to produce fructose.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199641A (en) * | 2011-03-05 | 2011-09-28 | 兰州理工大学 | Method for extracting sugar from enzymic hydrolysate of jerusalem artichoke residues |
| CN105087519A (en) * | 2015-09-24 | 2015-11-25 | 天津科技大学 | Genetic engineering inulase and method for preparing crystalline fructose with jerusalem artichoke as raw material |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
-
2002
- 2002-06-12 CN CNA021324468A patent/CN1465699A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199641A (en) * | 2011-03-05 | 2011-09-28 | 兰州理工大学 | Method for extracting sugar from enzymic hydrolysate of jerusalem artichoke residues |
| CN105087519A (en) * | 2015-09-24 | 2015-11-25 | 天津科技大学 | Genetic engineering inulase and method for preparing crystalline fructose with jerusalem artichoke as raw material |
| CN105087519B (en) * | 2015-09-24 | 2018-11-02 | 天津科技大学 | Gene engineering inulinase and its method that crystal diabetin is prepared as raw material using jerusalem artichoke |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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