CN1880459A - Plasmid pET28a(+)-P450BM3-gdh0310 capable of catalytic preparing indigo from indole, preparation process and use thereof - Google Patents
Plasmid pET28a(+)-P450BM3-gdh0310 capable of catalytic preparing indigo from indole, preparation process and use thereof Download PDFInfo
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- CN1880459A CN1880459A CN 200610050388 CN200610050388A CN1880459A CN 1880459 A CN1880459 A CN 1880459A CN 200610050388 CN200610050388 CN 200610050388 CN 200610050388 A CN200610050388 A CN 200610050388A CN 1880459 A CN1880459 A CN 1880459A
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- gdh0310
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- 239000013612 plasmid Substances 0.000 title claims abstract description 64
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 title claims abstract description 51
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 235000000177 Indigofera tinctoria Nutrition 0.000 title claims abstract description 24
- 229940097275 indigo Drugs 0.000 title claims abstract description 24
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 title claims abstract description 20
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 230000003197 catalytic effect Effects 0.000 title abstract description 3
- 238000002360 preparation method Methods 0.000 title description 5
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- 239000012634 fragment Substances 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
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- 238000013016 damping Methods 0.000 claims description 8
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
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- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 claims description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 2
- 102000008109 Mixed Function Oxygenases Human genes 0.000 abstract description 4
- 108010074633 Mixed Function Oxygenases Proteins 0.000 abstract description 4
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- 101000745610 Bacillus megaterium (strain ATCC 14581 / DSM 32 / JCM 2506 / NBRC 15308 / NCIMB 9376 / NCTC 10342 / NRRL B-14308 / VKM B-512) NADPH-cytochrome P450 reductase Proteins 0.000 abstract description 3
- 230000001580 bacterial effect Effects 0.000 abstract description 3
- COHYTHOBJLSHDF-BUHFOSPRSA-N indigo dye Chemical compound N\1C2=CC=CC=C2C(=O)C/1=C1/C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-BUHFOSPRSA-N 0.000 abstract 1
- 230000002195 synergetic effect Effects 0.000 abstract 1
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- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 5
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 101710088194 Dehydrogenase Proteins 0.000 description 4
- 230000001186 cumulative effect Effects 0.000 description 4
- 230000009089 cytolysis Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 241000194107 Bacillus megaterium Species 0.000 description 3
- -1 Hexose phosphate Chemical class 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 3
- 230000036983 biotransformation Effects 0.000 description 3
- 102200091217 c.563T>A Human genes 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
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- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000008859 change Effects 0.000 description 2
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- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a together-expressing P450BM3 and glucose dehydrogenase and plasmid pET28a (+)-P450BM3-gdh0310, making method and utility for catalyzing indole to synthesize indigo, which is characterized by the following: connecting P450BM3 gene and glucose dehydrogenase gene on the same expressing carrier pET28a(+); transferring the plasmid into E.coliBL21 to obtain bacterial strain to express cytochrome P450BM3 monooxygenase and glucose dehydrogenase simultaneously; catalyzing indole to synthesize indigo through synergic action; improving catalytic activity by 22-27 times; providing wide appliance prospect for biological transferring manufacturing indigo dye.
Description
Technical field
The present invention relates to the indigo technical field of genetic engineering bacterium production in the biochemical industry, relate in particular to a kind of energy catalyzing indole to generate indigo blue, plasmid pET28a (+)-P450BM3-gdh0310, the preparation method and its usage of energy coexpression Cytochrome P450 BM3 monooxygenase and Hexose phosphate dehydrogenase.
Background technology
Indigo is a kind of blue pigment, is one of natural dyestuff of finding the earliest.According to statistics, till 1998, the gross annual output amount of world's dyestuff is 800,000 tons, wherein indigoly accounts for 80,000 tons.Dyestuff is indigo to be extracted from plant at first, after its chemical structure in 1883 is illustrated, advantages such as chemical synthesis and preparation method is widely adopted, and it is easy to produce, raw material is sufficient, purity is high, easy to use are popularized rapidly, the very fast vegetable indigo that replaced.But the indigo environmental pollution of chemosynthesis is serious, and the potential carcinogenesis is arranged.Along with the reinforcement of people's environment protection and labour protection consciousness, adopt the biotechnological means synthesizing indigo to cause investigator's attention gradually.The bio-transformation synthesizing indigo is not only simplified technology, may reduce cost, and to eliminating potential carcinogen, prevent the pollution of the environment, and has more superiority.Therefore develop the biological process synthesizing indigo and be have promising.
P450 enzyme system is distributed widely in the intravital class metabolic enzyme of different biology such as animal, plant and microorganism to be, then has the characteristic light absorption peak to gain the name at the 450nm place because of its P450 albumen in mainly forming combines with CO.Studies show that up to now, it can the thousands of chemical reaction of catalysis, the principal reaction that participates in has the hydroxylation of alkyl, the epoxidation of alkyl, the oxidation of hydroxyl, dealkylation on ammonia, oxygen, the sulphur position, hydroxylation on the ammonia position and oxidation, the oxidation on the sulphur position, oxidisability deamination, dehydrogenation and dehalogenate, the C-C bond rupture of oxidisability and the reaction of some other reduction catalysts.Monograph is arranged even point out that it may be the multifarious biological catalyst of tool catalysis of occurring in nature, because character and the function and the effect in the vital movement process of this enzyme system, P450 enzyme system has been subjected to numerous investigators' great attention, and the importance of P450 enzyme system effect has become noticeable field in the contemporary biological study.External research work mainly is distributed in the aspects such as katalysis scale operation chiral drug that this enzyme is tied up to the effect in the living organism and utilizes this enzyme system, and at home, though have in a large number about the summary of this enzyme system and the property studied report, but research work only is confined to this enzyme and ties up to effect in the living organism, the katalysis that has not yet to see useful this enzyme system prepares the report of chiral drug, have the scholar to point out, major cause is the domestic an amount of enzyme of still failing to obtain at present.
In P450 enzyme system, P450 BM3 be from bacillus megaterium (B.megaterium) discovery have an active P450 enzyme of lipid acid hydroxylation, at NADPH and O
2Exist down, it has the quick catalysis chain length and is approximately C
12-C
18The monooxygenase activity of saturated fatty acid.Abroad, learn a skill the greatly R.D.Schmid of Biochemical Research institute leader's scientific research group of Stuttgart, Germany utilized the orthogenesis technology to obtain to have the P450 BM3 (Phe87Val in three mutational sites in 2000, Leu188Gln, Ala74Gly), they find this mutant enzyme energy catalyzing indole to generate indigo blue unexpectedly; See " Directed evolution of the fatty-acid hydroxylase P450 BM-3 into anindole-hydroxylating catalyst " " Chemistry-A European Journal " Li Q S for details, Schwaneberg U, Fischer P, Schmid R D.2000,6:1531-1536.
Then on this basis by professor Mei Lehe of bio-engineering research institute of Zhejiang University leader's scientific research group, by the further orthogenesis P450BM3 of fallibility round pcr (Phe87Val, Leu188Gln, Ala74Gly) variant gene obtains one and is higher than P450BM3 (Phe87Val, Leu188Gln, Ala74Gly) the active P450 BM3 of catalyzing indole mutant enzyme P450 BM3 (D168N, A225V K440N), has improved indigo output.See " the cytopigment 450BM-3 orthogenesis research of catalyzing indole to generate indigo blue " " biological chemistry and biophysics progress " Li Hongmei, plum happy, VladaUrlacher, Schmid RolfD.2005 for details, 32 (7): 1-6.
But P450 BM3 catalyzing indole to generate indigo blue needs the participation of reduced coenzyme NADPH, and this has just limited the practicality that P450 BM3 is used for synthesizing indigo.
Summary of the invention
The invention provides a kind of energy coexpression P450 BM3 and Hexose phosphate dehydrogenase, and plasmid pET28a (+)-P450BM3-gdh0310 of energy catalyzing indole synthesizing indigo.
The present invention also provides the preparation method of above-mentioned plasmid pET28a (+)-P450 BM3-gdh0310.
The present invention also provides plasmid pET28a (+)-P450 BM3-gdh0310 to be used for the purposes of catalyzing indole synthesizing indigo.
A kind of plasmid pET28a (+)-P450 BM3-gdh0310 of energy catalyzing indole to generate indigo blue is connected same expression vector pET28a (+) jointly by P450 BM3 gene and glucose dehydrogenase gene and makes up.
Preparation has the method for the reorganization bacterium of above-mentioned plasmid pET28a (+)-P450 BM3-gdh0310, may further comprise the steps:
(1) be that template obtains the gdh0310 gene fragment through pcr amplification with pQE30-gdh0310; , this gene fragment is connected on the pMD18-T plasmid, obtain pMD18-gdh0310;
(2) above-mentioned pMD18-gdh0310 is transformed among the E.coli JM109, and is applied on the LB agar plate that contains penbritin 37 ℃ of incubated overnight;
(3) selected clone is inoculated in the LB liquid nutrient medium that contains penbritin, 37 ℃ of incubated overnight amplification plasmids;
(4) extract plasmid after the amplification,, gained endonuclease bamhi orientation is connected on pET28a (+)-P450 BM3 that EcoRI and XhoI enzymolysis processing are crossed, make up and obtain pET28a (+)-P450 BM3-gdh0310 through EcoRI and XhoI enzymolysis;
(5) recombinant plasmid pET28a (+)-P450 BM3-gdh0310 is transformed among the E.coli DH5 α, is applied on the LB agar plate that contains kantlex 37 ℃ of incubated overnight;
(6) selected clone is inoculated in the LB liquid nutrient medium that contains kantlex, and 37 ℃ of incubated overnight are with the amplification plasmid;
(7) extract the amplification plasmid, recombinant plasmid pET28a (+)-P450BM3-gdh0310 is carried out double digestion, identify by electrophoresis with NcoI and XhoI;
(8) electrophoresis is identified correct plasmid is transformed among the E.coliBL21, obtained having the reorganization bacterium E.coliBL21 of plasmid (pET28a (+)-P450 BM3-gdh0310).
Above-mentioned plasmid pET28a (+)-P450 BM3-gdh0310 is used for catalyzing indole to generate indigo blue.
Its catalyzing indole to generate indigo blue may further comprise the steps:
(1) the reorganization bacterium E.coliBL21 positive colony that has plasmid pET28a (+)-P450 BM3-gdh0310 by LB agar plate picking is inoculated in the LB liquid nutrient medium that adds a certain amount of kantlex, 150-180r/min, 37 ℃ of shaking table incubated overnight 12h;
(2) add a certain amount of kantlex in the fermention medium, add an amount of glucose, the inoculum size of seed liquor being pressed 1-2% (v/v) inserts, 150-180r/min, and 37 ℃ of shaking tables are cultured to the dense OD of bacterium
600Be about 0.8-1.2, add IPTG and induce, add the appropriate amount of substrate indoles simultaneously, temperature is adjusted to 30 ℃ continues to cultivate 24h;
(3) with gained medium centrifugal 10min, harvested cell, and wash with water 2-3 time, adding an amount of N, dinethylformamide extracts blue material.
Catalyzing indole to generate indigo blue also can adopt following steps:
(1) the reorganization bacterium E.coliBL21 positive colony that has plasmid pET28a (+)-P450 BM3-gdh0310 by LB agar plate picking is inoculated in the LB liquid nutrient medium that adds a certain amount of kantlex, 150-180r/min, 37 ℃ of shaking table incubated overnight 12h;
(2) add a certain amount of kantlex in the fermention medium, the inoculum size of seed liquor being pressed 1-2% (v/v) inserts, 150-180r/min, and 37 ℃ of shaking tables are cultured to the dense OD of bacterium
600Be about 0.8-1.2, add IPTG and induce 6-8h, liquid amount is that 25-50ml/250ml shakes bottle;
(3) with the centrifugal 10min of gained fermented liquid, to collect thalline, and use the physiological saline washed twice, the thalline that obtains is preserved standby down in 4 ℃;
(4) take by weighing wet thallus, be suspended in the Tris-HCl damping fluid, add glucose and indoles, 30-35 ℃ of isothermal reaction 8h under the 100-200r/min oscillating condition;
(5) after reaction finishes, with the centrifugal 10min of gained reaction solution, add an amount of N, dinethylformamide extracts blue material.
The present invention is at mutant enzyme P450 BM3 (D168N, A225V, K440N) on the basis of mutator gene, this mutator gene and glucose dehydrogenase gene are connected on the same expression vector pET28a (+) jointly, change this plasmid over to E.coliBL21, the bacterial strain that obtains is express cell cytochrome p 450 BM3 monooxygenase and Hexose phosphate dehydrogenase simultaneously, and the catalyzing indole to generate indigo blue that can act synergistically, in reaction system, form the regenerating coenzyme recycle system, thereby increase substantially indigo output, make bio-transformation synthesizing indigo practicability more.Compare with the bacterial strain E.coli BL21 that can only express P450 BM3 (pET28a (+)-P450 BM3), its catalytic activity has improved 22-27 doubly, and wide application prospect is provided for bio-transformation production dyestuff is indigo.
Embodiment
With pQE30-gdh0310 is that template obtains the gdh0310 gene fragment through pcr amplification, gained gdh0310 gene fragment is cut glue reclaim the glucose dehydrogenase gene segment with electrophoresis detection and from sepharose, be connected on the pMD18-T plasmid by " A-T " mode of connection, obtain pMD18-gdh0310; Gained pMD18-gdh0310 is transformed among the E.coli JM109, and is applied on the LB agar plate that final concentration is 50 μ g/ml penbritins 37 ℃ of incubated overnight; Select about 8 clones and be inoculated into 3ml and contain in the LB liquid nutrient medium of 50 μ g/ml penbritins, 37 ℃ of incubated overnight are with the amplification plasmid; With the alkaline lysis method of extracting plasmid that increases, and with EcoRI and XhoI at 37 ℃ of enzymolysis, the endonuclease bamhi orientation that obtains is connected on pET28a (+)-P450 BM3 that handled with same double digestion, structure obtains pET28a (+)-P450 BM3-gdh0310.
Recombinant plasmid pET28a (+)-P450 BM3-gdh0310 is transformed into E.coli DH5 α, is applied on the LB agar plate that contains 30 μ g/ml kantlex 37 ℃ of incubated overnight; Select 8 clones and be inoculated into 3ml and contain in the LB liquid nutrient medium of 50 μ g/ml kantlex, 37 ℃ of incubated overnight are with the amplification plasmid; With the alkaline lysis method of extracting plasmid that increases, and be transformed among the E.coliBL21 (DE-3), promptly obtained the reorganization bacterium E.coliBL21 of band plasmid pET28a (+) P450 BM3-gdh0310.
Be inoculated into by the reorganization bacterium E.coliBL21 positive colony of dull and stereotyped picking band plasmid pET28a (+) P450 of LB BM3-gdh0310 that to add kantlex to final concentration be in the LB seed culture medium of 30 μ g/ml, 180r/min, 37 ℃ of shaking table incubated overnight 12h; Add kantlex in the fermention medium to final concentration 30 μ g/ml, add glucose to 100-200mM, the inoculum size of seed liquor being pressed 1-2% (v/v) inserts, 150-180r/min, and 37 ℃ of shaking tables are cultured to the dense OD of bacterium
600Be about 0.8-1.2, add IPTG and induce to final concentration 0.5-1.0mM, add the appropriate amount of substrate indoles simultaneously, temperature is adjusted to 30 ℃ continues to cultivate 24h, obtain blue cell, use N, dinethylformamide extraction blue material.
Perhaps the reorganization bacterium E.coliBL21 positive colony that has plasmid pET28a (+)-P450 BM3-gdh0310 by LB agar plate picking is inoculated in the LB liquid nutrient medium that adds a certain amount of kantlex, 150-180r/min, 37 ℃ of shaking table incubated overnight 12h; Add a certain amount of kantlex in fermention medium, the inoculum size of seed liquor being pressed 1-2% (v/v) inserts, 150-180r/min, and 37 ℃ of shaking tables are cultured to the dense OD of bacterium
600Be about 0.8-1.2, add IPTG and induce 6-8h, liquid amount is that 25-50ml/250ml shakes bottle; With the centrifugal 10min of gained fermented liquid, to collect thalline, and use the physiological saline washed twice, the thalline that obtains is preserved standby down in 4 ℃; Take by weighing wet thallus, be suspended in the Tris-HCl damping fluid, add glucose and indoles, 30-35 ℃ of isothermal reaction 8h under the 100-200r/min oscillating condition; Reaction with the centrifugal 10min of gained reaction solution, adds an amount of N after finishing, and dinethylformamide extracts blue material.
Sequence table
Sequence information
(a) sequence signature, this sequence does not comprise pET28 (+), P450 BM3 gene segment upstream is connected on the pET28a (+) by the NheI restriction enzyme site, the downstream is connected by the EcoRI restriction enzyme site with the glucose dehydrogenase gene segment, glucose dehydrogenase gene segment downstream is connected with pET28a (+) by the XhoI restriction enzyme site, this sequence is from the promotor of P450 BM3 gene, to glucose
The terminator of dehydrogenase gene finishes.
Length: 3946 bases
Type: nucleic acid
Chain: strand
Topological framework: linearity
(b) molecule type: cDNA
(c) initial source: P450 BM3 gene segment and glucose dehydrogenase gene all come from bacillus megaterium
(d) sequence description:
ATGACAATTAAAGAAATGCCTCAGCCAAAAACGTTTGGAGAG
CTTAAAAATTTACCGTTATTAAACACAGATAAACCGGTTCAAGCTTTG
ATGAAAATTGCGGATGAATTAGGAGAAATCTTTAAATTCGAGGCGCC
TGGTCGTGTAACGCGCTACTTATCAAGTCAGCGTCTAATTAAAGAAG
CATGCGATGAATCACGCTTTGATAAAAACTTAAGTCAAGGTCTTAAA
TTTGTACGTGATTTTGCAGGAGACGGGTTGGTTACAAGCTGGACGCA
TGAAAAAAATTGGAAAAAAGCGCATAATATCTTACTTCCAAGCTTCA
GTCAGCAGGCAATGAAAGGCTATCATGCGATGATGGTCGATATCGCC
GTGCAGCTTGTTCAAAAGTGGGAGCGTCTAAATGCAGATGAGCATAT
TGAAGTACCGGAAGACATGACACGTTTAACGCTTGATACAATTGGTC
TTTGCGGCTTTAACTATCGCTTTAACAGCTTTTACCGAGATCAGCCTC
ATCCATTTATTACAAGTATGGTCCGTGCACTGGATGAAGCAATGAAC
AAGCAGCAGCGAGCAAATCCAGACGACCCAGCTTATGATGAAAACA
AGCGCCAGTTTCAAGAAGATATCAAGGTGATGAACGACCTAGTAGAT
AAAATTATTGCAGATCGCAAAGCAAGCGGTGAACAAAGCGATGATTT
ATTAACGCATATGCTAAACGGAAAAGATCCAGAAACGGGTGAGCCG
CTTGATGACGAGAACATTCGCTATCAAATTATTACATTCTTAATTGCG
GGACACGAAACAACAAGTGGTCTTTTATCATTTGCGCTGTATTTCTTA
GTGAAAAATCCACATGTATTACAAAAAGCAGCAGAAGAAGCAGCAC
GAGTTCTAGTAGATCCTGTTCCAAGCTACAAACAAGTCAAACAGCTT
AAATATGTCGGCATGGTCTTAAACGAAGCGCTGCGCTTATGGCCAAC
TGCTCCTGCGTTTTCCCTATATGCAAAAGAAGATACGGTGCTTGGAG
GAGAATATCCTTTAGAAAAAGGCGACGAACTAATGGTTCTGATTCCT
CAGCTTCACCGTGATAAAACAATTTGGGGAGACGATGTGGAAGAGT
TCCGTCCAGAGCGTTTTGAAAATCCAAGTGCGATTCCGCAGCATGCG
TTTAAACCGTTTGGAAACGGTCAGCGTGCGTGTATCGGTCAGCAGTT
CGCTCTTCATGAAGCAACGCTGGTACTTGGTATGATGCTAAAACACT
TTGACTTTGAAGATCATACAAACTACGAGCTGGATATTAAAGAAACT
TTAACGTTAAAACCTGAAGGCTTTGTGGTAAAAGCAAAATCGAAAA
AAATTCCGCTTGGCGGTATTCCTTCACCTAGCACTGAACAGTCTGCT
AAAAAAGTATGCAAAAAGGCAGAAAACGCTCATAATACGCCGCTGC
TTGTGCTATACGGATCCAATATGGGAACAGCTGAAGGAACGGCGCGT
GATTTAGCAGATATTGCAATGAGCAAAGGATTTGCACCGCAGGTCGC
AACGCTTGATTCACACGCCGGAAATCTTCCGCGCGAAGGAGCTGTAT
TAATTGTAACGGCGTCTTATAACGGTCATCCGCCTGATAACGCAAAG
CAATTTGTCGACTGGTTAGACCAAGCGTCTGCTGATGAAGTAAAAGG
CGTTCGCTACTCCGTATTTGGATGCGGCGATAAAAACTGGGCTACTA
CGTATCAAAAAGTGCCTGCTTTTATCGATGAAACGCTTGCCGCTAAA
GGGGCAGAAAACATCGCTGACCGCGGTGAAGCAGATGCAAGCGAC
GACTTTGAAGGCACATATGAAGAATGGCGTGAACATATGTGGAGTGA
CGTAGCAGCCTACTTTAACCTCGACATTGAAAACAGTGAAGATAATA
AATCTACTCTTTCACTTCAATTTGTCGACAGCGCCGCGGATATGCCGC
TTGCGAAAATGCACGGTGCGTTTTCAACGAACGTCGTAGCAAGCAA
AGAACTTCAACAGCCAGGCAGTGCACGAAGCACGCGACATCTTGAA
ATTGAACTTCCAAAAGAAGCTTCTTATCAAGAAGGAGATCATTTAGG
TGTTATTCCTCGCAACTATGAAGGAATAGTAAACCGTGTAACAGCAA
GGTTCGGCCTAGATGCATCACAGCAAATCCGTCTGGAAGCAGAAGA
AGAAAAATTAGCTCATTTGCCACTCGCTAAAACAGTATCCGTAGAAG
AGCTTCTGCAATACGTGGAGCTTCAAGATCCTGTTACGCGCACGCAG
CTTCGCGCAATGGCTGCTAAAACGGTCTGCCCGCCGCATAAAGTAGA
GCTTGAAGCCTTGCTTGAAAGCAAGCCTACAAGACAAGTGCTGGCA
AACGTTTAACAATGCTTGAACTGCTTGAAAAATACCCGGCGTGTGAA
TGAAATTCAGCGAATTTATCGCCATTCTGCCAAGCATACGCCCGCGCT
ATTACTCGATTTCTTCATCACCTCGTGTCGATGAAAAACAAGCAAGC
ATCACGGTCAGCGTTGTCTCAGGAGAAGCGTGGAGCGGATATGGAG
AATATAAAGGAATTGCGTCGAACTATCTTGCCGAGCTGCAAGAAGGA
GATACGATTACGTGCTTTATTTCCACACCGCAGTCAGAATTTACGCTG
CCAAAAGACCCTGAAACGCCGCTTATCATGGTCGGACCGGGAACAG
GCGTCGCGCCGTTTAGAGGCTTTGTGCAGGCGCGCAAACAGCTAAA
AGAACAAGGACAGTCACTTGGAGAAGCACATTTATACTTCGGCTGC
CGTTCACCTCATGAAGACTATCTGTATCAAGAAGAGCTTGAAAACGC
CCAAAGCGAAGGCATCATTACGCTTCATACCGCTTTTTCTCGCATGCC
AAATCAGCCGAAAACATACGTTCAGCACGTAATGGAACAAGACGGC
AAGAAATTGATTGAACTTCTTGATCAAGGAGCGCACTTCTATATTTGC
GGAGACGGAAGCCAAATGGCACCTGCCGTTGAAGCAACGCTTATGA
AAAGCTATGCTGACGTTCACCAAGTGAGTGAAGCAGACGCTCGCTT
ATGGCTGCAGCAGCTAGAAGAAAAAGGCCGATACGCAAAAGACGTG
TGGGCTGGGTAAGAATTCGGATCCATGTATACAGATTTAAAAGATAA
AGTAGTAGTTGTAACAGGCGGATCAAAAGGATTGGGTCGCGCAATG
GCCGTTCGTTTTGGTCAAGAGCAGTCAAAAGTGGTTGTAAACTACC
GCAGCAATGAAGAAGAAGCGCTAGAAGTAAAAAAAGAAATTGAAC
AAGCTGGCGGCCAAGCAATTATTGTTCGAGGCGACGTAACAAAAGA
GGAAGACGTTGTGAATCTTGTAGAGACAGCTGTTAAAGAGTTTGGC
ACATTAGACGTTATGATTAACAATGCTGGTGTTGAAAACCCGGTTCC
TTCACATGAATTATCGTTAGAAAACTGGAATCAAGTAATCGATACAA
ACTTAACAGGCGCGTTTTTAGGAAGCCGCGAAGCGATTAAATATTTT
GTTGAAAATGATATTAAAGGAAACGTTATTAACATGTCCAGCGTTCA
CGAGATGATTCCTTGGCCACTATTTGTTCACTATGCAGCAAGTAAAG
GCGGTATGAAACTAATGACAGAAACATTGGCTCTTGAATATGCGCCA
AAAGGTATCCGCGTAAATAACATTGGACCAGGCGCGATCGATACGCC
AATCAACGCTGAAAAATTCGCAGATCCGGAACAGCGTGCAGACGTA
GAAAGCATGATTCCAATGGGCTACATCGGCAACCCGGAAGAAATTG
CATCAGTTGCAGCATTCTTAGCATCGTCACAAGCAAGCTACGTAACA
GGTATTACACTATTTGCTGATGGCGGTATGACAAAATATCCTTCTTTCC
AAGCGGGAAGAGGTTAATAA
Concrete operations adopt following steps to carry out:
1. the acquisition of glucose dehydrogenase gene:
With pQE30-gdh0310 is that template obtains the gdh0310 gene fragment through pcr amplification, designs corresponding upstream and downstream primer and is respectively:
5 '-AAAAGAATTCATGTATACAGATTTAAAAGATAAAGTAGTAG-3 ' and 5 '-AAAAACTCGAGTTATTAACCTCTTCCCGCTT-3 '.
For ease of connecting with pET28a (+)-P450 BM3,5 ' end of upstream and downstream primer has designed EcoRI and XhoI restriction enzyme site respectively.
The pcr amplification reaction system is: the dNTPs that adds 10nmol in the eppendorf pipe of 500 μ l, the upstream primer of each 10pmol and downstream primer, about 1ng plasmid pQE30-gdh0310 is as template DNA, 1.25U Taq Platinum polysaccharase, the PCR damping fluid of 5 μ l adds to cumulative volume 50 μ l with aseptic distilled water then.After the PCR reaction parameter is 94 ℃ of sex change 8min, 94 ℃/30s, 54 ℃/30s, 72 ℃/1min, circulate 30 times; 72 ℃ are extended 10min.
Gained gdh0310 gene fragment is cut glue reclaim the glucose dehydrogenase gene segment with electrophoresis detection and from sepharose, be connected on the pMD18-T plasmid by " A-T " mode of connection, obtain pMD18-gdh0310, gained pMD18-gdh0310 is transformed among the E.coli JM109, and be applied on the LB agar plate that final concentration is 50 μ g/ml penbritins 37 ℃ of incubated overnight; Selecting about 8 clones is inoculated into 3ml and contains in the LB liquid nutrient medium of 50 μ g/ml penbritins, 37 ℃ of incubated overnight are with the amplification plasmid, with the alkaline lysis method of extracting plasmid that increases,, behind 37 ℃ of enzymolysis, cut glue with electrophoresis detection and from sepharose and reclaim the glucose dehydrogenase gene segment through EcoRI and XhoI.
Enzyme is cut in the process, and the consumption of plasmid pMD18-gdh0310 and restriction enzyme is:
Plasmid pMD18-gdh0310 10 μ l
EcoRI 1μl
XhoI 1μl
Damping fluid (10 * H) 3 μ l
Sterilized water 15 μ l
Cumulative volume 30 μ l
2. the structure of expression vector pET28a (+)-P450 BM3-gdh0310:
To above-mentioned plasmid pMD18-gdh0310 EcoRI, the XhoI double digestion, the endonuclease bamhi orientation of acquisition is connected on pET28a (+)-P450 BM3 that handled with same double digestion, makes up to obtain pET28a (+)-P450 BM3-gdh0310.
Enzyme is cut in the process, and the consumption of plasmid pET28a (+)-P450 BM3 and restriction enzyme is:
Plasmid pET28a (+)-P450 BM3 10 μ l
EcoRI 1μl
XhoI 1μl
Damping fluid (10 * H) 3 μ l
Sterilized water 15 μ l
Cumulative volume 30 μ l
In the connection procedure, the consumption of ligase enzyme, glucose dehydrogenase gene segment and plasmid pET28a (+)-P450BM3 is:
Glucose dehydrogenase gene segment 4 μ l
Plasmid pET28a (+)-P450 BM3 4 μ l
T4 dna ligase 1 μ l
Damping fluid (10 *) 1 μ l
Cumulative volume 10 μ l
3. connect the evaluation of effect:
Recombinant plasmid transformed to E.coli DH5 α, is applied on the LB agar plate that contains 30 μ g/ml kantlex 37 ℃ of incubated overnight; Selecting about 8 clones is inoculated into 3ml and contains in the LB liquid nutrient medium of 50 μ g/ml kantlex, 37 ℃ of incubated overnight are with the amplification plasmid, with the alkaline lysis method of extracting plasmid that increases, and be transformed among the E.coliBL21 (DE-3), promptly obtained the reorganization bacterium E.coliBL21 of band plasmid pET28a (+)-P450 BM3-gdh0310; With NcoI and XhoI recombinant plasmid is carried out double digestion and identify utilize DNA electrophoresis detection glucose dehydrogenase gene fragment whether to be connected on pET28a (+)-P450 BM3.
4. it is indigo that whole-cell biological transforms production:
A) by the reorganization bacterium E.coliBL21 positive colony of LB agar plate picking band plasmid pET28a (+)-P450 BM3-gdh0310, being inoculated into interpolation kantlex to final concentration is in LB (pH7.0-7.5) seed culture medium of 30 μ g/ml, 150-180r/min, 37 ℃ of shaking table incubated overnight 12h; Add kantlex in the LB fermention medium to final concentration 30 μ g/ml, add glucose to 100-200mM, the inoculum size of seed liquor being pressed 1-2% (v/v) inserts, 150-180r/min, and 37 ℃ of shaking tables are cultured to the dense OD of bacterium
600Be about 0.8-1.2, add IPTG and induce to final concentration 0.5-1.0mM, adding an amount of final concentration simultaneously is the substrate indoles of 0.1-10mM, temperature is adjusted to 30 ℃ continues to cultivate 24h.
With the gained nutrient solution with the centrifugal 10min of 8000r/min, harvested cell, the gained cell washes with water 2-3 time, add an amount of N, dinethylformamide extracts blue material, and the centrifugal reservation supernatant liquor of 8000r/min is surveyed light absorption value, and calculate indigo content according to typical curve, obtaining indigo output is 49.98-915.3mg/L.
B) adding kantlex to final concentration in the seed culture medium is 30 μ g/ml, by the reorganization bacterium E.coliBL21 access of dull and stereotyped picking band plasmid pET28a (+)-P450 BM3-gdh0310,150-180r/min, 37 ℃ of shaking table incubated overnight 12h; Add kantlex in the LB fermention medium to final concentration 30 μ g/ml, the inoculum size of seed liquor being pressed 1-2% (v/v) inserts, 150-180r/min, and 37 ℃ of shaking tables are cultured to the dense OD of bacterium
600Be about 0.8-1.2, add IPTG and induce 6-8h to final concentration 0.5mM.Liquid amount is that 25-50ml/250ml shakes bottle.
After fermentation culture finished, fermented liquid was collected thalline in the centrifugal 10min of 8000r/min, and with 0.9% physiological saline washed twice, the thalline that obtains is preserved standby down in 4 ℃; Take by weighing the 0.2-0.4g wet thallus, be suspended in the 20mlTris-HCl damping fluid, adding 100-200mM glucose and a certain amount of final concentration is the indoles of 0.1-10mM, 30-35 ℃ of isothermal reaction 8h under the 100r/min oscillating condition; After reaction finished, reaction solution added an amount of N in the centrifugal 10min of 8000r/min, dinethylformamide extracts blue material, and the centrifugal reservation supernatant liquor of 8000r/min is surveyed light absorption value, and calculate indigo content according to typical curve, obtaining indigo output is 13.1-659mg/L.
Claims (5)
1. the plasmid pET28a (+) of an energy catalyzing indole to generate indigo blue-P450 BM3-gdh0310, be connected same expression vector pET28a (+) jointly by P450 BM3 gene and glucose dehydrogenase gene and go up structure, from the promotor of P450 BM3 gene, the gene order that finishes to the terminator of glucose dehydrogenase gene is:
ATGACAATTAAAGAAATGCCTCAGCCAAAAACGTTTGGAGAGCTTAAAAATTTACCGTTATTAAACACAGATAAACCGGTTCAAGCTTTGATGAAAATTGCGGATGAATTAGGAGAAATCTTTAAATTCGAGGCGCCTGGTCGTGTAACGCGCTACTTATCAAGTCAGCGTCTAATTAAAGAAGCATGCGATGAATCACGCTTTGATAAAAACTTAAGTCAAGGTCTTAAATTTGTACGTGATTTTGCAGGAGACGGGTTGGTTACAAGCTGGACGCATGAAAAAAATTGGAAAAAAGCGCATAATATCTTACTTCCAAGCTTCAGTCAGCAGGCAATGAAAGGCTATCATGCGATGATGGTCGATATCGCCGTGCAGCTTGTTCAAAAGTGGGAGCGTCTAAATGCAGATGAGCATATTGAAGTACCGGAAGACATGACACGTTTAACGCTTGATACAATTGGTCTTTGCGGCTTTAACTATCGCTTTAACAGCTTTTACCGAGATCAGCCTCATCCATTTATTACAAGTATGGTCCGTGCACTGGATGAAGCAATGAACAAGCAGCAGCGAGCAAATCCAGACGACCCAGCTTATGATGAAAACAAGCGCCAGTTTCAAGAAGATATCAAGGTGATGAACGACCTAGTAGATAAAATTATTGCAGATCGCAAAGCAAGCGGTGAACAAAGCGATGATTTATTAACGCATATGCTAAACGGAAAAGATCCAGAAACGGGTGAGCCGCTTGATGACGAGAACATTCGCTATCAAATTATTACATTCTTAATTGCGGGACACGAAACAACAAGTGGTCTTTTATCATTTGCGCTGTATTTCTTAGTGAAAAATCCACATGTATTACAAAAAGCAGCAGAAGAAGCAGCACGAGTTCTAGTAGATCCTGTTCCAAGCTACAAACAAGTCAAACAGCTTAAATATGTCGGCATGGTCTTAAACGAAGCGCTGCGCTTATGGCCAACTGCTCCTGCGTTTTCCCTATATGCAAAAGAAGATACGGTGCTTGGAGGAGAATATCCTTTAGAAAAAGGCGACGAACTAATGGTTCTGATTCCTCAGCTTCACCGTGATAAAACAATTTGGGGAGACGATGTGGAAGAGTTCCGTCCAGAGCGTTTTGAAAATCCAAGTGCGATTCCGCAGCATGCGTTTAAACCGTTTGGAAACGGTCAGCGTGCGTGTATCGGTCAGCAGTTCGCTCTTCATGAAGCAACGCTGGTACTTGGTATGATGCTAAAACACTTTGACTTTGAAGATCATACAAACTACGAGCTGGATATTAAAGAAACTTTAACGTTAAAACCTGAAGGCTTTGTGGTAAAAGCAAAATCGAAAAAAATTCCGCTTGGCGGTATTCCTTCACCTAGCACTGAACAGTCTGCTAAAAAAGTATGCAAAAAGGCAGAAAACGCTCATAATACGCCGCTGCTTGTGCTATACGGATCCAATATGGGAACAGCTGAAGGAACGGCGCGTGATTTAGCAGATATTGCAATGAGCAAAGGATTTGCACCGCAGGTCGCAACGCTTGATTCACACGCCGGAAATCTTCCGCGCGAAGGAGCTGTATTAATTGTAACGGCGTCTTATAACGGTCATCCGCCTGATAACGCAAAGCAATTTGTCGACTGGTTAGACCAAGCGTCTGCTGATGAAGTAAAAGGCGTTCGCTACTCCGTATTTGGATGCGGCGATAAAAACTGGGCTACTACGTATCAAAAAGTGCCTGCTTTTATCGATGAAACGCTTGCCGCTAAAGGGGCAGAAAACATCGCTGACCGCGGTGAAGCAGATGCAAGCGACGACTTTGAAGGCACATATGAAGAATGGCGTGAACATATGTGGAGTGACGTAGCAGCCTACTTTAACCTCGACATTGAAAACAGTGAAGATAATAAATCTACTCTTTCACTTCAATTTGTCGACAGCGCCGCGGATATGCCGCTTGCGAAAATGCACGGTGCGTTTTCAACGAACGTCGTAGCAAGCAAAGAACTTCAACAGCCAGGCAGTGCACGAAGCACGCGACATCTTGAAATTGAACTTCCAAAAGAAGCTTCTTATCAAGAAGGAGATCATTTAGGTGTTATTCCTCGCAACTATGAAGGAATAGTAAACCGTGTAACAGCAAGGTTCGGCCTAGATGCATCACAGCAAATCCGTCTGGAAGCAGAAGAAGAAAAATTAGCTCATTTGCCACTCGCTAAAACAGTATCCGTAGAAGAGCTTCTGCAATACGTGGAGCTTCAAGATCCTGTTACGCGCACGCAGCTTCGCGCAATGGCTGCTAAAACGGTCTGCCCGCCGCATAAAGTAGAGCTTGAAGCCTTGCTTGAAAGCAAGCCTACAAGACAAGTGCTGGCAAACGTTTAACAATGCTTGAACTGCTTGAAAAATACCCGGCGTGTGAATGAAATTCAGCGAATTTATCGCCATTCTGCCAAGCATACGCCCGCGCTATTACTCGATTTCTTCATCACCTCGTGTCGATGAAAAACAAGCAAGCATCACGGTCAGCGTTGTCTCAGGAGAAGCGTGGAGCGGATATGGAGAATATAAAGGAATTGCGTCGAACTATCTTGCCGAGCTGCAAGAAGGAGATACGATTACGTGCTTTATTTCCACACCGCAGTCAGAATTTACGCTGCCAAAAGACCCTGAAACGCCGCTTATCATGGTCGGACCGGGAACAGGCGTCGCGCCGTTTAGAGGCTTTGTGCAGGCGCGCAAACAGCTAAAAGAACAAGGACAGTCACTTGGAGAAGCACATTTATACTTCGGCTGCCGTTCACCTCATGAAGACTATCTGTATCAAGAAGAGCTTGAAAACGCCCAAAGCGAAGGCATCATTACGCTTCATACCGCTTTTTCTCGCATGCCAAATCAGCCGAAAACATACGTTCAGCACGTAATGGAACAAGACGGCAAGAAATTGATTGAACTTCTTGATCAAGGAGCGCACTTCTATATTTGCGGAGACGGAAGCCAAATGGCACCTGCCGTTGAAGCAACGCTTATGAAAAGCTATGCTGACGTTCACCAAGTGAGTGAAGCAGACGCTCGCTTATGGCTGCAGCAGCTAGAAGAAAAAGGCCGATACGCAAAAGACGTGTGGGCTGGGTAAGAATTCGGATCCATGTATACAGATTTAAAAGATAAAGTAGTAGTTGTAACAGGCGGATCAAAAGGATTGGGTCGCGCAATGGCCGTTCGTTTTGGTCAAGAGCAGTCAAAAGTGGTTGTAAACTACCGCAGCAATGAAGAAGAAGCGCTAGAAGTAAAAAAAGAAATTGAACAAGCTGGCGGCCAAGCAATTATTGTTCGAGGCGACGTAACAAAAGAGGAAGACGTTGTGAATCTTGTAGAGACAGCTGTTAAAGAGTTTGGCACATTAGACGTTATGATTAACAATGCTGGTGTTGAAAACCCGGTTCCTTCACATGAATTATCGTTAGAAAACTGGAATCAAGTAATCGATACAAACTTAACAGGCGCGTTTTTAGGAAGCCGCGAAGCGATTAAATATTTTGTTGAAAATGATATTAAAGGAAACGTTATTAACATGTCCAGCGTTCACGAGATGATTCCTTGGCCACTATTTGTTCACTATGCAGCAAGTAAAGGCGGTATGAAACTAATGACAGAAACATTGGCTCTTGAATATGCGCCAAAAGGTATCCGCGTAAATAACATTGGACCAGGCGCGATCGATACGCCAATCAACGCTGAAAAATTCGCAGATCCGGAACAGCGTGCAGACGTAGAAAGCATGATTCCAATGGGCTACATCGGCAACCCGGAAGAAATTGCATCAGTTGCAGCATTCTTAGCATCGTCACAAGCAAGCTACGTAACAGGTATTACACTATTTGCTGATGGCGGTATGACAAAATATCCTTCTTTCCAAGCGGGAAGAGGTTAATAA。
2. one kind prepares and has the method for the reorganization bacterium of plasmid pET28a (+)-P450BM3-gdh0310 according to claim 1, may further comprise the steps:
(1) be that template obtains the gdh0310 gene fragment through pcr amplification with pQE30-gdh0310; , this gene fragment is connected on the pMD18-T plasmid, obtain pMD18-gdh0310;
(2) above-mentioned pMD18-gdh0310 is transformed among the E.coli JM109, and is applied on the LB agar plate that contains penbritin 37 ℃ of incubated overnight;
(3) selected clone is inoculated in the LB liquid nutrient medium that contains penbritin, 37 ℃ of incubated overnight amplification plasmids;
(4) extract plasmid after the amplification,, gained endonuclease bamhi orientation is connected on pET28a (+)-P450 BM3 that EcoRI and XhoI enzymolysis processing are crossed, make up and obtain pET28a (+)-P450 BM3-gdh0310 through EcoRI and XhoI enzymolysis;
(5) recombinant plasmid pET28a (+)-P450 BM3-gdh0310 is transformed among the E.coli DH5 α, is applied on the LB agar plate that contains kantlex 37 ℃ of incubated overnight;
(6) select institute's DCRP and be inoculated in the LB liquid nutrient medium that contains kantlex, 37 ℃ of incubated overnight are with the amplification plasmid;
(7) extract the amplification plasmid, recombinant plasmid pET28a (+)-P450BM3-gdh0310 is carried out double digestion, identify by electrophoresis with NcoI and XhoI;
(8) electrophoresis is identified correct plasmid is transformed among the E.coli BL21, obtained having the reorganization bacterium E.coli BL21 of plasmid pET28a (+)-P450 BM3-gdh0310.
3. plasmid pET28a as claimed in claim 1 (+)-application of P450 BM3-gdh0310 in catalyzing indole to generate indigo blue.
4. application as claimed in claim 3 is characterized in that catalyzing indole to generate indigo blue may further comprise the steps:
(1) the reorganization bacterium E.coliBL21 positive colony that has plasmid pET28a (+)-P450 BM3-gdh0310 by LB agar plate picking is inoculated in the LB liquid nutrient medium that adds a certain amount of kantlex, 150-180r/min, 37 ℃ of shaking table incubated overnight 12h;
(2) add a certain amount of kantlex in fermention medium, and add an amount of glucose, the inoculum size of seed liquor being pressed 1-2% (v/v) inserts, 150-180r/min, and 37 ℃ of shaking tables are cultured to the dense OD of bacterium
600Be about 0.8-1.2, add IPTG and induce, add the appropriate amount of substrate indoles simultaneously, temperature is adjusted to 30 ℃ continues to cultivate 24h;
(3) with gained medium centrifugal 10min, harvested cell, and wash with water 2-3 time, adding an amount of N, dinethylformamide extracts blue material.
5. application as claimed in claim 4 is characterized in that catalyzing indole to generate indigo blue also can adopt following steps:
(1) the reorganization bacterium E.coliBL21 positive colony that has plasmid pET28a (+)-P450 BM3-gdh0310 by LB agar plate picking is inoculated in the LB liquid nutrient medium that adds a certain amount of kantlex, 150-180r/min, 37 ℃ of shaking table incubated overnight 12h;
(2) add a certain amount of kantlex in fermention medium, the inoculum size of seed liquor being pressed 1-2% (v/v) inserts, 150-180r/min, and 37 ℃ of shaking tables are cultured to the dense OD of bacterium
600Be about 0.8-1.2, add IPTG and induce 6-8h, liquid amount is that 25-50ml/250ml shakes bottle;
(3) with the centrifugal 10min of gained fermented liquid, to collect thalline, and use the physiological saline washed twice, the thalline that obtains is preserved standby down in 4 ℃;
(4) take by weighing wet thallus, be suspended in the Tris-HCl damping fluid, add glucose and indoles, 30-35 ℃ of isothermal reaction 8h under the 100-200r/min oscillating condition;
(5) after reaction finishes, with the centrifugal 10min of gained reaction solution, add an amount of N, dinethylformamide extracts blue material.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760736A (en) * | 2016-08-22 | 2018-03-06 | 中国科学院上海生命科学研究院 | A kind of method for promoting indigo biosynthesis conversion yield |
| CN109021608A (en) * | 2018-07-09 | 2018-12-18 | 陈岸瑛 | The preparation method of high quality indigo slurry based on lactobacillus-fermented |
| CN114369560A (en) * | 2021-12-30 | 2022-04-19 | 南京合谷生命生物科技有限公司 | Method for improving biological indigo yield |
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| MY126592A (en) * | 1999-07-27 | 2006-10-31 | Basf Ag | Novel cytochrome p450 monooxygenases and their use for the oxidation of organic compounds |
| CN1273585C (en) * | 2004-11-29 | 2006-09-06 | 浙江大学 | Cytochrome P450BM-3 monooxygehase varient gene and its use |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760736A (en) * | 2016-08-22 | 2018-03-06 | 中国科学院上海生命科学研究院 | A kind of method for promoting indigo biosynthesis conversion yield |
| CN107760736B (en) * | 2016-08-22 | 2020-11-27 | 中国科学院上海营养与健康研究所 | Method for promoting indigo biosynthesis conversion yield |
| CN109021608A (en) * | 2018-07-09 | 2018-12-18 | 陈岸瑛 | The preparation method of high quality indigo slurry based on lactobacillus-fermented |
| CN114369560A (en) * | 2021-12-30 | 2022-04-19 | 南京合谷生命生物科技有限公司 | Method for improving biological indigo yield |
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Granted publication date: 20081029 |