CN1283800C - P450BM-3Glu435 Asp varient gene capable of catalyzing indole to generate indigo blue and its use - Google Patents
P450BM-3Glu435 Asp varient gene capable of catalyzing indole to generate indigo blue and its use Download PDFInfo
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- 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
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Abstract
The present invention discloses a mutant gene capable of catalyzing indole to generate indigo and use thereof. The present invention carries out the screening procedure by error-prone PCR technologymediated random mutation to obtain a P450BM-3 mutant gene. A codon GAA at the 435-position in the P450BM-3 mutant gene is mutated into a codon GAT of aspartate; the sequence of the P450BM-3 mutant gene (Glu<435>-Asp) is an SEQ ID No. 1 nucleotide sequence, and the sequence encodes monooxygenase of cytochrome P450BM-3. The mutant gene can catalyze indole to generate a P450BM-3Glu435Asp mutant gene of indigo, P450BM-3 mutant ficin obtained by the expression of the P450BM-3Glu435Asp mutant gene can catalyze the indole to generate dye indigo. The present invention creates a P450BM-3 mutant gene having high catalyzing activity on the indole, and the mutational site of the mutant gene is Glu<435>-Asp. The mutant ficin expressed by the mutant gene can catalyze the indole to generate the dye indigo. Compared with P450BM-3(Phe87Val Leu188Gln, Ala74Gly) mutant ficin having three mutational sites, obtained by a scientific research group led by R. D. Schmid, the activity is increased by 1.5 to 1.7 times in a measured case. The mutant gene provides a wide application prospect for producing the dye indigo by biotransformation.
Description
Technical field
The present invention relates to the technical field that the genetic engineering bacterium in the biochemical industry is produced bipseudoindoxyl dye, relate in particular to P450BM-3Glu435Asp variant gene of a kind of energy catalyzing indole to generate indigo blue and uses thereof.
Background technology
The orthogenesis technology of enzyme molecule is exactly the artificial special evolution conditions of creation, simulate natural evolutionary mechanism, external gene is carried out random mutation, the evolution enzyme of expecting in advance by certain screening or the final acquisition of system of selection from one or more artificial mutation enzymes storehouse with some characteristic.Compare with evolving naturally.The orthogenesis process of enzyme molecule is evolved under artificial control fully, and the enzyme molecule is carried out towards the specific objective of people's expectation.In recent years along with such as fallibility PCR, The Application of Technology such as DNA reorganization, under the condition that destination gene expression is had the efficient detection screening decorum, although it be unclear that the characteristics such as structure of enzyme molecule, the new enzyme that the directional strategy of employing enzyme molecule can obtain to have the expection characteristic is realized the artificial tachytely of enzyme molecule.Many studies show that, suitably modify the amino-acid sequence that changes enzyme on by dna level under at present to enzyme molecular recognition is that all right ripe situation, and then the performance that changes enzyme is possible produce new enzyme in the reorganization biology, and obtain higher than natural enzyme vigor, stability and the catalytic performance enzyme of better evolving are to satisfy the needs of research and application.
P450 enzyme system is distributed widely in animal, and different biological intravital class metabolic enzymes such as plant and microorganism are.Because of P450 albumen in its main component with after CO combines, at the 450nm place characteristic light absorption peak is arranged and gains the name.Studies show that.It can catalysis be formed in chemical reaction up to ten thousand, and the principal reaction of participation has the hydroxylation of alkyl, the epoxidation of alkyl, the oxidation of hydroxyl.Ammonia, oxygen, dealkylation on the sulphur position, hydroxylation and oxidation on the ammonia position, 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 that occurring in nature has the multifarious biological catalyst of catalysis most.Because character and the function and the effect in the vital movement process of this enzyme system.P450 enzyme system is subjected to numerous investigators' extreme and payes attention to.The importance of the effect of P450 enzyme system 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 ties up to the effect in the living things system and utilizes this enzyme system.And at home.Though a large amount of reports about this enzyme summary that is and the property studied are arranged.But research work is only limited to this enzyme ties up to effect in the living organism.The katalysis that has not yet to see 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 BM-3 be from bacillus megaterium, find have an active enzyme of lipid acid hydroxylation, now as the important model of Mammals microsome P450 monooxygenase.P450BM-3 is a kind of soluble monooxygenase, molecular weight 117, and 641Da needs extra albumen unlike other P450 enzyme catalysis, and P450BM-3 is at NADPH and O
2Existence under, it has the monooxygenase activity that the quick catalysis chain length is the saturated fatty acid of C12-C18.Abroad, the Universitaet Stuttgart technology Biochemical Research R.D.Schmid of the institute leader's of Germany scientific research group utilized rite-directed mutagenesis orthogenesis technology to obtain to have the P450BM-3 (Phe87Val in three mutational sites in 1999, Leu188Gln, Ala74Gly), they find that unexpectedly this mutant enzyme unexpectedly can catalyzing indole to generate indigo blue.Therefore, the P450BM-3 (Phe87Val that the present invention just obtains with the further orthogenesis of fallibility round pcr in the scientific research group that R.D.Schmid leads with three mutational sites, Leu188Gln, Ala74Gly) on the basis of variant gene, by the further orthogenesis P450BM-3 of fallibility round pcr (Phe87Val, Leu188Gln, Ala74Gly) variant gene, obtain one and be higher than P450BM-3 (Phe87Val, Leu188Gln, Ala74Gly) the active P450BM-3 variant enzyme of catalyzing indole P450BM-3 (Glu
435→ Asp), thus improve indigo output, make the bio-transformation synthesizing indigo more near practicability.
Indigo (indigo) is a kind of bright-colored and competent blue dyes, is one of natural dyestuff of finding the earliest, and the whole world is 800 to the total demand of dyestuff, and 000t has indigoly just occupied 80,000t.Ancient times mainly is to extract from contain indigo plant.The synthesizing indigo that West Germany BASF in 1897 produces comes out, and it is easy to produce, raw material is sufficient, purity is high, easy storing, advantage such as easy to use are caught up from behind, and popularizes rapidly, thereby makes that to have historical vegetable indigo in several thousand overshadowed.After 1960's, natural indigoly make no public appearances finally withdrawed from the arena of history.Enter after the eighties, along with the raising day by day of social modernization's degree, environment protection and labour protection consciousness is popularized gradually, and people have begun to recognize synthetic chemical industry a series ofly impairs one's health, the disadvantage of contaminate environment.Can cause the acute and chronic poisoning of human body as aniline and the Tetra hydro Phthalic anhydride that uses in the synthesizing indigo, respiratory tract and nervus centralis and liver are all had certain infringement.And the harmful waste of the indigo generation of bio-transformation synthesis of natural lacks than chemical process, and is not only energy-conservation but also cheap, and the conservation of nature environment is kept aspects such as the eubiosis, all is significant undoubtedly.Therefore Many researchers think the biological bipseudoindoxyl dye of development biological process production be have promising.
Summary of the invention
The purpose of this invention is to provide P450BM-3Glu435Asp variant gene of a kind of energy catalyzing indole to generate indigo blue and uses thereof.
It is by the random mutation of fallibility round pcr mediation, the P450BM-3 variant gene that screening obtains, the codon GAA of the 435th L-glutamic acid in this P450BM-3 gene sports the codon GAT of aspartic acid, resulting P450BM-3 variant gene, this variant gene sequence is a SEQ ID No.1 nucleotide sequence, and the monooxygenase of Codocyte cytochrome p 450 BM-3.
P450BM-3Glu435 Asp variant gene that can catalyzing indole to generate indigo blue, express resultant P450BM-3 misfolded proteins enzyme can catalyzing indole to generate dyestuff indigo.
The invention a kind of indoles is had the P450BM-3 variant gene of higher catalysis activity, its mutational site is Glu
435→ Asp, it is indigo that the mutant enzyme energy structure catalyzing indole that this variant gene is expressed generates dyestuff, the P450BM-3 (Phe87Val that obtains with R.D.Schmid leader's scientific research group with three mutational sites, Leu188Gln, Ala74Gly) variant enzyme is compared, activity has improved 1.5-1.7 doubly in an example of measuring, and wide application prospect is provided for bio-transformation production dyestuff is indigo.
Description of drawings
The gene mapping of Fig. 1 plasmid pET28 α (+) P450BM-3;
The qualitative evaluation of TLC that Fig. 2 is indigo, among the figure: the indigo 2-catalysis of 1-standard produces indigo 3-catalysis and produces Indirubin;
Fig. 3 mutational site Glu
435The position of → Asp in tertiary protein structure;
Fig. 4 rite-directed mutagenesis principle schematic.
Embodiment
By changing some component concentrations in the conventional P CR reaction system, or use low fidelity TaqDNA polysaccharase etc., make base random error introducing acquisition P450BM-3 mutator gene library to a certain extent, and obtain P450BM-3 variant gene, i.e. Glu by certain screening method
435→ Asp:, and be implemented in efficiently expressing in the intestinal bacteria, through separation and purification, createing the P450BM-3 variant enzyme that indoles is had greater catalytic, this P450BM-3 variant enzyme can the catalyzing indole to generate indigo blue dyestuff.The mutant enzyme that the present invention obtains is produced the Environmentally-sound technology that bipseudoindoxyl dye may become a kind of instead of chemical dyestuff.
Technology contents of the present invention comprises:
By changing some component concentrations in the conventional P CR reaction system, or use low fidelity TaqDNA polysaccharase etc., make base random error introducing acquisition mutator gene library to a certain extent, and obtain a kind of variant gene by certain screening method, promptly, the present invention is according to above-mentioned design philosophy, the concentration by reducing a kind of or two kinds of dATP dGTP dCTP dTTP among the conventional P CR and add a certain amount of MnCl
2Adopt the TaqDNA polysaccharase of low fidelity simultaneously, gene can be suddenlyd change when amplification in creation at random, then with the fallibility pcr amplification product through being inserted into behind the NheI-BamHI double digestion with the linear plasmid pET28 α (+) behind the same enzyme double digestion, be transformed among the E.ColiBL21 then, be applied to and contain on the 30 μ g/ml kantlex LB agar plates 37 ℃ of incubated overnight.The clone of picking blueness from the flat board, and be inoculated into 200 μ l and contain in 96 microwell plates of 30 μ g/ml kanamycin LB liquid nutrient mediums, 37 ℃ of incubated overnight, get this incubated overnight liquid of 20 μ l and join 200 new μ l and contain in 96 microwell plates of 30 μ g/ml kantlex LB liquid nutrient mediums, be cultured to OD at 37 ℃
6000.5-0.7 in time, adds that IPTG (final concentration 0.5 μ M) induces and continues to cultivate 48 hours at 30 ℃, obtain blue cell, extract this blue material with THF, measure its absorption value at the 630nm place, the cultivation of 100ml scale is picked out and carried out to the bacterial strain that absorption value is higher than the parent, centrifugal collection thalline, carrying out ultrasonic bacteria breaking, keep supernatant liquor, this supernatant liquor contains the P450BM-3 variant enzyme, by measure measuring its enzymatic reaction kinetics curve, the bacterial strain that the vigor of filtering out is higher than protoenzyme is picked out and is extracted plasmid and carries out full-automatic dna sequencing, and the discovery mutational site is Glu
435→ Asp.
Be embodiments of the invention below:
1. the rite-directed mutagenesis sudden change obtains the BamHI restriction enzyme site in gene order 1460-1475 site
A) acquisition of rite-directed mutagenesis primer design and pcr amplification product
The T that selects the A of the 1469th of P450BM3 gene to replace with C and 1472 replaces with A, and this replacement belongs to silent mutation, designs a pair of mutant primer B1 and B2, is template with pET28 α (+) P450BM-3, uses QuikChang
TMThe method of rite-directed mutagenesis amplification is introduced point mutation to the P450BM-3 gene, as shown in Figure 4.
The dNTPs that in the eppendorf pipe of 500 μ l, adds 25pmol, upstream primer, each 10pmol of downstream primer, approximately 1ng plasmid pET28 α (+) BM3 is as template DNA, 2.5u pfuDNA polysaccharase, 5 μ l's contains MgCl
2PCR damping fluid (25mM) adds aseptic distilled water then to cumulative volume 50 μ l.The PCR reaction parameter is by 95 ℃ of sex change after 1 minute, at 95 ℃ of 30s, and 55 ℃ of 1min, each circulating temperature rises 0.7 ℃, 72 ℃ of 16min, 14 circulations altogether, 72 ℃ are extended 30s then.
B) has the acquisition of the variant gene of BamHI restriction enzyme site
Above-mentioned pcr amplification product is transformed among the E.coli DH5 α and is applied to after 2 hours with 1 μ l DpnI (10u/ μ l) digestion and contains on the 30 μ g/ml kantlex LB agar plates 37 ℃ of incubated overnight.Approximately select 12 clones and be inoculated into containing in the 30 μ g/ml kantlex LB liquid nutrient mediums of 5ml, 37 ℃ of incubated overnight are with the amplification plasmid.Extract plasmid and use BamHI and EcoRI behind 37 ℃ of enzymolysis, whether introduce in the mutational site that utilizes DNA electrophoresis and full-automatic dna sequencing to detect expectation.
2. the acquisition of fallibility PCR primer design and pcr amplification product:
According to the coding region design of the monooxygenase of the cDNA of P450BM-3, later operation is for convenience introduced BamHI and NheI site respectively at 5 of primer ' end.
The dNTPs that in the eppendorf pipe of 500 μ l, adds 100pmol, upstream primer, each 20pmol of downstream primer, about 1ng pET28 α (+) P450BM-3 (pET28 α (+) P450BM-3 that is had the BamHI restriction enzyme site by the rite-directed mutagenesis sudden change in gene order 1460-1475 site) is as template DNA, and the ultimate density scope is 0 to 0.20mM MnCl
2, 2.5u TaqDNA polysaccharase, 5 μ l the PCR damping fluid, the MgCl of 12.5 μ l 25mM
2, add aseptic distilled water then to cumulative volume 50 μ l.Reaction parameter is by 95 ℃ of sex change after 3 minutes, at 95 ℃ of 1min, and 47 ℃ of 2min, 72 ℃ of 2min, through 25 circulations, 72 ℃ are extended 2min then.
The sudden change library structure:
The fallibility pcr amplification product among the Transformed E .ColiBL21, is applied to and contains on the 30 μ g/ml kantlex LB agar plates 37 ℃ of incubated overnight then through being inserted into behind the NheI-BamHI double digestion with the linear plasmid pET28 α (+) behind the same enzyme double digestion.
The amount ratio of plasmid pET28 α (+) P450BM-3 and restriction enzyme when enzyme is cut:
Plasmid pET28 α (+) P450BM-3 5 μ l
NheI(10u/μl) 1μl
BamHI(10u/μl) 1μl
Buffer(10X) 5μl
Sterilized water 8 μ l
Cumulative volume 20 μ l
The proportioning of ligase enzyme and plasmid when plasmid pET28 α (+)-P450BM-3 connects:
Plasmid DNA pET28 α (+) segment 2 μ l
Gene P450BM-3 segment 4 μ l
T4DNA ligase enzyme 1 μ l
T4DNA ligase enzyme Buffer (10X) 2 μ l
Sterilized water 11 μ l
Cumulative volume 20 μ l
A variant gene that indoles is had a high catalytic activity screening
The clone of picking blueness from the flat board, and be inoculated into 200 μ l and contain in 96 microwell plates of 30 μ g/ml kantlex LB liquid nutrient mediums, 37 ℃ of incubated overnight, get this incubated overnight liquid of 20 μ l and join 200 new μ l and contain in 96 microwell plates of 30 μ g/ml kantlex LB liquid nutrient mediums, be cultured to OD at 37 ℃
6000.5-0.7 in time, adds that IPTG (final concentration 0.5 μ M) induces and continues to cultivate 48 hours at 30 ℃, obtain blue cell, extract this blue material with THF, measure its absorption value at the 630nm place, the bacterial strain that absorption value is higher than the parent is picked out and is inoculated in 100 milliliters of LB liquid nutrient mediums: seed liquor is the test tube nutrient solution, inoculum size 1%-3%, the volume of substratum is 100 milliliters, pH7.0-7.5, shaking speed be 150-200 change minute, adding IPTG (0.5-1.0um) when being cultured to OD0.5-0.7 for 37 ℃ induces, then temperature being transferred to 30 ℃ continues to cultivate 5-8 hour, nutrient solution with 80000 change minute centrifugal 10-20 minute, remove supernatant liquor, gather in the crops the cell after centrifugal, the pH7.550mM that is suspended in 5 milliliters contains in the phosphate buffered saline buffer of 0.5MNaCl and trace P MSF, carrying out ultrasonic bacteria breaking is 10 minutes (ultrasonic 60 seconds under the condition of ice bath, gap 60 seconds), centrifugal 4 ℃ of 8000rpm is centrifugal 15 minutes again, obtain containing the supernatant liquor of P450BM-3 variant enzyme, and measure its enzyme in order to following method and live:
At the 1ml damping fluid is that to add the 10mM-100mM indoles in the phosphoric acid buffer reaction solution of pH8.2 be substrate, adds 1.2nmol-2.4nmol P450BM-3 variant enzyme, cultivates 9 minutes under the room temperature, adds 20 μ l NADPH 5mg/ml and starts reaction (ε=6.2mM
-1Cml), measure its enzymatic reaction kinetics curve, the bacterial strain that the vigor of filtering out is higher than protoenzyme is picked out and is extracted plasmid and carries out full-automatic dna sequencing, and the discovery mutational site is Glu
435→ Asp.Determine this mutational site near avtive spot with the protein simulation technique, guess that this sudden change makes it allow substrate more near catalytic center, this sudden change all occurs in the hole of avtive spot
By the mensuration of enzyme kinetics curve, the P450BM-3 with three mutational sites that obtains with R.D.Schmid leader's scientific research group (Ala74Gly) compare for Phe87Val, Leu188Gln, and activity has improved 1.5-1.7 doubly in an example of measuring by variant enzyme.
5. full cells produce dyestuff is indigo
A) picking contains this P450BM-3 variant gene Glu
435The positive colony of → Asp is inoculated in the LB liquid nutrient medium: seed liquor is the test tube nutrient solution, inoculum size 1%-2%, the volume of substratum is 100 milliliters, pH7.0-7.5, shaking speed be 150-200 change minute, when being cultured to OD0.5-0.7 for 37 ℃, add IPTG (0.5-1.0um) and induce, then temperature is transferred to 30 ℃ and continue to cultivate 48 hours.
B) with nutrient solution with 4000-80000 change minute centrifugal 10-20 minute, abandon supernatant liquor, gather in the crops the cell after centrifugal.Cell after centrifugal washes 2-3 time with an amount of water, add an amount of tetrahydrofuran (THF) (THF) and extract blue material, 1000-2000 change minute centrifugal reservation supernatant liquor, use the Rotary Evaporators drying, add dehydrated alcohol extraction for several times then in the good blueness precipitation of drying, to the last an extracting solution does not have till the redness, and remaining blue material is dissolved with THF again.
What c) qualitative analysis of TLC thin-layer chromatography produced is indigo
Chromatographic condition: precoating silica gel: the silica gel 60F25TLC plate that TLC plate West Germany E.MERCK company produces: 10cm * 10cm * 2cm, activate 1 hour solvent systems tetrahydrofuran (THF) at 120 ℃ before using: gasoline ether=1: 2, dehydrated alcohol are the analytical pure method of deploying: ascending method was launched preceding saturated 5 minutes
Exhibition distance: 7.5cm
Temperature: room temperature
Test method:
Draw an amount of extracting solution apart from 1.5cm place, TLC plate base point sample with capillary vessel, treat that putting into the chromatography cylinder that has developping agent immediately after the solvent evaporates launches respectively, takes out airing, and scans immediately, as shown in Figure 2 when treating to forward position 7.5cm.
The scanning of the single wavelength reflection of the condition of scanning condition of scanning: 228nm square tooth, slit 04mm * 04
The blue material that extracts from fermented liquid as seen from Figure 2 and commercial standard of selling be indigo identical R
FValue all is 0.78, so the blue material that our this variant gene of obtaining produces is indigo.
Sequence table
(1) SEQ ID NO.1. information
(a) sequence signature:
Length: 3150 bases
Type: nucleic acid
Chain: strand
Topological framework: linearity
(b) molecule type: cDNA
(c) initial source: bacillus megaterium
(d) sequence description: SEQ ID NO.1:
ATGACAATTAAAGAAATGCCTCAGCCAAAAACGTTTGGAGAGCTTAAAAATTTACCGTTA
TTAAACACAGATAAACCGGTTCAAGCTTTGATGAAAATTGCGGATGAATTAGGAGAAATC
TTTAAATTCGAGGCGCCTGGTCGTGTAACGCGCTACTTATCAAGTCAGCGTCTAATTAAA
GAAGCATGCGATGAATCACGCTTTGATAAAAACTTAAGTCAAGCGCTTAAATTTGTACGT
GATTTTGCAGGAGACGGGTTATTTACAAGCTGGACGCATGAAAAAAATTGGAAAAAAGCG
CATAATATCTTACTTCCAAGCTTCAGTCAGCAGGCAATGAAAGGCTATCATGCGATGATG
GTCGATATCGCCGTGCAGCTTGTTCAAAAGTGGGAGCGTCTAAATGCAGATGAGCATATT
GAAGTACCGGAAGACATGACACGTTTAACGCTTGATACAATTGGTCTTTGCGGCTTTAAC
TATCGCTTTAACAGCTTTTACCGAGATCAGCCTCATCCATTTATTACAAGTATGGTCCGT
GCACTGGATGAAGCAATGAACAAGCTGCAGCGAGCAAATCCAGACGACCCAGCTTATGAT
GAAAACAAGCGCCAGTTTCAAGAAGATATCAAGGTGATGAACGACCTAGTAGATAAAATT
ATTGCAGATCGCAAAGCAAGCGGTGAACAAAGCGATGATTTATTAACGCATATGCTAAAC
GGAAAAGATCCAGAAACGGGTGAGCCGCTTGATGACGAGAACATTCGCTATCAAATTATT
ACATTCTTAATTGCGGGACACGAAACAACAAGTGGTCTTTTATCATTTGCGCTGTATTTC
TTAGTGAAAAATCCACATGTATTACAAAAAGCAGCAGAAGAAGCAGCACGAGTTCTAGTA
GATCCTGTTCCAAGCTACAAACAAGTCAAACAGCTTAAATATGTCGGCATGGTCTTAAAC
GAAGCGCTGCGCTTATGGCCAACTGCTCCTGCGTTTTCCCTATATGCAAAAGAAGATACG
GTGCTTGGAGGAGAATATCCTTTAGAAAAAGGCGACGAACTAATGGTTCTGATTCCTCAG
CTTCACCGTGATAAAACAATTTGGGGAGACGATGTGGAAGAGTTCCGTCCAGAGCGTTTT
GAAAATCCAAGTGCGATTCCGCAGCATGCGTTTAAACCGTTTGGAAACGGTCAGCGTGCG
TGTATCGGTCAGCAGTTCGCTCTTCATGAAGCAACGCTGGTACTTGGTATGATGCTAAAA
CACTTTGACTTTGAAGATCATACAAACTACGAGCTGGATATTAAAGATACTTTAACGTTA
AAACCTGAAGGCTTTGTGGTAAAAGCAAAATCGAAAAAAATTCCGCTTGGCGGTATTCCT
TCACCTAGCACTGAACAGTCTGCTAAAAAAGTACGCAAAAAGGCAGAAAACGCTCATAAT
ACGCCGCTGCTTGTGCTATACGGTTCAAATATGGGAACAGCTGAAGGAACGGCGCGTGAT
TTAGCAGATATTGCAATGAGCAAAGGATTTGCACCGCAGGTCGCAACGCTTGATTCACAC
GCCGGAAATCTTCCGCGCGAAGGAGCTGTATTAATTGTAACGGCGTCTTATAACGGTCAT
CCGCCTGATAACGCAAAGCAATTTGTCGACTGGTTAGACCAAGCGTCTGCTGATGAAGTA
AAAGGCGTTCGCTACTCCGTATTTGGATGCGGCGATAAAAACTGGGCTACTACGTATCAA
AAAGTGCCTGCTTTTATCGATGAAACGCTTGCCGCTAAAGGGGCAGAAAACATCGCTGAC
CGCGGTGAAGCAGATGCAAGCGACGACTTTGAAGGCACATATGAAGAATGGCGTGAACAT
ATGTGGAGTGACGTAGCAGCCTACTTTAACCTCGACATTGAAAACAGTGAAGATAATAAA
TCTACTCTTTCACTTCAATTTGTCGACAGCGCCGCGGATATGCCGCTTGCGAAAATGCAC
GGTGCGTTTTCAACGAACGTCGTAGCAAGCAAAGAACTTCAACAGCCAGGCAGTGCACGA
AGCACGCGACATCTTGAAATTGAACTTCCAAAAGAAGCTTCTTATCAAGAAGGAGATCAT
TTAGGTGTTATTCCTCGCAACTATGAAGGAATAGTAAACCGTGTAACAGCAAGGTTCGGC
CTAGATGCATCACAGCAAATCCGTCTGGAAGCAGAAGAAGAAAAATTAGCTCATTTGCCA
CTCGCTAAAACAGTATCCGTAGAAGAGCTTCTGCAATACGTGGAGCTTCAAGATCCTGTT
ACGCGCACGCAGCTTCGCGCAATGGCTGCTAAAACGGTCTGCCCGCCGCATAAAGTAGAG
CTTGAAGCCTTGCTTGAAAAGCAAGCCTACAAAGAACAAGTGCTGGCAAAACGTTTAACA
ATGCTTGAACTGCTTGAAAAATACCCGGCGTGTGAAATGAAATTCAGCGAATTTATCGCC
CTTCTGCCAAGCATACGCCCGCGCTATTACTCGATTTCTTCATCACCTCGTGTCGATGAA
AAACAAGCAAGCATCACGGTCAGCGTTGTCTCAGGAGAAGCGTGGAGCGGATATGGAGAA
TATAAAGGAATTGCGTCGAACTATCTTGCCGAGCTGCAAGAAGGAGATACGATTACGTGC
TTTATTTCCACACCGCAGTCAGAATTTACGCTGCCAAAAGACCCTGAAACGCCGCTTATC
ATGGTCGGACCGGGAACAGGCGTCGCGCCGTTTAGAGGCTTTGTGCAGGCGCGCAAACAG
CTAAAAGAACAAGGACAGTCACTTGGAGAAGCACATTTATACTTCGGCTGCCGTTCACCT
CATGAAGACTATCTGTATCAAGAAGAGCTTGAAAACGCCCAAAGCGAAGGCATCATTACG
CTTCATACCGCTTTTTCTCGCATGCCAAATCAGCCGAAAACATACGTTCAGCACGTAATG
GAACAAGACGGCAAGAAATTGATTGAACTTCTTGATCAAGGAGCGCACTTCTATATTTGC
GGAGACGGAAGCCAAATGGCACCTGCCGTTGAAGCAACGCTTATGAAAAGCTATGCTGAC
GTTCACCAAGTGAGTGAAGCAGACGCTCGCTTATGGCTGCAGCAGCTAGAAGAAAAAGGC
CGATACGCAAAAGACGTGTGGGCTGGGTAA
Claims (2)
1. the P450BM-3Glu435 Asp variant gene of an energy catalyzing indole to generate indigo blue, it is characterized in that: by the random mutation of fallibility round pcr mediation, the P450BM-3Glu435 Asp variant gene that screening obtains, the codon GAA of the 435th L-glutamic acid in this P450BM-3Glu435 Asp variant gene sports the codon GAT of aspartic acid, resulting P450BM-3Glu435 Asp variant gene, the nucleotides sequence of this variant gene is classified SEQ ID No.1 as, and the monooxygenase of Codocyte cytochrome p 450 BM-3Glu435 Asp.
One kind according to claim 1 can catalyzing indole to generate indigo blue the purposes of P450BM-3Glu435 Asp variant gene, it is characterized in that, express resultant P450BM-3Glu435 Asp misfolded proteins enzyme can catalyzing indole to generate dyestuff indigo.
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| CN100413968C (en) * | 2006-07-21 | 2008-08-27 | 浙江大学 | P450BM-3Glue435Thr variant gene capable of catalyzing indole to generate indigo blue and its use |
| CN101333521B (en) * | 2008-04-25 | 2010-12-15 | 浙江大学 | Variant enzyme of cytochrome P450BM-3D168S and method for preparing indirubin using the same |
| EP3097113B1 (en) * | 2014-01-20 | 2019-01-02 | Sanofi | Novel cytochrome p450 polypeptide with increased enzymatic activity |
| CN109423482A (en) * | 2017-08-23 | 2019-03-05 | 沈阳药科大学 | It is catalyzed the hydroxylated Cytochrome P450 BM3 mutant enzyme of cyperenoic acid |
| CN115109758B (en) * | 2021-03-17 | 2024-05-07 | 中国科学院青岛生物能源与过程研究所 | P450 mutant enzyme for catalyzing indigo biosynthesis |
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