CN101942497A - Method for quickly screening oxygenase microbes or oxygenase genes - Google Patents
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Abstract
The invention belongs to the technical field of microbes, and particularly relates to a method for quickly screening oxygenase microbes or oxygenase genes. The method for quickly screening oxygenase microbes comprises the following steps: using the only carbon source enriched environmental sample, coating the enriched environmental sample on the culture medium, culturing, separating and purifying to obtain the microbe strain of the target oxygenase. The method for quickly screening oxygenase genes comprises the following steps: constructing a metagenomic library of environmental samples, and screening the oxygenase gene from the metagenomic library. The invention has the advantage of high screening sensitivity, and can be used for conveniently and quickly screening oxygenase resources in the environmental sample in a high flux mode.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to the method for a kind of rapid screening oxygenase microorganism or oxygenase gene.
Background technology
Biocatalysis is with chemistry, zone, stereoselectivity and the reaction conditions gentleness of its height, and aftertreatment is easy, and energy consumption is low etc., and advantage becomes the trend of current Chemical Manufacture and correlative technology field, and has been successfully applied to many fields.Biological enzyme is the core of biocatalysis technology, and the acquisition of new and effective katalaze enzyme becomes the technical bottleneck in this field.At present, the development and use of oxygenase are also very limited, and wherein one of important reasons is exactly that the screening method of traditional oxygenase exists many limitations, make to be difficult to screen oxygenase bacterial strain and gene efficient, highly selective.
The oxygenase that the present invention mentions comprises cyclooxygenase and aromatic ring dioxygenase, and as naphthaline dioxygenase etc., its screening method mainly contains three classes at present.One class is by utilizing sole carbon source screening bacterial strain.Because environmental sample can utilize the microbial population of same carbon source very abundant, this method tends to obtain a large amount of microorganisms in screening process, the oxygenase bacterial strain is very difficult therefrom to screen and target is produced, particularly when handling the particular surroundings sample, as the abundant active sludge of oxygenase, very easily be subjected to the interference of other functional microorganisms, utilize the type bacterium as agar, false positive rate is very high.One class is directly to detect the catalysate of institute's sieve mesh mark oxygenase with analysis means such as thin plate chromatography, high-pressure liquid phase or gas phases, in the screening of epoxidation of styrene enzyme, detect (for example existing of enzyme by the generation of measuring product vinylbenzene epoxy, Park et al. (2005) Korean J.Chem.Eng., 22 (3), 418～424).This method flux is low, and length consuming time be the more important thing is, owing to adopt natural substrate, its oxidation products tends to be difficult to capture the catalysate of intermediary target oxygenase by the metabolism continuously rapidly of a plurality of enzymes in downstream in microbe, causes loss high.The 3rd class is the existence that comes the indirect proof enzyme according to other substrates that easily detect of oxygenase oxidation, as oxygenase (cyclooxygenase, P450 monooxygenases etc.) available 1-hydrogen indoles is a substrate in the screening, by detect color generate know the existing of oxygenase (as, Hellemond et al. (2007) Appl.Environ.Microbiol., 73 (18), 5832～5839).This method has been applied to the screening of recombinase, does not then appear in the newspapers as yet at environmental samples screening natural source microorganism.This method is comparatively intuitively easy, but with 1-hydrogen indoles is substrate, developing time is long, nearly more than 4 days, and product is indigo and mixture Indirubin, and what cause forming is blue shallow, the product molar absorptivity is lower, with be yellow-green colour after the yellow of coli somatic is mixed, not easy to identify, very easily cause the leakage sieve of the useful resource of oxygenase.There is bibliographical information 4-substituted indole steric hindrance reason owing to 4 behind oxidative coupling can't generate 4,4 '-two replace Indirubin, can only generate single product 4,4 '-two replacements are indigo, these characteristics demonstrate the application potential of 4-substituted indole aspect color reaction and are better than 1-hydrogen indoles (Polychronopoulos et al. (2004) J.Med.Chem., 47,935～946; Wu etal. (2005) Chem.Biodivers., 2,51～65).In the orthogenesis research of recombinase Cytochrome P450 2A6, reported and utilized the 4-chloro-indole to carry out the muton method for screening, but up to now, also do not appeared in the newspapers as yet at environmental sample screening natural source microorganism.
Summary of the invention
The object of the invention provide a kind of from environmental sample the method for rapid screening oxygenase, promptly in screening culture medium, add the 4-substituted indole, but blue material 4 through oxygenase oxidation generation direct viewing, 4 '-two replace indigo, thereby easy, quick, sensitive filters out the purpose oxygenase from environmental sample.
Concrete scheme of the present invention is:
1. the oxygenase bacterial strain is produced in screening in the environmental sample
Produce the oxygenase bacterial strain screening may further comprise the steps into:
1. enrichment environment sample: the cyclooxygenase enrichment is adopted vinylbenzene or tetrahydrobenzene or is sole carbon source to bromstyrol or vinyl toluene; It is sole carbon source that naphthalene or anthracene are selected in the naphthaline dioxygenase enrichment for use; And the interpolation inorganic salt, 30 ℃ of concussions were cultivated the enrichment environment sample 5～15 days.
2. the yeast powder with inorganic salt, sole carbon source, 4-substituted indole and 0.01%～0.1% prepares the agar solid medium, the environmental sample of enrichment is coated on the substratum, grew 1～5 day, blue colonies appears, the substratum of blue colonies with same composition separated, purifying, the microorganism strains of target oxygenase is produced in acquisition.
Inorganic salt can be M9 inorganic salt (molecular cloning experiment guide, Chinese edition, the third edition commonly used, volume two, 1595～1596) or the M12 inorganic salt of bibliographical information (Hartmans et al. (1989) Appl.Environ.Microbiol., 55 (11), 2850～2855) page number:.
The concentration of sole carbon source is 0.05～8mM.
4-substituted indole concentration is 0.05～1mM, and the 4-substituted indole is 4-skatole or 4-indolecarboxylic acid methyl esters or 4-chloro-indole or 4-bromo indole or 4-fluoro indole or 4-methoxyl group indoles.
2. screening the oxygenase gene in first genomic library that environmental sample makes up may further comprise the steps:
1. first genomic library of constructing environment sample: from environmental sample, extract total DNA with the CTAB method, and purifying, total DNA enzyme that purifying is good is cut, and behind 0.8% agarose gel electrophoresis, reclaims the fragment between 1～10Kb; Vector plasmid is cut with the same enzyme enzyme, and spends Starch phosphorylase (CIP) processing.The dna fragmentation that recovery is obtained between 1～10Kb is connected into carrier, connects product and handles through alcohol precipitation, uses deionized water dissolving, electric transformed into escherichia coli competent cell, constructing environment sample unit genomic library.
2. from first genomic library, screen the oxygenase gene:
Change first genomic library plasmid over to host e. coli, coat on the solid screening flat board.Nutrient media components is: add 4-substituted indole, IPTG 0.2～1mM and the corresponding microbiotic of carrier in the LB solid medium.Transformant was grown 1～2 day on the flat board or was placed 1～5 day in 4 ℃ again in screening, blue reorganization bacterium colony can occur, chose, and obtained containing the recon of oxygenase gene.
The environmental sample genomic library can be the library that the environmental sample genome DNA directly makes up, and also can be the target oxygenase library that makes up behind example enrichment.
The carrier of selecting for use in the library construction is pBluescript SK or pBluescript KS or pUC18 or pUC19 or pKK233 or pKK223 or pET-28 or pET-32 carrier; Host e. coli is DH1 or DH5 α or DH10B or JM109 or MC1061 or BL21 (DE3) or XL-Blue in the library screening.
The present invention utilizes many oxygenases can be with the oxidation of 4-substituted indole, generates single mazarine material 4,4 '-two and replaces indigoly, and its product is single, and therefore the molar absorptivity height has improved screening sensitivity greatly.Can high-throughput, easy, fast, the oxygenase resource that exists in the sensitive screening environmental sample.Present method can be used for screening cyclooxygenase and aromatic ring dioxygenase, as multiple oxygenases such as naphthaline dioxygenases.
Description of drawings
Fig. 1 is dull and stereotyped for screening;
Fig. 2 is the purifying flat board;
Fig. 3 is screening bacterial strain evolutionary tree;
Fig. 4 is screening bacterial strain cyclooxygenase evolutionary tree;
Fig. 5 is the extract photo behind the whole cell transformation 4-substituted indole, and wherein (1) is the 4-bromo indole, and (2) are the 4-fluoro indole, and (3) are 4-indolecarboxylic acid methyl esters, and (4) are 4-methoxyl group indoles, and (5) are the 4-skatole, and (6) are the 4-chloro-indole.
Embodiment
Embodiment 1 is that the cyclooxygenase bacterial strain is produced in the substrate screening with the 4-chloro-indole
Get the active sludge sample in certain sewage work, be enriched in the substratum that contains M12 inorganic salt and vinylbenzene (concentration is 2mM), 30 ℃, 225rpm cultivated 10 days.Enrichment culture liquid is diluted to dull and stereotyped go up (the M12+ vinylbenzene 2mM+4-chloro-indole 0.1mM+0.01% yeast powder+1.5% agar powder) of screening to be cultivated 3 days in 30 ℃, visible a plurality of blue bacterial plaques in blocks (Fig. 1) on every flat board, choose, continue purifying (Fig. 2), obtain the pure bacterial strain of 11 strains.
Sample and enriching method are with embodiment 1, enrichment culture liquid is diluted to dull and stereotyped go up (the M9+ vinylbenzene 2mM+4-skatole 0.2mM+0.05% yeast powder+1.5% agar powder) of screening to be cultivated 3 days in 30 ℃, blue bacterial plaque occurs, continue purifying, obtain the pure bacterial strain of 15 strains.
It is that the cyclooxygenase bacterial strain is produced in the substrate screening that embodiment 3 draws the diindyl methyl-formiate with 4-
Sample and enriching method are with embodiment 1, enrichment culture liquid is diluted to screening dull and stereotyped upward (M9+ vinylbenzene 3mM+4-draws diindyl methyl-formiate 0.3mM+0.06% yeast powder+1.5% agar powder) to be cultivated 4 days in 30 ℃, blue bacterial plaque occurs, continue purifying, obtain the pure bacterial strain of 12 strains.
Embodiment 4 is that the cyclooxygenase bacterial strain is produced in the substrate screening with the 4-bromo indole
Take from another sewage work's sample, enriching method is diluted to dull and stereotyped go up (the M9+ vinylbenzene 3mM+4-bromo indole 0.2mM+0.05% yeast powder+1.5% agar powder) of screening in 30 ℃ of cultivations 4 days with embodiment 1 with enrichment culture liquid, the mazarine bacterial plaque occurs, continue purifying, obtain the pure bacterial strain of 10 strains.
Embodiment 5 screening bacterial strain utilization of carbon source situations
The bacterial strain of embodiment 1,2,3,4 screenings is that sole carbon source is cultivated with vinylbenzene and vinylbenzene epoxy respectively, is respectively M12+ vinylbenzene 5mM; M12+ vinylbenzene epoxy 3mM.Behind the 24h, all bacterial strains all can be grown in two kinds of substratum, and promptly screening bacterial strain can be sole carbon source with vinylbenzene and vinylbenzene epoxy, has vinylbenzene to this pathways metabolism of vinylbenzene epoxy, therefore can produce the required cyclooxygenase of this approach.
The 16S rRNA that embodiment 6 produces the cyclooxygenase bacterial strain identifies
The bacterial strain of embodiment 1,2,3,4 screenings is to utilize vinylbenzene, vinylbenzene epoxy to grow for sole carbon source, and makes the 4-substituted indole become blue bacterial strain.For verifying that these bacterial strains are the purpose bacterial strain, it is carried out 16S rRNA identify.With bacterium universal primer amplification 16S rRNA sequence.Primer is F:5 '-AGAGTTTGATCCTCGCTCAG, and R:5 '-GGTCTACCTTGTTACGACTT obtains the amplified fragments size and is 1498bp.Through comparison, its gene order has the dozens of base difference.Wherein, embodiment 1 has three kinds of different bacterial strains of 16S rRNA sequence, embodiment 2,3 obtained strains and embodiment 1 identical (embodiment 1 and 2,3 is from same active sludge sample), and embodiment 4 then has two kinds of different bacterial strains of 16S rRNA sequence.Through compare of analysis, the bacterial strain that sieves all be Rhodopseudomonas.WLS4-1, the WLS4-9 of the three strain bacterium WLS1-1 that the 16S rRNA sequence of selecting embodiment 1,2,3 to screen is different, WLS1-5, WLS1-8 and embodiment 4 screenings do evolutionary tree analysis.Fig. 3 is screening bacterial strain evolutionary tree, wherein Pseudomonas sp.VLB 120 is the pseudomonas of having reported with cyclooxygenase function, the 16S rRNA gene order similarity reported of the bacterial strain that sieves and VLB 120 bacterial strains all＞99%, prove the bacterial strain that adopts the present invention's screening pseudomonas for the product cyclooxygenase.
The segmental checking of embodiment 7 cyclooxygenase gene expression characteristicses
Identify that through embodiment 6 the screening bacterial strain is a pseudomonas.For confirming that further these bacterial strains contain the cyclooxygenase gene order, with above-mentioned 5 strains screening bacterium amplification cyclooxygenase gene styA Partial Feature fragment.According to the pseudomonas cyclooxygenase sequence of reporting in the NCBI gene pool, design primer, F:5 '-ATGAAAAAGCGTATCGGTATTG; R:5 '-TTCTCGAAGGGCGAGTTTTC, pcr amplification obtain the 488bp fragment, called after styAsp.The similarity analysis of the cyclooxygenase that Fig. 4 originates for screening strain characteristics fragment styAsp-1 and styAsp-2 and the pseudomonas of having reported, its respective segments similarity is 97%～100%.Therefore, adopt the bacterial strain of the present invention's screening for producing the pseudomonad strain of cyclooxygenase (vinylbenzene monooxygenase).
Embodiment 8 is the bacterial strain that naphthaline dioxygenase is produced in the substrate screening with 4-methyl oxindoles
Sample source is enriched in the substratum that contains M12 inorganic salt and naphthalene (concentration is 7mM) with embodiment 1, and 30 ℃, 225rpm cultivated 12 days.Pregnant solution is diluted to dull and stereotyped go up (the M12+ naphthalene 7mM+ methoxyl group indoles 0.6mM+0.06% yeast powder+1.5% agar powder) of screening to be cultivated 7 days in 30 ℃, have blue bacterial plaque to occur on the flat board, separation, purifying obtain the pure bacterial strain of 23 strains (being numbered NPCL-1 to NPCL-23).
Embodiment 9 is the bacterial strain that naphthaline dioxygenase is produced in the substrate screening with the 4-fluoro indole
Sample source is with embodiment 4, and cultural method is with embodiment 8, and the screening substrate is 4-fluoro indole (concentration is 0.3mM), and blue bacterial plaque separation and purification is obtained 15 strain bacterial strains (being numbered NPF-1 to NPF-15).
Embodiment 10 screening bacterial strains are the sole carbon source growing state with the naphthalene
The bacterial strain of embodiment 8,9 screening is that sole carbon source is cultivated with the naphthalene, i.e. M12+ naphthalene 5mM, and the result shows that all bacterial strains all can be the sole carbon source growth with the naphthalene, can produce naphthaline dioxygenase.
The 16S rRNA that embodiment 11 produces the naphthaline dioxygenase bacterial strain identifies
The bacterial strain that obtains for checking embodiment 8 and 9 is the bacterial strain that produces naphthaline dioxygenase, and 4 strains that optional wherein label is NPCL-2, NPCL-5, NPF-7, NPF-11 are carried out 16S rRNA and identified.Wherein NPCL-5 is accredited as Pseudomonas aeruginosa genus bacterium, and NPCL-2, NPF-7, NPF-11 are Burkholder Pseudomonas bacterium.This two classes bacterium all has the report that produces naphthaline dioxygenase, proves that further the bacterial strain that adopts the inventive method to screen is the bacterial strain that produces naphthaline dioxygenase.
The segmental checking of embodiment 12 naphthaline dioxygenase gene expression characteristicses
Naphthaline dioxygenase gene to embodiment 11 described 4 strain bacterium is further verified, the PAH gene increases respectively, this gene is ring hydroxylation dioxygenase (ring-hydroxylating-dioxygenase, RHD) portion gene of α subunit of gram negative bacterium degraded PAHs compounds.
Degenerated primer, F:5 '-GAGATGCATACCACGTKGGTTGGA are adopted in PAH gene amplification; R:5 '-AGCTGTTGTTCGGGAAGAYWGTGCMGTT, expanding fragment length are 306bp.
The checking of table 1 screening bacterial strain PAH gene fragment
The conserved sequence of nahAc gene (pseudomonas naphthaline dioxygenase gene) among the NPCL-5 of further amplification Rhodopseudomonas.Degenerated primer, F:5 '-CCCYGGCGACTATGTTACC are adopted in NahAc gene amplification; R:5 '-CTCRGGCATGTCTTTTTCGAC.
Comparison after checking order, the fragment length that the NPCL-5 amplification obtains is 866bp, with the gene (AF491307) identical (100%) on the naphthalene dissimilation plasmid pDTG1 of strain Pseudomonas putida NCIB 9816-4 of report, and this bacterium is the naphthalene degradation bacteria of studying the earliest, the naphthaline dioxygenase of its generation (NDO) has substrate selective widely, can three-dimensionally select catalysis cis dihydroxylation reaction, single oxygenation, desaturation, O-and N-to take off alkyl effect, sulfoxidation, kind surplus the aromatic hydrocarbons substrate that can utilize reaches 60.
Therefore, the above-mentioned 4 strain bacterium of being identified contain the naphthaline dioxygenase gene, and the screening method that the present invention relates to can obtain the naphthaline dioxygenase microorganism strains.
The structure of embodiment 13 environmental samples unit genomic library
1, the extraction of the total DNA of active sludge
The CTAB method is adopted in the extraction of active sludge, referring to document Zhou et al. (1996) Appl.Environ.Microbiol., 62 (2), 316～322.
Concrete steps are: with damping fluid (100mM Tris-HCl, pH8.0; 100mM EDTA-Na
2100mM phosphoric acid salt, pH8.0; 1.5M NaCl; 1%CTAB, m/v), Proteinase K and active sludge sample place centrifuge tube, and half an hour is cultivated in 37 ℃ of concussions, adds 20%SDS behind 65 ℃ of processing 2h, and low-speed centrifugal is got supernatant; Once more with damping fluid in precipitation, add 20%SDS in 65 ℃ handle 15min after, low-speed centrifugal is collected supernatant; Add equal-volume phenol-chloroform mixing in the supernatant liquor of collecting, low-speed centrifugal is collected supernatant (if sample impurity is more, can heavily wash once resetting and adding the equal-volume chloroform on this), then adds the Virahol of 0.6～1 times of volume, standing over night under the greenhouse; Careful supernatant discarded adds 200～500uL ddH
2O, dissolving adheres to the DNA crude extract of centrifugal tube wall, obtains total DNA ,-20 ℃ of preservations.
2, the purifying of the total DNA of active sludge
The total DNA that extracts from active sludge contains material such as the humic acid of a lot of complicated components etc., can have a strong impact on follow-up molecule manipulation, enzyme cut be connected before, need carry out purifying.
Total DNA is through containing 5% hydroxylapatite (Fluka, Buchs, Switzerland) (Roh et al. (2005) behind the agarose gel electrophoresis purifying, " In-gelpatch electrophoresis ": A new method for environmental DNA purification.ELECTROPHORESIS, 26 (16), 3055～3061), cut glue reclaim purer total DNA use again TaKaRa RECOCHIP-Chip for DNA Recovery[TaKaRaBiotechnology (Dalian, China) Co.Ltd.] dna gel reclaims test kit and reclaims purifying.
3, the structure in environment DNA library
Total DNA that purifying is good cuts through the BamHI enzyme, behind 0.8% agarose gel electrophoresis, with the fragment between TaKaRa RECOCHIP recovery 1～10Kb.The pUC19 plasmid spends Starch phosphorylase (CIP) and handles after the BamHI enzyme is cut, prevent connecting certainly in follow-up connection.The dna fragmentation that recovery is obtained between 1～10Kb is connected into the pUC19 carrier, connects product and handles through alcohol precipitation, uses deionized water dissolving, electric transformed competence colibacillus cell DH10B (Bio-Rad MicroPulser electroporation apparatus, U.S. Bio-Rad).Obtain storage capacity about 10
4Active sludge unit genomic library.
Embodiment 14 screens the oxygenase gene from first genomic library
Screening cyclooxygenase gene from the constructed library of embodiment 13.Concrete grammar is: to DH5 α, to contain the LB plate screening of 4-chloro-indole, the LB flat board contains 4-chloro-indole 0.2mM with library plasmid chemical conversion, IPTG 0.4mM, penbritin 50 μ g/L.Transformant is directly coated on the above-mentioned flat board, cultivated after 1 day for 37 ℃, observe after 2 days, obtain 1 mazarine recon in 4 ℃ of placements.It is chosen, and subculture is on fresh above-mentioned flat board, and next day, the visible growth bacterium colony was blue entirely.With recon sample presentation order-checking, it inserts clip size is 4380bp, through the NCBI comparison, with the corresponding gene cluster sequence similarity of pseudomonas VLB120 (AF031161.1) epoxidation of styrene enzyme degree be 98%.Therefore, screening method of the present invention can sift out the oxygenase gene.
Embodiment 15 produces the whole cell transformation detection ring oxydase of cyclooxygenase bacterial strain
But bibliographical information transforms the indigo detection ring oxydase vigor that generates with 1-hydrogen indoles, and therefore the product cyclooxygenase bacterial strain that embodiment 1 is screened detects enzyme activity with whole cell transformation 4-substituted indole.
The strain culturing condition is: M12+0.05% yeast powder+0.1% glucose+2mM vinylbenzene, to cultivate after 10 hours, and thalli growth is good, takes out, and 4 ℃ of frozen centrifugations are collected thalline, and wash thalline 2 times with 100mM potassium phosphate buffer (pH7.0).Get thalline 0.2g and join in the 3ml damping fluid that contains 1% glucose, add 4-substituted indole 0.4mM, 240rpm, 37 ℃ were reacted 20 minutes, and reaction solution becomes mazarine.Therefore, the screening bacterial strain has higher cyclooxygenase vigor.
Fig. 5 is the resultant extract figure of the different substrates of the full cell transformation of 6 strain different strains.(1)～(6) representative strain is numbered, and the reaction substrate of left sample is a 1-hydrogen indoles, and the right is the 4-substituted indole, be respectively: (1) 4-bromo indole, (2) 4-fluoro indole, (3) 4-indolecarboxylic acid methyl esters, (4) 4-methoxyl group indoles, (5) 4-skatole, (6) 4-chloro-indole; Wherein (6) have showed two parallel sample.As seen from the figure, it is bigger that different strains transforms 1-hydrogen indoles color distortion, illustrates that different strains cyclooxygenase vigor differs greatly; Same bacterial strain transforms 1-hydrogen indoles and 4-substituted indole, and the former converted product color is more shallow, and 4-substituted indole converted product color is darker, illustrates that 4-substituted indole converted product molar absorptivity is higher, and detection sensitivity is higher.
SEQUENCE?LISTING
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Claims (6)
1. rapid screening oxygenase method of microorganism is characterized in that comprising following steps:
1. enrichment environment sample: the cyclooxygenase enrichment is adopted vinylbenzene or tetrahydrobenzene or is sole carbon source to bromstyrol or vinyl toluene; It is sole carbon source that naphthalene or anthracene are selected in the naphthaline dioxygenase enrichment for use; And the interpolation inorganic salt, 30 ℃ of concussions were cultivated the enrichment environment sample 5～15 days;
2. the yeast powder with inorganic salt, sole carbon source, 4-substituted indole and 0.01%-0.1% prepares the agar solid medium, the enrichment environment sample is coated on the substratum, grew 1-5 days, blue colonies appears, the substratum of blue colonies with same composition separated, purifying, the microorganism strains of target oxygenase is produced in acquisition.
2. the method for a rapid screening oxygenase gene is characterized in that comprising following steps:
1. first genomic library of constructing environment sample: from environmental sample, extract total DNA with the CTAB method, and purifying, total DNA enzyme that purifying is good is cut, and behind 0.8% agarose gel electrophoresis, reclaims the fragment between 1～10Kb; Vector plasmid is cut with the same enzyme enzyme, and spends Starch phosphorylase (CIP) processing; The dna fragmentation that recovery is obtained between 1～10Kb is connected into carrier, connects product and handles through alcohol precipitation, uses deionized water dissolving, electric transformed into escherichia coli competent cell, constructing environment sample unit genomic library;
2. from first genomic library, screen the oxygenase gene:
Change first genomic library plasmid over to intestinal bacteria, coat on the solid screening flat board; Nutrient media components is: add 4-substituted indole, IPTG 0.2-1mM and the corresponding microbiotic of carrier in the LB solid medium; Blue reorganization bacterium colony appears in transformant growth 1-2 days or placed again 1-5 days in 4 ℃ on the screening flat board, chooses this blueness reorganization bacterium colony, obtains containing the recon of oxygenase gene.
3. the described rapid screening oxygenase of claim 1 method of microorganism is characterized in that: M9 inorganic salt or the M12 inorganic salt of inorganic salt for using always; The concentration of sole carbon source is 0.05-8mM.
4. the described rapid screening oxygenase of claim 1 method of microorganism, it is characterized in that: 4-substituted indole concentration is 0.05-1mM, and the 4-substituted indole is 4-skatole or 4-indolecarboxylic acid methyl esters or 4-chloro-indole or 4-bromo indole or 4-fluoro indole or 4-methoxyl group indoles.
5. the method for the described rapid screening oxygenase of claim 2 gene, it is characterized in that: 4-substituted indole concentration is 0.05-1mM, and the 4-substituted indole is 4-skatole or 4-indolecarboxylic acid methyl esters or 4-chloro-indole or 4-bromo indole or 4-fluoro indole or 4-methoxyl group indoles.
6. the method for the described rapid screening oxygenase of claim 2 gene is characterized in that: the environmental sample genomic library is library or the target oxygenase library for making up behind example enrichment that the environmental sample genome DNA directly makes up; The carrier of selecting for use in the library construction is pBluescript SK or pBluescript KS or pUC18 or pUC19 or pKK233 or pKK223 or pET-28 or pET-32 carrier, and host e. coli is DH1 or DH5 α or DH10B or JM109 or MC1061 or BL21 (DE3) or XL-Blue.
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| CN104621106A (en) * | 2015-01-28 | 2015-05-20 | 云南省烟草公司玉溪市公司 | Application of 4-methoxy-indole solution in control of tobacco black shank, and preparation method of 4-methoxy-indole solution |
| CN110592110A (en) * | 2019-07-17 | 2019-12-20 | 中国科学院成都生物研究所 | (R) -selective styrene monooxygenase from streptomycete |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104621106A (en) * | 2015-01-28 | 2015-05-20 | 云南省烟草公司玉溪市公司 | Application of 4-methoxy-indole solution in control of tobacco black shank, and preparation method of 4-methoxy-indole solution |
| CN104621106B (en) * | 2015-01-28 | 2017-07-14 | 云南省烟草公司玉溪市公司 | Application and preparation method of the 4 methoxy-Indole solution in preventing and treating tobacco black shank |
| CN110592110A (en) * | 2019-07-17 | 2019-12-20 | 中国科学院成都生物研究所 | (R) -selective styrene monooxygenase from streptomycete |
| CN110592110B (en) * | 2019-07-17 | 2022-09-06 | 中国科学院成都生物研究所 | (R) -selective styrene monooxygenase from streptomycete |
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