CN102660484A - Rhodobacter azotoformans and culture method and application thereof - Google Patents

Rhodobacter azotoformans and culture method and application thereof Download PDF

Info

Publication number
CN102660484A
CN102660484A CN2012101567570A CN201210156757A CN102660484A CN 102660484 A CN102660484 A CN 102660484A CN 2012101567570 A CN2012101567570 A CN 2012101567570A CN 201210156757 A CN201210156757 A CN 201210156757A CN 102660484 A CN102660484 A CN 102660484A
Authority
CN
China
Prior art keywords
fixed nitrogen
rhodobacter azotoformans
neurosporene
red bacterium
dehydrogenation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN2012101567570A
Other languages
Chinese (zh)
Other versions
CN102660484B (en
Inventor
肖敏
张金华
卢丽丽
钱新民
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shandong University
Original Assignee
Shandong University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shandong University filed Critical Shandong University
Priority to CN201210156757.0A priority Critical patent/CN102660484B/en
Publication of CN102660484A publication Critical patent/CN102660484A/en
Application granted granted Critical
Publication of CN102660484B publication Critical patent/CN102660484B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Abstract

The invention relates to Rhodobacter azotoformans Y6 and a culture method and application thereof. The strain has the function of producing carotenoid C-3,4 dehydrogenase which can catalyze neurosporene and lycopene for C-3,4 dehydrogenation reaction to obtain 3,4-didehydrogenated neurosporene and 3,4-didehydrogenated lycopene. In addition, the invention also provides the strain and the characteristics of the Rhodobacter azotoformans, the culture method for the Rhodobacter azotoformans, and a catalytic property of the carotenoid C-3,4 dehydrogenase of the Rhodobacter azotoformans. Recombinant enzyme which is subjected to recombinant expression by a crtD gene derived from the stain in Escherichia coli can catalyze the neurosporene or the lycopene to be dehydrogenated to form the 3,4-didehydrogenated neurosporene or the 3,4-didehydrogenated lycopene respectively, so that a novel enzyme source is provided for the microbial fermentation production of the 3,4-didehydrogenated neurosporene and the 3,4-didehydrogenated lycopene.

Description

The red bacterium of one strain fixed nitrogen and cultural method and application
Technical field
The present invention relates to the carrotenoid C-3 that a strain produces catalysis neurosporene and Lyeopene dehydrogenation, the red bacterium of the fixed nitrogen of 4 desaturases (Rhodobacter azotoformans) Y6 and cultural method and application belong to microbial technology field.
Background technology
Carrotenoid is a kind of multiclass isoprenoid, extensively is present in mikrobe, plant, the animal body.Carrotenoid comprises special long-chain polyenoid conjugated structure, makes it have special physiologically active, comprises photoabsorption, anti-oxidant, cancellation radical etc.Plant extract is mainly leaned in carrotenoid production at present, and output is limited, can not satisfy the demands, and microbial fermentation production becomes the research focus.Can produce different types of carrotenoid through in engineering strain, expressing different carotenoid synthases; Seek the new microbe-derived carotenoid synthase that can obtain different catalytic characteristics, for the production of different sorts carrotenoid provides new way.
Carrotenoid C-3,4 desaturases (CrtD) they are desaturases in the carrotenoid route of synthesis, and usually catalysis 1-hydroxy kind Serlabo and 1 '-hydroxy kind Serlabo carry out C-3,4 and C-3 ', and 4 ' dehydrogenation reaction.Producing the oxygen photosynthetic bacterium---in the cyanobacteria, there are bibliographical information CrtD catalysis neurosporene and Lyeopene to carry out C-3, the reaction of 4 dehydrogenations, but do not find as yet that in the non-oxygen-production photosynthetic bacterium CrtD possesses this catalysis characteristics.
Through retrieval, there is not the red Production by Bacteria carrotenoid of fixed nitrogen C-3 at present in the world, the bibliographical information of 4 desaturases.
Summary of the invention
It is limited to the present invention is directed to existing mikrobe carotenoid synthase kind; Non-oxygen-production photosynthetic bacterium carrotenoid C-3 particularly; The blank of 4 desaturase catalysis neurosporenes and Lyeopene dehydrogenation research; The carrotenoid C-3 that provides a strain to produce catalysis neurosporene and Lyeopene dehydrogenation, the red bacterium of the fixed nitrogen of 4 desaturases (Rhodobacter azotoformans) Y6 and cultural method and application.
Summary of the invention
The red bacterium of fixed nitrogen of the present invention (Rhodobacter azotoformans) Y6 has the carrotenoid of producing C-3,4 desaturases; Can the catalysis neurosporene and Lyeopene carry out C-3,4 dehydrogenation reactions generate 3; 4-two dehydrogenation neurosporenes and 3, the function of 4-two dehydrogenation Lyeopenes.The present invention also provides characteristic, cultural method and the application of this fixed nitrogen Rhodobacter strain in addition.
Detailed Description Of The Invention
The red bacterium of one strain fixed nitrogen (Rhodobacter azotoformans) Y6 bacterial strain; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 8th, 2012; The address: No. 3 institutes of microbiology of the Chinese Academy of Sciences in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC No.6086.
The cultural method of the red bacterium of above-mentioned fixed nitrogen (Rhodobacter azotoformans) Y6, step is following:
Y6 is inoculated in the RCVBN substratum by 8 ~ 12wt% inoculum size with the red bacterium of fixed nitrogen (Rhodobacter azotoformans), is that constant temperature illumination is left standstill and cultivated 48 ~ 72 hours under 25 ~ 35 ℃ the condition in temperature, gets final product;
Above-mentioned RCVBN nutrient media components is following:
MgSO 47H 2O 0.2g/L, CaCl 20.056g/L, FeSO 47H 2O 0.012g/L, Na 2EDTA 0.02g/L, H 3BO 32.8 * 10 -3G/L, MnSO 41.6 * 10 -3G/L, ZnSO 47H 2O 2.4 * 10 -4G/L, Na 2MoO 42H 2O 7.52 * 10 -4G/L, Ca (NO3) 23H 2O 4 * 10 -5G/L, vitamin H 1.5 * 10 -5G/L, nicotinic acid 1 * 10 -3G/L, para-amino benzoic acid 1 * 10 -3G/L, vitamin 1 * 10 -3G/L, KH 2PO 40.6g/L, K 2HPO 43H 2O 0.9g/L, DL-oxysuccinic acid 4g/L, (NH 4) 2.SO 41g/L, pH6.8 sterilized 30 minutes for 115 ℃.
Preferred according to the present invention, said culture temperature is 30 ℃.
CrtD ability catalysis neurosporene and Lyeopene that the red bacterium Y6 of fixed nitrogen of the present invention bacterial strain produces carry out dehydrogenation reaction.
The application of the red bacterium of above-mentioned fixed nitrogen (Rhodobacter azotoformans) Y6 in neurosporene or Lyeopene dehydrogenation reaction.
Above-mentioned application, step is following:
Utilizing round pcr is template with the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 strain gene group DNA; Adopt upstream primer D-F:TGCCATATGATGAGACAGATTGTGCCGAA (SEQ ID NO.3) and downstream primer D-R:TCGAAGCTTCCTCGATCGCGAAGCAAGGT (SEQ ID NO.4); Amplification carrotenoid crtD gene; This gene is expressed in e. coli bl21 (DE3) through expression vector pET-22b; Collect the recombinant Bacillus coli cells supersonic wave wall breaking and prepare recombinant C rtD crude enzyme liquid, utilize recombinant C rtD crude enzyme liquid that neurosporene or Lyeopene are carried out external dehydrogenation reaction again, generate 3; 4-two dehydrogenation neurosporenes or 3,4-two dehydrogenation Lyeopenes.
Above operation steps, experiment condition and reagent all adopt this area routine operation and common agents if no special instructions.
Beneficial effect
Among the red bacterium of fixed nitrogen according to the invention (Rhodobacter azotoformans) Y6, contain carrotenoid C-3, the 4-desaturase; This enzyme ability catalysis neurosporene or Lyeopene carry out dehydrogenation reaction and generate 3 respectively, 4-two dehydrogenation neurosporenes or 3,4-two dehydrogenation Lyeopenes; This characteristic is discovery first in the non-oxygen-production photosynthetic bacterium; Be 3,4-two dehydrogenation neurosporenes or 3, the microbial fermentation production of 4-two dehydrogenation Lyeopenes provides new enzyme source.
Description of drawings
Fig. 1 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis neurosporene dehydrogenation product HPLC figure;
Wherein, 1,3,4-two dehydrogenation neurosporenes; 2, neurosporene;
Fig. 2 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis Lyeopene dehydrogenation product HPLC figure;
Wherein, 3,3,4-two dehydrogenation Lyeopenes; 4, Lyeopene;
Fig. 3 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis neurosporene dehydrogenation product 3,4-two dehydrogenation neurosporene optical absorption maps;
Fig. 4 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis Lyeopene dehydrogenation product 3,4-two dehydrogenation Lyeopene optical absorption maps;
Fig. 5 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis neurosporene dehydrogenation product 3,4-two dehydrogenation neurosporene mass-spectrograms;
Fig. 6 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis Lyeopene dehydrogenation product 3,4-two dehydrogenation Lyeopene mass-spectrograms.
Embodiment
Below in conjunction with embodiment the present invention is done further elaboration, but institute of the present invention protection domain is not limited only to this.
The red bacterium of fixed nitrogen described in the embodiment (Rhodobacter azotoformans) Y6; (address: No. 3 institutes of microbiology of the Chinese Academy of Sciences in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.6086 to be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 8th, 2012.
Na 2EDTA is available from U.S. Amresco company; Na 2MoO 42H 2O is available from Shanghai gelatinizing factory; Para-amino benzoic acid is available from Shanghai chemical reagent purchasing and supply station; The DL-oxysuccinic acid is available from Tianjin Da Mao chemical reagent factory; Vitamin H, nicotinic acid, vitamin are available from China Medicine (Group) Shanghai Chemical Reagent Co.; Peptone and yeast powder are available from Britain Oxoid company.
High performance liquid chromatograph and chromatographic column TC-C18 are all available from U.S. Agilent company.
Embodiment 1:
The red bacterium of one strain fixed nitrogen (Rhodobacter azotoformans) separates obtaining in the Xiaoqinghe River sewage of Jinan City, Shandong Province, have following biological property:
(1) morphological feature is observed
Gram-negative, cell is oval, and the thalline diameter is 0.6 μ m ~ 1.2 μ m; Under illumination anaerobism, dark aerobic and dark anaerobic condition, all can grow.Under the illumination anaerobic condition, culture is pale brown look; Under illumination half anaerobic condition, culture is red.
(2) physiological and biochemical property is observed
Under the illumination anaerobic condition, can utilize melampyrum to grow for sole carbon source, can not utilize soluble tartrate; Under dark anaerobism and saltpetre condition, can utilize fructose or wood sugar to grow for sole carbon source; Other characteristics see table 1 for details.
Table 1 fixed nitrogen red bacterium Y6 carbon source and electron donor utilization
Figure BDA00001658087100031
Annotate: +++, vigorous growth (OD660>1.0); ++, common growth (1.0; OD 660>0.2); +, faint growth (OD 660<0.2);-, do not grow
(3) 16S rDNA sequencing and homology analysis
Adopt bacterial genomes to extract test kit (available from BioFlux company) and extract the red bacterium Y6 of fixed nitrogen genomic dna, adopting general upstream primer 27f:AGAGTTTGATCMTGGCTCAG of bacterial 16 S rDNA (SEQ ID NO.1) and downstream primer 1525r:AAGGAGGTGATCCAGCC (SEQ ID NO.2) is template pcr amplification 16S rDNA sequence with the genomic dna.
PCR reaction system (50 μ L):
TaKaRa taq archaeal dna polymerase (2.5U), 10 * TaKaRataq polysaccharase buffer (5 μ L), MgCl 2(1.5mM), genomic dna (10ng), dNTP (200 μ M), primer (0.4 μ M).
Reaction conditions is:
94 ℃ of preparatory sex change 2min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 90s, 30 circulations; 72 ℃ are extended 10min.
The amplified production that is obtained is given birth to worker's biotechnology ltd by Shanghai and is carried out sequencing; Size is 1459bp; This sequence is carried out BLAST Nucleotide compare of analysis in the NCBI website, with 16S rDNA sequence (the GenBank number of including HM536622, the GU990617 of the red bacterium of 7 strain fixed nitrogen; GU990616, NR_029215 etc.) homology is 99%.
The GenBank number of including of the 16S rDNA of above-mentioned bacterial strains is JF738027; The most approaching with the red bacterium of fixed nitrogen on evolutionary relationship; Therefore with the red bacterium of this bacterial strain called after fixed nitrogen (Rhodobacter azotoformans) Y6 bacterial strain; And (address: No. 3 institutes of microbiology of the Chinese Academy of Sciences in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.6086 to be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 8th, 2012.
Embodiment 2:
The cultural method of the red bacterium of embodiment 1 said fixed nitrogen (Rhodobacter azotoformans) Y6, step is following:
The red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 is inoculated in the RCVBN substratum by the inoculum size of 10wt%, is that constant temperature illumination is left standstill and cultivated 48 ~ 72 hours under 30 ℃ the condition in temperature;
Above-mentioned RCVBN nutrient media components is following:
MgSO 47H 2O 0.2g/L, CaCl 20.056g/L, FeSO 47H 2O 0.012g/L, Na 2EDTA 0.02g/L, H 3BO 32.8 * 10 -3G/L, MnSO 41.6 * 10 -3G/L, ZnSO 47H 2O 2.4 * 10 -4G/L, Na 2MoO 42H 2O 7.52 * 10 -4G/L, Ca (NO3) 23H 2O 4 * 10 -5G/L, vitamin H 1.5 * 10 -5G/L, nicotinic acid 1 * 10 -3G/L, para-amino benzoic acid 1 * 10 -3G/L, vitamin 1 * 10 -3G/L, KH 2PO 40.6g/L, K 2HPO 43H 2O 0.9g/L, DL-oxysuccinic acid 4g/L, (NH 4) 2.SO 41g/L; Transfer pH to 6.8, sterilized 30 minutes for 115 ℃.
Embodiment 3:
The application of the red bacterium of above-mentioned fixed nitrogen (Rhodobacter azotoformans) Y6 in neurosporene or Lyeopene dehydrogenation reaction, concrete steps are following:
(a) adopt bacterial genomes to extract test kit (BioFlux) and extract the genomic dna that embodiment 2 cultivates the red bacterium Y6 of fixed nitrogen that obtains, adopt crtD upstream region of gene primer D-F:TGC CATATGATGAGACAGATTGTGCCGAA (SEQ ID NO.3), underscore is the NdeI restriction enzyme site) and downstream primer D-R:TCG AAGCTTCCTCGATCGCGAAGCAAGGT (SEQ ID NO.4), underscore is the HindIIII restriction enzyme site), be template pcr amplification crtD gene order with the genomic dna,
PCR reaction system (50 μ L):
TaKaRa LAtaq archaeal dna polymerase (2.5U), 2 * TaKaRa GC damping fluid (25 μ L), MgCl 2(1.5mM), genomic dna (10ng), dNTP (200 μ M), primer (0.4 μ M).
94 ℃ of preparatory sex change 2min; 94 ℃ of 30s, 61 ℃ of 30s, 72 ℃ of 90s, 30 circulations; 72 ℃ are extended 10min.
The final crtD gene that obtains, empirical tests, the GenBank number of including of the gene cluster at the crtD gene place of the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 is JF723980.
(b) (NEB USA) cut 1 hour in 37 ℃ of enzymes, carried out the DNA purifying, the gene fragment after making enzyme and cutting to add restriction enzyme NdeI, HindIII after the amplified production that step (a) is obtained reclaims;
Adopt same enzyme blanking method to handle the pET-22b plasmid vector, the pET-22b plasmid vector after making enzyme and cutting;
Gene fragment after enzyme cut and pET-22b plasmid vector after enzyme is cut 4:1 in molar ratio mix, and (NEB USA) connects 10 hours in 16 ℃, adopts CaCl then to adopt the T4 ligase enzyme 2Conversion method transforms host's bacterium e. coli bl21 (DE3), and the bacterium liquid coating amicillin resistance LB after the conversion is dull and stereotyped, 37 ℃ of incubated overnight.Cultivated 10 hours for 37 ℃ in the well-grown single bacterium colony of picking to the LB nutrient solution, extract plasmid,, obtain expressing the red bacterial carotenoid C-3 of fixed nitrogen, the intestinal bacteria reorganization bacterium of 4 desaturases through enzyme cutting method and PCR method checking positive transformant;
The dull and stereotyped composition of above-mentioned LB: peptone 10g/L, yeast powder 5g/L, NaCl 7g/L, agar 15g/L, pH7.0～7.5,121 ℃ sterilization 20 minutes.
Above-mentioned LB nutrient solution composition: peptone 10g/L, yeast powder 5g/L, NaCl 7g/L, pH7.0～7.5,121 ℃ sterilization 20 minutes.
(c) with expressing the red bacterial carotenoid C-3 of fixed nitrogen in the step (b), the intestinal bacteria reorganization bacterium of 4 desaturases is inoculated in 200mL LB nutrient solution, and 37 ℃ are cultured to OD 600Reach 0.5～0.9, add IPTG (sec.-propyl-β-D-sulfo-galactopyranoside) and induce, the IPTG final concentration is 0.5mmol/L, 25 ℃ of inducing culture 30 hours.Centrifugal collection somatic cells with the resuspended back of 100mM phosphoric acid buffer (pH 8.0) ultrasonic disruption, obtains recombinant C rtD crude enzyme liquid;
Above-mentioned LB nutrient solution composition: peptone 10g/L, yeast powder 5g/L, NaCl 7g/L, pH7.0～7.5,121 ℃ sterilization 20 minutes.
(d) get the recombinant C rtD crude enzyme liquid 400 μ L that obtain in the step (c), (available from CaroteNature company, Switzerland), reaction system is settled to 500 μ L with damping fluid to add 10 μ M neurosporenes or Lyeopene; With the ultrasonic disruption appearance mixing of U.S. Sonics company 30 seconds (22kHz, 100W); 30 ℃ of airtight oscillatory reactions of lucifuge 5 hours, mixed solution;
(e) in the mixed solution that step (d) makes, add 30 μ L 2mol/LNaOH and 30 μ L 10wt%SDS solution, mix; Add 300 μ L 3mol/L NaCH 3COOH (pH4.8) solution vibrated 5 seconds; Centrifugal 1 minute of 12000rpm discards supernatant, gets protein-based deposition;
(f) in the protein precipitation that step (e) makes, add 200 μ L acetone extraction carrotenoid, stirred 2 minutes, make the deposition homodisperse, obtain containing the acetone soln of carrotenoid;
(g) acetone soln that contains carrotenoid that step (f) is made centrifugal 1 minute through 12000rpm is got acetone soln, behind the 0.22 μ m membrane filtration; HPLC (performance liquid chromatography) method detection reaction product, chromatographic column is that TC-C18 is (available from Agilent company, USA); Mobile phase methanol/acetonitrile (volume ratio is 4/6), flow velocity 1mL/ minute, 30 ℃ of column temperatures; Detect wavelength 474nm, detected result is as shown in Figure 1.
(h) collect the carrotenoid component that step (g) HPLC detects, carry out the photoabsorption appearance (available from Thermo company, USA) with mass spectrograph (available from Kyoto company, Japan) analysis.Photoabsorption scanning wavelength scope is 350 ~ 600nm, solvent methanol/acetonitrile (volume ratio is 4/6); Mass ion source is ESI, positive ion mode, and moving phase is 98wt% acetonitrile (containing 0.05wt% formic acid).
Fig. 1 and Fig. 2 show that the red bacterium CrtD of above-mentioned reorganization fixed nitrogen catalysis neurosporene (component 2) or Lyeopene (component 4) have generated new carrotenoid (component 1 and 3).Component 1 has three main absorption peaks, and wavelength is respectively 444nm, 472nm and 504nm; Molecular weight is 536.4, and this carrotenoid is 3, and 4-two dehydrogenation neurosporenes (Fig. 3, Fig. 5).Component 3 has three main absorption peaks, and wavelength is respectively 469nm, 496nm and 528nm; Molecular weight is 534.4, and this carrotenoid is 3, and 4-two dehydrogenation Lyeopenes (Fig. 4, Fig. 6).

Claims (5)

1. the red bacterium of a strain fixed nitrogen (Rhodobacter azotoformans) Y6 bacterial strain; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 8th, 2012; The address: No. 3 institutes of microbiology of the Chinese Academy of Sciences in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC No.6086.
2. the cultural method of the red bacterium of the said fixed nitrogen of claim 1 (Rhodobacter azotoformans) Y6 is characterized in that step is following:
Y6 is inoculated in the RCVBN substratum by 8 ~ 12wt% inoculum size with the red bacterium of fixed nitrogen (Rhodobacter azotoformans), is that constant temperature illumination is left standstill and cultivated 48 ~ 72 hours under 25 ~ 35 ℃ the condition in temperature, gets final product;
Above-mentioned RCVBN nutrient media components is following:
MgSO 47H 2O 0.2g/L, CaCl 20.056g/L, FeSO 47H 2O 0.012g/L, Na 2EDTA 0.02g/L, H 3BO 32.8 * 10 -3G/L, MnSO 41.6 * 10 -3G/L, ZnSO 47H 2O 2.4 * 10 -4G/L, Na 2MoO 42H 2O 7.52 * 10 -4G/L, Ca (NO3) 23H 2O 4 * 10 -5G/L, vitamin H 1.5 * 10 -5G/L, nicotinic acid 1 * 10 -3G/L, para-amino benzoic acid 1 * 10 -3G/L, vitamin 1 * 10 -3G/L, KH 2PO 40.6g/L, K 2HPO 43H 2O 0.9g/L, DL-oxysuccinic acid 4g/L, (NH 4) 2.SO 41g/L, pH6.8 sterilized 30 minutes for 115 ℃.
3. the cultural method of the red bacterium Y6 of fixed nitrogen as claimed in claim 2 is characterized in that said temperature is 30 ℃.
4. the application of the red bacterium of the said fixed nitrogen of claim 1 (Rhodobacter azotoformans) Y6 in neurosporene or Lyeopene dehydrogenation reaction.
5. application as claimed in claim 4 is characterized in that step is following:
Utilizing round pcr is template with the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 strain gene group DNA; Adopt upstream primer D-F:TGCCATATGATGAGACAGATTGTGCCGAA and downstream primer D-R:TCGAAGCTTCCTCGATCGCGAAGCAAGGT; Amplification carrotenoid crtD gene is expressed through expression vector pET-22b this gene in e. coli bl21 (DE3), collect the recombinant Bacillus coli cells supersonic wave wall breaking and prepare recombinant C rtD crude enzyme liquid; Utilize recombinant C rtD crude enzyme liquid that neurosporene or Lyeopene are carried out external dehydrogenation reaction again; Generate 3,4-two dehydrogenation neurosporenes or 3,4-two dehydrogenation Lyeopenes.
CN201210156757.0A 2012-05-18 2012-05-18 Rhodobacter azotoformans and culture method and application thereof Active CN102660484B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201210156757.0A CN102660484B (en) 2012-05-18 2012-05-18 Rhodobacter azotoformans and culture method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201210156757.0A CN102660484B (en) 2012-05-18 2012-05-18 Rhodobacter azotoformans and culture method and application thereof

Publications (2)

Publication Number Publication Date
CN102660484A true CN102660484A (en) 2012-09-12
CN102660484B CN102660484B (en) 2014-06-04

Family

ID=46770066

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201210156757.0A Active CN102660484B (en) 2012-05-18 2012-05-18 Rhodobacter azotoformans and culture method and application thereof

Country Status (1)

Country Link
CN (1) CN102660484B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109400241A (en) * 2019-01-09 2019-03-01 苏州寰宝农业科技有限公司 A kind of cultivation bucket and its use the method for preparing organic selenium fertilizer
CN109438021A (en) * 2019-01-09 2019-03-08 苏州寰宝农业科技有限公司 A kind of organic selenium fertilizer and preparation method thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004003216A2 (en) * 2002-07-01 2004-01-08 Novozymes A/S Sterilization of a fermentation medium comprising hydrolysed n-source
CN101333509A (en) * 2008-07-07 2008-12-31 天辰神舟实业有限公司 Rhodobacter sphaeroides mutant of coenzyme Q10 and culturing method
CN102260692A (en) * 2011-07-08 2011-11-30 山东大学 Phytoene dehydrogenase gene and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004003216A2 (en) * 2002-07-01 2004-01-08 Novozymes A/S Sterilization of a fermentation medium comprising hydrolysed n-source
CN101333509A (en) * 2008-07-07 2008-12-31 天辰神舟实业有限公司 Rhodobacter sphaeroides mutant of coenzyme Q10 and culturing method
CN102260692A (en) * 2011-07-08 2011-11-30 山东大学 Phytoene dehydrogenase gene and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
XIAO M ET AL: "JF738027.1", 《GENBANK》 *
林觐勤: "固氮红细菌Y6光发酵产氢及其诱变的研究", 《中国优秀硕士学位论文全文数据库基础科学辑》 *
韩波: "光合细菌产类胡萝卜素的研究", 《中国优秀博硕士学位论文全文数据库（硕士）基础科学辑》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109400241A (en) * 2019-01-09 2019-03-01 苏州寰宝农业科技有限公司 A kind of cultivation bucket and its use the method for preparing organic selenium fertilizer
CN109438021A (en) * 2019-01-09 2019-03-08 苏州寰宝农业科技有限公司 A kind of organic selenium fertilizer and preparation method thereof

Also Published As

Publication number Publication date
CN102660484B (en) 2014-06-04

Similar Documents

Publication Publication Date Title
Hur et al. Highly efficient bioconversion of methane to methanol using a novel type I Methylomonas sp. DH‐1 newly isolated from brewery waste sludge
Deng et al. Metabolic engineering of Thermobifida fusca for direct aerobic bioconversion of untreated lignocellulosic biomass to 1-propanol
Shaw et al. Identification of the [FeFe]-hydrogenase responsible for hydrogen generation in Thermoanaerobacterium saccharolyticum and demonstration of increased ethanol yield via hydrogenase knockout
Du et al. Energy-efficient butanol production by Clostridium acetobutylicum with histidine kinase knockouts to improve strain tolerance and process robustness
Graham et al. Identification of the 7, 8-didemethyl-8-hydroxy-5-deazariboflavin synthase required for coenzyme F 420 biosynthesis
CN102816729B (en) Construction and application of polygene knockout strain of Halomonas sp. TD01
Wang et al. Improvement of stress tolerance and riboflavin production of Bacillus subtilis by introduction of heat shock proteins from thermophilic bacillus strains
CN101565709B (en) 3-sterone-9Alpha-hydroxylation enzyme gene, 3-sterone-9Alpha-hydroxylation enzyme reductase gene, relevant carriers, engineering bacteria and applications thereof
Jhala et al. Isolation and characterization of methane utilizing bacteria from wetland paddy ecosystem
Kawai et al. p-Hydroxycinnamic acid production directly from cellulose using endoglucanase-and tyrosine ammonia lyase-expressing Streptomyces lividans
Oslowski et al. Production of hydrogen from α-1, 4-and β-1, 4-linked saccharides by marine hyperthermophilic Archaea
CN107400652B (en) Construction method of engineering bacteria for dynamically regulating and controlling synthesis of 3-hydroxy propionic acid
Shang et al. Themoanaerobacterium calidifontis sp. nov., a novel anaerobic, thermophilic, ethanol-producing bacterium from hot springs in China
CN102808002B (en) The reconstitution cell of a kind of biological process synthesizing methyl acetoin and derivative thereof and method
CN104328148A (en) Method for preparing tert-butyl (3R, 5S)-6-chloro-3,5-dihydroxy hexanoate by using enzymatic method
CN101319219B (en) Method for preparing deoxidized violacein and special recombinant bacterium
CN102660484B (en) Rhodobacter azotoformans and culture method and application thereof
CN104480127B (en) Hyperthermophilic glucosides enzyme mutant and its application in prepared by Ginsenoside compound K
CN103555646B (en) A kind of coexpression L-arabinose isomerase gene and the genetic engineering bacterium of mannose-6-phosphate isomerase
CN104726435A (en) Beta-glucosaccharase mutant, recombinant expression plasmid thereof and transformed engineering strain
CN102382790A (en) Recombinant bacillus subtilis for high yield of catalase (CAT), and construction method and application thereof
CN101250539B (en) Method for preparing recombinant heat-proof beta-glucuronic acid enzyme
CN109694892B (en) Method and kit for preparing salidroside
Ichikawa et al. The expression of alternative sigma-I7 factor induces the transcription of cellulosomal genes in the cellulolytic bacterium Clostridium thermocellum
Ma et al. Construction of a novel expression system in Klebsiella pneumoniae and its application for 1, 3-propanediol production

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant