CN109486837A - One group of high-temperature biological Desulfurization gene and application - Google Patents
One group of high-temperature biological Desulfurization gene and application Download PDFInfo
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Abstract
The present invention relates to one group of high-temperature biological Desulfurization genes and application.This group of gene screens to obtain from the microbiologic population in the environmental sample by oil pollution；The gene that the present invention identifies, high temperature resistant have good thermal stability, can apply in the industrial production that fermentation etc. needs thermophilic enzyme condition；Its source strain group energy well-grown in reservoir media, by carrying out clonal expression, Validation in vitro to this group of gene, prove the ability that this group of gene has desulfurization, it is remarkably improved the biological desulphurization efficiency of fuel oil and effectively solves the problem of environmental pollution after use, has the good characteristic of industrial application, this group of gene has wide prospects for commercial application.
Description
Technical field
The present invention relates to macro genome and field of environment microorganism, and in particular to one group of high-temperature biological Desulfurization gene with answer
With.
Background technique
21 century is environmentally friendly century, and the energy industry as chemical industry main force all suffers from perhaps in environmental protection and process aspect
More challenges and opportunity, and the new branch of science that be thus born biochemical industry and environment protection field are intersected.With global industry
The fast development of process increases severely to the demand of the energy, currently, petroleum proportion is maximum in world energy sources structure, is
37.4%.There are sulphur compounds in oil product can cause environmental pollution using sulphur compound this product is contained, and produce to equipment
Raw corrosion.Therefore, desulfurization technology is the important subject of petrochemical industry and environmental protection.
Petroleum and products thereof deep desulfuration is the important class that China or even whole world petrochemical industry are faced in the new century
Topic.Sulphur in petroleum is mainly taken with mercaptan, thioether, thiophene, benzothiophene (BT), dibenzothiophenes (DBT) and their alkyl
Exist for the form of the organic sulfur compounds such as object and derivative, these compounds generate a large amount of rhodanide in burning, bring
A variety of harm.SO caused by wherein burning most serious of all2And SO3Acid rain easily is formed with vapor in the atmosphere chemical combination,
Serious pollution is caused, destroys the ecological balance, be detrimental to health (Monticello D J. desulfurization of
Fossil fuels.Ann Rev Microbiol.1985,39:371 ~ 389).In recent years, increasingly with biotechnology desulfurization
It is of interest for the people.Biocatalytic desulfurization (BDS) is a kind of at normal temperatures and pressures using aerobic, anaerobic bacteria removing petroleum containing thia
A kind of new technology that sulphur is combined in cycle compound, will be in fossil fuel in the way of biochemistry cracking by the distinctive enzyme of microorganism
Sulphur removes, and is to carry out in normal temperature and pressure under conditions of not needing hydrogen, and energy specificity is cut off carbon-sulfur bond and made in fossil fuel
Sulphur released in the form of sulfate, the purpose is to using the removing sulphur of microorganism existing for nature without destroying organic combustion
Expect the hydro carbons of value.Its main feature is that process costs are low, saving energy, process is simple, and reaction condition is mild, and clean environment meets life
The environmental-friendly development trend of state.Decades have been developed from appearance in BDS technology so far, so far still in developmental research rank
Section.Biocatalytic desulfurization technology is the removal of organic sulphur component in petroleum, is opened up a new way.And it passes
System biological desulfurizing technology haves the shortcomings that various very important: at high cost, operation difficulty is big, removing substrate kind has
Limit, removal effect be not significant, and the research report of existing related Desulfurization gene is almost all room temperature Desulfurization gene.
Gene of the invention belongs to high temperature desulfurizing gene, has the function of to petroleum sweetening, can be applied to petrochemical industry,
In environmental protection industry and the production of other related industries.
Summary of the invention
The main purpose of the present invention is to provide one group of high-temperature biological Desulfurization gene and its characterization and application, this group of gene is resistance to
High temperature has good thermal stability, can apply in the industrial production that fermentation etc. needs thermophilic enzyme condition；It can be effective
Remove the application of sulphur and the bacterial strain in the crude oil of sulfur-bearing and its fraction desulfurization in sulfur-containing compound in petroleum.
A second object of the present invention is to provide the recombinant plasmid (pET-28a- that one group can express recombinant high temperature Desulfurization gene
1,2,3,4, D).
Third object of the present invention is to provide one group of recombinant bacterium that can generate above-mentioned recombinant high temperature desulfurase (BL21-1,
2,3,4, D).
Fourth object of the present invention is to provide the method for element sulphur in bacterial strain removing sulfur-containing compound
Fifth object of the present invention is to provide a kind of methods that target gene can be screened in macro genome.
In order to realize appeal purpose, the present invention adopts the following technical scheme:
This group of high temperature desulfurizing gene provided by the present invention, is screened from the microbiologic population in the environmental sample by oil pollution
It arrives；And high temperature resistant, there is good thermal stability, can be answered in the industrial production that fermentation etc. needs thermophilic enzyme condition
With.Gene order is as shown in SEQ ID NO:1-5；Amino acid sequence corresponding to it is SEQ ID NO:6-10.
(1) we use the mode sulfurous pollutants dibenzothiophenes (DBT) in petroleum as induced pressure, from by petroleum wastewater
Then macro gene order-checking can have been carried out with the microbial flora of high temperature desulfurizing by having filtered out in the environmental sample of dye.By right
Macro genomic data carry out analysis and with it has been reported that the gene dszABCD of authenticated active room temperature degradation DBT compared
It is right, it was found that this group of high temperature desulfurizing gene (referring to table 1).
(2) the macro genome of desulfurization microbial flora is extracted as template, utilizes these bases of round pcr massive amplification
Cause is carrier with pET-28a, this 5 genes are transferred to BL21(DE3 respectively) carry out clonal expression.
(3) enzyme of clonal expression is purified, is then degraded with these enzymes to dibenzothiophenes (DBT), to sentence
Break the desulphurizing abilities of these enzymes.These enzymes are significant for the desulfuration in terms of petrochemical industry and environmental protection.
1 high temperature desulfurizing gene of table
| orf | Gene | |
| 60-DBT_C552551_6 | Alkylsulfonate monooxygenase | |
| 60-DBT_C552551_5 | Aliphatic sulfonate abc transport Binding Capacity albumen | |
| 60-DBT_C552551_3 | FMN dependent form monooxygenase | |
| 60-DBT_C552551_2 | Ethylene reductase | |
| 60-DBT_C549025_1 | Flavoxidoreductase |
The high temperature desulfurizing related gene is referring to table 1
SEQ ID NO:1-5 nucleotide sequence provided by the invention is as follows:
60-DBT_C552551_6 SEQ ID No.1:
ATGGTCGAATTTATCACAATGGCCCCAACATCAGGAGACAGTACACTTGTTGGGTTAGCCAATAACTCGTCAA
AACTGAATAGCTGGACAGGAACAGATGAAAATGCAGAACGCCCCCCTACCCAAGAATATATTAAAGCGATTGCCCAG
GCTGCTGAAAAAGGCGGATTCTCGACGCTTTTACTGCCGACTGGCACAGGATGCCTTGATTCTTTAGCCGTTGCTGC
CAATTTAATCGCCTATACAAGCAAATTAAAATTCCTATTTGCAATCCGCCCTGGTTTTATCGCACCTACTACCTTTG
CCAAACAATTTGCCACTGTGGATTATTGGTCAAATGGAAGAGCCTTGGTGAATATTGTTACCGGAGGTTCGCCGGTC
GAGTTAGCCAGCGAAGGGGATTATTTAGATCATGATACCCGTTATAAGCGGACACGCGAATACATGCAAATATTAAA
AAAACTATTTACTGAAGAAAGCGTTGATTACGAAGGGGAATTTTTTACATTAAAAGGCGCCTCATTATTTCCAAAAC
CAGTAAAAACGCCGCCAATCTATTTTGGTGGGGCATCAGAAATCGCCAAAGAAGTCGCGGCGGAAGAAGCAGATGTT
TATATGATGTGGGGGGAAACTTTTGAAAATACAAAACAAAGACTTGAAGAAATGAAGCAGAGAGCGGCCAAACATAA
CCGGACGCTGAGTTATAGCGTTTCGTTTCAAGTCATCCTCGGGAACACGGAAGAAGAAGCATGGGAAAAGGCAAACA
AGCTGATAAGCAAAGTGTCAGCATCCATATTAGCCAAAAAAGAGGAAATGATTGTGAAAGGAGATTCTATTGGCGCA
AAACGCCTGCATCAACTTATGGAAAGCAGCAAAGAACGTAACTTTCAAATTGGCCCGAATTTATGGGCGGGACTGAC
TCAAGTTTTATCCGGAAATTCTATTGCCCTTGTCGGCACTCCTGAGCAAATCGCTGAACGGATTGTCGAACTGGTGG
AATTAGGATTCGATAAAGTGTTGTTAAGAGGCTTCCCTCATTTGGAAACGATTGAGCAATTAGGAGAGCTAGTCATT
CCAAAAGTGAGAGAAAAACTCGCACAAAAACAACTTGTCAAATGA
60-DBT_C552551_5 SEQ ID No.2:
ATGAGGTCCATGAAGCGGAGCAAGCATATTCTTTGGATCATTCATTTCCTCGTATTTTCCCTTTTACTGTCAT
CCTGCGGGAAAGCGGAAGAAACGGGCGGGAAAAACAAAGAAATTCATATTGGATATCAAAAAAATGGCACTACCTTA
TTGTTAAAACATAAACAAGAACTGCAAAAAGAGTTGGAAAAAGAAGGATATAAAGTAACATGGTCAGAGTTTAACAC
CGGAAGCTCGATCCTTGAAGCCTTAAATGCTGGAAGTATTGATTTTGCCGGCGCAGGGGACATACCGTCCATCTTTG
CGCTGGAGAAAGGCAGTAATTTTAACTATATTGCTAGTGAGCCATCGTCTCCGTCTTCAGAGGGAATATTGGTGAGA
AAGGATTCGGGCATTCAATCACTGGAAGATTTAAAAGGAAAAAGAATCGCCTTTAATAAGGCTTCCATCGCTCAATA
TTTATTAACAAAAGCTTTAGATTCAGCAGGTTTATCGATGGATGATGTCGAGCCGGTGTACCTGAATCCTCCTGAAG
CAAGCATTGCTTTTGAACAAGGCGAAGTGGATGCGTGGGTCGTATGGGATCCTTATATGACGGTGGCCGAAAGCAAA
GGACACATCATTTTAAAAGATGCAACTGGAATTGTTCCGTATCGGACTTTTTATTTCAGCACTCCTGAAATAACGAA
AGAACATCCCGAAATAGTCAAAAAATTTGTGGAACATTTATCTAATATAGGAAAACAAATTAATCATGACCCTACCG
AAGCTGCCGCGCTGCTGCAAAAAGCCACCAATATCCCGGCGGAAACGTGGGAGAAAGTATTAAACAATAAAAAGTCA
GATGTTCACTTTATGGATGAAAAAGCTGTGCGTGATTTGCAAACAGAAGCAGATGATTTGTTAAAAATCGGGCTTAT
CAAAAAACAAGTGCAGATCGAAGATTACGTATGGTATCCAGAGAAATAA
60-DBT_C552551_3 SEQ ID No.3:
ATGAAAAAAAAGCAAATGAAATTGGGCGTGTTTCTCATGGGAACAGGCCACCATATCGCATCTTGGAGACATC
CTCATGTTCAAGCTGATGGATGTGAGGATTTCGCCTTTTTTCATAAAATAGCCAAAATAGCAGAAAAAGGAAAATTA
GATATACTGTTTTTAAGCGACGGGCTGTCTTTTAATGAACTTTCGCATCCGGCGGAATTAGTGAGATTTGAACCTAT
CACGTTATTGGCTGCGCTGTCTGTTGTCACCTCCCATATCGGGCTGGCAGCGACAGCGACGACCACCTATAACGAAC
CTTTCCATATCGCGAGAAAGTTTTCCTCTCTCGATCATTTAAGCAAAGGAAGAGCCGCATGGAATGTGGTGACTTCA
TACTACGAAGACGAAGCGAAAAATTTCAGCCAGGACGCTCATTTAGACCATCATCTTCGTTACGAGCGGGCAAAAGA
ATTCGTGGAGGTGGTGAAAGGACTGTGGGACAGCTGGGAAAAGGATGCACTCGTCCGCGATAAACAATCAGGCGTTT
ATTTCGATCCTAAAAAGCTGCATCCGTTAAACCATAAAGGCAAATATTTTTCTGTAAAAGGCCCGTTGAATTCTTCC
CGCTCCCCGCAAGGAAGGCCGGTCCTTATCCAAGCAGGGTCATCGGAAGACGGAATCAATTTTGCCGCACAAATTGC
AGACGTGATTTTCACCGCGCAACAAACATTGGAAGAAGCTCAGCATTTTTATCGAAAAGTGAAAACGAAAGCGGCGG
AATTCGGCAGAAATCCTGATGAAGTGATTATTATGCCCGGTGTTTCTCCATATATAGGCAATACAGAACAAGAAGCG
AGAGAAAAATATGAACAGCTGCAAGAGCTTATTGTTCCTGAAATCGGCTTGGCTTTTCTGTCTGACTACTTAGGGGG
CATCGATCTTTCTCGCTACTCATTAGATGATCCTTTGCCAGACGAAATTCCAGAAACCAATGGAAATAAAAGCAGAA
GAAAGTTAATCATTGATCTTGCAAGAAGAGAAAACTTAACGATCGGGGAGCTTTATAAGCGCATTGCCGGCTCGCGG
GGACACCGGATCATTTTCGGAACGCCAGAGCAAATCGCAGATCAGTTAGAAGAATGGATCATCCACGAAGGATCCGA
TGGCTTCAATCTGATGTTCCCGTATTATCCTGACGGCCTATCTGAATTTGTTGACCAAGTGATCCCGATCCTTCAAG
AAAGAGGACTGTTCAGAAAGGAATATGAAGGAACAACGTTACGGGAACATCTTGGATTGCCTGAGCCTGAATCAAGA
TATTCTCTGGCGCCCAGCCAATGA
60-DBT_C552551_2 SEQ ID No.4:
ATGTTAAGAAATTCGCAAAGCAGCGGAACCAGTTTTCTTCTGCCCCTTTTCAACTGGGCGCACGCGAAAGAAT
GCATTGCCGGTCAAAAATACAAGAAAGGAGAAATCATGTTGAGCATCGCAAGCAAAAAAACCCACCGCTCTTATTTA
TCTGACGAACTGTCAAGAAAGTTTGTCCAAAATGAACGCCAAGAATTTCTTTTACATCTTGCATCAGAACTTGCCGA
ACAATTTCACGAAACCGCCGACATCATAGACCAAGAAGGAAGATTCCCGTTTGAAAATTTCCAAAAATTAAAGGATT
GCAATTACCAATCACTAACCGTTCCTAAGCAATATGGCGGAGAAGAAATTTCACTTTATGAATTTCTTCTTATGCAA
GAGCGGTTAAGCCAAGGAGATGCTTCCACCGCCTTGTGCATCGGGTGGCATCTTGGGGTTATTTATGATCTCAGGGA
AAGACAAACATGGGACAGCGAAAAATTTCAATGGCTTTGCCATGAAGTTGTTCAAAATAAAGTGCTCATCAACCGGG
TGGCAACAGAGGACGGAACAGGAAGTCCAACAAGAGGCGGAAAACCTGAAACGGTAGCGGTCAAAAGAAATGGCAAA
TGGGTGATTACGGGCAGAAAATCCTTTGCGTCTATGGCGATAGCTCTTGATTATTCATTAGTAACAGCCACCATTCA
GGAATCAGGGAAAGTCGGATCTTTTTTAGTTGACCATCGCCTTCAAGGGGTTAGCGTCGAAGAAACTTGGGATATGA
TTGGAATGCGAGGCACACGGAGCGATGATCTGGTGCTAGATCAAGTGGAGTTGCCAGAAGATGCGCTGGTGGAATTG
GATCAATTGGAATCGCCGAAAGGCAATATGGGGAGAGCATGGCTACTTCATGTTCCGGCCTGCTTCCTTGGAATTGC
GATTGCCGCAAGAAATTATGCCATTTCCTTTGCTTCTGAATATCAGCCGAACAGCCTGCCCGGCCCAATCAAAGATG
TTCCAGAGGTGCAAAGAAAAATCGGCGAGATGGATTTAGAGCTGTTAAAAGCAAGACATACTCTTTATTCCGTCGCA
CACCGGTGGGATACGTACCCGGAAAAACGCATGGAAATGAGCGGAGAATTGGCGGCGGCGAAGCATATTGCCGTCAA
TAGTGCCAATAAAGTCGTTGATTTGGCGATGAGGATCGTAGGAGCCAGAAGCCTGCAAAAATCCAGCCCGTTGCAAC
GGTATTACAGAGATGTCAGAGCCGGGCTCCATAATCCGCCAATGGATGATGCCGTTATTTCGTTACTGGCAAAGCAA
GCATTGCAAAGTTTCCATTAA
60-DBT_C549025_1 SEQ ID No.5:
ATGGATGATCGTACATTTCGCAGAGCCATGGGAAAATTTGCGACCGGCGTGACGGTCGTGACAACGGAATATC
AAGGGGAAGCGAAAGGAATGACGGCGAACGCGTTTATGTCCGTTTCGCTCGACCCGAGACTTGTCGTCGTCTCCATT
GGCCATAAAGCGCGAATGCATGACATTGTCAAGCAAACGGGGAAATTTGCTGTCAACATTTTGCGGCGCGATCAAGA
GGAGTTGTCGCGCTTGTTTGCCGGCCAGTTGAAAGAAGAACGCAATGTTTCGTTTGATTGGGTGAACGGCCATCCGA
TTTTGCCGGAGGCGTTGGCGAATATTTTATGCAACGTCTATAGTTCGTACGTTGCCGGCGACCATACGTTGTATTTT
GGCGAAGTCACCGACATTTTCATGAAAGAGGAACCGGGCGACCCGCTTTTGTTTTTCGAAGGACAATACCGAAGCAT
CGGACAGTAA
Amino acid sequence SEQ ID NO:6-10 provided by the invention is as follows:
>60-DBT_C552551_6 SEQ ID No.6
MVEFITMAPTSGDSTLVGLANNSSKLNSWTGTDENAERPPTQEYIKAIAQAAEKGGFSTLLLPTGTGCLDSLA
VAANLIAYTSKLKFLFAIRPGFIAPTTFAKQFATVDYWSNGRALVNIVTGGSPVELASEGDYLDHDTRYKRTREYMQ
ILKKLFTEESVDYEGEFFTLKGASLFPKPVKTPPIYFGGASEIAKEVAAEEADVYMMWGETFENTKQRLEEMKQRAA
KHNRTLSYSVSFQVILGNTEEEAWEKANKLISKVSASILAKKEEMIVKGDSIGAKRLHQLMESSKERNFQIGPNLWA
GLTQVLSGNSIALVGTPEQIAERIVELVELGFDKVLLRGFPHLETIEQLGELVIPKVREKLAQKQLVKX
>60-DBT_C552551_5 SEQ ID No.7
MRSMKRSKHILWIIHFLVFSLLLSSCGKAEETGGKNKEIHIGYQKNGTTLLLKHKQELQKELEKEGYKVTWSE
FNTGSSILEALNAGSIDFAGAGDIPSIFALEKGSNFNYIASEPSSPSSEGILVRKDSGIQSLEDLKGKRIAFNKASI
AQYLLTKALDSAGLSMDDVEPVYLNPPEASIAFEQGEVDAWVVWDPYMTVAESKGHIILKDATGIVPYRTFYFSTPE
ITKEHPEIVKKFVEHLSNIGKQINHDPTEAAALLQKATNIPAETWEKVLNNKKSDVHFMDEKAVRDLQTEADDLLKI
GLIKKQVQIEDYVWYPEKX
>60-DBT_C552551_3 SEQ ID No.8
MKKKQMKLGVFLMGTGHHIASWRHPHVQADGCEDFAFFHKIAKIAEKGKLDILFLSDGLSFNELSHPAELVRF
EPITLLAALSVVTSHIGLAATATTTYNEPFHIARKFSSLDHLSKGRAAWNVVTSYYEDEAKNFSQDAHLDHHLRYER
AKEFVEVVKGLWDSWEKDALVRDKQSGVYFDPKKLHPLNHKGKYFSVKGPLNSSRSPQGRPVLIQAGSSEDGINFAA
QIADVIFTAQQTLEEAQHFYRKVKTKAAEFGRNPDEVIIMPGVSPYIGNTEQEAREKYEQLQELIVPEIGLAFLSDY
LGGIDLSRYSLDDPLPDEIPETNGNKSRRKLIIDLARRENLTIGELYKRIAGSRGHRIIFGTPEQIADQLEEWIIHE
GSDGFNLMFPYYPDGLSEFVDQVIPILQERGLFRKEYEGTTLREHLGLPEPESRYSLAPSQX
>60-DBT_C552551_2 SEQ ID No.9
MLRNSQSSGTSFLLPLFNWAHAKECIAGQKYKKGEIMLSIASKKTHRSYLSDELSRKFVQNERQEFLLHLASE
LAEQFHETADIIDQEGRFPFENFQKLKDCNYQSLTVPKQYGGEEISLYEFLLMQERLSQGDASTALCIGWHLGVIYD
LRERQTWDSEKFQWLCHEVVQNKVLINRVATEDGTGSPTRGGKPETVAVKRNGKWVITGRKSFASMAIALDYSLVTA
TIQESGKVGSFLVDHRLQGVSVEETWDMIGMRGTRSDDLVLDQVELPEDALVELDQLESPKGNMGRAWLLHVPACFL
GIAIAARNYAISFASEYQPNSLPGPIKDVPEVQRKIGEMDLELLKARHTLYSVAHRWDTYPEKRMEMSGELAAAKHI
AVNSANKVVDLAMRIVGARSLQKSSPLQRYYRDVRAGLHNPPMDDAVISLLAKQALQSFHX
>60-DBT_C549025_1 SEQ ID No.10
MDDRTFRRAMGKFATGVTVVTTEYQGEAKGMTANAFMSVSLDPRLVVVSIGHKARMHDIVKQTGKFAVNILRR
DQEELSRLFAGQLKEERNVSFDWVNGHPILPEALANILCNVYSSYVAGDHTLYFGEVTDIFMKEEPGDPLLFFEGQY
RSIGQX
The present invention further discloses one group of high-temperature biological desulfurization nucleotide answering in terms of preparing petrochemical industry and environmental protection
With；The petrochemical industry includes oil extraction in oil field, purification containing oil substance；Environmental protection includes microorganism remediation, fossil fuel
Deep desulfuration denitrogenation.Especially: the application of the element sulphur removing aspect in organic compounds containing sulfur.The present invention is using purifying enzyme drop
Solve the mode sulfur-containing compound dibenzothiophenes (DBT) in petroleum；Contained using DBT in the sample of internal standard method detection enzyme degradation front and back
Amount.The result shows that:
(1) this group of high temperature enzyme has significant desulfurization effect, and desulfurization effect can achieve 80% or more.
DBT/ saualane (internal standard) content is respectively 0.36,0.35 in (2) two groups of samples without high temperature enzymatic treatment, and two groups
DBT/ saualane (internal standard) content is respectively 0.062/0.067 after high temperature enzymatic treatment.
Specific embodiment:
Illustrate that the present invention, the scheme of embodiment described here do not limit the present invention, the professional people of this field below with reference to embodiment
Member's spirit according to the invention can make improvements and change, and the such modifications and variations are regarded as in the present invention
In the range of, the scope of the present invention and essence are defined by the claims；Wherein agents useful for same is commercially available.
Embodiment 1
(1) HMM(Hidden Markov Model is utilized) model discrimination high temperature desulfurizing gene
1, HMM(Hidden Markov Model) model foundation
Since the high temperature degradation gene of dibenzothiophenes is unknowable, and the room temperature bacterium degradation pathway of benzothiophene is also at and just rises
Step state.Therefore for the macro gene sequencing of 60-5-BT and 60-5-DBT as a result, can only be with room temperature dibenzothiophenes degradation bacteria
Gene in " 4S " approach operon carries out block building.We have searched in identified " 4S " approach operon for crossing function
DszABC gene, the mode sequences yet to be built of all same proteins is subjected to affiliation comparison, will be on chadogram in one
Sequence is as a family.Subsequent comparison is no longer participate in family's protein sequence of the protein sequence item number less than 4.Eventually for
DszA protein sequence totally 8 for establishing model, DszB protein sequence totally 9, DszC protein sequence totally 13.
2, model is compared with macro genomic data
By protein sequence with HMM construct block, respectively in " pro.fa " file of the macro genome of the 60-5-DBT group of extraction
In scan for.E value > 10-5 is set, and the sequence searched is compared in ncbi database.It is found all
Gene revert on the contigs being spliced that (DszB albumen is determined using the comparison result for not meeting threshold requirement
Position), it was found that by 60-DBT_C552551_6,60-DBT_C552551_5,60-DBT_C552551_3 and 60-DBT_
C552551_2(may be corresponded to respectivelydszA、dszB、dszAWithdszC) composition latent gene cluster.Since DszD is coenzyme, with
The enzyme is that the report of research object is more rare.Therefore the block using DszD as research object is not established in part research.We
It is compared with " pro.fa " file of the single sequence of the DszD reported and macro genome, filters out 60-DBT_C549025-1
This potential coenzyme gene.
(2) extraction of the macro genome of microbiologic population
The microbiologic population screened is cultivated in advance, then 4 DEG C, 5500 × g collection thallus.By thallus with 500 μ L 50mM
The Tris-HCl of pH 8.0 hangs, and 10000 × g is centrifuged 1 min, abandons supernatant.Rejoin 500 μ L 50mM pH's 8.0
Tris-HCl after sufficiently having hanged thallus, is added 20 μ L 0.4M EDTA and the mixing that is vortexed, reacts 20 in 37 DEG C of water-baths
Minute.20 μ L, 100 mg/mL lysozyme is added into system, is vortexed after concussion, is reacted 30 minutes in 37 DEG C of water-baths.
10 μ L, 20 mg/mL Proteinase K is added into system, after slight oscillatory mixes, 35 μ L 10%SDS are added, oscillation mixes,
It reacts 1-3 hours, was during which mixed by inversion every 10 minutes once in 56 DEG C of water-baths, until liquid is clarified.Add into system
Enter 10 μ L, 10 mg/mL RNase A, is vortexed after concussion, is reacted 60 minutes in 65 DEG C of water-baths.200 μ are added into system
L dehydrated alcohol is vortexed after concussion, and whole precipitating and liquid are transferred to the collection set of bacterial genomes DNA extraction kit
Guan Zhong, 10000 × g are centrifuged 90 s.To improve the rate of recovery, after can collecting again the liquid in casing once, collecting pipe is abandoned
In waste liquid.Then according to the requirement of kit specification, subsequent Genomic Purification processing is completed, treated, and genome use is suitable
Amount sterilizing MQ back dissolving, is stored in -80 DEG C of refrigerators.
(3) clone of high temperature desulfurizing gene and screening.
Take mentioned-above 0.5 μ L(about 10ng of macro genome solution) as template, with following oligonucleotide sequence difference
30 cycle P CR are carried out as primer, and by the PCR cycle parameter of following settings.
The PCR cycle parameter of setting is as follows:
95 DEG C, 2min；95 DEG C, 30s；55 DEG C, 45s；72 DEG C, 90s；72 DEG C, 10min；4 DEG C, 2hr
60-DBT_C552551_6:
Upstream primer: 5'CCGGAATTCATGGTCGAATTTATCACAATGGCC 3'
Downstream primer: 5'CCCAAGCTTGGTCATTTGACAAGTTGTTTTTGTGCG 3'
60-DBT_C552551_5:
Upstream primer: 5'CCGGAATTCATGAGGTCCATGAAGCGGAG 3'
Downstream primer: 5'CGAGCTCTTATTTCTCTGGATACCATACGTAA 3'
60-DBT_C552551_3:
Upstream primer: 5'CGAGCTCATGAAAAAAAAGCAAATGAAATTGG 3'
Downstream primer: 5'CCCAAGCTTGGTCATTGGCTGGGCGCCAGAGAATAT 3'
60-DBT_C552551_2:
Upstream primer: 5'CCGGAATTCATGTTAAGAAATTCGCAAAGCAGC 3'
Downstream primer: 5'CCCAAGCTTGGTTAATGGAAACTTTGCAATGCTT 3'
60-DBT_C549025_1:
Upstream primer: 5'CCGGAATTCATGGATGATCGTACATTTCGCAGAG 3'
Downstream primer: 5'CCCAAGCTTGGTTACTGTCCGATGCTTCGGTATTGT 3'
Above-mentioned five groups of PCR products after purification respectively with EcoR I/Hind III, EcoR I/Sac I, Sac I/Hind III,
EcoR I/Hind III, EcoR I/Hind III double digestion, respectively and through same restricted type restriction endonuclease enzymatic hydrolysis and gel extraction
Plasmid pET-28a (+) connection, after transformed competence colibacillus bacillus coli DH 5 alpha (preservation of this laboratory), be applied to containing 50 μ g/mL Kan
On the LB solid medium of (kalamycin).37 DEG C are cultivated 16~18 hours, picking monoclonal colonies identification, inserted with 60-
PET-28a (+) plasmid of the DNA sequence dna of DBT_C552551_2 coding is recombinant plasmid pET-28a-1, inserted with 60-DBT_
PET-28a (+) plasmid of the DNA sequence dna of C552551_3 coding is recombinant plasmid pET-28a-2, inserted with 60-DBT_
PET-28a (+) plasmid of the DNA sequence dna of C552551_5 coding is recombinant plasmid pET-28a-3, inserted with 60-DBT_
PET-28a (+) plasmid of the DNA sequence dna of C552551_6 coding is recombinant plasmid pET-28a-4, inserted with 60-DBT_
PET-28a (+) plasmid of the DNA sequence dna of C549025_1 coding is recombinant plasmid pET-28a-D.Using Sanger dideoxy
This DNA fragmentation is sequenced, the DNA sequence dna of sequencing result display insertion is correct.Then by recombinant plasmid pET-28a-1,
2,3,4, D is transformed into e. coli bl21 respectively, this e. coli bl21 is respectively designated as BL21-1,2,3,4, D.
(4) purifying of recombinant high temperature desulfurase
Above-mentioned recombinant bacterium monoclonal is respectively connected in LB culture medium of the 20mL containing 50 μ g/mL Kan, 37 DEG C, 180rpm/min training
Support 12 hours, then by culture by 1%(V/V) (totally 2 are shaken LB culture medium of the inoculum concentration access 200mL containing 50 μ g/mL Kan
Bottle), 37 DEG C, 220rpm/min cultivate A600 be 0.6 when, be added IPTG to final concentration of 0.1 mM, 37 DEG C, 180rpm/min
Induction 4 hours.Thalline were collected by centrifugation, is suspended from 50 mM Tris-Cl(pH8.0) in buffer, using ultrasonic disruption cell, from
Supernatant is the crude extract for recombinating high temperature desulfurizing enzyme.This supernatant is through chelating sepharose (Chelating
Sepharose) nickel affinity column chromatographic purifying, obtained enzyme preparation show a band on SDS-PAGE.And theoretically calculate
Molecular weight is consistent with SDS-PAGE testing result respectively.
(5) desulfurization effect
By purified 60-DBT_C552551_6,60-DBT_C552551_5,60-DBT_ in 1.5ml centrifuge tube
C552551_3,60-DBT_C552551_2, add 50ul, coenzyme 60-DBT_C549025_1 that 30ul, the FMN of 100mM is added to add respectively
The NADH of 2ul, 50mM add 20ul, and the DBT mother liquor of 80mM adds 7ul, the Tris-HCl buffer polishing of pH7.0 to one milliliter,
It is reacted five minutes in 60 DEG C of water-baths.It is added with the equimolar saualane of DBT in reaction solution as internal standard, then use respectively etc.
The pure n-hexane extraction of volumetric(al) chromatography.Sample extracted respectively takes 1ul to carry out gas chromatographic analysis.
Processing result is as follows:
Table 2: high temperature desulfurizing enzyme is to DBT disposition table
Conclusion: through high temperature enzyme, treated that DBT content declines to a great extent, and illustrates this group of high temperature enzyme significant desulfurization effect.
Embodiment 2:
(1) activation of recombinant bacterium: by the recombinant bacterium being deposited in glycerol stocks pipe (BL-1,2,3,4, D) by 1:1000(v/v) point
It is not seeded to through in autoclaved 50mL culture solution, 60 DEG C, 150rpm vortex stirring, closed (logical oxygen) is cultivated 18 hours.Its
In culture solution composition and weight percent are as follows: peptone 1.6%；Yeast extract 1%；Sodium chloride 0.5%；Water surplus.
(2) the expansion culture of recombinant bacterium: by the bacterium activated obtained by (1) respectively by 1:1000(v/v) it is seeded to and goes out through high pressure
In the 10L culture solution of bacterium, 60 DEG C, 150rpm vortex stirring, closed (logical oxygen) is cultivated 18 hours.The composition of culture solution therein and
Weight percent are as follows: peptone 1.6%；Yeast extract 1%；Sodium chloride 0.5%；Water surplus.
(3) recombinant bacterium handles high sulfur-containing diesel: will expand 5 kinds of 10L bacterium solutions after cultivating obtained by (2) and 20L high sulfur-containing diesel is mixed
It closes, 60 DEG C, 150rpm vortex stirring, confined reaction 24 hours；Bacteria-removing liquid, 60 DEG C of pure water, the whirlpool 150rpm are removed after the reaction was completed
Revolving stirring and washing, diesel oil is the low nitrogen diesel oil of low-sulfur to without obvious impurity, collecting that treated, is specifically shown in Table 3.
(4) it the measurement of present invention total sulfur content in oil sample system: is detected respectively with WK-2D type microcoulomb instrument without strain
The oil sample of processing and through total sulfur content in oil reservoir obtained by strain processed (3).
Processing result is as follows:
Table 3: recombinant bacterium is to high sulfur-containing diesel desulfurization situation table
The bacterial strain rebuild is 75.35% to the removal efficiency of the sulphur in high sulfur-containing diesel, and significant desulfurization effect, future has answers greatly very much
Use prospect.
Sequence table
<110>Nankai University
<120>one groups of high-temperature biological Desulfurization genes and application
<141> 2018-11-20
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1119
<212> DNA
<213>artificial sequence ()
<400> 1
atggtcgaat ttatcacaat ggccccaaca tcaggagaca gtacacttgt tgggttagcc 60
aataactcgt caaaactgaa tagctggaca ggaacagatg aaaatgcaga acgcccccct 120
acccaagaat atattaaagc gattgcccag gctgctgaaa aaggcggatt ctcgacgctt 180
ttactgccga ctggcacagg atgccttgat tctttagccg ttgctgccaa tttaatcgcc 240
tatacaagca aattaaaatt cctatttgca atccgccctg gttttatcgc acctactacc 300
tttgccaaac aatttgccac tgtggattat tggtcaaatg gaagagcctt ggtgaatatt 360
gttaccggag gttcgccggt cgagttagcc agcgaagggg attatttaga tcatgatacc 420
cgttataagc ggacacgcga atacatgcaa atattaaaaa aactatttac tgaagaaagc 480
gttgattacg aaggggaatt ttttacatta aaaggcgcct cattatttcc aaaaccagta 540
aaaacgccgc caatctattt tggtggggca tcagaaatcg ccaaagaagt cgcggcggaa 600
gaagcagatg tttatatgat gtggggggaa acttttgaaa atacaaaaca aagacttgaa 660
gaaatgaagc agagagcggc caaacataac cggacgctga gttatagcgt ttcgtttcaa 720
gtcatcctcg ggaacacgga agaagaagca tgggaaaagg caaacaagct gataagcaaa 780
gtgtcagcat ccatattagc caaaaaagag gaaatgattg tgaaaggaga ttctattggc 840
gcaaaacgcc tgcatcaact tatggaaagc agcaaagaac gtaactttca aattggcccg 900
aatttatggg cgggactgac tcaagtttta tccggaaatt ctattgccct tgtcggcact 960
cctgagcaaa tcgctgaacg gattgtcgaa ctggtggaat taggattcga taaagtgttg 1020
ttaagaggct tccctcattt ggaaacgatt gagcaattag gagagctagt cattccaaaa 1080
gtgagagaaa aactcgcaca aaaacaactt gtcaaatga 1119
<210> 2
<211> 969
<212> DNA
<213>artificial sequence ()
<400> 2
atgaggtcca tgaagcggag caagcatatt ctttggatca ttcatttcct cgtattttcc 60
cttttactgt catcctgcgg gaaagcggaa gaaacgggcg ggaaaaacaa agaaattcat 120
attggatatc aaaaaaatgg cactacctta ttgttaaaac ataaacaaga actgcaaaaa 180
gagttggaaa aagaaggata taaagtaaca tggtcagagt ttaacaccgg aagctcgatc 240
cttgaagcct taaatgctgg aagtattgat tttgccggcg caggggacat accgtccatc 300
tttgcgctgg agaaaggcag taattttaac tatattgcta gtgagccatc gtctccgtct 360
tcagagggaa tattggtgag aaaggattcg ggcattcaat cactggaaga tttaaaagga 420
aaaagaatcg cctttaataa ggcttccatc gctcaatatt tattaacaaa agctttagat 480
tcagcaggtt tatcgatgga tgatgtcgag ccggtgtacc tgaatcctcc tgaagcaagc 540
attgcttttg aacaaggcga agtggatgcg tgggtcgtat gggatcctta tatgacggtg 600
gccgaaagca aaggacacat cattttaaaa gatgcaactg gaattgttcc gtatcggact 660
ttttatttca gcactcctga aataacgaaa gaacatcccg aaatagtcaa aaaatttgtg 720
gaacatttat ctaatatagg aaaacaaatt aatcatgacc ctaccgaagc tgccgcgctg 780
ctgcaaaaag ccaccaatat cccggcggaa acgtgggaga aagtattaaa caataaaaag 840
tcagatgttc actttatgga tgaaaaagct gtgcgtgatt tgcaaacaga agcagatgat 900
ttgttaaaaa tcgggcttat caaaaaacaa gtgcagatcg aagattacgt atggtatcca 960
gagaaataa 969
<210> 3
<211> 1329
<212> DNA
<213>artificial sequence ()
<400> 3
atgaaaaaaa agcaaatgaa attgggcgtg tttctcatgg gaacaggcca ccatatcgca 60
tcttggagac atcctcatgt tcaagctgat ggatgtgagg atttcgcctt ttttcataaa 120
atagccaaaa tagcagaaaa aggaaaatta gatatactgt ttttaagcga cgggctgtct 180
tttaatgaac tttcgcatcc ggcggaatta gtgagatttg aacctatcac gttattggct 240
gcgctgtctg ttgtcacctc ccatatcggg ctggcagcga cagcgacgac cacctataac 300
gaacctttcc atatcgcgag aaagttttcc tctctcgatc atttaagcaa aggaagagcc 360
gcatggaatg tggtgacttc atactacgaa gacgaagcga aaaatttcag ccaggacgct 420
catttagacc atcatcttcg ttacgagcgg gcaaaagaat tcgtggaggt ggtgaaagga 480
ctgtgggaca gctgggaaaa ggatgcactc gtccgcgata aacaatcagg cgtttatttc 540
gatcctaaaa agctgcatcc gttaaaccat aaaggcaaat atttttctgt aaaaggcccg 600
ttgaattctt cccgctcccc gcaaggaagg ccggtcctta tccaagcagg gtcatcggaa 660
gacggaatca attttgccgc acaaattgca gacgtgattt tcaccgcgca acaaacattg 720
gaagaagctc agcattttta tcgaaaagtg aaaacgaaag cggcggaatt cggcagaaat 780
cctgatgaag tgattattat gcccggtgtt tctccatata taggcaatac agaacaagaa 840
gcgagagaaa aatatgaaca gctgcaagag cttattgttc ctgaaatcgg cttggctttt 900
ctgtctgact acttaggggg catcgatctt tctcgctact cattagatga tcctttgcca 960
gacgaaattc cagaaaccaa tggaaataaa agcagaagaa agttaatcat tgatcttgca 1020
agaagagaaa acttaacgat cggggagctt tataagcgca ttgccggctc gcggggacac 1080
cggatcattt tcggaacgcc agagcaaatc gcagatcagt tagaagaatg gatcatccac 1140
gaaggatccg atggcttcaa tctgatgttc ccgtattatc ctgacggcct atctgaattt 1200
gttgaccaag tgatcccgat ccttcaagaa agaggactgt tcagaaagga atatgaagga 1260
acaacgttac gggaacatct tggattgcct gagcctgaat caagatattc tctggcgccc 1320
agccaatga 1329
<210> 4
<211> 1326
<212> DNA
<213>artificial sequence ()
<400> 4
atgttaagaa attcgcaaag cagcggaacc agttttcttc tgcccctttt caactgggcg 60
cacgcgaaag aatgcattgc cggtcaaaaa tacaagaaag gagaaatcat gttgagcatc 120
gcaagcaaaa aaacccaccg ctcttattta tctgacgaac tgtcaagaaa gtttgtccaa 180
aatgaacgcc aagaatttct tttacatctt gcatcagaac ttgccgaaca atttcacgaa 240
accgccgaca tcatagacca agaaggaaga ttcccgtttg aaaatttcca aaaattaaag 300
gattgcaatt accaatcact aaccgttcct aagcaatatg gcggagaaga aatttcactt 360
tatgaatttc ttcttatgca agagcggtta agccaaggag atgcttccac cgccttgtgc 420
atcgggtggc atcttggggt tatttatgat ctcagggaaa gacaaacatg ggacagcgaa 480
aaatttcaat ggctttgcca tgaagttgtt caaaataaag tgctcatcaa ccgggtggca 540
acagaggacg gaacaggaag tccaacaaga ggcggaaaac ctgaaacggt agcggtcaaa 600
agaaatggca aatgggtgat tacgggcaga aaatcctttg cgtctatggc gatagctctt 660
gattattcat tagtaacagc caccattcag gaatcaggga aagtcggatc ttttttagtt 720
gaccatcgcc ttcaaggggt tagcgtcgaa gaaacttggg atatgattgg aatgcgaggc 780
acacggagcg atgatctggt gctagatcaa gtggagttgc cagaagatgc gctggtggaa 840
ttggatcaat tggaatcgcc gaaaggcaat atggggagag catggctact tcatgttccg 900
gcctgcttcc ttggaattgc gattgccgca agaaattatg ccatttcctt tgcttctgaa 960
tatcagccga acagcctgcc cggcccaatc aaagatgttc cagaggtgca aagaaaaatc 1020
ggcgagatgg atttagagct gttaaaagca agacatactc tttattccgt cgcacaccgg 1080
tgggatacgt acccggaaaa acgcatggaa atgagcggag aattggcggc ggcgaagcat 1140
attgccgtca atagtgccaa taaagtcgtt gatttggcga tgaggatcgt aggagccaga 1200
agcctgcaaa aatccagccc gttgcaacgg tattacagag atgtcagagc cgggctccat 1260
aatccgccaa tggatgatgc cgttatttcg ttactggcaa agcaagcatt gcaaagtttc 1320
cattaa 1326
<210> 5
<211> 468
<212> DNA
<213>artificial sequence ()
<400> 5
atggatgatc gtacatttcg cagagccatg ggaaaatttg cgaccggcgt gacggtcgtg 60
acaacggaat atcaagggga agcgaaagga atgacggcga acgcgtttat gtccgtttcg 120
ctcgacccga gacttgtcgt cgtctccatt ggccataaag cgcgaatgca tgacattgtc 180
aagcaaacgg ggaaatttgc tgtcaacatt ttgcggcgcg atcaagagga gttgtcgcgc 240
ttgtttgccg gccagttgaa agaagaacgc aatgtttcgt ttgattgggt gaacggccat 300
ccgattttgc cggaggcgtt ggcgaatatt ttatgcaacg tctatagttc gtacgttgcc 360
ggcgaccata cgttgtattt tggcgaagtc accgacattt tcatgaaaga ggaaccgggc 420
gacccgcttt tgtttttcga aggacaatac cgaagcatcg gacagtaa 468
<210> 6
<211> 373
<212> PRT
<213>high-temperature biological desulfurization core amino acid ()
<220>
<221> misc_feature
<222> (373)..(373)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> UNSURE
<222> (373)..(373)
<223> The 'Xaa' at location 373 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (373)..(373)
<223> The 'Xaa' at location 373 stands for Gln, Arg, Pro, or Leu.
<400> 6
Met Val Glu Phe Ile Thr Met Ala Pro Thr Ser Gly Asp Ser Thr Leu
1 5 10 15
Val Gly Leu Ala Asn Asn Ser Ser Lys Leu Asn Ser Trp Thr Gly Thr
20 25 30
Asp Glu Asn Ala Glu Arg Pro Pro Thr Gln Glu Tyr Ile Lys Ala Ile
35 40 45
Ala Gln Ala Ala Glu Lys Gly Gly Phe Ser Thr Leu Leu Leu Pro Thr
50 55 60
Gly Thr Gly Cys Leu Asp Ser Leu Ala Val Ala Ala Asn Leu Ile Ala
65 70 75 80
Tyr Thr Ser Lys Leu Lys Phe Leu Phe Ala Ile Arg Pro Gly Phe Ile
85 90 95
Ala Pro Thr Thr Phe Ala Lys Gln Phe Ala Thr Val Asp Tyr Trp Ser
100 105 110
Asn Gly Arg Ala Leu Val Asn Ile Val Thr Gly Gly Ser Pro Val Glu
115 120 125
Leu Ala Ser Glu Gly Asp Tyr Leu Asp His Asp Thr Arg Tyr Lys Arg
130 135 140
Thr Arg Glu Tyr Met Gln Ile Leu Lys Lys Leu Phe Thr Glu Glu Ser
145 150 155 160
Val Asp Tyr Glu Gly Glu Phe Phe Thr Leu Lys Gly Ala Ser Leu Phe
165 170 175
Pro Lys Pro Val Lys Thr Pro Pro Ile Tyr Phe Gly Gly Ala Ser Glu
180 185 190
Ile Ala Lys Glu Val Ala Ala Glu Glu Ala Asp Val Tyr Met Met Trp
195 200 205
Gly Glu Thr Phe Glu Asn Thr Lys Gln Arg Leu Glu Glu Met Lys Gln
210 215 220
Arg Ala Ala Lys His Asn Arg Thr Leu Ser Tyr Ser Val Ser Phe Gln
225 230 235 240
Val Ile Leu Gly Asn Thr Glu Glu Glu Ala Trp Glu Lys Ala Asn Lys
245 250 255
Leu Ile Ser Lys Val Ser Ala Ser Ile Leu Ala Lys Lys Glu Glu Met
260 265 270
Ile Val Lys Gly Asp Ser Ile Gly Ala Lys Arg Leu His Gln Leu Met
275 280 285
Glu Ser Ser Lys Glu Arg Asn Phe Gln Ile Gly Pro Asn Leu Trp Ala
290 295 300
Gly Leu Thr Gln Val Leu Ser Gly Asn Ser Ile Ala Leu Val Gly Thr
305 310 315 320
Pro Glu Gln Ile Ala Glu Arg Ile Val Glu Leu Val Glu Leu Gly Phe
325 330 335
Asp Lys Val Leu Leu Arg Gly Phe Pro His Leu Glu Thr Ile Glu Gln
340 345 350
Leu Gly Glu Leu Val Ile Pro Lys Val Arg Glu Lys Leu Ala Gln Lys
355 360 365
Gln Leu Val Lys Xaa
370
<210> 7
<211> 323
<212> PRT
<213>high-temperature biological desulfurization core amino acid ()
<220>
<221> misc_feature
<222> (323)..(323)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> UNSURE
<222> (323)..(323)
<223> The 'Xaa' at location 323 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (323)..(323)
<223> The 'Xaa' at location 323 stands for Gln, Arg, Pro, or Leu.
<400> 7
Met Arg Ser Met Lys Arg Ser Lys His Ile Leu Trp Ile Ile His Phe
1 5 10 15
Leu Val Phe Ser Leu Leu Leu Ser Ser Cys Gly Lys Ala Glu Glu Thr
20 25 30
Gly Gly Lys Asn Lys Glu Ile His Ile Gly Tyr Gln Lys Asn Gly Thr
35 40 45
Thr Leu Leu Leu Lys His Lys Gln Glu Leu Gln Lys Glu Leu Glu Lys
50 55 60
Glu Gly Tyr Lys Val Thr Trp Ser Glu Phe Asn Thr Gly Ser Ser Ile
65 70 75 80
Leu Glu Ala Leu Asn Ala Gly Ser Ile Asp Phe Ala Gly Ala Gly Asp
85 90 95
Ile Pro Ser Ile Phe Ala Leu Glu Lys Gly Ser Asn Phe Asn Tyr Ile
100 105 110
Ala Ser Glu Pro Ser Ser Pro Ser Ser Glu Gly Ile Leu Val Arg Lys
115 120 125
Asp Ser Gly Ile Gln Ser Leu Glu Asp Leu Lys Gly Lys Arg Ile Ala
130 135 140
Phe Asn Lys Ala Ser Ile Ala Gln Tyr Leu Leu Thr Lys Ala Leu Asp
145 150 155 160
Ser Ala Gly Leu Ser Met Asp Asp Val Glu Pro Val Tyr Leu Asn Pro
165 170 175
Pro Glu Ala Ser Ile Ala Phe Glu Gln Gly Glu Val Asp Ala Trp Val
180 185 190
Val Trp Asp Pro Tyr Met Thr Val Ala Glu Ser Lys Gly His Ile Ile
195 200 205
Leu Lys Asp Ala Thr Gly Ile Val Pro Tyr Arg Thr Phe Tyr Phe Ser
210 215 220
Thr Pro Glu Ile Thr Lys Glu His Pro Glu Ile Val Lys Lys Phe Val
225 230 235 240
Glu His Leu Ser Asn Ile Gly Lys Gln Ile Asn His Asp Pro Thr Glu
245 250 255
Ala Ala Ala Leu Leu Gln Lys Ala Thr Asn Ile Pro Ala Glu Thr Trp
260 265 270
Glu Lys Val Leu Asn Asn Lys Lys Ser Asp Val His Phe Met Asp Glu
275 280 285
Lys Ala Val Arg Asp Leu Gln Thr Glu Ala Asp Asp Leu Leu Lys Ile
290 295 300
Gly Leu Ile Lys Lys Gln Val Gln Ile Glu Asp Tyr Val Trp Tyr Pro
305 310 315 320
Glu Lys Xaa
<210> 8
<211> 443
<212> PRT
<213>high-temperature biological desulfurization core amino acid ()
<220>
<221> misc_feature
<222> (443)..(443)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> UNSURE
<222> (443)..(443)
<223> The 'Xaa' at location 443 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (443)..(443)
<223> The 'Xaa' at location 443 stands for Gln, Arg, Pro, or Leu.
<400> 8
Met Lys Lys Lys Gln Met Lys Leu Gly Val Phe Leu Met Gly Thr Gly
1 5 10 15
His His Ile Ala Ser Trp Arg His Pro His Val Gln Ala Asp Gly Cys
20 25 30
Glu Asp Phe Ala Phe Phe His Lys Ile Ala Lys Ile Ala Glu Lys Gly
35 40 45
Lys Leu Asp Ile Leu Phe Leu Ser Asp Gly Leu Ser Phe Asn Glu Leu
50 55 60
Ser His Pro Ala Glu Leu Val Arg Phe Glu Pro Ile Thr Leu Leu Ala
65 70 75 80
Ala Leu Ser Val Val Thr Ser His Ile Gly Leu Ala Ala Thr Ala Thr
85 90 95
Thr Thr Tyr Asn Glu Pro Phe His Ile Ala Arg Lys Phe Ser Ser Leu
100 105 110
Asp His Leu Ser Lys Gly Arg Ala Ala Trp Asn Val Val Thr Ser Tyr
115 120 125
Tyr Glu Asp Glu Ala Lys Asn Phe Ser Gln Asp Ala His Leu Asp His
130 135 140
His Leu Arg Tyr Glu Arg Ala Lys Glu Phe Val Glu Val Val Lys Gly
145 150 155 160
Leu Trp Asp Ser Trp Glu Lys Asp Ala Leu Val Arg Asp Lys Gln Ser
165 170 175
Gly Val Tyr Phe Asp Pro Lys Lys Leu His Pro Leu Asn His Lys Gly
180 185 190
Lys Tyr Phe Ser Val Lys Gly Pro Leu Asn Ser Ser Arg Ser Pro Gln
195 200 205
Gly Arg Pro Val Leu Ile Gln Ala Gly Ser Ser Glu Asp Gly Ile Asn
210 215 220
Phe Ala Ala Gln Ile Ala Asp Val Ile Phe Thr Ala Gln Gln Thr Leu
225 230 235 240
Glu Glu Ala Gln His Phe Tyr Arg Lys Val Lys Thr Lys Ala Ala Glu
245 250 255
Phe Gly Arg Asn Pro Asp Glu Val Ile Ile Met Pro Gly Val Ser Pro
260 265 270
Tyr Ile Gly Asn Thr Glu Gln Glu Ala Arg Glu Lys Tyr Glu Gln Leu
275 280 285
Gln Glu Leu Ile Val Pro Glu Ile Gly Leu Ala Phe Leu Ser Asp Tyr
290 295 300
Leu Gly Gly Ile Asp Leu Ser Arg Tyr Ser Leu Asp Asp Pro Leu Pro
305 310 315 320
Asp Glu Ile Pro Glu Thr Asn Gly Asn Lys Ser Arg Arg Lys Leu Ile
325 330 335
Ile Asp Leu Ala Arg Arg Glu Asn Leu Thr Ile Gly Glu Leu Tyr Lys
340 345 350
Arg Ile Ala Gly Ser Arg Gly His Arg Ile Ile Phe Gly Thr Pro Glu
355 360 365
Gln Ile Ala Asp Gln Leu Glu Glu Trp Ile Ile His Glu Gly Ser Asp
370 375 380
Gly Phe Asn Leu Met Phe Pro Tyr Tyr Pro Asp Gly Leu Ser Glu Phe
385 390 395 400
Val Asp Gln Val Ile Pro Ile Leu Gln Glu Arg Gly Leu Phe Arg Lys
405 410 415
Glu Tyr Glu Gly Thr Thr Leu Arg Glu His Leu Gly Leu Pro Glu Pro
420 425 430
Glu Ser Arg Tyr Ser Leu Ala Pro Ser Gln Xaa
435 440
<210> 9
<211> 442
<212> PRT
<213>high-temperature biological desulfurization core amino acid ()
<220>
<221> misc_feature
<222> (442)..(442)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> UNSURE
<222> (442)..(442)
<223> The 'Xaa' at location 442 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (442)..(442)
<223> The 'Xaa' at location 442 stands for Gln, Arg, Pro, or Leu.
<400> 9
Met Leu Arg Asn Ser Gln Ser Ser Gly Thr Ser Phe Leu Leu Pro Leu
1 5 10 15
Phe Asn Trp Ala His Ala Lys Glu Cys Ile Ala Gly Gln Lys Tyr Lys
20 25 30
Lys Gly Glu Ile Met Leu Ser Ile Ala Ser Lys Lys Thr His Arg Ser
35 40 45
Tyr Leu Ser Asp Glu Leu Ser Arg Lys Phe Val Gln Asn Glu Arg Gln
50 55 60
Glu Phe Leu Leu His Leu Ala Ser Glu Leu Ala Glu Gln Phe His Glu
65 70 75 80
Thr Ala Asp Ile Ile Asp Gln Glu Gly Arg Phe Pro Phe Glu Asn Phe
85 90 95
Gln Lys Leu Lys Asp Cys Asn Tyr Gln Ser Leu Thr Val Pro Lys Gln
100 105 110
Tyr Gly Gly Glu Glu Ile Ser Leu Tyr Glu Phe Leu Leu Met Gln Glu
115 120 125
Arg Leu Ser Gln Gly Asp Ala Ser Thr Ala Leu Cys Ile Gly Trp His
130 135 140
Leu Gly Val Ile Tyr Asp Leu Arg Glu Arg Gln Thr Trp Asp Ser Glu
145 150 155 160
Lys Phe Gln Trp Leu Cys His Glu Val Val Gln Asn Lys Val Leu Ile
165 170 175
Asn Arg Val Ala Thr Glu Asp Gly Thr Gly Ser Pro Thr Arg Gly Gly
180 185 190
Lys Pro Glu Thr Val Ala Val Lys Arg Asn Gly Lys Trp Val Ile Thr
195 200 205
Gly Arg Lys Ser Phe Ala Ser Met Ala Ile Ala Leu Asp Tyr Ser Leu
210 215 220
Val Thr Ala Thr Ile Gln Glu Ser Gly Lys Val Gly Ser Phe Leu Val
225 230 235 240
Asp His Arg Leu Gln Gly Val Ser Val Glu Glu Thr Trp Asp Met Ile
245 250 255
Gly Met Arg Gly Thr Arg Ser Asp Asp Leu Val Leu Asp Gln Val Glu
260 265 270
Leu Pro Glu Asp Ala Leu Val Glu Leu Asp Gln Leu Glu Ser Pro Lys
275 280 285
Gly Asn Met Gly Arg Ala Trp Leu Leu His Val Pro Ala Cys Phe Leu
290 295 300
Gly Ile Ala Ile Ala Ala Arg Asn Tyr Ala Ile Ser Phe Ala Ser Glu
305 310 315 320
Tyr Gln Pro Asn Ser Leu Pro Gly Pro Ile Lys Asp Val Pro Glu Val
325 330 335
Gln Arg Lys Ile Gly Glu Met Asp Leu Glu Leu Leu Lys Ala Arg His
340 345 350
Thr Leu Tyr Ser Val Ala His Arg Trp Asp Thr Tyr Pro Glu Lys Arg
355 360 365
Met Glu Met Ser Gly Glu Leu Ala Ala Ala Lys His Ile Ala Val Asn
370 375 380
Ser Ala Asn Lys Val Val Asp Leu Ala Met Arg Ile Val Gly Ala Arg
385 390 395 400
Ser Leu Gln Lys Ser Ser Pro Leu Gln Arg Tyr Tyr Arg Asp Val Arg
405 410 415
Ala Gly Leu His Asn Pro Pro Met Asp Asp Ala Val Ile Ser Leu Leu
420 425 430
Ala Lys Gln Ala Leu Gln Ser Phe His Xaa
435 440
<210> 10
<211> 156
<212> PRT
<213>high-temperature biological desulfurization core amino acid ()
<220>
<221> misc_feature
<222> (156)..(156)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> UNSURE
<222> (156)..(156)
<223> The 'Xaa' at location 156 stands for Gln, Arg, Pro, or Leu.
<220>
<221> UNSURE
<222> (156)..(156)
<223> The 'Xaa' at location 156 stands for Gln, Arg, Pro, or Leu.
<400> 10
Met Asp Asp Arg Thr Phe Arg Arg Ala Met Gly Lys Phe Ala Thr Gly
1 5 10 15
Val Thr Val Val Thr Thr Glu Tyr Gln Gly Glu Ala Lys Gly Met Thr
20 25 30
Ala Asn Ala Phe Met Ser Val Ser Leu Asp Pro Arg Leu Val Val Val
35 40 45
Ser Ile Gly His Lys Ala Arg Met His Asp Ile Val Lys Gln Thr Gly
50 55 60
Lys Phe Ala Val Asn Ile Leu Arg Arg Asp Gln Glu Glu Leu Ser Arg
65 70 75 80
Leu Phe Ala Gly Gln Leu Lys Glu Glu Arg Asn Val Ser Phe Asp Trp
85 90 95
Val Asn Gly His Pro Ile Leu Pro Glu Ala Leu Ala Asn Ile Leu Cys
100 105 110
Asn Val Tyr Ser Ser Tyr Val Ala Gly Asp His Thr Leu Tyr Phe Gly
115 120 125
Glu Val Thr Asp Ile Phe Met Lys Glu Glu Pro Gly Asp Pro Leu Leu
130 135 140
Phe Phe Glu Gly Gln Tyr Arg Ser Ile Gly Gln Xaa
145 150 155
Claims (4)
1. one group of high-temperature biological desulfurization nucleotide, which is characterized in that it can effectively remove the organic sulphur components in petroleum, and have
Good thermal stability；The nucleotide are as follows: shown in SEQ ID NO:1-5, the sequence of coding are as follows: SEQ ID NO:6-10
It is shown.
2. high-temperature biological desulfurization nucleotide described in claim 1, which is characterized in that specifically there is following gene:
。
3. one group of high-temperature biological desulfurization nucleotide answering in terms of for petrochemical industry and environmental protection described in claim 1
With；The petrochemical industry includes oil extraction in oil field, purification containing oil substance；Environmental protection includes microorganism remediation, fossil fuel
Deep desulfuration denitrogenation.
4. one group of high-temperature biological desulfurization nucleotide described in claim 1 is in the sulphur for removing in petroleum organic compounds containing sulfur
Application in terms of element.
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| CN110358743A (en) * | 2019-06-03 | 2019-10-22 | 华南理工大学 | A kind of monooxygenase DszC mutant and its preparation method and application |
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