CN109486837B

CN109486837B - High-temperature biological desulfurization genes and application

Info

Publication number: CN109486837B
Application number: CN201811380938.5A
Authority: CN
Inventors: 王威; 石钰琨
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2018-11-20
Filing date: 2018-11-20
Publication date: 2022-11-01
Anticipated expiration: 2038-11-20
Also published as: CN109486837A

Abstract

The invention relates to a group of high-temperature biological desulfurization genes and application thereof. The group of genes is obtained by screening microbial communities in an environment sample polluted by petroleum; the gene identified by the invention is high temperature resistant, has good thermal stability, and can be applied to industrial production requiring a thermal stability enzyme condition such as fermentation; the source flora can well grow in an oil reservoir environment, and the group of genes is proved to have the desulfurization capacity by cloning, expressing and in vitro verifying the group of genes, can obviously improve the biological desulfurization efficiency of fuel oil and effectively solve the problem of environmental pollution after use, has the excellent characteristics of industrial application, and has wide industrial application prospect.

Description

High-temperature biological desulfurization genes and application

Technical Field

The invention relates to the field of metagenome and environmental microorganism, in particular to a group of high-temperature biological desulfurization genes and application thereof.

Background

The 21 st century is an environmental protection century, and the energy industry, which is the leading force of the chemical industry, faces many challenges and opportunities in terms of environmental protection and technology, and thus creates an emerging discipline that intersects the biochemical and environmental protection fields. With the rapid development of the global industrialization process, the demand of energy is increased dramatically, and the proportion of petroleum in the world energy structure is 37.4% at present. The sulfur compounds exist in petroleum products, and the application of the product containing the sulfur compounds can cause pollution to the environment and corrode equipment. Therefore, desulfurization technology is an important research topic in petrochemical industry and environmental protection.

Deep desulfurization of petroleum and products thereof is a new technology in the petrochemical field of China and even all over the worldThe century faced an important issue. Sulfur in petroleum exists mainly as organic sulfides such as mercaptans, sulfides, thiophenes, benzothiophenes (BT), dibenzothiophenes (DBT), and alkyl substituents and derivatives thereof, and these compounds generate a large amount of thiocyanides during combustion, which are harmful to many kinds of plants. The most serious of them is SO produced by combustion₂And SO₃The water is easy to combine with water vapor in the atmosphere to form acid rain, which causes serious pollution, destroys ecological balance and endangers human health (Monticello D J. Depletion of folyl fuels. Ann Rev Microbiol.1985, 39. In recent years, desulfurization has become more and more of a concern to people with biotechnology. Biocatalytic Desulfurization (BDS) is a new technology for removing sulfur in petroleum sulfur-containing heterocyclic compounds by aerobic and anaerobic bacteria at normal temperature and pressure, which removes sulfur in fossil fuels by biochemical cracking by using enzymes specific to microorganisms, is carried out at normal temperature and pressure and under the condition of no need of hydrogen, can specifically cut carbon-sulfur bonds to release sulfur in the fossil fuels in the form of sulfate, and aims to remove sulfur by using microorganisms existing in the nature without damaging hydrocarbons with organic fuel value. The method has the characteristics of low process cost, energy consumption saving, simple flow, mild reaction conditions, clean environment and accordance with the development trend of ecological environment friendliness. The BDS technology has evolved over decades since its emergence and is still in the development stage. The biological catalytic desulfurization technology opens up a new way for removing organic sulfur components in petroleum. While the traditional biological desulfurization technology has various non-negligible disadvantages: high cost, large operation difficulty, limited types of removal substrates and insignificant removal effect, and the existing research reports of related desulfurization genes almost all refer to normal-temperature desulfurization genes.

The gene of the invention belongs to a high-temperature desulfurization gene, has the function of petroleum desulfurization, and can be applied to petrochemical industry, environmental protection industry and other related industrial production.

Disclosure of Invention

The invention mainly aims to provide a group of high-temperature biological desulfurization genes and characteristics and application thereof, wherein the group of genes is high-temperature resistant, has good thermal stability and can be applied to industrial production requiring a thermal stability enzyme condition such as fermentation; can effectively remove sulfur in sulfur-containing compounds in petroleum, and the application of the strain in sulfur-containing crude oil and fraction desulfurization.

The second purpose of the invention is to provide a group of recombinant plasmids (pET-28 a-1, 2, 3, 4, D) capable of expressing recombinant high-temperature desulfurization genes.

The third purpose of the invention is to provide a group of recombinant bacteria (BL 21-1, 2, 3, 4, D) capable of producing the recombinant high-temperature desulfurization enzyme.

The fourth purpose of the invention is to provide a method for removing sulfur element in sulfur-containing compounds by the strain

The fifth object of the present invention is to provide a method capable of screening a target gene in a metagenome.

In order to realize the purpose, the invention adopts the following technical scheme:

the group of high-temperature desulfurization genes provided by the invention is obtained by screening microbial communities in petroleum-polluted environmental samples; and the high-temperature resistant and high-temperature stable enzyme has good thermal stability, and can be applied to industrial production requiring thermal stability enzyme conditions such as fermentation and the like. The gene sequence is shown as SEQ ID NO 1-5; the corresponding amino acid sequence is SEQ ID NO 6-10.

(1) Dibenzothiophene (DBT) which is a mode sulfur-containing pollutant in petroleum is used as induction pressure, microbial flora capable of high-temperature desulfurization is screened from an environment sample polluted by petroleum, and then metagenome sequencing is carried out. The group of high temperature desulfurization genes was found by analyzing metagenome data and comparing with the reported gene dszABCD of the verified active normal temperature degradation DBT (see Table 1).

(2) Extracting metagenome of desulfurization microbial flora as template, amplifying several genes in large quantity by PCR technology, and transferring 5 genes into BL21 (DE 3) for cloning expression by using pET-28a as vector.

(3) The desulfurization ability of the enzymes was judged by purifying the clonally expressed enzymes and then degrading Dibenzothiophene (DBT) with these enzymes. The enzymes have important significance for desulfurization in petrochemical industry and environmental protection.

TABLE 1 high temperature desulfurization genes

orf	Gene
		60-DBT_C552551_6	Alkylsulfonate monooxygenase
60-DBT_C552551_5	Aliphatic sulfonate ABC transport substrate binding proteins
		60-DBT_C552551_3	FMN-dependent monooxygenases
60-DBT_C552551_2	acyl-CoA dehydrogenase
		60-DBT_C549025_1	Flavin oxidoreductase

The high temperature desulfurization-related genes are shown in Table 1

The nucleotide sequence of SEQ ID NO 1-5 provided by the invention is as follows:

60-DBT_C552551_6 SEQ ID No.1：

ATGGTCGAATTTATCACAATGGCCCCAACATCAGGAGACAGTACACTTGTTGGGTTAGCCAATAACTCGTCAAAACTGAATAGCTGGACAGGAACAGATGAAAATGCAGAACGCCCCCCTACCCAAGAATATATTAAAGCGATTGCCCAGGCTGCTGAAAAAGGCGGATTCTCGACGCTTTTACTGCCGACTGGCACAGGATGCCTTGATTCTTTAGCCGTTGCTGCCAATTTAATCGCCTATACAAGCAAATTAAAATTCCTATTTGCAATCCGCCCTGGTTTTATCGCACCTACTACCTTTGCCAAACAATTTGCCACTGTGGATTATTGGTCAAATGGAAGAGCCTTGGTGAATATTGTTACCGGAGGTTCGCCGGTCGAGTTAGCCAGCGAAGGGGATTATTTAGATCATGATACCCGTTATAAGCGGACACGCGAATACATGCAAATATTAAAAAAACTATTTACTGAAGAAAGCGTTGATTACGAAGGGGAATTTTTTACATTAAAAGGCGCCTCATTATTTCCAAAACCAGTAAAAACGCCGCCAATCTATTTTGGTGGGGCATCAGAAATCGCCAAAGAAGTCGCGGCGGAAGAAGCAGATGTTTATATGATGTGGGGGGAAACTTTTGAAAATACAAAACAAAGACTTGAAGAAATGAAGCAGAGAGCGGCCAAACATAACCGGACGCTGAGTTATAGCGTTTCGTTTCAAGTCATCCTCGGGAACACGGAAGAAGAAGCATGGGAAAAGGCAAACAAGCTGATAAGCAAAGTGTCAGCATCCATATTAGCCAAAAAAGAGGAAATGATTGTGAAAGGAGATTCTATTGGCGCAAAACGCCTGCATCAACTTATGGAAAGCAGCAAAGAACGTAACTTTCAAATTGGCCCGAATTTATGGGCGGGACTGACTCAAGTTTTATCCGGAAATTCTATTGCCCTTGTCGGCACTCCTGAGCAAATCGCTGAACGGATTGTCGAACTGGTGGAATTAGGATTCGATAAAGTGTTGTTAAGAGGCTTCCCTCATTTGGAAACGATTGAGCAATTAGGAGAGCTAGTCATTCCAAAAGTGAGAGAAAAACTCGCACAAAAACAACTTGTCAAATGA

60-DBT_C552551_5 SEQ ID No.2：

ATGAGGTCCATGAAGCGGAGCAAGCATATTCTTTGGATCATTCATTTCCTCGTATTTTCCCTTTTACTGTCATCCTGCGGGAAAGCGGAAGAAACGGGCGGGAAAAACAAAGAAATTCATATTGGATATCAAAAAAATGGCACTACCTTATTGTTAAAACATAAACAAGAACTGCAAAAAGAGTTGGAAAAAGAAGGATATAAAGTAACATGGTCAGAGTTTAACACCGGAAGCTCGATCCTTGAAGCCTTAAATGCTGGAAGTATTGATTTTGCCGGCGCAGGGGACATACCGTCCATCTTTGCGCTGGAGAAAGGCAGTAATTTTAACTATATTGCTAGTGAGCCATCGTCTCCGTCTTCAGAGGGAATATTGGTGAGAAAGGATTCGGGCATTCAATCACTGGAAGATTTAAAAGGAAAAAGAATCGCCTTTAATAAGGCTTCCATCGCTCAATATTTATTAACAAAAGCTTTAGATTCAGCAGGTTTATCGATGGATGATGTCGAGCCGGTGTACCTGAATCCTCCTGAAGCAAGCATTGCTTTTGAACAAGGCGAAGTGGATGCGTGGGTCGTATGGGATCCTTATATGACGGTGGCCGAAAGCAAAGGACACATCATTTTAAAAGATGCAACTGGAATTGTTCCGTATCGGACTTTTTATTTCAGCACTCCTGAAATAACGAAAGAACATCCCGAAATAGTCAAAAAATTTGTGGAACATTTATCTAATATAGGAAAACAAATTAATCATGACCCTACCGAAGCTGCCGCGCTGCTGCAAAAAGCCACCAATATCCCGGCGGAAACGTGGGAGAAAGTATTAAACAATAAAAAGTCAGATGTTCACTTTATGGATGAAAAAGCTGTGCGTGATTTGCAAACAGAAGCAGATGATTTGTTAAAAATCGGGCTTATCAAAAAACAAGTGCAGATCGAAGATTACGTATGGTATCCAGAGAAATAA

60-DBT_C552551_3 SEQ ID No.3：

ATGAAAAAAAAGCAAATGAAATTGGGCGTGTTTCTCATGGGAACAGGCCACCATATCGCATCTTGGAGACATCCTCATGTTCAAGCTGATGGATGTGAGGATTTCGCCTTTTTTCATAAAATAGCCAAAATAGCAGAAAAAGGAAAATTAGATATACTGTTTTTAAGCGACGGGCTGTCTTTTAATGAACTTTCGCATCCGGCGGAATTAGTGAGATTTGAACCTATCACGTTATTGGCTGCGCTGTCTGTTGTCACCTCCCATATCGGGCTGGCAGCGACAGCGACGACCACCTATAACGAACCTTTCCATATCGCGAGAAAGTTTTCCTCTCTCGATCATTTAAGCAAAGGAAGAGCCGCATGGAATGTGGTGACTTCATACTACGAAGACGAAGCGAAAAATTTCAGCCAGGACGCTCATTTAGACCATCATCTTCGTTACGAGCGGGCAAAAGAATTCGTGGAGGTGGTGAAAGGACTGTGGGACAGCTGGGAAAAGGATGCACTCGTCCGCGATAAACAATCAGGCGTTTATTTCGATCCTAAAAAGCTGCATCCGTTAAACCATAAAGGCAAATATTTTTCTGTAAAAGGCCCGTTGAATTCTTCCCGCTCCCCGCAAGGAAGGCCGGTCCTTATCCAAGCAGGGTCATCGGAAGACGGAATCAATTTTGCCGCACAAATTGCAGACGTGATTTTCACCGCGCAACAAACATTGGAAGAAGCTCAGCATTTTTATCGAAAAGTGAAAACGAAAGCGGCGGAATTCGGCAGAAATCCTGATGAAGTGATTATTATGCCCGGTGTTTCTCCATATATAGGCAATACAGAACAAGAAGCGAGAGAAAAATATGAACAGCTGCAAGAGCTTATTGTTCCTGAAATCGGCTTGGCTTTTCTGTCTGACTACTTAGGGGGCATCGATCTTTCTCGCTACTCATTAGATGATCCTTTGCCAGACGAAATTCCAGAAACCAATGGAAATAAAAGCAGAAGAAAGTTAATCATTGATCTTGCAAGAAGAGAAAACTTAACGATCGGGGAGCTTTATAAGCGCATTGCCGGCTCGCGGGGACACCGGATCATTTTCGGAACGCCAGAGCAAATCGCAGATCAGTTAGAAGAATGGATCATCCACGAAGGATCCGATGGCTTCAATCTGATGTTCCCGTATTATCCTGACGGCCTATCTGAATTTGTTGACCAAGTGATCCCGATCCTTCAAGAAAGAGGACTGTTCAGAAAGGAATATGAAGGAACAACGTTACGGGAACATCTTGGATTGCCTGAGCCTGAATCAAGATATTCTCTGGCGCCCAGCCAATGA

60-DBT_C552551_2 SEQ ID No.4：

ATGTTAAGAAATTCGCAAAGCAGCGGAACCAGTTTTCTTCTGCCCCTTTTCAACTGGGCGCACGCGAAAGAATGCATTGCCGGTCAAAAATACAAGAAAGGAGAAATCATGTTGAGCATCGCAAGCAAAAAAACCCACCGCTCTTATTTATCTGACGAACTGTCAAGAAAGTTTGTCCAAAATGAACGCCAAGAATTTCTTTTACATCTTGCATCAGAACTTGCCGAACAATTTCACGAAACCGCCGACATCATAGACCAAGAAGGAAGATTCCCGTTTGAAAATTTCCAAAAATTAAAGGATTGCAATTACCAATCACTAACCGTTCCTAAGCAATATGGCGGAGAAGAAATTTCACTTTATGAATTTCTTCTTATGCAAGAGCGGTTAAGCCAAGGAGATGCTTCCACCGCCTTGTGCATCGGGTGGCATCTTGGGGTTATTTATGATCTCAGGGAAAGACAAACATGGGACAGCGAAAAATTTCAATGGCTTTGCCATGAAGTTGTTCAAAATAAAGTGCTCATCAACCGGGTGGCAACAGAGGACGGAACAGGAAGTCCAACAAGAGGCGGAAAACCTGAAACGGTAGCGGTCAAAAGAAATGGCAAATGGGTGATTACGGGCAGAAAATCCTTTGCGTCTATGGCGATAGCTCTTGATTATTCATTAGTAACAGCCACCATTCAGGAATCAGGGAAAGTCGGATCTTTTTTAGTTGACCATCGCCTTCAAGGGGTTAGCGTCGAAGAAACTTGGGATATGATTGGAATGCGAGGCACACGGAGCGATGATCTGGTGCTAGATCAAGTGGAGTTGCCAGAAGATGCGCTGGTGGAATTGGATCAATTGGAATCGCCGAAAGGCAATATGGGGAGAGCATGGCTACTTCATGTTCCGGCCTGCTTCCTTGGAATTGCGATTGCCGCAAGAAATTATGCCATTTCCTTTGCTTCTGAATATCAGCCGAACAGCCTGCCCGGCCCAATCAAAGATGTTCCAGAGGTGCAAAGAAAAATCGGCGAGATGGATTTAGAGCTGTTAAAAGCAAGACATACTCTTTATTCCGTCGCACACCGGTGGGATACGTACCCGGAAAAACGCATGGAAATGAGCGGAGAATTGGCGGCGGCGAAGCATATTGCCGTCAATAGTGCCAATAAAGTCGTTGATTTGGCGATGAGGATCGTAGGAGCCAGAAGCCTGCAAAAATCCAGCCCGTTGCAACGGTATTACAGAGATGTCAGAGCCGGGCTCCATAATCCGCCAATGGATGATGCCGTTATTTCGTTACTGGCAAAGCAAGCATTGCAAAGTTTCCATTAA

60-DBT_C549025_1 SEQ ID No.5：

ATGGATGATCGTACATTTCGCAGAGCCATGGGAAAATTTGCGACCGGCGTGACGGTCGTGACAACGGAATATCAAGGGGAAGCGAAAGGAATGACGGCGAACGCGTTTATGTCCGTTTCGCTCGACCCGAGACTTGTCGTCGTCTCCATTGGCCATAAAGCGCGAATGCATGACATTGTCAAGCAAACGGGGAAATTTGCTGTCAACATTTTGCGGCGCGATCAAGAGGAGTTGTCGCGCTTGTTTGCCGGCCAGTTGAAAGAAGAACGCAATGTTTCGTTTGATTGGGTGAACGGCCATCCGATTTTGCCGGAGGCGTTGGCGAATATTTTATGCAACGTCTATAGTTCGTACGTTGCCGGCGACCATACGTTGTATTTTGGCGAAGTCACCGACATTTTCATGAAAGAGGAACCGGGCGACCCGCTTTTGTTTTTCGAAGGACAATACCGAAGCATCGGACAGTAA

the amino acid sequence SEQ ID NO provided by the invention is 6-10 as follows:

>60-DBT_C552551_6 SEQ ID No.6

MVEFITMAPTSGDSTLVGLANNSSKLNSWTGTDENAERPPTQEYIKAIAQAAEKGGFSTLLLPTGTGCLDSLAVAANLIAYTSKLKFLFAIRPGFIAPTTFAKQFATVDYWSNGRALVNIVTGGSPVELASEGDYLDHDTRYKRTREYMQILKKLFTEESVDYEGEFFTLKGASLFPKPVKTPPIYFGGASEIAKEVAAEEADVYMMWGETFENTKQRLEEMKQRAAKHNRTLSYSVSFQVILGNTEEEAWEKANKLISKVSASILAKKEEMIVKGDSIGAKRLHQLMESSKERNFQIGPNLWAGLTQVLSGNSIALVGTPEQIAERIVELVELGFDKVLLRGFPHLETIEQLGELVIPKVREKLAQKQLVKX

>60-DBT_C552551_5 SEQ ID No.7

MRSMKRSKHILWIIHFLVFSLLLSSCGKAEETGGKNKEIHIGYQKNGTTLLLKHKQELQKELEKEGYKVTWSEFNTGSSILEALNAGSIDFAGAGDIPSIFALEKGSNFNYIASEPSSPSSEGILVRKDSGIQSLEDLKGKRIAFNKASIAQYLLTKALDSAGLSMDDVEPVYLNPPEASIAFEQGEVDAWVVWDPYMTVAESKGHIILKDATGIVPYRTFYFSTPEITKEHPEIVKKFVEHLSNIGKQINHDPTEAAALLQKATNIPAETWEKVLNNKKSDVHFMDEKAVRDLQTEADDLLKIGLIKKQVQIEDYVWYPEKX

>60-DBT_C552551_3 SEQ ID No.8

MKKKQMKLGVFLMGTGHHIASWRHPHVQADGCEDFAFFHKIAKIAEKGKLDILFLSDGLSFNELSHPAELVRFEPITLLAALSVVTSHIGLAATATTTYNEPFHIARKFSSLDHLSKGRAAWNVVTSYYEDEAKNFSQDAHLDHHLRYERAKEFVEVVKGLWDSWEKDALVRDKQSGVYFDPKKLHPLNHKGKYFSVKGPLNSSRSPQGRPVLIQAGSSEDGINFAAQIADVIFTAQQTLEEAQHFYRKVKTKAAEFGRNPDEVIIMPGVSPYIGNTEQEAREKYEQLQELIVPEIGLAFLSDYLGGIDLSRYSLDDPLPDEIPETNGNKSRRKLIIDLARRENLTIGELYKRIAGSRGHRIIFGTPEQIADQLEEWIIHEGSDGFNLMFPYYPDGLSEFVDQVIPILQERGLFRKEYEGTTLREHLGLPEPESRYSLAPSQX

>60-DBT_C552551_2 SEQ ID No.9

MLRNSQSSGTSFLLPLFNWAHAKECIAGQKYKKGEIMLSIASKKTHRSYLSDELSRKFVQNERQEFLLHLASELAEQFHETADIIDQEGRFPFENFQKLKDCNYQSLTVPKQYGGEEISLYEFLLMQERLSQGDASTALCIGWHLGVIYDLRERQTWDSEKFQWLCHEVVQNKVLINRVATEDGTGSPTRGGKPETVAVKRNGKWVITGRKSFASMAIALDYSLVTATIQESGKVGSFLVDHRLQGVSVEETWDMIGMRGTRSDDLVLDQVELPEDALVELDQLESPKGNMGRAWLLHVPACFLGIAIAARNYAISFASEYQPNSLPGPIKDVPEVQRKIGEMDLELLKARHTLYSVAHRWDTYPEKRMEMSGELAAAKHIAVNSANKVVDLAMRIVGARSLQKSSPLQRYYRDVRAGLHNPPMDDAVISLLAKQALQSFHX

>60-DBT_C549025_1 SEQ ID No.10

MDDRTFRRAMGKFATGVTVVTTEYQGEAKGMTANAFMSVSLDPRLVVVSIGHKARMHDIVKQTGKFAVNILRRDQEELSRLFAGQLKEERNVSFDWVNGHPILPEALANILCNVYSSYVAGDHTLYFGEVTDIFMKEEPGDPLLFFEGQYRSIGQX

the invention further discloses application of a group of high-temperature biological desulfurization nucleotides in preparation of petrochemical industry and environmental protection; the petrochemical industry comprises oil extraction in oil fields and purification of petroleum-containing substances; the environmental protection comprises microbial remediation and deep desulfurization and denitrification of fossil fuel. In particular: the application of sulfur element in sulfur-containing organic compound. The invention adopts purified enzyme to degrade Dibenzothiophene (DBT) which is a mode sulfur-containing compound in petroleum; and detecting the DBT content in the sample before and after the enzyme degradation by adopting an internal standard method. The results show that:

(1) The group of high-temperature enzymes has obvious desulfurization effect which can reach more than 80%.

(2) The DBT/squalane (internal standard) content in two groups of samples which are not treated by high-temperature enzyme is 0.36 and 0.35 respectively, and the DBT/squalane (internal standard) content in two groups of samples which are treated by high-temperature enzyme is 0.062/0.067 respectively.

The specific implementation mode is as follows:

the present invention is described below with reference to examples, which are not intended to limit the present invention, and modifications and variations may be made thereto by those skilled in the art in light of the spirit of the present invention, and these modifications and variations are to be considered as within the scope of the present invention, the scope and spirit of which are defined by the appended claims; the reagents used therein are all commercially available.

Example 1

HMM (Hidden Markov Model) Model is utilized to screen high-temperature desulfurization genes

1. Establishment of HMM (Hidden Markov Model) Model

Because the high-temperature degradation gene of dibenzothiophene is unknown, the degradation pathway of benzothiophene in normal temperature bacteria is just started. Therefore, for the macro gene sequencing results of 60-5-BT and 60-5-DBT, the model sequence construction can be carried out only by the gene on the operon of the '4S' way of the normal-temperature dibenzothiophene degrading bacteria. We searched dszABC genes on operons of the identified functional "4S" pathway, and performed genetic relationship alignment on the sequences to be modeled of all homologous proteins, and the sequences in one branch of the evolutionary tree were taken as a family. Protein sequences of less than 4 protein sequences of the same family are not involved in subsequent alignment. The total number of DszA protein sequences, 9 DszB protein sequences and 13 DszC protein sequences used for establishing the model.

2. Model and metagenomic data comparison

HMM is used for constructing module sequences of the protein sequences, and the protein sequences are respectively searched in the extracted pro.fa file of the metagenome of the 60-5-DBT group. Setting E value>10-5, and aligning the searched sequences in NCBI database. All found genes were regressed onto spliced contigs (DszB proteins were mapped using alignment not meeting the threshold requirements) and found to be composed of 60-DBT _ C552551_6, 60-DBT _ C552551_5, 60-DBT _ C552551_3 and 60-DBT _ C552551_2 (which may correspond to each other)dszA、dszB、dszAAnddszC) The potential gene cluster of composition. Because DszD is a coenzyme, reports on the enzyme as a research object are rare. This part of the study therefore did not establish a model order for DszD as the subject. We compare the reported DszD single sequence with the macro genome 'pro.fa' file, and screen out the potential coenzyme gene 60-DBT _ C549025-1.

(II) extraction of microbial community Macro-genome

The screened microbial colonies were cultured in advance, and then the cells were collected at 5500 Xg at 4 ℃. The mycelia were suspended in 500. Mu.L of 50mM Tris-HCl pH8.0, centrifuged at 10000 Xg for 1 min, and the supernatant was discarded. mu.L of 50mM Tris-HCl pH8.0 was added again, and after the cells were sufficiently suspended, 20. Mu.L of 0.4M EDTA was added thereto and vortexed, and reacted in a water bath at 37 ℃ for 20 minutes. mu.L of 100 mg/mL lysozyme was added to the system, vortexed, and reacted in a 37 ℃ water bath for 30 minutes. Adding 10 μ L of 20 mg/mL proteinase K to the system, mixing by gentle shaking, adding 35 μ L of 10% SDS, mixing by shaking, reacting in 56 deg.C water bath for 1-3 hours, and mixing by inversion every 10 minutes until the solution is clear. 10 μ L of 10 mg/mL RNase A was added to the system, vortexed and reacted in a 65 ℃ water bath for 60 minutes. Adding 200 mu L of absolute ethyl alcohol into the system, after vortex shaking, transferring all precipitates and liquid into a collecting sleeve of a bacterial genome DNA extraction kit, and centrifuging 90 s at 10000 Xg. In order to improve the recovery rate, the waste liquid in the collecting pipe can be discarded after the liquid in the sleeve pipe is collected once again. Then according to the requirements of the kit specification, finishing the subsequent genome purification treatment, and redissolving the treated genome by using a proper amount of sterilized MQ and storing in a refrigerator at the temperature of-80 ℃.

(III) cloning and screening the high-temperature desulfurization gene.

Using 0.5. Mu.L (about 10 ng) of the above-mentioned metagenomic solution as a template, 30 cycles of PCR were carried out using the following oligonucleotide sequences as primers, respectively, according to the PCR cycle parameters set forth below.

The PCR cycle parameters were set as follows:

95℃，2min；95℃，30s；55℃，45s；72℃，90s；72℃，10min；4℃，2hr

60-DBT_C552551_6：

an upstream primer: 5 'CCGGAATTCATGGTCGAATTTATCACAATGGCC'

A downstream primer: 5 'CCCAAGCTTGGTCATTTGACAAGTTGTTTTTGTGCG'

60-DBT_C552551_5：

An upstream primer: 5 'CCGGAATTCATGAGGTCCATGAAGCGGAG'

A downstream primer: 5 'CGAGCTCTTATTTCTCTGGATACCATACGTAA'

60-DBT_C552551_3：

An upstream primer: 5 'CGAGCTCATGAAAAAAAAGCAAATGAAATTGG'

A downstream primer: 5 'CCCAAGCTTGGTCATTGGCTGGGCGCCAGAGAATAT'

60-DBT_C552551_2：

An upstream primer: 5 'CCGGAATTCATGTTAAGAAATTCGCAAAGCAGC'

A downstream primer: 5 'CCCAAGCTTGGTTAATGGAAACTTTGCAATGCTT'

60-DBT_C549025_1：

An upstream primer: 5 'CCGGAATTCATGGATGATCGTACATTTCGCAGAG'

A downstream primer: 5 'CCCAAGCTTGGTTACTGTCCGATGCTTCGGTATTGT'

After the five groups of PCR products are purified, the products are respectively cut by EcoR I/Hind III, ecoR I/Sac I, sac I/Hind III, ecoR I/Hind III and EcoR I/Hind III, and are respectively connected with plasmid pET-28a (+) which is subjected to enzymolysis by the same restriction endonuclease and gel cutting recovery, transformed into competent Escherichia coli DH5 alpha (stored in the laboratory), and then smeared on LB solid culture medium containing 50 mu g/mL Kan (kalamycin). Culturing at 37 ℃ for 16-18 hours, picking out single clone colonies for identification, wherein the plasmid pET-28a (+) inserted with the DNA sequence coded by 60-DBT _ C552551_2 is a recombinant plasmid pET-28a-1, the plasmid pET-28a (+) inserted with the DNA sequence coded by 60-DBT _ C552551_3 is a recombinant plasmid pET-28a-2, the plasmid pET-28a (+) inserted with the DNA sequence coded by 60-DBT _ C552551_5 is a recombinant plasmid pET-28a-3, the plasmid pET-28a (+) inserted with the DNA sequence coded by 60-DBT _ C552551_6 is a recombinant pET-28a-4, and the plasmid pET-28a (+) inserted with the DNA sequence coded by 60-DBT _ C549025_1 is a pET-28a-D. The DNA fragment was sequenced by Sanger dideoxy method, and the sequencing result showed that the inserted DNA sequence was correct. Then, the recombinant plasmids pET-28a-1, 2, 3, 4 and D are respectively transformed into Escherichia coli BL21, and the Escherichia coli BL21 is respectively named as BL21-1, 2, 3, 4 and D.

(IV) purification of recombinant high temperature desulfurization enzyme

The recombinant bacteria are respectively inoculated into 20mL LB culture medium containing 50 mug/mL Kan, cultured for 12 hours at 37 ℃ under 180rpm/min, then the culture is inoculated into 200mL LB culture medium containing 50 mug/mL Kan according to the inoculation amount of 1% (V/V) (2 shake flasks in total), when A600 is cultured at 37 ℃ under 220rpm/min and is 0.6, IPTG is added until the final concentration is 0.1 mM, and the induction is carried out for 4 hours at 37 ℃ under 180 rpm/min. The cells were collected by centrifugation, suspended in 50mM Tris-Cl (pH8.0) buffer, and the cells were disrupted by ultrasonic wave, and the supernatant was centrifuged to give a crude extract of recombinant high-temperature desulfurization enzyme. The supernatant was purified by a Chelating Sepharose (Chelating Sepharose) nickel affinity column chromatography, and the resulting enzyme preparation showed a band on SDS-PAGE. And the theoretically calculated molecular weights are respectively consistent with the detection result of SDS-PAGE.

(V) desulfurization Effect

Purified 60-DBT _ C552551_6, 60-DBT _ C552551_5, 60-DBT _ C552551_3, 60-DBT _ C552551_2 were added to 50ul, coenzyme 60-DBT _ C549025_1 plus 30ul,100mM FMN plus 2ul,50mM NADH plus 2ul, 80mM DBT mother liquor plus 7ul, and Tris-HCl buffer solution at pH7.0 in a 1.5ml centrifuge tube, and reacted in a60 ℃ water bath for five minutes. And adding squalane in the same mole as DBT into the reaction solution as an internal standard, and extracting by using isovolumetric chromatogram pure hexane respectively. 1ul of each extracted sample was analyzed by gas chromatography.

The treatment results are given in the following table:

table 2: table of DBT treatment conditions by high temperature desulfurization enzyme

And (4) conclusion: the DBT content after the high-temperature enzyme treatment is greatly reduced, which shows that the group of high-temperature enzymes has remarkable desulfurization effect.

Example 2:

(1) Activation of the recombinant bacteria: the recombinant bacteria (BL-1, 2, 3, 4, D) stored in the glycerol storage tube were inoculated into 50mL of an autoclaved culture medium at 1 (v/v) and subjected to vortex stirring at 150rpm at 60 ℃ for 18 hours in a closed state (aerobic culture). The culture solution comprises the following components in percentage by weight: 1.6 percent of peptone; 1% of yeast extract; 0.5 percent of sodium chloride; the balance of water.

(2) And (3) carrying out expanded culture on the recombinant bacteria: the activated bacteria obtained in (1) were inoculated into 10L of autoclaved culture medium at 1. The culture solution comprises the following components in percentage by weight: peptone 1.6%; 1% of yeast extract; 0.5 percent of sodium chloride; the balance of water. .

(3) Treating the high-sulfur diesel oil by using recombinant bacteria: mixing 10L of the bacterial liquid obtained in the step (2) after the expanded culture with 20L of high-sulfur diesel oil, carrying out vortex stirring at 60 ℃ and 150rpm, and carrying out closed reaction for 24 hours; after the reaction is finished, removing the bacterial liquid, carrying out vortex stirring and cleaning on purified water at 60 ℃ and 150rpm until no obvious impurity exists, and collecting the treated diesel oil, namely the low-sulfur low-nitrogen diesel oil, which is shown in table 3.

(4) The invention is used for measuring the total sulfur content in an oil sample system: and (3) respectively detecting the total sulfur content in the oil layer obtained by the oil sample which is not treated by the strain and the oil layer obtained by the oil layer treated by the strain (3) by using a WK-2D type micro coulometer.

The treatment results are given in the following table:

table 3: table of desulfurization conditions of recombinant bacteria on high-sulfur diesel oil

The removal rate of the reconstructed bacterial strain to sulfur in high-sulfur diesel oil is 75.35%, the desulfurization effect is obvious, and the method has a great application prospect in the future.

Sequence listing

<110> university of southern kayak

<120> a group 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 of nuclear amino acids ()

<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 of nuclear amino acids ()

<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 of nuclear amino acids ()

<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 of nuclear amino acids ()

<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 of nuclear amino acids ()

<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

1. A group of separated nucleotide sequences for coding high-temperature biological desulfurization genes, which is characterized in that the nucleotide sequences can effectively remove organic sulfur components in petroleum and have good thermal stability; the nucleotide sequence is as follows: 1-5, and the coded sequence is as follows: SEQ ID NO 6-10.

2. The use of the set of isolated nucleotide sequences encoding a hyperthermophilic biodesulfurization gene according to claim 1 in petrochemical industry and environmental protection; the petrochemical industry comprises oil extraction in oil fields and purification of petroleum-containing substances; the environmental protection comprises microbial remediation and deep desulfurization and denitrification of fossil fuel.

3. The use of the set of isolated nucleotide sequences encoding a high temperature biodesulfurization gene of claim 1 for the removal of elemental sulfur from petroleum sulfur-containing organic compounds.