An alkane-degrading gene, protein and their applications

Abstract

The present invention belongs to the fields of genetic engineering and enzyme engineering, and particularly relates to a petroleum hydrocarbon degradation gene and its application. The present invention provides a gene alkane-1-monooxygenase gene that can be used for degrading petroleum hydrocarbons alk B1_3, and the alkane-1-monooxygenase gene alk B1_3 is derived from the petroleum hydrocarbon-degrading bacterium Rhodococcus sp. RHZ01, and the alk nucleotide sequence of B1_3 is shown in SEQ IN NO.1. The present invention constructs a recombinant vector pMBP1- alk B1_3 containing the alkane-1-monooxygenase encoding gene alk B1_3, and expresses it in the prokaryotic host cell Escherichia coli BL21(DE3). Experiments prove that after the gene is expressed in the prokaryotic host cell, the obtained purified monooxygenase has the activity of catalyzing the hydrolysis of alkane substrates, and has great application potential in petroleum hydrocarbon degradation and bioremediation of contaminated soil.

Description

Technical Field

[0001] The present invention belongs to the fields of genetic engineering and enzyme engineering, and particularly relates to an alkane-degrading gene, protein capable of degrading petroleum-polluted soil and their applications. Background Art

[0002] With the development of industry, the world's demand for petroleum continues to grow, resulting in a large amount of petroleum pollutants pouring into the environment, affecting the physical and chemical properties of the soil and disrupting the biological balance, thus affecting the growth of various plants. Due to the high toxicity and carcinogenic, mutagenic and teratogenic potential of petroleum pollution, it also poses a certain threat to the health of animals and humans. Microbial remediation is regarded as an eco-friendly, low-cost and effective alternative to physical and chemical methods. The isolation, screening and application of highly efficient petroleum hydrocarbon-degrading bacteria have become the main research directions for the degradation of petroleum pollution. Among them, Rhodococcus can degrade environmental pollutants such as alkanes, aromatic hydrocarbons and polycyclic aromatic hydrocarbons in petroleum, and can also decompose lipase, nitrile hydratase and cholesterol oxidase, etc., and has great application prospects for the remediation of petroleum hydrocarbons.

[0003] In recent years, with the rapid development of various omics technologies, starting from different levels such as genomics, transcriptomics, proteomics and metabolomics, analyzing the reasons for the unique physiological and biochemical characteristics of Rhodococcus and excavating the corresponding genes can lay a foundation for the genetic transformation of strains. Reported alkane-degrading genes obtained from Rhodococcus include n-alkane monooxygenase, cytochrome P450-type alkane hydroxylase, dioxygenase, catechol dioxygenase and polycyclic aromatic hydrocarbon hydroxylase dioxygenase, etc. A strain of Rhodococcus qingshengii RHZ01 was isolated from petroleum-polluted soil in Dongying Shengli Oilfield, Shandong Province, China, which has a strong degradation effect on petroleum hydrocarbons. There is no report on the related degradation mechanism and molecular biology research of related functions of this strain. In view of this problem, the present invention obtained an alkane monooxygenase and its encoding gene by studying the genome of Rhodococcus qingshengii RHZ01, which is of great significance for the future research on the bioremediation mechanism of petroleum hydrocarbon-polluted sites and improving the remediation effect. Summary of the Invention

[0004] The present invention provides a petroleum hydrocarbon-degrading gene, alkane-1-monooxygenase gene alk B1. The present invention screened a petroleum hydrocarbon-degrading bacterium Rhodococcus RHZ01 from the soil in Dongying Shengli Oilfield, Shandong Province, China, which has been long-term polluted by petroleum. By analyzing the whole genome sequencing to excavate alkane-degrading functional genes, an alkane-1-monooxygenase gene alk B1_3 was obtained.

[0005] The present invention also provides an application of the above-mentioned degradation gene, alkane-1-monooxygenase gene alk B1_3 in degrading petroleum hydrocarbons.

[0006] The technical solution adopted by the present invention to achieve the above object is as follows:

[0007] The present invention provides a degradation gene alkane-1-monooxygenase gene in petroleum-contaminated soil alk B1_3, characterized in that the alkane-1-monooxygenase gene alk B1_3 is derived from the petroleum hydrocarbon-degrading bacterium Rhodococcus qingshengii RHZ01, and the alk nucleotide sequence of B1_3 is shown in SEQ IN NO.1.

[0008] Furthermore, the protein sequence of the alkane-1-monooxygenase gene alk B1_3 provided by the present invention in petroleum-contaminated soil is shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant cloning plasmid pMBP1- alk containing the above-mentioned degradation gene alkane-1-monooxygenase gene in petroleum-contaminated soil alk B1_3.

[0010] Another object of the present invention is to provide an Escherichia coli engineering strain alk BL21(DE3) / pMBP1- E.coli containing the above recombinant cloning plasmid pGM-T_ alk B1_3.

[0011] Preferably, the petroleum hydrocarbon is n-hexadecane or diesel, or an alkane or petroleum hydrocarbon having a structure similar to that of n-hexadecane or diesel.

[0012] The present invention provides an application of the induced enzyme solution of the above-mentioned recombinant engineering strain in degrading alkanes in petroleum-contaminated soil; preferably, the alkane is n-hexadecane and diesel; preferably, after the recombinant engineering strain is induced, the alkane-1-monooxygenase gene alk B1_3 can be highly expressed in the recombinant engineering strain, and after cell disruption and centrifugation, the supernatant is collected, and the monooxygenase is purified by specific adsorption of the target protein His tag.

[0013] The present invention has at least the following beneficial effects: The present invention constructs a recombinant vector pMBP1- alk containing the alkane-1-monooxygenase encoding gene alk B1_3, expresses it in the prokaryotic host cell Escherichia coli BL21(DE3), and experiments prove that after the gene is expressed in the prokaryotic host cell, the obtained purified monooxygenase has the activity of catalyzing the hydrolysis of alkane substrates and has great application potential in petroleum hydrocarbon degradation and bioremediation of contaminated soil. Description of the Drawings

[0014] Figure 1 : Electron microscopic morphology and colony plate morphology of strain RHZ01;

[0015] Figure 2 : Alkane-1-monooxygenase encoding gene alk PCR amplification of B1_3 and PCR detection map of recombinant vector;

[0016] Figure 3 : Recombinant vector pMBP1- alk Construction flow chart of B1_3;

[0017] Figure 4 : SDS-PAGE gel electrophoresis detection;

[0018] Figure 5 : E. coli Degradation of n-hexadecane by BL21 / pMBP1 and E.coli BL21 / pMBP1- alk B1_3 enzyme solution;

[0019] Figure 6 : E. coli Degradation of diesel by BL21 / pMBP1 and E.coli BL21 / pMBP1- alk B1_3 enzyme solution. Specific implementation mode

[0020] The following further describes the present invention in detail with reference to the attached drawings, so that those skilled in the art can implement it according to the description in the specification. The plasmids, strains and objects of microbial catalytic degradation cited in the following examples are only used to further describe the present invention in detail, and do not limit the essence of the present invention. All conditions not specified in the specific experiments are conventional conditions well known to those skilled in the art or conditions recommended by the manufacturer. The sources of the plasmids, strains and various molecular reagents cited in the examples are as follows:

[0021] Cloning vector pGM-T: A commercially available product of Tiangen Biochemical Technology (Beijing) Co., Ltd.;

[0022] Expression plasmid pMBP1: Provided by Jinan Anbot Biotechnology Co., Ltd.;

[0023] Escherichia coli Top10, BL21(DE3): Commercially available products of Tiangen Biochemical Technology (Beijing) Co., Ltd.;

[0024] Restriction endonucleases, DNA ligases, Taq enzymes, etc. were purchased from TaKaRa Company, and DNA extraction, purification, gel recovery kits, etc. were all purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0025] Example 1 Screening of Petroleum Hydrocarbon-Degrading Bacteria

[0026] Soil samples were collected from the soil of the Shengli Oilfield in Dongying, Shandong Province, China, which had been contaminated by petroleum for a long time. In an inorganic salt medium with diesel as the sole carbon source, a petroleum hydrocarbon-degrading bacterium RHZ01 capable of degrading diesel was isolated through enrichment culture. The degradation rate of this bacterium for diesel with a concentration of 4139.5 mg / L could reach over 60%. Through morphological observation, determination of physiological and biochemical indexes, and 16S rRNA analysis, it was preliminarily identified as Rhodococcus qingshengii ( Rhodococcus qingshengii ). The different conditions for the degradation of diesel by strain RHZ01 were studied through single-factor experiments. Strain RHZ01 grew well in MSM medium with diesel as the sole carbon source, and the growth curve reached its maximum on the 8th day. Optimization studies on various nitrogen sources showed that NH4NO3 was the best nitrogen source suitable for the growth of strain RHZ01. This strain grew best in MSM supplemented with Mg 2+ , and the degradation rate of diesel was 67.78%. When Cu 2+ was used to replace Mg 2+ , the growth rate of strain RHZ01 was slow and no diesel was degraded. The optimal pH value for the growth of the strain was 7.5. The growth and diesel degradation effect of this strain were the best in the range of 20 - 30 °C.

[0027] The electron microscopy morphology and colony plate morphology of the screened strain RHZ01 are as shown in Figure 1 .

[0028] The above-mentioned inorganic salt liquid medium (MSM): 1 g of NH4NO3, 1 g of NaCl, 1.5 g of K2HPO4, 0.5 g of KH2PO4, 0.2 g of MgSO4·7H2O; 1000 mL of distilled water; adjust the pH to 7.0 and autoclave at 121 °C for 20 min.

[0029] Example 2 Cloning of Alkane-1-Monooxygenase Gene

[0030] Activate Rhodococcus qingshengii RHZ01 in LB solid medium. Inoculate a single colony into LB liquid medium and culture it overnight at 30 °C on a shaker at 150 rpm. Inoculate the bacterial liquid into fresh LB liquid medium at a volume ratio of 1% and continue to culture it on a shaker until the logarithmic growth phase. Extract genomic DNA using the Bacterial Genomic DNA Rapid Extraction Kit from Sangon Biotech (Shanghai) Co., Ltd. The extracted genomic DNA sample of the strain was sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for whole-genome sequencing, assembly, and analysis. The results showed that the genome of Rhodococcus qingshengii RHZ01 was 6,506,318 bp in length and contained only one circular chromosome. The G+C content of the genome was 62.48%. Through genome annotation, a series of genes related to petroleum hydrocarbon degradation were found in this strain, including 5 alkane monooxygenase-encoding genes. Alkane monooxygenase is the key enzyme acting on the first step of the alkane degradation reaction. By comparing with the alkane monooxygenase-encoding genes in other Rhodococcus strains, it is speculated that these five alkane monooxygenase-encoding genes are involved in the petroleum hydrocarbon degradation process. Among these 5 encoding genes, only alk B1_1 and alk B1_3 are followed by two rubredoxin-encoding genes, and among them, the alk thermostability of the protein encoded by B1_3 is stronger than that of alk the protein encoded by B1_1.

[0031] Extract the total RNA of the bacteria, obtain cDNA through reverse transcription, and the transcriptome was sequenced and analyzed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. According to the analysis of the petroleum hydrocarbon degradation pathway of the transcriptome results, it was found that alk B1_3 in the medium with diesel and n-hexadecane as the sole carbon source showed a significant increase in induced expression. This indicates that this gene plays an important role in the metabolism of diesel or n-hexadecane. Combining the fact that the thermostability of the alkane-1-monooxygenase encoded by alkB1_3 is stronger than that of alk the protein encoded by B1_1, subsequent research work was carried out on the alkane monooxygenase-encoding alk B1_3.

[0032] According to the genomic information of Rhodococcus qingshengii RHZ01, through GenBank sequence alignment, using the Rhodococcus qingshengii genomic DNA of RHZ01 as a template, primers alk B1_3 were designed according to the alkane-1-monooxygenase gene alk B1_3 F and alkFor the target gene, PCR amplification was performed using B1_3 R to obtain a 1.2 kb fragment. After gel extraction of the PCR product, it was cloned onto the vector pGM-T, named pGM-T_alkB1, and verified by sequencing. Then, the alkB1 gene with sticky ends and the pMBP1 vector containing the Lac promoter were obtained by double digestion with HindⅢ / Xhol to construct the Escherichia coli expression vector pMBP-alkB1. This expression vector was transformed into Escherichia coli BL21(DE3). After PCR, enzyme digestion, and sequencing to verify that the inserted sequence was correct, this strain was named E.coli BL21(DE3) / pMBP1- alk B1_3. Escherichia coli BL21(DE3) containing the pMBP1 control empty plasmid was named E.coli BL21(DE3) / pMBP1.

[0033] In the PCR reaction system of the above example, the primer sequences were as follows:

[0034] alk B1_3 F: 5'-CG AAGCTT GTGTCGACGCACGTGGACTCGCAA-3'

[0035] alk B1_3 R: 5'-CG CTCGAG CGCCGCTCCGTACCGAGCAAG-3'

[0036] The underlined parts were the restriction sites of the restriction enzymes HindⅢ and Xhol respectively.

[0037] The PCR amplification of the alkane-1-monooxygenase encoding gene and the PCR detection of the recombinant vector are shown in Figure 2 Figure [Figure number not provided in the original].

[0038] Example 3: Induced expression of the recombinant vector pMBP- alk B1_3

[0039] The construction flowchart of the recombinant vector pMBP1- alk B1_3 is shown in Figure 3 Figure [Figure number not provided in the original]; the specific operation is as follows:

[0040] The expression host bacterium E. coli BL21(DE3) containing the recombinant plasmid was inoculated into a liquid LB medium with ampicillin and cultured in a constant temperature shaking incubator at 37 °C and 220 rpm until OD600 = 0.5 - 0.6. IPTG with a final concentration of 0.5 mM was added to induce expression for 3.5 hours at 20 °C and 150 rpm. The cells were collected, rinsed twice with phosphate buffer and then resuspended. The cells were lysed using an ultrasonic cell disruptor, and the expression was detected by SDS-PAGE protein electrophoresis. As Figure 4 shown, it was found that after induction with IPTG, an obvious specific protein band appeared at around 75 kDa in the strain containing the recombinant plasmid. The protein was expressed in both the supernatant and inclusion bodies, and soluble expression purification could be continued.

[0041] In the above example, the LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 ml of distilled water, pH 7.2 - 7.5, autoclaved at 121 °C for 20 min.

[0042] LB solid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 20 g of agar, 1000 ml of distilled water, pH 7.2 - 7.5, autoclaved at 121 °C for 20 min.

[0043] In the above example, the storage concentration of ampicillin was 100 mg / ml, which was added after the medium was cooled to room temperature, and its working concentration was 100 μg / ml.

[0044] Example 4: E.coli Degradation of n-hexadecane by BL21(DE3) / pMBP-alkB1_3

[0045] Pick E. coli BL21 / pMBP1-alkB1_3 and E. coliSingle colonies of BL21(DE3) / pMBP1 were separately inoculated into 50 mL of LB liquid medium containing ampicillin and cultured overnight. Expression was carried out according to Example 3. The bacterial liquid was poured into a centrifuge tube and centrifuged at 8000 rpm for 5 min at 4 °C. The supernatant was discarded. PBS was added to the centrifuge tube to wash the bacterial cell precipitate, and it was shaken and centrifuged, repeating 3 times. It was suspended in PBS according to a certain ratio. The bacterial cell suspension was put into an ultrasonic cell disruptor for cell disruption. The disruption conditions were 30%, 17 min, mode 02, φ06, ultrasound for 2.0 s, and interval for 2.0 s. After cell disruption, it was centrifuged and the supernatant (enzyme solution) was taken. The n-hexadecane substrate, enzyme solution, and PBS buffer were mixed according to a certain volume ratio. Among them, the initial concentration of n-hexadecane was 3867 mg / L. Under the condition of 37 °C, after the enzyme solution degradation reaction for 4 h, it was extracted with n-hexane, filtered by suction, rotary evaporated, and finally quantified to 900 uL with n-hexane. The content of n-hexadecane was detected by gas phase, and the removal rate of n-hexadecane was calculated. The experiment was repeated three times, and the treatment without bacteria was used as the control strain. The results showed that E. coli the enzyme solutions of BL21 / pMBP-alkB1_3 and E. coli BL21(DE3) / pMBP1 could both degrade n-hexadecane, and their relative degradation rates were 15.84% and 4.26% respectively. E. coli BL21 / pMBP1 had a certain removal effect on C16 and might be involved in the metabolic cycle of n-hexadecane. E. coli BL21 / pMBP1- alk B1_3 contained the target gene fragment, and its removal effect on n-hexadecane was stronger than that of the strain E.coli BL21 / pMBP1, indicating that the target gene fragment alk B1_3 was successfully expressed in the strain E.coli BL21 / pMBP1- alk B1_3; the specific degradation was as Figure 5 shown.

[0046] Example 5: E.coli Degradation of diesel by BL21(DE3) / pMBP1-alkB1_3

[0047] According to the method of Example 4, the removal effect of E. coli BL21 / pMBP1-alkB1_3 on diesel was detected, and the initial concentration of diesel was 4139.5 mg / L. The results showed that the enzyme solutions of E. coli BL21 / pMBP-alkB1_3 and E.coli BL21(DE3) / pMBP1 could both degrade diesel, and their relative degradation rates were 43.70% and 13.30% respectively.E.coli BL21 / pMBP1 has a certain degradation effect on diesel. E.coli Since BL21 / pMBP1-alkB1_3 contains the target gene fragment, its diesel removal effect is stronger than that of the strain E. coli BL21 / pMBP1. Meanwhile, the enzyme solution prepared from the bacteria containing this fragment has a higher diesel degradation effect than its own degradation effect on n-hexadecane, which has important application value in petroleum hydrocarbon contaminated soil; the specific degradation is as Figure 6 shown.

[0048] Sequence Listing

[0049] (1) alk The nucleotide sequence of the B1 gene.

[0050] GTGTCGACGC ACGTGGACTC GCAATCCGGC CAGACGCTCC CGCCCGAGCA GTGGCGTGAC

[0051] AAGAAGAGAT ATCTCTGGCT GCTCGGCCTC GTGCCGCCGA CGGCGGTCTT CATCGCCGTC

[0052] GGATTGGTCG CGTTGTTCAA CAGTCTCGGA TGGAATGCGG TCTCGCCGGT GTGGTGGTGG

[0053] ATCGGGCCGC TGCTCGTCTA CATCCTGCTC CCGATCCTCG ACGTCTTCTT CGGCCCGGAC

[0054] GGCGAGAATC CGCCGGACGA AGTGATGGAG CGCCTCGAGA ACGACAAGTA CTACCGGTAT

[0055] TGCACCTACA TCTACATACC GTTCCAGTTG GTCAGCCTGG TCCTGGCCTG CTACCTCTGG

[0056] TCGGCGACCG ATCTGTCCTG GCTCGGAATC GACGGGGGAC TTGGGCTGAT CTCCAAGATC

[0057] GGTCTGGCGA TCAGTATCGG TTGTGTCGCA GGAATCGGGA TCAACACCGC ACACGAGCTC

[0058] GGTCACAAGA AGGACGATCT CGAACGCTGG TTGTCGAAGA TCACTCTGGC GCAGTCGTTT

[0059] TACGGCCACT TCTACATCGA GCACAATCGT GGACACCACG TGCGCGTCGC CACGCCCGAG

[0060] GATCCGGCGT CGTCTCGCTT CGGTGAGAGC TTCTGGACCT TCCTGCCGCG CAGTGTGTGG

[0061] GGATCTCTGC GCTCGTCGTG GTCCCTGGAA AAGGCCAGAC TCGATCGACT GGGTAAGAAG

[0062] CCGTGGACAA TTCGCAACGA CGTCCTGCAT TCGTGGTTGA TGTCCGTCGT ACTGTTCGGT

[0063] GTCCTCGTCG CCGTCTTCGG CCTCTCGGTA CTGCCGTTCC TGGTGCTGCA GGCCGTGTTC

[0064] GGATTCTGTC TGCTCGAAAC CGTCAACTAC CTCGAACATT ACGGACTGAA GCGTCGTCGT

[0065] CTGGACAGCG GGCGGTACGA GCGTGCAGCA CCGGAGCACA GTTGGAACAG TGATCACATC

[0066] TGCACCAACA TCTTCCTGTA TCACCTCCAG CGTCACAGCG ATCATCACGC GAACCCGACG

[0067] CGTCGTTATC AGACGCTTCG AAGCATGGAC GGTGCGCCCA ACCTGCCCAG TGGGTACGCG

[0068] AGCATGATCA TCCTCGCCTA CGTTCCACCG CTGTGGCGAA AAGTGATGGA TCCCAAAGTC

[0069] CTTGCGCACT ACGGCGGCGA CATCACCCGC GTCAACGTTC AGCCGTCCAA GCGTGATCGC

[0070] ATCCTTGCTC GGTACGGAGC GGCGTGA

[0071] (2) Amino acid sequence of AlkB1 protein.

[0072] 1 Val Ser Thr His Val Asp Ser Gln Ser GlyGlnThrLeu ProProGluGlnTrpArg Asp 20

[0073] 21 Lys LysArg Tyr LeuTrpLeuLeuGlyLeu Val Pro ProThr Ala Val PheIle Ala Val 40

[0074] 41 GlyLeu Val Ala LeuPheAsn Ser LeuGlyTrpAsn Ala Val Ser Pro ValTrpTrpTrp 60

[0075] 61 Ile Gly Pro LeuLeu Val Tyr Ile LeuLeu Pro Ile Leu Asp ValPhePheGly Pro Asp 80

[0076] 81 GlyGluAsn Pro Pro Asp Glu Val Met GluArgLeuGluAsn Asp Lys TyrTyrArg Tyr 100

[0077] 101 CysThr Tyr Ile Tyr Ile Pro PheGlnLeu Val Ser Leu Val Leu AlaCys Tyr LeuTrp 120

[0078] 121 Ser Ala Thr Asp Leu Ser TrpLeuGly Ile Asp GlyGlyLeuGlyLeu IleSer Lys Ile 140

[0079] 141 GlyLeu Ala Ile Ser Ile GlyCys Val Ala Gly Ile Gly Ile AsnThrAla His GluLeu 160

[0080] 161 Gly His Lys Lys Asp AspLeuGluArgTrpLeu Ser Lys Ile ThrLeu AlaGln Ser Phe 180

[0081] 181 Tyr Gly His Phe Tyr Ile Glu His AsnArgGly His His Val Arg ValAla Thr Pro Glu 200

[0082] 201 Asp Pro Ala Ser SerArgPheGlyGlu Ser PheTrpThrPheLeu Pro Arg SerVal Trp 220

[0083] 221 Gly Ser LeuArg Ser SerTrp Ser LeuGlu Lys Ala ArgLeu AspArgLeuGly Lys Lys 240

[0084] 241 Pro TrpThr Ile ArgAsn Asp Val Leu His Ser TrpLeu Met Ser ValValLeuPheGly 260

[0085] 261 Val Leu Val Ala Val PheGlyLeu Ser Val Leu Pro PheLeu Val LeuGlnAla Val Phe 280

[0086] 281 GlyPheCysLeuLeuGluThr Val Asn Tyr LeuGlu His Tyr GlyLeu LysArgArgArg 300

[0087] 301 Leu Asp Ser GlyArg Tyr GluArg Ala Ala Pro Glu His Ser TrpAsnSer Asp His Ile 320

[0088] 321 CysThrAsn Ile PheLeu Tyr His LeuGlnArg His Ser Asp His His AlaAsn Pro Thr 340

[0089] 341 ArgArg Tyr GlnThrLeuArg Ser Met Asp Gly Ala Pro AsnLeu Pro SerGly Tyr Ala 360

[0090] 361 Ser Met Ile IleLeu Ala Tyr Val Pro ProLeuTrpArg Lys Val Met AspPro Lys Val 380

[0091] 381 Leu Ala His Tyr GlyGly Asp Ile ThrArg Val Asn Val Gln Pro SerLys Arg Asp Arg 400

[0092] 391 Ile Leu Ala Arg Tyr Gly Ala Ala End

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent substitutions, and these modifications or equivalent substitutions cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An alkane-1-monooxygenase gene alk The application of B1_3 in petroleum hydrocarbon degradation, characterized in that The alkane-1-monooxygenase gene alk B1_3 is derived from the petroleum hydrocarbon-degrading bacterium Rhodococcus sp. RHZ01, and alk the nucleotide sequence of B1_3 is shown in SEQ ID NO.1; its protein sequence is shown in SEQ ID NO.

2.

2. A recombinant vector pMBP1 - alk B1_3 containing the alkane - 1 - monooxygenase gene described in claim 1 alk Application of B1_3 in alkane degradation.

3. A recombinant engineering strain E.coli BL21(DE3) / pMBP1- alk The application of B1_3 in alkane degradation, characterized in that The recombinant vector pMBP1- described in claim 2 alk B1_3 is introduced into the host bacterium by heat shock to obtain the recombinant engineering strain E.coli BL21(DE3) / pMBP1_ alk B1_3.

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