Alkane hydroxylase from delftia tsuruhatensis and its encoding gene and application

Abstract

The present application relates to the field of biotechnology, disclose a kind of alkane hydroxylase derived from orange ditzia and its coding gene and application.The present application obtains the coding gene of alkane hydroxylase from a strain of orange ditzia, is expressed into pseudomonas fluorescens and foul-smelling pseudomonas by being transferred, can obviously improve the ability of recipient bacteria to degrade alkane, therefore, the alkane hydroxylase of the present application has good application prospect in microbial oil recovery and oil pollution control.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a novel alkane hydroxylase derived from Dictamnus orangeis, its encoding gene, and its applications. Background Technology

[0002] Microbial degradation of alkanes can occur through various pathways, such as terminal oxidation, subterminal oxidation, ω-oxidation, and β-oxidation [Trotsenko YA, Murrell JC. 2008, Metabolic aspects of aerobic obligatemethanotrophy. [J] Advances in Applied Microbiology, 63: 183-229]. Terminal oxidation is the most common pathway, where microorganisms use monooxygenases to oxidize the methyl group at the end of the alkane, converting it to an alcohol. The corresponding alcohol dehydrogenase then converts it to an aldehyde and fatty acid. The fatty acid undergoes β-oxidation to generate acetyl-CoA, which enters the central metabolic pathway and is converted into CO2 and H2O. In ω-oxidation, fatty acids undergo double-terminal oxidation to become dicarboxylic acids before entering β-oxidation. Subterminal oxidation differs from terminal oxidation in that the alkane first becomes a secondary alcohol, then a methyl ketone, which is converted to a primary alcohol via acetyl esterification, and finally into fatty acids before entering β-oxidation. In all three pathways, alkane hydroxylases catalyze the first step of the reaction, making them key enzymes in alkane degradation. The activity of these enzymes determines the efficiency of the entire degradation process.

[0003] Long-chain alkanes typically refer to alkanes with more than 10 carbon atoms. Due to their long carbon chains, stable chemical bonds, and strong hydrophobicity, they are not easily biodegraded. Currently discovered long-chain alkane-degrading enzymes mainly include alkane hydroxylases and cytochrome P450s; both enzymes degrade alkanes through terminal oxidation. As early as 1998, Ratajczak A et al. discovered the alkane hydroxylase AlkM in the Acinetobacter baylyi ADP1 strain [Ratajczak A, W, Hillen W. 1998, Alkane hydroxylase from Acinetobacter sp. ADP1 is encoded by alkM and belongs to a new family of bacterial integral-membrane hydrocarbon hydroxylases [J]. Applied and Environmental Microbiology, 64(4): 1175-1179], which can catalyze the degradation of C12-C36 alkanes. Subsequently, in 2001, Tani A et al. discovered two homologous genes of alkM in Acinetobacter sp. M-1 strain, named alkMa and alkMb respectively. This strain can degrade C20-C44 alkanes [Tani A, Ishige T, Sakai Y, et al. 2001, Gene structures and regulation of the alkane hydroxylase complex in Acinetobacter sp. strain M-1 [J]. Journal of Bacteriology, 183(5): 1819-1823]. The CYP116B5 enzyme in the cytochrome P450 family enables Acinetobacter radioresistens strain to grow using C14-C36 as the sole carbon source [Minerdi D, Sadeghi SJ, Di Nardo G, et al. 2015, CYP116B5: a new class VII catalytically self-sufficient cytochrome P450 from Acinetobacter radioresistens that enables growth on alkanes [J]. Molecular Microbiology, 95(3): 539-554].In addition, there are two representative genes for degrading solid long-chain alkanes, namely almA and ladA. almA, from Acinetobacter sp. strain DSM 17874, can specifically degrade genes with carbon chains exceeding C30 [Throne-Holst M, Wentzel A, Ellingsen TE, et al. 2007, Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. strain DSM17874 [J]. Applied and Environmental Microbiology, 73(10): 3327-3332]; the thermophilic enzyme LadA, from Bacillus thermophilus strain NG80-2, can perform terminal oxidation of C15-C36 alkanes [Li L, Liu XQ, Yang W, et al. 2008, Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unlocking the long-chain alkane hydroxylase[J]. Journal of Molecular Biology, 376(2):453-465].

[0004] Increased long-chain alkanes in crude oil lead to higher heavy component content, increased crude oil viscosity, and thus reduced fluidity, thereby limiting crude oil extraction [Van Hamme J, Singh A, Ward OP. 2003, Recent advances in petroleum microbiology [J]. Microbiology and Molecular Biology Reviews, 67(4): 503-549]. Microbial-enhanced heavy crude oil extraction was proposed in the 1920s and received increasing attention as a microbial enhanced oil recovery (MEOR) technology in the 1980s [Donaldson, EC, GV Chilingarian and TF Yen. 1989. Introduction, p. 1–15. In. EC Donaldson, GV Chilingarian and TF Yen (ed.), Microbial enhanced oil recovery. Elsevier, New York, NY]. The ability of microorganisms to degrade long-chain alkanes is one of the important factors affecting the application effect of MEOR technology. Furthermore, the pollution caused by petroleum hydrocarbons during the extraction, transportation, or storage of petroleum resources cannot be ignored [Atlas RM, Bartha R. 1992. Hydrocarbon biodegradation and oil spill bioremediation. In: Marshall KC, eds. Advances in Microbial Ecology. New York: Springer US, 287-338]. Currently, the main technologies for treating petroleum pollution include physical, chemical, and biological methods. Bioremediation utilizes highly efficient petroleum hydrocarbon-degrading microorganisms to remediate petroleum pollution. Although microbial remediation has advantages such as low cost, convenience, non-toxicity, and no secondary pollution compared to physical and chemical methods, making it one of the most promising remediation methods, the degradation of structurally stable long-chain alkane pollution still faces many challenges. Researching new alkane hydroxylases with excellent performance and improving the degradation capacity of bacterial strains has significant application prospects. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a new alkane hydroxylase, its encoding gene, and its applications.

[0006] To achieve the above objectives, the present invention provides an alkane hydroxylase, which is (a) or (b):

[0007] (a) An alkane hydroxylase consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0008] (b) A protein derived from (a) whose amino acid sequence shown in SEQ ID NO: 2 has been substituted, deleted or added with one or more amino acids and whose enzyme activity remains unchanged, or a protein with a tagged amino acid sequence attached to the amino terminus and / or carboxyl terminus of SEQ ID NO: 2.

[0009] Secondly, the present invention provides a gene capable of encoding the alkane hydroxylase described in the first aspect.

[0010] Thirdly, the present invention provides a recombinant vector containing the gene described in the second aspect.

[0011] Fourthly, the present invention provides a recombinant strain containing the recombinant vector described in the third aspect.

[0012] Fifthly, the present invention provides a method for preparing alkane hydroxylase, the method comprising: (1) culturing the recombinant strain described in the fourth aspect and inducing the expression of a gene encoding alkane hydroxylase; and (2) isolating and purifying the expressed alkane hydroxylase.

[0013] In a sixth aspect, the present invention provides a composition for degrading alkanes, the composition containing the alkane hydroxylase described in the first aspect as an active ingredient, wherein the content of the alkane hydroxylase is 10-90% by weight based on the total weight of the composition.

[0014] In a seventh aspect, the present invention provides the use of the alkane hydroxylase described in the first aspect, the gene described in the second aspect, the recombinant vector described in the third aspect, the recombinant strain described in the fourth aspect, and the composition described in the sixth aspect in improving the cellular alkane utilization capacity or degrading alkane.

[0015] After the encoding gene of the alkane hydroxylase (AlkW) of the present invention was introduced into recipient cells (recipient bacteria), the ability of the recipient cells to degrade alkanes was significantly improved. Therefore, the alkane hydroxylase of the present invention has good application prospects in microbial enhanced oil recovery and oil pollution control. Attached Figure Description

[0016] Figure 1 Agarose gel electrophoresis image of the expression plasmid pCom8-alkW constructed in this invention;

[0017] Figure 2 GCMS map of long-chain alkanes degraded by the host bacteria expressing the alkW gene of this invention;

[0018] Figure 3The degradation rate of alkanes of different chain lengths by the host bacterium KOB2Δ1, which expresses the AlkW of the present invention;

[0019] Figure 4 The relative enzyme activity of the AlkW enzyme protein of the present invention;

[0020] Figure 5 GCMS spectrum of long-chain alkanes by KT2440, the host bacterium expressing the AlkW of the present invention. Detailed Implementation

[0021] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0022] The alkane hydroxylase provided by this invention is (a) or (b):

[0023] (a) An alkane hydroxylase consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0024] (b) A protein derived from (a) whose amino acid sequence shown in SEQ ID NO: 2 has been substituted, deleted, or added with one or more amino acids without altering its enzyme activity; or a protein with the amino acid sequence shown in SEQ ID NO: 2 tagged at its amino terminus and / or carboxyl terminus. Wherein, altered enzyme activity means that, under the same assay conditions, the percentage (relative activity) between the ability of the protein derived from (a) to degrade alkanes and the ability of (a) to degrade alkanes is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%).

[0025] The 20 amino acid residues that make up proteins can be divided into four categories according to the polarity of their side chains: 1. Nonpolar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro); 2. Polar, uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine ​​(Cys), aspartic acid (Asn), glutamine (Gln), and tyrosine (Tyr); 3. Positively charged amino acids: arginine (Arg), lysine (Lys), and histidine (His); 4. Negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu) (see "Biochemistry" (Second Edition), Volume 1, Shen Tong and Wang Jingyan, pp. 82-83, Higher Education Press, December 1990). If a substitution of an amino acid residue belonging to the same category occurs in a protein, such as Arg replacing Lys or Leu replacing Ile, the function of that residue in the protein's structural domain (e.g., providing a positive charge or forming a hydrophobic sac structure) remains unchanged. Therefore, the protein's three-dimensional structure is not affected, and the protein can still perform its function. This substitution of an amino acid residue belonging to the same category can occur at any amino acid residue position in the aforementioned alkane hydroxylases.

[0026] As previously stated, the alkane hydroxylase provided by this invention can be further modified or mutated to obtain derived proteins. The term "derived protein" in this invention refers to an alkane hydroxylase having an amino acid sequence difference from the one described above, or a difference in modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. The induced variants can be obtained through various techniques, such as random mutations induced by radiation or mutagens, or through techniques such as site-directed mutagenesis or other known molecular biology techniques. The "derived protein" also includes analogs containing residues of naturally occurring L-type amino acids (such as D-type amino acids), and analogs containing non-naturally occurring or synthetic amino acids (such as β-amino acids, γ-amino acids, etc.).

[0027] Modifications (typically without altering the primary structure, i.e., without changing the amino acid sequence) include chemically derived forms of proteins, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those occurring during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to glycosylating enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Furthermore, modifications can also be applied to proteins that improve their resistance to proteolysis or optimize their solubility.

[0028] To facilitate purification, (a) can be modified using tags commonly used in the art. For example, (b) can be obtained by attaching a tag (such as at least one of Poly-Arg, Poly-His, FLAG, Strep-tagⅡ, and c-myc) to the amino and / or carboxyl terminus of (a). The tag does not affect the activity of the alkane hydroxylase of the present invention, and in practical applications, the addition of a tag can be selected as needed.

[0029] The aforementioned alkane hydroxylases can be obtained through artificial synthesis, or by first synthesizing their encoding genes and then obtaining them through biological expression.

[0030] The present invention also provides a gene capable of encoding the above-mentioned alkane hydroxylase. Accordingly, the gene may be (1) or (2) as follows:

[0031] (1) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 1;

[0032] (2) A DNA molecule that hybridizes to the DNA sequence defined in (1) under stringent conditions and whose encoding alkane hydroxylase activity remains unchanged. The stringent conditions may be: hybridization at 65°C in a solution of 6×SCC, 0.5% SDS, followed by washing once each with 2×SCC, 0.1% SDS and 1×SCC, 0.1% SDS. Unchanged enzyme activity means that, under the same assay conditions, the percentage (relative activity) between the ability of the protein encoded by (2) to degrade alkanes and the ability of the protein encoded by (1) to degrade alkanes is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%).

[0033] As is well known in the art, of the 20 different amino acids that make up proteins, except for Met (ATG) or Trp (TGG), which are encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see Appendix D, page 950). That is, due to the degeneracy of the genetic codon, most amino acids are determined by more than one codon. The substitution of the third nucleotide in a triplet codon often does not change the amino acid composition; therefore, genes encoding the same protein can have different nucleotide sequences. Based on known codon tables, those skilled in the art can deduce the nucleotide sequences of genes encoding these proteins from the amino acid sequences disclosed in this invention, and from amino acid sequences that do not change the activity of alkane hydroxylases obtained from said amino acid sequences. These nucleotide sequences can be obtained through biological methods (such as PCR, mutation methods) or chemical synthesis methods; therefore, all such nucleotide sequences should be included within the scope of this invention. Conversely, using the DNA sequence disclosed herein, one can also obtain an amino acid sequence consistent with the alkane hydroxylase activity described herein by means of methods known in the art, such as those of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989), by modifying the nucleic acid sequence provided by the present invention.

[0034] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO: 1.

[0035] As described above, the 5′ and / or 3′ ends of the nucleotide sequence may also be linked with the coding sequence of the tag shown above.

[0036] The nucleotide sequences provided by this invention can generally be obtained using polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. For example, those skilled in the art can easily obtain templates and primers based on the nucleotide sequences provided by this invention, and use PCR to amplify the relevant sequences.

[0037] Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities using recombinant methods. Typically, the obtained nucleotide sequence is cloned into a vector, then transferred into genetically engineered bacteria, and finally isolated from the proliferated host cells using conventional methods.

[0038] In addition, known methods of artificial chemical synthesis can be used to synthesize the relevant nucleotide sequences.

[0039] The recombinant vector provided by this invention contains the gene provided by this invention.

[0040] The recombinant vector is preferably the recombinant plasmid pCom8-alkW. The "vector" used in the recombinant vector can be any vector known in the art, such as commercially available plasmids, granules, bacteriophages, and retroviruses; the present invention preferably uses the pCom8 plasmid. The recombinant vector can be constructed by digesting it with various endonucleases (such as NdeI and HindIII) that have cleavage sites at the vector's multiple cloning site to obtain a linear plasmid, which is then ligated with a gene fragment digested with the same endonuclease to obtain the recombinant plasmid. The present invention preferably uses NdeI and HindIII to double-digest pCom8 and the gene fragment ligated thereto, followed by ligation with a ligase to construct the recombinant vector pCom8-alkW.

[0041] The recombinant strain provided by this invention contains the recombinant vector provided by this invention.

[0042] The recombinant vector can be transformed, transduced, or transfected into host cells (strains) using conventional methods in the art, such as chemical transformation using calcium chloride or high-voltage electroporation, with electroporation being preferred. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably a Pseudomonas bacterium, such as Pseudomonas fluorescens or Pseudomonas putida.

[0043] The method for preparing alkane hydroxylase provided by this invention includes: culturing the recombinant strain provided by this invention to induce the expression of a gene encoding alkane hydroxylase; and isolating and purifying the expressed alkane hydroxylase. The culture conditions are conventional, such as using an inorganic salt medium and culturing at 28-32°C for approximately 12-15 days. Because the recombinant strain provided by this invention contains a gene encoding alkane hydroxylase, it can efficiently express alkane hydroxylase. After culture, high-purity alkane hydroxylase can be obtained through isolation and purification. Isolation and purification can be performed using methods known to those skilled in the art, which will not be elaborated here.

[0044] The composition for degrading alkanes provided by this invention contains the alkane hydroxylase of this invention as an active ingredient, and the content of the alkane hydroxylase is 10-90% by weight, based on the total weight of the composition. The composition may also contain solvents (such as glycerol, sugars and protein protectants such as protease inhibitors), agonists (such as NiCl2, calcium chloride), etc., which are known to those skilled in the art.

[0045] The present invention also provides the application of the above-mentioned alkane hydroxylase, gene, recombinant vector, recombinant strain and composition of the present invention in improving the cellular alkane utilization capacity or degrading alkane.

[0046] In this invention, the method for degrading alkanes using the alkane hydroxylase may include contacting an alkane sample with the alkane hydroxylase. The alkane sample may be alkanes from various sources, particularly long-chain alkanes (alkanes with more than 10 carbon atoms), such as C16-C32 long-chain alkanes. The alkane may be a straight-chain alkane or a branched-chain alkane. Examples of the alkane include, but are not limited to, hexadecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, triadecane, tridecane, and dodecane.

[0047] The present invention will be described in detail below through examples. Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and biological materials described are commercially available unless otherwise specified. All alkanes used in the examples are straight-chain alkanes.

[0048] Example 1

[0049] Cloning of the alkane hydroxylase gene alkW

[0050] A strain of *Dietzia aurantiaca* BLG92, exhibiting excellent ability to degrade long-chain alkanes, was isolated from the Baolig oilfield in North China Oilfield. Freshly cultured wet cells were collected, and genomic DNA was obtained using an Omega bacterial genome extraction kit. Genome sequencing and gene annotation were then performed. Through searching and comparison, a hydroxylase gene, alkW, was found in the BLG92 genome. The gene is 1512 bp in size (sequence shown in SEQ ID NO: 1) and encodes a protein of 503 amino acids (sequence shown in SEQ ID NO: 2). The molecular weight is 56 kDa. The closest amino acid sequence is the fatty acid dehydrogenase sequence of Dietziacinnamea (WP_131885604.1), with a similarity of 95.43%. The next closest sequences are the alkane hydroxylase sequence of Dietziacinnamea E1 (ACN62569.1), with a similarity of 95.23%, and the fatty acid dehydrogenase sequence of Dietzia lutea (WP_108846117.1), with a similarity of 94.04%. This indicates that the gene is different from the currently reported alkane hydroxylases.

[0051]

[0052] MSSTEYIRPADGAGEHEAPHAHHDPHTHAHADVEPYAWTDAKRYLWLLGVIPAMGLFLSMPFVAGFNALGWEIPATVAWYLLPLLVYVAIPLGDLAIGEDGENPPDEVMDKLEADPFYRWCTYLYIPLQYGSLVAACYLWTADDLSWLGYDGGLGIAASVGVAWTVAITGGIGINTAHELGHKIAGSEKWLSKVALATTGYGHFFIEHNRGHHARVATPEDPASSRFGESFWAFLPRSVVGSLRSAWSLESERLARLGKSPWTLRNDNLNAWLMTVVLFGALIAVFGWEVAPWLIVQAIFGFSLLEVVNYLEHYGLLRQKTSAGRYQRCRPEHSWNSDHLVTNIFLYHLQRHSDHHANPMRRYQMLRSFEQAPQLPSGYATMMVIAYVPPLWRRVMDKRVLAHYDGDITRANIQPSKREKILARYGAGTTAVVEEVVADTDIATDQTSPTGEYVCPNCGNHYSEAAGLPREGFPPGTPWSAIPATWQCSDCGVRDKVDFLPVK(SEQ ID NO:2)

[0053] Using the genomic DNA of this bacterium as a template, primers were designed according to the sequence shown in SEQ ID NO: 1: 92alkW-F5'-atttaataaaaattggagaattccatatgatgtccagcaccgagtacatcc-3' (SEQ ID NO: 3) and 92alkW-R 5'-tctctcatccgccaaaacagaagctttcacttcacgggcaggaagt-3' (SEQ ID NO: 4). PCR amplification was performed using a total PCR reaction volume of 50 μL. The reaction conditions were: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 1 min, 60℃ annealing for 30 s, 72℃ extension for 2.5 min, for a total of 30 cycles; followed by a 72℃ extension for 7 min. After PCR products were detected by agarose gel electrophoresis, they were purified and recovered using the Omega Cycle Pure Kit. The products were then ligated into the linear vector pCom8 (obtained by NdeI and HindIII digestion) using the Gibson method. The Gibson ligation system (10 μl) consisted of 5 μl of 2x clone Express Mix, 3.5 μl of pCom8 DNA, and 1.5 μl of alkW DNA, and reacted at 50 °C for 30 min. 5 μl of the Gibson ligation product was transformed into 50 μl of competent E. coli DH5α cells, gently mixed, and incubated on ice for 30 min. This was followed by heat shock at 42 °C for 45 s, followed by an ice bath for 2 min. Then, 500 μl of antibiotic-free LB agar was added, and the cells were incubated at 37 °C for 1 h. An appropriate amount of the recovery solution was plated onto LB agar plates containing gentamicin Gm and incubated upside down at 37 °C. Single clones were picked and inoculated into test tubes, cultured at 37°C, and then plasmids were extracted and PCR was performed. Positive clones were identified by agarose gel electrophoresis. The plasmids of positive clones were sent for sequencing to verify the correctness of the sequence and to prevent frameshifts, mutations, and other issues.

[0054] Example 2

[0055] Determination of the degradation ability of the alkW-recovery defective strain of *Pseudomonas fluorescens* KOB2Δ1 in this invention.

[0056] *Pseudomonas fluorescens* KOB2Δ1 is a defective strain of *Pseudomonas fluorescens* CHA0 with one alkane hydroxylase gene, alkB2, knocked out. It no longer utilizes C12-C16 alkanes. When alkane hydroxylase genes from other sources are inserted into the expression plasmid pCom8 and transformed into the *Pseudomonas fluorescens* KOB2Δ1 defective strain, the host strain KOB2Δ1 regains its ability to degrade C12-C16 alkanes. Therefore, the expression plasmid pCom8 and the host strain *Pseudomonas fluorescens* KOB2Δ1 become a universal system for verifying the function of alkane hydroxylase genes (see Smits, THM, SBBalada, B. Witholt, and JBvanBeilen. 2002. Functional analysis of alkane hydroxylases from Gram-negative and Gram-positive bacteria. J. Bacteriol. 184: 1733–1742).

[0057] The correctly constructed expression plasmid was electroporated into *Pseudomonas fluorescens* KOB2Δ1 cells as follows: 1 ml of overnight KOB2Δ1 culture was centrifuged at 8000 rpm for 2 min, washed twice with ice-cold 300 mM sucrose solution, and finally resuspended in 100 μl of 300 mM sucrose solution to obtain competent cells, which were then placed on ice. 2 μl of plasmid was added to the competent cells and gently mixed, then transferred to an ice-cold electroporation cuvette and electroporated at 2000 V. After adding 500 μl of antibiotic-free LB agar, the cells were recovered at 30°C and 200 rpm for 1 h. An appropriate amount of the recovery solution was plated onto LB agar plates containing gentamicin Gm and incubated upside down at 30°C overnight. Four positive clones were randomly selected for plasmid extraction and PCR verification. Figure 1 Add 15% glycerol to the successfully prepared expression strain and store at -80℃ for later use.

[0058] Fluorescent Pseudomonas KOB2Δ1 containing pCom8-alkW expression plasmid and pCom8 empty vector, respectively, were inoculated into LB medium and cultured overnight. After centrifugation at 6000 rpm, the bacterial cells were washed twice with sterile physiological saline and resuspended to obtain the inoculum. Then, at an inoculum volume of 5-10%, the inoculum was inoculated into E2 medium (E2 inorganic salt medium (L)) containing different alkanes. -1): NaNH4HPO4·4H2O, 3.5g; K2HPO4·3H2O, 7.5g; KH2PO4, 3.7g; trace element solution 1ml; vitamin solution 1ml; 10ml 100mM MgSO4 solution; FeSO4·7H2O, 100mg; rhamnolipid 0.1g; Gm, 50mg. Incubate at 30℃ and 200rpm for 2 weeks. The degradation of various alkanes by the strain was detected by GC-MS as follows: 20 μl of phenanthrene internal standard solution was added to the sample to be extracted and mixed well; HPLC-grade n-hexane was added, and the unused alkanes were extracted by shaking and dissolved in the organic phase, followed by centrifugation; the organic phase was transferred to a new EP tube, and the n-hexane extraction was repeated once. The organic phases were combined into the EP tube, and anhydrous Na2SO4 was added to remove any possible residual water; after filtration through a 0.22 μm organic filter membrane, GC-MS was performed to detect the alkanes. The peak area was used to characterize the alkanes content, and the degradation rate relative to the empty pack was calculated.

[0059] GC-MS detection results ( Figure 2 and Figure 3 The results showed that after the defective strain of *Pseudomonas fluorescens* KOB2Δ1 expressed the AlkW protein of the present invention, the expressed strain could significantly degrade long-chain alkanes of C16-C31, with a degradation rate close to 100% compared to the empty hexadecane, and a degradation rate of over 80% for C22-C29 alkanes, indicating that the AlkW of the present invention has a very good ability to degrade long-chain alkanes.

[0060] Example 3

[0061] Expression and enzyme activity detection of the alkane hydroxylase gene alkW from BLG92 of Dizzyella orange

[0062] Strains KOB2Δ1 / pCom8-alkW and KOB2Δ1 / pCom8, containing the expression plasmid and empty vector respectively, were inoculated into LB medium and cultured overnight. After centrifugation at 6000 rpm, the bacterial cells were washed twice with sterile physiological saline and resuspended to obtain the inoculum. This inoculum was then inoculated into E2 inorganic salt medium containing 0.5% sodium pyruvate and cultured at 30°C until OD... 600 When the bacterial concentration was 0.4-0.6, 0.05% dicyclopropyl ketone (DCPK) was added to induce culture for 48 hours. An appropriate amount of bacterial culture was then subjected to SDS-PAGE electrophoresis to verify protein expression. The SDS-PAGE results showed that KOB2Δ1 / pCom8-alkW exhibited a specific band between 50-63 kDa on the protein marker, approximately the theoretical value of 56.2 kDa, indicating that AlkW was correctly expressed.

[0063] Take 2 ml of the cultured bacterial solution, centrifuge at 12000 rpm for 2 min, and add 100 μl of Millipore's [product name - likely a brand name] solution to the bacterial cells. Mix the Master Mix lysis buffer thoroughly and incubate at room temperature for 30 min; centrifuge at 6000 rpm for 2 min, and use the supernatant as crude enzyme solution (protein content approximately 2 mg / ml) for later use. Enzyme activity assay: 190 μl of reaction system consisting of 100 mM Tris-HCl (pH 7.4) containing 5% glycerol, 100 μM NADH2 (nicotinamide adenine dinucleotide), and 0.5% hexadecane, with 10 μl of crude enzyme solution added to start the reaction. The reaction temperature was controlled at 30℃, and OD was measured at 0 and 10 min. 340 Absorbance value. The corresponding enzyme activity was calculated based on the amount of NADH2 consumed; that is, enzyme activity is defined as the amount of protein required to consume 1 μmol of NADH2 per minute. The results showed ( Figure 4 Compared to the crude enzyme solution containing empty pCom8, the crude enzyme solution containing AlkW showed significantly higher alkane hydroxylase activity, nearly five times greater.

[0064] Example 4

[0065] The alkyl hydroxylase gene alkW of this invention is used to modify *Pseudomonas putida* KT2440 to degrade long-chain alkanes.

[0066] The recombinant bacteria were constructed using a method similar to that in Example 2, except that *Pseudomonas fluorescens* KOB2Δ1 was replaced with *Pseudomonas putida* KT2440 (purchased from ATCC, ATCC number ATCC 47054). *Pseudomonas putida* KT2440 does not have the ability to utilize and degrade alkanes. In this invention, *Pseudomonas putida* KT2440 was selected as the host bacterium, and its ability to degrade long-chain alkanes was enabled by expressing alkW.

[0067] An expression vector for strain KT2440 was constructed. The alkW gene fragment was amplified by PCR and inserted into the pFsC1G vector to construct a vector that can express alkW in strain KT2440; then, it was electroporated into strain KT2440 to construct the recombinant strain KT2440 / pFsC1G-alkW. The growth of the recombinant strain and GC-MS detection were then performed. Figure 5 Analysis revealed that the recombinant strain KT2440 / pFsC1G-alkW possessed the ability to degrade long-chain alkanes, achieving a degradation rate of approximately 10% for C16-C26 alkanes compared to the empty-vectored recombinant strain.

[0068] The above experiments demonstrate that the AlkW of the present invention has excellent long-chain alkane degradation ability and can be used to modify other strains to improve their alkane degradation ability.

[0069] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention. SEQUENCE LISTING <110> Institute of Microbiology, Chinese Academy of Sciences <120> Alkane hydroxylases derived from Dictamnus orange and their encoding genes and applications <130> 2021 <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 1509 <212> DNA <213> Dietzia aurantiaca <400> 1 atgtccagca ccgagtacat ccgccccgcc gacggggccg gtgagcacga ggcgcctcac 60 gcgcaccatg accctcacac ccacgcgcac gccgacgtcg agccctacgc gtggaccgac 120 gccaagcgtt acctctggct cctcggcgtc atccccgcga tgggcctgtt cctgtccatg 180 ccgttcgtcg ccggcttcaa cgccctcggc tgggagatcc ccgccaccgt cgcgtggtac 240 ctcctgccgc tcctcgtcta cgtcgccatc ccgctcggcg acctcgcgat cggcgaggac 300 ggggagaacc cgcccgacga ggtcatggac aagctcgagg cggacccctt ctaccgctgg 360 tgcacctacc tgtacatccc gctgcagtac ggctccctcg tcgcggcctg ctacctgtgg 420 accgccgacg acctgtcctg gctcgggtac gacggcgggc tgggtatcgc cgcctccgtc 480 ggcgtcgcct ggaccgtcgc gatcaccggc ggcatcggca tcaacaccgc ccacgagctg 540 ggacacaaga tcgccggcag cgagaagtgg ctgtccaagg tggcgctggc caccaccggc 600 tacgggcact tcttcatcga gcacaaccgc gggcaccacg cgcgggtcgc caccccggag 660 gaccccgcca gctcgcgctt cggcgagtcg ttctgggcgt tcctgccccg cagtgtggtg 720 ggctcgctcc gctcggcgtg gtcgctggag tccgagcgcc tcgcccggct gggcaagagc 780 ccctggactc tgcgcaacga caacctgaac gcctggctca tgaccgtcgt gctgttcggc 840 gcgctcatcg cggtgttcgg ctgggaggtc gccccctggc tcatagtcca ggcgatcttc 900 gggttctccc tgctcgaggt ggtcaactac ctcgagcact acggcctgct gcgccagaag 960 acctccgccg gccggtacca gcggtgccgc cccgagcact cgtggaactc ggaccacctg 1020 gtgaccaaca tcttcctgta ccacctgcag cgccactcgg accaccacgc caacccgatg 1080 cgccgctacc agatgctccg cagcttcgag caggccccgc agctgccgtc cgggtacgcc 1140 acgatgatgg tcatcgccta cgtcccgccg ctgtggcgca gggtcatgga caagcgcgtc 1200 ctggcccact acgacgggga catcacccgc gccaacatcc agccgtccaa gcgggagaag 1260 atcctcgccc ggtacggggc cggcacgacc gcggtggtcg aggaggtcgt cgcggacacc 1320 gacatcgcca ccgaccagac ctcgcccacc ggtgagtacg tgtgccccaa ctgcggcaat 1380 cactactccg aggccgcggg cctgccccgg gaggggttcc cgcccggcac cccgtggtcg 1440 gccatccccg ccacctggca gtgctccgac tgcggcgtgc gtgacaaggt ggacttcctg 1500 cccgtgaag 1509 <210> 2 <211> 503 <212> PRT <213> Auranthian diet <400> 2 Met Ser Ser Thr Glu Tyr Ile Arg Pro Ala Asp Gly Ala Gly Glu His 1 5 10 15 Glu Ala Pro His Ala His His Asp Pro His Thr His Ala His Ala Asp 20 25 30 Val Glu Pro Tyr Ala Trp Thr Asp Ala Lys Arg Tyr Leu Trp Leu Leu 35 40 45 Gly Val Ile Pro Ala Met Gly Leu Phe Leu Ser Met Pro Phe Val Ala 50 55 60 Gly Phe Asn Ala Leu Gly Trp Glu Ile Pro Ala Thr Val Ala Trp Tyr 65 70 75 80 Leu Leu Pro Leu Leu Val Tyr Val Ala Ile Pro Leu Gly Asp Leu Ala 85 90 95 Ile Gly Glu Asp Gly Glu Asn Pro Pro Asp Glu Val Met Asp Lys Leu 100 105 110 Glu Ala Asp Pro Phe Tyr Arg Trp Cys Thr Tyr Leu Tyr Ile Pro Leu 115 120 125 Gln Tyr Gly Ser Leu Val Ala Ala Cys Tyr Leu Trp Thr Ala Asp Asp 130 135 140 Leu Ser Trp Leu Gly Tyr Asp Gly Gly Leu Gly Ile Ala Ala Ser Val 145 150 155 160 Gly Val Ala Trp Thr Val Ala Ile Thr Gly Gly Ile Gly Ile Asn Thr 165 170 175 Ala His Glu Leu Gly His Lys Ile Ala Gly Ser Glu Lys Trp Leu Ser 180 185 190 Lys Val Ala Leu Ala Thr Thr Gly Tyr Gly His Phe Phe Ile Glu His 195 200 205 Asn Arg Gly His His Ala Arg Val Ala Thr Pro Glu Asp Pro Ala Ser 210 215 220 Ser Arg Phe Gly Glu Ser Phe Trp Ala Phe Leu Pro Arg Ser Val Val 225 230 235 240 Gly Ser Leu Arg Ser Ala Trp Ser Leu Glu Ser Glu Arg Leu Ala Arg 245 250 255 Leu Gly Lys Ser Pro Trp Thr Leu Arg Asn Asp Asn Leu Asn Ala Trp 260 265 270 Leu Met Thr Val Val Leu Phe Gly Ala Leu Ile Ala Val Phe Gly Trp 275 280 285 Glu Val Ala Pro Trp Leu Ile Val Gln Ala Ile Phe Gly Phe Ser Leu 290 295 300 Leu Glu Val Val Asn Tyr Leu Glu His Tyr Gly Leu Leu Arg Gln Lys 305 310 315 320 Thr Ser Ala Gly Arg Tyr Gln Arg Cys Arg Pro Glu His Ser Trp Asn 325 330 335 Ser Asp His Leu Val Thr Asn Ile Phe Leu Tyr His Leu Gln Arg His 340 345 350 Ser Asp His His Ala Asn Pro Met Arg Arg Tyr Gln Met Leu Arg Ser 355 360 365 Phe Glu Gln Ala Pro Gln Leu Pro Ser Gly Tyr Ala Thr Met Met Val 370 375 380 Ile Ala Tyr Val Pro Pro Leu Trp Arg Arg Val Met Asp Lys Arg Val 385 390 395 400 Leu Ala His Tyr Asp Gly Asp Ile Thr Arg Ala Asn Ile Gln Pro Ser 405 410 415 Lys Arg Glu Lys Ile Leu Ala Arg Tyr Gly Ala Gly Thr Thr Ala Val 420 425 430 Val Glu Glu Val Val Ala Asp Thr Asp Ile Ala Thr Asp Gln Thr Ser 435 440 445 Pro Thr Gly Glu Tyr Val Cys Pro Asn Cys Gly Asn His Tyr Ser Glu 450 455 460 Ala Ala Gly Leu Pro Arg Glu Gly Phe Pro Pro Gly Thr Pro Trp Ser 465 470 475 480 Ala Ile Pro Ala Thr Trp Gln Cys Ser Asp Cys Gly Val Arg Asp Lys 485 490 495 Val Asp Phe Leu Pro Val Lys 500 <210> 3 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 3 atttaataaa aattggagaa ttccatatga tgtccagcac cgagtacatc c 51 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 4 tctctcatcc gccaaaacag aagctttcac ttcacgggca ggaagt 46

Claims

1. An alkane hydroxylase, characterized in that, The alkane hydroxylase consists of an amino acid sequence represented by SEQ ID NO:

2.

2. A gene capable of encoding the alkane hydroxylase according to claim 1.

3. The gene of claim 2, wherein, The nucleotide sequence of the gene is represented by SEQ ID NO:

1.

4. A recombinant vector, characterized in that, The recombinant vector contains the gene according to claim 2 or 3.

5. A recombinant bacterial strain, characterized in that, The recombinant strain contains the recombinant vector according to claim 4.

6. The recombinant strain of claim 5, wherein, The strain is Pseudomonas.

7. The recombinant strain of claim 5 or 6, wherein, The strain is Pseudomonas fluorescens or Pseudomonas putida.

8. A method of preparing an alkane hydroxylase, characterized in that, The method comprises the following steps: (1) Culturing the recombinant strain according to any one of claims 5-7 to induce expression of the gene encoding the alkane hydroxylase; (2) Separating and purifying the expressed alkane hydroxylase.

9. A composition for degrading alkanes, characterized by, The composition contains the alkane hydroxylase according to claim 1 as an active ingredient, and the content of the alkane hydroxylase is 10-90% by weight based on the total weight of the composition.

10. Use of the alkane hydroxylase according to claim 1, the gene according to claim 2 or 3, the recombinant vector according to claim 4, the recombinant strain according to any one of claims 5-7, and the composition according to claim 9 in improving the ability of cells to utilize or degrade alkanes.

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