Halogenated alkane dehalogenase HLD-7113, its encoding gene and application

Abstract

The application discloses a kind of halogenated alkane dehalogenase HLD-7113 and its coding gene and application, belong to the field of biotechnology.The halogenated alkane dehalogenase is a new type of halogenated alkane dehalogenase that is excavated from the southwest Indian Ocean deep-sea sediment metagenome.The enzyme exhibits super-high catalytic efficiency to short-chain halogenated hydrocarbon.The substrate specificity is as follows: the catalytic activity of 4-bromobutyronitrile, n-butane, 1,5-dichloropentane and 1-chlorohexane with carbon chain length ≥3 is higher, while the activity of 1,2-dibromoethane, 1,2-dichloroethane, chlorocyclohexane, bromocyclohexane, bromomethylcyclohexane with cyclic structure and 1-bromo-2-methylpropane with branched chain is lower.In addition, HLD-7113 has degradation potential for stubborn pollutants such as DDT, hexachlorocyclopentadiene, and vesicant mustard gas simulant 2-cees, providing a new tool for contaminated site remediation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a haloalkane dehalogenase HLD-7113, its encoding gene, and its applications. Background Technology

[0002] Industrial and agricultural production processes inevitably generate large quantities of haloalkanes as byproducts. These byproducts not only waste resources, but a significant portion of them are also toxic and polluting, posing a serious threat to human health and the ecological environment. Therefore, developing efficient and environmentally friendly haloalkane treatment technologies is of great practical significance.

[0003] Methods for purifying haloalkane byproducts include chemical methods and microbial dehalogenation. Chemical methods often use toxic and corrosive reagents, making them unsuitable for use on personnel, clothing, and precision instruments. Microbial dehalogenation primarily utilizes the induction and release of dehalogenases by bacterial strains. Different dehalogenases have different specific substrates, including chlorinated, brominated, and iodinated organic compounds with varying chain lengths. Substrate preference depends on the catalytic mechanism of each dehalogenase, but dehalogenases also have the potential to catalyze a broad spectrum of substrates. The main catalytic mechanism of dehalogenation is nucleophilic substitution. Based on the dehalogenation pathway, dehalogenases can be classified into eight categories, including oxidative, reducing, and hydrolytic dehalogenates. Based on substrate type, dehalogenases can be classified into haloacid dehalogenases (EC3.8.1.2, Haloacid dehalogenase, HAD), haloalkane dehalogenases (EC3.8.1.5, Haloalkane dehalogenase, HLD), 4-chlorobenzoyl-CoA dehalogenases, and haloacrylic acid dehalogenases, among others. The two most common types of dehalogenases are haloic acid dehalogenases and haloalkane dehalogenases, both of which are hydrolytic dehalogenases.

[0004] Halogenated alkane dehalogenases have attracted much attention due to their unique catalytic mechanism and broad substrate specificity. They are currently used in the recovery of byproducts from chemical processes, bioremediation of toxic environmental pollutants, decontamination of chemical warfare agents, biosensing of environmental pollutants, protein tagging for protein analysis, and cell imaging. Therefore, developing novel dehalogenases with superior properties has significant application value and social importance. Summary of the Invention

[0005] The purpose of this invention is to provide a haloalkane dehalogenase HLD-7113, its encoding gene, and its applications, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a haloalkane dehalogenase HLD-7113, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The present invention provides a nucleic acid molecule encoding the above-mentioned haloalkane dehalogenase HLD-7113.

[0008] Optionally, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.1.

[0009] This invention provides biomaterials containing the aforementioned nucleic acid molecules.

[0010] Optionally, the biological material includes a recombinant vector, a recombinant microorganism, or an expression cassette.

[0011] This invention provides the use of the above-described haloalkane dehalogenase HLD-7113, the above-described nucleic acid molecule, or the above-described biological material in any of the following: (1) Degradation of haloalkanes; (2) Preparation of haloalkane degrading agents; (3) Degrading pesticides; (4) Preparation of pesticide degradation agents; (5) A mustard gas simulant for degrading vesicant; (6) Preparation of mustard gas simulant degradation agent for vesicant.

[0012] Optionally, the haloalkane includes one or more of the following: 1-iodopropane, 1-iodohexane, 1,3-dibromopropane, 4-bromobutonitrile, bromobutane, bromomethylcyclohexane, 1,3-dichloropropane, 1-chlorobutane, 1-chlorohexane, 1,2-dichloroethane, 1,2-dibromoethane, 1,2,3-trichloropropane, 1,2-dichloropropane, 1-bromo-2-methylpropane, 1,5-dichloropentane, bromocyclohexane, and chlorocyclohexane. The pesticides include DDT and / or hexachlorocyclohexane (HCH); The vesicant mustard gas analogue degrader includes 2-cees.

[0013] The present invention provides a degradation agent comprising the above-mentioned haloalkane dehalogenase HLD-7113, the above-mentioned nucleic acid molecule, or the above-mentioned biological material.

[0014] The present invention provides a method for degrading haloalkanes, comprising the step of treating the subject with the haloalkane dehalogenase HLD-7113 or the degradation agent described above.

[0015] The present invention provides a method for degrading pesticides and / or mustard gas mimics, comprising the step of treating the subject with the haloalkane dehalogenase HLD-7113 or the degrading agent described above.

[0016] The present invention discloses the following technical effects: The haloalkane dehalogenase HLD-7113 provided by this invention is a novel haloalkane dehalogenase discovered from the metagenomics of deep-sea sediments in the southwestern Indian Ocean. This enzyme exhibits ultra-high catalytic efficiency for short-chain haloalkanes (especially 1,3-dibromopropane). Its substrate specificity is as follows: it exhibits high catalytic activity for 4-bromobutyronitrile, bromobutane, 1,5-dichloropentane, and 1-chlorohexane with carbon chain lengths ≥3, while its activity is lower for shorter-chain halogenated hydrocarbons such as 1,2-dibromoethane, 1,2-dichloroethane, as well as cyclic cyclohexane, bromocyclohexane, bromomethylcyclohexane, and branched 1-bromo-2-methylpropane. Its optimal temperature is 40℃, and its optimal pH is 8.2. Furthermore, HLD-7113 shows potential for degrading persistent pollutants such as DDT, hexachlorocyclohexane (HCH), and the vesicant mustard gas mimic 2-cees, providing a new tool for contaminated site remediation. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Soluble expression of the haloalkane dehalogenase HLD-7113; where M is the marker, 1-6 are the whole bacterial culture of HLD-7113 without soluble tagged culture (HLD7113-pET21a-BL21(DE3) culture), the supernatant of HLD-7113 without soluble tagged culture (HLD7113-pET21a-BL21(DE3) culture supernatant), the whole bacterial culture of Escherichia coli blank control, the supernatant of Escherichia coli blank control, the whole bacterial culture of HLD-7113 with soluble tagged culture (pET-7113-GDDD [BL21(DE3)] culture), and the supernatant of HLD-7113 with soluble tagged culture (pET-7113-GDDD [BL21(DE3)] culture supernatant). Figure 2 Purification of the haloalkane dehalogenase HLD-7113; where M is the marker and 1 is the purified band of HLD-7113; Figure 3This demonstrates substrate specificity for the haloalkane dehalogenase HLD-7113. Figure 4 The optimal temperature for the dehalogenase HLD-7113 of haloalkanes; Figure 5 The optimal pH for the dehalogenase HLD-7113 of haloalkane; Figure 6 Temperature stability of the haloalkane dehalogenase HLD-7113; Figure 7 Organic solvent tolerance of the haloalkane dehalogenase HLD-7113; Figure 8 The kinetic equation for the enzymatic reaction of the haloalkane dehalogenase HLD-7113 is fitted. Figure 9 A double reciprocal plot of the substrate saturation curve for the haloalkane dehalogenase HLD-7113; Figure 10 This study describes the degradation effect of pesticides by the haloalkane dehalogenase HLD-7113. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1 1. Obtaining the HLD-7113 protein and its encoding gene This haloalkane dehalogenase gene was obtained from samples collected during the 39th voyage of the research vessel "Dayang Yihao" (Ocean One). Six deep-sea sediment samples (TVG05, TVG06, TVG07, TVG08, TVG10, and TVG12) were acquired using a television grab bucket. Metagenomic high-throughput sequencing was performed on these samples, and the raw sequences from all samples were mixed and assembled. Functional genes were predicted using ORF, and the amino acid sequences of their encoded proteins were obtained. The mixed assembly of the six sediment samples yielded 7,495,897 protein-coding gene sequences, approximately 5.34 Gb of nucleotide sequence data. DhlA is the first biochemically validated haloalkane dehalogenase, derived from *Xanthobacter autotrophicus* GJ10. Using this enzyme as a template, and based on amino acid homology sequence alignment (BLASTP), a new dehalogenase Gene3407113_TVG06_41369_4 was obtained from the aforementioned Southwest Indian Ocean deep-sea sediment metagenomic database. This protein has the highest sequence similarity to the known haloalkane dehalogenase [Myxococcales bacterium] (HTO99229.1) on NCBI, with an amino acid sequence similarity of 71.81%. After codon optimization and de novo gene synthesis, the coding gene for this protein was synthesized and named HLD-7113. Then, the coding gene fragment of the protein was obtained through codon optimization and whole-genome synthesis, and cloned into the pET21a vector at the NdeⅠ and HindⅢ cloning sites. The final stop codon was removed during plasmid construction, and the stop codon on the vector was used to retain the six histidine histidines at the C-terminus of the His-tag affinity tag. Finally, the recombinant vector was transformed into the E. coli BL21(DE3) genetically engineered bacteria to obtain the recombinant expression strain HLD7113-pET21a-BL21(DE3), which was then sequenced and identified.

[0025] The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2. The protein encoded by the gene is named haloalkane dehalogenase HLD-7113.

[0026] SEQ ID NO.1: ATGATCGAAACGCTGCGCACCCCAGACAACCGCTTCGAGGGCCTGCCGGGCTTCGATTTCGCACCGCACTATGTTGATCAGCTGGACGGCTTCCCGGGTCTCCGCCTGCATTACCTGGATGAAGGTCCGGCTGGTGCGGAGCACACCTTTCTGTGTCTGCATGGCGAACCAACCTGGTCTTATCTGTACCGCAAGATGATTCCGGTTTTCACCGCGGCCGGTCACCGTGTTGTAGCTCCAGATTACTTCGGTTTCGGCCGCAGCGACAAGCCGGTTGACGACGCGACTTACACCTTCACTTTTCATCGCGCATCGCTCCTGCGCCTCATCGAACATCTCGACCTGGCAAATCTCACTCTGGTAGTTCAGGATTGGGGTGGTCTGCTGGGTCTGACCCTGCCGATTGATGTTCCGGAACGTATTTCTCGCCTGGTGGTAATGAACACCGCCCTGGGTACGGGCGACATGCCGCTGGGTGAAGGCTTCCTGGCTTGGCGCGCGTATTGCCGTCAGAACCCGGATCTCGACGTCGCAGCGCTGATGGGCCGTGCGTGTCCGGACCTCTCCCCGGCTGAATGTGCAGCTTACGCTGCGCCGTTCCCGGATGTTCGTTCCAAAGCGGGTGTTCGCCGTTTCCCGGATATGGTTCCGGACAACCCAGACGCTGACGGTGCTGCACTCTCCCGTCGTGCCCGCGATTTCTGGCAGAACCAGTGGTCCGGTCAGTCCTTCATGGCGATCGGTATGCAAGACCCGGTGCTGGGTCCACCTGCGATGGTCGCGCTGCGTCGTCTGATTAAAGGTTGCCCGGAGCCTCTGGAACTGGCTGACGCAGGTCACTTTGTTCAGGAGTCTGGTGAACAGGTTGCCGCACGTGCACTGGAAGCGTTCGGT。

[0027] SEQ ID NO.2: MIETLRTPDNRFEGLPGFDFAPHYVDQLDGFPGLRLHYLDEGPAGAEHTFLCLHGEPTWSYLYRKMIPVFTAAGHRVVAPDYFGFGRSDKPVDDATYTFTFHRASLLRLIEHLDLANLTLVVQDWGGLLGLTLPIDVPERISRLVVMNT ALGTGDMPLGEGFLAWRAYCRQNPDLDVAALMGRACPDLSPAECAAYAAPFPDVRSKAGVRRFPDMVPDNPDADGAALSRRARDFWQNQWSGQSFMAIGMQDPVLGPPAMVALRRLIKGCPEPLELADAGHFVQESGEQVAARALEAFG.

[0028] 2. Obtaining the dehalogenase HLD-7113 for haloalkanes 2.1 Obtaining the recombinant vector The PCR amplification product (SEQ ID NO.1) and pET-21a(+) vector obtained above were double-digested with restriction endonucleases Nde I and Hind III, ligated, and the recombinant vector pET-7113 was obtained and sent for sequencing.

[0029] Sequencing results showed that pET-7113 replaced the DNA molecule shown in SEQ ID NO.1 with the DNA molecule in the pET21a(+) vector that contains the coding region of the multiple cloning site between NdeⅠ (CATATG) and HindⅢ (AAGCTT), while keeping the other sequences of the pET-21a(+) vector unchanged. The resulting recombinant vector contained the DNA molecule shown in SEQ ID NO.1, and the T7 promoter of the vector was used to start the expression of the recombinant protein HLD-7113 shown in SEQ ID NO.2 using the DNA molecule shown in SEQ ID NO.1.

[0030] 2.2 Obtaining Recombinant Bacteria The recombinant vector pET-7113 obtained in step "2.1, Obtaining the recombinant vector" was transformed into Escherichia coli BL21(DE3) using the calcium chloride chemical transformation method to obtain recombinant bacteria. The recombinant bacteria were screened and cultured in 2×YT medium containing ampicillin (100 μg / mL). Single colonies were picked, plasmids were extracted and verified, and the finally obtained positive recombinant bacteria were named pET-7113[BL21(DE3)].

[0031] 2.3 Soluble expression of haloalkane dehalogenase HLD-7113 To convert the partially soluble expression of the haloalkane dehalogenase HLD-7113 into a fully soluble expression, the coding sequence for the (GDDD)3 (SEQ ID NO.3) lysis-promoting tag needs to be added to the end of the HLD-7113 gene sequence in the original expression vector to construct the HLD-7113 lysis-promoting expression vector. Using PET-21a as a template, the linear fragment was amplified using primers designed and synthesized for polymerase chain reaction, resulting in the linearized HLD-7113 vector containing the lysis-promoting tag.

[0032] The primers for amplifying the target gene are as follows: Upstream primer: AAGAAGGAGATATACATATGATCGAAACGCTGCGCACC, SEQ ID NO.4; Downstream primer: TTGTCGTCGTCACCGTCGTCGTCACCGTCGTCGTCACCACCGAACG CTTCCAGTGCA, SEQ ID NO.5.

[0033] The primers for amplifying the PET21a vector fragment are as follows: Fragment 1: Upstream primer: ACGACGGTGACGACGACAAGCTTCACCACCACCACCACCA, SEQ ID NO.6; Downstream primer: GAGCGGTATCAGCTCACTCAAAGGCGGTAA, SEQ ID NO.7; Fragment 2: Upstream primer: TGAGTGAGCTGATACCGCTCGCC, SEQ ID NO.8; Downstream primer: CATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGG, SEQ ID NO.9.

[0034] The amplification system is shown in Table 1.

[0035] Table 1 Amplification System The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 72℃ extension for 10 s, 30 cycles; 72℃ complete extension for 1 min; and storage at 4℃ after PCR.

[0036] The target gene fragment and two successfully PCR-generated vector fragments (fragment 1 and fragment 2) were purified by gel extraction to remove impurities. The template plasmid from the PCR reaction was then removed by DpnI digestion. The purified DNA fragment was obtained after gel extraction. The three linear fragments were ligated together using seamless cloning and transformed into *E. coli* Trans1-T1 competent cells. The following day, single clones were picked from ampicillin-containing plates and transferred to 2×YT liquid medium containing ampicillin. The plasmids extracted from the bacterial culture were sequenced using universal T7 primers. Plasmids from positive strains were extracted and transformed into *E. coli* BL21(DE3) competent cells. The specific steps were as follows: 1 μL of each of the three fragments was added, followed by 2 μL of sterile water and 5 μL of the seamless cloning enzyme. The mixture was incubated at 50°C for 15 minutes. The ligated fragments were then transformed into Trans1-T1 competent cells. After successful sequencing, the constructed plasmid was extracted and transformed into *E. coli* BL21(DE3) cells for expression. Single clones were picked and cultured in 2×YT liquid medium containing ampicillin. A portion of the bacterial culture was then stored in a 20% glycerol tube at -80°C. The final lysing expression vector pET-21a-HLD7113-GDDD for the dehalogenase HLD-7113 was obtained.

[0037] Repeat step "2.2, Obtaining Recombinant Bacteria" to obtain a positive recombinant bacterium named pET-7113-GDDD [BL21(DE3)]. After adding the solubilization tag (GDDD)3, the theoretical molecular weight of HLD-7113 changed from 32.7 kDa to 35.1 kDa. After protein expression was induced, the protein sample was analyzed by SDS-PAGE.

[0038] 2.4 Obtaining the dehalogenase HLD-7113 for haloalkanes A single colony of pET-7113-GDDD [BL21(DE3)] was picked and inoculated into 2×YT medium containing ampicillin (100 μg / mL) and cultured overnight at 37°C. The overnight culture was then inoculated into 100 mL of 2×YT medium containing ampicillin (100 μg / mL) and cultured at 37°C with shaking (200 rpm) until the OD of the fermentation broth was reached. 600When the pH reaches approximately 0.6-0.8, add IPTG (final concentration 1 mM) to the fermentation system and incubate at 16℃ for another 24 h. After fermentation, centrifuge at 8000 rpm for 20 minutes, discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells in non-denaturing nickel column binding buffer I, sonicate (4 s sonication, 7 s interval, 50 W power, 50 cycles), and centrifuge at 12,000 rpm for 15 min. Collect the supernatant, which is the crude enzyme solution containing recombinant protein HLD-7113. The target recombinant protein HLD-7113 has a band size of 35.1 kDa, and this protein is the recombinant protein HLD-7113 shown in sequence 2. Figure 1 and Figure 2 ).

[0039] 2.5 Purification of dehalogenase HLD-7113 The composition of non-denaturing nickel column binding buffer I is: 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, with water as the solvent and pH 7.5.

[0040] The composition of non-denaturing nickel column elution buffer II is: 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, with water as the solvent and pH 7.5.

[0041] The desalting buffer composition is: 50 mM Gly-NaOH, pH 8.2.

[0042] Purification was performed using nickel affinity chromatography. The supernatant was purified using a 1 mL HiTrap chelating HP column (nickel column) in an Amersham AKTA FPLC system. The purification column was equilibrated with non-denaturing nickel column binding buffer I and loaded with 10 mL of protein at a flow rate of 1 mL / min. The protein was washed with non-denaturing nickel column binding buffer I to remove non-specifically bound proteins. Elution was performed with non-denaturing nickel column elution buffer at a linear gradient of 0%-100%, and the eluent containing the elution peak was collected. The eluent was then purified using a Hitrap Q desalting column (5 mL) to obtain the purified recombinant protein HLD-7113 at a concentration of 0.915 mg / mL.

[0043] 3. Methods for determining dehalogenase activity 3.1 Methods for determining enzyme activity Dehalogenases catalyze the dehalogenation of haloalkanes to produce haloalcohols, halide ions, and protons. Currently, the most commonly used technique for detecting the concentration of halide ions generated in the dehalogenation reaction is the mercuric thiocyanate-ferric ammonium sulfate colorimetric method. This method works by utilizing the halide ions produced in the dehalogenation reaction to displace the thiocyanate ions in the mercuric thiocyanate, and then detecting the halide ions by forming an orange-red complex with the iron ions.

[0044] R-CH2X + H2O → R-CH2OH + H - +X, X=Cl - , Br - I - …; .

[0045] The dehalogenase activity unit is defined as the amount of enzyme required to catalyze the dehalogenation reaction of 1 μmol of substrate per minute under optimal conditions.

[0046] (1) Mercuric thiocyanate-ferric ammonium sulfate colorimetric method: Prepare the following two solutions: Mercuric thiocyanate solution: 0.3 g mercuric thiocyanate dissolved in 100 mL 95% ethanol.

[0047] Ferric ammonium sulfate solution: 6 g of ferric ammonium sulfate is dissolved in 6 M nitric acid.

[0048] One-tenth of the volume of mercuric thiocyanate solution and one-fifth of the original sample volume of ammonium ferric sulfate solution were added to a certain volume of sample, mixed well, and reacted for more than 10 minutes.

[0049] The absorbance of the reaction mixture was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0050] Halogen ion standard solutions of different concentrations were prepared, and a halide ion concentration standard curve was constructed based on the concentration of halide ions in the sample and the standard solutions. The halide ion concentration in the sample was calculated according to the absorbance value and the halide ion standard curve. The standard curves for each halide ion are as follows: I - Ion concentration standard curve: y = 1.49x + 0.1026; Br - Concentration standard curve: y = 1.5128x + 0.168; Cl - Concentration standard curve: y = 0.2632x + 0.2635.

[0051] Colorimetric reaction buffer: 50 mM Gly-NaOH, pH 8.2.

[0052] Experimental group: The purified recombinant protein HLD-7113 obtained above was diluted to 100 μg / mL with desalting buffer (50 mM Gly-NaOH, pH 8.2). 100 μL of the diluted enzyme was added to 900 μL of colorimetric reaction buffer (containing 10 mM substrate), thoroughly mixed, and immediately reacted at the optimal temperature for 30 min. Then, 500 μL of the reaction solution was taken out, and 50 μL of 30% nitric acid, 55 μL of mercuric thiocyanate solution, and 110 μL of ferric ammonium sulfate solution were added. The reaction was allowed to proceed for at least 10 minutes, and the absorbance at 450 nm was measured using a microplate reader.

[0053] Control group: The difference from the experimental group is that no enzyme is added.

[0054] 3.2 Substrate specificity of haloalkane dehalogenase HLD-7113 Choose from 1-iodopropane, 1-iodohexane, 1,3-dibromopropane, 4-bromobutonitrile, 1-bromobutane, bromomethylcyclohexane, 1,3-dichloropropane, 1-chlorobutane, 1-chlorohexane, 1,2-dichloroethane, and 1,2-dibromoethane. Seventeen alkyl halogens, including 1,2,3-trichloropropane, 1,2-dichloropropane, 1-bromo-2-methylpropane, 1,5-dichloropentane, bromocyclohexane, and chlorocyclohexane, were used as substrates to study the substrate specificity of the alkyl halogen dehalogenase HLD-7113. The determination was performed colorimetrically at a reaction temperature of 40°C. The enzyme activity at the optimal substrate was defined as 100%, and the relative activities catalyzing other substrates were calculated.

[0055] The results are as follows Figure 3As shown, the optimal substrate for the haloalkane dehalogenase HLD-7113 is 1,3-dibromopropane. HLD-7113 exhibits certain specificity for the type of halogen and carbon chain length. It demonstrates high catalytic activity for 4-bromobutyronitrile, bromobutane, 1,5-dichloropentane, and 1-chlorohexane with carbon chain lengths ≥3, at 70.74%, 49.49%, 86.41%, and 67.57% of the optimal substrate 1,3-dibromopropane, respectively. The activity was low for 1,2-dibromoethane and 1,2-dichloroethane with shorter carbon chains, as well as for cyclic cyclohexane, bromocyclohexane, bromomethylcyclohexane, and branched 1-bromo-2-methylpropane, with only 27.13%, 18.76%, 20.86%, 4.5%, 17.56%, and 19.6% of the optimal substrate 1,3-dibromopropane, respectively. Compared to iodinated and chlorinated substrates, HLD-7113 shows a greater preference for brominated substrates. For 1-iodohexane (longer carbon chain) and 1-iodopropane (shorter carbon chain), HLD-7113 exhibits low catalytic activity, only 7.21% and 36.11% of the optimal substrate, 1,3-dibromopropane, respectively. While 1,2-dichloropropane and 1,3-dichloropropane have the same carbon chain length as 1,3-dibromopropane, their activity is significantly reduced due to different halogen types, reaching only 36.4% and 19.4% of the optimal substrate, respectively. It also shows some activity for the more difficult-to-degrade 1,2,3-trichloropropane, at 22.73% of the optimal substrate. The haloalkane dehalogenase HLD-7113 can degrade most haloalkane substrates, especially brominated substrates with a carbon chain ≥3, and also exhibits some catalytic activity for the more difficult-to-degrade chlorinated substrates.

[0056] 3.3 Method for determining the optimal temperature of haloalkane dehalogenase HLD-7113 The experiment was conducted using a colorimetric method at reaction temperatures of 4℃, 25℃, 30℃, 40℃, 45℃, 50℃, 60℃, and 70℃; the substrate was 1,3-dibromopropane. The enzyme activity at the optimal temperature was defined as 100%, and the relative activities at other reaction temperatures were calculated.

[0057] The results are as follows Figure 4As shown, the optimal temperature for the purified recombinant protein HLD-7113 is 40℃, and its relative activity is defined as 100%. At lower temperatures, HLD-7113 exhibits low catalytic activity, with a relative activity of 17.71%. Within the range of 4℃ to 40℃, enzyme activity gradually increases with increasing temperature. However, after 40℃, enzyme activity decreases with increasing temperature. Within the range of 40℃ to 45℃, HLD-7113 exhibits high enzyme activity, with a relative activity exceeding 89%. At a reaction temperature of 70℃, the specific activity of HLD-7113 is only 11.45% of that at 40℃. Above 70℃, its relative activity gradually decreases until it loses its catalytic activity.

[0058] 3.4. Method for determining the pH stability of haloalkane dehalogenase HLD-7113 Citrate-sodium citrate buffers at pH 5.0 and 6.0, Tris-H₂SO₄ buffers at pH 7.0 and 9.0, and sodium carbonate-sodium bicarbonate buffers at pH 10.0 and 11.0 were prepared. The haloalkane dehalogenases were placed in these buffers and treated at room temperature for 5 h. Activity was then determined colorimetrically using 1,3-dibromopropane as the substrate at 40 °C. The enzyme activity of the untreated group (pH 8.2, incubated at room temperature for 5 h) was defined as 100%. The relative activities of enzymes treated at other pH conditions were calculated. Three replicates were designed for each experimental group.

[0059] Citric acid-sodium citrate buffer: The final concentration of sodium citrate is 100 mM, which is then diluted with distilled water and adjusted to the pH required for the experiment.

[0060] Tris-H2SO4 buffer: The final concentration of Tris is 100 mM. The solution is diluted with distilled water and adjusted to the pH required for the experiment.

[0061] Sodium carbonate-sodium bicarbonate buffer: The final concentration of sodium bicarbonate is 100 mM, which is then diluted with distilled water and adjusted to the pH required for the experiment.

[0062] The results are as follows Figure 5 As shown, the optimal pH value is 8.2, and its relative activity is defined as 100%. After treatment under conditions of pH 6.0 to pH 8.2, it can still maintain high catalytic activity, with a relative activity of over 80%. However, the tolerance of the haloalkane dehalogenase HLD-7113 is significantly reduced when the pH value is lower than 6.0 or higher than 9.0. Specifically, when the pH value is lowered to 5.0, the relative activity is 34.99%, and when the pH value is raised to 9.0, the relative activity is 63.50%. When the pH value is lowered to 4 or raised to 10, the enzyme activity drops sharply, and the relative enzyme catalytic activity is less than 5%. With the continued decrease or increase of pH, the enzyme activity continues to decrease until it basically loses its catalytic activity.

[0063] 3.5 Method for determining the thermal stability of dehalogenases for haloalkanes Within the temperature range of 40℃ to 70℃, the haloalkane dehalogenase HLD-7113 was treated at different temperatures for 5 min, 10 min, 20 min, 30 min, 60 min, and 90 min, respectively. The specific activity of the treated enzymes was then determined, with the relative activity of the untreated haloalkane dehalogenase HLD-7113 defined as 100%. The activity of the treated enzymes was determined colorimetrically using 1,3-dibromopropane as the substrate. The enzyme activity of the untreated control group was defined as 100%.

[0064] The results are as follows Figure 6 As shown, after heat treatment at 40℃, 50℃, 60℃, and 70℃ for 5 min, the relative activities of enzymes in some experimental groups decreased to varying degrees. The enzyme activity at 50℃, 60℃, and 70℃ decreased the fastest, reaching only 35.67%, 16.17%, and 11.19% of the control group's activity, respectively. After treatment at 50℃, 60℃, and 70℃ for 20 min, the activity of HLD-7113 was gradually lost. However, after treatment at 40℃ for 5 min, the relative activity of the enzyme did not decrease significantly. After treatment at this temperature for 30 min, the enzyme activity still reached 61.97%. After heat treatment for 60 min, the enzyme activity rapidly decreased to 37.96%, and with continued heat treatment time, the activity of the haloalkane dehalogenase HLD-7113 was gradually lost.

[0065] 3.6 Method for determining the organic solvent stability of the haloalkane dehalogenase HLD-7113 Different groups were set up, and the purified recombinant protein HLD-7113 was placed in methanol aqueous solution, ethanol aqueous solution, DMSO aqueous solution, and acetonitrile aqueous solution with volume percentages of 10%, 20%, 30%, 40%, and 50%, respectively. After treatment at room temperature for 1 h, its activity was determined by colorimetry. The substrate was 1,3-dibromopropane, and the reaction temperature was 40 °C. The relative activity of the control group without organic solvent treatment was defined as 100%.

[0066] The results are as follows Figure 7As shown, HLD-7113 exhibited high stability in all four organic solvents at a volume percentage of 10%, with relative enzyme activity remaining above 80% after treatment. HLD-7113 showed the best tolerance to DMSO, with no impact on enzyme activity at 10% DMSO treatment. Even when the DMSO concentration increased to 30%, the relative enzyme activity still reached over 70%. It showed the worst tolerance to acetonitrile, with relative activity rapidly decreasing to 7.88% at a low concentration of 20%. At acetonitrile concentrations above 20%, HLD-7113 essentially lost its catalytic activity. Under low concentrations (10-20%) of ethanol and methanol, the relative enzyme activity was less affected, but as the organic solvent concentration further increased, the enzyme activity gradually decreased.

[0067] 3.7 Determination of Enzymatic Reaction Kinetic Parameters of Haloalkane Dehalogenase HLD-7113 Optimal substrate 1,3-dibromopropane was prepared at different concentrations using ethanol, namely 0.1 mM, 0.15 mM, 0.2 mM, 0.3 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 5 mM, and 10 mM. Colorimetric experiments were conducted using different optimal substrate concentrations as experimental groups. A graph was plotted with the reciprocal of substrate concentration (1 / [S]) on the x-axis and the reciprocal of reaction rate (1 / v) on the y-axis. The slope of the resulting straight line was... K m / V max The intercept of the resulting line is 1 / V max .

[0068] The calculated enzyme kinetic parameters are as follows: Using 1,3-dibromopropane as a substrate, the maximum reaction rate of this enzyme was determined to be Vmax = 0.2941 mM / min, and the Michaelis constant was... K m = 1.761 mM, conversion number k cat = 1.6 s -1 , k cat / K m It is 0.909 mM -1 ·s -1 ( Figure 8 and Figure 9 ).

[0069] 3.8 Degradation of traditional pesticides and vesicant mustard gas mimics by the haloalkane dehalogenase HLD-7113 To investigate the degradation ability of the haloalkane dehalogenase HLD-7113 on traditional pesticides, DDT (p,p'-DDT), β-HCH (hexachlorocyclohexane), and the vesicant mustard gas mimic 2-cees at a final concentration of 0.5 mM were used as the sole substrates. Crude enzyme solution from *E. coli* (containing recombinant protein HLD-7113) served as a blank control. The supernatant after centrifugation of *E. coli* cell lysate containing recombinant protein HLD-7113 (crude enzyme solution) and purified HLD-7113 protein were both used in the activity assay system at a final concentration of 0.01 mg / mL. The catalytic activities against the traditional organochlorine pesticides p,p'-DDT and β-HCH, and the vesicant mustard gas mimic 2-cees were calculated.

[0070] The results showed that the substrate autohydrolysis of the vesicant mustard gas analog 2-cees was strong, while HLD-7113 exhibited poor degradation ability against 2-cees. However, HLD-7113 showed some degradation ability against p,p'-DDT and β-hexachlorocyclohexane (HCH), with HLD-7113 showing the most significant degradation effect on p,p'-DDT. Figure 10 ).

[0071] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A haloalkane dehalogenase HLD-7113, characterized in that, The amino acid sequence of the haloalkane dehalogenase HLD-7113 is shown in SEQ ID NO.

2.

2. A nucleic acid molecule encoding the haloalkane dehalogenase HLD-7113 of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

1.

4. Biomaterials containing the nucleic acid molecules of claim 2 or 3.

5. The biomaterial according to claim 4, characterized in that, The biomaterials include recombinant vectors, recombinant microorganisms, or expression cassettes.

6. The use of the haloalkane dehalogenase HLD-7113 according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the biomaterial according to claim 4 or 5 in any of the following: (1) Degradation of haloalkanes; (2) Preparation of haloalkane degrading agents; (3) Degrading pesticides; (4) Preparation of pesticide degradation agents; (5) A mustard gas simulant for degrading vesicant; (6) Preparation of mustard gas simulant degradation agent for vesicant.

7. The application according to claim 6, characterized in that, The haloalkanes include one or more of the following: 1-iodopropane, 1-iodohexane, 1,3-dibromopropane, 4-bromobutyronitrile, bromo-n-butane, bromomethylcyclohexane, 1,3-dichloropropane, 1-chlorobutane, 1-chlorohexane, 1,2-dichloroethane, 1,2-dibromoethane, 1,2,3-trichloropropane, 1,2-dichloropropane, 1-bromo-2-methylpropane, 1,5-dichloropentane, bromocyclohexane, and chlorocyclohexane. The pesticides include DDT and / or hexachlorocyclohexane (HCH); The vesicant mustard gas analogue degrader includes 2-cees.

8. A degradation agent, characterized in that, The degrading agent includes the haloalkane dehalogenase HLD-7113 of claim 1, the nucleic acid molecule of claim 2 or 3, or the biomaterial of claim 4 or 5.

9. A method for degrading haloalkanes, characterized in that, The step includes treating the substance to be treated with the haloalkane dehalogenase HLD-7113 as described in claim 1 or the degradation agent as described in claim 8.

10. A method for degrading pesticides and / or mustard gas simulants, characterized in that, The step includes treating the substance to be treated with the haloalkane dehalogenase HLD-7113 as described in claim 1 or the degradation agent as described in claim 8.

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