Halogenated alkane dehalogenase hld-1405, encoding gene and application thereof

Abstract

This invention discloses a haloalkane dehalogenase HLD-1405, its encoding gene, and its applications, belonging to the field of biotechnology. The amino acid sequence of this haloalkane dehalogenase HLD-1405 is shown in SEQ ID NO.2, and its encoding gene is shown in SEQ ID NO.1. This haloalkane dehalogenase HLD-1405 exhibits high catalytic activity towards 1,3-dibromopropane, with optimal operating conditions of 40℃ / pH 9.0, and retains 100% activity in 20% methanol. This haloalkane dehalogenase HLD-1405 can efficiently degrade short-chain haloalkanes and also has the ability to degrade pollutants such as DDT and hexachlorocyclohexane (HCH), showing great application potential in the treatment of stubborn pollutants and suitable for environmental bioremediation and industrial catalysis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a haloalkane dehalogenase HLD-1405, its encoding gene, and its applications. Background Technology

[0002] Halogenated organic compounds (HOCs) have wide applications in many fields. Their extensive use in industrial enterprises and daily consumer goods has made them a significant class of environmental pollutants. Due to their highly stable chemical bonds, these HOCs have consistently caused environmental problems in the biosphere for many years. HOCs are among the most used and studied organic compounds due to their relatively simple synthetic pathways. However, because of their generally high stability and toxicity to microorganisms that could potentially degrade them, their biodegradability has been poor for many years. Excessive exposure to these HOCs by humans or the natural environment can lead to serious environmental and health consequences. Currently, microbial remediation technology is the main means of controlling environmental pollution caused by HOCs.

[0003] Microbial dehalogenation primarily utilizes the induction and release of dehalogenases by microbial strains. Different dehalogenases have different specific substrates, including chlorinated, brominated, and iodinated organic compounds, and varying chain lengths. The 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 dehalogenases. 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, haloacrylic acid dehalogenases, etc. The two most common types of dehalogenases are haloacid 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-1405, its encoding gene, and its applications, to address the problems existing in the prior art. This haloalkane dehalogenase HLD-1405 exhibits significant substrate specificity for short-chain haloalkane. The enzyme demonstrates excellent stability at pH 7.0-10.0, especially showing no loss of activity in 20% methanol, overcoming the limitation of traditional dehalogenases being easily inactivated in organic solvents. Furthermore, its ability to degrade DDT, hexachlorocyclohexane (HCH), and 2-cees expands its application potential in the treatment of stubborn pollutants.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides an isolated protein having the amino acid sequence shown in SEQ ID NO.2; or a protein having at least 98% identity with SEQ ID NO.2 and having haloalkane dehalogenase activity.

[0008] The present invention also provides a nucleic acid molecule encoding the said protein.

[0009] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.1.

[0010] The present invention provides recombinant vectors, recombinant microorganisms, or expression cassettes containing the said nucleic acid molecules.

[0011] The present invention also provides a method for preparing the protein, comprising: culturing the recombinant microorganism, inducing expression, and purifying the protein.

[0012] The present invention also provides the use of the protein as a dehalogenase for haloalkane.

[0013] The present invention also provides a method for degrading haloalkanes, comprising: using the protein to catalyze the dehalogenation of haloalkanes; wherein the haloalkanes include 1,3-dibromopropane, 1,2-dibromoethane, or 1,3-dichloropropane.

[0014] The present invention also provides a method for degrading DDT, hexachlorocyclohexane (HCH), or 2-cees, comprising: using the protein to catalyze the DDT, HCH, or 2-cees.

[0015] The present invention discloses the following technical effects:

[0016] The haloalkane dehalogenase HLD-1405 provided by this invention is a novel haloalkane dehalogenase discovered from the metagenomics of deep-sea sediments in the southwestern Indian Ocean. Its core technological value lies in its unique substrate preference and environmental adaptability. This enzyme exhibits ultra-high catalytic efficiency for short-chain haloalkanes (especially 1,3-dibromopropane), with an activity reaching 12957.99 U / mg, far exceeding most known HLD enzymes. Its substrate specificity is demonstrated by >98% activity for haloalkanes with a carbon chain length ≤3 (such as 1,2-dibromoethane), while activity drops sharply to <28% for long-chain (such as 1,5-dichloropentane) or cyclic substrates (bromocyclohexane), revealing its precise recognition mechanism of substrate configuration within its catalytic pocket. Furthermore, it possesses excellent organic solvent tolerance: maintaining 100% activity in 20% methanol and retaining >80% activity in 30% methanol, overcoming the bottleneck of traditional dehalogenases' easy inactivation in low-concentration organic solvents. This property makes it suitable for treating industrial wastewater containing organic solvents. Furthermore, HLD-1405 maintains high stability within a pH range of 7.0-10.0 and a temperature range of 30-40°C, and possesses the potential to degrade stubborn pollutants such as DDT and hexachlorocyclohexane (HCH), 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 Electrophoresis diagram of HLD-1405 protein expression; where 1 and 2 are the purified bands of the enzyme;

[0019] Figure 2 The specificity of HLD-1405 protein for different substrates;

[0020] Figure 3 Determination of the optimal temperature for HLD-1405 protein;

[0021] Figure 4 Determination of the optimal pH stability of HLD-1405 protein;

[0022] Figure 5 To determine the temperature stability of HLD-1405 protein;

[0023] Figure 6 To determine the stability of HLD-1405 protein in organic solvents;

[0024] Figure 7This study aims to determine the enzyme activity of HLD-1405 against DDT, hexachlorocyclohexane (HCH), and 2-cees. Detailed Implementation

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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 obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] 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.

[0030] Example 1

[0031] 1. Obtaining the HLD-1405 protein and its encoding gene

[0032] The 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. ORF prediction was used to identify the functional genes and the amino acid sequences of their encoded proteins. The mixed assembly of the six sediment samples yielded 7,495,897 protein-coding gene sequences, approximately 5.34 Gb of nucleotide sequence data. Using the highly active haloalkane dehalogenase LINB_SPHIB (A0A1L5BTC1.1, 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase) from the strain Sphingobium indicum B90A, which is capable of degrading hexachlorocyclohexane, as a template, a new dehalogenase Gene2251405_TVG10_602697_1 was obtained from the aforementioned Southwest Indian Ocean deep-sea sediment metagenomic database based on amino acid homology sequence alignment (BLASTP). The protein sequence with the highest known sequence similarity on NCBI is haloalkanedehalogenase [Alphaproteobacteriabacterium] (TMJ02448.1) from the Mediterranean steppe soil microbial community metagenomic database, with an amino acid sequence similarity of 75.85%. 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 HLD1405-pET21a-BL21(DE3), which was then sequenced and identified.

[0033] The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the protein sequence encoded by the gene is shown in SEQ ID NO.2. The protein encoded by the gene is named HLD-1405.

[0034] SEQ ID NO.1:

[0035] ATGCCGCTGTCTGCTGAACCGTTTGCGGCTAAAAAGTTCGCGGAAATCAAAGGCCGTCGTATGGCCTACATTGATGTTGGTGAAGGCGATGCTATTGTCTTTCAGCACGGCAACCCAACTTCCTCTTACCTGTGGCGCAACATCATGCCGCACTGTCAGGGTCTGGGCCGTCTGATCGCATGCGACCTGATCGGTATGGGCGACTCTGATCGTCTGCCTGATCCGGGTCCGGGCTCCTACGGTCTGGCAGAACACCAGGACTATCTGGATGCTCTGTGGGATCACATCGGTCTGGGTGATAACGTCGTTCTGGTTGTTCACGACTGGGGTTCCGCCCTGGGCTTTGACTGGGCGCACCGTCACCGTGGTCGTGTTCAAGGTATCGCTTACATGGAGGCGATGGTGACCCCGCTGGGCTGGGACGACTGGCCGGAAGCTGCGCGCCGCGCGTTCCAGGGTTTCCGCTCCGACGCAGGTGAGGACATGATCCTGGAACGTAACATGTTCGTCGAACGTATTCTGCCGTCGGCAGTGCTGCGTGAACTGACTGATGCGGAAATGGCGGTATACCGTGCCCCGTATCAGTCTCCGGGCGAAGATCGCCGTCCGACCCTGACTTGGCCTCGCCAGATTCCGATTGATGGTGAACCGGCAGACGTTGTTGCAACCGTGGAAGCGTATGGCGCGTGGCTGGCTGAGTCCGATGTAGCCAAACTGTTCGTCAATGCAGAGCCGGGTTCGATTCTGACGGGTCGTCAGCGTGAGGTCTGCCGTGCGTGGCCGAATCAAACTGAAGTTACTGTTCCAGGCGCTCACTTCGTCCAGGAAGATTCTCCGGACGAGATTGGCCGTGCGATCGCAGATTTCGTTCGCGGCCTGCGCGGTCAGGCCGTAGACTCC;

[0036] SEQ ID NO.2:

[0037] MPLSAEPFAAKKFAEIKGRRMAYIDVGEGDAIVFQHGNPTSSYLWRNIMPHCQGLGRLIACDLIGMGDSDRLPDPGPGSYGLAEHQDYLDALWDHIGLGDNVVLVVHDWGSALGFDWAHRHRGRVQGIAYMEAMVTPLGWDDWPEAARRA FQGFRSDAGEDMILERNMFVERILPSAVLRELTDAEMAVYRAPYQSPGEDRRPTLTWPRQIPIDGEPADVVATVEAYGAWLAESDVAKLFVNAEPGSILTGRQREVCRAWPNQTEVTVPGAHFVQEDSPDEIGRAIADFVRGLRGQAVDS.

[0038] 2. Obtaining the haloalkane dehalogenase HLD-1405

[0039] 2.1 Obtaining the recombinant vector

[0040] The gene coding sequence of HLD-1405 was synthesized using in vitro de novo gene synthesis technology. The optimized nucleotide sequence (SEQ ID NO.1) was obtained through E. coli codon optimization and cloned into the DNA region containing the multiple cloning site between NdeⅠ(CATATG) and HindⅢ(AAGCTT) in the pET21a(+) vector. The other sequences of the pET-21a(+) vector remained unchanged, resulting in a recombinant vector containing the DNA molecule shown in SEQ ID NO.1. This plasmid vector containing the target gene was transformed into E. coli strain BL21(DE3), and the T7 promoter of the vector was used to initiate the expression of the recombinant protein HLD-1405 shown in SEQ ID NO.1.

[0041] 2.2 Obtaining recombinant bacteria

[0042] The recombinant vector pET-1405 obtained above 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 2XYT 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-1405[BL21(DE3)].

[0043] 2.3 Obtaining the dehalogenase HLD-1405

[0044] Single colonies of pET-D1[BL21(DE3)] were picked and inoculated into 2XYT medium containing ampicillin (100 μg / ml) and cultured overnight at 37°C. The overnight culture was then inoculated into 100 mL of 2XYT medium containing ampicillin (100 μg / ml) and cultured at 37°C with shaking (200 rpm) until the OD of the fermentation broth reached [value missing]. 600 Once the pH reaches approximately 0.6-0.8, add IPTG (final concentration 1 mM) to the fermentation system and incubate at 16°C for another 24 hours. After fermentation, centrifuge at 8000 rpm for 20 minutes, discard the supernatant, and collect the bacterial cells. Resuspend the cells in non-denaturing nickel column binding buffer I, sonicate (4 s, 7 s interval, 50 W power, 50 cycles), and centrifuge at 12,000 rpm for 15 minutes. Collect the supernatant, which is the crude enzyme solution containing recombinant protein HLD-1405. The target recombinant protein HLD-1405 band size is 33.2 kDa (this protein is the recombinant protein HLD-1405 shown in SEQ ID NO.2). Figure 1 ).

[0045] 2.4 Purification of dehalogenase HLD-1405

[0046] The composition of non-denaturing nickel column binding buffer I is as follows: 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, water as solvent, pH 7.5.

[0047] The composition of non-denaturing nickel column elution buffer II is as follows: 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, water as solvent, pH 7.5.

[0048] The desalting buffer composition is as follows: 50 mM Gly-NaOH, pH 8.2.

[0049] Purification was performed using nickel affinity chromatography. The supernatant was purified using a 1 mL HiTrapchelating HPcolumn (nickel column) in an Amersham AKTA FLC system. The 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. Washing with non-denaturing nickel column binding buffer I removed 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 5 mL Hitrap Q desalting column to obtain the purified recombinant protein HLD-1405 at a concentration of 1.107 mg / mL.

[0050] 3. Methods for determining dehalogenase activity

[0051] 3.1 Methods for determining enzyme activity

[0052] 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 dehalogenation reactions 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.

[0053] R-CH2X + H2O → R-CH2OH + H + +X(X=Cl - ,Br - ,I - …)

[0054] 6X - +2Hg(CNS)2→HgX2+HgX4 2- +4CNS -

[0055] CNS - +FE 3+ →Fe(CNS) 2+ →Detect absorbance at 460nm(red color)

[0056] (X - =Cl - , Br - , and I - )

[0057] 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.

[0058] Mercuric thiocyanate-ferric ammonium sulfate colorimetric method: Prepare the following two solutions: Mercuric thiocyanate solution: Dissolve mercuric thiocyanate in 100 ml of 95% ethanol. Ferric ammonium sulfate solution: Dissolve 6 g of ferric ammonium sulfate in 6 M nitric acid. Add one-tenth of the original volume of mercuric thiocyanate solution and one-fifth of the original sample volume of ferric ammonium sulfate solution to a given sample volume, mix well, and react for at least ten minutes. Measure the absorbance of the reaction mixture at 450 nm using an ELISA reader.

[0059] Prepare halide ion standard solutions of different concentrations, and simultaneously construct a halide ion concentration standard curve based on the concentration of the standard solutions while detecting the halide ion concentration in the sample. Calculate the halide ion concentration in the sample based on the absorbance value and the halide ion standard curve. (Br) - Concentration standard curve: y = 1.5128x + 0.168; Cl -Concentration standard curve: y = 0.2632x + 0.2635.

[0060] Colorimetric reaction buffer: 50mM Gly-NaOH, pH 8.2.

[0061] Experimental group: The purified recombinant protein HLD-1405 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 ten minutes, and the absorbance at 450 nm was measured using a microplate reader.

[0062] The control group for the above substrate self-hydrolysis differs from the experimental group in that no enzyme is added.

[0063] 3.2 Substrate specificity of dehalogenase HLD-1405

[0064] 1,3-Dibromopropane, 1,3-Dichloropropane, 1,2-Dibromoethane, 1-Bromo-2-methylpropane, 1-Chloropentane, 1,5-Dichloropentane, Bromocyclohexane, Chlorocyclohexane, DDT, HCH, and 2-cees were selected as reaction substrates. The enzyme activity was determined 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. Three replicates were designed for each experimental group.

[0065] The results are as follows Figure 2As shown, the optimal substrate for HLD-1405 is 1,3-dibromopropane, with a specific activity of 12957.99 U / mg, and its relative activity is defined as 100%. HLD-1405 exhibits high specificity for carbon chain length. Besides the optimal substrate 1,3-dibromopropane, it also shows relatively high catalytic activity (98.99% and 75.57%) for 1,2-dibromoethane and 1,3-dichloropropane, which have shorter carbon chains and similar lengths. However, for the relatively longer-chain haloalkanes such as 1,5-dichloropentane, and cyclic cyclohexane and chlorocyclohexane, its relative activity is very low, only 27.6%, 10.84%, and 4.61% of that of the optimal substrate 1,3-dibromopropane, respectively. Meanwhile, 1-bromo-2-methylpropane, containing branched chains, exhibits a significantly different spatial structure from 1,3-dibromopropane. Despite having the same main chain length, its relative activity (31.7%) is significantly lower than that of the optimal substrate. Furthermore, it also shows some catalytic activity against the highly hazardous traditional pesticides DDT and hexachlorocyclohexane, as well as the neurotoxic agent mustard gas mimic 2-cees. Figure 7 The relative activity was 6.07 U / mg, 5.87 U / mg and 10.95 U / mg, respectively.

[0066] 3.3 Determination of the optimal temperature for dehalogenase HLD-1405

[0067] The experiments were conducted using a colorimetric method at reaction temperatures of 4℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, respectively; 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. Three replicates were designed for each experimental group.

[0068] The results are as follows Figure 3 As shown, the optimal temperature for the purified recombinant protein HLD-1405 is 40℃, with a specific activity of 15501.14 U / mg, and its relative activity is defined as 100%. At lower temperatures, HLD-1405 exhibits low catalytic activity, with a relative activity of 10.99%. Within the temperature range of 4℃ to 40℃, its relative activity significantly increases with increasing reaction temperature. However, above 40℃, its relative activity rapidly decreases with further increases in reaction temperature. At 50℃, the specific activity of HLD-1405 is only 9.09% of that at 40℃. Above 50℃, its relative activity gradually decreases until it loses its catalytic activity.

[0069] 3.4 Method for determining the pH stability of dehalogenases

[0070] 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 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 9, incubated at room temperature for 5 h) was defined as 100%, with a specific activity of 15757.29 U / mg. The relative activities of the enzymes treated at other pH conditions were calculated. Three replicates were designed for each experimental group.

[0071] Citric acid-sodium citrate buffer: The final concentration of sodium citrate is 100mM, which is then diluted with distilled water and adjusted to the pH required for the experiment.

[0072] Tris-H2SO4 buffer: The final concentration of Tris is 100mM. Dissolve Tris in distilled water and adjust to the pH required for the experiment.

[0073] Sodium carbonate-sodium bicarbonate buffer: The final concentration of sodium bicarbonate is 100mM, which is then diluted with distilled water and adjusted to the pH required for the experiment.

[0074] The results are as follows Figure 4 As shown, HLD-1405 maintained high catalytic activity after treatment at pH 7.0 to pH 10.0, with a relative activity of over 80%. However, the tolerance of HLD-1405 decreased significantly at pH values ​​below 7.0 or above 10.0. Specifically, the relative activity was 2.55% at pH 5.0 and 34.82% at pH 11.0. The optimal pH value was 9, with a relative activity of 100%.

[0075] 3.5 Method for determining the thermal stability of dehalogenases

[0076] The selected treatment temperatures were 40℃, 50℃, 60℃, and 70℃. Different groups were set up, and the purified recombinant protein HLD-1405 was heat-treated at different temperatures for 30 min, 60 min, and 90 min, respectively. The enzyme activity was determined colorimetrically using 1,3-dibromopropane as the substrate. The enzyme activity of the untreated control group was defined as 100%, with a specific activity of 7986.31 U / mg. The relative activities of the different experimental groups were calculated. Three replicates were designed for each experimental group.

[0077] The results are as follows Figure 5As shown, after heat treatment at temperatures of 40℃, 50℃, 60℃, and 70℃ for 30 minutes, the relative activity of the enzyme decreased to varying degrees. The enzyme activity decreased most rapidly in the 60℃ and 70℃ experimental groups, reaching only 35.68% and 22.79% of the control group's activity, respectively. At 40℃ for up to 90 minutes, the enzyme activity was only half that of the control group and gradually stabilized. However, at 70℃, the specific activity of the enzyme rapidly decreased to less than 30% of its initial activity within 30 minutes. With further extension of the treatment time, HLD-1405 gradually lost its activity.

[0078] 3.6 Method for determining the stability of dehalogenases in organic solvents

[0079] Different groups were set up, and the purified recombinant protein HLD-1405 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℃. The relative activity of the control group without organic solvent treatment was defined as 100%, and the specific activity was 9600.63 U / mg. The relative activities of the experimental groups were calculated. Three replicates were designed for each experimental group.

[0080] The results are as follows Figure 6 As shown, HLD-1405 exhibits the worst tolerance to acetonitrile; under low concentration (10%-20%) treatment conditions, its relative activity rapidly decreases to 7.09%. When the acetonitrile concentration exceeds 30%, HLD-1405 essentially loses its catalytic activity. Under low concentrations of ethanol and DMSO, the relative activity of the enzyme is less affected, but its specific activity decreases rapidly with further increases in organic solvent concentration. HLD-1405 shows the best tolerance to methanol; its activity remains unaffected when treated with a 10%-20% concentration.

[0081] 3.7 Methods for determining the kinetic parameters of dehalogenases

[0082] Optimal substrate 1,3-dibromopropane was prepared at different concentrations using the organic solvent ethanol, resulting in optimal substrate concentrations of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM. Colorimetric experiments were conducted using these different optimal substrate concentrations as experimental groups. The reaction temperature was 40 °C, and each substrate concentration was measured in triplicate. A graph was plotted with the reciprocal of the substrate concentration (1 / [S]) on the x-axis and the reciprocal of the reaction rate (1 / v) on the y-axis. The slope of the resulting line was K. m / V max The intercept of the resulting line is 1 / V maxThen, based on the concentration and molecular weight of the dehalogenase, the kinetic parameter V of the enzyme-catalyzed reaction is calculated. max K m k cat and k cat / K m Results: The maximum reaction rate of this enzyme, determined using 1,3-dibromopropane as a substrate, was V. max =0.1077 mM / min, Michaelis constant K m =0.496mM, transformation number k cat =5.96s -1 k cat / K m 12.01mM -1 ·s -1 .

[0083] 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. An isolated protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. A nucleic acid molecule encoding the protein of claim 1.

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

1.

4. A recombinant vector, recombinant microorganism, or expression cassette containing the nucleic acid molecule of claim 2 or 3.

5. A method for preparing the protein of claim 1, characterized in that, include: The recombinant microorganism of claim 4 is cultured, and the protein is induced to express and purified.

6. A method for degrading haloalkanes, characterized in that, include: The protein catalyzes the dehalogenation of haloalkanes according to claim 1; wherein the haloalkanes are 1,3-dibromopropane, 1,2-dibromoethane, or 1,3-dichloropropane.

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