Antarctic krill dehalogenase and its application in degradation of halogenated alkane compounds

Abstract

The present invention provides an Antarctic krill dehalogenase and its application in the degradation of halogenated alkane compounds. The amino acid sequence of the Antarctic krill dehalogenase provided is SEQ ID NO: 2. The Antarctic krill dehalogenase provided by the present invention can efficiently degrade bis-2-chloroethyl sulfide under mild conditions to generate chloride ions and non-toxic thiodiglycol, which can replace traditional chemical decontamination methods and show great application potential. Moreover, the stability protective agent of the Antarctic krill dehalogenase provided can always maintain the activity of the dehalogenase above 88%.

Description

Technical Field

[0001] The invention belongs to the technical field of functional genes, and in particular relates to an Antarctic krill dehalogenase and an application thereof in the degradation of halogenated alkane compounds. Background Art

[0002] Since a large amount of halogenated alkane byproducts are inevitably produced in the industrial and agricultural production process, these byproducts not only cause a waste of resources, but also some byproducts have certain toxicity and pollution, causing harm to the human body and the environment. Haloalkane dehalogenases have attracted much attention due to their unique catalytic mechanism and wide substrate specificity. They not only have important theoretical value, but have also been partially applied in the fields of recycling byproducts of chemical processes, bioremediation of toxic environmental pollutants, decontamination of chemical warfare agents, biosensing of environmental pollutants, protein labeling for protein analysis and cell imaging. Especially in the application field of chemical warfare agents, mustard gas (bis-2-chloroethyl sulfide) is a conventional chemical warfare agent and also a corrosive agent, which can alkylate genetic material in organisms, thereby causing blistering, erosion and necrosis of skin and tissues.

[0003] Traditionally, chemical decontamination methods are used to purify halogenated alkane byproducts, but most of these chemical reagents are toxic and corrosive, and are not suitable for use on personnel, clothing, precision instruments, etc. Dehalogenases can effectively degrade bis-2-chloroethyl sulfide under mild conditions to generate chloride ions and non-toxic thiodiglycol, which is expected to replace traditional chemical decontamination methods and show great application potential. Since the discovery of the first haloalkane dehalogenase (HLDs) in 1985, they have been found in marine bacteria, pathogens, archaea, and bacterial strains of plant symbionts and plant parasites. The first reported haloalkane dehalogenase was DhlA discovered in Xanthobacter autotrophicus GJ10 in 1985, which can hydrolyze 1,2-dichloroethane. In 1993 and 1997, LinB and DhaA were discovered, which can hydrolyze 1,3,4,6-tetrachloro-1,4-cyclohexane and 1-chlorobutane, respectively. They were found in Sphingomonas paucimobilis UT26 and Rhodococcus rhodochrous, respectively. The crystal structures and substrate specificities of these three haloalkane dehalogenases have been widely studied. According to phylogenetic analysis, HLDs can be divided into three subfamilies: HLD-I, HLD-II and HLD-III. The three subfamilies have different catalytic pentads, namely Asp-His-Asp+Trp-Trp, Asp-His-Glu+Asn-Trp, and Asp-His-Asp+Asn-Trp. Most of the HLDs identified by biochemistry belong to HLD-II. According to substrate specificity, they are divided into four groups, namely SSG-I, SSG-II, SSG-III and SSG-IV. SSG-I can transform many difficult-to-degrade chlorinated substrates; HLDs that prefer brominated and iodinated compounds are classified as SSG-IV.

[0004] There is no report on dehalogenases from marine animals, especially Antarctic krill. Antarctic krill is a key species in the Antarctic ecosystem, with a population density of 10,000-30,000 per cubic meter. In terms of biomass energy, they may be the animal species with the largest biomass on Earth (about 500 million tons in total). Antarctic krill itself is in extreme conditions such as low ultraviolet light above Antarctica, and produces genes and biological enzyme systems different from those of marine organisms in other seas. The present invention has discovered a dehalogenase with good degradation effect on bis-2-chloroethyl sulfide from Antarctic krill, and has achieved efficient expression and preparation in yeast cells. Summary of the invention

[0005] The present invention provides an Antarctic krill dehalogenase and application thereof in the degradation of halogenated alkane compounds. The provided Antarctic krill dehalogenase can catalyze the degradation of halogenated alkane compounds such as chlorinated, brominated, iodinated alkanes, cycloalkanes, esters, ethers, epoxides, etc.

[0006] The present invention first provides an Antarctic krill dehalogenase, which comprises:

[0007] 1) a protease having an amino acid sequence of SEQ ID NO: 2;

[0008] 2) an isozyme of the protease in 1) amplified from Antarctic krill;

[0009] The isozymes described herein are proteases derived from 1) by substituting, deleting or adding one or more amino acids to the amino acid sequence of SEQ ID NO: 2;

[0010] In yet another aspect, the present invention also provides a gene encoding the above-mentioned Antarctic krill dehalogenase, one sequence of which is SEQ ID NO: 1.

[0011] The present invention further protects a recombinant expression vector comprising the above gene fragment.

[0012] The present invention also protects a recombinant engineering strain, which contains the above-mentioned recombinant expression vector.

[0013] As a specific description of an embodiment of the present invention, the recombinant bacteria are Pichia pastoris cells.

[0014] The present invention also protects one use of the Antarctic krill dehalogenase, which is the use in degrading and purifying halogenated alkane compounds.

[0015] The present invention also provides a method for degrading and purifying halogenated alkane compounds, which uses the screened Antarctic krill dehalogenase to degrade;

[0016] The halogenated alkane compound, as a specific record of an embodiment of the present invention, is bis-2-chloroethyl sulfide.

[0017] In another aspect, the present invention also provides the protective agent for Antarctic krill dehalogenase, the composition ratio of which is as follows: 0.02 g / ml borax, 0.03 g / ml calcium chloride, and 5% (v / v) glycerol.

[0018] The Antarctic krill dehalogenase provided by the present invention can efficiently degrade bis-2-chloroethyl sulfide under mild conditions to generate chloride ions and non-toxic thiodiglycol, and can replace traditional chemical decontamination methods, showing great application potential. Moreover, the stability protective agent of the Antarctic krill dehalogenase provided can always maintain the activity of the dehalogenase above 88%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Electrophoresis of Antarctic krill dehalogenase gene nucleic acid, wherein lane M is D2000 marker, and lane 1 is Antarctic krill dehalogenase gene;

[0020] Figure 2 : Electrophoresis of Antarctic krill Antarctic krill dehalogenase protein, wherein lane M is Marker (10-180Kda), lane 1 is blank control, lane 2 is empty vector control yeast cell supernatant; lane 3 is recombinant Antarctic krill dehalogenase (39kDa);

[0021] Figure 3 : Fermentation growth and enzyme activity curve of recombinant bacteria of Antarctic krill dehalogenase;

[0022] Figure 4 :Antarctic krill dehalogenase stability experiment diagram. DETAILED DESCRIPTION

[0023] The enzyme discovered by the present invention is classified as a haloalkane dehalogenase, which is a type of hydrolase that can catalyze the cleavage of the carbon-halogen bond of a haloalkane compound. It belongs to the α / β hydrolase superfamily together with lipase, esterase, carboxypeptidase, etc. The haloalkane compound is converted into the corresponding alcohol, halogen ion and proton after being hydrolyzed, and the whole enzymatic reaction process does not require the participation of coenzyme / cofactor or oxygen. Its substrate spectrum is relatively wide, and it can catalyze the hydrolysis of haloalkane compounds such as chlorinated, brominated, iodinated alkanes, cycloalkanes, esters, ethers, epoxides, etc.

[0024] The present invention is described in detail below in conjunction with embodiments and drawings.

[0025] Example 1: Cloning of Antarctic krill dehalogenase gene and construction of expression vector

[0026] Using the Antarctic krill cDNA genome sequence as a template, the Antarctic krill dehalogenase gene was amplified using HeF / HeR primers (primers contain EcoRI-NotI restriction sites). The agarose gel electrophoresis results of the PCR-amplified Antarctic krill dehalogenase are shown in Figure 2. Figure 1 As shown, the gene fragment is 1083bp in length (including restriction enzyme cutting sites). The primers are as follows:

[0027] HeF 5,-CGGGCCGATGGCGGGCTTGAAGTTCTATTTGA-3,

[0028] HeR 5,-GCGGCCTATGATAGTTTTCCATCAACAAAG-3,

[0029] The gene to be screened was determined, and the sequence of its nucleotide fragment was as follows:

[0030] ATGGCGGGCTTGAAGTTCTATTTGATACGGCTGTTGGTACTACCCATCCTTATCATCAAAGTTCAAGCGCTACAAATGGT

[0031] GAACATTTTACTGAGCATCGCATCAAAGTTATTTATCACAGGAAAGAAACAAATGATAGTTCGTACCCCGGAAGAAAACT

[0032] TTTCTAACTTGGATAAAGTTGGCTACACCTTCAAAGCTAACTACGTTGAACTTCCAATAGGTGGGGGAAAAGCACTGCCA

[0033] AGGGTGCACTATGTTGATGAAGGCCCACGAGATGCCATGGAAACAATCCTATGTCTTCATGGGGAGCCAAGTTGGTCTTT

[0034] CCTCTACCGCCACATGATCCCCATTCTTGCCAAGGCTGGTTACCGTGTTATTGCTCCAGACTTCATAGGCTTTGGGAAAT

[0035] CTGATAAATACACATATATGGAATGTTACACTCATGAAATGCATACTCAGACTCTTCGACTGCTTCTAGATTATTTAAAG

[0036] GTGTCAAATGTTACAGTGGTTGGGCAGGATTGGGGTGGTCTTATCGGTTGCTCCGTGTTGAAGGATTCTCCAGATAAATT

[0037] CTCTCGTCTTGTTGTCATGAACACCGGCGTCCCTGATGGCATCATAAAAAAGTTCACCCCATATGTCATAGCCAATGCTA

[0038] CACCTGTTATACTCTGGCAGGCAGTTGTTCAGCTTCTTGGAGTATGGTTACCAATAGGTCTCTTGTTCAAACACATCTTG

[0039] AAGAATAACCCCTCTAAGGATGTAATAAAGGGTTACTGTGCCCCATTCCCTTCTGCACTCTACAAGGCCGGTGCAGCAGC

[0040] ATGGCCACTGAATGTTCCAGTCCTCCCTGGTGGGCCTATTGCTGCTGATATGAAGATAGCACAAACCTTCCTGAAAAGAT

[0041] GGAAGAATCCTGTGCTTATTATGTTCTCAGATAGTGACCCTATCTCTGCCTCATGGAAATACACTTTTGAAGAGTTATGT

[0042] CCTGCGGCACACCAGAAGACAATTGTAGGTGCTGGTCACATGCTGCAGGAAGAGAAGGGAGAAGAGATCGCTTACAACAT

[0043] TGTCAGCTTTGTTGATGGAAAACTATCATAA(SEQ ID NO:1)。

[0044] The amino acid sequence of its encoded protein is as follows:

[0045] MAGLKFYLIRLLVLPILIIKVQALQMVNILLSIASKLFITGKKQMIVRTPEENFSNLDKVGYTFKANYVELPIGGGKALP

[0046] RVHYVDEGPRDAMETILCLHGEPSWSFLYRHMIPILAKAGYRVIAPDFIGFGKSDKYTYMECYTHEMHTQTLRLLLDYLK

[0047] VSNVTVVGQDWGGLIGCSVLKDSPDKFSRLVVMNTGVPDGIIKKFTPYVIANATPVILWQAVVQLLGVWLPIGLLFKHIL

[0048] KNNPSKDVIKGYCAPFPSALYKAGAAAWPLNVPVLPGGPIAADMKIAQTFLKRWKNPVLIMFSDSDPISASWKYTFEELC

[0049] PAAHQKTIVGAGHMLQEEKGEEIAYNIVSFVDGKLS (SEQ ID NO: 2).

[0050] The amino acid sequence of the haloalkane dehalogenase obtained by the present invention is compared on NCBI, and the result shows that the enzyme sequence has a low similarity with the haloalkane dehalogenase protein sequence reported so far, wherein the similarity with the haloalkane dehalogenase from Chinese shrimp (Penaeus chinensis) (NCBI accession number XP_047472246.1) is the highest, which is only 59.1%, and the similarity with the haloalkane dehalogenase from American lobster (American lobster) (NCBI accession number XP_042239770.1) is 58.99%. It shows that the haloalkane dehalogenase obtained by screening is a new type of dehalogenase.

[0051] The PGAPZαA vector and the Antarctic krill dehalogenase gene were double-digested with EcoI and NotI, and the digestion products were recovered with a DNA product purification kit. The Antarctic krill dehalogenase gene and the double-digested PGAPZαA vector were recombined using a ligase and transformed into Pichia pastoris cells. After 48 hours, a single bacterium was picked and the positive recombinant clone was verified by bacterial liquid PCR. The positive clone was then sequenced by DNA to verify the correctness of the gene sequence.

[0052] Example 2: Preparation of recombinant Antarctic krill dehalogenase

[0053] Take 100 μL of the positive recombinant Pichia pastoris GS115 / PGAPZαA-dehalogenase bacterial solution identified by sequencing, inoculate it into 5.0 mL of YPD liquid medium containing bleomycin (glycerol 2%, yeast powder 1%, peptone 2%), and culture it at 28°C overnight. The next day, take 500.0 μL of seed solution and inoculate it into 50.0 mL of YPD liquid medium containing bleomycin and continue to culture it at 28°C for 54 hours. Centrifuge it at 8000g for 5 minutes, collect the supernatant, and concentrate it 20 times through an ultrafiltration concentration membrane with a molecular weight cutoff of 10Kda to obtain the recombinant Antarctic krill dehalogenase crude enzyme solution. The supernatant was detected by SDS-PAGE ( Figure 2 ). When the amount of inducer glycerol added was 2%, after 54 hours of induction, a recombinant dehalogenase with a molecular weight of 39Kda was obtained in the fermentation supernatant.

[0054] Example 3 Activity determination of Antarctic krill dehalogenase

[0055] Enzyme activity determination method: bis(2-chloroethyl) ether is used as substrate, the total reaction system is 500μl, the buffer is 0.1mol / L Gly-NaOH, the final concentration of the substrate is 10mmol / L, and the amount of enzyme added is 10μl crude enzyme solution. After reacting at 40℃ for 20min, 200μl is added to a centrifuge tube containing 20μl 30% (volume fraction) HNO3 to terminate the reaction, and then 55μl Hg(SCN)2 solution and 110μl NH4Fe(SO4)2 solution are added. After mixing and standing for 10min, 200μl is added to a 96-well plate, and the absorbance at 600nm is measured using an enzyme reader.

[0056] Enzyme activity definition: Under certain conditions, 1 minute of catalysis of substrate produces 1 μmol Cl - The amount of enzyme required was defined as 1 unit of enzyme activity.

[0057] Using 2.0% glycerol as an inducer, recombinant dehalogenase was secreted into the extracellular space after 8 h of shake flask fermentation ( Figure 3 ), sampling was performed at 54h when the enzyme production was the highest, and the degradation activity of the recombinant enzyme on bis(2-chloroethyl) ether reached 2000U / mL (Table 1).

[0058] Table 1: Recombinant Antarctic krill dehalogenase activity and comparison table

[0059]

[0060] The prepared bis(2-chloroethyl) ether was used as a substrate. When the substrate concentration was 100 mmol / L and the temperature was 40°C, the recombinant halogenated alkane dehalogenase was added to measure the degradation efficiency at different times. The results showed that after 0.5 h, 1 h, 2 h, 3 h, and 5 h, the degradation rates of bis(2-chloroethyl) ether reached 7.9%, 18.3%, 37.6%, 53.7%, and 63.2%, respectively. The results show that the recombinant halogenated alkane dehalogenase obtained by the present invention can effectively decompose the substrate under mild conditions.

[0061] Example 4 Stability Protectant for Antarctic Krill Dehalogenase

[0062] Enzyme inactivation is an important factor limiting the industrial production and application of enzyme preparations. There are three main methods for stabilizing enzymes, namely, adding protective agents, chemical modification and immobilization. The present invention provides an optimal dosage of a chemical protective agent for Antarctic krill dehalogenase. Borax 0.02 g / ml, calcium chloride 0.03 g / ml, glycerol 5% (v / v). After being stored at 30 degrees for 90 days, the activity of the recombinant dehalogenase was always maintained at more than 88% ( Figure 4 ).

[0063] In summary, the present invention provides an Antarctic krill dehalogenase gene, protein sequence and preparation method thereof. The provided dehalogenase comes from Antarctic krill. The enzyme has a wide substrate spectrum and can catalyze the breaking of carbon-halogen bonds of halogenated alkane compounds such as chlorinated, brominated, and iodinated alkanes, cycloalkanes, esters, ethers, and epoxides. It has application prospects in the fields of biocatalysis, bioremediation, biosensing and cell imaging.

Claims

1. An Antarctic krill dehalogenase, characterized in that The amino acid sequence of the Antarctic krill dehalogenase is SEQ ID NO:

2.

2. A gene, characterized in that The gene encodes the Antarctic krill dehalogenase according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

1.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid fragment of the gene according to claim 2.

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

6. The recombinant engineered strain according to claim 5, characterized in that The recombinant engineering strain is a Pichia pastoris cell.

7. Use of the Antarctic krill dehalogenase according to claim 1 in the degradation and purification of halogenated alkane compounds, wherein the compound is bis-2-chloroethyl sulfide.

8. A method for degrading and purifying halogenated alkane compounds, characterized in that: The method is to use the Antarctic krill dehalogenase described in claim 1 to degrade halogenated alkane compounds, and the compound is bis-2-chloroethyl sulfide.

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