Burkholderia lipase with four mutations of P113A, Q88S, T132G and L287S as well as recombinant expression vector and application thereof

Abstract

The invention relates to the technical field of bioengineering, and discloses Burkholderia lipase with four mutations of P113A, Q88S, T132G and L287S as well as a recombinant expression vector and application of the Burkholderia lipase. Proline of the 113th amino acid sequence of the LipA of the wild burkholderia lipase mutates into alanine, glutamine of the 88th amino acid sequence of the LipA of the wild burkholderia lipase mutates into serine, threonine of the 132th amino acid sequence of the LipA of the wild burkholderia lipase mutates into glycine, and leucine of the 287th amino acid sequence of the LipA of the wild burkholderia lipase mutates into serine. The yield of the mutant enzyme is improved by 63%, the enzyme activity is improved by 34%, the hydrolysis capacity on ethyl ester type fish oil is improved by 38%, the selectivity on EPA is obviously enhanced, and the EPA purification and enrichment effect is excellent. According to the mutant, the preparation cost is reduced, the hydrolysis capability and selectivity are improved, and the mutant has great popularization and application values.

Description

[0001] This invention is a divisional application of the invention with the application number 2025101891393, the application date of February 20, 2025, the application type of invention, and the invention title of "Mutants of Burkholderia lipase, Their Recombinant Expression Vectors and Applications". Technical Field

[0002] This invention relates to the field of bioengineering technology, specifically to Burkholderia lipase with four mutations of P113A, Q88S, T132G and L287S, its recombinant expression vector and applications. Background Art

[0003] The statements in this section only provide background information related to the disclosure of this application and may not constitute prior art.

[0004] Eicosapentaenoic acid (EPA), as an omega-3 series polyunsaturated fatty acid, plays a crucial role in human health, but the human body cannot synthesize it by itself. Research has confirmed that EPA has positive multiple effects in maintaining cardiovascular health, anti-tumor activity, regulating glucose and lipid metabolism, and anti-inflammation. Currently, EPA mainly comes from marine fish and has been widely used in many fields such as food, health products, and disease treatment. However, the content of polyunsaturated fatty acids (PUFAs) such as EPA and docosahexaenoic acid (DHA) in fish oil is relatively low (5% - 26%), which cannot meet the market demand for health products. Therefore, how to effectively and selectively enrich EPA from fish oil faces challenges and has become one of the hotspots of concern for researchers.

[0005] The key problem that unsaturated fatty acids in fish oil are easily degraded and oxidized during molecular distillation still needs to be further solved. Enzymatic enrichment of EPA is considered a promising approach because of its mild reaction conditions, high efficiency, and strong specificity, which can effectively reduce oxidation and isomerization. Lipase belongs to hydrolases and can hydrolyze triglycerides into diglycerides, monoglycerides, and fatty acids. These enzymes also exhibit esterification, transesterification, and alcoholysis. However, lipases usually show different selectivities for medium-chain free fatty acids (FFAs) or long-chain free fatty acids, as well as saturated or unsaturated free fatty acids. Currently, researchers mainly focus on the total amount of DHA and EPA. Currently, some people use Geotrichum sp. lipase to hydrolyze n-3 PUFA and selectively enrich the total content of EPA (1.53% - 1.85%) and DHA (24.1% - 30.9%) in fish oil, etc. However, the selectivity and catalytic stability of these lipases in enriching EPA from fish oil are relatively low.

[0006] Studies have shown that Burkholderia cepacia lipase (BCL) has good selectivity for EPA, increasing the EPA in fish oil by 22.4%. However, this content is still far from meeting the market demand. Summary of the Invention

[0007] The object of the present invention is to provide a mutant of Burkholderia cepacia lipase, its recombinant expression vector and application, aiming at the problem that the effect of enriching EPA in fish oil by wild-type Burkholderia cepacia lipase is limited. It can significantly improve the effect of enriching EPA in fish oil and increase the EPA content in fish oil by 33%.

[0008] The technical solution of the present invention is as follows: On the one hand, the present invention provides a mutant of Burkholderia cepacia lipase, and the mutant has at least any one or more of the following mutations: P131S: The proline at the 131st amino acid of LipA of wild-type Burkholderia cepacia lipase is mutated to serine; T132A: The threonine at the 132nd amino acid of LipA of wild-type Burkholderia cepacia lipase is mutated to alanine; T251A: The threonine at the 251st amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to alanine; T251S: The threonine at the 251st amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to serine; P131A: The proline at the 131st amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to alanine; P131Q: The proline at the 131st amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to glutamine; T132G: The threonine at the 132nd amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to glycine; Q88S: The glutamine at the 88th amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to serine; P113A: The proline at the 113th amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to alanine.

[0009] On the other hand, the present invention provides a mutant of Burkholderia cepacia lipase, and the mutant has the following 2 mutations: Q88S: The glutamine at the 88th amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to serine; P113A: The proline at the 113th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to alanine.

[0010] On the other hand, the present invention provides a mutant Burkholderia lipase, and the mutant has any one of the following mutation combinations: Combination 1: Q88S: The glutamine at the 88th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to serine; P113A: The proline at the 113th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to alanine; T132G: The threonine at the 132nd amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to glycine; L287S: The leucine at the 287th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to serine; Combination 2: Q88S: The glutamine at the 88th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to serine; P113A: The proline at the 113th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to alanine; P131S: The proline at the 131st amino acid of LipA of wild-type Burkholderia lipase is mutated to serine; L287S: The leucine at the 287th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to serine; Combination 3: Q88S: The glutamine at the 88th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to serine; P113A: The proline at the 113th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to alanine; P131S: The proline at the 131st amino acid of LipA of wild-type Burkholderia lipase is mutated to serine; T251A: The threonine at the 251st amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to alanine.

[0011] According to a preferred embodiment, the mutation is induced and prepared by mutant primers. The mutant primer for P131S includes P131S-F shown in SEQ ID NO.3; and P131S-R shown in SEQ ID NO.4.

[0012] According to a preferred embodiment, the mutation is induced and prepared by mutation primers. The mutation primer for T132A includes T132A-F shown in SEQ ID NO.13; and T132A-R shown in SEQ ID NO.14.

[0013] According to a preferred embodiment, the mutation is induced and prepared by mutation primers. The mutation primer for T251A includes T251A-F shown in SEQ ID NO.9; and T251A-R shown in SEQ ID NO.10.

[0014] According to a preferred embodiment, the mutation is induced and prepared by mutation primers. The mutation primer for T251S includes T251S-F shown in SEQ ID NO.11; and T251S-R shown in SEQ ID NO.12.

[0015] According to a preferred embodiment, the mutation is induced and prepared by mutation primers. The mutation primer for P131A includes P131A-F shown in SEQ ID NO.1; and P131A-R shown in SEQ ID NO.2.

[0016] According to a preferred embodiment, the mutation is induced and prepared by mutation primers. The mutation primer for P131Q includes P131N-F shown in SEQ ID NO.17; and P131M-R shown in SEQ ID NO.18.

[0017] According to a preferred embodiment, the mutation is induced and prepared by mutation primers. The mutation primer for T132G includes T132N-F shown in SEQ ID NO.19; and T132N-R shown in SEQ ID NO.20.

[0018] According to a preferred embodiment, the mutation is induced and prepared by mutation primers. The mutation primer for Q88S includes Q88S-F shown in SEQ ID NO.5; and Q88S-R shown in SEQ ID NO.6.

[0019] According to a preferred embodiment, the mutation is induced and prepared by mutation primers. The mutation primer for P113A includes P113A-F shown in SEQ ID NO.7; and P113A-R shown in SEQ ID NO.8.

[0020] According to a preferred embodiment, the mutation is induced and prepared by a mutant primer, and the mutant primer for L287S includes L287S-F shown in SEQ ID NO.15; and L287S-R shown in SEQ ID NO.16.

[0021] On the other hand, the present invention provides the application of a Burkholderia lipase mutant as described above in the purification and enrichment of EPA.

[0022] On the other hand, the present invention provides a recombinant expression vector of Burkholderia lipase, which includes a plasmid backbone and a nucleotide sequence encoding a Burkholderia lipase mutant as described above inserted into the plasmid backbone.

[0023] According to a preferred embodiment, the plasmid backbone is pETDuet, pET-28a, pET-22b, pET-32a or pET-25.

[0024] According to a preferred embodiment, the nucleotide sequence encoding a Burkholderia lipase mutant as described above is inserted upstream of the f1 ori site of the pETDuet plasmid.

[0025] According to a preferred embodiment, the nucleotide sequence encoding a Burkholderia lipase mutant as described above includes the lipA and lipB genes, the lipB, lipA and the f1 ori on the pETDuet plasmid are in series in turn, and the lipA gene sequence has the mutation as described above.

[0026] The wild type of the Burkholderia lipase is the lipase (Lipase) derived from Burkholderia sp. ZYB002 with the NCBI accession number 573762. This lipase contains two genes, lipA (NCBI Gene ID: 56661727) and its corresponding chaperone protein lipB (NCBI Gene ID: 56662904). lipA has a total of 993 nucleotides and 330 amino acids.

[0027] Compared with the existing technology, the beneficial effects of the present invention are as follows: 1. For the mutants of Burkholderia lipase, multiple Burkholderia lipase mutants with significant effects on the purification and enrichment of EPA are discovered by means of induced mutation, which respectively involve single-site mutation or combined mutation at multiple sites. The mutants improve the protein yield of Burkholderia lipase, and all have better enzyme activity (the highest increase is 57%) and the efficiency of catalyzing the hydrolysis reaction of ethyl ester type fish oil; they have good effects on the purification and enrichment of EPA; 2. After constructing an expression vector for heterologous expression with a mutant of Burkholderia lipase, the mutant can be heterologously expressed in other fields, achieving a more extensive effect and a more extensive production source, and reducing the production cost of the mutant of Burkholderia lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the recombinant expression vector of the lipase-encoding gene in the present invention; Figure 2 is the standard curve graph of the protein solution concentration in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0029] The specific examples listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific examples described below. For those skilled in the art, any equivalent modifications and substitutions to the examples described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. Those not specified in the examples are carried out under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer can be purchased commercially as conventional products. To better illustrate the present invention, numerous specific details are given in the following detailed description. Those skilled in the art should understand that the present invention can be implemented without some specific details. In other examples, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Unless otherwise specified, the units used in this specification are international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production. Unless otherwise specified, the methods used in the present invention are conventional methods in the art; unless otherwise specified, the reagents used in the present invention are commercially available.

[0031] The features and properties of the present invention will be further described in detail below in conjunction with the examples.

[0032] Example 1 I. Construction of Mutant lipA and lipB of Burkholderia (synthesized by Beijing Tsingke Biotechnology Co., Ltd.) were used with Figure 1The construction is carried out on the pETDuet plasmid (purchased from EMD Biosciences (Novagen)) vector in the order shown. The mutant plasmid is constructed using the Novoprotein MutExpress II Fast Mutagenesis Kit V2. Log in to the Novoprotein official website to design site-directed mutagenesis primers. The designed primers are shown in Table 1 below. The target plasmid is amplified using Phanta Max Super-Fidelity DNA Polymerase. After the amplified product is recombined and circularized, it is transformed into E. coli DE3 (BL21) (purchased from Beijing Tsingke Biotechnology Co., Ltd.) competent cells. Take 70 μL of the bacterial solution and spread it on an LB solid plate medium containing Ampicillin (Amp, 50 mg / L) antibiotic using glass beads, and incubate it upside down overnight in a 37 °C incubator. Recombinant product identification: Randomly pick 5 single colonies and add them to a 1.5 mL or 2 mL centrifuge tube containing 600 μL of LB liquid medium containing Amp antibiotic, shake the bacteria at 37 °C and 220 rpm for 5 - 6 h. The bacterial solution will become turbid. Aliquot 100 μL and send it for sequencing. Store the remaining bacterial solution at 4 °C. If the sequencing result is correct, preserve the mutant strain using 60% glycerol (LB medium formula: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, 15 g / L agar powder needs to be added for solid medium).

[0033] Table 1 Site-directed mutagenesis primers

[0034] II. Expression and collection of mutant proteins Mother liquor preparation: Inoculate a single colony and culture it overnight; Inoculate the mother liquor into TB medium (2% volume ratio); (TB medium formula: peptone 20 g / L, yeast extract 24 g / L, glycerol 4 g / L, dipotassium hydrogen phosphate 12.25 g / L, potassium dihydrogen phosphate 2.3 g / L.) Cultivate for about 4 h until OD 600 reaches 0.7 - 0.9, add IPTG (0.1 mmol / L), collect the bacteria after expressing for 48 h (180 rpm, 20 °C); Centrifuge at 4 °C and 4000 rpm for 10 min to collect the bacteria; wash the bacteria once with about 100 mL of pure water and PBS respectively; Add PBS buffer (with a mass ratio of 1:10 to the bacterial cells), mix well, and perform ultrasonic disruption (15%, ultrasonic for 4 seconds, pause for 3 seconds, 5 minutes), centrifuge twice (10,000 rpm, 8 minutes); the supernatant is the protein solution containing the mutant enzyme, that is, the enzyme solution. PBS buffer formula: NaCl (sodium chloride): 8 g / L, KCl (potassium chloride): 0.2 g / L, Na2HPO4 (disodium hydrogen phosphate): 1.44 g / L, KH2PO4 (potassium dihydrogen phosphate): 0.24 g / L, adjust the pH to 7.4.

[0035] III. Protein content detection Prepare standard protein solutions with different concentrations: Take 7 ordinary clean test tubes. Tube 1 contains 0.1 mL of deionized water as the blank group. For tubes 2 - 6, use a micropipette to add 0.02, 0.04, 0.06, 0.08, and 0.10 mL of 1.0 mg / mL BSA solution respectively, and then fill each tube to 0.1 mL with deionized water. Additionally, tube 7 contains 0.1 mL of the protein solution to be tested. Vortex all the test tubes well. Add the staining reagent: Use a pipette or a burette to add 3.0 mL of Coomassie Brilliant Blue G250 reagent to the above test tubes respectively, and mix gently and quickly with a vortex mixer. Colorimetry: The combination of Coomassie Brilliant Blue reagent and protein is fast and can be completed in 2 - 5 minutes and remains stable within 1 hour. Therefore, the light absorbance value A of the solution in each test tube at a wavelength of 595 nm can be measured on a spectrophotometer after the solution has been left standing for 5 minutes. 595 value; Make a standard curve: Use the concentration of the standard protein solution (mg / mL) as the abscissa and the absorbance value A 595 as the ordinate to plot a graph, and a straight - line fitting can be performed to obtain a standard curve; the results are as Figure 2 shown; Calculate the concentration of the protein to be tested: Compare the light absorbance A of the protein solution to be tested measured by the spectrophotometer with the standard curve, and the concentration of the protein to be tested can be calculated. The results are shown in Table 2. 595 value.

[0036] Table 2 Protein concentration

[0037] According to the results shown in Table 2, these mutants effectively increased the protein yield and can greatly reduce the production cost of Burkholderia lipase in industrial production.

[0038] IV. Protein purification First, equilibrate the HisTrapTM HP affinity chromatography column with 5 column volumes of nickel column equilibration buffer at a flow rate of 0.5 mL / min; then load 50 mL of crude enzyme solution filtered through a 0.22 μm filter membrane into the HisTrapTM HP affinity chromatography column at a flow rate of 0.3 mL / min; after sample loading, first wash the affinity chromatography column with more than 10 column volumes of HisTrapTM HP affinity chromatography column equilibration buffer at a flow rate of 0.5 mL / min to remove unbound proteins; finally, elute the HisTrapTM HP affinity chromatography column with a gradient of HisTrapTM HP affinity chromatography column equilibration buffer and HisTrapTM HP affinity chromatography column elution buffer. The elution conditions are as follows: the total elution volume is 20 mL, the flow rate is 0.3 mL / min, and the eluate is collected in fractions of 2 mL / tube.

[0039] V. Enzyme Activity Assay The method for measuring lipase activity by the olive oil titration method is as follows: Take 4 g of polyvinyl alcohol and add it to 180 mL of deionized water. Stir slowly at 90 °C until completely dissolved, and cool and make up the volume to 200 mL. Take 150 mL of the solution, add 50 mL of olive oil, and emulsify it on a tissue homogenizer for 3 - 5 min to obtain an olive oil emulsion. Take 4 mL of the olive oil emulsion, add 5 mL of 50 mM PB buffer solution at pH 7.5. The blank control group adds 15 mL of ethanol in advance, then incubate it in a water bath at 30 °C for 15 min, quickly add 1 mL of enzyme solution, stir magnetically at 800 r / min and start timing the reaction for 15 min. Add 15 mL of ethanol to the sample group to terminate the reaction. Finally, add 2 drops of 1% phenolphthalein indicator, and titrate with a 100 mM sodium hydroxide solution to measure the enzyme activity. The results are shown in Table 3.

[0040] Table 3 Enzyme Activity

[0041] According to the results shown in Table 3, among them, Q88S, P113A, P131S, T251A had a 57% increase in enzyme activity compared to WT, Q88S, P113A, T132G, L287S had a 34% increase, and Q88S, P113A, P131S, L287S had a 34.5% increase. The increase in the enzyme activity of the mutants indicates that these mutants have a stronger ability to catalyze subsequent reactions.

[0042] VI. Inspection of Ethyl Ester - type Fish Oil Hydrolysis Reaction Use ethyl ester type fish oil with an initial EPA content of 56% as the reaction raw material: Take 15 mL of fish oil, add the prepared enzyme solution, and then add water to make up to 22.5 mL (1.5 times the volume of fish oil). The rotation speed is 400 rpm, and at the same time, perform a vacuum pumping operation. After reacting for 2 - 3 h, perform an acid value detection.

[0043] Acid value detection: Take about 5 g of the upper layer of oil, add 20 mL of acid value reagent (ethanol and ether are mixed at 1:1 (V / V)), then add 3 drops of phenolphthalein, and titrate with 0.1 mol / L sodium hydroxide standard titration solution until the sample solution turns light pink and does not fade after shaking for 30 s, and record the volume of sodium hydroxide titrant consumed.

[0044] Acid value (NaOH mg / g) = 56.11 * V * C / W, where: W - the weight of the sample, g; V - V1 - VO (the volume of the titrant consumed by the sample minus the volume of the titrant consumed by the blank), mL.

[0045] C - the concentration of the sodium hydroxide standard titration solution, mol / L.

[0046] The acid value is a measurement standard for the number of free carboxylic acid groups in a compound (such as fatty acids) or a mixture. In this reaction, it can reflect the ability of the enzyme to hydrolyze ethyl ester type fish oil into ethanol and free fatty acids.

[0047] The acid value detection results are shown in Table 4 below: Table 4 Acid value detection result table

[0048] According to the results shown in Table 4, the acid values of the constructed mutants are all higher than that of the starting strain wild type WT, indicating that these mutants have a stronger ability to catalyze this hydrolysis reaction.

[0049] When the acid value is greater than 20, take more than 10 g of oil for caustic refining operation: Calculation of NaOH addition amount: (0.714 * mass of oil (g) * acid value) / 1000.

[0050] Add NaOH to the oil, stir with a homogenizer until the reaction is complete, centrifuge to collect the oil, and wash it three times with hot water until the oil is clear and transparent.

[0051] Detection of each component content: Take 30 mg of the sample to be tested in a 10 mL volumetric flask, dissolve and dilute to the mark with the internal standard solution. Accurately pipette 2.0 mL into a glass test tube, and slowly blow off the solvent with nitrogen. Add 1.5 mL of 2% (w / v) sodium hydroxide - methanol solution, cover tightly with a lid lined with polytetrafluoroethylene, mix well and heat in a boiling water bath for 7 min. Cool, add 2 mL of boron trichloride - methanol solution, cover the bottle stopper tightly with nitrogen, mix well, heat in a boiling water bath for 30 min, cool to 40 - 50 °C, add 1 mL of isooctane, cover tightly, vortex mix or shake for at least 30 s, and immediately add 5 mL of saturated sodium chloride solution (containing 1 volume of sodium chloride and 2 volumes of water). Fill with nitrogen, cover the stopper, vortex mix or shake for at least 15 s. After standing for phase separation, transfer the supernatant to a test tube, shake and separate layers again with 1 mL of isooctane, combine the supernatant with the previous one, then wash twice with water, 1 mL each time, dry the extract with anhydrous sodium sulfate, and transfer the supernatant to a 2 mL brown injection vial for GC detection of the product.

[0052] The chromatographic column material is a fused silica capillary column with a size of 0.25 mm × 25 m, the stationary phase is a polyethylene glycol stationary liquid, the detector temperature is 270 °C, the injection port temperature is 250 °C, the column temperature is 170 °C for the first 2 min, then increases at a rate of 3 °C per minute to 240 °C and holds for 15 min, the carrier gas is nitrogen / helium, the split ratio is 1:200, and the flow rate is 1 mL / min.

[0053] The GC results are shown in Table 5 below. Compared with the starting strain WT, the EPA content of each mutant strain has been significantly improved. Among them, the EPA content of the optimal mutant strain P131S reaches 73.18%, which is 33% higher than the initial EPA content and 11.7% higher than that of WT.

[0054] Table 5 EPA content table

[0055] Therefore, it can be seen that the Burkholderia lipase mutant provided by the present invention shows higher selectivity for EPA, which can enrich and provide more possibilities for the industrial production of EPA, and also provides some insights for the engineering transformation of other multi - domain proteins.

[0056] The LipA amino acid and nucleotide sequences of the wild - type Burkholderia lipase are as follows: LipA amino acid sequence - SEQ ID No. 21: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. LipA nucleotide sequence - SEQ ID NO.22: GCCGATGGCTACGCGGCGACGCATTACCCGATCATCCTCGTTCACGGGCTCTCGGGTACCGACAAGTACGCGGGCGTGCTCGAGTATTGGTACGGCATCCAGGAGGACCTGCAACAGAACGGTGCGACCGTCTACGTCGCGAACCTGTCGGGTTTCCAGAGCGACGACGGCCCGAACGGGCGCGGCGAACAGTTGCTCGCTTACGTGAAGACGGTGCTCGCGGCGACGGGGGCGACCAAGGTGAATCTCGTCGGCCACAGCCAGGGCGGCCTCTCGTCGCGCTATGTTGCTGCCGTCGCGCCCGATCTCGTTGCGTCGGTGACGACGATCGGCACGCCGCATCGCGGCTCGGAATTCGCCGACTTCGTGCAGAACGTGCTGGCGTACGATCCGACCGGGCTTTCGTCATCGGTGATCGCCGCGTTCGTCAATGTGTTCGGAATCCTGACGAGCAGCAGCCACAACACCAACCAGGACGCGCTCGCCGCACTGCAGACGCTGACCACCGCACGGGCCGCCACGTACAACCAGAACTATCCGAGCGCGGGCCTGGGTGCGCCGGGCAGTTGCCAGACCGGCGCGCCGACCGAAACCGTCGGCGGCAACACGCACCTGCTGTATTCGTGGGCCGGCACGGCGGTCCAGCCGACGCTCTCCGTGTTCGGCGTCACGGGTGCGACGGACACGAGCACCCTTCCGCTCGTCGATCCGGCGAACGTGCTCGACCTGTCGACGCTCGCGCTGTTCGGCACCGGAACGGTGATGATCAACCGCGGCTCCGGACAGAACGACGGGCTCGTGTCGAAGTGCAGTGCGCTGCACGGCAAGGTGCTGAGCACGAGCTACAAGTGGAACCACCTCGACGAGATCAACCAGCTGCTCGGCGTGCGCGGCGCGTATGCTGAAGATCCGGTCGCGGTGGTCCGCACGCATGTGAACCGGCTGAAGCTGGCGGGCGTGTAA. The amino acid sequences of lipA of each mutant are as follows: P131S: SEQ ID NO.23: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDSTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T132A: SEQ ID NO.24: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPAGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T251A: SEQ ID NO.25: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGAGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T251S: SEQ ID NO.26: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGSGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. P131A: SEQ ID NO.27: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDATGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. P131Q: SEQ ID NO.28: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDQTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T132G: SEQ ID NO.29: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPGGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. P113A: SEQ ID NO.30: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Q88S: SEQ ID NO.31: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Double mutant Q88S, P113A: SEQ ID NO.32: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Quadruple mutant Q88S, P113A, T132G, L287S: SEQ ID NO.33: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPGGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHSDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Quadruple mutants Q88S, P113A, P131S, L287S: SEQ ID NO.34: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDsTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHSDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Quadruple mutants Q88S, P113A, P131S, T251A: SEQ ID NO.35: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDsTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGAGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. The above-described embodiments merely represent specific implementation manners of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application.

Claims

1. There is a Burkholderia lipase with four mutations of P113A, Q88S, T132G and L287S, characterized in that the Burkholderia lipase has the following mutations: P113A: The proline at the 113th amino acid sequence of LipA of the wild-type Burkholderia lipase is mutated to alanine; and Q88S: The glutamine at the 88th amino acid sequence of LipA of the wild-type Burkholderia lipase is mutated to serine; and T132G: The threonine at the 132nd amino acid sequence of LipA of the wild-type Burkholderia lipase is mutated to glycine; and L287S: The leucine at the 287th amino acid sequence of LipA of the wild-type Burkholderia lipase is mutated to serine; The sequence of LipA of the wild-type Burkholderia lipase is shown in SEQ ID NO.

21.

2. Use of the Burkholderia lipase with four mutations of P113A, Q88S, T132G and L287S as described in claim 1 in the purification or enrichment of EPA.

3. A recombinant expression vector of Burkholderia lipase, characterized in that, It includes a plasmid backbone and a nucleotide sequence encoding the Burkholderia lipase with four mutations of P113A, Q88S, T132G and L287S as described in claim 1 inserted into the plasmid backbone.

4. The recombinant expression vector of Burkholderia lipase according to claim 3, wherein The plasmid backbone is pETDuet, pET-28a, pET-22b, pET-32a or pET-25.

5. A recombinant expression vector of Burkholderia lipase according to claim 4, characterized in that, The nucleotide sequence encoding the Burkholderia lipase with four mutations of P113A, Q88S, T132G and L287S as described in claim 1 is inserted upstream of the f1 ori site of the pETDuet plasmid.

6. The recombinant expression vector of Burkholderia lipase according to claim 5, characterized in that, The nucleotide sequence encoding the Burkholderia lipase with four mutations of P113A, Q88S, T132G and L287S as described in claim 1 includes the lipB gene and the mutated lipA gene, and the lipB, lipA and f1ori on the pETDuet plasmid are connected in series in turn.

7. Use of a recombinant expression vector of a Burkholderia lipase as described in any one of claims 3-6 in the preparation of a Burkholderia lipase.

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