Mutants of Burkholderia lipase, recombinant expression vectors thereof and applications

Abstract

The present invention relates to the field of bioengineering technology. The present invention discloses mutants of Burkholderia lipase, their recombinant expression vectors and applications. The mutants include single mutations of P131S, T132A, T251A, T251S, P131A, P131Q, T132G, Q88S, P113A; double mutations of Q88S and P113A; and multi-site mutation combinations: Combination 1: Q88S, P113A, T132G, L287S; Combination 2: Q88S, P113A, P131S, L287S; Combination 3: Q88S, P113A, P131S, T251A. Based on experimental findings, each mutant can not only increase the protein yield, but also improve the enzyme activity, with the highest increase of 57%. The EPA enrichment experiment found that the mutant can increase the EPA content by up to 33% at most, and has excellent EPA purification and enrichment effects.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a mutant of Burkholderia cepacia lipase, a recombinant expression vector thereof, and an application thereof. Background Art

[0002] The statements in this section only provide background information related to the disclosure of the present application and may not constitute prior art.

[0003] 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 glycolipid metabolism, and anti-inflammation. At present, 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.

[0004] The key problem that unsaturated fatty acids in fish oil are easily degraded and oxidized during the molecular distillation process 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 the class of 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. At present, 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 in fish oil are relatively low.

[0005] Research shows that Burkholderia cepacia lipase (BCL) has good selectivity for EPA and can increase the EPA content in fish oil by 22.4%. However, this content is still far from meeting the market demand. Summary of the Invention

[0006] The object of the present invention is to: in view of the limited effect of wild-type Burkholderia cepacia lipase in enriching EPA in fish oil at present, provide a mutant of Burkholderia cepacia lipase, its recombinant expression vector and application, which can significantly improve the effect of enriching EPA in fish oil and can increase the content of EPA in fish oil by 33%.

[0007] The technical solution of the present invention is as follows:

[0008] 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:

[0009] P131S: The proline at the 131st amino acid of LipA of wild-type Burkholderia cepacia lipase is mutated to serine;

[0010] T132A: The threonine at the 132nd amino acid of LipA of wild-type Burkholderia cepacia lipase is mutated to alanine;

[0011] T251A: The threonine at the 251st amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to alanine;

[0012] T251S: The threonine at the 251st amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to serine;

[0013] P131A: The proline at the 131st amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to alanine;

[0014] P131Q: The proline at the 131st amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to glutamine;

[0015] T132G: The threonine at the 132nd amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to glycine;

[0016] Q88S: The glutamine at the 88th amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to serine;

[0017] P113A: The proline at the 113th amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to alanine.

[0018] On the other hand, the present invention provides a mutant of Burkholderia cepacia lipase, and the mutant has the following 2 mutations:

[0019] Q88S: The glutamine at the 88th amino acid sequence of LipA of wild-type Burkholderia cepacia lipase is mutated to serine;

[0020] and P113A: The proline at the 113th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to alanine.

[0021] On the other hand, the present invention provides a mutant Burkholderia lipase, and the mutant has any one of the following mutation combinations:

[0022] Combination 1:

[0023] Q88S: The glutamine at the 88th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to serine;

[0024] P113A: The proline at the 113th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to alanine;

[0025] T132G: The threonine at the 132nd amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to glycine;

[0026] L287S: The leucine at the 287th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to serine;

[0027] Combination 2:

[0028] Q88S: The glutamine at the 88th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to serine;

[0029] P113A: The proline at the 113th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to alanine;

[0030] P131S: The proline at the 131st amino acid of LipA of wild-type Burkholderia lipase is mutated to serine;

[0031] L287S: The leucine at the 287th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to serine;

[0032] Combination 3:

[0033] Q88S: The glutamine at the 88th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to serine;

[0034] P113A: The proline at the 113th amino acid sequence of LipA of wild-type Burkholderia lipase is mutated to alanine;

[0035] P131S: The proline at the 131st amino acid of LipA of wild-type Burkholderia lipase is mutated to serine;

[0036] T251A: The 251st amino acid sequence of LipA of wild-type Burkholderia lipase is mutated from threonine to alanine.

[0037] According to a preferred embodiment, the mutation is induced and prepared by a mutagenic primer. The mutagenic primer for P131S includes P131S-F shown in SEQ ID NO.3; and P131S-R shown in SEQ ID NO.4.

[0038] According to a preferred embodiment, the mutation is induced and prepared by a mutagenic primer. The mutagenic primer for T132A includes T132A-F shown in SEQ ID NO.13; and T132A-R shown in SEQ ID NO.14.

[0039] According to a preferred embodiment, the mutation is induced and prepared by a mutagenic primer. The mutagenic primer for T251A includes T251A-F shown in SEQ ID NO.9; and T251A-R shown in SEQ ID NO.10.

[0040] According to a preferred embodiment, the mutation is induced and prepared by a mutagenic primer. The mutagenic primer for T251S includes T251S-F shown in SEQ ID NO.11; and T251S-R shown in SEQ ID NO.12.

[0041] According to a preferred embodiment, the mutation is induced and prepared by a mutagenic primer. The mutagenic primer for P131A includes P131A-F shown in SEQ ID NO.1; and P131A-R shown in SEQ ID NO.2.

[0042] According to a preferred embodiment, the mutation is induced and prepared by a mutagenic primer. The mutagenic primer for P131Q includes P131N-F shown in SEQ ID NO.17; and P131M-R shown in SEQ ID NO.18.

[0043] According to a preferred embodiment, the mutation is induced and prepared by a mutagenic primer. The mutagenic primer for T132G includes T132N-F shown in SEQ ID NO.19; and T132N-R shown in SEQ ID NO.20.

[0044] According to a preferred embodiment, the mutation is induced and prepared by a mutagenic primer. The mutagenic primer for Q88S includes Q88S-F shown in SEQ ID NO.5; and Q88S-R shown in SEQ ID NO.6.

[0045] According to a preferred embodiment, the mutation is induced and prepared by mutant primers. The mutant primer for P113A includes P113A-F shown in SEQ ID NO.7; and P113A-R shown in SEQ ID NO.8.

[0046] According to a preferred embodiment, the mutation is induced and prepared by mutant primers. The mutant primer for L287S includes L287S-F shown in SEQ ID NO.15; and L287S-R shown in SEQ ID NO.16.

[0047] On the other hand, the present invention provides the application of a Burkholderia lipase mutant as described above in purifying and enriching EPA.

[0048] On the other hand, the present invention provides a recombinant expression vector of Burkholderia lipase, which comprises a plasmid backbone and a nucleotide sequence encoding a Burkholderia lipase mutant as described above inserted into the plasmid backbone.

[0049] According to a preferred embodiment, the plasmid backbone is pETDuet, pET-28a, pET-22b, pET-32a or pET-25.

[0050] 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.

[0051] 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 connected in series in turn, and the lipA gene sequence has the mutation as described above.

[0052] 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.

[0053] Compared with the existing technology, the beneficial effects of the present invention are:

[0054] 1. Mutants of Burkholderia lipase: Through induced mutation, multiple mutants of Burkholderia lipase with significant effects on purifying and enriching EPA were discovered, involving single-site mutations or combined mutations at multiple sites. These mutants increased the protein yield of Burkholderia lipase and all had better enzyme activity (up to 57% increase) and the efficiency of catalyzing the hydrolysis reaction of ethyl ester-type fish oil; they had good effects on purifying and enriching EPA.

[0055] 2. After constructing an expression vector for heterologous expression with the mutants of Burkholderia lipase, the mutants can be heterologously expressed in other fields, achieving a wider range of effects and production sources, and reducing the production cost of the mutants of Burkholderia lipase. Brief Description of the Drawings

[0056] Figure 1 It is the recombinant expression vector of the lipase-encoding gene in the present invention;

[0057] Figure 2 It is the standard curve graph of the protein solution concentration in the present invention. Detailed Embodiments

[0058] 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. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer can be obtained through regular commercial purchases. To better illustrate the present invention, numerous specific details are given in the following detailed embodiments. Those skilled in the art should understand that the present invention can still be implemented without some specific details. In other embodiments, 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.

[0059] 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. Without special instructions, the units used in this specification are all 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. Without special instructions, the methods used in the present invention are conventional methods in the art; without special instructions, the reagents used in the present invention are all commercially available.

[0060] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0061] Example 1

[0062] 1. Construction of mutants

[0063] The lipA and lipB of Burkholderia (synthesized by Beijing Qingke Biotechnology Co., Ltd.) were used to Figure 1 The sequence shown was constructed on the pETDuet plasmid (purchased from EMD Biosciences (Novagen)). The mutant plasmid was constructed using the Novagen MutExpress II Fast Mutagenesis Kit V2. Log in to the Novagen official website to design primers for site-directed mutagenesis. The designed primers are shown in Table 1 below. The target plasmid was amplified using Phanta Max Super-Fidelity DNA Polymerase. The amplified product was recombinantly circularized and transformed into E. coli DE3 (BL21) (purchased from Beijing Qingke Biotechnology Co., Ltd.) competent cells. 70 μL of the bacterial solution was taken and coated with glass beads on LB solid plate culture medium containing Ampicillin (Amp, 50 mg / L) antibiotics, and inverted overnight in a 37°C incubator. Identification of recombinant products: Randomly pick 5 single colonies and add them to a 1.5mL or 2mL centrifuge tube containing 600 μL LB liquid medium containing Amp antibiotics. Shake the bacteria at 37℃ and 220 rpm for 5-6 h. When the bacterial solution becomes turbid, aliquot 100 μL for sequencing and store the remaining bacterial solution at 4℃. If the sequencing results are correct, use 60% glycerol to preserve the mutant strain (LB medium formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 15 g / L agar powder should be added to the solid culture medium).

[0064] Table 1 Site-directed mutagenesis primers

[0065]

[0066] 2. Expression and collection of mutant proteins

[0067] Stock solution preparation: Inoculate overnight culture of a single bacterial colony;

[0068] The mother solution was inoculated into TB medium (2% volume ratio);

[0069] (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.)

[0070] Culture for about 4 h until OD 600Reach 0.7 - 0.9, add IPTG (0.1 mmol / L), collect the bacterial cells after expressing for 48 h (180 rpm, 20 °C);

[0071] Centrifuge at 4000 rpm for 10 min at 4 °C to collect the bacterial cells; wash the bacterial cells once with about 100 mL of pure water and once with PBS;

[0072] Add PBS buffer (with a mass ratio of 1:10 to the bacterial cells), mix well, ultrasonically disrupt (15%, ultrasonic for 4 s, pause for 3 s, 5 min), centrifuge twice (10000 rpm, 8 min); the supernatant is the protein solution containing the mutant enzyme, i.e., the enzyme solution. PBS buffer formulation: 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.

[0073] III. Protein content detection

[0074] Prepare standard protein solutions of different concentrations: Take 7 ordinary clean test tubes. Tube 1 takes 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, 0.10 mL of 1.0 mg / mL BSA solution respectively, and then supplement each tube to 0.1 mL with deionized water. In addition, tube 7 takes 0.1 mL of the protein solution to be tested. Mix all the test tubes well with a vortex mixer;

[0075] 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;

[0076] Colorimetry: The combination of Coomassie Brilliant Blue reagent and protein is fast, and it 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 stands for 5 minutes; 595 value;

[0077] 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;

[0078] Calculate the concentration of the protein to be tested: Compare the light absorbance A 595 value 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 obtained are shown in Table 2.

[0079] Table 2 Protein Concentration

[0080]

[0081] As shown in the results of Table 2, these mutants effectively increased the protein yield, which can greatly reduce the production cost of Burkholderia lipase in industrial production.

[0082] IV. Protein Purification

[0083] 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 the 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.

[0084] V. Enzyme Activity Assay

[0085] The method for measuring lipase activity by the olive oil titration method is as follows: Add 4 g of polyvinyl alcohol 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 30 °C water bath for 15 min, quickly add 1 mL of the 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, and 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.

[0086] Table 3 Enzyme Activity

[0087]

[0088] According to the results in Table 3, the enzyme activities of Q88S, P113A, P131S, and T251A are increased by 57% compared with the WT enzyme. The enzyme activities of Q88S, P113A, T132G, and L287S are increased by 34%. The enzyme activities of Q88S, P113A, P131S, and L287S are increased by 34.5%. The increase in the enzyme activities of the mutants indicates that these mutants have stronger abilities to catalyze the subsequent reactions.

[0089] VI. Inspection of the hydrolysis reaction of ethyl ester type fish oil

[0090] Using 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 the fish oil). The rotation speed is 400 rpm, and at the same time, a vacuum operation is carried out. After reacting for 2 - 3 h, the acid value is detected.

[0091] 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 the sodium hydroxide titrant consumed.

[0092] Acid value (NaOH mg / g) = 56.11 * V * C / W, where:

[0093] W - the weight of the sample, g;

[0094] V - V1 - VO (the volume of the titrant consumed by the sample minus the volume of the titrant consumed by the blank), mL.

[0095] C - the concentration of the sodium hydroxide standard titration solution, mol / L.

[0096] 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 this enzyme to hydrolyze ethyl ester type fish oil into ethanol and free fatty acids.

[0097] The acid value detection results are shown in Table 4 below:

[0098] Table 4 Acid value detection result table

[0099]

[0100] According to the results in Table 4, the acid values of the constructed mutants are all higher than those of the starting strain wild type WT, indicating that these mutants have stronger abilities to catalyze this hydrolysis reaction.

[0101] When the acid value is greater than 20, take more than 10 g of oil for alkali refining operation:

[0102] Calculation of the amount of NaOH added: (0.714 * mass of oil (g) * acid value) / 1000.

[0103] Add NaOH to the oil, stir with a homogenizer until the reaction is complete, centrifuge to collect the oil, and wash three times with hot water until the oil is clear and transparent.

[0104] Detection of the content of each component:

[0105] 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, 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, 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, transfer the supernatant to a 2 mL brown injection vial and use GC to detect the product.

[0106] 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 starts at 170 °C for 2 min, then rises at a rate of 3 °C per minute to 240 °C and is maintained for 15 min, the carrier gas is nitrogen / helium, the split ratio is 1:200, and the flow rate is 1 mL / min.

[0107] 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.

[0108] Table 5 EPA content table

[0109]

[0110] 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.

[0111] The LipA amino acid and nucleotide sequences of the wild-type Burkholderia lipase are as follows:

[0112] LipA amino acid sequence - SEQ ID No. 21:

[0113] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0114] LipA nucleotide sequence - SEQ ID NO. 22:

[0115] GCCGATGGCTACGCGGCGACGCATTACCCGATCATCCTCGTTCACGGGCTCTCGGGTACCGACAAGTACGCGGGCGTGCTCGAGTATTGGTACGGCATCCAGGAGGACCTGCAACAGAACGGTGCGACCGTCTACGTCGCGAACCTGTCGGGTTTCCAGAGCGACGACGGCCCGAACGGGCGCGGCGAACAGTTGCTCGCTTACGTGAAGACGGTGCTCGCGGCGACGGGGGCGACCAAGGTGAATCTCGTCGGCCACAGCCAGGGCGGCCTCTCGTCGCGCTATGTTGCTGCCGTCGCGCCCGATCTCGTTGCGTCGGTGACGACGATCGGCACGCCGCATCGCGGCTCGGAATTCGCCGACTTCGTGCAGAACGTGCTGGCGTACGATCCGACCGGGCTTTCGTCATCGGTGATCGCCGCGTTCGTCAATGTGTTCGGAATCCTGACGAGCAGCAGCCACAACACCAACCAGGACGCGCTCGCCGCACTGCAGACGCTGACCACCGCACGGGCCGCCACGTACAACCAGAACTATCCGAGCGCGGGCCTGGGTGCGCCGGGCAGTTGCCAGACCGGCGCGCCGACCGAAACCGTCGGCGGCAACACGCACCTGCTGTATTCGTGGGCCGGCACGGCGGTCCAGCCGACGCTCTCCGTGTTCGGCGTCACGGGTGCGACGGACACGAGCACCCTTCCGCTCGTCGATCCGGCGAACGTGCTCGACCTGTCGACGCTCGCGCTGTTCGGCACCGGAACGGTGATGATCAACCGCGGCTCCGGACAGAACGACGGGCTCGTGTCGAAGTGCAGTGCGCTGCACGGCAAGGTGCTGAGCACGAGCTACAAGTGGAACCACCTCGACGAGATCAACCAGCTGCTCGGCGTGCGCGGCGCGTATGCTGAAGATCCGGTCGCGGTGGTCCGCACGCATGTGAACCGGCTGAAGCTGGCGGGCGTGTAA.

[0116] The amino acid sequences of lipA of each mutant are as follows:

[0117] P131S: SEQ ID NO.23:

[0118] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDSTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0119] T132A: SEQ ID NO.24:

[0120] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPAGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0121] T251A: SEQ ID NO.25:

[0122] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGAGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0123] T251S: SEQ ID NO.26:

[0124] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGSGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0125] P131A: SEQ ID NO.27:

[0126] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDATGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0127] P131Q: SEQ ID NO.28:

[0128] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDQTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0129] T132G: SEQ ID NO.29:

[0130] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPGGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0131] P113A: SEQ ID NO.30:

[0132] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0133] Q88S: SEQ ID NO.31:

[0134] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0135] Double mutant Q88S, P113A: SEQ ID NO.32:

[0136] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0137] Quadruple mutant Q88S, P113A, T132G, L287S: SEQ ID NO.33:

[0138] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPGGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHSDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0139] Quadruple mutants Q88S, P113A, P131S, L287S: SEQ ID NO.34:

[0140] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDsTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHSDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0141] Quadruple mutants Q88S, P113A, P131S, T251A: SEQ ID NO.35:

[0142] ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDsTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGAGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV.

[0143] The above-described embodiments only represent the specific implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on 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. A Burkholderia lipase mutant, characterized in that The mutant only has the following mutations: P131S: The proline at the 131st amino acid of LipA of the wild-type Burkholderia lipase is mutated to serine; the amino acid sequence of LipA of the wild-type Burkholderia lipase is shown in SEQ ID No.

21.

2. A Burkholderia lipase mutant, characterized in that The mutant only has any one of the following mutation combinations: Combination 2: Q88S: glutamine at position 88 of the amino acid sequence of LipA of the wild-type Burkholderia lipase is mutated to serine; P113A: the proline at amino acid position 113 of the wild-type Burkholderia lipase LipA was mutated to alanine; P131S: the proline at amino acid 131 of the wild-type Burkholderia lipase LipA was mutated to serine; L287S: the leucine at amino acid position 287 of the wild-type Burkholderia lipase LipA was mutated to serine; Combination three: Q88S: glutamine at position 88 of the amino acid sequence of wild-type Burkholderia lipase LipA is mutated to serine; P113A: the proline at amino acid position 113 of the wild-type Burkholderia lipase LipA was mutated to alanine; P131S: the proline at amino acid 131 of the wild-type Burkholderia lipase LipA was mutated to serine; T251A: The threonine in the 251st amino acid sequence of the wild-type Burkholderia lipase LipA was mutated to alanine; The amino acid sequence of LipA of the wild-type Burkholderia lipase is shown in SEQ ID No.

21.

3. Use of a Burkholderia lipase mutant as described in any one of claims 1-2 in purifying or enriching EPA.

4. A recombinant expression vector for Burkholderia lipase, characterized in that: The invention comprises a plasmid backbone and a nucleotide sequence encoding a Burkholderia lipase mutant as claimed in any one of claims 1 to 2 and inserted into the plasmid backbone.

5. The Burkholderia lipase recombinant expression vector according to claim 4, characterized in that: The plasmid backbone is pETDuet, pET-28a, pET-22b, pET-32a or pET-25.

6. The Burkholderia lipase recombinant expression vector according to claim 5, characterized in that: The nucleotide sequence encoding a Burkholderia lipase mutant as described in any one of claims 1-2 is inserted upstream of the f1 ori site of the pETDuet plasmid.

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