Mutant of Burkholderia lipase as well as recombinant expression vector and application thereof

Abstract

The invention relates to the technical field of bioengineering, and discloses a mutant of Burkholderia lipase as well as a recombinant expression vector and application of the mutant, the mutant comprises P131S, T132A, T251A, T251S, P131A, P131Q, T132G, Q88S and P113A single processes; and a Q88S double process and a P113A double process; and a multi-site mutation combination: a combination I: Q88S, P113A, T132G and L287S, and a combination II: Q88S, P113A, T132G and L287S; a combination II: Q88S, P113A, P131S and L287S; and a combination III: Q88S, P113A, P131S and T251A. Based on experiments, the invention finds that each mutant can not only improve the protein yield, but also improve the enzyme activity by 57% at most; ePA enrichment experiments find that the EPA content of the mutant can be increased by 33% at most, and the mutant has excellent EPA purification and enrichment effects.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a mutant of Burkholderia lipase and a recombinant expression vector and application thereof. Background Art

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

[0003] Eicosapentaenoic acid (EPA), as an omega-3 polyunsaturated fatty acid, plays a vital role in human health, but the human body cannot synthesize it on its own. Studies have confirmed that EPA has multiple positive effects in maintaining cardiovascular health, anti-tumor activity, regulating glucose and lipid metabolism, and anti-inflammation. At present, EPA is mainly derived 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 (PUFA) such as EPA and docosahexanoic 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 hot topics of researchers' attention.

[0004] 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 to be a promising approach because of its mild reaction conditions, high efficiency, strong specificity, and ability to effectively reduce oxidation and isomerization. Lipases belong to the class of hydrolases, which can hydrolyze triglycerides into diglycerides, monoglycerides, and fatty acids. These enzymes also perform esterification, transesterification, and alcoholysis. However, lipases usually show different selectivity for medium-chain free fatty acids (FFA) or long-chain free fatty acids, as well as saturated or unsaturated free fatty acids. At present, researchers mainly focus on the total amount of DHA and EPA. At present, some people use Geotrichum sp. lipase to hydrolyze n-3 PUFA, selectively enriching the total content of EPA (1.53%~1.85%) and DHA (24.1%~30.9%) in fish oil. However, these lipases have low selectivity and catalytic stability in enriching EPA in fish oil.

[0005] Studies have shown 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 far from meeting market demand. Summary of the invention

[0006] The purpose of the present invention is to address the problem that the current wild-type Burkholderia cepacia lipase has limited effect in enriching EPA in fish oil, and to provide a mutant of Burkholderia cepacia lipase and a recombinant expression vector and application thereof, which can significantly improve the effect of enriching EPA in fish oil and can increase the EPA content in fish oil by 33%.

[0007] The technical solution of the present invention is as follows: In one aspect, the present invention provides a Burkholderia lipase mutant, wherein the mutant has at least one or more of the following mutations: P131S: the proline at amino acid 131 of the wild-type Burkholderia lipase LipA was mutated to serine; T132A: The threonine at amino acid 132 of the wild-type lipase LipA was mutated to alanine; T251A: The threonine in the 251st amino acid sequence of the wild-type lipase LipA was mutated to alanine; T251S: The threonine in the 251st amino acid sequence of the wild-type lipase LipA was mutated to serine; P131A: the proline in the 131st amino acid sequence of the wild-type lipase LipA was mutated to alanine; P131Q: the proline in the 131st amino acid sequence of the wild-type lipase LipA was mutated to glutamine; T132G: The threonine in the 132nd amino acid sequence of the wild-type lipase LipA was mutated to glycine; Q88S: glycine at position 88 of the amino acid sequence of wild-type lipase LipA is mutated to serine; P113A: The proline at the 113th amino acid sequence of the wild-type lipase LipA is mutated to serine.

[0008] Another aspect of the present invention provides a Burkholderia lipase mutant, wherein the mutant has two mutations as shown below: Q88S: glycine at position 88 of the amino acid sequence of the wild-type lipase is mutated to serine; and P113A: the proline at amino acid position 113 of the wild-type lipase is mutated to serine.

[0009] Another aspect of the present invention provides a Burkholderia lipase mutant, wherein the mutant has any one of the following mutation combinations: Combination 1: Q88S: glycine at position 88 of the amino acid sequence of the wild-type lipase is mutated to serine; P113A: the proline at amino acid position 113 of the wild-type lipase was mutated to serine; T132G: The threonine in the 132nd amino acid sequence of the wild-type lipase was mutated to glycine; L287S: Lysine at position 287 of the amino acid sequence of LipA of the wild-type Burkholderia lipase is mutated to serine; Combination 2: Q88S: glycine at position 88 of the amino acid sequence of the wild-type lipase is mutated to serine; P113A: the proline at amino acid position 113 of the wild-type lipase was mutated to serine; P131S: the proline at amino acid 131 of the wild-type Burkholderia lipase was mutated to serine; L287S: Lysine at position 287 of the amino acid sequence of LipA of the wild-type Burkholderia lipase is mutated to serine; Combination three: Q88S: glycine at position 88 of the amino acid sequence of the wild-type lipase is mutated to serine; P113A: the proline at amino acid position 113 of the wild-type lipase was mutated to serine; P131S: the proline at amino acid 131 of the wild-type Burkholderia lipase was mutated to serine; T251A: The threonine in the 251st amino acid sequence of the wild-type lipase is mutated to alanine.

[0010] According to a preferred embodiment, the mutation is prepared by inducing mutation primers, and the mutation primers of P131S include P131A-F as shown in SEQ ID NO.8; and P131A-R as shown in SEQ ID NO.9.

[0011] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the mutation primer of T132A includes T132A-F as shown in SEQ ID NO.10; and T132A-R as shown in SEQ ID NO.11.

[0012] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the mutation primer of T251A includes T251A-F as shown in SEQ ID NO.12; and T251A-R as shown in SEQ ID NO.13.

[0013] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the mutation primer of T251S includes T251S-F as shown in SEQ ID NO.14; and T251S-R as shown in SEQ ID NO.15.

[0014] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the mutation primers of P131A include P131A-F as shown in SEQ ID NO.16; and P131A-R as shown in SEQ ID NO.17.

[0015] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the mutation primer of P131Q includes P131N-F as shown in SEQ ID NO.26; and P131M-R as shown in SEQ ID NO.27.

[0016] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the mutation primer of T132G includes T132N-F as shown in SEQ ID NO.28; and T132N-R as shown in SEQ ID NO.29.

[0017] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the mutation primers of Q88S include Q88S-F as shown in SEQ ID NO.30; and Q88S-R as shown in SEQ ID NO.31.

[0018] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the mutation primer of P113A includes P113A-F as shown in SEQ ID NO.32; and P113A-R as shown in SEQ ID NO.33.

[0019] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the mutation primer of L287S includes L287S-F as shown in SEQ ID NO.34; and L287S-R as shown in SEQ ID NO.35.

[0020] Another aspect of the present invention provides the use of the Burkholderia lipase mutant as described above in purifying and enriching EPA.

[0021] Another aspect of the present invention provides a Burkholderia lipase recombinant expression vector, comprising a plasmid backbone and a nucleotide sequence encoding a Burkholderia lipase mutant as described above and inserted into the plasmid backbone.

[0022] According to a preferred embodiment, the plasmid backbone is pETDuet, pET-28a, pET-22b, pET-32a or pET-25.

[0023] 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.

[0024] According to a preferred embodiment, the nucleotide sequence encoding a Burkholderia lipase mutant as described above includes lipA and lipB genes, and the lipB, lipA and f1 ori on the pETDuet plasmid are connected in series in sequence, and the lipA gene sequence has the mutation as described above.

[0025] The wild type of the Burkholderia lipase is a lipase (Lipase) derived from Burkholderia sp. ZYB002, with an NCBI accession number of 573762. The lipase comprises two genes, lipA (NCBI Gene ID: 56661727) and its corresponding chaperone protein lipB (NCBI Gene ID: 56662904), and lipA has a total of 993 nucleotides and 330 amino acids.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. Mutants of Burkholderia lipase. Several mutants of Burkholderia lipase with significant effects on purifying and enriching EPA were discovered by induced mutation, involving single mutations or combined mutations at multiple sites. The mutants increased the protein yield of Burkholderia lipase, and all had better enzyme activity (up to 57%) and efficiency in catalyzing the hydrolysis of ethyl ester fish oil; they had better effects on purifying and enriching EPA; 2. After constructing a heterologous expression vector using a mutant of Burkholderia lipase, the mutant can be heterologously expressed in other fields, achieving a wider range of effects and production sources, and reducing the production cost of the mutant of Burkholderia lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a recombinant expression vector of the lipase encoding gene in the present invention; Figure 2 It is a standard curve diagram of protein solution concentration in the present invention. DETAILED DESCRIPTION

[0028] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Unless otherwise specified, 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. 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 all commercially available.

[0030] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0031] Example 1 1. Construction of mutants The lipA and lipB of Burkholderia (synthesized by Beijing Qingke Biotechnology Co., Ltd.) were used to Figure 1The 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).

[0032] Table 1 Site-directed mutagenesis primers

[0033] 2. Expression and collection of mutant proteins Stock solution preparation: Inoculate overnight culture of a single bacterial colony; The mother solution was inoculated 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.) Culture for about 4 h until OD 600 When the concentration reached 0.7-0.9, IPTG (0.1 mmol / L) was added, and the cells were collected after expressing for 48 h (180 rpm, 20°C); Collect the cells by centrifugation at 4°C, 4000 rpm, 10 min; wash the cells once with about 100 mL of pure water and once with PBS; Add PBS buffer (1:10 ratio to bacterial mass), mix well, ultrasonically disrupt (15%, ultrasonic for 4 seconds, stop for 3 seconds, 5 minutes), centrifuge twice (10000 rpm, 8 minutes); the supernatant is the protein solution containing mutant enzymes, i.e. enzyme solution. PBS buffer formula: NaCl (sodium chloride): 8g / L, KCl (potassium chloride): 0.2g / L, Na2HPO4 (disodium hydrogen phosphate): 1.44g / L, KH2PO4 (potassium dihydrogen phosphate): 0.24g / L, adjust pH to 7.4.

[0034] 3. Protein content detection Prepare standard protein solutions of different concentrations: Take 7 ordinary clean test tubes, put 0.1 mL of deionized water in tube No. 1 as a blank group, use a micropipette to add 0.02, 0.04, 0.06, 0.08, and 0.10 mL of 1.0 mg / mL BSA solution to tubes No. 2 to 6, respectively, and then add deionized water to each tube to 0.1 mL, and put 0.1 mL of the protein solution to be tested in test tube No. 7, and mix all test tubes thoroughly with a vortex mixer; Add staining reagent: Use a pipette or pipette to add 3.0 mL of Coomassie Brilliant Blue G250 reagent to each of the above test tubes, and mix gently and quickly with a vortex mixer; Colorimetric: Coomassie Brilliant Blue reagent binds to protein quickly, which can be completed in 2 to 5 minutes, and remains stable within 1 hour. Therefore, the light absorption value A of each test tube solution at a wavelength of 595 nm can be measured on a spectrophotometer after the solution has been allowed to stand for 5 minutes. 595 value; Make a standard curve: use the concentration of standard protein solution (mg / mL) as the horizontal axis and the absorbance value A as the horizontal axis. 595 Plotting with y as the ordinate, a straight line fitting can be performed to obtain a standard curve; the result is as follows Figure 2 As shown; Calculate the concentration of the protein to be tested: The light absorbance A of the protein solution to be tested measured by the spectrophotometer 595 The protein concentration can be calculated by comparing the value with the standard curve. The results are shown in Table 2.

[0035] Table 2 Protein concentration

[0036] According to the results shown in Table 2, these mutants effectively increased protein production and can greatly reduce the production cost of Burkholderia lipase in industrial production.

[0037] 4. Protein Purification First, the HisTrapTMHP affinity column was equilibrated with 5 column volumes of nickel column equilibration buffer at a flow rate of 0.5 mL / min; then, 50 mL of crude enzyme solution filtered through a 0.22 μm filter membrane was loaded into the HisTrapTMHP affinity column at a flow rate of 0.3 mL / min; after the loading was completed, the affinity column was first rinsed with more than 10 column volumes of HisTrapTMHP affinity column equilibration buffer at a flow rate of 0.5 mL / min to remove unbound proteins; finally, the HisTrapTMHP affinity column was gradient eluted with HisTrapTMHP affinity column equilibration buffer and HisTrapTMHP affinity column elution buffer. The elution conditions were: total elution volume 20 mL, flow rate 0.3 mL / min, and 2 mL / tube fractions of the eluate were collected.

[0038] 5. Enzyme activity determination The olive oil titration method for measuring lipase activity is as follows: 4 g of polyvinyl alcohol is added to 180 mL of deionized water, slowly stirred at 90 °C until completely dissolved, and cooled to 200 mL. Take 150 mL of the solution, add 50 mL of olive oil to it, and emulsify it on a tissue mixer for 3-5 min to obtain an olive oil emulsion. Take 4 mL of olive oil emulsion, add 5 mL of 50 mM pH 7.5 PB buffer solution, add 15 mL of ethanol to the blank control group in advance, and then keep it warm in a 30 °C water bath for 15 min, quickly add 1 mL of enzyme solution, stir magnetically at 800 r / min and start timing reaction for 15 min, add 15 mL of ethanol to the sample group to terminate the reaction, and finally drop 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.

[0039] Table 3 Enzyme activity

[0040] According to the results in Table 3, the enzyme activity of Q88S, P113A, P131S, and T251A increased by 57% compared with WT, Q88S, P113A, T132G, and L287S increased by 34%, and Q88S, P113A, P131S, and L287S increased by 34.5%. The increase in the enzyme activity of the mutants indicates that these mutants have a stronger ability to catalyze subsequent reactions.

[0041] 6. Ethyl ester fish oil hydrolysis reaction test Use ethyl ester 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 speed is 400 rpm, and vacuum operation is performed at the same time. After reacting for 2~3 hours, the acid value is tested.

[0042] Acid value test: Take about 5 g of the upper layer of oil, add 20 mL of acid value reagent (ethanol and ether mixed at a ratio of 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 oscillation for 30 seconds, and record the volume of sodium hydroxide titration solution consumed.

[0043] Acid value (NaOH mg / g) = 56.11*V*C / W, where: W--the weight of the sample, g; V--V1-VO (the volume of titrant consumed by the sample minus the volume of titrant consumed by the blank), mL.

[0044] C--Concentration of sodium hydroxide standard titration solution, mol / L.

[0045] The acid number is a measure of the number of free carboxylic acid groups in a compound (eg fatty acid) or mixture and, in this reaction, reflects the ability of the enzyme to hydrolyze ethyl ester fish oil into ethanol and free fatty acids.

[0046] The acid value test results are shown in Table 4 below: Table 4 Acid value test results

[0047] According to the results shown in Table 4, the acid values ​​of the constructed mutants were all higher than that of the starting strain wild type WT, indicating that these mutants have a stronger ability to catalyze the hydrolysis reaction.

[0048] When the acid value is greater than 20, take more than 10g of oil for alkali refining operation: Calculation of NaOH addition amount: (0.714*oil mass (g)*acid value) / 1000.

[0049] Add NaOH to the oil, stir with a homogenizer until fully reacted, collect the oil by centrifugation, and wash with hot water three times until the oil is clear and transparent.

[0050] Detection of content of each component: Take 30 mg of the sample to be tested in a 10 mL volumetric flask, dissolve it with the internal standard solution and dilute to the mark. Accurately transfer 2.0 mL to a glass test tube and slowly blow nitrogen to remove the solvent. Add 1.5 mL of 2% (w / v) sodium hydroxide-methanol solution, cover it with a cap lined with tetrafluoroethylene, mix well, heat it in a boiling water bath for 7 min, cool it, add 2 mL of boron trichloride-methanol solution, fill it with nitrogen, cover it with a cap, mix well, heat it in a boiling water bath for 30 min, cool it to 40-50°C, add 1 mL of isooctane, cover it tightly, vortex mix it or shake it 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 it with nitrogen, cover it with a cap, vortex mix it or shake it for at least 15 s. After standing for stratification, transfer the supernatant to a test tube, shake again with 1 mL of isooctane to separate the layers, combine the supernatant with the previous one, and 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 bottle to detect the product using GC.

[0051] The chromatographic column material is a quartz capillary column with a size of 0.25 mm×25 m. The stationary phase is 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 increased to 240°C at an increase of 3°C per minute and maintained for 15 min. The carrier gas is nitrogen / helium with a split ratio of 1:200 and a flow rate of 1 mL / min.

[0052] The GC results are shown in Table 5 below. Compared with the starting strain WT, the EPA content of each mutant strain was significantly increased, among which the EPA content of the optimal mutant strain P131S reached 73.18%, an increase of 33% over the initial EPA content and an increase of 11.7% compared with WT.

[0053] Table 5 EPA content

[0054] Therefore, it can be seen that the Burkholderia lipase mutant provided by the present invention exhibits higher selectivity for EPA, can enrich EPA, provide more possibilities for industrial production, and also provide some insights for the engineering transformation of other multi-domain proteins.

[0055] The amino acid and nucleotide sequences of the wild-type Burkholderia lipase LipA are as follows: LipA ​​amino acid sequence - SEQ ID No. 24: 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.1: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDSTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T132A:SEQ ID NO.2: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPAGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T251A:SEQ ID NO.3: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGAGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T251S:SEQ ID NO.4: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGSGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. P131A:SEQ ID NO.5: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDATGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. P131Q:SEQ ID NO.6: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDQTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T132G:SEQ ID NO.7: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPGGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. P113A:SEQ ID NO.37: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Q88S:SEQ ID NO.36: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Double mutant Q88S, P113A: SEQ ID NO.38: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Quadruple mutant Q88S, P113A, T132G, L287S: SEQ ID NO.39: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPGGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHSDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Quadruple mutant Q88S, P113A, P131S, L287S: SEQ ID NO.40: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDsTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHSDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Quadruple mutant Q88S, P113A, P131S, T251A: SEQ ID NO.41: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDsTGLSSSVIAAFVNVFGILTSSSHNTNQDA LAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGAGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A Burkholderia lipase mutant, characterized in that The mutant has at least one or more mutations as shown below: P131S: the proline at amino acid 131 of the wild-type Burkholderia lipase LipA was mutated to serine; T132A: The threonine at amino acid 132 of the wild-type lipase LipA was mutated to alanine; T251A: The threonine in the 251st amino acid sequence of the wild-type lipase LipA was mutated to alanine; T251S: The threonine in the 251st amino acid sequence of the wild-type lipase LipA was mutated to serine; P131A: the proline in the 131st amino acid sequence of the wild-type lipase LipA was mutated to alanine; P131Q: the proline in the 131st amino acid sequence of the wild-type lipase LipA was mutated to glutamine; T132G: The threonine in the 132nd amino acid sequence of the wild-type lipase LipA was mutated to glycine; Q88S: glycine at position 88 of the amino acid sequence of wild-type lipase LipA is mutated to serine; P113A: The proline at the 113th amino acid sequence of the wild-type lipase LipA is mutated to serine.

2. A Burkholderia lipase mutant according to claim 1, characterized in that: The mutation is prepared by inducing a mutation primer, and the mutation primers of P131S include P131S-F as shown in SEQ ID NO.8 and P131S-R as shown in SEQ ID NO.

9.

3. A Burkholderia lipase mutant according to claim 1, characterized in that: The mutation is prepared by inducing a mutation primer, and the mutation primer of T132A includes T132A-F as shown in SEQ ID NO.10 and T132A-R as shown in SEQ ID NO.

11.

4. The Burkholderia lipase mutant according to claim 1, characterized in that: The mutation is prepared by inducing a mutation primer, and the mutation primer of T251A includes T251A-F as shown in SEQ ID NO.12 and T251A-R as shown in SEQ ID NO.

13.

5. The Burkholderia lipase mutant according to claim 1, characterized in that: The mutation is prepared by inducing a mutation primer, and the mutation primers of T251S include T251S-F as shown in SEQ ID NO.14 and T251S-R as shown in SEQ ID NO.

15.

6. The Burkholderia lipase mutant according to claim 1, characterized in that: The mutation is prepared by inducing mutation primers, and the mutation primers of P131A include P131A-F as shown in SEQ ID NO.16 and P131A-R as shown in SEQ ID NO.

17.

7. The Burkholderia lipase mutant according to claim 1, characterized in that: The mutation is prepared by inducing mutation primers, and the mutation primers of P131Q include P131N-F as shown in SEQ ID NO.26 and P131M-R as shown in SEQ ID NO.

27.

8. The Burkholderia lipase mutant according to claim 1, characterized in that: The mutation is prepared by inducing a mutation primer, and the mutation primer of T132G includes T132N-F as shown in SEQ ID NO.28 and T132N-R as shown in SEQ ID NO.

29.

9. The Burkholderia lipase mutant according to claim 1, characterized in that: The mutation is prepared by inducing mutation primers, and the mutation primers of Q88S include Q88S-F as shown in SEQ ID NO.30 and Q88S-R as shown in SEQ ID NO.

31.

10. The Burkholderia lipase mutant according to claim 1, characterized in that: The mutation is prepared by inducing mutation primers, and the mutation primers of P113A include P113A-F as shown in SEQ ID NO.32 and P113A-R as shown in SEQ ID NO.

33.

11. A Burkholderia lipase mutant, characterized in that The mutant has two mutations as shown below: Q88S: glycine at position 88 of the amino acid sequence of the wild-type lipase is mutated to serine; and P113A: the proline at amino acid position 113 of the wild-type lipase is mutated to serine.

12. A Burkholderia lipase mutant, characterized in that The mutant has any one of the following mutation combinations: Combination 1: Q88S: glycine at position 88 of the amino acid sequence of the wild-type lipase is mutated to serine; P113A: the proline at amino acid position 113 of the wild-type lipase was mutated to serine; T132G: The threonine in the 132nd amino acid sequence of the wild-type lipase was mutated to glycine; L287S: Lysine at position 287 of the amino acid sequence was mutated to serine; Combination 2: Q88S: glycine at position 88 of the amino acid sequence of the wild-type lipase is mutated to serine; P113A: the proline at amino acid position 113 of the wild-type lipase was mutated to serine; P131S: the proline at amino acid 131 of the wild-type Burkholderia lipase was mutated to serine; L287S: Lysine at position 287 of the amino acid sequence was mutated to serine; Combination three: Q88S: glycine at position 88 of the amino acid sequence of the wild-type lipase is mutated to serine; P113A: the proline at amino acid position 113 of the wild-type lipase was mutated to serine; P131S: the proline at amino acid 131 of the wild-type Burkholderia lipase was mutated to serine; T251A: The threonine in the 251st amino acid sequence of the wild-type lipase is mutated to alanine.

13. Use of a Burkholderia lipase mutant according to any one of claims 1 to 12 in purifying and enriching EPA.

14. 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 according to any one of claims 1 to 12 and inserted into the plasmid backbone.

15. The Burkholderia lipase recombinant expression vector according to claim 14, characterized in that: The plasmid backbone is pETDuet, pET-28a, pET-22b, pET-32a or pET-25.

16. The Burkholderia lipase recombinant expression vector according to claim 15, characterized in that: The nucleotide sequence encoding a Burkholderia lipase mutant as described in any one of claims 1 to 12 is inserted upstream of the f1 ori site of the pETDuet plasmid.

17. The Burkholderia lipase recombinant expression vector according to claim 16, characterized in that: The nucleotide sequence encoding a Burkholderia lipase mutant as described in any one of claims 1-12 includes lipA and lipB genes, and the lipB, lipA and f1 ori on the pETDuet plasmid are connected in series in sequence, and the lipA gene sequence has a mutation as described in any one of claims 1-12.

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