Thermally stable lipase mutant and application thereof

Abstract

The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to a lipase mutant with high thermal stability and application thereof. The stable burkholderia lipase mutant is obtained through rational design and site-specific mutagenesis, the T5010 value of the stable burkholderia lipase mutant is increased by 5-10 DEG C, the stable burkholderia lipase mutant is constructed into an expression vector pBBRMCS2, efficient expression (4.94 KU / L) in stable burkholderia is achieved, and after being purified, an expression product can be used for developing related kits for cholesterol content detection and triglyceride detection.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a thermally stable lipase mutant and application thereof. Background Art

[0002] Lipase (EC 3.1.1.13), also known as glycerol ester hydrolase or triacylglycerol hydrolase, belongs to the α / β hydrolase fold superfamily. Lipases catalyze the hydrolysis of triglycerides, converting them into glycerol and fatty acids at the oil-water interface. Based on their regioselectivity, lipases can be divided into three categories: (1) nonspecific lipases, which catalyze the conversion of triglycerides into free fatty acids and glycerol, with monoglycerides and diglycerides as intermediates, and fatty acids can be removed from any position of the substrate under lipase catalysis; (2) 1,3-specific lipases, which release fatty acids from the first and third positions of triglycerides and cannot hydrolyze the ester bond at the second position; (3) fatty acid-specific lipases, which show fatty acid selectivity and catalyze the hydrolysis of esters with long-chain fatty acids. In addition, lipases can also catalyze the hydrolysis of cholesterol esters. Due to their wide range of catalytic properties, lipases are widely used as biocatalysts in different industries such as agrochemicals, pharmaceuticals, detergents, leather making, food and surfactant production.

[0003] At present, lipases used in commerce are mainly from bacteria. Stable Burkholderia lipase is a non-specific lipase that can be used to measure serum cholesterol levels by enzymatic methods coupled with cholesterol esterase and cholesterol oxidase, and has been widely used in clinical practice. However, the thermal stability of wild-type lipase is poor, which affects the long-term stability of the reagent. Therefore, it is of great significance to improve the thermal stability of wild-type lipase by protein engineering. Summary of the invention

[0004] The present invention aims to transform the lipase from Burkholderia stablea through rational design, obtain mutants with improved thermal stability, and produce proteins that meet the needs of clinical reagents.

[0005] A highly thermostable lipase mutant, the mutant being formed by amino acid mutations in the lipase shown in SEQ ID NO.1, wherein the amino acid mutations are selected from one or more mutations of proline at position 273, leucine at position 274, valine at position 275, alanine at position 280 or phenylalanine at position 289.

[0006] The "highly thermostable lipase mutant" described in the present invention refers to a mutated lipase having higher cholesterol esterase activity after heat treatment than wild lipase.

[0007] Preferably, the mutation is selected from one or more of P273D, L274P, V275S, A280Y or F289L.

[0008] More preferably, the mutation is selected from P273D / L274P / V275S, A280Y or F289L.

[0009] Preferably, the mutation A280Y or F289L also has glycerol esterase activity.

[0010] The glyceride lipase activity mentioned in the present invention refers to catalyzing the hydrolysis of triglycerides to generate glycerol and fatty acids.

[0011] In another aspect, the present invention discloses a biological material related to the lipase mutant, characterized in that:

[0012] The biological material is any one of the following C1) to C5):

[0013] C1) a nucleotide sequence encoding the lipase mutant;

[0014] C2) an expression cassette containing the nucleotide sequence described in C1);

[0015] C3) a recombinant vector containing the nucleotide sequence described in C1), or a recombinant vector containing the expression cassette described in C2);

[0016] C4) a recombinant microorganism containing the nucleotide sequence described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3);

[0017] C5) A transgenic cell line comprising the nucleotide sequence described in C1), or a transgenic cell line comprising the expression cassette described in C2), wherein the transgenic cell line does not include propagation material.

[0018] In the above-mentioned biological material, the vector can be a plasmid, a cosmid, a phage or a viral vector. Preferably, the recombinant vector is a pBBR-lipA / lipB recombinant vector. More preferably, the pBBR-lipA / lipB recombinant vector contains a trimethoprim resistance gene, a promoter, the nucleotide sequence and the folding enzyme sequence LipB required for its folding.

[0019] In the above biological material, the microorganism may be yeast, bacteria, algae or fungi. Preferably, the microorganism is Burkholderia.

[0020] Another aspect of the present invention discloses a reagent or a kit, wherein the reagent or the kit contains the lipase mutant.

[0021] Another aspect of the present invention discloses a method for preparing the lipase mutant, comprising introducing the coding nucleotide sequence of the lipase mutant into biological cells to express the coding gene of the lipase mutant to obtain the lipase mutant.

[0022] In another aspect, the present invention discloses any of the following applications:

[0023] E1) Use of the lipase mutant as a biocatalyst;

[0024] E2) Use of any of the above biological materials in the preparation of lipase mutants;

[0025] E3) Use of the lipase mutant in the determination of cholesterol esterase activity;

[0026] E4) use of the lipase mutant A280Y or F289L in the determination of glycerol lipase activity;

[0027] E5) Use of the lipase mutant in preparing a cholesterol content detection and triglyceride detection kit;

[0028] E6) Use of any of the above biological materials in the preparation of cholesterol content detection and triglyceride detection products.

[0029] Beneficial effects:

[0030] The present invention obtains a stable Burkholderia lipase mutant through rational design and site-directed mutagenesis. The thermal stability of the lipase mutant is greatly improved compared with the wild-type lipase, making its T 50 10 The value was increased by 5-10℃, and it was constructed into the expression vector pBBRMCS2 to achieve efficient expression (4.94KU / L) in stable Burkholderia. The expression product performed well in the reagent after purification, which can meet the clinical determination of serum triglycerides and cholesterol lipids and the development of related kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Plasmid map of the stable Burkholderia expression vector pBBR-lipA / lipB;

[0032] Figure 2 : LipA-A280Y mutant purification test results;

[0033] Figure 3 :Wild-type lipase and its mutant T 50 10 Determination of value. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the present disclosure clearer, the technical scheme in the embodiments of the present disclosure will be described clearly and completely below. If no specific conditions are specified in the examples or embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0036] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0037] Example 1 Recombinant expression host and vector modification

[0038] The stable Burkholderia used for expression testing was purchased from Beina Bio or China General Microbiological Culture Collection Center. Through antibiotic screening experiments, it was determined that this type of bacteria was insensitive to common antibiotics (ampicillin, kanamycin, chloramphenicol, etc.), and could not grow in a medium containing ≥50 mg / L trimethoprim. Therefore, the trimethoprim resistance gene (Genebank accession number: WP_015060228.1) expression cassette as shown in SEQ ID NO.2 was synthesized, and the resistance gene of pBBRMCS2 was replaced by seamless cloning, and the resulting expression vector was named pBBR_Tpr.

[0039] According to the Burkholderia lipase (Genebank accession number: BAX62350.1) and its chaperone protein (Genebank accession number: BAX62349.1) published by Genebank, the sequence shown in SEQ ID NO.3 was synthesized and constructed into the multiple cloning site of pBBR_Tpr by seamless cloning to obtain a recombinant expression plasmid, as shown in Figure 1 As shown, it was named pBBR_LipAB.

[0040] Example 2 Design and construction of LipA mutants

[0041] The free energy changes before and after the point mutation were calculated using FoldX and Rosetta software. The results are shown in Table 1.

[0042] Table 1 Calculation results of free energy changes of LipA mutants

[0043]

[0044] Through homologous sequence alignment, it was speculated that P273D / L274P / V275S had an effect on the stability of the enzyme.

[0045] The pBBR_LipAB plasmid was used as a template and the primer pairs in Table 2 were used to amplify the full-length mutant by PCR.

[0046] Table 2 Primer pairs used for mutant construction

[0047]

[0048] Example 3 Preparation of stable Burkholderia BS 352 competent cells

[0049] Take out the BS 352 strain from the -80℃ refrigerator, thaw on ice, streak on non-resistant LB solid medium, incubate at 30℃ overnight, pick a single clone in 5mL LB liquid medium, and incubate at 30℃ overnight; pipette 500μL of bacterial solution into 50mL fresh LB liquid medium, and incubate at 30℃ in a shaker until OD 600 = about 0.8, immediately ice-bath for 10 min, centrifuge at 5000 r / min for 10 min at 4°C to collect the cells, wash three times with pre-cooled electroporation buffer, resuspend in 800 μL electroporation buffer, and dispense 100 μL per tube and store BS 352 electroporation competent cells at -80°C.

[0050] Example 4 Construction of LipA expression strain

[0051] Take 50 ng of the plasmid constructed in Example 2 and add it to the BS 352 competent cells prepared in Example 3, mix well and place in an ice bath for 10 minutes, transfer to a pre-cooled 2 mm inner diameter electroporation cup, set the parameters C = 25 μF, PC = 200Ω, V = 2.5 kV, and immediately add 1 mL of SOC medium after electroporation in an electroporator. After culturing at 37°C in a shaking incubator for 2 hours, take 100 μL of the bacterial solution and spread it on LB solid medium containing 50 mg / L trimethoprim resistance and culture it in an inverted oven at 30°C overnight; the single colony grown on the resistance plate the next day is the successfully transformed lipase production strain.

[0052] Example 5 Lipase activity determination method

[0053] (I) Cholesterol esterase activity assay

[0054] Lipase catalyzes the hydrolysis of cholesterol esters into free cholesterol and free fatty acids. Cholesterol generates Δ4-cholestenone and H2O2 under the action of cholesterol oxidase (COO). H2O2 reacts in the presence of 4-aminoantipyrine and TODB to generate red quinone compounds. The activity of the enzyme is determined by colorimetric determination at 500nm and the change in absorbance per minute. Enzyme activity unit: The amount of lipase required to produce 1μmol H2O2 (or 0.5μmol quinone imine coloring substance) per minute at 37℃ with linoleic acid cholesterol ester as substrate is defined as one unit of lipase enzyme activity, and the unit is U.

[0055] (1) Main reagents:

[0056] a) 0.2 M potassium phosphate solution (A): prepare 0.2 M K2HPO4 and KH2PO4 solutions respectively, mix and adjust to pH 7.0, and keep at room temperature for use;

[0057] b) Cholesteryl linoleate solution (B): Weigh 19.5 mg of cholesterol linoleate, add 2 mL of isopropanol, and heat to completely dissolve. Mix with 40 mL of 1.0% (v / v) Triton X-100 solution (preheated at 72-74°C), and stir in a hot water bath (72-74°C) for 30 minutes. The solution will first become clear and then turbid. Slowly stir and cool to room temperature, add 0.3 g of sodium cholate and dissolve. The above solution is diluted to 50 mL with 1.0% (v / v) Triton X-100 solution. The solution can be stably stored at 4°C for 5 days;

[0058] c) 1.76% 4-AA solution (C): weigh 0.352 g of 4-aminoantipyrine and dissolve it in 20 mL of water. Store it in a brown bottle at 4°C.

[0059] d) 6.0% TODB solution (D): weigh 1.2 g TODB and dissolve it in 20 mL water. Store it in a brown bottle at 4°C.

[0060] e) 150 U / mL peroxidase solution (E): Prepare 150 PU / mL horseradish peroxidase (Toyobo, Grade III) with 0.1 M potassium phosphate solution (pH 7.0) and use it immediately;

[0061] f) Cholesterol oxidase (COO) solution (F): According to the activity of cholesterol oxidase (Toyobo, Grade III), prepare 300 U / mL COO solution with pre-cooled pure water and use it immediately after preparation;

[0062] g) Enzyme diluent (G): 20 mM potassium phosphate solution (pH 7.0, containing 2.0 mM MgCl2, 0.5 mM EDTA-Na3 and 0.2% BSA);

[0063] h) Working solution: Accurately measure 7.5 mL of (A) solution, 5 mL of (B) solution, 250 μL of (C) solution, 500 μL of (D) solution, and 500 μL of (E) solution into a brown bottle and use immediately.

[0064] (2) Sample determination: Pipette 165 μL of working solution into the well of the ELISA plate, equilibrate at 37°C for 5 min, add 6 μL of COO (F) solution, mix well, equilibrate at 37°C for 2 min, add 6 μL of enzyme solution, mix gently, and immediately place in the ELISA reader to measure the OD500nm value at 37°C for 4 min. At the same time, make a sample blank tube and add an equal amount of blank diluent (G).

[0065] (3) Vitality calculation formula:

[0066]

[0067] ΔA 500nm = ΔOD 4min -ΔOD 3min

[0068] C: protein concentration after dilution mg / mL

[0069] (Di) Glycerol lipase activity assay

[0070] In order to test the substrate selectivity of the wild type and mutants, the purchased Zhongyuan Huiji lipase detection kit was selected to detect the activity of lipase. The detection principle is: lipase can hydrolyze the substrate 1,2-o-dilauryl-glycerol-3-glutaric acid-(6-methylresorufin)-ester in the presence of co-lipase and bile acid to generate 1,2-o-dilauryl-racemic-glycerol and glutaric acid-(6-methylresorufin)-ester, which continues to decompose under alkaline conditions to form glutaric acid and red methylresorufin. The increase in absorbance caused by this red dye is proportional to the activity of lipase. Specific tests are carried out in accordance with the reagent instructions.

[0071] Example 6 Screening of thermal stability mutants

[0072] A single colony was picked from the transformation plate in Example 4 and cultured in 20 mL YS medium (30 g / L yeast powder, 30 g / L sorbitol, containing 50 mg / L trimethoprim) at 30°C and 250 rpm for 48 h-72 h, and centrifuged at 10,000 rpm for 20 min. The fermentation supernatant was transferred to an eight-tube PCR tube and placed on a PCR instrument for heat treatment at 65°C for 15 min. After the heat treatment, the tube was immediately transferred to ice and allowed to stand for 10 min, cooled at room temperature for 30 min, and cholesterol esterase activity was detected according to the method in Example 5 (I) to calculate the residual activity.

[0073] The results are shown in Table 3 below. Table 3 shows that after heat treatment at 65°C for 15 minutes, the residual activity of the wild-type lipase was 56.92%, the residual activities of mutants T46A, A64F, and V124I were all 50%, the residual activities of mutants A200F, Q302P, and S321D were close to those of the wild type, and the residual activities of mutants P273D / L274P / V275S, A280Y, and F289L were higher, at 81.73%, 69.74%, and 68.00%, respectively.

[0074] Table 3 Residual activity determination of LipA mutants after heat treatment at 65°C for 15 min

[0075]

[0076] Based on Table 3, we further explored mutations with better activity, changed the original mixed mutations into single mutations, combined the original single mutations, and explored the activity effects.

[0077] The required primers were designed according to the basic method of primer design, which is not specifically demonstrated in this example. The lipase-producing strain was prepared and the activity was determined according to the operation method in the previous example.

[0078] The results are shown in Table 4. It can be seen that when the three sites P273D / L274P / V275S mutate simultaneously, the residual activity of the lipase is higher than that of P273D, L274P, and V275S mutations alone. The simultaneous mutations of P273D / L274P / V275S / A280Y, P273D / L274P / V275S / A280Y, and A280Y / F289L are higher than their individual mutations. However, it can be seen that the individual mutations of P273D, L274P, V275S, A280Y, and F289L are significantly higher than the residual activity of the wild-type lipase, indicating that the individual mutations or combined mutations of the five sites P273D, L274P, V275S, A280Y, and F289L can improve the thermal stability of the lipase, and the thermal stability of the simultaneous mutations of multiple sites is higher than that of the single-point mutations.

[0079] Table 4 Determination of residual activity of the better mutants after heat treatment alone or in combination

[0080]

[0081] Example 7 LipA mutant purification

[0082] The purification methods of various mutants are similar. The present invention does not limit the specific operation of the purification method, as long as the purpose of purification can be achieved. This example takes the purification of LipA-A280Y mutant as an example.

[0083] A single colony was picked from the transformation plate in Example 4 and placed in 1LYS medium. The culture was carried out at 30° C. and 250 rpm for 48 h. The active yield was 4.94 KU / L.

[0084] The specific purification steps of the present invention are as follows):

[0085] 1. Add 50% saturated ammonium sulfate precipitation (25°C, 3h) to the fermentation broth supernatant, centrifuge at 12000rpm and 4°C for 30min, discard the supernatant and collect the precipitate;

[0086] 2. Redissolve the precipitate with 150 mL Buffer A, adjust the conductivity to 105 ms / cm by adding solid ammonium sulfate or Buffer B, and filter with a 0.6-0.8 μm filter membrane;

[0087] 3. Use 5mL Octyl FF filler (GE) to pack the column, equilibrate with 15mL Buffer A, and then load the above-dissolved protein solution at a flow rate of 5mL / min. Collect the flow-through during the process;

[0088] 4. After loading, use 15mL Buffer B for balance at a flow rate of 5mL / min;

[0089] 5. Use 100mL (0-100%) Buffer B for elution at a flow rate of 5mL / min;

[0090] 6. Collect the target protein eluate and dialyze into storage buffer at a ratio of 1:10. Dialyze twice at 4°C, with each time >6h; stir at 400rpm.

[0091] The relevant reagent Buffer used in this example is as follows:

[0092] Buffer A: 20mM Tris-HCl, pH 8.0, 2mM MgCl2, 1M (NH4)2SO4;

[0093] Buffer B: 20mM Tris-HCl, pH 8.0, 2mM MgCl2;

[0094] Storage Buffer: 20 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 50 mM NaCl.

[0095] The results showed that after ammonium sulfate precipitation and Octyl FF purification, the LipA target protein with high purity (such as Figure 2 shown).

[0096] Example 8 Evaluation of glycerol esterase activity and stability of wild-type and mutant LipA

[0097] Wild-type LipA and its mutants were purified by the method of Example 7, and the glycerol esterase activity was determined according to the method of Example 5 (ii), and the results are shown in Table 5. The specific activity of mutant A280Y was 128.2U / mg, and the specific activity of F289L was 172.6U / mg; compared with the commercially available Toyobo lipase LPL-311, the specific activities of mutants A280Y and F289L were increased by 62.7% and 119.0%, respectively. However, mutant P273D / L274P / V275S was inactive, indicating that the substrate specificity was improved after the 273, 274, and 275 sites were mutated to aspartic acid, proline, and serine, respectively, resulting in its inability to hydrolyze three ester bonds. When applied to total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol detection kits, mutant P273D / L274P / V275S could not react with triglycerides in serum samples, thereby avoiding the adverse effects of serum triglycerides on the test. Therefore, putting the modified lipase into production can generate extremely high economic benefits.

[0098] Table 5 Specific activity determination of wild-type LipA and its mutants

[0099]

[0100] The enzyme solution with the same protein concentration was placed at 40-70°C for 10 min, ice-bathed for 10 min, cooled at room temperature for 30 min, and the enzyme activity was determined according to the method of Example 5. The enzyme activity of the untreated lipase was defined as 100%, and the temperature corresponding to 50% residual activity was T 50 10 value.

[0101] T of wild-type lipase and positive mutant with positive effect 50 10 The results of the determination of the value are as follows Figure 3 The wild-type lipase LipA-WT T 50 10 The value was 50℃, and that of F289L was 56℃, which was 6℃ higher, while the T of LipA-A280Y 50 10 The value was 60℃, which was 10℃ higher than that of the wild type.

[0102] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A highly thermostable lipase mutant, the mutant being formed by amino acid mutations in the lipase shown in SEQ ID NO.1, wherein the amino acid mutations are selected from one or more mutations of proline at position 273, leucine at position 274, valine at position 275, alanine at position 280, or phenylalanine at position 289.

2. The lipase mutant according to claim 1, characterized in that The amino acid mutation is selected from one or more of P273D, L274P, V275S, A280Y or F289L.

3. The lipase mutant according to claim 2, characterized in that The mutation is selected from one or more of P273D / L274P / V275S, A280Y or F289L.

4. The lipase mutant according to any one of claims 1 to 3, characterized in that: The mutation A280Y or F289L also has glycerol esterase activity.

5. The biomaterial related to the lipase mutant according to any one of claims 1 to 4, characterized in that: The biological material is any one of the following C1) to C5): C1) a nucleotide sequence encoding the lipase mutant; C2) an expression cassette containing the nucleotide sequence described in C1); C3) a recombinant vector containing the nucleotide sequence described in C1), or a recombinant vector containing the expression cassette described in C2); C4) a recombinant microorganism containing the nucleotide sequence described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) A transgenic cell line comprising the nucleotide sequence described in C1), or a transgenic cell line comprising the expression cassette described in C2), wherein the transgenic cell line does not include propagation material.

6. The biomaterial related to the lipase mutant according to claim 5, characterized in that: The recombinant vector is a pBBR-lipA / lipB recombinant vector. More preferably, the pBBR-lipA / lipB recombinant vector contains a trimethoprim resistance gene, a promoter, the nucleotide sequence and a folding enzyme sequence LipB necessary for its folding.

7. The biomaterial related to the lipase mutant according to claim 5, characterized in that: The recombinant microorganism is Burkholderia.

8. A reagent or a kit, characterized in that: The reagent or kit contains the lipase mutant according to any one of claims 1-4.

9. The method for preparing the lipase mutant according to any one of claims 1 to 4, comprising introducing the coding nucleotide sequence of the lipase mutant into biological cells to express the coding gene of the lipase mutant to obtain the lipase mutant.

10. Any of the following applications is disclosed: E1) Use of the lipase mutant according to any one of claims 1 to 4 as a biocatalyst; E2) Use of the biomaterial according to any one of claims 5 to 7 in the preparation of a lipase mutant; E3) Use of the lipase mutant according to any one of claims 1 to 4 in the determination of cholesterol esterase activity; E4) Use of the lipase mutant according to claim 4 in the determination of glycerol lipase activity; E5) Use of the lipase mutant according to any one of claims 1 to 4 in preparing a cholesterol content detection and triglyceride detection kit; E6) Use of the biomaterial as described in any one of claims 5 to 7 in the preparation of products for cholesterol content detection and triglyceride detection.

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